REGULATION MECHANISMS OF DROSOPHILA RETINOBLASTOMA
PROTEIN STABILITY AND REPRESSION ACTIVITY
By
Liang Zhang

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Cell and Molecular Biology – Doctor of Philosophy
2013

ABSTRACT
REGULATION MECHANISMS OF DROSOPHILA RETINOBLASTOMA PROTEIN
STABILITY AND REPRESSION ACTIVITY
By
Liang Zhang
The retinoblastoma (RB) family proteins are pivotal transcriptional corepressors involved
in diverse cellular events. As a potent tumor suppressor, RB plays a variety of important
roles including regulating cell cycle, promoting cellular differentiation, inducing cell
senescence and balancing apoptotic death, many of which are controlled by specific
post-translational modifications of RB. We identified an evolutionarily conserved
instability element (IE) in the C-terminus of Drosophila RB-related protein Rbf1 that
simultaneously regulates degradation and repression activity. Paradoxically, when the IE is
deleted, increased protein levels do not cause enhanced repression activity. Rather, these
mutations diminish repression activity of Rbf1, indicating a linkage between Rbf1 activity
and instability. I found that the IE is an independent module which may serve as an
interaction domain for multiple cofactors linking protein turnover and transcriptional
repression. I showed that loss of the IE promotes cell growth, a disastrous consequence
favorable to cancer progression. To better understand the control of this unique bifunctional
element of Rbf1, I investigated the effects of phosphorylation by Cyclin-Cdk complexes. I
show that protein stability and activity governed by the IE are cleanly separable, possibly
by coordinated interactions with E2F transcription factors and E3 ubiquitin ligases. A
highly conserved lysine residue K774 in the IE, frequently mutated in RB family protein
p130 in lung cancers, is critical for Cyc-Cdk-mediated phosphorylation. My data suggest

that the IE governs distinct functional outputs of Rbf1 through post-translational
modifications and potentially cofactor binding. The similarities with mammalian systems
suggest that parallel processes may regulate RB family proteins in human cancers.

iii

ACKNOWLEDGMENTS

I would like to thank all people who helped me during my PhD study and contributed to
this work. I am especially grateful to my mentor Dr. David Arnosti for his intelligence and
patience in shaping my creative thinking and leading me to the correct direction over the
course of the scientific training. I would like to thank my committee members Dr. Henry,
Dr. Kroos, Dr. Xiao and Dr. Conrad for their guidance and suggestions. Also, I would like
to thank all the former and present members of the Arnosti lab and the Henry lab,
especially members of our RB group, who have brought great ideas to the table and
inspired my work.
I am deeply grateful to my parents, Bing Zhang and Xiaoqiong Liang, and my wife
Jingyu Zou for their constant love and support. Without them, my success in PhD training
would be unimaginable. I also want to thank my friends at MSU and everywhere in the
world for their care and help.

TABLE OF CONTENTS

LIST OF TABLES ...................................................................................................... ix
LIST OF FIGURES ..................................................................................................... x
KEY TO ABBREVIATIONS ...................................................................................xii
CHAPTER 1 ................................................................................................................. 1
Introduction .................................................................................................................. 1
Retinoblastoma family proteins .............................................................................. 2
General properties of retinoblastoma family proteins .......................................... 2
Drosophila retinoblastoma family proteins .......................................................... 5
Functions of RB proteins ......................................................................................... 7
Cell cycle regulation .............................................................................................. 7
Cell division and genomic stability........................................................................ 8
Apoptosis .............................................................................................................. 10
Tumor suppression functions of p107 and p130 ................................................ 11
Binding partners of RB proteins ........................................................................... 12
Binding of E2F family proteins .......................................................................... 12
Binding of transcription cofactors ...................................................................... 13
Binding of E3 ubiquitin ligases........................................................................... 13
Binding of Cyclin-Cdk and phosphatase ............................................................ 14
Regulation of RB proteins ..................................................................................... 14
Viral oncoprotein binding and inactivation ....................................................... 14
Phosphorylation ................................................................................................... 15
Ubiquitination and degradation .......................................................................... 16
Other post-translational modifications ............................................................... 17
Genome-wide regulation by RB proteins ............................................................. 18
Genome-wide binding and functional targets of RB .......................................... 18
Genome-wide binding and functional targets of Rbf1 ....................................... 19
Dissertation overview ............................................................................................. 19
REFERENCES ....................................................................................................... 22
CHAPTER 2 ............................................................................................................... 32
Paradoxical instability-activity relationship defines a novel regulatory pathway for
Retinoblastoma proteins ............................................................................................ 32
Abstract ................................................................................................................... 32
Introduction ............................................................................................................ 34
Results ..................................................................................................................... 37
The Rbf1 C-terminal region encodes an instability element.............................. 37
Critical roles of lysine residues within instability element ................................. 38
v

The Rbf1 instability element contributes to repression potency ........................ 46
The Rbf1 IE is not essential for E2F interactions and promoter binding......... 48
The Rbf1 IE is a dual-function regulator of repressor potency......................... 62
Conserved instability domain of mammalian p107 ............................................ 63
Discussion ................................................................................................................ 64
Materials and Methods .......................................................................................... 75
REFERENCES ....................................................................................................... 81
CHAPTER 3 ............................................................................................................... 86
Ubiquitination of Retinoblastoma family protein 1 potentiates gene-specific repression
function ....................................................................................................................... 86
Abstract ................................................................................................................... 86
Introduction ............................................................................................................ 87
Results ..................................................................................................................... 89
A modular degron influences Rbf1 ubiquitination and stability ....................... 89
The Rbf1-IE can function independently in transcriptional repression ........... 99
Context-dependent repression by Rbf1-IE regulatory domain ........................ 100
Rbf1 ubiquitination stimulates repressor potency ............................................ 107
Discussion .............................................................................................................. 112
Materials and Methods ........................................................................................ 113
APPENDIX ........................................................................................................... 117
REFERENCES ..................................................................................................... 122
CHAPTER 4 ............................................................................................................. 127
Rbf1 degron dysfunction enhances cellular DNA replication.............................. 127
Abstract ................................................................................................................. 127
Introduction .......................................................................................................... 129
The RB/E2F regulatory nexus .......................................................................... 129
The ubiquitin-proteasome system and RB/E2F regulation ............................. 130
Results & Discussion ............................................................................................ 133
The COP9 signalosome regulates the Rbf1/E2F1 pathway ............................. 133
Drosophila Rbf1 enhances dE2F1 levels .......................................................... 134
Rbf degron mutations enhance cellular S-phase entry .................................... 144
Materials and Methods ........................................................................................ 152
REFERENCES ..................................................................................................... 156
CHAPTER 5 ............................................................................................................. 164
Differential phosphorylation events govern stability-activity linkage of Drosophila
retinoblastoma protein Rbf1 ................................................................................... 164
Abstract ................................................................................................................. 164
Introduction .......................................................................................................... 166
Results ................................................................................................................... 170
The instability element (IE) harbors phosphorylation sites critical for controlling
Rbf1 stability and activity .................................................................................. 170

vi

The N-terminal T356 plays a dominant role in Cyc-Cdk-mediated stabilization of
Rbf1 .................................................................................................................... 178
T356 is critical for Cyc-Cdk-mediated inactivation of Rbf1 ............................ 183
Phosphorylation mediates Rbf1 stabilization independently of the lysine residues in
the IE .................................................................................................................. 183
Rbf1 responsiveness to Cyc-Cdk-mediated inactivation determines Rbf1 potency in
Drosophila eye development .............................................................................. 184
K774 in the IE is critical for response to Cyc-Cdk-mediated phosphorylation185
Discussion .............................................................................................................. 196
Materials and Methods ........................................................................................ 202
APPENDIX ........................................................................................................... 204
REFERENCES ..................................................................................................... 206
CHAPTER 6 ............................................................................................................. 211
Future directions ...................................................................................................... 211
REFERENCES ..................................................................................................... 215

vii

LIST OF TABLES

Table 2-1. Rbf1 repression, stability, and localization. ........................................ 42
Table 2-2. rbf

14

rescued by transgenic Rbf1. ....................................................... 52

viii

LIST OF FIGURES

Figure 1-1. Schematic diagram of RB structure..................................................... 6
Figure 2-1. Identification of an instability element (IE) in Rbf1. ........................ 39
Figure 2-2. Conserved lysine residues in IE play critical roles in accumulation and
stability of Rbf1. ............................................................................................ 43
Figure 2-3. Expression of wild-type and IE mutant forms of Rbf1 in the Drosophila
larva................................................................................................................ 50
Figure 2-4. Rbf1 requires the IE for transcriptional repression. .......................... 53
Figure 2-5. Rbf1 IE is not essential for E2F interactions and promoter binding. 58
Figure 2-6. Rbf1 IE harbors positive and negative regulatory elements.............. 66
Figure 2-7. Severe developmental consequences of expression of hyperactive Rbf1.
........................................................................................................................ 69
Figure 2-8. Mutations in the conserved IE of p107 enhance expression. ............ 72
Figure 3-1. The instability element (IE) of Rbf1 is a modular degron. ............... 92
Figure 3-2. Rbf1 is degraded via an ubiquitin-proteasome dependent pathway. . 95
Figure 3-3. The Rbf1 instability element enhances protein ubiquitination. ......... 97
Figure 3-4. Rbf1 IE functions as a transcriptional repression domain. .............. 101
Figure 3-5. Context dependence of the Rbf1-IE for transcriptional repression. 104
Figure 3-6. Rbf1 ubiquitination enhances gene specific repression activity. ..... 109
Figure 3-S1. Identification of the Rbf1 nuclear localization sequence (NLS)... 118
Figure 3-S2. The Rbf1 C-terminal lysines (K732, K739, K740 and K754) contribute to
degron function in GFP degradation. .......................................................... 119
Figure 3-S3. Lysine residues within the Rbf1 C-terminal degron influence Rbf1
ubiquitination. ............................................................................................. 120
ix

Figure 3-S4. Lysine residues within the Rbf1 C-terminal degron participate in
enhanced GFP ubiquitination. ..................................................................... 121
Figure 4-1. Dual roles of the COP9 signalosome in regulation of the Rbf1-dE2F1
network. ...................................................................................................... 137
Figure 4-2. The Rbf1 pocket domain contributes to dE2F1 protection from
proteasome-mediated degradation. ............................................................. 139
Figure 4-3. Mutant Rbf1 lacking IE function stabilizes dE2F1 but cannot fully repress
its transcriptional activity. ........................................................................... 141
Figure 4-4. The IE region contributes to Rbf1-mediated G1 arrest. .................. 147
Figure 4-5. Mutation of the RB-family degron positively influences DNA replication
frequency. .................................................................................................... 149
Figure 5-1. Drosophila Rbf1 is subject to Cyc-Cdk-mediated inactivation and
stabilization. ................................................................................................ 173
Figure 5-2. Three serine residues in the IE are critical for Rbf1 protein stability and
repression activity. ...................................................................................... 175
Figure 5-3. N-terminal T356 is a key residue for the regulation of Rbf1 protein levels.
.................................................................................................................... 179
Figure 5-4. T356 is critical for Cyc-Cdk-mediated inactivation of transcriptional
repression activity. ...................................................................................... 186
Figure 5-5. Conserved serine residues within the IE influence Rbf1 stability
independently of the lysine residues in the IE. ........................................... 189
Figure 5-6. Dramatic developmental defects induced by Cyc-Cdk-resistant Rbf1
proteins........................................................................................................ 191
Figure 5-7. K774 influences Cyc-Cdk control of both Rbf1 protein levels and
transcriptional activity. ................................................................................ 193
Figure 5-8. Model for the phosphorylation-mediated functional inactivation and
stabilization of Rbf1.................................................................................... 200
Figure 5-S1. The IE alone is insufficient for Cyc-Cdk-mediated stabilization.. 205

x

KEY TO ABBREVIATIONS
c-Abl

Cellular Abelson murine leukemia

c-Myc

Cellular Myelocytomatosis viral oncogene C

CBP

CREB-Binding Protein

cdc2

cell division cycle 2

Cdk

Cyclin dependent kinase

CNS

Central Nervous System

COP9

Constitutive Photomorphogenic 9

CSN

COP9 Signalosome

dCAP-D3

Chromosome Associated Protein D3

DNA

Deoxynucleic Acid

DP

E2F Dimerization Partner

E3

Ubiquitin ligase enzyme 3

E1A

Early region 1A

E2F

E2 promoter binding Factor

EID-1

EP300-interacting Inhibitor of Differentiation 1

H3K9

Histone 3 Lysine 9

H3K18

Histone 3 Lysine 18

HAT

Histone Acetyltransferase

HDAC

Histone Deacetylase Complex

HP1

Heterochromatin Protein 1

HPV E7

Human Papilloma Virus Early 7
xi

JAK/STAT

Janus Kinase and Signal Transducer and Activator of Transcription

JNK

c-Jun N-terminal Kinase

L3MBTL1

Lethal 3 Malignant Brain Tumor-Like 1

MAP

Mitogen-Activated Protein

Mdm2

Murine double minute 2

PCNA

Proliferating Cell Nuclear Antigen

PP1

Protein Phosphatases type 1

RB

Retinoblastoma protein

Rbf1

Retinoblastoma family protein 1

Rbf2

Retinoblastoma family protein 2

RING

Really Interesting New Gene

RNR2

Ribonucleotide Reductase 2

S2 cells

Schneider 2 cells

SCF

Skp/Cullin/F-box complex

Skp

S-phase kinase-associated protein

SMYD2

SET and MYND domain containing 2

SUV39H1

Suppressor of Variegation 3-9 Homolog 1

SV40

Simian Virus 40

SWI/SNF

Switch/Sucrose Nonfermenting complex

Tip60

TAT interacting protein 60

VP-16

Virion Protein 16

xii

CHAPTER 1

Introduction

The most important lesson scientists have learned in the field of cancer research over the
last decades is that a diverse spectrum of genetic and epigenetic changes occur and
accumulate to promote cancer in different ways. This heterogeneity hinders the development
of efficient therapeutic strategies. To better understand underlying properties of the disease
state, one essential task is to use model systems to understand the basic properties of
regulatory molecules that are affected in cancer, to identify general principles that can be
applied to systems of higher complexity.
In 1971, Knudson proposed a “two-hit hypothesis” to explain occurrences of sporadic and
inherited forms of retinoblastoma, a juvenile retinal cancer. He suggested that this type of
cancer was initiated by separately occurring lesions in each allele of the same gene, either
inherited from a parent or acquired during somatic development (Knudson, 1971). This rather
simple hypothesis, supported by clinical observations, ignited extensive research on tumor
suppressor genes and cancer development. The retinoblastoma protein (RB) was identified as
the first tumor suppressor due to its loss of function being associated with tumorigenesis.
After decades of biochemical and physiological studies on the retinoblastoma family
proteins, RB and the structurally related proteins p107 and p130, scientists have arrived at an
understanding of their tumor suppressor functions, which include controlling cell cycle arrest,
inducing cellular differentiation, maintaining genomic stability and inducing cell senescence
(Burkhart and Sage, 2008). Consistent with these important cellular roles of RB, the protein
is frequently inactivated in a broad range of human cancers other than retinoblastoma,
including lung cancer, melanoma, prostate cancer, breast cancer, bladder cancer, leukemia,

1

brain cancer, oesophageal cancer and liver cancer (Burkhart and Sage, 2008). Moreover,
upstream regulators of RB family proteins are also frequently mutated, attesting the
functional importance of RB and its tight regulation (Knudsen and Knudsen, 2008).
Despite diverse roles of RB in many cellular events, a common molecular mechanism is
thought to be involved; RB serves as a recruiter to assemble protein-DNA complexes or
protein-protein complexes in transcriptional-dependent or -independent manners. Numerous
partner proteins have been found to associate with RB (Morris and Dyson, 2001).
Understanding these physical and physiological interactions in different pathways can
illuminate the functional importance of RB in tumor suppression.
Due to its diverse regulatory roles, RB is subject to tight spatial and temporal control by
diverse post-translational modifications. Phosphorylation of RB is a well-studied mechanism
of manipulating RB structure and activity in response to cellular and environmental stimuli
(Rubin, 2013). Other post-translational modifications, including methylation, acetylation and
ubiquitination, are involved in different regulation pathways that control RB functional
outputs in diverse physiological conditions (Munro et al., 2012).
In this introduction, I will focus on mammalian RB family proteins and their Drosophila
counterparts. I discuss their functions, binding partners and regulatory mechanisms revealed
in diverse studies over the decades. I will also discuss the identification of physical and
functional targets of RB on a genome-wide scale, which points to exciting newly discovered
roles of RB that will be the focus of future research.

Retinoblastoma family proteins
General properties of retinoblastoma family proteins
After identification of a genetic locus on human chromosome 13 that was associated with
2

retinoblastoma, the retinoblastoma susceptibility gene was successfully cloned and found to
encode a nuclear phosphoprotein that is bound to chromatin and may regulate other genes
(Friend et al., 1986; Lee et al., 1987a; Lee et al., 1987b). The first cellular function identified
for RB is its negative regulation of the cell cycle, which provides a simple explanation for RB
tumor suppression (Goodrich et al., 1991). RB was reported as an important target of viral
oncoproteins such as adenovirus E1A, SV40 T antigen and human papilloma virus E7 protein;
mutant viral oncoproteins incapable of RB binding lose their ability to transform host cells
into tumor cells (Helin and Ed, 1993). Thus, a clear correlation between DNA tumor viruses
and RB regulation of cell proliferation has been established.
Further experimentation identified a key player in RB biology: the E2F1 transcription
factor, which was the first cellular target of RB to be identified (Helin et al., 1992; Kaelin et
al., 1992). E2F family proteins bind to and activate a variety of genes important for
progression through the cell cycle, including Cyclin A, Cyclin E, Cdc2, c-Myc and DNA
polymerase α (Helin, 1998). The current model suggests that RB cell cycle regulation is
mediated by repressive interactions with E2F family proteins, in which histone modifiers and
chromatin remodelers are recruited to promoters to inhibit E2F-dependent transcription of
cell cycle genes (Brehm and Kouzarides, 1999). RB proteins are also thought to directly
antagonize the activation domain of certain E2F proteins.
RB-mediated cell cycle regulation is controlled by Cyclin-dependent kinases (Cdks).
Phosphorylation of RB disrupts association between RB and E2F and releases transcription of
E2F-dependent genes (Buchkovich et al., 1989). Therefore, RB itself is subject to a cell
cycle-controlled regulatory network. Genetic lesions in these upstream regulators cause a
broad range of tumors, indicating that disruption of the RB pathway is a common hotspot in
tumorigenesis.
Further research has demonstrated that the cellular functions of RB as a potent tumor

3

suppressor also include genes in many categories other than cell cycle regulation, including
DNA repair, DNA replication and apoptosis, some of which involve E2F-independent
functions (Burkhart and Sage, 2008). It is widely considered that the diverse roles of RB all
contribute to its tumor suppression and may govern cell type-specific and tumor
stage-specific functions.
Identification of homologous genes containing a conserved region similar to the RB
“pocket”, the portion of the RB binding domain targeted by viral oncoproteins, revealed two
RB-related proteins, p107 and p130. Cloning of these two genes suggest that RB, p107 and
p130 share sequence homology especially in the central pocket domain that mediates
interactions with viral oncoproteins. These proteins are collectively called the pocket proteins.
RB family proteins share not only sequence similarities but also biochemical similarities.
Like RB, p107 and p130 also associate with E2F family proteins to repress E2F-dependent
transcription by recruiting common cofactors such as HDAC. Their cell cycle activities are
also regulated by Cdks (Classon and Dyson, 2001).
The highly conserved central pocket domain shared by RB family proteins contains two
well-ordered subdomains separated by an unstructured spacer (Fig. 1-1). Each subdomain
bears cyclin box folds which are well-characterized structures for protein-protein interactions
(Kim and Cho, 1997). The N-terminal domain of RB also contains cyclin fold-like structures
(Hassler et al., 2007). The less structured C-terminal domain is not well conserved in RB
family proteins but may account for some functional differences in pocket proteins.
Despite sequence and functional similarities, significant differences exist between p107,
p130 and RB. Expression levels of the pocket proteins and their association with E2F
proteins are clearly different depending on cell types. RB interacts with E2F1-4 in both
quiescent and dividing cells, whereas p107 and p130 primarily interact with E2F4 and 5.
p107 binds to E2F in the S phase of cell cycle and p130 interacts with E2F in quiescent cells

4

(Classon and Dyson, 2001). These biochemical differences indicate that the pocket proteins
may govern differential functional outputs at different stages of the cell cycle and in different
cell types.

Drosophila retinoblastoma family proteins
The pocket proteins are highly conserved in vertebrates, invertebrates and plants. The RB
homologues have been extensively studied in Drosophila melanogaster due to its simpler
RB-E2F network. Studies of RB family proteins in this model organism can provide insights
into control of diverse biological functions, such as cell cycle regulation, differentiation and
apoptosis, in different organisms.
Drosophila contains two retinoblastoma family proteins, Rbf1 and Rbf2, two E2F family
proteins E2F1 and E2F2, and one DP partner for E2F binding (van den Heuvel and Dyson,
2008). Rbf1 interacts with E2F1, a potent transcriptional activator, and E2F2, a
transcriptional repressor, while Rbf2 exclusively interacts with E2F2 (Stevaux et al., 2002).
Rbf1-knockout embryos show constitutive expression of PCNA and RNR2, two
E2F-regulated genes for DNA replication, and ectopic S-phase entry, indicating the
importance of Rbf1 for regulating E2F-dependent transcription and cell cycle progression
during embryogenesis (Du and Dyson, 1999). Rbf1 is expressed relatively uniformly at all
stages of embryonic development, while Rbf2 is dynamically expressed during development
and peaks during early embryogenesis, indicating that these two factors may have redundant
and unique roles (Keller et al., 2005; Stevaux et al., 2002). rbf1 is essential for Drosophila
development, whereas rbf2 nulls are viable, although fertility is impaired. Rbf2 has also been
found to govern tissue-specific regulation of E2F2 target genes (Stevaux et al., 2005). As
with mammalian RB proteins, Drosophila Rbf proteins are subject to phosphorylation by
Cyclin-Cdk complexes (Xin et al., 2002).

5

Figure 1-1. Schematic diagram of RB structure.

RB contains three domains: the N-terminal domain, the pocket domain and the C-terminal
domain. Two subdomains in the pocket are separated by the pocket linker (PL). Five
phosphorylation sites important for controlling conformational change of RB are indicated.
Regions in RB for protein binding are indicated by lines.

6

These studies in Drosophila do not directly address cancer-related questions, but they
illuminate essential biological functions of the well-conserved RB pathway and help us better
understand potential roles and regulation of RB in human development and cancer
progression.

Functions of RB proteins
The fact that RB is functionally inactivated in a broad range of human cancers supports the
general notion that the protein is a tumor suppressor. It was originally believed that RB tumor
suppression function was largely due to its cell cycle regulation during G1-S transition by
inhibiting E2F-dependent transcription of cell cycle genes. Now it is generally considered
that RB has many functional roles in addition to serving as a G1 checkpoint. RB might
suppress tumorigenesis by inducing cellular differentiation, promoting cell senescence and
preserving genomic stability in different cell types. RB is also associated with controlling
angiogenesis and metastasis, which are not well understood. A further complexity of RB
regulation is the dual context-dependent pro-apoptotic and anti-apoptotic functions (Burkhart
and Sage, 2008).

Cell cycle regulation
The most thoroughly studied function of RB is its interaction with sequence-specific DNA
binding E2F transcription factors in the regulation of cell proliferation. The pocket proteins
negatively regulate E2F-dependent transcription through at least two mechanisms. First, the
transactivation domain of E2F1 initiates transcription by recruiting general transcription
factors and histone acetyltransferases (Emili and Ingles, 1995; Hagemeier et al., 1993; Lang
et al., 2001; Pearson and Greenblatt, 1997). RB binding with E2F is primarily through the

7

interaction between the pocket domain of RB and the transactivation domain of E2F,
interfering with the transactivation domain’s ability to engage the transcriptional machinery
(Rubin, 2013). Second, when bound to E2F, RB also recruits a large set of histone modifiers
and

chromatin

remodeling

complexes

including

histone

deacetylases,

histone

methyltransferases, histone demethylases, DNA methyltransferases and SWI/SNF complexes
(Frolov and Dyson, 2004). However, the molecular details of these two mechanisms for gene
expression control in cell cycle are not well understood.
Histone acetylation and deacetylation seem to be a key mechanism for regulation of
E2F-dependent transcription. E2F recruits several histone acetyltransferseas (HAT) as
coactivators such as CBP, p300 and Tip60 to E2F-regulated promoters. Histone deacetylases
(HDAC) recruited by RB family proteins reverse the histone marks placed by E2F activation
complexes. RB also interacts with the histone methyltransferase SUV39H1, which methylates
H3K9 and recruits heterochromatin protein HP1. RB serves as an adaptor protein to tether
histone modifiers to the promoter and revamp the chromatin status (Munro et al., 2012).
H3K9 methylation and HP1 binding are considered to be heterochromatin markers, indicating
that RB-mediated repression may drive some E2F-regulated genes into a permanently
repressed status. In Drosophila, some genes are stably repressed in proliferating cells by
E2F2-Rbf complexes, which are resistant to cell cycle regulation (Dimova et al., 2003).
These RB-bound genes are likely involved in cellular events other than cell cycle
progression.

Cell division and genomic stability
Loss of RB leads to upregulation of E2F, which drives cells into ectopic S phase followed
by apoptosis due to genomic instability, suggesting that RB plays an essential role in
maintaining mitotic fidelity (Chau and Wang, 2003). This specific role of RB is appealing

8

because it might explain why inactivation of RB contributes to both cancer initiation and
progression.
Development of aneuploidy has been long considered as a key transition in metastatic
progression (Hartwell, 1992). Genomic instability, including deletions, inversions and
chromosomal rearrangements, caused by inappropriate mitotic chromosome segregation may
introduce mutations in tumor suppressor genes and oncogenes, which is conducive to cancer
development. This notion is well supported by identification of chromosomal alterations in
many human cancers (Albertson et al., 2003).
Inactivation of RB increases mitotic defects and genomic instability, leading to
tumorigenesis. Loss of RB function results in supernumerary centrosomes, centromeric
defects and merotelic kinetochore attachments during mitosis, which is associated with
chromosome mis-segregation (Manning and Dyson, 2011). However, there is no evidence
supporting a direct relationship between RB and mitosis. It is more likely that loss of RB
causes defects in early preparation for mitosis.
An early defect includes misregulated E2F-dependent transcription of genes required for
chromosome segregation during mitosis. One example is that expression of Mad2, an E2F
target, is upregulated by inactivation of RB. The overexpression of Mad2 is sufficient to
induce chromosome mis-segregation (Hernando et al., 2004). E2F-independent functions of
RB are also important for maintaining genomic stability. Loss of RB causes defects in
recruitment of cohesin and condensin to the centromere of chromosomes, which promotes
merotelic kinetochore attachments and chromosome mis-segregation. In Drosophila, Rbf1
also colocalizes with condensin II protein dCAP-D3 on polytene chromosomes in an
E2F-independent manner (Longworth et al., 2008).
Chromosomal instability drives tumor evolution by gradual acquisition of deleterious
mutations in tumor suppressor genes and oncogenes. Based on the mitotic defects observed

9

following inactivation of RB, it is likely that the normal tumor suppressive role of RB
involves the preservation of genomic stability.

Apoptosis
RB is involved in apparently mutually exclusive cellular events: cell proliferation and
apoptotic death. This raises one important question of how cells coordinate growth and death.
Many cancers inactivate RB by phosphorylation, rather than destabilizing or eliminating the
protein entirely, possibly because of the inhibitory function of RB on apoptosis during cancer
initiation (Knudsen and Knudsen, 2008).
One model to explain the anti-apoptotic function of RB is that the increased apoptosis in
RB-null cells is an indirect outcome caused by defects in ectopic S phase entry correlated
with loss of RB. However, some studies suggest that E2F1 is a pro-apoptotic transcription
factor and that RB represses E2F1-regulated promoters to play a negative role in apoptosis
(Field et al., 1996; Leone et al., 2001). Loss of RB in mice sensitizes cells to apoptosis in the
nervous systems, lens and skeletal muscles, which requires the activity of E2F1 and p53
(Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992). For example, apoptosis protease
activating factor-1 (Apaf1), a common target for transcriptional regulation by E2F1 and p53,
is deregulated in RB-null CNS neurons (Moroni et al., 2001). Therefore, RB has a direct role
in inhibiting apoptosis.
E2F1 is not the only mediator for the anti-apoptotic function of RB. RB can directly bind
to c-Abl tyrosine kinase and inhibit its activity, which promotes apoptosis (Wang, 2000;
Welch and Wang, 1993). The JNK kinase is also involved in stress-induced apoptosis and it
seems to be inhibited by RB (Shim et al., 2000). Taken together, RB is able to inhibit
apoptosis through interactions with other cellular proteins in an E2F1-independent manner.
However, the role of RB in apoptosis is rather complicated as an RB-dependent

10

proapoptotic function has also been reported. In response to DNA damage and oncogenic
stress, the RB-E2F1 complex binds to apoptotic genes that are transcriptionally active and RB
is required to achieve maximal apoptotic response (Ianari et al., 2009; Knudsen et al., 1999).
Taken together, RB is able to either suppress or promote apoptosis, depending on the cellular
context.

Tumor suppression functions of p107 and p130
Viral oncoproteins do not only bind to RB but also interact with the other two family
members, p107 and p130, suggesting potential functional roles of these two RB-related
proteins in tumor suppression (Dyson et al., 1989; Harlow et al., 1986). However, due to the
functional overlap within the RB family, it is difficult to distinguish the individual
contribution of RB family members in diverse cellular events.
Genetic lesions in the upstream regulators, such as Cyclin, Cdk and Cdk inhibitors, of RB
family proteins functionally inactivate all three family members by hyperphosphorylation,
suggesting that all RB family members contribute to tumor suppression (Canepa et al., 2007;
Pei and Xiong, 2005). It is known that p107 and p130 are weak tumor suppressors, since
mutations in them are rarely found in human tumors (Beroukhim et al., 2010; Burkhart and
Sage, 2008). But in the context of RB loss, p107 and p130 contribute some tumor suppression
functions. For example, mice with mutations in both RB and p107, but not mutation in RB
only, develop papillomatous lesions and squamous cell carcinomas in the epidermis (Lara et
al., 2008). RB/p107 and RB/p130-deficient mice develop many types of tumors, a larger
spectrum than tumorigenesis in RB-deficient mice, suggesting p107 and p130 contribute to
tissue-specific functional compensation in response to the loss of RB (Dannenberg et al.,
2004).

11

Binding partners of RB proteins
Consistent with the diverse cellular roles of RB, the protein has been found to associate
with numerous factors involved in different cellular events(Morris and Dyson, 2001).
Nevertheless, despite the ability of RB to associate with different partners, it is thought that
RB has a common activity as an adaptor protein to assemble protein-protein complexes and
protein-DNA complexes (Fig. 1-1). Its major binding partner E2F is involved in
transcriptional regulation of many types of genes by RB. RB exerts full repression potency by
recruiting numerous cofactors including histone modifiers and chromatin remodeling
complexes to the promoter. Moreover, because this protein has pleiotropic activities, RB itself
is tightly controlled by upstream regulators through different pathways. RB is subject to
phosphorylation and dephosphorylation mediated by Cyclin-Cdk and phosphatase.
Ubiquitination of RB targeted by Mdm2 and Skp2 is also a key mechanism for proper
depletion of RB in a timely fashion. Understanding how RB is involved in different cellular
events and is regulated through different pathways requires thorough investigation of
protein-protein interactions on a structural basis.

Binding of E2F family proteins
The best characterized interactions of RB are with E2F family proteins. E2F
transactivation domain (TD) binds to the cleft between two subdomains of the pocket (Lee et
al., 2002; Xiao et al., 2003). The RB C-terminus also makes an important second interaction
with the Marked Box (MB) domain of E2F and its heterodimer binding partner DP. Two
separate regions in the C-terminus are involved in the second interaction (Rubin, 2013).
Phosphorylation induces global conformational changes in RB to disrupt E2F association
involving at least three mechanisms. Phosphorylation of S608/S612 in the pocket linker (PL)
induces PL binding to the pocket domain to compete with E2F TD binding. Phosphorylation
12

of T373 in the N-terminus creates a hydrophobic surface to promote RBN-pocket binding
(Burke et al., 2012). Phosphorylation of S788/S795 in the C-terminus inhibits that domain
from binding to the E2F MB, while phosphorylation of T821/T826 induces RBC binding to
the pocket domain (Rubin et al., 2005).

Binding of transcription cofactors
A binding motif LXCXE is found in viral oncoproteins such as adenovirus E1A, SV40
large T-antigen and HPV E7 (Moran, 1993). Structural studies show that this motif binds to a
shallow groove in the B subdomain of the pocket domain (Lee et al., 1998). Interestingly,
histone modifiers, chromatin remodeling complexes, and the condensin II complex all bind to
RB in an LXCXE-dependent manner (Brehm and Kouzarides, 1999; Longworth et al., 2008;
Morris and Dyson, 2001). At least some of the components in these complexes have the
LXCXE sequence. For example, HDAC contains an LXCXE motif that is required for RB
binding (Brehm et al., 1998; Kouzarides, 1999; Luo et al., 1998; Magnaghi-Jaulin et al.,
1998).

Binding of E3 ubiquitin ligases
The Mdm2 E3 ubiquitin ligase physically interacts with RB and inhibits RB growth
regulatory function, and as an oncoprotein, Mdm2 is frequently overexpressed in a variety of
human cancers (Xiao et al., 1995). Mdm2 contains a central acidic region that interacts with
the C-terminus of RB (Sdek et al., 2004). The C-terminal RING finger domain of Mdm2
functions as a ubiquitin ligase to target substrate proteins, including p53 and RB. Interestingly,
Mdm2

binds

preferentially

to

hypophosphorylated

RB,

suggesting

a

potential

phosphorylation-mediated regulation of Mdm2-RB interaction (Sdek et al., 2004). In contrast,
p130 is regulated by a distinct E3 ubiquitin ligase complex, Skp1/Cul1/Skp2, which binds

13

and promotes p130 degradation, a process which requires prior phosphorylation by Cdks
(Bhattacharya et al., 2003).

Binding of Cyclin-Cdk and phosphatase
The C-terminus of RB contains a docking site for CycA/E-Cdk2 and CycD-Cdk4 (Adams
et al., 1999; Pan et al., 2001). Interestingly, it overlaps with the binding site for protein
phosphatase 1 (PP1). Competition by PP1 with Cyc-Cdk for RB binding is sufficient to block
inactivation of RB and cell cycle progression (Hirschi et al., 2010; Tamrakar and Ludlow,
2000).
Unlike RB, p107 and p130 share a conserved spacer region before A and B subdomains in
the pocket. Importantly, this region provides a high-affinity binding site for CycA/E-Cdk2
(Hannon et al., 1993; Lees et al., 1992; Li et al., 1993). This alternative mode of Cyc-Cdk
binding may provide a discriminatory regulation of RB family proteins in response to
different stimuli. Additionally, the N-terminal region of p107 and p130 seems to inhibit the
activities of Cyc-Cdk (Woo et al., 1997).

Regulation of RB proteins
Viral oncoprotein binding and inactivation
DNA tumor viruses transform host cells into proliferating cancer cells by inactivating
tumor suppressor proteins such as p53 and RB (DeCaprio, 2009). Specifically, viral
oncoproteins, such as adenovirus E1A, SV40 large T-antigen and HPV E7, sequester RB
from E2F and disrupt its function of cell cycle regulation (Helin and Ed, 1993). In addition,
RB can be phosphorylated by viral proteins mimicking cyclin-CDK function (Hume et al.,
2008).
14

Phosphorylation
The best-studied regulation mechanism of RB is phosphorylation mediated by Cyclin-Cdk
family kinases. A simple model is that during G1-S transition, RB is phosphorylated by
Cyc-Cdk and dissociates from E2F, resulting in progression of the cell into S phase (Brehm
and Kouzarides, 1999). However, structural and functional studies have provided evidence to
support that RB physically interacts with numerous proteins and that it is progressively
phosphorylated and inactivated by multiple Cyc-Cdk complexes (Mittnacht, 1998).
Several Cyc-Cdk complexes have been reported to regulate RB, namely CycD-Cdk4,
CycE-Cdk2 and CyCA-Cdk2, each of which is responsible for RB phosphorylation at a
specific time point in the cell cycle. CycD-Cdk4 is active in mid to late G1, CycE-Cdk2 in
late G1 and CycA-Cdk2 in S phase (Munro et al., 2012). The relative contribution of different
Cyc-Cdk complexes is not fully understood, however.
One indication of differential regulation functions by different Cyc-Cdk complexes comes
from the finding that they phosphorylate different sites on RB. Different Cyc-Cdk kinases
have different preferred target sites in vitro. For example, CycD-Cdk4 preferentially
phosphorylates S780 (Kitagawa et al., 1996). T826 is also specifically targeted by
CycD-Cdk4 (Zarkowska and Mittnacht, 1997). Interestingly, these two sites are different
from the typical Cdk concensus S/TPXK/R, suggesting that site selection by different
Cyc-Cdk might be determined by chemical specificity.
Differential phosphorylation patterns have distinct effects on RB functions. As described
above, several key residues in the N-terminus, the pocket and the C-terminus are
phosphorylated to induce conformational changes for the release of RB from E2F (Rubin,
2013). Moreover, phosphorylation of T821 and T826 in the C-terminus of RB is required for
dissociation of LXCXE binding proteins whereas phosphorylation of S807 and S811 is
required to disrupt c-Abl binding (Knudsen and Wang, 1996).
15

The phosphorylation mechanism is utilized for the regulation of RB not only in the context
of cell cycle progression but also during other cellular events. Under conditions of DNA
damage, RB is phosphorylated by p38 MAP kinase, which is activated by a signaling cascade
in response to stress stimuli (Delston et al., 2011). Additionally, the checkpoint kinases Chk1
and Chk2 regulate RB function on apoptotic genes during DNA damage response (Inoue et
al., 2007). These examples demonstrate that phosphorylation mediated by multiple kinases
modulates RB functions involved in different pathways, generating distinct molecular
outputs.

Ubiquitination and degradation
In addition to phosphorylation control, RB protein levels are tightly controlled by
proteolysis, which is less understood. A variety of viral oncoproteins, such as HPV E7,
human cytomegalovirus pp71 and hepatitis C virus NS5B, can bind to RB and target it for
degradation through a proteasome-dependent pathway (Boyer et al., 1996; Kalejta and Shenk,
2003; Munakata et al., 2005). As mentioned above, Mdm2, a RING finger protein, serves as a
ubiquitin E3 ligase to promote proteasome-dependent degradation of RB, and in cancers
overexpressing Mdm2, RB levels are diminished (Sdek et al., 2005; Uchida et al., 2005).
Moreover, Mdm2 mediates both ubiquitin-dependent and ubiquitin-independent pathways, as
has also been described for the degradation of p53. Mdm2-mediated degradation of RB is an
important mechanism to block RB-E2F formation and facilitate E2F-dependent transcription.
Unlike RB and p107, p130 protein levels are dramatically reduced when the protein is
hyperphosphorylated during G1-S transition in cell cycle (Tedesco et al., 2002). In this
process, Skp1-Cul1-Skp2 SCF E3 ubiquitin ligase mediates ubiquitination and degradation of
hyperphosphorylated p130 through a proteasome-dependent pathway, suggesting an
important mechanism for negative regulation of p130 transcriptional activity (Bhattacharya et

16

al., 2003).

Other post-translational modifications
Acetylation of lysine residues is also a well-studied regulation mechanism of RB. The
p300/CBP histone acetyltransferase acetylates RB on K873 and K874 during cell cycle
progression and differentiation. This acetylation hinders Cdk-mediated phosphorylation of
RB and enhances the binding of Mdm2, suggesting that acetylation may be involved in
regulating protein-protein interactions (Chan et al., 2001a). Consistent with this, acetylation
of RB during myogenesis is associated with RB hypophosphorylation. Acetylated RB recruits
Mdm2 to target EID-1, a differentiation inhibitor, for degradation to promote terminal
differentiation (Nguyen et al., 2004).
Interestingly, K873 and K874 fall within the binding motif for Cdks, providing a possible
explanation of antagonistic effects of acetylation to Cdk-mediated phosphorylation and
inactivation. Moreover, in response to DNA damage, acetylation of K873/K874 releases the
E2F1 Marked Box domain from the C-terminal interaction domain of RB, indicating that
acetylation of RB C-terminus has gene-specific regulation of RB activity (Markham et al.,
2006).
Unlike RB, acetylation of K1079 in p130 enhances Cdk4-mediated phosphorylation in
vitro, suggesting that interplay between acetylation and phosphorylation may contribute to
differential functions of the pocket proteins (Saeed et al., 2012).
The C-terminal lysine residues are also subject to methylation control. Methylation at
K873 in RB by the methyltransferase Set7/9 creates a binding site for the heterochromatin
protein HP1 involved in RB-dependent cell cycle arrest and transcriptional repression (Munro
et al., 2010). Similarly, methylation of K860 by SMYD2 provides a binding site for the
methyl-binding domain of the transcriptional repressor L3MBTL1 (Saddic et al., 2010). In

17

response to DNA damage, K810 in the C-terminus of RB can be methylated to block the
interaction between RB and Cdk4, inhibiting phosphorylation of the protein, suggesting an
interplay between methylation and phosphorylation (Carr et al., 2011).

Genome-wide regulation by RB proteins
By performing genome-wide analysis of the RB binding profile and RB-regulated
transcription in recent years, researchers have started to reveal previously less understood
functions of RB family proteins, especially for nonoverlapping cellular roles between
different RB proteins.
Genome-wide binding and functional targets of RB
Adenovirus oncoprotein E1A binding to RB family proteins is an essential step in the
transformation process of the host cells. Upon transformation, E1A induces large-scale
epigenetic and transcriptional reprogramming through relocalization of RB, p107, p130 and
the p300/CBP histone acetyltransferase, driving cells into replication mode and inhibiting
antiviral responses and cellular differentiation. This whole process is in a temporal order. E1A
first depletes RB from cell cycle genes and recruits p300/CBP to the promoters, causing
enrichment of H3K18Ac and transcriptional activation. Subsequently, RB and p130 bind to
and repress antiviral genes, induced by E1A binding. E1A also associates with p107 on
development and differentiation genes to inhibit these target genes (Ferrari et al., 2008;
Ferrari et al., 2012). These data reveal distinct roles of RB family proteins involved in
transformation-induced transcriptional reprogramming associated with epigenetic activities,
which is also possibly used in nonviral mechanisms. This idea is supported by the study of
the unique role of RB during senescence. RB plays a nonredundant role of targeting

18

E2F-dependent cell cycle genes in oncogene-induced senescence (Chicas et al., 2010).

Genome-wide binding and functional targets of Rbf1
In our recent study, we performed a genome-wide survey of Rbf1 targets in Drosophila
embryos. We found that in addition to cell cycle genes Rbf1 also targets many components of
the insulin, Hippo, JAK/STAT, Notch and other signaling pathways. Interestingly, many Rbf1
target genes identified in our study lack canonical E2F sites, suggesting that Rbf1 might be
recruited by other transcription factors involved in different regulatory programs (Acharya et
al., 2012a). However, Rbf1 binding on E2F-regulated promoters and promoters lacking E2F
sites requires DP, the binding partner of E2F, suggesting that the binding of E2F-DP
complexes to the promoter is an essential step in the recruitment of Rbf1 and that they may
bind to weak E2F sites or bind through other DNA binding proteins (Korenjak et al., 2012b).
Consistent with genome-wide binding profiles of Rbf1, both cell cycle-dependent and cell
cycle-independent functional targets are found in the genome-wide gene expression analysis
in S2 cells. Rbf1-E2F2 and Rbf2-E2F2 complexes repress a subset of genes in proliferating
cells, indicating possible functions of Rbf proteins beyond cell cycle control (Dimova et al.,
2003).

Dissertation overview
Chapter 2 follows our initial observations that Rbf1 is specifically destabilized when the
COP9 signalosome is inactivated. In this study, we characterized mutant forms of the Rbf1
protein in Drosophila and identified an instability element (IE) in the C-terminal region that
is responsible for the instability of Rbf1. Paradoxically, when the IE is deleted, increased
protein levels do not cause enhanced repression activity. Rather, these mutations diminish
repression activity of Rbf1, indicating a linkage between Rbf1 activity and instability.
19

In Chapter 3, I focus on determining how the instability element (IE) influences Rbf1
stability and activity. By assaying the IE in the context of chimeric GFP proteins, I find that
the IE is a modular degron that is able to direct the turnover of a heterologous protein through
the ubiquitination pathway. More importantly, the IE itself is a repression domain when
directly tethered to the promoter, suggesting that the IE may serve as an interaction domain
for multiple cofactors linking protein turnover and transcriptional repression. Ubiquitination
of Rbf1 not only enhances protein turnover, but also promote the repression activity of Rbf1
in a gene-specific manner. Interestingly, the IE domain of Rbf1 is not required for
transcriptional repression of certain promoters, indicating that the IE is not the sole repression
domain and that Rbf1 exerts functions through distinct mechanisms possibly by interacting
with different transcription factors.
In Chapter 4, I show that COP9 may play a dual role in regulation of E2F1, through
cullin-based turnover of E2F1, as well as control of Rbf1 levels that influence E2F1 stability.
Transcriptionally inactive Rbf1 proteins that lack the IE can still stabilize E2F1, supporting a
model that disruption of the functions of the IE may promote activity of E2F1. By performing
BrdU incorporation assays in S2 cells, I show that expression of Rbf1∆IE drives cells into
ectopic S phase while the wild-type Rbf1 causes G1 arrest, suggesting the mutant protein
confers an uncontrolled growth advantage, which provides a possible explanation to human
cancer development upon loss of RB functions.
In Chapter 5, I focus on the regulation of the Rbf1 IE by Cyclin-Cdk complexes, and
demonstrate that protein stability and activity are separable, suggesting that the
multifunctional IE acts in parallel regulatory roles involving interactions with E2F
transcription factors and E3 ubiquitin ligases. An N-terminal threonine contributes
independently to control of turnover and activity. Disruption of IE-mediated regulation of
Rbf1 induces dramatic developmental phenotypes in the Drosophila eye. Our data show

20

evidence for a complex phosphorylation code that may have gene- or stage-specific
implications for RB protein function.
In Chapter 6, I describe my current understanding of the regulation mechanisms of Rbf1
and propose research directions for our future study. We will look for potential proteins which
interact with the IE and identify IE-dependent and IE-independent functional targets. We will
further characterize the physical interaction between Rbf1 IE and E2F and investigate the
functional consequences of loss of the IE.

21

REFERENCES

22

REFERENCES

Acharya, P., Negre, N., Johnston, J., Wei, Y., White, K.P., Henry, R.W., and Arnosti, D.N.
(2012). Evidence for autoregulation and cell signaling pathway regulation from genome-wide
binding of the Drosophila retinoblastoma protein. G3 (Bethesda) 2, 1459-1472.
Adams, P.D., Li, X., Sellers, W.R., Baker, K.B., Leng, X., Harper, J.W., Taya, Y., and Kaelin,
W.G., Jr. (1999). Retinoblastoma protein contains a C-terminal motif that targets it for
phosphorylation by cyclin-cdk complexes. Mol Cell Biol 19, 1068-1080.
Albertson, D.G., Collins, C., McCormick, F., and Gray, J.W. (2003). Chromosome
aberrations in solid tumors. Nat Genet 34, 369-376.
Beroukhim, R., Mermel, C.H., Porter, D., Wei, G., Raychaudhuri, S., Donovan, J., Barretina,
J., Boehm, J.S., Dobson, J., Urashima, M., et al. (2010). The landscape of somatic
copy-number alteration across human cancers. Nature 463, 899-905.
Bhattacharya, S., Garriga, J., Calbo, J., Yong, T., Haines, D.S., and Grana, X. (2003). SKP2
associates with p130 and accelerates p130 ubiquitylation and degradation in human cells.
Oncogene 22, 2443-2451.
Boyer, S.N., Wazer, D.E., and Band, V. (1996). E7 protein of human papilloma virus-16
induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway.
Cancer Res 56, 4620-4624.
Brehm, A., and Kouzarides, T. (1999). Retinoblastoma protein meets chromatin. Trends
Biochem Sci 24, 142-145.
Brehm, A., Miska, E.A., McCance, D.J., Reid, J.L., Bannister, A.J., and Kouzarides, T. (1998).
Retinoblastoma protein recruits histone deacetylase to repress transcription. Nature 391,
597-601.
Buchkovich, K., Duffy, L.A., and Harlow, E. (1989). The retinoblastoma protein is
phosphorylated during specific phases of the cell cycle. Cell 58, 1097-1105.
Burke, J.R., Hura, G.L., and Rubin, S.M. (2012). Structures of inactive retinoblastoma
protein reveal multiple mechanisms for cell cycle control. Genes Dev 26, 1156-1166.
Burkhart, D.L., and Sage, J. (2008). Cellular mechanisms of tumour suppression by the
retinoblastoma gene. Nat Rev Cancer 8, 671-682.
23

Canepa, E.T., Scassa, M.E., Ceruti, J.M., Marazita, M.C., Carcagno, A.L., Sirkin, P.F., and
Ogara, M.F. (2007). INK4 proteins, a family of mammalian CDK inhibitors with novel
biological functions. IUBMB Life 59, 419-426.
Carr, S.M., Munro, S., Kessler, B., Oppermann, U., and La Thangue, N.B. (2011). Interplay
between lysine methylation and Cdk phosphorylation in growth control by the retinoblastoma
protein. EMBO J 30, 317-327.
Chan, H.M., Krstic-Demonacos, M., Smith, L., Demonacos, C., and La Thangue, N.B. (2001).
Acetylation control of the retinoblastoma tumour-suppressor protein. Nat Cell Biol 3,
667-674.
Chau, B.N., and Wang, J.Y. (2003). Coordinated regulation of life and death by RB. Nat Rev
Cancer 3, 130-138.
Chicas, A., Wang, X., Zhang, C., McCurrach, M., Zhao, Z., Mert, O., Dickins, R.A., Narita,
M., Zhang, M., and Lowe, S.W. (2010). Dissecting the unique role of the retinoblastoma
tumor suppressor during cellular senescence. Cancer Cell 17, 376-387.
Clarke, A.R., Maandag, E.R., van Roon, M., van der Lugt, N.M., van der Valk, M., Hooper,
M.L., Berns, A., and te Riele, H. (1992). Requirement for a functional Rb-1 gene in murine
development. Nature 359, 328-330.
Classon, M., and Dyson, N. (2001). p107 and p130: versatile proteins with interesting
pockets. Exp Cell Res 264, 135-147.
Dannenberg, J.H., Schuijff, L., Dekker, M., van der Valk, M., and te Riele, H. (2004).
Tissue-specific tumor suppressor activity of retinoblastoma gene homologs p107 and p130.
Genes Dev 18, 2952-2962.
DeCaprio, J.A. (2009). How the Rb tumor suppressor structure and function was revealed by
the study of Adenovirus and SV40. Virology 384, 274-284.
Delston, R.B., Matatall, K.A., Sun, Y., Onken, M.D., and Harbour, J.W. (2011). p38
phosphorylates Rb on Ser567 by a novel, cell cycle-independent mechanism that triggers
Rb-Hdm2 interaction and apoptosis. Oncogene 30, 588-599.
Dimova, D.K., Stevaux, O., Frolov, M.V., and Dyson, N.J. (2003). Cell cycle-dependent and
cell cycle-independent control of transcription by the Drosophila E2F/RB pathway. Genes
Dev 17, 2308-2320.
Du, W., and Dyson, N. (1999). The role of RBF in the introduction of G1 regulation during
24

Drosophila embryogenesis. EMBO J 18, 916-925.
Dyson, N., Buchkovich, K., Whyte, P., and Harlow, E. (1989). The cellular 107K protein that
binds to adenovirus E1A also associates with the large T antigens of SV40 and JC virus. Cell
58, 249-255.
Emili, A., and Ingles, C.J. (1995). Promoter-dependent photocross-linking of the acidic
transcriptional activator E2F-1 to the TATA-binding protein. J Biol Chem 270, 13674-13680.
Ferrari, R., Pellegrini, M., Horwitz, G.A., Xie, W., Berk, A.J., and Kurdistani, S.K. (2008).
Epigenetic reprogramming by adenovirus e1a. Science 321, 1086-1088.
Ferrari, R., Su, T., Li, B., Bonora, G., Oberai, A., Chan, Y., Sasidharan, R., Berk, A.J.,
Pellegrini, M., and Kurdistani, S.K. (2012). Reorganization of the host epigenome by a viral
oncogene. Genome Res 22, 1212-1221.
Field, S.J., Tsai, F.Y., Kuo, F., Zubiaga, A.M., Kaelin, W.G., Jr., Livingston, D.M., Orkin,
S.H., and Greenberg, M.E. (1996). E2F-1 functions in mice to promote apoptosis and
suppress proliferation. Cell 85, 549-561.
Friend, S.H., Bernards, R., Rogelj, S., Weinberg, R.A., Rapaport, J.M., Albert, D.M., and
Dryja, T.P. (1986). A human DNA segment with properties of the gene that predisposes to
retinoblastoma and osteosarcoma. Nature 323, 643-646.
Frolov, M.V., and Dyson, N.J. (2004). Molecular mechanisms of E2F-dependent activation
and pRB-mediated repression. J Cell Sci 117, 2173-2181.
Goodrich, D.W., Wang, N.P., Qian, Y.W., Lee, E.Y., and Lee, W.H. (1991). The
retinoblastoma gene product regulates progression through the G1 phase of the cell cycle.
Cell 67, 293-302.
Hagemeier, C., Cook, A., and Kouzarides, T. (1993). The retinoblastoma protein binds E2F
residues required for activation in vivo and TBP binding in vitro. Nucleic Acids Res 21,
4998-5004.
Hannon, G.J., Demetrick, D., and Beach, D. (1993). Isolation of the Rb-related p130 through
its interaction with CDK2 and cyclins. Genes Dev 7, 2378-2391.
Harlow, E., Whyte, P., Franza, B.R., Jr., and Schley, C. (1986). Association of adenovirus
early-region 1A proteins with cellular polypeptides. Mol Cell Biol 6, 1579-1589.

25

Hartwell, L. (1992). Defects in a cell cycle checkpoint may be responsible for the genomic
instability of cancer cells. Cell 71, 543-546.
Hassler, M., Singh, S., Yue, W.W., Luczynski, M., Lakbir, R., Sanchez-Sanchez, F., Bader, T.,
Pearl, L.H., and Mittnacht, S. (2007). Crystal structure of the retinoblastoma protein N
domain provides insight into tumor suppression, ligand interaction, and holoprotein
architecture. Mol Cell 28, 371-385.
Helin, K. (1998). Regulation of cell proliferation by the E2F transcription factors. Curr Opin
Genet Dev 8, 28-35.
Helin, K., and Ed, H. (1993). The retinoblastoma protein as a transcriptional repressor. Trends
Cell Biol 3, 43-46.
Helin, K., Lees, J.A., Vidal, M., Dyson, N., Harlow, E., and Fattaey, A. (1992). A cDNA
encoding a pRB-binding protein with properties of the transcription factor E2F. Cell 70,
337-350.
Hernando, E., Nahle, Z., Juan, G., Diaz-Rodriguez, E., Alaminos, M., Hemann, M., Michel,
L., Mittal, V., Gerald, W., Benezra, R., et al. (2004). Rb inactivation promotes genomic
instability by uncoupling cell cycle progression from mitotic control. Nature 430, 797-802.
Hirschi, A., Cecchini, M., Steinhardt, R.C., Schamber, M.R., Dick, F.A., and Rubin, S.M.
(2010). An overlapping kinase and phosphatase docking site regulates activity of the
retinoblastoma protein. Nat Struct Mol Biol 17, 1051-1057.
Hume, A.J., Finkel, J.S., Kamil, J.P., Coen, D.M., Culbertson, M.R., and Kalejta, R.F. (2008).
Phosphorylation of retinoblastoma protein by viral protein with cyclin-dependent kinase
function. Science 320, 797-799.
Ianari, A., Natale, T., Calo, E., Ferretti, E., Alesse, E., Screpanti, I., Haigis, K., Gulino, A.,
and Lees, J.A. (2009). Proapoptotic function of the retinoblastoma tumor suppressor protein.
Cancer Cell 15, 184-194.
Inoue, Y., Kitagawa, M., and Taya, Y. (2007). Phosphorylation of pRB at Ser612 by Chk1/2
leads to a complex between pRB and E2F-1 after DNA damage. EMBO J 26, 2083-2093.
Jacks, T., Fazeli, A., Schmitt, E.M., Bronson, R.T., Goodell, M.A., and Weinberg, R.A.
(1992). Effects of an Rb mutation in the mouse. Nature 359, 295-300.
Kaelin, W.G., Jr., Krek, W., Sellers, W.R., DeCaprio, J.A., Ajchenbaum, F., Fuchs, C.S.,
Chittenden, T., Li, Y., Farnham, P.J., Blanar, M.A., et al. (1992). Expression cloning of a
26

cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell 70, 351-364.
Kalejta, R.F., and Shenk, T. (2003). Proteasome-dependent, ubiquitin-independent
degradation of the Rb family of tumor suppressors by the human cytomegalovirus pp71
protein. Proc Natl Acad Sci U S A 100, 3263-3268.
Keller, S.A., Ullah, Z., Buckley, M.S., Henry, R.W., and Arnosti, D.N. (2005). Distinct
developmental expression of Drosophila retinoblastoma factors. Gene Expr Patterns 5,
411-421.
Kim, H.Y., and Cho, Y. (1997). Structural similarity between the pocket region of
retinoblastoma tumour suppressor and the cyclin-box. Nat Struct Biol 4, 390-395.
Kitagawa, M., Higashi, H., Jung, H.K., Suzuki-Takahashi, I., Ikeda, M., Tamai, K., Kato, J.,
Segawa, K., Yoshida, E., Nishimura, S., et al. (1996). The consensus motif for
phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin
A/E-Cdk2. EMBO J 15, 7060-7069.
Knudsen, E.S., and Knudsen, K.E. (2008). Tailoring to RB: tumour suppressor status and
therapeutic response. Nat Rev Cancer 8, 714-724.
Knudsen, E.S., and Wang, J.Y. (1996). Differential regulation of retinoblastoma protein
function by specific Cdk phosphorylation sites. J Biol Chem 271, 8313-8320.
Knudsen, K.E., Weber, E., Arden, K.C., Cavenee, W.K., Feramisco, J.R., and Knudsen, E.S.
(1999). The retinoblastoma tumor suppressor inhibits cellular proliferation through two
distinct mechanisms: inhibition of cell cycle progression and induction of cell death.
Oncogene 18, 5239-5245.
Knudson, A.G., Jr. (1971). Mutation and cancer: statistical study of retinoblastoma. Proc Natl
Acad Sci U S A 68, 820-823.
Korenjak, M., Anderssen, E., Ramaswamy, S., Whetstine, J.R., and Dyson, N.J. (2012). RBF
binding to both canonical E2F targets and noncanonical targets depends on functional
dE2F/dDP complexes. Mol Cell Biol 32, 4375-4387.
Kouzarides, T. (1999). Histone acetylases and deacetylases in cell proliferation. Curr Opin
Genet Dev 9, 40-48.
Lang, S.E., McMahon, S.B., Cole, M.D., and Hearing, P. (2001). E2F transcriptional
activation requires TRRAP and GCN5 cofactors. J Biol Chem 276, 32627-32634.

27

Lara, M.F., Santos, M., Ruiz, S., Segrelles, C., Moral, M., Martinez-Cruz, A.B., Hernandez,
P., Martinez-Palacio, J., Lorz, C., Garcia-Escudero, R., et al. (2008). p107 acts as a tumor
suppressor in pRb-deficient epidermis. Mol Carcinog 47, 105-113.
Lee, C., Chang, J.H., Lee, H.S., and Cho, Y. (2002). Structural basis for the recognition of the
E2F transactivation domain by the retinoblastoma tumor suppressor. Genes Dev 16,
3199-3212.
Lee, E.Y., Chang, C.Y., Hu, N., Wang, Y.C., Lai, C.C., Herrup, K., Lee, W.H., and Bradley, A.
(1992). Mice deficient for Rb are nonviable and show defects in neurogenesis and
haematopoiesis. Nature 359, 288-294.
Lee, J.O., Russo, A.A., and Pavletich, N.P. (1998). Structure of the retinoblastoma
tumour-suppressor pocket domain bound to a peptide from HPV E7. Nature 391, 859-865.
Lee, W.H., Bookstein, R., Hong, F., Young, L.J., Shew, J.Y., and Lee, E.Y. (1987a). Human
retinoblastoma susceptibility gene: cloning, identification, and sequence. Science 235,
1394-1399.
Lee, W.H., Shew, J.Y., Hong, F.D., Sery, T.W., Donoso, L.A., Young, L.J., Bookstein, R., and
Lee, E.Y. (1987b). The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein
associated with DNA binding activity. Nature 329, 642-645.
Lees, E., Faha, B., Dulic, V., Reed, S.I., and Harlow, E. (1992). Cyclin E/cdk2 and cyclin
A/cdk2 kinases associate with p107 and E2F in a temporally distinct manner. Genes Dev 6,
1874-1885.
Leone, G., Sears, R., Huang, E., Rempel, R., Nuckolls, F., Park, C.H., Giangrande, P., Wu, L.,
Saavedra, H.I., Field, S.J., et al. (2001). Myc requires distinct E2F activities to induce S
phase and apoptosis. Mol Cell 8, 105-113.
Li, Y., Graham, C., Lacy, S., Duncan, A.M., and Whyte, P. (1993). The adenovirus
E1A-associated 130-kD protein is encoded by a member of the retinoblastoma gene family
and physically interacts with cyclins A and E. Genes Dev 7, 2366-2377.
Longworth, M.S., Herr, A., Ji, J.Y., and Dyson, N.J. (2008). RBF1 promotes chromatin
condensation through a conserved interaction with the Condensin II protein dCAP-D3. Genes
Dev 22, 1011-1024.
Luo, R.X., Postigo, A.A., and Dean, D.C. (1998). Rb interacts with histone deacetylase to
repress transcription. Cell 92, 463-473.

28

Magnaghi-Jaulin, L., Groisman, R., Naguibneva, I., Robin, P., Lorain, S., Le Villain, J.P.,
Troalen, F., Trouche, D., and Harel-Bellan, A. (1998). Retinoblastoma protein represses
transcription by recruiting a histone deacetylase. Nature 391, 601-605.
Manning, A.L., and Dyson, N.J. (2011). pRB, a tumor suppressor with a stabilizing presence.
Trends Cell Biol 21, 433-441.
Markham, D., Munro, S., Soloway, J., O'Connor, D.P., and La Thangue, N.B. (2006).
DNA-damage-responsive acetylation of pRb regulates binding to E2F-1. EMBO Rep 7,
192-198.
Mittnacht, S. (1998). Control of pRB phosphorylation. Curr Opin Genet Dev 8, 21-27.
Moran, E. (1993). DNA tumor virus transforming proteins and the cell cycle. Curr Opin
Genet Dev 3, 63-70.
Moroni, M.C., Hickman, E.S., Lazzerini Denchi, E., Caprara, G., Colli, E., Cecconi, F.,
Muller, H., and Helin, K. (2001). Apaf-1 is a transcriptional target for E2F and p53. Nat Cell
Biol 3, 552-558.
Morris, E.J., and Dyson, N.J. (2001). Retinoblastoma protein partners. Adv Cancer Res 82,
1-54.
Munakata, T., Nakamura, M., Liang, Y., Li, K., and Lemon, S.M. (2005). Down-regulation of
the retinoblastoma tumor suppressor by the hepatitis C virus NS5B RNA-dependent RNA
polymerase. Proc Natl Acad Sci U S A 102, 18159-18164.
Munro, S., Carr, S.M., and La Thangue, N.B. (2012). Diversity within the pRb pathway: is
there a code of conduct? Oncogene 31, 4343-4352.
Munro, S., Khaire, N., Inche, A., Carr, S., and La Thangue, N.B. (2010). Lysine methylation
regulates the pRb tumour suppressor protein. Oncogene 29, 2357-2367.
Nguyen, D.X., Baglia, L.A., Huang, S.M., Baker, C.M., and McCance, D.J. (2004).
Acetylation regulates the differentiation-specific functions of the retinoblastoma protein.
EMBO J 23, 1609-1618.
Pan, W., Cox, S., Hoess, R.H., and Grafstrom, R.H. (2001). A cyclin D1/cyclin-dependent
kinase 4 binding site within the C domain of the retinoblastoma protein. Cancer Res 61,
2885-2891.

29

Pearson, A., and Greenblatt, J. (1997). Modular organization of the E2F1 activation domain
and its interaction with general transcription factors TBP and TFIIH. Oncogene 15,
2643-2658.
Pei, X.H., and Xiong, Y. (2005). Biochemical and cellular mechanisms of mammalian CDK
inhibitors: a few unresolved issues. Oncogene 24, 2787-2795.
Rubin, S.M. (2013). Deciphering the retinoblastoma protein phosphorylation code. Trends
Biochem Sci 38, 12-19.
Rubin, S.M., Gall, A.L., Zheng, N., and Pavletich, N.P. (2005). Structure of the Rb
C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F
release. Cell 123, 1093-1106.
Saddic, L.A., West, L.E., Aslanian, A., Yates, J.R., 3rd, Rubin, S.M., Gozani, O., and Sage, J.
(2010). Methylation of the retinoblastoma tumor suppressor by SMYD2. J Biol Chem 285,
37733-37740.
Saeed, M., Schwarze, F., Loidl, A., Meraner, J., Lechner, M., and Loidl, P. (2012). In vitro
phosphorylation and acetylation of the murine pocket protein Rb2/p130. PLoS One 7,
e46174.
Sdek, P., Ying, H., Chang, D.L., Qiu, W., Zheng, H., Touitou, R., Allday, M.J., and Xiao, Z.X.
(2005). MDM2 promotes proteasome-dependent ubiquitin-independent degradation of
retinoblastoma protein. Mol Cell 20, 699-708.
Sdek, P., Ying, H., Zheng, H., Margulis, A., Tang, X., Tian, K., and Xiao, Z.X. (2004). The
central acidic domain of MDM2 is critical in inhibition of retinoblastoma-mediated
suppression of E2F and cell growth. J Biol Chem 279, 53317-53322.
Shim, J., Park, H.S., Kim, M.J., Park, J., Park, E., Cho, S.G., Eom, S.J., Lee, H.W., Joe, C.O.,
and Choi, E.J. (2000). Rb protein down-regulates the stress-activated signals through
inhibiting c-Jun N-terminal kinase/stress-activated protein kinase. J Biol Chem 275,
14107-14111.
Stevaux, O., Dimova, D., Frolov, M.V., Taylor-Harding, B., Morris, E., and Dyson, N. (2002).
Distinct mechanisms of E2F regulation by Drosophila RBF1 and RBF2. EMBO J 21,
4927-4937.
Stevaux, O., Dimova, D.K., Ji, J.Y., Moon, N.S., Frolov, M.V., and Dyson, N.J. (2005).
Retinoblastoma family 2 is required in vivo for the tissue-specific repression of dE2F2 target
genes. Cell Cycle 4, 1272-1280.
30

Tamrakar, S., and Ludlow, J.W. (2000). The carboxyl-terminal region of the retinoblastoma
protein binds non-competitively to protein phosphatase type 1alpha and inhibits catalytic
activity. J Biol Chem 275, 27784-27789.
Tedesco, D., Lukas, J., and Reed, S.I. (2002). The pRb-related protein p130 is regulated by
phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2). Genes
Dev 16, 2946-2957.
Uchida, C., Miwa, S., Kitagawa, K., Hattori, T., Isobe, T., Otani, S., Oda, T., Sugimura, H.,
Kamijo, T., Ookawa, K., et al. (2005). Enhanced Mdm2 activity inhibits pRB function via
ubiquitin-dependent degradation. EMBO J 24, 160-169.
van den Heuvel, S., and Dyson, N.J. (2008). Conserved functions of the pRB and E2F
families. Nat Rev Mol Cell Biol 9, 713-724.
Wang, J.Y. (2000). Regulation of cell death by the Abl tyrosine kinase. Oncogene 19,
5643-5650.
Welch, P.J., and Wang, J.Y. (1993). A C-terminal protein-binding domain in the
retinoblastoma protein regulates nuclear c-Abl tyrosine kinase in the cell cycle. Cell 75,
779-790.
Woo, M.S., Sanchez, I., and Dynlacht, B.D. (1997). p130 and p107 use a conserved domain
to inhibit cellular cyclin-dependent kinase activity. Mol Cell Biol 17, 3566-3579.
Xiao, B., Spencer, J., Clements, A., Ali-Khan, N., Mittnacht, S., Broceno, C., Burghammer,
M., Perrakis, A., Marmorstein, R., and Gamblin, S.J. (2003). Crystal structure of the
retinoblastoma tumor suppressor protein bound to E2F and the molecular basis of its
regulation. Proc Natl Acad Sci U S A 100, 2363-2368.
Xiao, Z.X., Chen, J., Levine, A.J., Modjtahedi, N., Xing, J., Sellers, W.R., and Livingston,
D.M. (1995). Interaction between the retinoblastoma protein and the oncoprotein MDM2.
Nature 375, 694-698.
Xin, S., Weng, L., Xu, J., and Du, W. (2002). The role of RBF in developmentally regulated
cell proliferation in the eye disc and in Cyclin D/Cdk4 induced cellular growth. Development
129, 1345-1356.
Zarkowska, T., and Mittnacht, S. (1997). Differential phosphorylation of the retinoblastoma
protein by G1/S cyclin-dependent kinases. J Biol Chem 272, 12738-12746.

31

CHAPTER 2

Paradoxical instability-activity relationship defines a novel regulatory pathway for
Retinoblastoma proteins

Abstract
The Retinoblastoma (RB) transcriptional corepressor and related family of pocket proteins
play central roles in cell cycle control and development, and the regulatory networks
governed by these factors are frequently inactivated during tumorigenesis. During normal
growth, these proteins are subject to tight control through at least two mechanisms. First,
during cell cycle progression, repressor potential is downregulated by Cdk-dependent
phosphorylation, resulting in repressor dissociation from E2F family transcription factors.
Second, RB proteins are subject to proteasome-mediated destruction during development. To
better understand the mechanism for RB family protein instability, we characterized Rbf1
turnover in Drosophila, and the protein motifs required for its destabilization. We show that
specific point mutations in a conserved C-terminal instability element strongly stabilize Rbf1,
but strikingly, these mutations also cripple repression activity. Rbf1 is destabilized
specifically in actively proliferating tissues of the larva, indicating that controlled degradation
of Rbf1 is linked to developmental signals. The positive linkage between Rbf1 activity and its
destruction indicates that repressor function is governed in a fashion similar to that described
by the degron theory of transcriptional activation. Analogous mutations in the mammalian
RB family member p107 similarly induce abnormal accumulation, indicating substantial
conservation of this regulatory pathway.

32

This work was published as the following manuscript:
Acharya, P.*, Raj, N.*, Buckley, M. S.*, Zhang, L., Duperon, S., Williams, G., Henry, R. W.,
and Arnosti, D. N. (2010). Paradoxical instability-activity relationship defines a novel
regulatory pathway for retinoblastoma proteins. Mol. Biol. Cell. 21, 3890-3901. (*These
authors contributed equally to this work.)

My contribution to this study was the demonstration that destabilization of Rbf1 occurs
specifically in proliferating tissues of the larva, indicating that Rbf1 degradation is tightly
regulated and is likely triggered by developmental signals.

33

Introduction
Originally identified as an important player in juvenile retinal cancer, and the first example
of a tumor suppressor protein, the retinoblastoma (RB) gene product has been recognized as a
key regulator of the eukaryotic cell cycle. RB is also inactivated in a significant proportion of
adult onset human cancers (Classon and Harlow, 2002; Knudson, 1978) attesting to the
centrally important role for RB in proliferation control. Further analyses in mammals have
revealed that other RB related proteins, p130 and p107, contribute to cell cycle governance, but
the partitioning of cell cycle duties among family members is not well defined. Nonetheless,
the RB family and their cognate regulatory networks are well conserved among metazoans,
substantiating the physiological significance of RB family function (van den Heuvel and
Dyson, 2008).
As potent regulators of cellular proliferation, the activities of RB family proteins are tightly
regulated. The canonical pathway for RB family regulation is mediated by cyclin/Cdk
complexes that phosphorylate pocket proteins at key points during the cell cycle. In response,
phospho-RB dissociates from E2F binding partners, and transcription of cell cycle related
genes such as PCNA can initiate at the G1/S phase transition (Dyson, 1998). In addition to
phosphorylation control, RB protein activities are also regulated by proteolysis. During in vitro
differentiation of 3T3-L1 adipocytes, p130 levels are transiently decreased relative to p107 by
a proteasome-mediated pathway, and this switch is associated with successful differentiation
(Prince et al., 2002). RB levels can be regulated by the Mdm2 ubiquitin ligase, better known
for its control of levels of the p53 tumor suppressor, and in cancers overexpressing Mdm2, RB
levels are diminished (Sdek et al., 2005; Uchida et al., 2005). The idea that altered RB protein
levels contribute to disease etiology is further highlighted during infection by certain
oncogenic viruses that hijack the proteolytic process and induce RB family member turnover to
relieve host control of cellular proliferation (Boyer et al., 1996; Stubdal et al., 1997). Together,
34

these examples demonstrate that regulation of RB family protein levels are important for
normal cellular growth, but that these processes are often deregulated in disease.
In Drosophila, the RB family (Rbf) is comprised of two members, Rbf1 and Rbf2, and like
their mammalian counterparts, these proteins function as transcriptional corepressors that
interact with the E2F family of transcription factors (Sutcliffe et al., 2003). The Drosophila Rbf
proteins provide canonical cell cycle control functions, and they are similarly regulated by
phosphorylation involving cyclin/cdk complexes (Frolov et al., 2005; Swanhart et al., 2007;
Xin et al., 2002). Rbf proteins are further subjected to influence of their turnover rates. Our
recent studies indicated that proteasome-mediated turnover of both Rbf1 and Rbf2 is prevented
through an association with the COP9 signalosome (Ullah et al., 2007). This linkage may
contribute to COP9 control of cell cycle and development in plants and animals (Wei et al.,
2008). The COP9 signalosome consists of 8 subunits (CSN1-8), many of which exhibit limited
similarity to subunits of the 19S regulatory lid of the proteasome, suggesting that the COP9
signalosome may play a direct role in modulating protein stability, possibly via interactions
with the catalytic 20S core proteasome (Chang and Schwechheimer, 2004; Su et al., 2003). The
COP9 signalosome may also control protein degradation through interactions with and
subsequent deneddylation of the cullin subunits of SCF ubiquitin E3 ligase complexes (Wei et
al., 2008). Multiple subunits of the COP9 signalosome were found to physically associate with
Rbf proteins, and the depletion of any of these subunits lead to destabilization of both Rbf1 and
Rbf2 in cultured cells and embryos (Ullah et al., 2007), suggesting that the entire complex is
involved in stabilizing Rbf proteins. However, it is not known whether the COP9 regulation of
Rbf proteins is a constitutive process, or whether this control is regulated during development.
The CSN4 subunit of the COP9 signalosome co-occupies cell cycle regulated genes
simultaneously with Rbf proteins, suggesting that processes affecting repressor stability are
spatially and temporally linked to repressor function during gene regulation (Ullah et al.,

35

2007).
While proteasome-mediated destruction of cellular proteins is clearly linked to
downregulation of factor activity, the converse relationship has also been described, notably,
that the potency of transcriptional regulatory proteins is directly linked to processes that
mediate their destruction. This somewhat paradoxical relationship has been described for a
variety of eukaryotic transcriptional activator proteins, including c-Jun, c-Fos, Myc, E2F1, and
Gal4, all of which harbor degradation signals in regions closely overlapping with their
activation domains (Salghetti et al., 2001; Salghetti et al., 1999; Salghetti et al., 2000).
Synthetic constructs with multiple degradation domains exhibit higher levels of transcriptional
activation, suggesting that the correspondence is not just coincidental (Salghetti et al., 1999;
Salghetti et al., 2000). One proposed explanation for the tight correlation between protein
lability and increased transcriptional potency, posits that the proteasome, which is essential for
turnover of ubiquitylated substrates, also mediates transcriptional activation functions directly
(Ferdous et al., 2007; Gonzalez et al., 2002). A second mechanism suggests that activator
ubiquitylation serves to recruit co-activator proteins, such as P-TEFb, to increase RNA
polymerase elongation while simultaneously increasing the susceptibility of the activator to
proteasome-mediated destruction (Collins and Tansey, 2006; Daulny et al., 2008; Lee et al.,
2005; Muratani and Tansey, 2003). Although this effect has been observed for transcriptional
activator proteins, no transcriptional repressor has been reported as potentiated by proteolytic
susceptibility. In this study, we provide evidence that the lability of the Drosophila RB-related
factor Rbf1 is tightly linked to its function as a transcriptional repressor, and that this
evolutionarily conserved feature may provide an additional level of developmental control of
the cell cycle.

36

Results
The Rbf1 C-terminal region encodes an instability element
Our previous studies demonstrated that endogenous Rbf1 and Rbf2 proteins are dependent
on the presence of the COP9 signalosome for stability; depletion of COP9 subunits resulted
in a loss of Rbf protein, which was prevented by the addition of proteasome inhibitors,
indicating the involvement of the 26S proteasome pathway (Ullah et al., 2007). To identify
regions involved in Rbf turnover as first step towards understanding the process of Rbf
stabilization, we examined the stability of epitope-tagged, transfected Rbf1 proteins in S2
cells. We focused on Rbf1 because this protein represents the predominant functional RB
family member in Drosophila; rbf1 null mutations are lethal, while rbf2 null mutants have
only very modest phenotypes (Stevaux et al., 2005). Furthermore, previous data suggested
that endogenous Rbf1 levels fluctuate during embryogenesis (Keller et al., 2005; Stevaux et
al., 2005). We initially examined the importance of the conserved central pocket domain, as
well as the less-conserved N- and C-terminal regions (Fig. 2-1A; Table 2-1). In this process,
we identified a region in the C-terminus of the protein as an instability element (IE); proteins
lacking residues 728-786 accumulated to high levels, and these levels were not further
increased by treatment with the proteasome inhibitor MG132 (Fig. 2-1B). In contrast, Rbf1
proteins containing the IE were expressed at lower levels, and these levels were enhanced by
proteasome inhibition. Rbf1 stability was sensitive to growth conditions; Rbf1 ∆IE proteins
were expressed at higher levels than proteins containing this domain under conditions of
higher cell density, longer periods of cell culture, or with low amounts of transfected DNA
(Fig. 2-1C). This last observation suggested that the system for Rbf1 turnover can be
saturated, and indeed we observed greater differences between the wild-type and mutant Rbf1
∆IE proteins in cells expressing lower levels of each protein (not shown). We conclude that

37

the C-terminal region encompassing amino acids 728-786 harbors element(s) that contribute
to Rbf1 instability and proteasome responsiveness.

Critical roles of lysine residues within instability element
The striking accumulation of wild-type Rbf1 protein in cells treated with the proteasome
inhibitor MG132 indicated that this protein, but not the mutant forms lacking the IE, is
subject to active degradation. We hypothesized that the Rbf1 IE may serve as a target for
protein ubiquitylation as one mechanism explaining the contribution of this region to
proteasome-mediated turnover. Protein ubiquitylation of lysine residues often directs
processing by the 26S proteasome, therefore we tested whether the lysine residues in the IE
are involved in the stability of Rbf1 (Fig. 2-2; Table 2-1). Mutant Rbf1 in which three, four,
or all of the six lysines were converted to alanine (K to A) were assessed for expression. All
three of these mutant forms accumulated to significantly higher levels than the wild-type
protein. In contrast, mutant Rbf1 proteins harboring charge-conserving lysine-to-arginine
substitutions in the same residues did not over accumulate, suggesting that the positive charge
of the side chain, rather than its ability to be ubiquitylated, is important for low steady state
levels (Fig. 2-2A). To determine whether the change in steady state levels is due to altered
stability, we next tested whether the half-life of wild-type and mutant (4KA) Rbf1 proteins
differed by treating S2 cells with the translational inhibitor cycloheximide. Three days after
transfection at a point when our previous data indicated that Rbf1 (4KA) mutant protein was
expressed at higher levels than wild type Rbf1, S2 cells were treated with cycloheximide and
Rbf1 protein levels subsequently measured at 0, 6, and 12 hours (Fig. 2-2B, 2-2C). By 6
hours, levels of the wild-type Rbf1 protein, but not the mutant Rbf1 (4KA), were
significantly decreased, confirming that the heightened accumulation of Rbf1 proteins
lacking the IE is caused by reduced rate of Rbf1 degradation (Fig. 2-2D).

38

Figure 2-1. Identification of an instability element (IE) in Rbf1.

39

Figure 2-1 (cont’d)

40

Figure 2-1 (cont’d)
(A) Schematic diagram of Rbf1 proteins expressed in Drosophila S2 cells. The N and C termini
are indicated in dark and light gray respectively; the black box represents the instability
element; the E2f-binding pocket domain is in white. (B) Effect of proteasome inhibitor MG132
on Rbf1 protein levels. Cells were transfected to express the indicated proteins and treated for
1-8 hours with MG132, and protein levels assayed by Western blot using antibodies to
C-terminal Flag epitope tag. The wild-type 1-845 and mutants lacking the extreme C terminus
(∆787-845) or the pocket domain deletion mutant (∆376-727) were expressed at lower levels,
and were strongly stabilized by this drug, while the mutants lacking the IE (Δ728-786 and
1-727) were expressed at higher levels and were not much further stabilized by MG132
treatment. (C) Effects of cell density and culture time on differential expression of wild-type
Rbf1 and IE mutant. 400 ng of Rbf1 expression plasmid was transfected into S2 cells. At lower
6

initial cell densities (0.75 x 10 /ml) and shorter growth times (3 days), expression of wild-type
Rbf1 (1-845) and a deletion mutant lacking the IE (Δ728-786) accumulate to similar levels.
Normalized protein levels are shown below the lanes containing Rbf1. Cells at higher initial
6

densities (1.5-3 x 10 /ml) grown for longer times (5 days) show higher levels of the mutant
protein relative to the wild-type form. Levels of transfected CtBP protein, and endogenous
tubulin protein, are shown as controls.

41

Table 2-1. Rbf1 repression, stability, and localization.
Rbf1
construct

Repression
activity ± stdev

1-845
1-375
376-845
1-727
Δ728-786
Δ787-845
K754A
K754R
K774A
K774R
3K-A.1
3K-R.1
4K-A.1
4K-R.1
6K-A.1
6K-R.1

Protein
stability

100 ± 9
12 ± 1
42 ± 3
16 ± 2
16 ± 4
107 ± 14
65 ± 6
81 ± 9
151 ± 15
125 ± 22
35 ± 11
105 ± 26
22 ± 5
86 ± 7
36 ± 9
110 ± 9

Nuclear
localization

+
+

+
+
+

Constructs marked (-) for nuclear localization were not exclusively nuclear.

42

+
+
+
+
+
+
+
+
+
+
+
+
+

Figure 2-2. Conserved lysine residues in IE play critical roles in accumulation and stability of
Rbf1.

43

Figure 2-2 (Cont’d)

44

Figure 2-2 (Cont’d)
(A) Mutation of multiple lysine residues within the IE leads to increased protein accumulation.
Lysine residues were changed to alanine (K732A, K739A, K740A for 3K-A; also K754A for
4K-A; also K774A and K782A for 6K-A) or to arginine. Rbf1 over-accumulation is not
6

observed with the lysine to arginine substitution. 1.5 x 10 S2 cells were transfected with 100
ng of Rbf1 expression plasmid and grown for five days. The data shown is representative of
three biological experiments. (B, C) Half-life measurements of unstable wild-type and stable
IE mutant Rbf1 proteins. Three days after transfection, cells were treated with cycloheximide
and harvested at the indicated times. Rbf1 protein levels were quantitated by photon-capture
analysis with a Fuji LAS-3000 Imager and normalized to tubulin levels. (D) Bar graphs
showing averaged normalized flag:tubulin ratios for the Rbf1 wild-type and 4K-A mutant
proteins at the 6 hour time point from three biological replicates. At this time point, the
difference between the wild-type and the 4K-A mutant protein levels was statistically
significant (p=0.05).

45

To assess whether the Rbf1 IE functions as an instability element in the context of normal
Drosophila development, we devised a rescue construct that expresses epitope-tagged Rbf1
under the control of the endogenous rbf1 regulatory sequences. Developmental expression of
the wild-type Rbf1 and Rbf1 ∆IE (∆728-786) proteins was then assessed by Western blotting.
As shown in Fig 2-3A (left panel), the overall levels of both proteins were similar in
third-instar larval extracts, suggesting that the deletion mutant accumulated to wild-type
levels. However, a very different picture emerged when we measured protein expression in
imaginal disc tissue from third-instar larvae as shown through Western blots in Fig. 2-3A
(right panel) and imaginal disc staining in Fig. 2-3B-J. Steady state levels of wild-type Rbf1
were far lower than Rbf1 ∆IE in eye, wing and leg imaginal discs; this observation was
consistent for three independent lines of each construct (Fig. 2-3B-J). The relationship
between this effect and previously characterized Rbf1 function is especially evident in the eye
imaginal disc. The terminally differentiating cells of the posterior eye disc normally have no
transcription of rbf1 and low or nonexistent levels of Rbf1 (Keller et al., 2005), but the Rbf1
∆IE mutant also shows staining in these posterior cells, suggesting an abnormal perdurance of
the protein (Fig. 2-3C, D). The marked difference between the steady-state levels of the two
proteins in these contexts indicates that the wild-type Rbf1 protein is specifically destabilized
in the proliferating and differentiating tissue of the imaginal discs. The tissue-specific
stability of the Rbf1 wild-type and mutant proteins suggests that turnover of Rbf1 is a
regulated event, and is likely triggered by developmental signals. The cell-density-dependent
difference in protein accumulation for wild-type and IE-deleted Rbf1 proteins as described in
Fig. 2-1C also supports this hypothesis.

The Rbf1 instability element contributes to repression potency

In the previous experiment, the rbf1-Flag transgene rescued an rbf
46

14

null mutant,

substituting for both zygotic and maternal Rbf1 protein as demonstrated by its ability to
support viable flies for generations (Table 2-2 and data not shown). In contrast, the similar
construct expressing Rbf1 (∆IE)–Flag protein was not capable of rescuing the mutation,
despite robust expression in imaginal discs and wild type expression at the third-instar larval
stage. We therefore hypothesized that the IE is required for Rbf1’s role in regulating activity.
To test this hypothesis, S2 cells were co-transfected with expression plasmids encoding wild
type or mutant Rbf1 proteins and the effect on repression potency was determined using
PCNA-luciferase reporter construct, which sensitive to repression by Rbf1 (Stevaux et al.,
2002). As expected, proteins lacking the central pocket domain were inactive; this region of
the protein is required for interaction with the E2F transcription factors that recruit Rbf1 to
the promoter (Fig. 2-4A). Removal of the N-terminal portion of the protein had only a mildly
deleterious effect on repression, consistent with previous studies that suggested it is not
required for transcriptional activity in vivo and in vitro (Hiebert et al., 1992). In contrast,
removal of portions of the entire C-terminus revealed multiple effects. First, deletion of the
IE region alone had a strong inhibitory effect on transcriptional repression, and this effect was
just as severe as removal of the critical pocket domain. The Rbf1 ∆IE and pocket deletion
mutant proteins did not exhibit aberrant localization, but remained in the nucleus (Fig. 2-4B).
Second, loss of the adjacent C-terminal 59 amino acids (∆787-845) did not abolish repression,
but did change its sub-cellular localization so that the protein was no longer strictly nuclear.
This data indicates that this region harbors a nuclear targeting element governing Rbf1
cytoplasmic/nuclear distribution. As observed for deletion of the entire IE (∆728-786),
removal of portions of this fifty-nine amino acid region in blocks of twenty was sufficient to
inhibit repression activity, suggesting that the function of the IE is distributed over numerous
residues throughout this region (data not shown).
Our previous data indicated that multiple lysine residues within the Rbf1 IE contributed to
47

Rbf1 stability, thus we tested whether these same residues were involved in the
transcriptional repression mediated by Rbf1. Indeed, as shown in Fig. 2-4C, Rbf1 proteins
bearing multiple lysine to alanine substitutions were less effective repressors, even though
these proteins were more stable than the wild-type Rbf1. This effect was most notable for the
Rbf1 4KA mutant whose repression capability was similar to that mediated by Rbf1 lacking
the IE. Surprisingly, alanine substitution of two additional lysine resides (6KA) reproducibly
improved the function of Rbf1 in repression. This observation raised the possibility that this
region harbors elements that throttle Rbf1 repressor potency, as discussed further below. In
contrast to alanine substitution, Rbf1 proteins harboring multiple lysine to arginine
substitutions did not over-accumulate, and significantly, were just as potent as wild type Rbf1
for transcriptional repression. Based on these data, we conclude that these residues contribute
both to Rbf1 instability and to repressor function. These data further indicate that
modification of these residues is not essential to either process. To test whether the effects on
transcriptional repression of these Rbf1 mutations were evident in other contexts, we
compared transcriptional repression of wild-type and mutant Rbf1 proteins on the Polα
promoter, which has somewhat different requirements for E2F and DP activation compared to
the PCNA promoter (Fig. 2-4D) (Dimova et al., 2003). Deletion of the IE or point mutations
within this region similarly reduced the repression activity on this promoter as well,
indicating that the relationship between protein activity and instability is independent of
promoter context. Taken together, these data strongly indicate that the ability of the Rbf1
protein to act as a transcriptional repressor is tightly associated with its instability, and that
the IE in the Rbf1 C terminus is multifunctional, linking these two features.

The Rbf1 IE is not essential for E2F interactions and promoter binding
Previous studies have shown that both the pocket domain as well as the carboxy-terminus

48

of the human RB protein can make molecular contacts with E2F1 (Lee et al., 2002; Rubin et
al., 2005; Xiao et al., 2003). We reasoned that the reduced activity of the Rbf1 instability
element mutants might be a direct result of their inability to physically associate with the E2F
transcription factors. Therefore, we performed GST pull-down and co-immunoprecipitation
(Co-IP) assays to test for interactions between Rbf1 and E2f proteins. In the GST pull-down
assays, both GST-Rbf1 1-845 and the IE mutant (∆728-786) displayed similar binding ability
to in vitro translated E2f1 and E2f2 proteins (Fig. 2-5A, lanes 5 and 6). No interaction was
observed with beads alone or GST protein (Fig. 2-5A, lanes 3 and 4). Similarly in Co-IP assays
from Drosophila S2 cells, Myc-tagged E2f1 co-precipitated with Rbf1 1-845 and two IE
mutants (∆728-786 and 4K-A.1) but not with the pocket domain deletion mutant (∆376-727)
(Fig. 2-5B; top panel, lanes 3-6). These results show that the IE mutants retain a capacity to
interact with both E2f1 and E2f2 proteins.
To assess whether the IE plays a role in Rbf1 promoter occupancy we performed chromatin
immunoprecipitation (ChIP) assays using embryos expressing the Flag-tagged Rbf1
wild-type or ∆IE mutant to test for promoter binding of these proteins at the DNA primase
promoter (Fig. 2-5C). Binding at an intergenic locus and a non-target gene (sloppy paired 1)
promoter was assessed as negative controls. Interestingly, the DNA primase promoter was
found to be enriched in immunoprecipitates from chromatin derived from embryos
expressing both the wild-type Rbf1 as well as the Rbf1 IE mutant proteins indicating that the
Rbf1 IE mutant can still occupy promoters (Fig. 2-5C; top panel). Binding of the IE mutant at
this locus was slightly reduced compared to the wild-type Rbf1 although the association was
significantly above background as no enrichment was observed at an intergenic locus (middle
panel) or the non-target sloppy paired 1 promoter (bottom panel). It appears that, unlike the
Rbf1 pocket deletion mutant, the reduced activity of the Rbf1 IE mutants cannot be attributed
simply to their inability to interact with E2F proteins or target gene promoters.
49

Figure 2-3. Expression of wild-type and IE mutant forms of Rbf1 in the Drosophila larva.

50

Figure 2-3 (cont’d)
Indicated proteins were expressed from the endogenous rbf1 promoter, and expression levels
were assayed in total larval extracts as well as in imaginal discs. (A) Western blot showing
expression of Flag-tagged Rbf1 from third-instar larvae (left panel) and pooled imaginal discs
(right panel) carrying homozygous copies of rbf1 genomic constructs. Equivalent levels of
proteins were noted in whole larval extracts whereas the mutant protein was found to
accumulate to about 4-fold of the wild-type protein in the imaginal discs. The Western blot of
whole larval extracts is representative of four biological replicates for the two lines shown in C,
F, I, and D, G, J; the average difference in protein levels in total larval extracts was 13% +/- 2%.
(B-J) Rbf1 expression in third-instar larval imaginal discs. (B-D) eye discs, (E-G) wing discs,
and (H-J) leg discs. Weak background staining was observed in non-transgenic yw flies (B, E,
and H), and specific, but weak staining was evident in discs expressing wild-type Rbf1 protein
(C, F, and I). Strong expression was noted in flies expressing the inactive Rbf1 ∆ 728 -786 IE
mutant protein (D, G, and J). The imaginal disc staining is representative of stainings of three
different lines for each construct; in all cases, the IE mutant protein was expressed at higher
levels. For interpretation of the references to color in this and all other figures, the reader is
referred to the electronic version of this dissertation.

51

Table 2-2. rbf

14

rescued by transgenic Rbf1.

14

rbf mutant male flies rescued by rbf1 transgene
Strain
Genotype (%)
14
14
FM7/Y
rbf /Y
rbf /+
Rbf1 L1
3.7
19.1
41.2
Rbf1 L2
3.6
22.6
39.8
Rbf1Δ728-786
0
30.0
37.4

n
FM7/+
36.0
34.0
32.6

1116
1163
697

14

rbf mutant female flies rescued by rbf1 transgene
Strain
Genotype (%)
14
14
14
FM7/Y
rbf /Y
rbf /rbf
Rbf1 L1
6.1
39.6
9.8
Rbf1 L2
1.1
36.7
8.5

n
14

rbf /FM7
44.5
53.7

164
188

L1 and L2 are two independent transgenic lines expressing wild-type Rbf1 protein.
14
Rbf1Δ728-786 expresses a nonfunctional, proteolytically stabilized form of Rbf1. rbf is a
14

complete deletion mutant of Rbf1. FM7 represents an X-chromosome balancer. rbf /Y
14

14

represents rescued males; rbf /rbf represents rescued females. The larger percentage of
flies carrying the wild-type (+) or balancer (FM7) X-Chromosome indicates that some flies
are not rescued.

52

Figure 2-4. Rbf1 requires the IE for transcriptional repression.

53

Figure 2-4 (cont’d)

54

Figure 2-4 (cont’d)

55

Figure 2-4 (cont’d)

56

Figure 2-4 (cont’d)
(A) Deletion of the IE (Δ728-786) or E2F binding pocket (Δ376-727) compromises
transcriptional repression activity of Rbf1 proteins measured on the PCNA-luciferase reporter
gene (bar graph). Under these transfection conditions, proteins were expressed at similar levels
(Western blot). (B) Subcellular localization of wild-type (1-845) and deletion mutants. DAPI
staining indicates DNA in nucleus, and FITC staining the Rbf1 proteins. Proteins lacking
residues 787-845, which include the presumptive nuclear localization signal, are found
predominantly in the cytoplasm. (C) Transcriptional activity of Rbf1 IE deletion and point
mutant proteins assayed on PCNA-luciferase reporter. Mutant proteins lacking the IE, or with
multiple lysine to alanine mutations, were compromised for transcriptional repression activity.
Lysine to arginine mutant proteins exhibited wild-type repression activity. Error bars indicate
standard deviation, and asterisks indicate p < 0.05. (D) Rbf1 repression of Drosophila
Polα-luciferase reporter. Deletion of the IE largely inactivates the protein for transcriptional
repression (top panel). Data in 4A represent two biological replicates, each with three technical
replicates, except for 1-845 and ∆ 728 -786, which represent 16 and 9 biological replicates.
Other transfections include data from at least three biological replicates. Firefly luciferase
activity is expressed relative to Renilla luciferase control.

57

Figure 2-5. Rbf1 IE is not essential for E2F interactions and promoter binding.

58

Figure 2-5 (cont’d)

59

Figure 2-5 (cont’d)
(A) GST-Rbf1 and E2f interaction assay. Indicated GST fusion proteins were bound to
radio-labeled E2f proteins and bound proteins were analysed by SDS-PAGE and
autoradiography. GST-Rbf1 1-845 and ∆IE mutant displayed similar binding ability to both in
vitro translated E2f1 and E2f2 proteins (compare lanes 5 and 6). No interaction was observed
with beads alone and GST protein (lanes 3 and 4). Coomassie stained gel showing equal
amounts of GST fusion proteins used in binding assays (bottom panel). The data shown is
representative of three biological replicates. (B) Co-immunoprecipitation assay. Rbf1/E2f1
interactions in co-transfected S2 cells. Cells were co-transfected with Myc-tagged E2f1 and
Flag-tagged Rbf1 expression constructs. Whole cell lysates were used for Flag
immunoprecipitations (IP) and the samples were assayed using Western blots with anti-Myc
antibody (top panel). Myc-tagged E2f1 co-precipitated with Rbf1 1-845 and two IE mutants
(∆728-786 and 4K-A.1) but not with the pocket domain deletion mutant (∆376-727) (top panel,
lanes 3-6). Mock is IP performed using cell lysate from untransfected cells (lane 7). The
asterisk indicates a non-specific band that is contributed by the Flag M2 beads since it appeared
in the no extract control where IP was performed in the absence of any cell lysate (lane 8).
Equivalent levels of the heavy chain IgG (marked as HC) were seen in all samples indicating
the use of equal amount of antibody for each IP reaction. The IP samples were also blotted with
the anti-Flag antibody (bottom panel) to verify the amount of Flag-tagged protein that was
captured in each assay. The data shown is representative of two biological replicates. (C)
Promoter occupancy by Flag-tagged Rbf1 wild-type and Rbf1 IE mutant proteins measured by
chromatin immunoprecipitation. Formaldehyde cross-linked chromatin was prepared from 0 to
20 hour embryos expressing the wild-type or mutant Rbf1 protein and immunoprecipitated
using the indicated antibodies. Enrichment of the Rbf-regulated promoter (DNA primase) was
observed by anti-Flag antibody immunoprecipitation reactions with both wild-type and IE

60

Figure 2-5 (cont’d)
mutant fly embryos but not in reactions using pre-immune IgG (top panel) or at an intergenic
locus (middle panel) and a non-target gene promoter (sloppy paired 1 ) (bottom panel).

61

The Rbf1 IE is a dual-function regulator of repressor potency
Our data indicates that the Rbf1 IE region influences Rbf1 instability and contributes to
Rbf1 repression potency, providing a link between these two activities. However, during
these analyses we additionally observed that Rbf1 (6KA), harboring substitutions of all lysine
residues within the IE was reproducibly a more potent repressor than Rbf1 (4KA), harboring
substitutions of only the four most N-terminal lysine residues within the IE. This observation
raised the possibility that while most of the lysines play a positive role in Rbf1 repression,
one or both of the C-terminal-most lysine residues (K774, K782) play a negative role,
restricting Rbf1 activity. Therefore, to determine whether the lysine residues within the IE
contribute to both positive and negative regulation of Rbf1 function, we tested the repression
activities of Rbf1 proteins with individual alanine substitutions of each lysine residue within
the IE. A subset of these results is shown in Fig. 2-6A, revealing three outcomes. In one case
(K732), alanine substitution did not affect repressor potency, and was indistinguishable from
wild type Rbf1. The second class of mutants were hypomorphic (K739, K740, K754)
exhibiting modest but reproducible inhibitory effects on repression, consistent with these
residues contributing a positive influence on repressor potency (Fig. 2-6A, B). In contrast,
three mutants, K774A, K774R, and K782A exhibited hypermorphic phenotypes with modest
but reproducibly higher repression activity than the wild-type Rbf1 protein, suggesting that
these residues are involved in a negative control of repressor activity (Fig. 2-6A, B). In cases
where lysine to arginine substitution did not moderate activity to wild type levels, such as
with K754 and K774, it is possible that the lysine in question is a target of modification, as a
positive charge is not the sole important feature. However, for mutants with only single point
mutations, we did not observe the robust stabilization of mutant proteins compared to the
wild-type protein (not shown). Together, these data also indicate that the IE exerts both
positive and negative influences on transcriptional activity. Those mutant forms of Rbf1
62

lacking all lysines exhibited intermediate repression phenotypes because of two distinct and
opposite effects, with decreased activity caused by mutations in K739, 740, and 754 partially
offset by increased activity mediated by the mutation of K774 and K782.
To test the physiological importance of these positively and negatively-acting residues for
repressor regulation in Drosophila, we expressed Rbf1 isoforms in the developing eye
imaginal disc using an eyeless-Gal4 driver system (Fig. 2-7A-H). As noted in previous
studies, misexpression of the wild-type Rbf1 protein induced rough eyes in a large percentage
of offspring. The mutant form of Rbf1 (∆728-786) lacking the IE was completely inert,
despite robust expression of the protein in the fly (not shown), consistent with a role for the
IE in repression. Individual point mutations that had modest effects on repression in cell
culture assays similarly showed modest effects on eye development, exhibiting milder
phenotypes, and lower penetrance than the wild-type Rbf1. In contrast, the hypermorphic
K774A mutant, which exhibited elevated repression activity in cell culture assays, induced
dramatic phenotypes (Fig. 2-7E-H). A large percentage of offspring expressing this protein
exhibited very severe eye defects, including complete loss of the eye or developmental
abnormalities including antennal outgrowths and fewer transgenic individuals were recovered
relative to non-expressing controls, suggesting lethality (Fig. 2-7I, J). Thus, the effects of the
mutant forms of Rbf1 on eye development mirror exactly the relative potencies of these
proteins as measured in cell-based repression assays indicating that Rbf1 is subjected to both
positive and negative regulation of repressor potency via the C-terminal IE in vivo. This
result additionally demonstrates the importance of limiting Rbf1 repression activity during
development.

Conserved instability domain of mammalian p107
The correlation between Rbf1 activity and instability in Drosophila prompted us to

63

examine whether similar regulation affects mammalian RB proteins. The overall level of
amino acid conservation is highest between the “pocket” domains of RB family members, but
there are clearly conserved blocks of residues in the C-terminal region. The primary structure
of the C-terminus of Rbf1 most closely resembles that of p107, including the amino acids
residues located in the instability element of Rbf1 (Fig. 2-8A). To directly compare Rbf1 and
p107, we transfected S2 cells with wild-type p107 and mutant forms in which conserved
lysine and arginine residues were replaced with alanine, as well as a deletion of the region
most similar to the Rbf1 IE (amino acids 964-1024). Similar to the stabilization effects noted
with Rbf1, mutant p107 exhibited increased accumulation compared to the wild-type protein
(Fig. 2-8B), suggesting that the C-terminal region of p107 harbors an instability element that
funnels p107 into similar turnover pathways even in this heterologous system.

Discussion
During Drosophila development, cell-cycle regulation deviates considerably from the
classical four-stage G1/S/G2/M pattern, exhibiting rapid direct S-M cycling early in
development, stepwise acquisition of G2 and G1 phases, and endoreplication. These
alternative cycles involve a variety of regulatory features, including constitutive inactivation of
Rbf proteins by phosphorylation, transcriptional regulation of the rbf1 and rbf2 genes, and
regulated degradation of the E2F1 protein. Here, we provide evidence that this regulatory
richness also includes a novel developmentally-triggered degradation of Rbf1 that
paradoxically appears to be required for repression activity. Our study indicates that Rbf1
lability is tightly linked to repression activity, both in a cellular as well as a whole organismal
context. The IE identified in the C-terminus of this protein appears to be a complex domain
with dual functions, so that even a few lysine to alanine mutations can dramatically enhance

64

protein stability while inhibiting transcriptional activity, while other lesions enhance the
protein’s activity (Figs. 2-1, 2-3, and 2-4).
Not only is the turnover of Rbf1 required for effective gene regulation, but it appears that
this turnover can be developmentally cued, presumably to be coordinated with the
engagement of Rbf1 with regulation of the cell cycle (Fig. 2-3). Highly proliferative imaginal
disc tissue appears to provide one such context, where levels of wild-type, but not an
instability element mutant, Rbf1 protein decrease sharply, presumably in response to the
engagement of this protein during cell cycling. In the eye imaginal disc, the Rbf1 protein
levels drop sharply in the posterior, where cells are becoming terminally differentiated.
Presumably, Rbf1 is activated and consumed in the coordinated cell divisions that occur in
the two stripes flanking the morphogenetic furrow; the absence of any further transcription
leads to global depletion of Rbf1. The Rbf1 protein lacking the IE accumulates
inappropriately in differentiating cells.
How might the repression activity of Rbf1 be linked to protein turnover? Protein lability
has previously been found to underlie the action of some eukaryotic transcriptional activators
(Kim et al., 2003; Salghetti et al., 2001). The activation domain of the VP16 protein was
found to be subject to modification by ubiquitylation, enhancing the transcriptional potency
of this factor as well as destabilizing it. This process is thought to affect other transcriptional
activators as well (Salghetti et al., 2000). The exact mechanism by which ubiquitylation
enhances transcriptional activation is poorly understood. The ubiquitin tag may serve a dual
purpose of facilitating interactions with the transcriptional machinery as well as attracting the
26S proteasome. Alternatively, the proteasome itself, or portions of this multi-protein
complex, may directly enhance transcription; chromatin immunoprecipitation experiments
have placed the “lid” of the proteasome on specific genomic locations (Ferdous et al., 2007;
Gonzalez et al., 2002).

65

Figure 2-6. Rbf1 IE harbors positive and negative regulatory elements.

66

Figure 2-6 (cont’d)

67

Figure 2-6 (cont’d)
(A) Transcriptional repression activity of Rbf1 lysine point mutant proteins. Examples of
mutant proteins that show either enhanced or reduced repression activity. Mutation of K754
to alanine or arginine attenuates repression activity while K774 to alanine mutant exhibited
enhanced repression activity with respect to the wild-type protein (top panel). Under these
transfection conditions, proteins were expressed at similar levels (lower panel). Error bars
indicate standard deviations, and asterisks indicate p < 0.05 compared to wild-type Rbf1. (B)
The lysine point mutants were classified as neutral, hypo- or hypermorphic based on the
indicated t-test results. (C) Schematic representation of the Rbf1 IE indicating the location of
lysine residues that play a positive or negative role in Rbf1-mediated repression.

68

Figure 2-7. Severe developmental consequences of expression of hyperactive Rbf1.

69

Figure 2-7 (cont’d)

70

Figure 2-7 (cont’d)
cDNAs of rbf1 wild-type and IE hypermorphic and hypomorphic mutants were misexpressed
in the eye imaginal disc using the eye-Gal4 driver. (A-H) representative eyes exhibiting
wild-type, mild, moderate, severe, and four very severe phenotypes. (I) Bar graphs
representing frequency with which flies carrying the eye-Gal4 driver and UAS-rbf1 gene were
recovered, as well as frequency with which these latter flies exhibited a phenotype (“WT”
normal eye, “RE” rough eye of any degree of severity, “Cy wings” indicates flies that lacked
the Gal4 driver, did not express the rbf1 transgene, and had wild-type eyes). Note that
∆728-786 and 1-727, which lack the IE and were inactive in cell culture, never showed a
phenotype, and that the hyperactive K774 mutants exhibited a partially lethal phenotype, as
judged by lower recovery of flies containing the eye-Gal4 driver. (J) Severity of eye phenotype
in flies exhibiting rough eyes. Mutants are shown in order of increasing severity; point
mutations in the IE that decreased function in cell culture assays also exhibited weaker eye
phenotypes, and hypermorphic K774 alleles exhibited much stronger phenotypes.

71

Figure 2-8. Mutations in the conserved IE of p107 enhance expression.

72

Figure 2-8 (cont’d)
(A) Similarities between Rbf1 IE and homologous region of p107, which is most similar to
Rbf1. Asterisks mark basic residues mutated in each protein to stabilize expression. (B) Genes
for Flag-tagged wild-type p107 or IE mutants were transfected into S2 cells and expression
quantitated by Western blot. The 60 amino acid region deleted from p107 in
∆964 -1024 is
similar to the Rbf1 IE. Endogenous tubulin levels are shown as controls.

73

Until now, there have been no examples of a connection between transcriptional repression
and turnover. If it is the modification of the protein with ubiquitin that potentiates Rbf1’s
repressor activity, this moiety may allow efficient interaction with the transcriptional
machinery, similar to the manner in which SUMOylation of PPAR-γ enhances interaction
with NCoR corepressors to silence inflammatory genes (Pascual et al., 2005). Ubiquitylation
would in this case attract the 26S proteasome in a competing, parallel reaction that enables
Rbf1 turnover. Alternatively, Rbf1 recruitment of the proteasome may allow this complex to
directly mediate repression, in a way opposite to that produced by activation domains.
The C-terminus of Rbf1 appears to represent a regulatory nexus for this protein; in addition
to the instability/repression activity described here, key residues appear to provide a damper
to modulate its overall activity (Fig. 2-6), and phosphorylation within this region by cyclin
kinases can inactivate the protein (Xin et al., 2002). The deep conservation of residues within
the Rbf1 IE argues strongly for similar activities in mammalian pocket proteins; indeed,
mutations of key residues in p107, the closest homolog to Rbf1, strongly stabilize the levels
of this protein (Fig. 2-8). In addition, the spectrum of mutations associated with the human
retinoblastoma gene indicates that the C-terminal region correlating to the Rbf1 IE may
similarly contain critical functions for the mammalian RB protein. One common class of
genetic lesion associated with retinoblastomas are nonsense mutations that cause a truncation
of the C-terminus of the RB protein, and several cancer-associated missense mutations have
similarly been mapped to the region corresponding to the Rbf1 IE (Lohmann, 1999).
Previous studies have shown that the RB C-terminus interacts with the E3 ligase Skp2 and
the anaphase promoting complex (APC/C) to regulate turnover of the p27 cyclin kinase
inhibitor (Binne et al., 2007; Ji et al., 2004). This pathway has been suggested to represent a
transcription-independent mechanism by which RB controls the cell cycle, and indeed RB
was shown not to be subject to APC/C degradation (Binne et al., 2007). Our results indicate

74

that a clean separation of transcription and proteolytic control in the context of RB proteins
may be oversimplified; here we see evidence for a separate route of proteolytic regulation
that modulates transcriptional regulatory potential and protein stability of Rbf1, and possibly
related mammalian pocket proteins. Interestingly, the regulation of this pathway may involve
the evolutionarily conserved COP9 signalosome. Our previous biochemical studies indicated
that the COP9 signalosome regulatory complex is physically associated with Rbf proteins and
limits turnover of these repressors (Ullah et al., 2007). From the results of the current study,
we postulate that COP9 antagonizes the function of the Rbf1 IE, perhaps by blocking the
access of ubiquitin-modifying E3 ligases that would otherwise potentiate Rbf1 activity and
turnover. Alternatively, inhibition of E3 ligases may involve the enzymatic activity of COP9,
whereby this complex downregulates E3 ligases by deneddylation of their cullin subunits
(Wei et al., 2008). How the instability of pocket proteins potentiates their activities, and how
these processes relate to developmental control of retinoblastoma family proteins and cancer,
will be an area of active investigation.

Materials and Methods
Expression Constructs and Transgenic Lines
To express Rbf1 proteins under control of the endogenous regulatory sequences, an 8.8 kbp
genomic locus of Rbf1 was cloned, extending from 2.4 kb upstream of first exon to 2.4 kb
downstream stop (2.1 kb downstream end of last exon) into pCaSpeR (Schejter and Shilo, 1989)
between KpnI and XhoI sites, in three steps using PCR amplification of genomic DNA. Two
Flag epitope tags were inserted immediately 5’ of the rbf1 stop codon into an XbaI site. The
genomic construct of Rbf1 ∆728-786 was made by site-directed mutagenesis. For genes used
in S2 cell culture transfection, rbf1 cDNA was PCR amplified and various mutants produced by

75

site-directed mutagenesis were cloned from pLD02906 (Keller et al., 2005) into KpnI and XbaI
sites of pAX vector (Ryu and Arnosti, 2003). Two Flag epitope tags were inserted 5’ of the stop
codon. For misexpression in the fly, the constructs were cloned into KpnI and XbaI sites of
pUAST (Brand and Perrimon, 1993). For bacterial expression of GST fusion proteins, the
pRSF Duet-1 vector (Novagen) was modified to introduce a GST ORF followed by a ligation
independent cloning (LIC) site into its multiple cloning site I (MCS I) to generate the pRSF
GST-Tb/LIC vector. rbf1 cDNA was PCR amplified and cloned into this LIC site to generate
the pRSF GST-Rbf1 1-845 construct. The pRSF GST-Rbf1 Δ728-786 construct was generated
by site-directed mutagenesis. For expression of human p107 in S2 cells, the cDNA and various
mutants produced by site-directed mutagenesis were cloned into the pAX vector and modified
with a C-terminal double Flag epitope. The pCaSpeR and pUAST plasmids were used to
generate transgenic flies by P-element mediated germline transformation of yw flies. The
transgenic flies were then balanced with SM2 CyO or TM3 Sb balancers.
Luciferase Reporter Assay
Drosophila S2 cells were transfected using Effectene transfection reagent (Qiagen)
according to the manufacturer’s protocol. Typically, 1.5 million cells were transfected with 1
µg of PCNA-Luciferase reporter, 0.25 µg of pRL-CMV Renilla luciferase reporter (Promega)
and 0.2 µg of one of pAX-rbf1 constructs. Cells were harvested 72 hours after transfection and
luciferase activity was measured using the Dual-Glo Luciferase assay system (Promega) and
quantified using the Veritas microplate luminometer (Turner Biosystems). Firefly luciferase
activity was normalized to renilla luciferase activity.
Immunocytochemistry
Drosophila S2 cells were transfected with 400ng of each rbf mutant using the Effectene
transfection reagent (Qiagen) according to the manufacturer’s protocol. Cells were grown
directly on cover slips pre-treated with 0.01% poly-L-Lysine (Sigma). Three days after

76

transfection, cells were washed once in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4,
1.47 mM KH2PO4, pH 7.4) and fixed in 4% paraformaldehyde (in PBS) for 30 min. at room
temperature. Cells were then washed four times in PBS, permeabilized in PBS+Triton-X-100
(0.4%v/v) for 10 min at room temperature, and blocked with 1% bovine serum albumin (in
PBS). Cells were then incubated with M2 anti-Flag antibody (Sigma; final concentration 20
g/ml) in 1% w/v BSA in PBS buffer, washed three times in TBST (10 mM Tris-HCl, pH 8.0,
0.15 M NaCl, 0.05% Tween-20) for 5 min at room temperature and incubated for 1 h at room
temperature with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin
G (1:500 dilution) (Boehringer Mannheim and Invitrogen). Cells were then washed three times
in TBST and mounted in Vectashield mounting medium (Vector laboratories) containing 1.5
µg/ml 4’,6-diamidino-2-phenylindole (DAPI) and incubated overnight at room temperature.
Cells were visualized using an Olympus BX51 fluorescent microscope.
Western Blot Analysis
To measure protein expression in larval tissue, third-instar larvae were collected from
transgenic lines expressing Flag-tagged Rbf1 and Rbf1 ∆728-786, mashed with a plastic pestle
and sonicated (3 cycles of 12 pulses each) in lysis buffer (50 mM HEPES, pH 7.9, 150 mM
NaCl, 10% glycerol, 0.1 mM EDTA, 12.5 mM MgCl2, Complete mini-EDTA free protease
inhibitor cocktail, Roche). Imaginal discs were dissected out from ten third-instar larvae and
extracts were prepared in lysis buffer. Extracts were run on 10% SDS-PAGE gels and analysed
by Western blotting using M2 anti-Flag (mouse monoclonal, 1:10,000, 5 mg/ml Sigma; F3165).
Antibody incubation was performed in TBST (20 mM Tris-Cl, pH 7.5, 120 mM NaCl, 0.1%
Tween-20) with 5% non-fat dry milk. Blots were developed using HRP-conjugated secondary
antibodies (Pierce) and SuperSignal West Pico chemiluminescent substrate (Pierce). To
measure protein expression in cell culture, 50 µg S2 cell lysates were resolved by SDS-PAGE,
transferred to a PVDF membrane and probed with M2 anti-Flag mouse monoclonal at 1:10,000

77

dilution, mouse monoclonal anti-tubulin (Iowa Hybridoma Bank) at 1:20,000 dilution.
Treatments with MG132 Proteasome Inhibitor and Cycloheximide
For proteasome inhibitor treatments, S2 cells were transfected with 0.5 µg of pAXrbf1
constructs using the calcium phosphate transfection method. The cells were grown for 5 days,
then treated with 50 µg/ml MG132 (Sigma-Aldrich) or the vehicle DMSO for the indicated
times. For determination of Rbf1 protein half-life, 1.5 million S2 cells were transfected using
Effectene transfection reagent (Qiagen) with 10 ng of pAXrbf1 1-845 or 4K-A.1 genes. 72 hrs
post-transfection the cells were treated with 100 µM cycloheximide for the indicated times.
Protein-protein Interaction Studies
For the expression of GST fusion proteins, the appropriate expression constructs were
transformed into Rosetta2 (DE3) E.coli cells (Novagen). Protein expression was induced by
0.5 mM IPTG for 3 hours at 37 °C. The proteins were purified on Glutathione sepharose
beads (GE Healthcare). The [35S]-Met labeled E2f proteins were generated using the TNT T7
Quick for PCR DNA Kit (Promega). In vitro translated proteins were bound to ~1 µg of
preincubated immobilized GST fusion proteins for 3 hours at room temperature. The beads
were washed three times with HEMGT-150 buffer (25 mM HEPES, 0.1 mM EDTA, 12.5 mM
MgCl2, 10% Glycerol, 0.1% Tween-20, 150 mM KCl). Bound proteins were eluted by boiling
in 1X Laemmli sample buffer and analysed by SDS-PAGE and autoradiography. For the
co-immunoprecipitation assays, 200 ng Myc-tagged E2f1 and 200 ng of various Flag-tagged
Rbf1 constructs were co-transfected into S2 cells using Effectene transfection reagent
(Qiagen). Cells were grown for 3 days after which whole cell extracts were prepared and Flag
immunoprecipitation reactions were performed (Anti-Flag M2 affinity gel, Sigma) followed
by anti-Myc Western blotting (mouse monoclonal, 1:3000 dilution, 5 mg/ml, Roche).
Chromatin Immunoprecipitation
Chromatin was prepared and analysed from 0-20 hour old embryos as described previously

78

(Martinez and Arnosti, 2008) except that the chromatin (1 ml) was incubated with 5 µl (5 µg)
of Flag antibody (Sigma; F7425) or 2 µl H3 antibody (Abcam; 0.4 µg/µl) overnight at 4 °C.
The recovered DNA was dissolved in 40 µl water. 2 µl of each ChIP sample was used for 28
cycles of PCR. The oligos used for PCR were 5’- CCGCAAGCATCGATAATGAGCAGA-3’
and

5’-AGTTGTGCGGGTACTTGGTTTC

C-3’ for

5’-TGTGGGCTCTCTTCGTGTAGACTT-3’

the

DNA primase

promoter;
and

5’-TGGTTTCTGATTCTCACACACGAC-3’ for the sloppy paired 1 promoter and
5’-GTTGAGAATGTGAGAAAGCGG-3’ and 5’-CGAAAAAGGAGAAGGCACAAAG-3’
for an intergenic region.
Fly Assays
Flies harboring the wild-type or mutant rbf1 forms in the pUAST vector were crossed with
flies containing an eyeless-Gal4 / CyO driver (Gilbert et al., 2006) and the offspring were
screened for eye phenotypes. The rbf14 mutant (stock number 7435) was obtained from the
Bloomington Stock Center.
Immunohistochemical Staining of Imaginal Discs
Imaginal discs were dissected in chilled PBS from third-instar larvae of rbf1 and
rbf1Δ728-786 flies and fixed in 3.7% formaldehyde in 10 mM potassium phosphate, pH 6.8;
15 mM NaCl; 45 mM KCl; 2 mM MgCl2 for 30 minutes at room temperature. Antibody
detection was performed by diaminobenzadine staining using the Vectastain kit (Vector Labs).
Primary M2 α-Flag dilution was 1:1500. Following the horseradish peroxidase reaction, discs
were mounted in 70% glycerol.

Acknowledgments
We thank Nick Dyson for sharing the PCNA and Polα luciferase reporter genes, Maxim
Frolov for providing the Myc-E2f1 construct, Stefan Gaubatz for providing the p107

79

expression construct, Min-Hao Kuo and Jianjun Luo for aid with fluorescence microscopy,
Jaclyn Peraino for generating Rbf1 single lysine point mutants, and John Wang for reagents
and friendly advice on protein localization assays. We also appreciate the assistance from
Satyaki Sengupta for his helpful discussions. This work was supported by an undergraduate
summer training grant from the MSU Genetics Program to SD, the MSU Gene Expression in
Development and Disease Group, and a grant from the National Institutes of Health to DNA
and RWH.

80

REFERENCES

81

REFERENCES

Binne, U.K., Classon, M.K., Dick, F.A., Wei, W., Rape, M., Kaelin, W.G., Jr., Naar, A.M.,
and Dyson, N.J. (2007). Retinoblastoma protein and anaphase-promoting complex physically
interact and functionally cooperate during cell-cycle exit. Nat Cell Biol 9, 225-232.
Boyer, S.N., Wazer, D.E., and Band, V. (1996). E7 protein of human papilloma virus-16
induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway.
Cancer Res 56, 4620-4624.
Brand, A.H., and Perrimon, N. (1993). Targeted gene expression as a means of altering cell
fates and generating dominant phenotypes. Development 118, 401-415.
Chang, E.C., and Schwechheimer, C. (2004). ZOMES III: the interface between signalling
and proteolysis. Meeting on The COP9 Signalosome, Proteasome and eIF3. EMBO Rep 5,
1041-1045.
Classon, M., and Harlow, E. (2002). The retinoblastoma tumour suppressor in development
and cancer. Nat Rev Cancer 2, 910-917.
Collins, G.A., and Tansey, W.P. (2006). The proteasome: a utility tool for transcription? Curr
Opin Genet Dev 16, 197-202.
Daulny, A., Geng, F., Muratani, M., Geisinger, J.M., Salghetti, S.E., and Tansey, W.P. (2008).
Modulation of RNA polymerase II subunit composition by ubiquitylation. Proc Natl Acad Sci
U S A 105, 19649-19654.
Dimova, D.K., Stevaux, O., Frolov, M.V., and Dyson, N.J. (2003). Cell cycle-dependent and
cell cycle-independent control of transcription by the Drosophila E2F/RB pathway. Genes
Dev 17, 2308-2320.
Dyson, N. (1998). The regulation of E2F by pRB-family proteins. Genes Dev 12, 2245-2262.
Ferdous, A., Sikder, D., Gillette, T., Nalley, K., Kodadek, T., and Johnston, S.A. (2007). The
role of the proteasomal ATPases and activator monoubiquitylation in regulating Gal4 binding
to promoters. Genes Dev 21, 112-123.
Frolov, M.V., Moon, N.S., and Dyson, N.J. (2005). dDP is needed for normal cell
proliferation. Mol Cell Biol 25, 3027-3039.

82

Gilbert, M.K., Tan, Y.Y., and Hart, C.M. (2006). The Drosophila boundary
element-associated factors BEAF-32A and BEAF-32B affect chromatin structure. Genetics
173, 1365-1375.
Gonzalez, F., Delahodde, A., Kodadek, T., and Johnston, S.A. (2002). Recruitment of a 19S
proteasome subcomplex to an activated promoter. Science 296, 548-550.
Hiebert, S.W., Chellappan, S.P., Horowitz, J.M., and Nevins, J.R. (1992). The interaction of
RB with E2F coincides with an inhibition of the transcriptional activity of E2F. Genes Dev 6,
177-185.
Ji, P., Jiang, H., Rekhtman, K., Bloom, J., Ichetovkin, M., Pagano, M., and Zhu, L. (2004).
An Rb-Skp2-p27 pathway mediates acute cell cycle inhibition by Rb and is retained in a
partial-penetrance Rb mutant. Mol Cell 16, 47-58.
Keller, S.A., Ullah, Z., Buckley, M.S., Henry, R.W., and Arnosti, D.N. (2005). Distinct
developmental expression of Drosophila retinoblastoma factors. Gene Expr Patterns 5,
411-421.
Kim, S.Y., Herbst, A., Tworkowski, K.A., Salghetti, S.E., and Tansey, W.P. (2003). Skp2
regulates Myc protein stability and activity. Mol Cell 11, 1177-1188.
Knudson, A.G., Jr. (1978). Retinoblastoma: a prototypic hereditary neoplasm. Semin Oncol 5,
57-60.
Lee, C., Chang, J.H., Lee, H.S., and Cho, Y. (2002). Structural basis for the recognition of the
E2F transactivation domain by the retinoblastoma tumor suppressor. Genes Dev 16,
3199-3212.
Lee, D., Ezhkova, E., Li, B., Pattenden, S.G., Tansey, W.P., and Workman, J.L. (2005). The
proteasome regulatory particle alters the SAGA coactivator to enhance its interactions with
transcriptional activators. Cell 123, 423-436.
Lohmann, D.R. (1999). RB1 gene mutations in retinoblastoma. Hum Mutat 14, 283-288.
Martinez, C.A., and Arnosti, D.N. (2008). Spreading of a corepressor linked to action of
long-range repressor hairy. Mol Cell Biol 28, 2792-2802.
Muratani, M., and Tansey, W.P. (2003). How the ubiquitin-proteasome system controls
transcription. Nat Rev Mol Cell Biol 4, 192-201.

83

Pascual, G., Fong, A.L., Ogawa, S., Gamliel, A., Li, A.C., Perissi, V., Rose, D.W., Willson,
T.M., Rosenfeld, M.G., and Glass, C.K. (2005). A SUMOylation-dependent pathway
mediates transrepression of inflammatory response genes by PPAR-gamma. Nature 437,
759-763.
Prince, A.M., May, J.S., Burton, G.R., Lyle, R.E., and McGehee, R.E., Jr. (2002).
Proteasomal degradation of retinoblastoma-related p130 during adipocyte differentiation.
Biochem Biophys Res Commun 290, 1066-1071.
Rubin, S.M., Gall, A.L., Zheng, N., and Pavletich, N.P. (2005). Structure of the Rb
C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F
release. Cell 123, 1093-1106.
Ryu, J.R., and Arnosti, D.N. (2003). Functional similarity of Knirps CtBP-dependent and
CtBP-independent transcriptional repressor activities. Nucleic Acids Res 31, 4654-4662.
Salghetti, S.E., Caudy, A.A., Chenoweth, J.G., and Tansey, W.P. (2001). Regulation of
transcriptional activation domain function by ubiquitin. Science 293, 1651-1653.
Salghetti, S.E., Kim, S.Y., and Tansey, W.P. (1999). Destruction of Myc by
ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize Myc.
EMBO J 18, 717-726.
Salghetti, S.E., Muratani, M., Wijnen, H., Futcher, B., and Tansey, W.P. (2000). Functional
overlap of sequences that activate transcription and signal ubiquitin-mediated proteolysis.
Proc Natl Acad Sci U S A 97, 3118-3123.
Schejter, E.D., and Shilo, B.Z. (1989). The Drosophila EGF receptor homolog (DER) gene is
allelic to faint little ball, a locus essential for embryonic development. Cell 56, 1093-1104.
Sdek, P., Ying, H., Chang, D.L., Qiu, W., Zheng, H., Touitou, R., Allday, M.J., and Xiao, Z.X.
(2005). MDM2 promotes proteasome-dependent ubiquitin-independent degradation of
retinoblastoma protein. Mol Cell 20, 699-708.
Stevaux, O., Dimova, D., Frolov, M.V., Taylor-Harding, B., Morris, E., and Dyson, N. (2002).
Distinct mechanisms of E2F regulation by Drosophila RBF1 and RBF2. EMBO J 21,
4927-4937.
Stevaux, O., Dimova, D.K., Ji, J.Y., Moon, N.S., Frolov, M.V., and Dyson, N.J. (2005).
Retinoblastoma family 2 is required in vivo for the tissue-specific repression of dE2F2 target
genes. Cell Cycle 4, 1272-1280.

84

Stubdal, H., Zalvide, J., Campbell, K.S., Schweitzer, C., Roberts, T.M., and DeCaprio, J.A.
(1997). Inactivation of pRB-related proteins p130 and p107 mediated by the J domain of
simian virus 40 large T antigen. Mol Cell Biol 17, 4979-4990.
Su, H.W., Qu, L.J., Chen, Z.L., and Gu, H.Y. (2003). Evolution of COP9 signalosome and
proteasome lid complex. Acta Botanica Sinica 45, 523-529.
Sutcliffe, J.E., Korenjak, M., and Brehm, A. (2003). Tumour suppressors--a fly's perspective.
Eur J Cancer 39, 1355-1362.
Swanhart, L.M., Sanders, A.N., and Duronio, R.J. (2007). Normal regulation of Rbf1/E2f1
target genes in Drosophila type 1 protein phosphatase mutants. Dev Dyn 236, 2567-2577.
Uchida, C., Miwa, S., Kitagawa, K., Hattori, T., Isobe, T., Otani, S., Oda, T., Sugimura, H.,
Kamijo, T., Ookawa, K., et al. (2005). Enhanced Mdm2 activity inhibits pRB function via
ubiquitin-dependent degradation. EMBO J 24, 160-169.
Ullah, Z., Buckley, M.S., Arnosti, D.N., and Henry, R.W. (2007). Retinoblastoma protein
regulation by the COP9 signalosome. Mol Biol Cell 18, 1179-1186.
van den Heuvel, S., and Dyson, N.J. (2008). Conserved functions of the pRB and E2F
families. Nat Rev Mol Cell Biol 9, 713-724.
Wei, N., Serino, G., and Deng, X.W. (2008). The COP9 signalosome: more than a protease.
Trends Biochem Sci 33, 592-600.
Xiao, B., Spencer, J., Clements, A., Ali-Khan, N., Mittnacht, S., Broceno, C., Burghammer,
M., Perrakis, A., Marmorstein, R., and Gamblin, S.J. (2003). Crystal structure of the
retinoblastoma tumor suppressor protein bound to E2F and the molecular basis of its
regulation. Proc Natl Acad Sci U S A 100, 2363-2368.
Xin, S., Weng, L., Xu, J., and Du, W. (2002). The role of RBF in developmentally regulated
cell proliferation in the eye disc and in Cyclin D/Cdk4 induced cellular growth. Development
129, 1345-1356.

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CHAPTER 3

Ubiquitination of Retinoblastoma family protein 1 potentiates gene-specific repression
function

Abstract
The Retinoblastoma (RB) tumor suppressor family functions as a regulatory node
governing cell cycle progression, differentiation and apoptosis. Post-translational
modifications play a critical role in modulating RB activity, but additional levels of control,
including protein turnover, are also essential for proper function. The Drosophila RB
homolog Rbf1 is subjected to developmentally cued proteolysis mediated by an instability
element (IE) present in this protein’s C-terminus. Paradoxically, instability mediated by the
IE is also linked to Rbf1 repression potency, suggesting that proteolytic machinery may also
be directly involved in transcriptional repression. We show that the Rbf1 IE is an autonomous
degron that stimulates both Rbf1 ubiquitination and repression potency. Importantly, Rbf1 IE
function is promoter-specific, contributing to repression of cell cycle responsive genes but
not to repression of cell signaling genes. The multifunctional IE domain thus provides Rbf1
flexibility for discrimination between target genes embedded in divergent cellular processes.

This work was published as the following manuscript:
Raj, N.*, Zhang, L.*, Wei, Y, Arnosti, D. N., and Henry, R. W. (2012). Ubiquitination of
retinoblastoma family protein 1 potentiates gene-specific repression function. J. Biol. Chem.
287, 41835-41843. (*These authors contributed equally to this work.)

86

Introduction
The RB tumor suppressor protein functions as a crucial regulator of the G1/S transition
during cell cycle progression, and thus plays a central role in restricting cellular proliferation
(Burkhart and Sage, 2008). Consistent with this property, the RB1 gene is inactivated in a
broad range of human cancers, often as a seminal event contributing to both cancer initiation
and progression (Hanahan and Weinberg, 2011). RB has been further implicated in the
governance of diverse physiological processes, including differentiation and apoptosis, and as
a central hub connecting these processes, RB activity is subjected to strict control by
post-translational modification during normal growth and development (Nguyen and
McCance, 2005; Skapek et al., 2006). Indeed, in many tumor types, upstream regulatory
pathways governing RB are inactivated with similar frequencies as inactivation of RB itself,
attesting to the importance of close supervision over RB function (Wikenheiser-Brokamp,
2006a).
In an intricate network of gene control, RB and its related family members, p107 and p130,
function as transcriptional repressors of diverse gene sets through interactions with members
of the E2F family of transcriptional activator proteins (Genovese et al., 2006; Morris and
Dyson, 2001). RB family members govern apparently mutually exclusive physiological
processes, notably cell cycle progression and apoptosis, thus distinct regulatory mechanisms
must ensure that RB-mediated induction of apoptosis does not ensue, even as RB proteins are
periodically activated on cell cycle genes during normal proliferation (Delston and Harbour,
2006). Canonical regulation of RB activity is governed by cyclin/cdk regulatory kinases
(Chen et al., 1989; Hinds et al., 1992; Kato et al., 1993; Lin and Wang, 1992). Timely
phosphorylation blocks RB/E2F association, and unleashes waves of E2F-mediated
transcription that contribute to cell cycle progression (Knudsen et al., 1998). However, RB
continues to reside at a number of genomic sites after cyclin/cdk-mediated deactivation
87

(Wells et al., 2000; Wells et al., 2003), revealing that cyclin/cdk activity does not universally
de-repress all RB target genes. Indeed, RB phosphorylation by p38MAPK at a site that is not
a target for cyclin/cdks can modulate RB-mediated repression of apoptotic response genes
(Delston and Harbour, 2006; Delston et al., 2011). This model suggests that RB is subjected
to a protein-modification code that enables gene specific outcomes, namely cyclin/cdk
kinases regulate cell cycle-responsive promoters and stress responsive kinases regulate
apoptosis-responsive promoters.
In Drosophila, RB family proteins Rbf1 and Rbf2 interact with E2F transcription factors as
corepressors, similar to their mammalian counterparts. Drosophila Rbf proteins are also
controlled by a canonical phosphorylation mechanism through cyclin-cdk complexes (Du et
al., 1996a; Du et al., 1996b). Mutant rbf1 embryos show constitutive expression of PCNA
and RNR2, two E2F1-regulated genes for DNA replication, and ectopic S-phase entry,
indicating the importance of Rbf1 for arresting cells in G1 phase (Du and Dyson, 1999). Rbf1
associates at numerous canonical E2F cell cycle-regulated genes in the early embryo
(Acharya et al., 2010; Stevaux and Dyson, 2002), indicating that key components of the RB
regulatory pathway are evolutionarily conserved. However, in the embryo, Rbf1 also
associates with numerous E2F1-independent target genes beyond the canonical cadre of
E2F1-dependent target genes (Acharya et al., 2012b; Korenjak et al., 2012a). Many of these
candidate E2F1-independent target genes encode components of signaling pathways,
exemplified by the insulin receptor (InR), and whose expression is regulated independently of
the cell cycle. Thus, Drosophila Rbf regulatory influence during development appears to
extend beyond cell cycle progression and apoptosis to include cellular signaling, although in
a mechanism likely independent of E2F1.
In addition to regulation by phosphorylation, Rbf proteins are subject to developmental
regulation of their proteolytic turnover. Developmental regulation occurs in imaginal disc

88

tissue (Acharya et al., 2010) with stability controlled by the COP9 signalosome (Ullah et al.,
2007), a developmentally regulated complex that controls proteasome-mediated protein
degradation via modulation of E3 ubiquitin ligase activity (Bech-Otschir et al., 2002;
Chamovitz and Glickman, 2002). The COP9 signalosome is physically associated with Rbf1
and Rbf2, and depletion of COP9 subunits stimulates Rbf1 turnover (Ullah et al., 2007). Rbf1
stability is influenced by a C-terminal instability element (IE) that positively contributes to
both repressor destruction and potency (Acharya et al., 2010). The conservation of the IE in
mammalian RB family proteins suggests that these pathways operate in higher eukaryotes,
however the function of the IE in integrating protein turnover and transcriptional control is
poorly understood. Here, we show that the Rbf1 IE is sufficient to facilitate ubiquitination
and turnover, and directly mediates transcriptional repression. Strikingly, Rbf1 ubiquitination
enhances E2F1-dependent PCNA repression but not E2F1-independent repression of InR
transcription. Thus, the IE is a key protein motif directing promoter-specific activity of Rbf1.
These studies reveal a novel level of regulatory discrimination within the RB protein
modification code that enables gene-specific repression during development.

Results
A modular degron influences Rbf1 ubiquitination and stability
Drosophila Rbf proteins are subjected to developmentally regulated turnover, exhibiting
tissue-specific modulation in both the developing embryo and larvae (Acharya et al., 2010;
Keller et al., 2005). To understand the mechanism underlying this regulation, we tested
whether the Rbf1-IE can autonomously control protein stability by fusing the IE region
(728-786) to GFP (Fig. 3-1A), and measuring the half-lives of GFP and GFP-Rbf1-IE
chimeras in S2 cells after cycloheximide treatment. Steady state levels of GFP-Rbf1-IE, but
89

not GFP, were substantially decreased by 12 hours after cyclohexamide challenge, indicating
that the IE directly enhanced GFP turnover (Fig. 3-1B). Thus, the IE region can function
autonomously as a degron, and independently of other domains within Rbf1. This ability is
consistent with the previously discovered role of the IE in control of full-length Rbf1 stability
during development (Acharya et al., 2010).
Previous models of degron function indicate that subcellular location of substrate proteins
influences turnover (Hammond-Martel et al., 2012). Therefore, to examine the effect of
substrate localization on Rbf1-degron function, the Rbf1 nuclear localization signal (NLS,
Fig. 3-S1) was appended to GFP-Rbf1-IE, largely confining the chimera protein to the
nucleus (Fig. 3-1C). Accumulation of the GFP chimera proteins was then measured; testing
lysine-to-alanine substitutions within the IE that were previously shown to both inactivate
and stabilize wild type Rbf1 (Acharya et al., 2010). In all experiments, both GFP-Rbf1-IE
(-NLS) and GFP-Rbf1-C (+NLS) behaved similarly, with K to A mutants accumulating to
levels approximately three fold higher than those of their wild-type counterparts. Consistent
with these observations, the GFP-Rbf1-IE 4K-A mutant displayed a significantly longer
half-life compared to GFP-Rbf-IE (Fig. 3-S2). The steady state levels of both GFP-Rbf1-IE
and GFP-Rbf1-C were unaffected by lysine-to-arginine substitution of the same amino acids,
indicating that the positive charges of the side chains are important for IE substrate
destabilization and that these lysine residues are unlikely targets for ubiquitination (Fig.
3-1D). These data indicate that the function of the IE as a modular degron is unaffected by its
preferential nuclear localization, and is consistent with a model wherein some components of
the Rbf1 degradation pathway occur in the nucleus.
Regulated protein turnover often involves the activity of the 26S proteasome, which
interacts with substrates that have been modified with ubiquitin, but also in some cases
proteins that are not ubiquitinated. In mammals, RB and p107 are substrates of E3 ubiquitin

90

ligases and are turned over in a proteasome-dependent manner (Barbash et al., 2007; Sdek et
al., 2004; Uchida et al., 2005; Ying and Xiao, 2006). Rbf1 is likewise dependent on the
proteasome pathway, but there are no reports of ubiquitination of this protein. To test whether
Rbf1 is ubiquitinated in vivo, we expressed Flag-tagged Rbf1 and HA-tagged ubiquitin
proteins in S2 cells, and immunoprecipitated the Rbf1 proteins. As shown in Fig. 3-2A,
poly-ubiquitinated Rbf1 species were detected in heat denatured extracts prepared from cells
co-expressing both Flag-Rbf1 and HA-ubiquitin. Ubiquitinated species were not observed in
mock-transfected samples, in samples containing only one of the two proteins, or in extracts
containing Rbf1 and HA-ubiquitin from denatured extracts containing individually expressed
HA-Ub or Flag-Rbf1 proteins that were mixed together prior to immunoprecipitation. In the
presence of the MG132 proteasome inhibitor, higher levels of polyubiquitinated Rbf1 were
observed (Fig. 3-2B). We conclude that the Rbf1 protein was ubiquitinated in vivo, and is
targeted for proteasome-mediated turnover, an outcome that is consistent with previous
observations linking the COP9 signalosome to protection of Rbf1 from destruction by the
proteasome (Ullah et al., 2007). Interestingly, Rbf1 lacking the IE region (Rbf1-∆IE)
exhibited a substantial reduction, but not complete loss, of Rbf1 ubiquitination (Fig. 3-3A), a
result that was also observed for Rbf1-4KA (Fig. 3-S3), suggesting that the IE enhances
ubiquitination, but is not essential for all modification events. We tested whether the IE is
sufficient to independently drive ubiquitination by co-expressing HA-tagged ubiquitin and
the GFP-IE chimera. Indeed, as shown in Fig. 3-3B, levels of poly-ubiquitinated GFP were
substantially increased by appending the Rbf1-IE region as compared to levels observed for
untagged GFP. GFP-Rbf1 IE ubiquitination was reduced by the introduction of the 4K-A
substitutions (Fig. 3-S4). Together, these data show that one function of the Rbf1 IE is to
facilitate substrate ubiquitination.

91

Figure 3-1. The instability element (IE) of Rbf1 is a modular degron.

92

Figure 3-1 (cont’d)

93

Figure 3-1 (cont’d)
(A) Schematic diagram of GFP-fusion proteins expressed in Drosophila S2 cells. (B)
Presence of the IE increases protein turnover. Half-lives of GFP-fusion proteins were
measured by Western blot after cycloheximide (CHX) treatment (error bars are standard
deviation, p<0.01). Inset Western blot shows the steady-state levels of GFP and GFP IE
fusion protein before CHX treatment. (C) Subcellular localization of GFP and GFP-fusion
proteins as measured by confocal microscopy. (D) IE function modulates GFP stability.
Indicated GFP-fusion proteins were expressed in S2 cells for 3 or 5 days and measured by
Western blot with antibodies against the Flag epitope. Lysine residues (K732, K739, K740
and K754) were changed to alanine or to arginine. Protein levels were quantitated by
photon-capture analysis with a Fuji LAS-3000 Imager and normalized to tubulin levels. Error
bars indicate standard deviation, and asterisks indicate p < 0.01. Western blot data is a
representative from the 5-day set of experiments.

94

Figure 3-2. Rbf1 is degraded via an ubiquitin-proteasome dependent pathway.

95

Figure 3-2 (cont’d)
(A) Rbf1 is ubiquitinated in vivo. S2 cells were transfected with Flag-tagged Rbf1 and
HA-tagged ubiquitin expression constructs. Denatured protein extracts were used for Flag
immunoprecipitation (IP) and recovered samples were assayed by anti-HA Western blot
analysis (top panel). The asterisk indicates a non-specific band and “m” indicates reaction
performed using mixed samples from those in lanes 2 and 3. The IP samples were also blotted
with anti-Flag antibody (bottom panel) to verify equivalent Rbf1 recovery (lanes 3-5). The
numbers underneath the HA Western blot panel represent the ratios of HA/Flag signals. The
data shown are representative of three biological replicates. (B) Rbf1 ubiquitination is
sensitive to proteasome inhibition. Samples were treated as in (A) except that they were
treated with MG132, a proteasome inhibitor.

96

Figure 3-3. The Rbf1 instability element enhances protein ubiquitination.

97

Figure 3-3 (cont’d)
(A) The Rbf1 IE enhances ubiquitination. Wild type and mutant Rbf1 lacking the IE
(Rbf1-∆IE) were compared for ubiquitination as performed in Figure 2. (B) The Rbf1 IE is
sufficient to drive the ubiquitination of a heterologous protein, GFP. Fusion of the Rbf1-IE to
GFP led to a substantial increase in the levels of its ubiquitination as compared to the levels
observed for GFP as measured by co-transfection and CO-IP/Western analysis.

98

The Rbf1-IE can function independently in transcriptional repression
We showed previously that in addition to influencing protein stability, the IE region is
critical for Rbf1 repressor activity on E2F1-dependent promoters, such as PCNA and Polα
(Acharya et al., 2010). We therefore hypothesized that the Rbf1 degron functions as a bona
fide transcriptional repression domain. To test this hypothesis, the Rbf1 degron alone or
degron plus NLS was fused to the Tet repressor, and the activity of these proteins was
assayed on an Actin5C reporter harboring two Tet binding sites (Fig. 3-4A). Indeed, when
directly tethered to its target promoter in the absence of doxycycline, both Tet-Rbf1-IE and
Tet-Rbf1-C showed strong repression activity at levels approaching that observed with
Tet-Knirps, a potent short-range repressor that was included as a positive control on this
reporter (Fig. 3-4B). As expected, treatment with doxycycline to inhibit DNA binding also
diminished repression (not show). The Tet repressor DNA binding domain alone lacked
notable repression activity. These data are consistent with a direct role for the IE in
transcriptional repression. Interestingly, both Tet-Rbf1-C and Tet-Rbf1-IE harboring the K-A
substitutions repressed transcription to similar levels as observed for the wild type
Tet-Rbf1-IE chimera. Thus, these lysine residues that influence repression in the context of
full-length Rbf1 are not essential in this context (Acharya et al., 2010).
The ability of the IE to independently repress transcription next prompted us to examine
whether the IE is an essential element within full-length Rbf1 when targeted to a promoter
independently of E2F1. Strikingly, the Tet-Rbf1 chimera lacking the IE (Tet-Rbf1-∆IE) was
not compromised for activity; the protein repressed transcription from the Actin5C-Tet
reporter as effectively as did the wild type Tet-Rbf1 chimera, indicating that the IE is not
essential in this context (Fig. 3-4C). When assayed on the PCNA reporter that lacks Tet
binding sites but utilizes E2F1 to recruit Rbf1, the Tet-Rbf1-∆IE chimera was compromised
for repression, consistent with previous observations that the IE is important for Rbf1
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repression of cell cycle genes (Acharya et al., 2010). Therefore, this outcome suggests that
the mechanism of promoter targeting does influence whether the IE region functions in
repression. Interestingly, both Tet-Rbf1-C (4KA) and Tet-Rbf1-IE (4KA) were expressed at
similar levels as their wild type counterparts, and under conditions wherein the same alanine
substitutions increased Tet-full-length Rbf1 steady state levels (Fig. 3-4D). These
observations suggest that the function of these IE-lysine residues is context dependent for
both repression and stability.

Context-dependent repression by Rbf1-IE regulatory domain
The substantial repression exhibited by the Rbf1-∆IE mutant protein when directly
recruited to the Tet promoter demonstrated that this protein is not inherently defective. This
observation also raised the interesting possibility that the IE provides gene specific repression
capability. To examine the possibility that the IE provides repression capability specifically in
the context of E2F1-regulated promoters, the repression potency of wild type Rbf1 was
compared to Rbf1-∆IE on E2F1-regulated promoters (PCNA, Polα, and Mcm7) (Fig. 3-5A)
and non-canonical E2F1-independent promoters (InR, wts, Pi3K68D) (Fig. 3-5B). The InR,
wts, and Pi3K68D gene promoters are devoid of recognizable E2F1 binding sites and were
refractory to activation by E2F1, but are directly bound by Rbf1 in the embryo (Acharya et al.,
2012b). On the canonical target genes, Rbf1-∆IE was much weaker than wild-type Rbf1 for
E2F1-dependent gene repression, but both repressors exhibited similar potency on the
non-canonical Rbf1 reporter genes. As previous data showed that Rbf1-∆IE can interact with
E2F1 and associate with endogenous E2F1 target genes (Acharya et al., 2012b), the IE may
provide post-recruitment functions that are dispensable when Rbf1 is recruited independently
of E2F1.

100

Figure 3-4. Rbf1 IE functions as a transcriptional repression domain.

101

Figure 3-4 (cont’d)

102

Figure 3-4 (cont’d)
(A) Schematic representation of the E2F1-independent and E2F1-dependent reporter genes
used in this study (B) Transcriptional activities of Tet-fusion proteins were assayed on the
Actin5C-Tet-luc reporter. The IE with or without the NLS repressed the target gene when
directly tethered to the promoter compared to reactions lacking Rbf1 fusion proteins (*,
p<0.05). Both the WT and 4KA mutant versions repressed transcription equivalently. A
Knirps fusion protein (Tet-Knirps) and Tet protein alone (Tet-Stop) served as positive and
negative controls, respectively. (C) Transcriptional activities of the Tet-Rbf1 WT and
Tet-Rbf1 ∆IE chimeras were compared on the Actin5C-Tet-luc and PCNA-luc reporters. Data
are from at least three biological replicates. (D) Levels of the indicated Tet-Rbf1 fusion
proteins were determined by anti-Flag Western blot analysis 3 days after transfection. Lysine
to alanine substitution did not affect steady state levels of the Tet-Rbf1-IE and Tet-Rbf1-C
proteins under conditions wherein Tet-Rbf1 levels were increased. Tubulin levels are shown
as a loading control.

103

Figure 3-5. Context dependence of the Rbf1-IE for transcriptional repression.

104

Figure 3-5 (cont’d)

105

Figure 3-5 (cont’d)
(A, B) Rbf1 WT and Rbf1 ∆IE showed dissimilar repression activities on the E2F1 dependent
reporters as compared to the E2F1 independent promoters. Transcriptional activity was
measured as described in Figure 4. Data are from at least three biological replicates.

106

Rbf1 ubiquitination stimulates repressor potency
The function of the instability element as both a repression domain and a degron that
stimulates Rbf1 ubiquitination, suggested that ubiquitin might function directly in
Rbf1-mediated repression. We showed above that MG132 treatment substantially increases
the levels of ubiquitinated Rbf1. Therefore, we measured Rbf1-mediated repression of the
PCNA reporter in the presence or absence of MG132 (Fig. 3-6A). A modest but reproducible
enhancement in repression potency of wild type Rbf1 was observed within 2 hours of drug
treatment, an effect that was not observed with the Rbf1-∆IE mutant. This data is consistent
with IE-directed ubiquitination influencing repression activity.

Although MG132 affected

only the wild type Rbf1, a general concern remained that global proteasome inhibition may
induce pleiotropic effects (Deroo and Archer, 2002). Therefore, to directly assess the effect of
ubiquitin on Rbf1 function, repression assays were performed using chimera proteins
containing ubiquitin fused to the N-terminus of full length Rbf1. As ubiquitin attachment
markedly destabilized full-length Rbf1 (see also Fig. 3-6C) consistent with this modification
directing Rbf1 for proteasome destruction, repression assays were performed using differing
amounts of expression plasmids to equalize repressor concentration. Under conditions
wherein both Rbf1 and Ub-Rbf1 were expressed at comparable levels, the presence of
ubiquitin markedly improved Rbf1 repression activity on the PCNA promoter on average 4-5
fold (Fig. 3-6B). This outcome supports the hypothesis that ubiquitin can contribute directly
to target gene repression.
The potent role of ubiquitin in Rbf1 target gene repression noted above allowed the
possibility to examine whether poly-ubiquitination at this site is essential for enhanced
repressor potency. To test this possibility, K48R and K63R substitutions were incorporated
within the N-terminal ubiquitin at positions expected to impede poly-ubiquitination. Indeed,
as shown in Fig. 3-6C, Rbf1 appended with mutant ubiquitin (K48R, K63R) was maintained
107

at higher steady state levels than Rbf1 fused to wild type ubiquitin when expressed using
comparable amounts of expression plasmid. Thus, the N-terminal ubiquitin was functional in
the proteasome-mediated turnover of Rbf1. When compared to wild type Rbf1 lacking
ubiquitin, Rbf1 harboring mutant ubiquitin remained a more potent repressor of PCNA
transcription. This result suggests that while ubiquitination at the Rbf1 N-terminus can
contribute to repression potency, poly-ubiquitination at this site is not essential for this
enhancement. Nonetheless, in all experiments, Rbf1 containing wild type ubiquitin did
exhibit improved specific activity, suggesting that higher order ubiquitination can contribute
to repression.
Based on the observation that Rbf1-∆IE is defective for repression on E2F1 target genes,
whether the forced ubiquitination of Rbf1-∆IE could stimulate repression potency was tested.
In this experiment, higher levels of Rbf1-∆IE were tested to ensure that active proteins were
being compared. Under these conditions, and despite substantially lower steady state protein
levels associated with forced ubiquitination, Rbf1-∆IE harboring the appended wild type
ubiquitin exhibited increased repression ability of PCNA transcription (Fig. 3-6D). However,
ubiquitin did not enhance Rbf1-∆IE repression of the InR reporter, suggesting that the effect
of this modification is restricted to certain types of target genes. These observations imply
that insufficient ubiquitination observed with IE deletion underlies the loss of repression
activity at cell cycle regulated genes.

108

Figure 3-6. Rbf1 ubiquitination enhances gene specific repression activity.

109

Figure 3-6 (cont’d)

110

Figure 3-6 (cont’d)
(A) Proteasome inhibition by MG132 influences transcriptional repression activity of Rbf1
on the PCNA-luc reporter. Repression potency of WT Rbf1 on the PCNA-luc reporter (set to
100%), but not the ∆IE mutant was significantly enhanced after MG132 treatment (*, p<0.01)
(B) Ubiquitin enhances Rbf1 repression potency. In this experiment, wild type Rbf1
expression was adjusted to match that of the unstable Ub-Rbf1 chimera (3 ng pAX-Rbf1 WT
vs. 1000 ng pAX-Ub-Rbf1 WT) for testing using the PCNA-luc reporter (upper panel). At
comparable levels of repressor, as detected by Flag Western analysis (lower panel), ubiquitin
improved Rbf1 specific activity 3-4 fold. Tubulin levels are shown as a loading control. (C)
Poly-ubiquitination of the N-terminal ubiquitin is not essential for enhanced repression. K to
R substitutions at positions 48 and 63 within the N-terminal ubiquitin tag increased Rbf1
steady state levels as compared to wild type ubiquitin-Rbf1 chimeras in transfection
experiments using equal amounts of DNA (lower panel). At comparable protein levels, the
mutant Ub-Rbf1 chimera repressed transcription better than Rbf1 lacking the ubiquitin tag (*,
n=3, p<0.05) and to levels similar as observed for the Rbf1 chimera harboring the wild type
ubiquitin tag. (D) Ubiquitin fusion partially restores transcriptional repression activity to
Rbf1-∆IE on the PCNA-luc reporter (p<0.05) but not on the InR-luc reporter using equal
amounts of DNA during transfection. In these experiments, the faster migrating protein
observed with the Ub-Rbf1-∆IE fusion protein (lower panel) is likely due to substantial
cleavage of the ubiquitin tag.

111

Discussion
The RB family of proteins governs diverse physiological processes including cell cycle,
apoptosis, and differentiation. An important question remains how these factors maintain
differential influence over mutually exclusive pathways. Previous studies demonstrated that
mammalian RB phosphorylation by cell cycle dependent kinases or stress responsive kinases
can distinguish between cell cycle arrest or apoptotic responses (Delston et al., 2011). In this
study of the Drosophila Rbf1 protein, we uncovered a direct role for ubiquitination in
differential gene regulation. In particular, the C-terminal regulatory domain of Rbf1 was
found to harbor an independently acting degron that directs Rbf1 ubiquitination.
Post-translational modification by ubiquitin improved Rbf1 transcriptional repression,
directly linking repressor potency to ubiquitin-mediated turnover pathways. Furthermore,
Rbf1 lacking the degron was also debilitated for repression of cell cycle regulated PCNA,
Polα, and Mcm7 promoters, but not for regulation of non-canonical Rbf1 target genes, thus
highlighting a role for ubiquitination in differential regulation of Rbf target genes. These
findings point to distinct modes of transcriptional repression depending upon the promoters
targeted. Recent genomic studies have shown that Rbf1 association at many non-canonical
promoters, including the InR locus, is independent of E2F1 but is dependent upon the general
E2F partner, DP1 (Acharya et al., 2012b; Korenjak et al., 2012a). Thus, it remains possible
that the Rbf1 degron functions primarily when recruited by E2F1/DP1 and not when recruited
by E2F2/DP1. This concept is consistent with structural studies of human RB that show the
corresponding region located within the RB C-terminus is important for interactions with
E2F1/DP1 complexes (Rubin et al., 2005). As the Rbf1 degron sequence is highly conserved
within the mammalian RB homologs p107 and p130, degron function in differential gene
repression may be evolutionarily conserved.
While ubiquitin clearly enhanced Rbf1 activity towards the PCNA promoter, the molecular
112

mechanism by which ubiquitination is associated with transcriptional repression is unknown.
In one model, repression is enhanced by direct proteasome recruitment to a promoter through
interactions mediated by ubiquitin. In a second model, ubiquitination serves two roles,
recruiting essential cofactors to a promoter, and separately interacting with the protein
degradation machinery. Aspects of this mechanism are analogous to the degron theory of
gene activation previously described for the c-Myc proto-oncoprotein (Geng et al., 2012;
Salghetti et al., 2001; Salghetti et al., 1999; Salghetti et al., 2000). During activation,
ubiquitin can function for co-factor recruitment, such as described for recruitment of p-TEFb
by the viral activator VP16 (Kurosu and Peterlin, 2004), and thus ubiquitin may similarly
contribute to RB co-repressor recruitment. As our studies demonstrate that the C-terminal
degron may recruit an E3 ligase, a direct role for these enzymes in Rbf1 gene regulation is
possible. Such a direct role for E3 ligases in repression was observed for BRCA1-mediated
transcriptional regulation (Horwitz et al., 2007); however, in that example, ubiquitin
interfered with assembly of the preinitiation complex. Alternatively, Rbf1-mediated E3
recruitment could promote E2F1 ubiquitination. However, the IE region does not appear to
influence Rbf1-mediated E2F1 stabilization (42). Whether E3 ligases participate directly in
Rbf1-mediated repression is unknown, nonetheless, observations that the COP9 signalosome,
an evolutionarily conserved complex that functions to inhibit E3 ligase activity, was directly
found at Rbf1 target genes simultaneously with the Rbf1 repressor (Ullah et al., 2007)
suggests that a complex network of feedback regulation is proximally available at Rbf1 target
gene promoters.

Materials and Methods
Expression Constructs

113

Generation of Rbf1 WT and mutant expression constructs was described previously
(Acharya et al., 2010). To generate GFP fusion proteins, eGFP cDNA was PCR-amplified
from phs-eGFP and cloned into KpnI site of pAX vector. Two Flag epitope tags were inserted
5’ of the stop codon. The C-terminus and the IE of Rbf1 were made by site-directed
mutagenesis. To minimize the differences among mRNAs transcribed from GFP fusion
protein constructs, the first two amino acids of the IE were mutated into stop codons to
generate GFP alone constructs. Tet fusion protein expression constructs were generated as
described previously (Ryu and Arnosti, 2003). Rbf1 WT and mutants were digested from
pAX-rbf1 vector and ligated into KpnI and XbaI sites of pAX-Tet vector. The C-terminus and
the IE were amplified with KpnI and XbaI on the ends and inserted into pAX-Tet vector. To
generate ubiquitin fusion proteins, the ubiquitin coding sequence was amplified using
oligonucleotides with KpnI sites on both ends, and the amplicon was inserted into the KpnI
site of the pAX vector. The C-terminal glycine residues at the junction were initially mutated
to alanine to prevent ubiquitin removal by isopeptidases (Ub-Rbf1-∆IE, Fig. 3-6D) and then
to isoleucine (Ub-Rbf1, Figs. 3-6B, 3-6C) to provide a more complete block to cleavage.
Luciferase Reporter Assay
Drosophila S2 cells were transfected using Effectene transfection reagent (Qiagen)
according to the manufacturer’s protocol. Typically, 1.5 million cells were transfected with
100 ng of Ac5C2T50-Luciferase reporter, 0.25 µg of pRL-CMV Renilla luciferase reporter
(Promega) and 20 ng of one of pAX-Tet-rbf1 constructs. For PCNA-luciferase assay, 1.5
million cells were transfected with 1 µg of PCNA-Luciferase reporter, 250 ng of pRL-CMV
Renilla luciferase reporter (Promega) and 200 ng of pAX Rbf1-WT, pAX Rbf1-∆IE, or
pAX-Ub-Rbf1-∆IE constructs. 1000 ng of pAX-Ub-Rbf1-WT and 3 ng of pAX Rbf1-WT
was used in Fig. 3-6B. Cells were harvested 3 days after transfection and luciferase activity
was measured using the Dual-Glo Luciferase assay system (Promega) and quantified using
114

the Veritas microplate luminometer (Turner Biosystems). Firefly luciferase activity was
normalized to Renilla luciferase activity except when analyzing Rbf1 activity on the InR
promoter. For doxycycline treatment (1µg/ml), the drug was added to the media immediately
after transfection.
Western Blot Analysis
To measure protein levels in S2 cell culture, cells were harvested 3 or 5 days after
transfection and lysed by freeze-and-thaw cycles three times in lysis buffer (50mM Tris-HCl
pH8.0, 150mM NaCl, 1% Triton X-100). Typically, 50 µg S2 cell lysates were separated by
12.5% SDS-PAGE, transferred to PVDF membrane for analysis using M2 anti-Flag (mouse
monoclonal, 1:10,000, Sigma, F3165), anti-GFP (mouse monoclonal, 1:1,000, Santa Cruz
Biotechnology, sc-9996) and anti-tubulin (mouse monoclonal, 1:20,000, Iowa Hybridoma
Bank). Antibody incubation was performed in TBST (20 mM Tris-HCl, pH 7.5, 120 mM
NaCl, 0.1% Tween-20) with 5% non-fat dry milk. Blots were developed using
HRP-conjugated secondary antibodies (Pierce) and SuperSignal West Pico chemiluminescent
substrate (Pierce).
Stability Assays
For determination of GFP fusion protein half-life, 1.5 million S2 cells were transfected
with 200 ng of pAX-GFP-Rbf1-IE or 400ng of pAX-GFP. After 3-day incubation, cells were
treated with 100 µM cycloheximide for the indicated times. For proteasome inhibitor
treatments in Figs. 3-2B and 3-6A, seventy-two hours post-transfection, cells were treated
with DMSO or DMSO containing 50 µg/ml MG132 (Sigma-Aldrich) for 2 hours.
In vivo Ubiquitination Assay
In experiments shown in Fig. 3-2A and 3-2B, S2 cells were co-transfected with 250 ng of
pAX Rbf1-WT, 250 ng of pAcGal4 and 250 ng of UAS-Ub constructs using Effectene
transfection reagent (Qiagen, Valencia, CA). In Fig. 3-3A, cells were transfected with 50 ng

115

pAX Rbf1-WT or pAX Rbf1-∆IE, 50 ng of pAcGal4 and 50 ng of UAS-Ub constructs. In Fig.
3-3B, cells were transfected with 200 ng of Rbf1 WT, 400 ng of pAX-GFP-flag and 200 ng of
pAX GFP-Rbf1-IE constructs. In all cases, cells were grown for 3 days after which extracts
were prepared using SDS lysis buffer (2% SDS, 150mM NaCl, 10mM Tris-HCl, pH 8.0). The
extracts were heat denatured and sonicated followed by a 10-fold dilution using dilution
buffer (10mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100). Flag
immunoprecipitation reactions were performed (Anti-Flag M2 affinity gel, Sigma) followed
by anti-HA Western blotting (mouse polyclonal, 1:5000 dilution).

Acknowledgements
We thank Nick Dyson for sharing the PCNA-luciferase reporter gene, Cathie M. Pfleger
for the HA-Ubiquitin construct, and Rupinder Sayal for aid with confocal microscopy. This
work was supported by the MSU Gene Expression in Development and Disease Group, and
the National Institutes of Health (1R01GM079098, 1R01CA142644).

116

APPENDIX

117

Figure 3-S1. Identification of the Rbf1 nuclear localization sequence (NLS).

The indicated Rbf1 proteins were expressed in Drosophila S2 cells for subcellular
localization assessment by immunostaining (FITC). DNA within the nucleus was measured
by DAPI staining. The amino acids required for nuclear localization are contained within
787-808 of the C-terminus of Rbf1 - key residues are indicated in bold (bottom).

118

Figure 3-S2. The Rbf1 C-terminal lysines (K732, K739, K740 and K754) contribute to
degron function in GFP degradation.

Steady state levels of GFP fusion proteins were measured after cycloheximide treatment for
the indicated times. Lysine to alanine substitutions (GFP-IE 4K-A) in the IE resulted in a
significant extension of protein half-life compared to the GFP-IE protein. Error bars
indicate standard deviation, and asterisks indicate p<0.05.
119

Figure 3-S3. Lysine residues within the Rbf1 C-terminal degron influence Rbf1
ubiquitination.

S2 cells were transfected with HA-tagged ubiquitin and the indicated Flag-tagged Rbf1
expression constructs. After 3 or 5 days, denatured protein extracts were prepared for Flag
immunoprecipitation (IP). Recovered samples were assayed by anti-HA Western blot
analysis to detect ubiquitinated species and anti-Flag analysis to estimate recovery of Rbf1
proteins. The amounts of each species were quantified and the ratio of ubiquitin to Rbf1
was calculated.

In each experiment, the ratio for wild type Rbf1 was set to 1.0.

120

Figure 3-S4. Lysine residues within the Rbf1 C-terminal degron participate in enhanced
GFP ubiquitination.

GFP-IE and GFP-IE 4K-A were compared for ubiquitination as performed in Figure 3B.
Under conditions wherein expression levels of GFP-IE and GFP-IE (4KA) were comparable,
the presence of the 4K-A substitutions decreased ubiquitination as compared to the
ubiquitination levels observed for GFP-IE, as measured by co-transfection and
Co-IP/Western analysis.

121

REFERENCES

122

REFERENCES

Acharya, P., Negre, N., Johnston, J., Wei, Y., White, K.P., Henry, R.W., and Arnosti, D.N.
(2012). Evidence for autoregulation and cell signaling pathway regulation from
genome-wide binding of the Drosophila retinoblastoma protein.
Acharya, P., Raj, N., Buckley, M.S., Zhang, L., Duperon, S., Williams, G., Henry, R.W.,
and Arnosti, D.N. (2010). Paradoxical instability-activity relationship defines a novel
regulatory pathway for retinoblastoma proteins. Mol Biol Cell 21, 3890-3901.
Barbash, O., Lin, D.I., and Diehl, J.A. (2007). SCF Fbx4/alphaB-crystallin cyclin D1
ubiquitin ligase: a license to destroy. Cell division 2, 2.
Bech-Otschir, D., Seeger, M., and Dubiel, W. (2002). The COP9 signalosome: at the
interface between signal transduction and ubiquitin-dependent proteolysis. J Cell Sci 115,
467-473.
Burkhart, D.L., and Sage, J. (2008). Cellular mechanisms of tumour suppression by the
retinoblastoma gene. Nat Rev Cancer 8, 671-682.
Chamovitz, D.A., and Glickman, M. (2002). The COP9 signalosome. Curr Biol 12, R232.
Chen, P.L., Scully, P., Shew, J.Y., Wang, J.Y., and Lee, W.H. (1989). Phosphorylation of the
retinoblastoma gene product is modulated during the cell cycle and cellular differentiation.
Cell 58, 1193-1198.
Delston, R.B., and Harbour, J.W. (2006). Rb at the interface between cell cycle and
apoptotic decisions. Curr Mol Med 6, 713-718.
Delston, R.B., Matatall, K.A., Sun, Y., Onken, M.D., and Harbour, J.W. (2011). p38
phosphorylates Rb on Ser567 by a novel, cell cycle-independent mechanism that triggers
Rb-Hdm2 interaction and apoptosis. Oncogene 30, 588-599.
Deroo, B.J., and Archer, T.K. (2002). Proteasome inhibitors reduce luciferase and
beta-galactosidase activity in tissue culture cells. J Biol Chem 277, 20120-20123.
Du, W., and Dyson, N. (1999). The role of RBF in the introduction of G1 regulation during
123

Drosophila embryogenesis. EMBO J 18, 916-925.
Du, W., Vidal, M., Xie, J.E., and Dyson, N. (1996a). RBF, a novel RB-related gene that
regulates E2F activity and interacts with cyclin E in Drosophila. Genes Dev 10, 1206-1218.
Du, W., Xie, J.E., and Dyson, N. (1996b). Ectopic expression of dE2F and dDP induces cell
proliferation and death in the Drosophila eye. EMBO J 15, 3684-3692.
Geng, F., Wenzel, S., and Tansey, W.P. (2012). Ubiquitin and proteasomes in transcription.
Annu Rev Biochem 81, 177-201.
Genovese, C., Trani, D., Caputi, M., and Claudio, P.P. (2006). Cell cycle control and
beyond: emerging roles for the retinoblastoma gene family. Oncogene 25, 5201-5209.
Hammond-Martel, I., Yu, H., and Affar el, B. (2012). Roles of ubiquitin signaling in
transcription regulation. Cell Signal 24, 410-421.
Hanahan, D., and Weinberg, R.A. (2011). Hallmarks of cancer: the next generation. Cell
144, 646-674.
Hinds, P.W., Mittnacht, S., Dulic, V., Arnold, A., Reed, S.I., and Weinberg, R.A. (1992).
Regulation of retinoblastoma protein functions by ectopic expression of human cyclins.
Cell 70, 993-1006.
Horwitz, A.A., Affar el, B., Heine, G.F., Shi, Y., and Parvin, J.D. (2007). A mechanism for
transcriptional repression dependent on the BRCA1 E3 ubiquitin ligase. Proc Natl Acad Sci
U S A 104, 6614-6619.
Kato, J., Matsushime, H., Hiebert, S.W., Ewen, M.E., and Sherr, C.J. (1993). Direct binding
of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the
cyclin D-dependent kinase CDK4. Genes Dev 7, 331-342.
Keller, S.A., Ullah, Z., Buckley, M.S., Henry, R.W., and Arnosti, D.N. (2005). Distinct
developmental expression of Drosophila retinoblastoma factors. Gene Expr Patterns 5,
411-421.
Knudsen, E.S., Buckmaster, C., Chen, T.T., Feramisco, J.R., and Wang, J.Y. (1998).
Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression.
124

Genes Dev 12, 2278-2292.
Korenjak, M., Anderssen, E., Ramaswamy, S., Whetstine, J.R., and Dyson, N.J. (2012).
RBF binding to both canonical E2F targets and non-canonical targets depends on functional
dE2F/dDP complexes. Mol Cell Biol.
Kurosu, T., and Peterlin, B.M. (2004). VP16 and ubiquitin; binding of P-TEFb via its
activation domain and ubiquitin facilitates elongation of transcription of target genes. Curr
Biol 14, 1112-1116.
Lin, B.T., and Wang, J.Y. (1992). Cell cycle regulation of retinoblastoma protein
phosphorylation. Ciba Foundation symposium 170, 227-241; discussion 241-223.
Morris, E.J., and Dyson, N.J. (2001). Retinoblastoma protein partners. Adv Cancer Res 82,
1-54.
Nguyen, D.X., and McCance, D.J. (2005). Role of the retinoblastoma tumor suppressor
protein in cellular differentiation. J Cell Biochem 94, 870-879.
Rubin, S.M., Gall, A.L., Zheng, N., and Pavletich, N.P. (2005). Structure of the Rb
C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F
release. Cell 123, 1093-1106.
Ryu, J.R., and Arnosti, D.N. (2003). Functional similarity of Knirps CtBP-dependent and
CtBP-independent transcriptional repressor activities. Nucleic Acids Res 31, 4654-4662.
Salghetti, S.E., Caudy, A.A., Chenoweth, J.G., and Tansey, W.P. (2001). Regulation of
transcriptional activation domain function by ubiquitin. Science 293, 1651-1653.
Salghetti, S.E., Kim, S.Y., and Tansey, W.P. (1999). Destruction of Myc by
ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize
Myc. EMBO J 18, 717-726.
Salghetti, S.E., Muratani, M., Wijnen, H., Futcher, B., and Tansey, W.P. (2000). Functional
overlap of sequences that activate transcription and signal ubiquitin-mediated proteolysis.
Proc Natl Acad Sci U S A 97, 3118-3123.
Sdek, P., Ying, H., Zheng, H., Margulis, A., Tang, X., Tian, K., and Xiao, Z.X. (2004). The
125

central acidic domain of MDM2 is critical in inhibition of retinoblastoma-mediated
suppression of E2F and cell growth. J Biol Chem 279, 53317-53322.
Skapek, S.X., Pan, Y.R., and Lee, E.Y. (2006). Regulation of cell lineage specification by
the retinoblastoma tumor suppressor. Oncogene 25, 5268-5276.
Stevaux, O., and Dyson, N.J. (2002). A revised picture of the E2F transcriptional network
and RB function. Curr Opin Cell Biol 14, 684-691.
Uchida, C., Miwa, S., Kitagawa, K., Hattori, T., Isobe, T., Otani, S., Oda, T., Sugimura, H.,
Kamijo, T., Ookawa, K., et al. (2005). Enhanced Mdm2 activity inhibits pRB function via
ubiquitin-dependent degradation. EMBO J 24, 160-169.
Ullah, Z., Buckley, M.S., Arnosti, D.N., and Henry, R.W. (2007). Retinoblastoma protein
regulation by the COP9 signalosome. Mol Biol Cell 18, 1179-1186.
Wells, J., Boyd, K.E., Fry, C.J., Bartley, S.M., and Farnham, P.J. (2000). Target gene
specificity of E2F and pocket protein family members in living cells. Mol Cell Biol 20,
5797-5807.
Wells, J., Yan, P.S., Cechvala, M., Huang, T., and Farnham, P.J. (2003). Identification of
novel pRb binding sites using CpG microarrays suggests that E2F recruits pRb to specific
genomic sites during S phase. Oncogene 22, 1445-1460.
Wikenheiser-Brokamp, K.A. (2006). Retinoblastoma family proteins: insights gained
through genetic manipulation of mice. Cell Mol Life Sci 63, 767-780.
Ying, H., and Xiao, Z.X. (2006). Targeting retinoblastoma protein for degradation by
proteasomes. Cell Cycle 5, 506-508.

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CHAPTER 4

Rbf1 degron dysfunction enhances cellular DNA replication

Abstract

The E2F family of transcription factors contributes to oncogenesis through activation of
multiple genes involved in cellular proliferation, a process that is opposed by the
Retinoblastoma tumor suppressor protein (RB). RB also increases E2F1 stability by
inhibiting its

proteasome-mediated

degradation,

but

the

consequences

of

this

post-translational regulation of E2F1 remain unknown. To better understand the mechanism
of E2F stabilization and its physiological relevance, we examined the streamlined
Rbf1-dE2F1 network in Drosophila. During embryonic development, Rbf1 is insulated
from ubiquitin-mediated turnover by the COP9 signalosome, a multi-protein complex that
modulates E3 ubiquitin ligase activity. Here, we report that the COP9 signalosome also
protects the Cullin4-E3 ligase that is responsible for dE2F1 proteasome-mediated
destruction. This dual role of the COP9 signalosome may serve to buffer E2F levels,
enhancing its turnover via Cul4 protection and its stabilization through protection of Rbf1.
We further show that Rbf1-mediated stabilization of dE2F1 and repression of dE2F1
cell-cycle target genes are distinct properties. Removal of an evolutionarily conserved Rbf1
C-terminal degron disabled Rbf1 repression without affecting dE2F1 stabilization. This
mutant form of Rbf1 also enhanced G1-to-S phase progression when expressed in
Rbf1-containing S2 embryonic cells, suggesting that such mutations may generate
gain-of-function properties relevant to cellular transformation. Consistent with this idea,
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several studies have identified mutations in the homologous C-terminal domains of RB and
p130 in human cancer.

This work was published as the following manuscript:
Raj, N.*, Zhang, L.*, Arnosti, D. N., and Henry, R. W. (2012). Rbf1 degron dysfunction
enhances cellular DNA replication. Cell Cycle 11, 3731-3738. (*These authors contributed
equally to this work.)

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Introduction

The RB/E2F regulatory nexus

The Retinoblastoma family of proteins consists of the RB, p107, and p130 members that
control multiple processes associated with cellular proliferation, including cell cycle,
differentiation, apoptosis, and cellular biosynthetic potential (Henley and Dick, 2012).
Consistent with their regulatory governance of these processes, RB family members are
frequently inactivated in human cancers (Lohmann, 2010; Lohmann and Gallie, 2004). In
some diseases, such as retinoblastoma and small cell lung carcinoma, mutations in the RB1
gene itself are potentially causative for disease. In other cancer types, deregulation is
accomplished through altered function of upstream regulatory factors, including the
cyclin-dependent kinases (cdk) and cyclin/cdk inhibitors, with effects encompassing all RB
family members (Wikenheiser-Brokamp, 2006b). Together, these genetic changes are so
pervasive as to be recognized as a hallmark of cancer (Hanahan and Weinberg, 2000, 2011).
One important target for RB family members in gene regulation is the E2F family of
transcription factors that like RB are tightly linked to growth control. In humans, at least
eight different E2F species (E2F1-E2F8) have been identified, and are classified as either
transcriptional activators (E2F1-3) or repressors (E2F4-8) based on their sequence
homology and functional properties (Dimova and Dyson, 2005; van den Heuvel and Dyson,
2008). In Drosophila, these pathways are streamlined with two RB family proteins, Rbf1
and Rbf2, contributing to regulation of two E2F proteins, dE2F1 and dE2F2 (Stevaux et al.,
2002). During G0 and early G1 of cell-cycle progression, RB family members directly bind

129

to different sets of E2F factors (Goodrich et al., 1991; Takahashi et al., 2000), and at least
for E2F1, RB association reverses regulatory polarity from activation to repression
(Flemington et al., 1993; Helin et al., 1993; Hiebert et al., 1992; Takahashi et al., 2000;
Weintraub et al., 1995; Weintraub et al., 1992). Cyclin-cdk kinase mediated
phosphorylation of RB in late G1 causes RB/E2F1 dissociation, allowing E2F to activate
numerous proliferation genes that drive entry into S phase (Dynlacht et al., 1994; Ewen et
al., 1993; Hinds et al., 1992; Kato et al., 1993; Nevins et al., 1991). In human cancer,
increased E2F activity is frequently observed (Chen et al., 2009; Eymin et al., 2001; Imai et
al., 2004; Saberwal et al., 2004), and is associated with poor prognosis, particularly in
melanoma and breast cancer (Alla et al., 2010; Baldini et al., 2006; Hallett and Hassell,
2011; Vuaroqueaux et al., 2007), highlighting the importance of imposing regulatory curbs
on E2F1 expression and activity.

The ubiquitin-proteasome system and RB/E2F regulation

In addition to limitation through cyclin/cdk-mediated phosphorylation, the RB/E2F axis
is governed by the ubiquitin-proteasome system. Indeed, inappropriate RB turnover
contributes to disease as demonstrated during cellular immortalization by viral proteins
leading to enhanced RB ubiquitination (Boyer et al., 1996). Although RB levels often
appear stable in actively proliferating cells (Classon and Harlow, 2002; Haberichter et al.,
2007), steady state fluctuations have been correlated with phosphorylation changes during
cellular stress (Tedesco et al., 2002), suggesting that a negative correlation exists between
RB levels and its activity in certain contexts. For example, in response to nocodazole

130

blockade, U2OS osteosarcoma cells exhibit marked elevation of RB levels in the G2/M
phase of the cell cycle, and upon release into early G1, RB destabilization reestablishes
lower baseline steady state levels (not shown). In seminal experiments linking RB to cell
cycle control, microinjected RB induced cellular G1 arrest only when introduced during the
window of time immediately after nocodazole release and not when injected in
asynchronously proliferating cells (Goodrich et al., 1991), suggesting that RB function is
correlated with conditions in early G1 amenable to its diminishing steady state levels. An
inverse relationship between steady state levels and repressor potency was also observed
for the Drosophila Retinoblastoma family member Rbf1 wherein unstable Rbf1 proteins
were potent for target gene repression while stable mutant proteins were impotent (Acharya
et al., 2010). A tight activity-instability linkage may ensure that RB repression programs
remain dynamic and sensitive to growth conditions, such as previously suggested for
dynamic p53 fluctuation in response to DNA damage (Batchelor et al., 2011; Batchelor et
al., 2008). Similar to RB, both p107 and p130 exhibit differential expression during the cell
cycle with p107 levels peaking in S phase and p130 levels highest in G0 (Beijersbergen et
al., 1995; Classon and Harlow, 2002; Tedesco et al., 2002), and thus multiple mechanisms
likely influence turnover of these different RB family members. Interestingly, cyclin/cdk
kinase activity is correlated with changes in RB family member levels (Beijersbergen et al.,
1995; Tedesco et al., 2002), suggesting that the cyclin/cdk and ubiquitin/proteasome
regulatory arms crosstalk to govern both RB family activity and stability.
In previous studies of the Drosophila embryo, we observed that the Rbf1 and Rbf2
proteins associate with the COP9 signalosome (Ullah et al., 2007), a developmentally

131

regulated complex that controls proteasome-mediated degradation of many proteins through
interactions with SCF (SKP1/cullin/F-box) E3 ubiquitin ligase complexes (Wei et al., 2008).
CSN5, the catalytic core of COP9, contains a metalloprotease motif termed JAMM
(Jab1/MPN domain-associated metalloisopeptidase) that removes Nedd8 from the cullin
subunits (Cope et al., 2002). As cullin neddylation activates E3 ligase activity, the COP9
signalosome thus serves to protect substrates from turnover. Indeed, both Rbf1 and Rbf2 are
destabilized in the absence of COP9 function (Ullah et al., 2007), connecting the regulation
of Rbf protein turnover to the ubiquitin proteasome system. The involvement of a specific
ubiquitin ligase remains unknown, although in mammals, RB and p130 turnover has been
Skp2

linked to MDM2 (Sdek et al., 2005; Xiao et al., 1995) and SCF

(Tedesco et al., 2002),

respectively. As with the RB family, E2F family members are degraded through
ubiquitin-mediated turnover, both at defined points during the cell cycle (Peart et al., 2010)
and in response to DNA damage (Blattner et al., 1999; Hofferer et al., 1999) with E2F1
Skp2

subjected to ubiquitination via the S-phase specific F-box protein SCF

and degradation

in the S/G2 phases of the cell cycle (Marti et al., 1999; Zhang et al., 2005). Other ubiquitin
ligases including APC/C (Peart et al., 2010) and ROC-Cullin ligases (Ohta and Xiong,
2001) likely contribute to E2F1 degradation in these contexts. In contrast, a protective role
is suggested for the MDM2 ubiquitin ligase (Zhang et al., 2005), and consistently,
ARF

p19

-mediated inhibition of MDM2 encourages E2F1 turnover (Chang et al., 2007;

Martelli et al., 2001). Interestingly, a key determinant of E2F1 degradation turns out to be
RB itself (Hateboer et al., 1996; Hofmann et al., 1996; Ikeda et al., 1996; Martelli and
Livingston, 1999). RB can bind to a carboxy-terminal instability element in E2F1, and may

132

stabilize E2F1 by occluding the cellular ubiquitination machinery (Campanero and
Flemington, 1997). In Drosophila, E2F1 destruction is mediated by the Cul4

Cdt2

E3-Ubiquitin ligase (Shibutani et al., 2008; Zielke et al., 2011), suggesting that both Rbf1
and COP9 may coordinately influence E2F1 stability. Herein, we show that dE2F1 levels
are indeed influenced by Rbf1 and COP9, but through distinct mechanisms. While Rbf1
can stabilize dE2F1 through pocket-domain dependent protein-protein interactions, the
COP9 signalosome complex down regulates dE2F1 levels through modulation of the Cul4
E3 ligase. Rbf1-mediated dE2F1 stabilization and repression activity are separate
properties as select mutant Rbf1 forms lacking dE2F1 repression capability retained their
capacity to stabilize dE2F1. We further show that this class of repression-inactive Rbf1
mutants enhanced the rate of S-phase entry perhaps through their inappropriate stimulation
of dE2F1 levels.

Results & Discussion

The COP9 signalosome regulates the Rbf1/E2F1 pathway

To test whether loss of COP9 function is associated with destabilized E2F1, endogenous
dE2F1 steady-state levels were examined in S2 embryonic cells that were depleted of the
largest subunit of the COP9 complex, CSN1, using dsRNA. This treatment strongly
reduced levels of transfected flag-tagged RBF1 and endogenous RBF2, as expected, while
E2F1 levels were increased significantly (Fig. 4-1A). This result shows that the COP9
signalosome stabilizes Rbf proteins, as previously noted (Ullah et al., 2007), but instead of
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protecting E2F1, the COP9 complex contributes to its turnover. Similar results were
obtained during CSN4 and CSN5 knockdowns (not shown), suggesting that the COP9
complex rather than individual COP9 subunits contribute to E2F modulation. Previous
studies showed that the COP9 signalosome can stabilize cullin E3 ligase-containing SCF
complexes (Wu et al., 2005), and a Cul4-containing SCF complex contributes to dE2F1
ubiquitination in S2 cells(Shibutani et al., 2008). Therefore, Cul4 levels were examined in
Csn1 knockdown cells, ascertaining that Cul4 was indeed diminished during COP9
knockdown. Direct knockdown of Cul4 but not Cul5 also led to increased levels of E2F1,
consistent with previous reports (Shibutani et al., 2008). Thus, a pathway emerges wherein
the Cul4 E3 ligase responsible for E2F1 turnover is stabilized by the COP9 signalosome
(Fig. 4-1B). Direct modulation of Cul4 and Cul5 levels had no discernable effect on Rbf
levels, indicating that Rbf1 and E2F1 are ubiquitinated through distinct pathways. We
conclude that COP9 signalosome is associated with opposing roles for Rbf1 and E2F1,
contributing to Rbf1 stabilization but E2F1 destabilization.

Drosophila Rbf1 enhances dE2F1 levels

The data presented above highlights that a complicated network governs E2F stability with
the COP9 signalosome contributing to low E2F1 levels during normal function. Previous
studies have shown that in humans, RB can stabilize E2F1 (Campanero and Flemington,
1997; Hateboer et al., 1996; Hofmann et al., 1996). We therefore examined dE2F1 levels
using Drosophila S2 cells that harbor wild type COP9 function in the absence or presence
of increased Rbf1 expression (Fig. 4-2A). Three days post-transfection, steady-state protein

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levels of Myc-tagged dE2F1 were measured in the presence or absence of the MG132
proteasome inhibitor. Consistent with previous studies on mammalian E2F1, increased
levels of Drosophila dE2F1 were observed during Rbf1 expression and at levels
comparable to those observed with MG132 proteasome inhibition (Fig. 4-2A). Under the
conditions selected for this experiment, Rbf1 was relatively stable and its levels largely
unaffected by MG132 treatment, although under different growth conditions Rbf1 is
proteasome sensitive (Acharya et al., 2010). These data indicate that dE2F1 is targeted by
the ubiquitin-proteasome pathway and is responsive to the steady-state levels of Rbf1.
We next tested whether the direct binding of Rbf1 is required for dE2F1 stabilization.
The conserved RB-family pocket domain is the primary site for E2F1 interaction (Lee et al.,
2002; Rubin et al., 2005), therefore a deletion mutant of Rbf1 lacking this domain was
tested. Unlike with wild type Rbf1, dE2F1 levels were unaffected by this mutant form of
RBF1, while retaining responsiveness to proteasome inhibition. The pocket domain alone
was sufficient to confer at least partial stabilization on dE2F1, suggesting that this domain
is necessary but not sufficient for complete stabilization (Fig. 4-2B). These data are
consistent with a model wherein dE2F1 is stabilized by direct contacts with Rbf1.
Combined with our previous analysis of COP9 function, we conclude that the COP9
signalosome complex influences dE2F1 levels through two separate pathways, positively
by stabilization of Rbf1, which binds dE2F1 to enhance cellular levels, and negatively by
stabilizing the E3 ligase Cul4.
Our previous studies of Rbf1 indicated that an evolutionarily conserved C-terminal
instability element (IE) functions as an autonomous degron that stimulates both Rbf1

135

ubiquitination and repression potency (Acharya et al., 2010; Raj et al., 2012b). RB family
proteins require multiple domains to mediate gene repression, including the pocket domain
that facilitates E2F interaction and co-factor recruitment (Chan et al., 2001b; Ferreira et al.,
1998; Luo et al., 1998; Magnaghi-Jaulin et al., 1998), and the C-terminal region that
harbors the IE (Acharya et al., 2010), and may provide additional dE2F1 contacts, as was
shown for human RB and p107 (Rubin et al., 2005). Interestingly, Rbf1 degron deletion
mutants retained their capacity to physically associate with dE2F1 at target gene promoters
(Acharya et al., 2010). To determine whether Rbf1 repression activity and dE2F1
stabilization are biochemically separable, we generated a series of Rbf1 deletion constructs
that were tested for both properties (Fig. 4-3A). Consistent with previous studies,
transcription from the PCNA-promoter was activated by dE2F1, but was repressed upon
co-expression with wild type Rbf1 (Fig. 4-3B). Also consistent, wild type Rbf1 (1-845)
robustly stabilized dE2F1. Mutant forms of Rbf1 lacking the pocket domain (1-375 and
376-727) were inactive for both repression and dE2F1 stabilization, attesting to the
importance of this domain for both these properties. Significantly, three different mutant
forms of Rbf1 that lacked the IE entirely or had mutations in four key lysines were
defective for repression, but continued to stabilize E2F1. Together, these data identify one
class of Rbf1 mutations that disable repression without affecting dE2F1 stabilization, and
another class that disables both repression and stabilization.

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Figure 4-1. Dual roles of the COP9 signalosome in regulation of the Rbf1-dE2F1 network.

137

Figure 4-1 (cont’d)
(A) The COP9 signalosome complex governs both Rbf and E2F1 stability. S2 cells were
treated with indicated dsRNA and proteins were measured by western blot analyses.
Endogenous dE2F1 levels were dramatically increased due to reduced Cul4 levels when
Cul4 (lane 4 and 6) or its upstream regulator COP9 (CSN1 subunit, lane 3) were depleted.
Flag-Rbf1 and endogenous Rbf2 levels were substantially decreased by the CSN1
knockdown, but were not affected by Cullin knockdowns. (B) COP9 is a dual-functional
regulator of dE2F1 stability. First, COP9 plays a protective role on Rbf1, which in turn
stabilizes dE2F1. Second, COP9 restrains dE2F1 level by maintaining a Cul4-based E3
ligase, which targets dE2F1 for degradation.

138

Figure 4-2. The Rbf1 pocket domain contributes to dE2F1 protection from
proteasome-mediated degradation.

139

Figure 4-2 (cont’d)
(A) dE2F1 is sensitive to proteasome inhibition and is robustly stabilized by Rbf1 WT
protein but not by forms of Rbf1 lacking the central pocket domain. Under these
experimental conditions, Rbf1 WT and ΔPocket forms were expressed at equivalent levels
and both are insensitive to proteasome inhibition. Endogenous tubulin levels are shown as
loading controls. The experiment shown is representative of three biological replicates. (B)
The Rbf1 pocket domain is insufficient for robust dE2F1 stabilization. The Rbf1 WT
protein stabilizes dE2F1 protein, whereas at equivalent levels of expression, the Rbf1
pocket deletion mutant is incapable of stabilizing dE2F1 protein, while the Rbf1
pocket-only mutant provides only partial stabilization. Endogenous tubulin levels are
shown as loading controls.

140

Figure 4-3. Mutant Rbf1 lacking IE function stabilizes dE2F1 but cannot fully repress its
transcriptional activity.

141

Figure 4-3 (cont’d)

142

Figure 4-3 (cont’d)
(A) Schematic representation of Rbf1 proteins used for functional testing showing the
relative positions of the pocket domain and the IE region. (B) Functional characterization
of Rbf1. Mutations in the IE (728-786 and 4K-A) compromise transcriptional repression
activities of Rbf1 proteins measured on the PCNA-luciferase reporter gene (bar graph) but
do not affect dE2F1 stabilization property (anti-Myc Western blot). Under these
transfection conditions, Rbf proteins were expressed at similar levels (anti-Flag Western
blot). Data represents at least three biological replicates.

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Rbf degron mutations enhance cellular S-phase entry

Previous studies have shown that elevated E2F levels are associated with increased cellular
proliferation, and therefore we hypothesized that enhanced dE2F1 stabilization enabled by
repression-incompetent Rbf1 would facilitate ectopic S phase and contribute to deregulated
cell growth. To test this possibility, GFP-Rbf1 WT and the Rbf1 ∆IE mutant were
expressed in S2 cells and the effect on cell cycle progression was examined by FACS
analysis (Fig. 4-4). Consistent with the established role of Rbf1 in G1-to-S phase transition,
GFP-Rbf1 WT induced a strong G1 arrest in the transfected cells (GFP positive) that was
not observed in untransfected cells (GFP negative) from the same culture (not shown) or in
cells expressing the GFP-Rbf1 ∆IE mutant. The lack of G1 arrest by GFP-Rbf1 ∆IE is
consistent with a parallel lack of dE2F1 repression potency associated with IE loss.
Interestingly, GFP-Rbf1 ∆IE-expressing cells also displayed a modest increase in their
S-phase percentage, as estimated by Modfit analysis (Fig. 4-4, inset). Therefore, as a direct
measure of the ability of this mutant form of Rbf1 to stimulate S-phase entry, we performed
BrdU incorporation assays. In this assay, cells expressing wild type or mutant Rbf1 were
visualized by anti-Flag epitope immunofluorescence, and cells undergoing de novo DNA
synthesis were identified by BrdU staining (Fig. 4-5A). To assess the effect of transfected
proteins on cell cycle, we calculated a proliferation index comparing the percentage of
BrdU-positive cells in the transfected Rbf1-expressing population to the total population of
cells. If a transfected protein exhibits no effect on cell cycle entry, the index should be
equal to one, whereas the index should be less than one should the transfected protein cause
cell cycle arrest. A protein that induces ectopic S phase entry should result in an index
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greater than one. As shown in Fig. 4-5B, expression of Rbf1 lacking the pocket domain had
no effect on DNA synthesis (P.I. = 1.03), consistent with observations that this protein is
defective for repression and is unable to interact with and stabilize dE2F1. Therefore, in the
context of S2 cells that express endogenous Rbf1, this defective Rbf1 protein had no effect.
Cells expressing wild type Rbf1 experienced substantial cell cycle arrest (P.I. = 0.63),
suggesting that repression-competent Rbf1 down regulated key genes required for S phase
entry. Importantly, expression of the repression-defective Rbf1 ∆IE protein resulted in a
substantial increase in the percentage of cells undergoing DNA replication (P.I. = 1.24),
presumably through effects on dE2F1 stabilization. We conclude that expression of mutant
Rbf1 harboring alterations to IE function confers a distinct growth advantage due to
increased rate of S phase entry. In the model proposed in Fig. 4-5C, dE2F1 activity
increases during early G1 concomitant with increases in its steady state levels mediated, in
part, by either wild type or mutant Rbf1. However, loss of Rbf1-mediated repression
associated with IE mutation permits premature S phase entry, a process that would
normally be delayed in the presence of wild type Rbf1 until licensing by cyclin/cdk
phosphorylation. Based on these observations, we hypothesized that mutations in the IE
domain of human RB family members may be selected for in human cancers. Indeed, in
one study of non-small lung carcinoma, a substantial percentage of patients (84%) were
found to harbor mutations in the p130 IE region (Claudio et al., 2000). We previously
showed that mutant Rbf1 harboring alanine substitutions of lysine residues within the IE
diminish repressor potency but do not eliminate Rbf1-dE2F1 protein-protein interactions
(Acharya et al., 2010). It is interesting to note that many of the p130 IE cancer-associated

145

mutations were observed in these conserved basic residues. Together, these studies suggest
an unexpected role for RB family turnover in cellular proliferation and cancer.

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Figure 4-4. The IE region contributes to Rbf1-mediated G1 arrest.

147

Figure 4-4 (cont’d)
Wild type or mutant GFP-Rbf1∆IE proteins were expressed in Drosophila S2 cells and the
effect on cell cycle was determined by propidium iodide staining and FACS analyses. An
overlay of the DNA content histograms for wild type GFP-Rbf1 (grey) and mutant
GFP-Rbf1∆IE-expressing cells (solid unfilled) shows that the loss of IE function is
correlated with a diminished proportion of cells in the G1 phase. (Inset) Bar graph shows
the ratios of total S-phase percentages for GFP positive versus GFP negative populations
for GFP-Rbf1 WT and GFP-Rbf1∆IE transfected samples. In two separate experiments,
GFP-Rbf1∆IE expressing cells exhibited an increased proportion of cells in S phase, as
estimated using Modfit analysis.

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Figure 4-5. Mutation of the RB-family degron positively influences DNA replication
frequency.

149

Figure 4-5 (cont’d)

150

Figure 4-5 (cont’d)
(A) Rbf1 ∆IE enhances S -phase entry. S2 cells were transfected with the indicated Rbf1
proteins and the effect on DNA synthesis was monitored by BrdU incorporation.
Transfected cells and BrdU-positive cells were visualized by immunofluorescent staining
with anti-Flag and anti-BrdU antibodies. Arrows indicate representative cells that are
transfected/BrdU negative (green) or transfected/BrdU positive (yellow). Rbf1 ∆IE
expression was associated with increased BrdU positive staining compared to Rbf1 WT
expressing cells. (B) The effect of Rbf1 proteins on DNA replication was indicated as a
proliferation index calculated as a ratio of the percentage of BrdU-positive cells in
transfected cells to that in the total population. The Rbf1∆Pocket mutant that is unable to
both stabilize and repress dE2F1 also showed no effect on BrdU incorporation. Rbf1 WT
expressing cells exhibited diminished BrdU incorporation consistent with increased G1
arrest, whereas Rbf1 IE expressing cells exhibited enhanced S phase. Data from three
∆
biological replicates were analyzed. Error bars indicate standard deviation, and asterisks
indicate p < 0.05. (C) A model for the regulation of E2F function and stability during cell
cycle progression. In this model, E2F levels are stabilized during early G1 by both wild
type and mutant Rbf1 proteins. The COP9 signalosome contributes to enhanced dE2F1
steady state levels during this stage by stabilization of Rbf1. However, dE2F1 activity is
not restrained by mutant Rbf1 leading to premature S phase entry. After Rbf1-dE2F1
estrangement mediated by cyclin cdk phosphorylation, the COP9 signalosome contributes
to dE2F1 destruction via protection of Cul4.

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Materials and Methods

Expression Constructs
Generation of Rbf1 WT and mutant expression constructs was described previously
(Acharya et al., 2010). To generate GFP-Rbf1 fusion proteins, eGFP cDNA was amplified
from phs-eGFP and inserted into KpnI site of pAX-Rbf1 WT and pAX-Rbf1 728 -786
vectors. The Myc-E2F1 expression construct was a gift from Dr. Maxim Frolov, Univ. of
Chicago).
E2F1 Stabilization Assay
1.5 million Drosophila S2 cells were transfected with 0.2 µg of pAXRbf1 WT or
mutants and 0.2 µg of pIE-E2F1 constructs using the Effectene transfection reagent
(Qiagen) according to the manufacturer’s protocol. The cells were grown for 3 days after
which protein levels were analyzed through Western blotting. For proteasome inhibition in
Fig. 4-2, the cells were treated with 50 µg/ml MG132 (Sigma-Aldrich) or the vehicle
DMSO for 2 hours.
Western Blot Analysis
To measure protein levels in Drosophila S2 cell culture, cells were harvested 3 days
post-transfection and lysed by three freeze-thaw cycles in lysis buffer (50mM Tris-HCl
pH8.0, 150mM NaCl, 1% Triton X-100, protease inhibitors). 50 µg of S2 cell lysates were
run on 12.5% SDS-PAGE gels, transferred to a PVDF membrane, and probed with M2
anti-Flag (mouse monoclonal, 1:10,000, Sigma; F3165), anti-Myc (mouse monoclonal,

152

1:3000, Roche; 9E10), anti-tubulin (mouse monoclonal, 1:20,000, Iowa Hybridoma Bank),
anti-Cul4 (1:1000, a gift from Dr. Robert Duronio), anti-E2f1 (1:1000, gift from Dr. Maki
Asano), anti-Rbf2 (rabbit polyclonal, 1:5000, R2C1) and anti-CtBP (rabbit polyclonal,
1:5000, DNA208).
Luciferase Reporter Assay
Drosophila S2 cells were transfected using the Effectene transfection reagent (Qiagen)
according to the manufacturer’s protocol. Typically, 1.5 million cells were transfected with
1 µg of PCNA-Luciferase reporter, 0.25 µg of pRL-CMV Renilla luciferase reporter
(Promega), 200 ng of pIE-E2F1 and 200 ng of one of pAXRbf1 constructs. Cells were
harvested 72 hours after transfection and luciferase activity was measured using the
Dual-Glo luciferase assay system (Promega) and quantified using the Veritas microplate
luminometer (Turner Biosystems). Firefly luciferase activity was normalized to renilla
luciferase activity.
Fluorescence-activated Cell Sorting and Cell Cycle Analysis
To analyze the effects of Rbf1 proteins on Drosophila S2 cell cycle, FACS analyses were
performed using cells expressing GFP-tagged Rbf1 proteins. 1.5 million cells were
transfected with 1 µg of pAXGFP-Rbf1 WT or pAXGFP-Rbf1 ∆IE constructs using the
Effectene transfection reagent (Qiagen). Cells were harvested four days post-transfection
and analyzed by flow cytometry to separate the GFP positive and GFP negative populations.
Sorted cells were fixed with 70% ethanol and stained with propidium iodide (PI) for DNA
content measurements using a BD Bioscience Vantage SE flow cytometer. The cell cycle
data was analyzed through ModFit LT v3.3 (Verity Software House).

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BrdU Incorporation Assay
Cell proliferation was assessed by examining bromodeoxyuridine (BrdU) incorporation
20 h after the addition of BrdU to the S2 cell cultures that were transiently transfected with
the indicated pAXRbf1 expression constructs. 2 million S2 cells were plated on polylysine
coated glass coverslips and transfected with 400ng pAX vector expressing Flag-tagged
Rbf1 WT or mutants. Cells were incubated at 25 °C for one day and then incubated in
medium with 100uM BrdU for 20 hours. Cells were washed once with PBS and fixed in 4%
paraformaldehyde for 15 min at room temperature. Cells were permeabilized with 0.4%
Triton X-100 for 10 min and blocked with 1% BSA for 1 hour. Cells were then incubated
with rabbit polyclonal anti-Flag (1:250, F7425, Sigma-Aldrich) for 1 hour. After two
washes with TBST (20 mM Tris pH 7.5, 120 mM NaCl, 0.1% Tween 20), cells were
incubated with Alexa488-cojugated chicken anti-rabbit (1:500). For detection of BrdU,
cells were fixed again in 4% paraformaldehyde for 30 min, treated with 2M HCl for 30 min
and blocked with 1% BSA for 30 min. cells were then incubated with mouse monoclonal
anti-BrdU (1:250, BD Pharmingen) for 1 hour. After two washes with TBST, cells were
incubated with Alexa555-cojugated goat anti-mouse (1:500). Cover slips were mounted in
Vectashield mounting medium containing 1.5ug/ml DAPI.
RNA Interference
Double-stranded RNAs were transcribed with MEGAscript T7 High Yield Transcription
Kit (Ambion). S2 cells were maintained in Sf-900 II serum free medium (GIBCO)
supplemented with 0.5% Penicillin-Streptomycin. 1.5 million cells were incubated with
fresh medium containing 15 µg dsRNA for 30 min and then transfected with 200 ng

154

pAX-Rbf1. Cells were grown for 5 days at 25 °C.

Acknowledgements
We thank Nick Dyson for sharing the PCNA-luciferase reporter gene, Maxim Frolov for
the pIE-E2F1 construct, Robert Duronio for the anti-Cul4 antibody, Maki Asano for the
anti-E2F1 antibody, Louis King for expert assistance with flow cytometry, and Satyaki
Sengupta for his thoughtful comments. This work was supported by the MSU Gene
Expression in Development and Disease Group, and a grant from the NIH to DNA and
RWH (1R01GM079098).

155

REFERENCES

156

REFERENCES

Acharya, P., Raj, N., Buckley, M.S., Zhang, L., Duperon, S., Williams, G., Henry, R.W.,
and Arnosti, D.N. (2010). Paradoxical instability-activity relationship defines a novel
regulatory pathway for retinoblastoma proteins. Mol Biol Cell 21, 3890-3901.
Alla, V., Engelmann, D., Niemetz, A., Pahnke, J., Schmidt, A., Kunz, M., Emmrich, S.,
Steder, M., Koczan, D., and Putzer, B.M. (2010). E2F1 in melanoma progression and
metastasis. J Natl Cancer Inst 102, 127-133.
Baldini, E., Camerini, A., Sgambato, A., Prochilo, T., Capodanno, A., Pasqualetti, F.,
Orlandini, C., Resta, L., Bevilacqua, G., and Collecchi, P. (2006). Cyclin A and E2F1
overexpression correlate with reduced disease-free survival in node-negative breast cancer
patients. Anticancer Res 26, 4415-4421.
Batchelor, E., Loewer, A., Mock, C., and Lahav, G. (2011). Stimulus-dependent dynamics
of p53 in single cells. Mol Syst Biol 7, 488.
Batchelor, E., Mock, C.S., Bhan, I., Loewer, A., and Lahav, G. (2008). Recurrent initiation:
a mechanism for triggering p53 pulses in response to DNA damage. Mol Cell 30, 277-289.
Beijersbergen, R.L., Carlee, L., Kerkhoven, R.M., and Bernards, R. (1995). Regulation of
the retinoblastoma protein-related p107 by G1 cyclin complexes. Genes Dev 9, 1340-1353.
Blattner, C., Sparks, A., and Lane, D. (1999). Transcription factor E2F-1 is upregulated in
response to DNA damage in a manner analogous to that of p53. Mol Cell Biol 19,
3704-3713.
Boyer, S.N., Wazer, D.E., and Band, V. (1996). E7 protein of human papilloma virus-16
induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway.
Cancer Res 56, 4620-4624.
Campanero, M.R., and Flemington, E.K. (1997). Regulation of E2F through
ubiquitin-proteasome-dependent degradation: stabilization by the pRB tumor suppressor
protein. Proc Natl Acad Sci U S A 94, 2221-2226.
Chan, H.M., Smith, L., and La Thangue, N.B. (2001). Role of LXCXE motif-dependent
157

interactions in the activity of the retinoblastoma protein. Oncogene 20, 6152-6163.
Chang, D.L., Qiu, W., Ying, H., Zhang, Y., Chen, C.Y., and Xiao, Z.X. (2007). ARF
promotes accumulation of retinoblastoma protein through inhibition of MDM2. Oncogene
26, 4627-4634.
Chen, H.Z., Tsai, S.Y., and Leone, G. (2009). Emerging roles of E2Fs in cancer: an exit
from cell cycle control. Nat Rev Cancer 9, 785-797.
Classon, M., and Harlow, E. (2002). The retinoblastoma tumour suppressor in development
and cancer. Nat Rev Cancer 2, 910-917.
Claudio, P.P., Howard, C.M., Pacilio, C., Cinti, C., Romano, G., Minimo, C., Maraldi, N.M.,
Minna, J.D., Gelbert, L., Leoncini, L., et al. (2000). Mutations in the retinoblastoma-related
gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by
retrovirus-mediated gene transfer. Cancer Res 60, 372-382.
Cope, G.A., Suh, G.S., Aravind, L., Schwarz, S.E., Zipursky, S.L., Koonin, E.V., and
Deshaies, R.J. (2002). Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of
Nedd8 from Cul1. Science 298, 608-611.
Dimova, D.K., and Dyson, N.J. (2005). The E2F transcriptional network: old acquaintances
with new faces. Oncogene 24, 2810-2826.
Dynlacht, B.D., Flores, O., Lees, J.A., and Harlow, E. (1994). Differential regulation of
E2F transactivation by cyclin/cdk2 complexes. Genes Dev 8, 1772-1786.
Ewen, M.E., Sluss, H.K., Sherr, C.J., Matsushime, H., Kato, J., and Livingston, D.M.
(1993). Functional interactions of the retinoblastoma protein with mammalian D-type
cyclins. Cell 73, 487-497.
Eymin, B., Karayan, L., Seite, P., Brambilla, C., Brambilla, E., Larsen, C.J., and Gazzeri, S.
(2001). Human ARF binds E2F1 and inhibits its transcriptional activity. Oncogene 20,
1033-1041.
Ferreira, R., Magnaghi-Jaulin, L., Robin, P., Harel-Bellan, A., and Trouche, D. (1998). The
three members of the pocket proteins family share the ability to repress E2F activity
through recruitment of a histone deacetylase. Proc Natl Acad Sci U S A 95, 10493-10498.

158

Flemington, E.K., Speck, S.H., and Kaelin, W.G., Jr. (1993). E2F-1-mediated
transactivation is inhibited by complex formation with the retinoblastoma susceptibility
gene product. Proc Natl Acad Sci U S A 90, 6914-6918.
Goodrich, D.W., Wang, N.P., Qian, Y.W., Lee, E.Y., and Lee, W.H. (1991). The
retinoblastoma gene product regulates progression through the G1 phase of the cell cycle.
Cell 67, 293-302.
Haberichter, T., Madge, B., Christopher, R.A., Yoshioka, N., Dhiman, A., Miller, R.,
Gendelman, R., Aksenov, S.V., Khalil, I.G., and Dowdy, S.F. (2007). A systems biology
dynamical model of mammalian G1 cell cycle progression. Mol Syst Biol 3, 84.
Hallett, R.M., and Hassell, J.A. (2011). E2F1 and KIAA0191 expression predicts breast
cancer patient survival. BMC Res Notes 4, 95.
Hanahan, D., and Weinberg, R.A. (2000). The hallmarks of cancer. Cell 100, 57-70.
Hanahan, D., and Weinberg, R.A. (2011). Hallmarks of cancer: the next generation. Cell
144, 646-674.
Hateboer, G., Kerkhoven, R.M., Shvarts, A., Bernards, R., and Beijersbergen, R.L. (1996).
Degradation of E2F by the ubiquitin-proteasome pathway: regulation by retinoblastoma
family proteins and adenovirus transforming proteins. Genes Dev 10, 2960-2970.
Helin, K., Harlow, E., and Fattaey, A. (1993). Inhibition of E2F-1 transactivation by direct
binding of the retinoblastoma protein. Mol Cell Biol 13, 6501-6508.
Henley, S.A., and Dick, F.A. (2012). The retinoblastoma family of proteins and their
regulatory functions in the mammalian cell division cycle. Cell Div 7, 10.
Hiebert, S.W., Chellappan, S.P., Horowitz, J.M., and Nevins, J.R. (1992). The interaction of
RB with E2F coincides with an inhibition of the transcriptional activity of E2F. Genes Dev
6, 177-185.
Hinds, P.W., Mittnacht, S., Dulic, V., Arnold, A., Reed, S.I., and Weinberg, R.A. (1992).
Regulation of retinoblastoma protein functions by ectopic expression of human cyclins.
Cell 70, 993-1006.

159

Hofferer, M., Wirbelauer, C., Humar, B., and Krek, W. (1999). Increased levels of
E2F-1-dependent DNA binding activity after UV- or gamma-irradiation. Nucleic Acids Res
27, 491-495.
Hofmann, F., Martelli, F., Livingston, D.M., and Wang, Z. (1996). The retinoblastoma gene
product protects E2F-1 from degradation by the ubiquitin-proteasome pathway. Genes Dev
10, 2949-2959.
Ikeda, M.A., Jakoi, L., and Nevins, J.R. (1996). A unique role for the Rb protein in
controlling E2F accumulation during cell growth and differentiation. Proc Natl Acad Sci U
S A 93, 3215-3220.
Imai, M.A., Oda, Y., Oda, M., Nakanishi, I., and Kawahara, E. (2004). Overexpression of
E2F1 associated with LOH at RB locus and hyperphosphorylation of RB in non-small cell
lung carcinoma. J Cancer Res Clin Oncol 130, 320-326.
Kato, J., Matsushime, H., Hiebert, S.W., Ewen, M.E., and Sherr, C.J. (1993). Direct binding
of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the
cyclin D-dependent kinase CDK4. Genes Dev 7, 331-342.
Lee, C., Chang, J.H., Lee, H.S., and Cho, Y. (2002). Structural basis for the recognition of
the E2F transactivation domain by the retinoblastoma tumor suppressor. Genes Dev 16,
3199-3212.
Lohmann, D. (2010). Retinoblastoma. Adv Exp Med Biol 685, 220-227.
Lohmann, D.R., and Gallie, B.L. (2004). Retinoblastoma: revisiting the model prototype of
inherited cancer. Am J Med Genet C Semin Med Genet 129C, 23-28.
Luo, R.X., Postigo, A.A., and Dean, D.C. (1998). Rb interacts with histone deacetylase to
repress transcription. Cell 92, 463-473.
Magnaghi-Jaulin, L., Groisman, R., Naguibneva, I., Robin, P., Lorain, S., Le Villain, J.P.,
Troalen, F., Trouche, D., and Harel-Bellan, A. (1998). Retinoblastoma protein represses
transcription by recruiting a histone deacetylase. Nature 391, 601-605.
Martelli, F., Hamilton, T., Silver, D.P., Sharpless, N.E., Bardeesy, N., Rokas, M., DePinho,
R.A., Livingston, D.M., and Grossman, S.R. (2001). p19ARF targets certain E2F species
for degradation. Proc Natl Acad Sci U S A 98, 4455-4460.
160

Martelli, F., and Livingston, D.M. (1999). Regulation of endogenous E2F1 stability by the
retinoblastoma family proteins. Proc Natl Acad Sci U S A 96, 2858-2863.
Marti, A., Wirbelauer, C., Scheffner, M., and Krek, W. (1999). Interaction between
ubiquitin-protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation.
Nat Cell Biol 1, 14-19.
Nevins, J.R., Chellappan, S.P., Mudryj, M., Hiebert, S., Devoto, S., Horowitz, J., Hunter, T.,
and Pines, J. (1991). E2F transcription factor is a target for the RB protein and the cyclin A
protein. Cold Spring Harb Symp Quant Biol 56, 157-162.
Ohta, T., and Xiong, Y. (2001). Phosphorylation- and Skp1-independent in vitro
ubiquitination of E2F1 by multiple ROC-cullin ligases. Cancer Res 61, 1347-1353.
Peart, M.J., Poyurovsky, M.V., Kass, E.M., Urist, M., Verschuren, E.W., Summers, M.K.,
Jackson, P.K., and Prives, C. (2010). APC/C(Cdc20) targets E2F1 for degradation in
prometaphase. Cell Cycle 9, 3956-3964.
Raj, N., Zhang, L., Wei, Y., Arnosti, D.N., and Henry, R.W. (2012). Ubiquitination of
retinoblastoma family protein 1 potentiates gene-specific repression function. J Biol Chem
287, 41835-41843.
Rubin, S.M., Gall, A.L., Zheng, N., and Pavletich, N.P. (2005). Structure of the Rb
C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F
release. Cell 123, 1093-1106.
Saberwal, G., Lucas, S., Janssen, I., Deobhakta, A., Hu, W.Y., Galili, N., Raza, A., and
Mundle, S.D. (2004). Increased levels and activity of E2F1 transcription factor in
myelodysplastic bone marrow. Int J Hematol 80, 146-154.
Sdek, P., Ying, H., Chang, D.L., Qiu, W., Zheng, H., Touitou, R., Allday, M.J., and Xiao,
Z.X. (2005). MDM2 promotes proteasome-dependent ubiquitin-independent degradation of
retinoblastoma protein. Mol Cell 20, 699-708.
Shibutani, S.T., de la Cruz, A.F., Tran, V., Turbyfill, W.J., 3rd, Reis, T., Edgar, B.A., and
Duronio, R.J. (2008). Intrinsic negative cell cycle regulation provided by PIP box- and
Cul4Cdt2-mediated destruction of E2f1 during S phase. Dev Cell 15, 890-900.
Stevaux, O., Dimova, D., Frolov, M.V., Taylor-Harding, B., Morris, E., and Dyson, N.
161

(2002). Distinct mechanisms of E2F regulation by Drosophila RBF1 and RBF2. EMBO J
21, 4927-4937.
Takahashi, Y., Rayman, J.B., and Dynlacht, B.D. (2000). Analysis of promoter binding by
the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression.
Genes Dev 14, 804-816.
Tedesco, D., Lukas, J., and Reed, S.I. (2002). The pRb-related protein p130 is regulated by
phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2). Genes
Dev 16, 2946-2957.
Ullah, Z., Buckley, M.S., Arnosti, D.N., and Henry, R.W. (2007). Retinoblastoma protein
regulation by the COP9 signalosome. Mol Biol Cell 18, 1179-1186.
van den Heuvel, S., and Dyson, N.J. (2008). Conserved functions of the pRB and E2F
families. Nat Rev Mol Cell Biol 9, 713-724.
Vuaroqueaux, V., Urban, P., Labuhn, M., Delorenzi, M., Wirapati, P., Benz, C.C., Flury, R.,
Dieterich, H., Spyratos, F., Eppenberger, U., et al. (2007). Low E2F1 transcript levels are a
strong determinant of favorable breast cancer outcome. Breast Cancer Res 9, R33.
Wei, N., Serino, G., and Deng, X.W. (2008). The COP9 signalosome: more than a protease.
Trends Biochem Sci 33, 592-600.
Weintraub, S.J., Chow, K.N., Luo, R.X., Zhang, S.H., He, S., and Dean, D.C. (1995).
Mechanism of active transcriptional repression by the retinoblastoma protein. Nature 375,
812-815.
Weintraub, S.J., Prater, C.A., and Dean, D.C. (1992). Retinoblastoma protein switches the
E2F site from positive to negative element. Nature 358, 259-261.
Wikenheiser-Brokamp, K.A. (2006). Retinoblastoma regulatory pathway in lung cancer.
Curr Mol Med 6, 783-793.
Wu, J.T., Lin, H.C., Hu, Y.C., and Chien, C.T. (2005). Neddylation and deneddylation
regulate Cul1 and Cul3 protein accumulation. Nat Cell Biol 7, 1014-1020.
Xiao, Z.X., Chen, J., Levine, A.J., Modjtahedi, N., Xing, J., Sellers, W.R., and Livingston,
162

D.M. (1995). Interaction between the retinoblastoma protein and the oncoprotein MDM2.
Nature 375, 694-698.
Zhang, Z., Wang, H., Li, M., Rayburn, E.R., Agrawal, S., and Zhang, R. (2005).
Stabilization of E2F1 protein by MDM2 through the E2F1 ubiquitination pathway.
Oncogene 24, 7238-7247.
Zielke, N., Kim, K.J., Tran, V., Shibutani, S.T., Bravo, M.J., Nagarajan, S., van Straaten, M.,
Woods, B., von Dassow, G., Rottig, C., et al. (2011). Control of Drosophila endocycles by
E2F and CRL4(CDT2). Nature 480, 123-127.

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CHAPTER 5

Differential phosphorylation events govern stability-activity linkage of Drosophila
retinoblastoma protein Rbf1

Abstract

The retinoblastoma (RB) family proteins are transcriptional corepressors that play
diverse roles in many cellular events to exert potent tumor suppression functions. Precise
regulation of activity and turnover of these proteins is effected through a variety of
post-translational modifications. We identified an evolutionarily conserved C-terminal
instability element (IE) in the Drosophila RB-related protein Rbf1 that simultaneously
regulates degradation and repression activity. Surprisingly, stabilizing mutations in the IE
are less, not more, active in repression, suggesting that instability is tightly linked to Rbf1
function. To better understand the control of this unique bifunctional element of Rbf1, we
investigated the effects of phosphorylation by Cyclin-Cdk complexes. We show by directed
in vivo phosphorylation of Rbf1 and mutagenesis of target sites that protein stability and
activity are cleanly separable, suggesting that the multifunctional IE domain can act in
parallel regulatory roles, possibly by coordinated interactions with E2F transcription factors
and E3 ubiquitin ligases. A separate phosphorylation event in the N-terminus of the protein
contributes independently to overall turnover control. Dramatic developmental phenotypes
are observed in Drosophila eyes for all Rbf1 mutations that affect protein activity,
independent of protein lability. Included in this class of mutants are those that affect K774,
a conserved residue in RB family proteins mutated in human tumors. Our data suggests that
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a phosphorylation code governs distinct functional outputs, which may have gene- or
stage-specific implications in disease-related RB family mutations.

165

Introduction

The retinoblastoma protein RB, recognized as a key tumor suppressor, has a plethora of
cellular roles in diverse pathways, including cell cycle control, induction of cellular
differentiation, regulation of apoptosis and maintenance of genomic stability (Burkhart and
Sage, 2008). Functional inactivation of RB in a broad range of human cancers contributes
to both cancer initiation and progression, suggesting cell type-specific and tumor
stage-specific functions of RB as a potent tumor suppressor. Consistent with diverse
regulatory roles, RB is subject to tight controls through multiple levels of regulation
mechanisms which are often severely disrupted under disease conditions (Chau and Wang,
2003).
Phosphorylation of RB, and related family members p107 and p130, is recognized as a
pivotal mechanism which significantly influences RB functions under different cellular
conditions including cell cycle regulation, apoptosis and DNA repair (Classon and Dyson,
2001; Munro et al., 2012). A canonical phosphorylation pathway of RB involves activity of
cyclin-dependent kinases (Cdk) during cell cycle progression. Inactivation of RB during
G1-S transition requires sequential phosphorylation events by different Cyclin-Cdk
complexes (Mittnacht, 1998). Under conditions of DNA damage, RB is phosphorylated by
p38 MAP kinase which is activated by a signaling cascade in response to stress stimuli
(Delston et al., 2011). Additionally, phosphorylation of RB mediated by checkpoint kinases,
Chk1 and Chk2, regulates RB function on apoptotic genes during DNA damage response
(Inoue et al., 2007). These examples demonstrate that phosphorylation mediated by
multiple kinases modulates RB functions involved in different pathways, generating
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distinct molecular outputs.
Numerous phosphorylation sites have been mapped to the RB protein, supporting the
idea that phosphorylation events throughout the protein participate in the regulation of RB
functions (Adams, 2001). More importantly, differentially phosphorylated forms of RB
controlled by different kinases have distinct regulatory functions. For example, Cyclin-Cdk
complexes target overlapping as well as unique phosphorylation sites involved in different
cell cycle stages (Zarkowska and Mittnacht, 1997). p38 phosphorylates RB at a specific site
S567 to modulate RB repression function on apoptotic genes (Delston et al., 2011). S612 of
RB is phosphorylated by checkpoint kinases in response to DNA damage to strengthen
rather than alleviate RB anti-apoptotic activity (Inoue et al., 2007). Therefore,
phosphorylation is not a unimodal modification for the diverse cellular functions of RB on
its target genes. It has been proposed that the phosphorylation code in RB translates
regulatory inputs into signals for recruitment of cofactors and modulation of gene activity
(Munro et al., 2012).
A mechanism by which differential phosphorylation affects RB is that modification of
specific sites causes intramolecular conformational changes, affecting interactions with RB
binding partners. At least three key phosphorylation events induce structural changes in
different domains of RB to inhibit RB binding to E2F family proteins (Rubin, 2013). The
association between the pocket domain in RB and the transactivation domain (TD) in E2F
is inhibited by phosphorylation of S608/S612 in the pocket domain (Burke et al., 2012).
T373 phosphorylation induces the binding of RB N-terminus to the pocket domain to
disrupt E2F binding (Burke et al., 2012). Phosphorylation of the C-terminal region in RB

167

has two major structural functions: inhibiting C-terminus binding to the marked box (MB)
domain of E2F and inducing RB C-terminus binding to the pocket domain (Rubin et al.,
2005). Additionally, phosphorylation also regulates association between RB and cofactors
such as HDAC (Rubin et al., 2005). Conformational changes in RB caused by
phosphorylation provide a platform on which different phosphorylation events can be
translated into different functional consequences.
In addition to phosphorylation control of activity, RB is also subject to proteolysis
through a proteasome-dependent pathway in the context of normal cellular growth. RB can
be regulated by Mdm2, a well known E3 ubiquitin ligase for its regulation of the p53 tumor
suppressor (Sdek et al., 2005; Uchida et al., 2005). Additionally, certain viral proteins can
induce RB protein turnover to promote host cell proliferation, supporting the idea that
control of RB stability is a key process in normal cell growth (Boyer et al., 1996; Stubdal et
al., 1997). Interestingly, several studies of cell cycle regulation by RB family proteins show
that RB protein levels fluctuate in corresponding to phosphorylation status, suggesting an
intimately connected network between phosphorylation control of RB activity and stability
(Buchkovich et al., 1989; Tedesco et al., 2002).
In Drosophila, the RB-E2F network and its upstream regulation pathways are well
conserved in cellular events including cell cycle regulation and apoptosis, attesting the
physiological importance of phosphorylation and proteolysis controls (van den Heuvel and
Dyson, 2008). The Drosophila RB family protein Rbf1 is subject to the canonical
phosphorylation mechanism mediated by Cyclin-Cdk (Xin et al., 2002). Additionally, Rbf1
protein level is controlled by a developmentally regulated turnover process which involves

168

the function of the COP9 signalosome, a conserved complex that controls E3 ligase activity
and proteasome-dependent protein degradation (Ullah et al., 2007). Our previous studies
indicate that a C-terminal instability element (IE) of Rbf1 influences its turnover rate and
contributes to Rbf1 repression activity, suggesting a tight correlation between Rbf1 stability
and activity (Acharya et al., 2010; Raj et al., 2012b). Post-translational modifications
including ubiquitination and phosphorylation are likely to be involved to bridge potential
crosstalk between different regulation pathways. However, the molecular details of the IE
as a regulatory module in integrating protein turnover and transcriptional control are not
well understood.
In this study, we show that N-terminal and C-terminal phosphorylation sites contribute to
Cyclin-Cdk-mediated regulation of Rbf1 stability and activity, but functional importance of
phosphorylation in different regulation pathways is not equally distributed among these
phosphorylation sites, supporting the idea of the phosphorylation code translated into
functional consequences. Interestingly, a key conserved lysine residue in the Rbf1 IE
appears to influence phosphorylation effects; the homologous residue is found to be
mutated in p130 in lung cancers. These studies demonstrate the functions of the IE as a
nexus connecting regulatory power of phosphorylation involved in multiple pathways.

169

Results

The instability element (IE) harbors phosphorylation sites critical for controlling Rbf1
stability and activity

Drosophila

Rbf1

protein

is

subject

to

a

phosphorylation

control

by

Cyclin-Cyclin-dependent kinase (Cyc-Cdk) during cell cycle progression, leading to cell
cycle-dependent modulation of its transcriptional repression activity (Xin et al., 2002).
Studies of mammalian RB family proteins suggest a tight correlation between their
phosphorylation status, activity and stability (Tedesco et al., 2002). To understand the
phosphorylation mechanism underlying the activity-stability connection, we first asked
whether the IE mediates phosphorylation-induced stabilization. We first examined the
function of exogenous Cyclin-Cdk complexes on wild-type Rbf1 in Drosophila S2 cells by
cotransfecting CycD-Cdk4 or CycE-Cdk2 pairs with Flag-epitope tagged Rbf1 (Fig. 5-1A).
The activity of Rbf1 under these conditions was assayed on a PCNA-luciferase reporter. As
expected, Rbf1 was inactivated substantially by both of the Cyc-Cdk complexes, consistent
with the known function of Cyc-Cdk-mediated phosphorylation on Rbf1. Intriguingly, the
steady-state level of Rbf1 was increased under the condition of Cyc-Cdk overexpression
(Fig. 5-1B). In addition to higher levels of the original Rbf1 protein, slower-migrating band
was enhanced in the presence of overexpressed Cyc-Cdk, indicating a hyperphosphorylated
species. The IE functions as an autonomous degron, destabilizing the levels of a
heterologous GFP protein to which it is fused (Raj et al., 2012b); we did not observe
stabilization of this chimera upon expression of Cyc-Cdk kinases, suggesting that the IE

170

alone is insufficient to mediate interaction of these enzymes (Fig. 5-S1).
The common effects on protein stability and activity suggested that the C-terminal IE,
which is important for both of these effects, may be directly involved in the regulation by
phosphorylation (Raj et al., 2012b). Structural studies of RB demonstrate that multiple
phosphorylation events in the C-terminus can disrupt RB binding to E2F1, as well as
induce a conformational change in RB (Rubin et al., 2005). We compared the sequence of
Rbf1 IE with human RB family proteins and noted three highly conserved SP sites (S728,
S760 and S771), which are potential phosphorylation targets by Cyc-Cdk (Fig. 5-2A).
Previous studies have indicated that the sites in Rbf1 or RB family members can be
phosphorylated in vivo (Canhoto et al., 2000; Cantin et al., 2008; Dephoure et al., 2008;
Gauci et al., 2009; Leng et al., 2002; Mayya et al., 2009; Zhai et al., 2008). We examined
whether these three serine residues are critical for Rbf1 stability by mutating them to either
aspartates or alanines. The mutant Rbf1 harboring phosphomimetic Ser-to-Asp mutations
accumulated to about twofold of the wild-type Rbf1 protein, whereas the mutant with
unphosphorylatable Ser-to-Ala mutations was expressed at a lower level than the wild-type
protein (Fig. 5-2B). These results indicate that the serine residues influence Rbf1 stability,
possibly as targets of phosphorylation, providing a regulatable switch to turn up or down
Rbf1 protein levels. Our previous study of the IE domain indicated that there was a close
positive association between Rbf1 lability and activity, and earlier work has indicated that
phosphorylation of RB family proteins downregulates their transcriptional activity
(Acharya et al., 2010; Classon and Dyson, 2001; Mittnacht, 1998). We therefore
hypothesized that the phosphomimetic Ser-to-Asp mutations in the Rbf1 IE would

171

inactivate Rbf1. We assayed the activity of the stabilized Rbf1 3SD and destabilized Rbf1
3SA mutants on the PCNA-luciferase reporter gene (Fig. 5-2C). Surprisingly, both mutants
showed strong repression activity at levels similar to the wild-type Rbf1. As expected, the
stabilized Rbf1 4KA, included as a negative control, was a much weaker repressor in this
assay. Thus, the C-terminal phosphorylation sites that influence Rbf1 stability are not
essential for repression activity in this context. One possibility is that other sites are
important for phosphorylation-mediated regulation of Rbf1 activity, and these sites only
affect stability. We tested this notion by overexpression of Cyc-Cdk, and noted that Rbf1
3SD and 3SA were less effectively inactivated than Rbf1 WT, suggesting that the serine
residues within the IE are indeed important for the phosphorylation control of activity (Fig.
5-2D). The partial responsiveness of the Rbf1 mutants does indicate that additional
phosphorylation sites may be involved.
The comparison between Rbf1 4KA and Rbf1 3SD enabled us to revisit our previous
model that Rbf1 exhibits strong repression potency when it becomes labile and ready for
degradation (Raj et al., 2012b). Both sets of mutations stabilize Rbf1, but they lead to
distinct outcomes in repression activity; Lys-to-Ala mutations abolish Rbf1 activity
whereas Ser-to-Asp mutations show little effect in the same context. This inconsistency
with the model suggests that at least two distinct mechanisms for Rbf1 activity are utilized
to govern regulations by different post-translational modifications.

172

Figure 5-1. Drosophila Rbf1 is subject to Cyc-Cdk-mediated inactivation and stabilization.

173

Figure 5-1 (cont’d)
(A) The wild-type Rbf1 was coexpressed with CycD-Cdk4 or CycE-Cdk2 in S2 cells and
its repression activity was assayed on the PCNA-luciferase reporter gene. Rbf1-mediated
repression is reduced by Cyc-Cdk. Bar graph shows average of four biological replicates. (B)
The wild-type Rbf1 protein level with or without coexpression of Cyc-Cdk was assayed by
western blotting using anti-Flag antibody in this and subsequent experiments. Rbf1 levels
are increased in the presence of overexpressed Cyc-Cdk. Bar graph represents average of
eight biological replicates. Protein levels were quantitated by photon-capture analysis with a
Fuji LAS-3000 Imager and normalized to tubulin levels. In subsequent figures, data
represent at least three biological replicates in all luciferase activity and western blot assays.
Error bars indicate standard deviation.

174

Figure 5-2. Three serine residues in the IE are critical for Rbf1 protein stability and
repression activity.

175

Figure 5-2 (cont’d)

176

Figure 5-2 (cont’d)
(A) Schematic diagram of the wild-type Rbf1 and sequence alignment of the IE in Rbf1
and human RB family proteins. The three serine residues subject to mutagenesis are
indicated by numbers (728, 760 and 771). Identical residues are colored in blue and similar
residues are in aquamarine. Known phosphorylation sites are indicated in orange. The four
lysine resides in the IE critical for protein stability are shown in boldface. (B) Western blot
measuring expression levels of Rbf1 proteins, comparing wild-type (WT), a stabilized 4KA
mutant, and Ser-to-Asp or Ser-to-Ala mutants.

Phosphomimetic S-D mutations stabilize

Rbf1, whereas unphosphorylatable S-A mutations lead to lower expression levels. (C) Rbf1
3SD and 3SA activities were assayed on the PCNA-luciferase reporter gene. Both mutants
retain wild-type repression activity. The 4KA mutant exhibits low activity, as previously
shown (Acharya et al., 2010). (D) Responsiveness of 3SD and 3SA to Cyc-Cdk
overexpression was assayed on the PCNA-luciferase reporter gene. Rbf1 WT is inactivated
by Cyc-Cdk, while 3SD and 3SA are partially resistant to Cyc-Cdk-mediated inactivation.
Asterisks indicate p < 0.01.

177

The N-terminal T356 plays a dominant role in Cyc-Cdk-mediated stabilization of Rbf1

Studies of the N-terminus of RB show that phosphorylation of T373 induces a
conformational change that allows the N-terminus to bind to the pocket region, blocking
E2F association and relieving RB repression (Burke et al., 2012). In Rbf1, T356 is located
in a conserved region homologous to that in which the RB T373 is found (Fig. 5-3A). We
first assessed the role of T356 in Rbf1 stability by mutating this residue to either aspartate
or alanine (Fig. 5-3B). Unlike the distinct effects of Ser-Ala and Ser-Asp mutations in the
IE, both Thr-to-Asp and Thr-to-Ala destabilized Rbf1, suggesting that T356 also affects
Rbf1 stability, and that both mutations may block phosphorylation to affect the steady-state
levels. (T356D is apparently not phosphomimetic in this context). To test whether enhanced
phosphorylation would further affect Rbf1 proteins in which T356 is mutated, we assayed
Rbf1 response to overexpressed Cyc-Cdk with T356D alone or in combination with the
three Ser-to-Asp mutations in the IE (Fig. 5-3C). Strikingly, mutants with T356D alone or
all four mutations were resistant to Cyc-Cdk-mediated stabilization, consistent with a
model that phosphorylation of T356 in Rbf1 stabilization plays a key step that may involve
facilitating phosphorylation of other targets on Rbf1 (Fig. 5-3D). The weak residual
response of Rbf1 T356D to Cyc-Cdk overexpression suggests that phosphorylation of
additional sites may contribute to Rbf1 stabilization. We conclude that Cyc-Cdk stabilizes
Rbf1 through two distinct regions containing phosphorylation sites; phosphorylation of
T356 is predominant and may facilitate the downstream phosphorylation events.

178

Figure 5-3. N-terminal T356 is a key residue for the regulation of Rbf1 protein levels.

179

Figure 5-3 (cont’d)

180

Figure 5-3 (cont’d)

181

Figure 5-3 (cont’d)
(A) Protein sequence alignment of the N-terminal region harboring T356 in Rbf1 and
human RB family proteins. The threonine residue is indicated by number 356. Identical
residues are colored in blue and similar residues are in aquamarine. Known
phosphorylation sites are indicated in orange. (B) Both mutants with Thr-to-Asp or
Thr-to-Ala are destabilized in S2 cells, shown in western blot assays. (C) Schematic
diagram of Rbf1 mutants with the N-terminal Thr-to-Asp mutation and the C-terminal
Ser-to-Asp mutations. (D) Rbf1 WT and 3SD levels are increased by expression of
Cyc-Cdk, whereas Rbf1 mutants with a Thr-to-Asp mutation (T356D and 4D) show much
less responses. The difference between the fold increase of WT and T356D or WT and 4D
under the condition of CycE-Cdk2 overexpression is significant (p = 0.01).

182

T356 is critical for Cyc-Cdk-mediated inactivation of Rbf1

Our ability to separate control of turnover and transcriptional activity with respect to the
IE led us to ask whether the regulation mediated by T356 had similar or disparate effects on
Rbf1 repression activity. We measured the transcriptional activity of Rbf1 T356D and Rbf1
4D in response to Cyc-Cdk overexpression (Fig. 5-4A). Strikingly, like the wild-type
protein, the T356D was still highly responsive to Cyc-Cdk inactivation, whereas the Rbf1
mutant bearing all four mutations of phosphorylation sites was completely resistant to
Cyc-Cdk inactivation. In this assay of transcriptional activity, the nature of the mutation at
T356 was critical; unlike the similar effects of Thr-Ala and Thr-Asp on stability, Rbf1
T356A responsiveness to Cyc-Cdk was significantly reduced, while Rbf1 4A was
completely resistant under these conditions (Fig. 5-4B). These results suggest that
phosphorylation of T356 is critical to induce inactivation of Rbf1 and that all four
phosphorylation sites are necessary for this process. In this case, the T356D mutation does
appear to be phosphomimetic with respect to inactivation, although not with respect to
stabilization. Phosphorylation of T356 plays distinct roles in the stability and activity
pathways: it is a key event in phosphorylation-mediated stabilization but only contributes
partially to phosphorylation-mediated inactivation, reflecting separable pathways.

Phosphorylation mediates Rbf1 stabilization independently of the lysine residues in the
IE

Mutations of Ser-to-Asp and Lys-to-Ala within the IE both stabilize Rbf1, therefore we
asked whether the two sets of residues contribute in an additive fashion to regulate Rbf1
183

degradation. We compared steady-state levels of proteins with 4KA (stabilizing) mutations
to those with 4KA and 3SA (destabilizing) mutations. Both were overexpressed to a similar
extent, raising the possibility that the lysines of the IE may regulate modifications of the
adjacent serines (Fig. 5-5A, B). However, when in vivo phosphorylation was stimulated by
overexpression of Cyc/Cdk kinases, the 4KA mutant was expressed at higher levels, but not
the 4KA3SA mutant, indicating that modification of these serines is still possible, and can
contribute to protein lability independently, (Fig. 5-5C). Together, these data suggest that
lysines and serines are involved in distinct regulation mechanisms both of which influence
Rbf1 stability in a nonredundant but cumulative manner.

Rbf1 responsiveness to Cyc-Cdk-mediated inactivation determines Rbf1 potency in
Drosophila eye development

To assess the physiological importance of the phosphorylation sites under developmental
settings, we expressed these mutant forms of Rbf1 in eye imaginal discs using an
eyeless-Gal4 driver system (Fig. 5-6A). As shown in our previous studies, the wild-type
Rbf1 induced mild and moderate eye phenotypes, and the mutant form of Rbf1 lacking the
IE had no effect on eye development (Fig. 5-6B). The T356D mutant, which like the
wild-type protein was inactivated by Cyc/Cdk in luciferase assays, induced slightly more
severe phenotypes than wild-type Rbf1. In contrast, Rbf1 3SD and 4D mutants exhibited
very severe phenotypes, including complete loss of the eye. These proteins were noted to
be resistant to Cyc-Cdk inactivation in cell culture assays. Similarly, the T356A mutant
caused severe and very severe phenotypes. Further mutation of serines in the IE did not
appreciably alter this strong phenotype. Overall, the spectrum of eye phenotypes correlates
184

well with the responsiveness of each mutant protein to Cyc-Cdk-mediated inactivation,
supporting the idea that Cyc-Cdk-mediated phosphorylation is a key mechanism to restrain
Rbf1 activity under physiological conditions. In contrast, Rbf1 mutants that either showed
enhanced stabilization (3SD) or destabilization (T356A) were equally active in this
overexpression assay.

K774 in the IE is critical for response to Cyc-Cdk-mediated phosphorylation

The hypermorphic 3SD and 4D mutants phenocopy a previous identified mutant K774A,
suggesting a potential link between the K774A mutation and effects of phosphorylation
(Fig. 5-6B). Therefore, we tested whether K774 is important for response of Rbf1 to
Cyc-Cdk overexpression. To test this hypothesis, we examined K774A mutant protein level
and activity in response to Cyc-Cdk overexpression (Fig. 5-7A, B). Whereas the wild-type
Rbf1 was stabilized and inactivated by Cyc-Cdk overexpression, the K774A mutant
showed a muted response, suggesting that the K774A mutation reduces susceptibility of
Rbf1 to Cyc-Cdk control. As shown in Figure 4 and Figure 5, N-terminal and IE
phosphorylation sites play different roles in Cyc-Cdk mediated modulation of Rbf1
stability and activity. The phenotype exhibited by the K774A mutation indicates K774
might be critical for effects mediated by both N-terminal and IE residues.

185

Figure 5-4. T356 is critical for Cyc-Cdk-mediated inactivation of transcriptional repression
activity.

186

Figure 5-4 (cont’d)

187

Figure 5-4 (cont’d)
(A) PCNA-luciferase assays were used to measure Rbf1 repression activity. Rbf1 WT and
Rbf1 T356D are inactivated by Cyc-Cdk, whereas Rbf1 4D shows no response to Cyc-Cdk
overexpression. (B) Rbf1 T356A is partially resistant to Cyc-Cdk inactivation, whereas
Rbf1 4A is completely resistant. Asterisks indicate p < 0.01.

188

Figure 5-5. Conserved serine residues within the IE influence Rbf1 stability independently
of the lysine residues in the IE.

189

Figure 5-5 (cont’d)
(A) Schematic diagram of Rbf1 mutants with Lys-to-Ala and Ser-to-Ala mutations in the IE.
(B) Western blot of Rbf1 proteins expressed in S2 cells. S-A mutations do not further
significantly change level of stabilized Rbf1 4KA mutant. (C) Protein levels of stabilized
Rbf1 4KA are further increased by Cyc-Cdk overexpression. However, mutant with
combined K-A and S-A mutations is unresponsive to Cyc-Cdk overexpression. Asterisks
indicate p < 0.05.

190

Figure 5-6. Dramatic developmental defects induced by Cyc-Cdk-resistant Rbf1 proteins.

191

Figure 5-6 (cont’d)
(A) Representative Drosophila eyes exhibiting wild-type, mild, moderate, severe and very
severe phenotypes induced by expression of rbf1 genes in the eye imaginal disc. (B) Bar
graphs represent percentage of flies exhibiting different eye phenotypes caused by Rbf1
overexpression. A mutant lacking the IE (∆IE) has no effect on eye development, while the
wild-type Rbf1 (WT) causes a high percentage of mild and moderate phenotypes. The T356
mutant exhibited a slightly stronger phenotype than Rbf1 WT, and other mutants had much
more pronounced effects. 70-400 flies were scored for each of these lines.

192

Figure 5-7. K774 influences Cyc-Cdk control of both Rbf1 protein levels and
transcriptional activity.

193

Figure 5-7 (cont’d)

194

Figure 5-7 (cont’d)
(A) Western blot analysis of Rbf1 WT and K774A mutant. The latter is partially resistant to
Cyc-Cdk-mediated stabilization. The Rbf1 WT and K774A have similar levels of
expression without Cyc-Cdk overexpression. (B) PCNA-luciferase assay to measure Rbf1
WT and K774A mutant. Repression activity of K774A is partially resistant to Cyc-Cdk
overexpression. Asterisks indicate p < 0.05.

195

Discussion

RB family proteins play significant roles in diverse cellular events including cell cycle
progression, cellular differentiation and apoptotic death (Burkhart and Sage, 2008). As
described for posttranslational modifications of histone proteins and transcription factors
such as p53, a biochemical regulatory code of conduct has been proposed for RB based on
extensive research on RB phosphorylation and other modifications (Munro et al., 2012). In
particular, phosphorylation serves as a key mechanism of transmitting upstream regulatory
signals into functional outputs, which allows the channeling of RB activity into diverse
pathways. However, the roles of specific phosphorylation patterns are not yet fully
understood. In this study, we provide evidence for essential phosphorylation sites in two
regions involved in regulation of Drosophila Rbf1 stability and activity, two key features
frequently disrupted in human diseases.
The N-terminal T356 phosphorylation site functions as a critical switch for Rbf1 protein
stability and repression activity, presumably by serving as a priming site for other
phosphorylation events, for instance, in the IE. This highly conserved threonine residue
(T373) in RB, when phosphorylated, induces a global conformational change to facilitate
the N-terminus binding to the pocket domain, which blocks association of the pocket
domain with E2F transactivation domain (TD) (Burke et al., 2012). Consistent with the
structural importance of T373, its phosphorylation is sufficient to inactivate RB in human
and rodent cells (Lents et al., 2006). This similarity between the involvements of this single
phosphorylation site in the inactivation mechanism suggests that phosphorylation of T356
in Rbf1 may also cause a conformational change. This level of regulation may facilitate
196

Cdk targeting of additional phosphorylation sites in the pocket domain or the IE, disrupting
protein-protein interactions important for function and stability (Fig. 5-8).
Our previous studies demonstrate that the C-terminal IE in Rbf1 is crucial for both
protein turnover and repression activity (Acharya et al., 2010; Raj et al., 2012b). This
multifunctional region may serve as a surface for protein-protein interactions that regulate
contacts with E2F factors and E3 ligases. Mutations of the lysines would both weaken E3
ligase binding to block the degradation pathway and impair E2F binding, attenuating
transcriptional repression. In this study of phosphorylation control, we uncovered a second
set of residues involved in stability-activity linkage through the IE. Phosphorylation of the
serine residues in the IE may block E3 and E2F associations to stabilize and inactivate the
protein respectively (Fig. 5-8). The IE of Rbf1 is highly conserved with RB family
members, p107 and p130, implying a similar IE-mediated control of stability and activity.
Indeed, a p107 mutant lacking the IE, when expressed in S2 cells, accumulates to a much
higher level than the wild-type p107 (Acharya et al., 2010), supporting one aspect of the
regulation mechanisms mediated by the IE. The conservation of the three serines in the IE
indicates that the phosphorylation control through the IE may also be a conserved
mechanism. In mammalian RB, the C-terminus is required for RB activity due to its
essential association with the Marked Box (MB) of E2F. Structural studies of RB
C-terminus reveal that phosphorylation in the conserved IE region destabilizes association
of C-terminus with the E2F MB and induces an intramolecular interaction between the RB
C-terminus and the pocket (Rubin et al., 2005). A similar mechanism may apply to Rbf1
inactivation, and additionally the conformational change caused by these phosphorylation

197

events may also influence E3 binding (Fig. 5-8).
Our previous model of paradoxical instability-activity relationship suggests that these
two processes are tightly linked, and that ubiquitination driven by the IE contributes to both
protein degradation and repression activity (Raj et al., 2012b). However, in this study, we
identified a way to separate these two pathways that converge on the IE. Ser-to-Asp
mutations only affect the Rbf1 degradation pathway, but do not disrupt repression activity.
We propose that these mutations specifically block E3 ligase binding to Rbf1 to reduce the
level of ubiquitination, but are not disruptive to E2F interactions. Phosphorylation control
of E3 ligase binding is also observed for RB: the Mdm2 E3 ligase binds preferentially to
the C-terminal region of hypophosphorylated RB. The ability to disengage turnover and
repression activity suggests that high levels of ubiquitination are not essential for Rbf1
activity in every context. In rapidly dividing cells, Rbf1 activity may be only controlled by
phosphorylation and dephosphorylation so that it can be recycled and reused efficiently. In
contrast, in differentiating cells Rbf1 may be ubiquitinated and degraded to ensure a rapid
depletion when it is no longer needed. In the mammalian system, Mdm2 targets RB for
degradation to release E2F-dependent transcription, suggesting a conserved degradation
pathway. However, there may be specific contexts in which the linkage between
ubiquitination-mediated degradation and activity of Rbf1 is important. We previously
showed that a single ubiquitin tag is able to enhance Rbf1 activity, therefore this
modification may allow Rbf1 to exert different levels of activity on the same genes
depending on the cellular context. The ubiquitin tag may help recruit transcription cofactors
to achieve the maximal repression activity of Rbf1, and even some components of the

198

degradation machinery, such as the proteasome, may be directly involved in transcriptional
repression.
Within the Rbf1 IE, lysine and serine residues appear to each contribute to the regulation
of the IE, however, it is possible that there is functional interplay between the two sets of
residues. Of particular interest are recent findings that in response to DNA damage, K810
in the C-terminus of RB can be methylated to block phosphorylation of serine sites by
impeding the interaction between RB and Cdk4 (Carr et al., 2011). In addition, a recent
study shows that acetylation of K1079 (equivalent to Rbf1 K774) in p130 enhances
Cdk4-mediated phosphorylation in vitro (Saeed et al., 2012). This particular residue of
p130 is found to be mutated in lung cancers, which suggests that there may be specific
growth advantages to disruption of physiological controls of stability and activity (Claudio
et al., 2000).

199

Figure 5-8. Model for the phosphorylation-mediated functional inactivation and
stabilization of Rbf1.

200

Figure 5-8 (cont’d)
Unphosphorylated Rbf1 binds to E2f1 through Pocket-Transactivation domain (TD) and C
domain-Marked Box (MB) interactions. Phosphorylation of T356 by Cdk induces a partial
conformational change which may serve as a priming event for other phosphorylation
events in the pocket or in the IE. When the C-terminus is phosphorylated, Rbf1 dissociates
from E2f1 and loses its repression activity and repels E3 binding to be stabilized. A key
lysine K774 in the IE is critical for Cdk targeting on phosphorylation sites to affect Rbf1
sensitivity toward Cyc-Cdk for stability and activity.

201

Materials and Methods

Expression Constructs
Generation of Rbf1 WT and mutant expression constructs was described previously
(Acharya et al., 2010). The mutations of phosphorylation sites in Rbf1 were made by
site-directed mutagenesis using a Quick-Change TM strategy (Stratagene). To generate
Cyclin and Cdk expression constructs, Cyclin D, Cyclin E, Cdk2 and Cdk4 cDNA were
PCR-amplified from respective pOT construct (DGRC) and cloned into the KpnI and NotI
or NotI and XbaI sites of pAX vector (Ryu and Arnosti, 2003). Two Flag epitope tags were
inserted 5’ of the stop codon. To generate Rbf1 expression constructs used in the fly eye
assays, Rbf1 WT and mutants were cloned into pUASTattB (Bischof et al., 2007). The
plasmids were then injected by Rainbow Transgenics into the 51D site of yw flies to generate
transgenic lines.
Western Blot Analysis
Drosophila S2 cells were transfected using Effectene transfection reagent (Qiagen)
according to the manufacturer’s protocol. 1.0 million cells were transfected with 100ng of
pAX-Rbf1. For the Cyc-Cdk overexpression assays, 100ng of pAX-Rbf1 was cotransfected
with 200ng of pAX-CyclinE or D and 400ng of pAX-Cdk2 or 4. Cells were harvested 5 days
after transfection and lysed by freeze-and-thaw cycles three times in lysis buffer (50mM
Tris-Cl pH8.0, 150mM NaCl, 1% Triton X-100). Total protein levels were measured by
Bradford assays. To measure Rbf1 protein levels, 50 µg S2 cell lysates were run on 12.5%
SDS-PAGE gels, transferred to PVDF membranes and probed with M2 anti-Flag antibody
(mouse monoclonal, 1:10,000, Sigma, F3165) and anti-tubulin (mouse monoclonal, 1:10,000,
202

Iowa Hybridoma Bank). Antibody incubation was performed in TBST (20 mM Tris-Cl, pH 7.5,
120 mM NaCl, 0.1% Tween-20) with 5% non-fat dry milk. Blots were developed using
HRP-conjugated secondary antibodies (Pierce) and SuperSignal West Pico chemiluminescent
substrate (Pierce).
Luciferase Reporter Assays
For PCNA-luciferase assays, 1.5 million Schneider S2 cells were transfected with 600ng of
PCNA-luciferase reporter (Frolov et al., 2003), 200ng of pAX-Rbf1 WT, 200ng of
pAX-CyclinE or D and 400ng of pAX-Cdk2 or 4. Cells were harvested 3 days after
transfection and luciferase activity was measured using the Dual-Glo Luciferase assay system
(Promega) and quantified using the Veritas microplate luminometer (Turner Biosystems).
Fly Assays
Heterozygous lines harboring the wild-type or mutant rbf1 forms in the pUAST vector
were crossed with flies containing an eyeless-Gal4 / CyO driver (Gilbert et al., 2006) and the
offspring containing both driver and UAS-rbf1 transgenes (70-400 flies per construct) were
screened for eye phenotypes.

Acknowledgments
We thank Nick Dyson for sharing PCNA-luciferase reporter construct and Abigail
Vanderberg for aid with scanning electronic microscopy. We thank the Drosophila
Genomics Resource Center for plasmids and the Bloomington Stock Center for Drosophila
lines. This study was supported by a dissertation completion fellowship from the College of
Natural Sciences, MSU to LZ and by GM 079098 to RWH and DNA.

203

APPENDIX

204

Figure 5-S1. The IE alone is insufficient for Cyc-Cdk-mediated stabilization.

GFP-IE protein level is not affected by overexpression of Cyc-Cdk when the wild-type
Rbf1 is stabilized under these conditions.

205

REFERENCES

206

REFERENCES

Acharya, P., Raj, N., Buckley, M.S., Zhang, L., Duperon, S., Williams, G., Henry, R.W.,
and Arnosti, D.N. (2010). Paradoxical instability-activity relationship defines a novel
regulatory pathway for retinoblastoma proteins. Molecular biology of the cell 21,
3890-3901.
Adams, P.D. (2001). Regulation of the retinoblastoma tumor suppressor protein by
cyclin/cdks. Biochim Biophys Acta 1471, M123-133.
Bischof, J., Maeda, R.K., Hediger, M., Karch, F., and Basler, K. (2007). An optimized
transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl
Acad Sci U S A 104, 3312-3317.
Boyer, S.N., Wazer, D.E., and Band, V. (1996). E7 protein of human papilloma virus-16
induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway.
Cancer Res 56, 4620-4624.
Buchkovich, K., Duffy, L.A., and Harlow, E. (1989). The retinoblastoma protein is
phosphorylated during specific phases of the cell cycle. Cell 58, 1097-1105.
Burke, J.R., Hura, G.L., and Rubin, S.M. (2012). Structures of inactive retinoblastoma
protein reveal multiple mechanisms for cell cycle control. Genes Dev 26, 1156-1166.
Burkhart, D.L., and Sage, J. (2008). Cellular mechanisms of tumour suppression by the
retinoblastoma gene. Nat Rev Cancer 8, 671-682.
Canhoto, A.J., Chestukhin, A., Litovchick, L., and DeCaprio, J.A. (2000). Phosphorylation
of the retinoblastoma-related protein p130 in growth-arrested cells. Oncogene 19,
5116-5122.
Cantin, G.T., Yi, W., Lu, B., Park, S.K., Xu, T., Lee, J.D., and Yates, J.R., 3rd (2008).
Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient
phosphoproteomic analysis. J Proteome Res 7, 1346-1351.
Carr, S.M., Munro, S., Kessler, B., Oppermann, U., and La Thangue, N.B. (2011). Interplay
between lysine methylation and Cdk phosphorylation in growth control by the
207

retinoblastoma protein. EMBO J 30, 317-327.
Chau, B.N., and Wang, J.Y. (2003). Coordinated regulation of life and death by RB. Nat
Rev Cancer 3, 130-138.
Classon, M., and Dyson, N. (2001). p107 and p130: versatile proteins with interesting
pockets. Exp Cell Res 264, 135-147.
Claudio, P.P., Howard, C.M., Pacilio, C., Cinti, C., Romano, G., Minimo, C., Maraldi, N.M.,
Minna, J.D., Gelbert, L., Leoncini, L., et al. (2000). Mutations in the retinoblastoma-related
gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by
retrovirus-mediated gene transfer. Cancer Res 60, 372-382.
Delston, R.B., Matatall, K.A., Sun, Y., Onken, M.D., and Harbour, J.W. (2011). p38
phosphorylates Rb on Ser567 by a novel, cell cycle-independent mechanism that triggers
Rb-Hdm2 interaction and apoptosis. Oncogene 30, 588-599.
Dephoure, N., Zhou, C., Villen, J., Beausoleil, S.A., Bakalarski, C.E., Elledge, S.J., and
Gygi, S.P. (2008). A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S
A 105, 10762-10767.
Frolov, M.V., Stevaux, O., Moon, N.S., Dimova, D., Kwon, E.J., Morris, E.J., and Dyson,
N.J. (2003). G1 cyclin-dependent kinases are insufficient to reverse dE2F2-mediated
repression. Genes Dev 17, 723-728.
Gauci, S., Helbig, A.O., Slijper, M., Krijgsveld, J., Heck, A.J., and Mohammed, S. (2009).
Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined
SCX-based approach. Anal Chem 81, 4493-4501.
Gilbert, M.K., Tan, Y.Y., and Hart, C.M. (2006). The Drosophila boundary
element-associated factors BEAF-32A and BEAF-32B affect chromatin structure. Genetics
173, 1365-1375.
Inoue, Y., Kitagawa, M., and Taya, Y. (2007). Phosphorylation of pRB at Ser612 by Chk1/2
leads to a complex between pRB and E2F-1 after DNA damage. EMBO J 26, 2083-2093.
Leng, X., Noble, M., Adams, P.D., Qin, J., and Harper, J.W. (2002). Reversal of growth
suppression by p107 via direct phosphorylation by cyclin D1/cyclin-dependent kinase 4.
Mol Cell Biol 22, 2242-2254.
208

Lents, N.H., Gorges, L.L., and Baldassare, J.J. (2006). Reverse mutational analysis reveals
threonine-373 as a potentially sufficient phosphorylation site for inactivation of the
retinoblastoma tumor suppressor protein (pRB). Cell Cycle 5, 1699-1707.
Mayya, V., Lundgren, D.H., Hwang, S.I., Rezaul, K., Wu, L., Eng, J.K., Rodionov, V., and
Han, D.K. (2009). Quantitative phosphoproteomic analysis of T cell receptor signaling
reveals system-wide modulation of protein-protein interactions. Sci Signal 2, ra46.
Mittnacht, S. (1998). Control of pRB phosphorylation. Curr Opin Genet Dev 8, 21-27.
Munro, S., Carr, S.M., and La Thangue, N.B. (2012). Diversity within the pRb pathway: is
there a code of conduct? Oncogene 31, 4343-4352.
Raj, N., Zhang, L., Wei, Y., Arnosti, D.N., and Henry, R.W. (2012). Ubiquitination of
retinoblastoma family protein 1 potentiates gene-specific repression function. J Biol Chem
287, 41835-41843.
Rubin, S.M. (2013). Deciphering the retinoblastoma protein phosphorylation code. Trends
Biochem Sci 38, 12-19.
Rubin, S.M., Gall, A.L., Zheng, N., and Pavletich, N.P. (2005). Structure of the Rb
C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F
release. Cell 123, 1093-1106.
Ryu, J.R., and Arnosti, D.N. (2003). Functional similarity of Knirps CtBP-dependent and
CtBP-independent transcriptional repressor activities. Nucleic Acids Res 31, 4654-4662.
Saeed, M., Schwarze, F., Loidl, A., Meraner, J., Lechner, M., and Loidl, P. (2012). In vitro
phosphorylation and acetylation of the murine pocket protein Rb2/p130. PLoS One 7,
e46174.
Sdek, P., Ying, H., Chang, D.L., Qiu, W., Zheng, H., Touitou, R., Allday, M.J., and Xiao,
Z.X. (2005). MDM2 promotes proteasome-dependent ubiquitin-independent degradation of
retinoblastoma protein. Mol Cell 20, 699-708.
Stubdal, H., Zalvide, J., Campbell, K.S., Schweitzer, C., Roberts, T.M., and DeCaprio, J.A.
(1997). Inactivation of pRB-related proteins p130 and p107 mediated by the J domain of
simian virus 40 large T antigen. Molecular and cellular biology 17, 4979-4990.

209

Tedesco, D., Lukas, J., and Reed, S.I. (2002). The pRb-related protein p130 is regulated by
phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2). Genes
Dev 16, 2946-2957.
Uchida, C., Miwa, S., Kitagawa, K., Hattori, T., Isobe, T., Otani, S., Oda, T., Sugimura, H.,
Kamijo, T., Ookawa, K., et al. (2005). Enhanced Mdm2 activity inhibits pRB function via
ubiquitin-dependent degradation. EMBO J 24, 160-169.
Ullah, Z., Buckley, M.S., Arnosti, D.N., and Henry, R.W. (2007). Retinoblastoma protein
regulation by the COP9 signalosome. Mol Biol Cell 18, 1179-1186.
van den Heuvel, S., and Dyson, N.J. (2008). Conserved functions of the pRB and E2F
families. Nat Rev Mol Cell Biol 9, 713-724.
Xin, S., Weng, L., Xu, J., and Du, W. (2002). The role of RBF in developmentally regulated
cell proliferation in the eye disc and in Cyclin D/Cdk4 induced cellular growth.
Development 129, 1345-1356.
Zarkowska, T., and Mittnacht, S. (1997). Differential phosphorylation of the retinoblastoma
protein by G1/S cyclin-dependent kinases. J Biol Chem 272, 12738-12746.
Zhai, B., Villen, J., Beausoleil, S.A., Mintseris, J., and Gygi, S.P. (2008). Phosphoproteome
analysis of Drosophila melanogaster embryos. J Proteome Res 7, 1675-1682.

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CHAPTER 6

Future directions

Here I discuss how my work on the Drosophila retinoblastoma protein Rbf1 has changed
our understanding of RB biology, leading to future perspectives and suggested research
directions in this area.
My studies have demonstrated that the C-terminal instability element (IE) of Rbf1 has
key functions in the regulation of Rbf1 stability and activity through post-translational
modifications (Acharya et al., 2010; Raj et al., 2012b). One plausible model to explain the
central role of the IE is that it serves as an interaction domain for proteins and cofactors
involved in diverse regulation pathways (Chapter 5). To further investigate the roles of the
IE, one essential step will be to identify potential binding proteins to the IE region. I have
generated a chimeric protein with the minimal IE fused to a heterologous GFP, and showed
that in this context, the IE is sufficient to direct destabilization of the chimera. This
epitope-tagged chimeric protein can be used in immunoprecipitation and mass
spectrometry assays to identify physically associated proteins in S2 cells and flies. A
similar assay to identify Rbf2 interaction proteins was performed in the Henry and Arnosti
laboratories previously (Ullah et al., 2007).
One class of proteins that we will be looking for is E3 ubiquitin ligases that may bind to
the IE and target the protein for ubiquitination and degradation. Although my preliminary
RNAi experiments failed to reveal any potential E3 ligases among the Cullin family that
influences Rbf1 protein levels, I am confident that such activity exists. For example,
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studies of mammalian RB demonstrate that the E3 ligase Mdm2 binds to the conserved
C-terminus and promotes RB degradation (Sdek et al., 2004). Therefore, the question about
candidate E3 ligases remains open. Another class of proteins of particular interest is
transcription cofactors that interact with the IE and contribute to the repression activity of
Rbf1 at the target promoter. The interaction domain for many RB binding proteins,
especially LXCXE-containing proteins, has been mapped to the pocket domain, but the
C-terminus may still be important for certain aspects of protein binding. For example,
HDAC1 binds to the C-terminus of p130 and enhances its repression on the E2F-dependent
Cyclin A promoter (Stiegler et al., 1998). More importantly, the C-terminus may provide a
control of gene-specific activities of RB. C-terminal binding of RB with c-Jun promotes
c-Jun-dependent transcription, showing a role of RB as a transcriptional activator in early
G1 during keratinocyte differentiation (Nead et al., 1998). Consistent with this, in our
studies, we show that the IE is required for Rbf1 activity on cell cycle genes, but not on
other classes of genes, such as the ones in signaling pathways, suggesting functional
importance of the IE as a module to discriminate distinct roles of RB in different pathways.
Related to the above goal, another interesting and essential direction is to identify
IE-dependent and IE-independent functional targets of Rbf1. Our lab has generated stable
S2 cell lines to express the wild-type Rbf1 and the ΔIE mutant under an inducible promoter.
We will first test a select group of previously identified Rbf1 target genes to validate this
assay and then perform a genome-wide survey of functional targets by microarrays. We
anticipate that IE-dependent and IE-independent genes may fall into different gene
ontology categories, showing distinct requirements of the IE for different Rbf1 regulation

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mechanisms. In this case, regulation of Rbf1 by modification of the IE may impact certain
functional classes of target genes preferentially, permitting alternative Rbf1 regulation of a
subset of the regulon.
A third long-term goal worth pursuing is to characterize physical protein-protein
interactions between the IE and E2f and some of the binding partners identified in the
above assays. My work indicates that the IE may have critical contacts with E2F, which are
subject to Cyc-Cdk-mediated phosphorylation (Chapter 5). The structure and the
phosphorylation control of the RB C-terminus have been well studied, but how these
structural features contribute to RB functions remains elusive (Rubin, 2013). I have
generated a cohort of Rbf1 mutants with mutations on key lysine and serine residues
important for Rbf1 activity and stability. A structural study of these mutants with E2F
binding would elucidate the importance of post-translational modifications in the IE for
E2F interaction and possible functional outputs. In particular, K1079 (equivalent to Rbf1
K774) is frequently mutated in lung cancers and it governs an interplay between its
acetylation and cdk4-mediated phosphorylation (Claudio et al., 2000; Saeed et al., 2012).
This class of mutations may greatly affect RB-E2F interactions and therefore the
transcriptional regulation of genes involved in cancer progression. Structural studies may
provide insights into the basis by which such mutations are beneficial to transformed cells.
Our cell culture-based assays show that overexpression of the Rbf1∆IE mutant promotes
DNA replication, a phenotype favorable to cancer growth (Raj et al., 2012a). It will be
interesting to investigate if this mutant is capable of disrupting normal cell growth and
driving excess DNA replication under developmental settings. However, overexpression of

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Rbf1∆IE in the fly eye imaginal disc does not cause any observable abnormal eye
phenotype, suggesting that additional cofactors may be required to mediate the effect of
Rbf1∆IE in regulating the cell cycle (Acharya et al., 2010). Consistent with this idea, a
previous study showed that overexpression of E2F1 alone has a modest eye phenotype,
whereas when it is overexpressed with DP, a more pronounced rough eye phenotype is
observed (Du et al., 1996b). Therefore, we will overexpress Rbf1∆IE with DP in the eye
imaginal disc to look for eye morphology defects and monitor DNA replication by BrdU
staining. We expect to observe similar phenotypes as with E2F1 and DP overexpression.
This assay would provide insights into the importance of the IE for controlling cell cycle
arrest and gain of function in cell proliferation when it is mutated in human cancers.

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REFERENCES

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REFERENCES

Acharya, P., Raj, N., Buckley, M.S., Zhang, L., Duperon, S., Williams, G., Henry, R.W.,
and Arnosti, D.N. (2010). Paradoxical instability-activity relationship defines a novel
regulatory pathway for retinoblastoma proteins. Mol Biol Cell 21, 3890-3901.
Claudio, P.P., Howard, C.M., Pacilio, C., Cinti, C., Romano, G., Minimo, C., Maraldi, N.M.,
Minna, J.D., Gelbert, L., Leoncini, L., et al. (2000). Mutations in the retinoblastoma-related
gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by
retrovirus-mediated gene transfer. Cancer Res 60, 372-382.
Du, W., Xie, J.E., and Dyson, N. (1996). Ectopic expression of dE2F and dDP induces cell
proliferation and death in the Drosophila eye. EMBO J 15, 3684-3692.
Nead, M.A., Baglia, L.A., Antinore, M.J., Ludlow, J.W., and McCance, D.J. (1998). Rb
binds c-Jun and activates transcription. EMBO J 17, 2342-2352.
Raj, N., Zhang, L., Wei, Y., Arnosti, D.N., and Henry, R.W. (2012a). Rbf1 degron
dysfunction enhances cellular DNA replication. Cell Cycle 11, 3731-3738.
Raj, N., Zhang, L., Wei, Y., Arnosti, D.N., and Henry, R.W. (2012b). Ubiquitination of
retinoblastoma family protein 1 potentiates gene-specific repression function. J Biol Chem
287, 41835-41843.
Rubin, S.M. (2013). Deciphering the retinoblastoma protein phosphorylation code. Trends
Biochem Sci 38, 12-19.
Saeed, M., Schwarze, F., Loidl, A., Meraner, J., Lechner, M., and Loidl, P. (2012). In vitro
phosphorylation and acetylation of the murine pocket protein Rb2/p130. PLoS One 7,
e46174.
Sdek, P., Ying, H., Zheng, H., Margulis, A., Tang, X., Tian, K., and Xiao, Z.X. (2004). The
central acidic domain of MDM2 is critical in inhibition of retinoblastoma-mediated
suppression of E2F and cell growth. J Biol Chem 279, 53317-53322.
Stiegler, P., De Luca, A., Bagella, L., and Giordano, A. (1998). The COOH-terminal region
of pRb2/p130 binds to histone deacetylase 1 (HDAC1), enhancing transcriptional
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repression of the E2F-dependent cyclin A promoter. Cancer Res 58, 5049-5052.
Ullah, Z., Buckley, M.S., Arnosti, D.N., and Henry, R.W. (2007). Retinoblastoma protein
regulation by the COP9 signalosome. Mol Biol Cell 18, 1179-1186.

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