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Michigan State
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This is to certify that the

dissertation entitled

The Regulation of the Mixed-Lineage Kinase

SPRK/MLK-3 by the Small GTPase Cdc42

presented by

Barbara Carola Bock

has been accepted towards fulfillment
of the requirements for

Ph .D. degree in Physiology

 

jam“ ,4 . «SW’

Major professor

Date $4101)

MS U it an Affirmative Action/Equal Opportunity Institution 0-12771

 

PLACE IN RETURN Box to remove this checkout from your record.
To AVOID FINES return on or before date due.
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DATE DUE

DATE DUE

DATE DUE

 

W 1i 2002

 

 

OCT M.

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THE REGULATION OF THE MD(ED-LINEAGE KINASE

SPRK/MLK-3 BY THE SMALL GTPase Cdc42
By

Barbara Carola Bock

A DISSERTATION
Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of
DOCTOR OF PHILOSOPHY
Department of Physiology

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ABSTRACT
THE REGULATION OF THE MIXED-LINEAGE KIN ASE SPRK/MLK-3 BY
THE SMALL GTPase Cdc42
By

Barbara Carola Bock

Src homology 3 domain containing proline-rich protein kinase (SPRK), also
called mixed-lineage kinase (MLK)-3 is a serine/threonine kinase which upon
overexpression in mammalian cells activates the c-Jun NHz-terminal kinase
(JNK)/stress activated kinase (SAPK) pathway. The 95 kDa protein bears, in
addition to its kinase domain, various motifs mediating protein-protein interaction.
However the mechanism by which SPRK activity is regulated still remains unclear.
This thesis work examines the role of the small GTPase Cdc42 in the regulation of
SPRK activity.

Coexpression of SPRK with Cdc42 in 293 cells results in an increase in
SPRK's catalytic activity. Mutational analysis of the Cdc42/Rac interactive-binding
(CRIB) motif, a consensus motif required for binding of small GTPases, shows that
SPRK contains a functional CRIB motif, since mutations within this motif abrogate
association of SPRK with Cdc42, and thus Cdc42-induced SPRK activation. Further
experiments using a SPRK variant which lacks the zipper region/basic stretch
suggest that not only the CRIB motif is important for Cdc42 binding, but that also

the zipper/basic stretch contributes to Cdc42 binding. Interestingly, SPRK activation

by Cdc42 cannot be recapitulated in an in vitro system using recombinant, purified
proteins. This suggests that Cdc42-induced activation of SPRK requires the cellular
environment. Comparative two-dimensional in vivo phosphopeptide mapping
indicated that coexpression of Cdc42 changes SPRK's in vivo phosphorylation
pattern, supporting the idea that Cdc42 changes the in viva phosphorylation of
SPRK. Analysis of the subcellular distribution of SPRK showed that the majority of
SPRK upon overexpression in COS-7 cells colocalizes with the Golgi apparatus.
Coexpression of SPRK and Cdc42 induces targeting of SPRK to a plasma
membrane-enriched subcellular fraction. The targeting requires prenylation of
Cdc42, suggesting the requirement for Cdc42 in this process.

The present study demonstrates that Cdc42 is an activator of SPRK and
provides important insight into the structural requirements for Cdc42-induced SPRK
activation. The observation that Cdc42 changes SPRK's subcellular distribution
suggests a role for Cdc42 in membrane targeting, although it was not possible to

correlate the subcellular targeting of SPRK with an increase in kinase activity.

iv

To my family

ACKNOWELEDGEMENTS

I would like to thank my committee members Drs. Kathleen Gallo, Susan
Conrad, Laura McCabe, Richard Miksicek, and William Spielman. I would
especially like to thank my mentor Dr. Kathleen Gallo for the constant
encouragement, guidance, and support throughout my graduate career. Furthermore
I would like to thank Drs. Susan Conrad and Walter Esselman for the useful
discussions during the Friday afternoon meetings. I would also like to thank Drs.
Joanne Whallon and Shirley Owens for the help with confocal imaging at the Laser
Scanning Microscope Laboratory at Michigan State University.

I would also like to take this opportunity to thank my friends in the lab,
especially Panayiotis Vacratsis, Erion Qamirani, Ritesh Agrawal, Hua Zhang, Mary
Chao and David Allton. I am grateful to Panayiotis Vacratsis for his support during
this project and constructive discussions. I would like to sincerely thank the office

staff for all the help rendered to me.

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TABLE OF CONTENTS

Page
List of Tables .............................................................................................................. viii
List of Figures .............................................................................................................. .ix
Key to Abbreviations .................................................................................................. .xii
I. Introduction ............................................................................................................... .1
11. Literature Review ...................................................................................................... 4
1. The Regulation of Protein Kinases ................................................... 5
1.1 Regulation of protein kinases by activators and inhibitors............... . . . . . . . .5
1.2 Regulation of protein kinases by phosphorylation ..................................... 7
1.3 Regulation of protein kinases by proteolytic cleavage.................. . ..........7
1.4 Regulation of protein kinases by subcellular distribution .......................... 8
2. Small GTPases of the Rho family .................................................................. .14
3. The MAPK Pathways ..................................................................................... .22
4. The Physiological Importance of the IN K Pathway ...................................... .30
4.1 JN K signaling in Drosophila melanogaster ........................................... .30
4.2 INK signaling in mammals ...................................................................... 32
5. The Mixed-Lineage Kinase Family ................................................................. 37
III. Cdc42-Induced Activation of the Mixed-Lineage Kinase SPRK
in Vivo: Requirement of the Cdc42/Rac Interactive Binding Motif
and Changes in Phosphorylation .......................................................................... .46
1. Abstract ........................................................................................................... 46
2. Introduction ..................................................................................................... 47
3. Materials and Methods ................................................................................... .49
3.1 Construction of mammalian expression vectors and mutagenesis.... . . . . . .49
3.2 Expression and purification of recombinant SPRK. ............................... . 51
3.3 Cell lines and transfections ........................................................................ 52
3.4 Cell lysis and immunoprecipitations ....................................................... .52
3.5 Gel electrophoresis and Western blot analysis ........................................ .53
3.6 In vitro kinase assays ............................................................................... .54
3.7 In vivo phosphopeptide mapping ............................................................. .55

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4. Results ............................................................................................................ .57
4.1 Association of activated Cdc42 with SPRK does not require

SPRK kinase activity ............................................................................... .57
4.2 SPRK's CRIB motif is necessary for association with Cdc42
and for Cdc42-induced activation of SPRK ............................................. 60
4.3 Effects of deleting the COOH-terminal portion of SPRK's
zipper domain ........................................................................................... 61
4. 4 SPRK“? fails to activate INK ............................................................... .63
4. 5 Activated Cdc42 fails to activate SPRK in vitro ..................................... .64
4. 6 Cdc42 alters the in vivo phosphorylation pattern of SPRK .................... .65
5. Discussion ........................................................................................................ 84

IV. Cdc42 Changes the Subcellular Distribution of the

Mixed—Lineage Kinase SPRK. .............................................................................. 91
1. Abstract ........................................................................................................... 91
2. Introduction ..................................................................................................... 92
3. Materials and Methods ................................................................................... .95
3.1 Construction of mammalian expression vectors and
site-directed mutagenesis ..................................................................... .95
3.2 Cell lines and transfections ...................................................................... 96
3.3 Cell lysis, membrane fractionation, and irnmunoprecipitation................ 96
3.4 Gel electrophoresis and Western blot analysis ........................................ 98
3.5 In vitro kinase assays and in vivo phosphopeptide mapping ................... 99
3.6 Immunofluorescence and microscopy .................................................... 100
3.7 Protein Markers ..................................................................................... 101
4. Results ........................................................................................................... 102
4.1 Analysis of cellular fractions ................................................................. .102
4.2 Activated Cdc42 targets SPRK to cellular membranes .......................... 104
4.3 Analysis of the subcellular distribution of SPRK by
confocal microscopy .......................................................................... 105
4.4 Analysis of SPRK distribution by biochemical fractionation ................. 109
4.5 Effect of membrane targeting on SPRK kinase activity ..................... 113

5. Discussmnl38
V. Summary and Conclusnon 145

VI. List of References 150

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LIST OF TABLES

Table Page
1. Analysis of subcellular fractions by marker proteins and

biotynilation .......................................................................... 1 l7
2. Estimated distribution of total cellular non-nuclear protein

among biochemical fractions ...................................................... 127
3. Estimated distribution of SPRK protein among biochemical

fractions in presence and absence of Cdc42VI ................................. 128

viii

LIST OF FIGURES

Figure Page
1. MAPK scaffold complexes in Saccharomyces cervisiae ..................................... .12
2. Mammalian scaffold complexes ........................................................................... 13
3. Regulation of small GTPases ................................................................................ 21
4. MAPK pathway module ....................................................................................... .23
5. Parallel mammalian MAPK pathways .................................................................. 29
6. Domain arrangement of SPRK ............................................................................. .45
7. Schematic of SPRK ............................................................................................. .68
8. Alignment of CRIB motifs and SPRK variants .................................................... 69
9. Coimmunoprecipitation of SPRK with wild type Cdc42 or Cdc42V'2 ................. 7O
10. Coimmunoprecipitation of SPRKK|44A with activated Cdc42V12 ......................... .71
11. In vitro kinase assays of SPRK and SPRKKIM ................................................... 72

12. Effects of mutations in the CRIB motif on association of SPRK with
Cdc42Vl2 .............................................................................................................. . 73

13. Effects of mutations in the CRIB motif on SPRK in vitro kinase activity........... 74

14. Quantitation of in vitro kinase assays of SPRK and SPRK‘492A'S493A .................. 75
15. Effects of partial deletion of the zipper/basic stretch on association
with Ccia42Vlz ...................................................................................................... . 76
16. Effects of partial deletion of the zipper/basic stretch on SPRK in vitro
kinase activity ........................................................................................................ 77
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l7. Quantitation of in vitra kinase assays of SPRK and SPRK“ip ............................ 78
18. Effects of SPRKAzip on JNK activity ................................................................... . 79
19. Quantitation of IN K in vitra kinase assay ........................................................... . 80
20. In vitra kinase assay using purified Cdc42 .......................................................... . 81
21. Phosphopeptide mapping of tryptic peptides derived from in viva

phosphorylated SPRK. ......................................................................................... .82
22. Alignment of various Cdc42 variants .................................................. 118
23. Analysis of subcellular fractions ........................................................ 119
24. Subcellular distribution of SPRK ....................................................... 120
25. Subcellular localization of SPRK ....................................................... 121
26. Time course of SPRK and Cdc42 expression ....................................... 122
27. Subcellular distribution of coexpressed SPRK and Cdc42V12 ...................... 123
28. Subcellular distribution of Cdc42V12 and Cdc42V'2‘3'885 ............................ 124
29. Subcellular localization of coexpressed SPRK and Cdc42“20888 ................ 125
30. SPRK's subcellular distribution u n coexpression with

Cdc42w2'C1885 and Cdc42“2088 ”’40 ............................................... 126
31. Coirnmunoprecipitation of SPRK and Cdc42 variants ............................... 129
32. Subcellular distribution of SPRKWA and SPRKM" ................................ 130
33. Subcellular distribution of endogenous SPRK ........................................ 131
34. Electrophoretic mobility shift of SPRK ............................................... 132
35. Phosphopeptide mapping of in viva phosphorylated SPRK ........................ 133
36. SPRK in vitra kinase activity upon coexpression with

Cdc42V12 or ct1c42‘“2‘C1888 .............................................................. 134

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38. Kinase activity of membrane-targeted SPRK ......................................... 136
39. In vitra kinase assay of endogenous, membrane-targeted SPRK .................. 137

xi

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ATP
cAMP
GAP
GDP
GEF
cGMP
GST
GTP

GTPyS

DLK
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ERK

HPK
IKK
IKKK

JN K
LZK
MAPK
MAPKK

MAPKKK
MEK
MLK
NAK
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PBS
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PKC
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KEY TO ABBREVIATIONS

Activated Cdc42HS-associated kinase
Acquired immune deficiency syndrome
Adenosine 5' -triphosphate

Adenosine 3' -5' cyclic monophosphate
GTPase activating protein

Guanosine 5' -diphosphate

Guanine nucleotide exchange factor
Guanosine 3' -5' cyclic monophosphate
Glutathione S-transferase

Guanosine 5' -triphosphate

Guanosine 5' -3-0-(thio)triphosphosphate
Cdc42/Rac interactive binding

Dual leucine zipper-bearing kinase
Embryonic stem cell

Extracellular signal-regulated protein kinase
Human immune deficiency virus
Human hematopoietic kinase
IKB-kinase

IKB-kinase kinase

Interleukin

Interferon

JN K interacting protein

c-Jun NHz-terminal kinase

Leucine zipper bearing kinase
Mitogen-activated kinase
Mitogen-activated kinase kinase

Mitogen-activated kinase kinase kinase
MAPK kinase

Mixed lineage-kinase

Nef associated kinase

Negative factor

NCK interacting kinase
p38/reactivating kinase
Polyacrylamide gel electrophoresis
p21-activated kinase
Phosphate-buffered saline

Polymerase chain reaction

Protein kinase A

Protein kinase C

Ste-homology 3 domain-containing proline-rich protein
kinase

xii

 

SRF
TCF
lGF
TLC

TNT

WASP
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Serum response factor

Ternary complex factor
Transforming growth factor

Thin layer chromatography

Thin layer electrophoresis

Tumor necrosis factor
Thymocyte helper cell

Wiskott Aldrich syndrome protein
Wild type

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I. Introduction

Protein kinases regulate a variety of cellular processes, ranging from
transcription to long term memory. Since these enzymes control so many different
processes it is clear that protein kinases themselves have to be highly regulated.
Binding of activating or inhibitory molecules, posttranslational modifications, and
subcellular targeting are mechanisms that regulate protein kinases.

Src-homology 3 domain-containing proline-rich protein kinase (SPRK), also
called mixed-lineage kinase (MLK)-3 (Ing et al., 1994), or protein tyrosine kinase
(PTK)-1 (Ezoe et al., 1994), is a member of the mixed-lineage kinase family.
Despite the similarity of SPRK’s kinase domain to both serine/threonine and tyrosine
kinases, only serine/threonine kinase activity has been demonstrated in vitra (Gallo
et al., 1994). SPRK, with a predicted molecular weight of 95 kDa, contains several
domains that may mediate protein-protein interactions, including a Src-homology
(SH) 3 domain, a closely, spaced leucine/isoleucine zipper motif, a Cdc42/Rac
interactive binding (CRIB) motif, and a proline/serine/threonine-rich COOH-
terminal region. The CRIB motif is a sequence of 14-16 amino acids, which is
required for binding of Rho family GTPases (Burbelo et al., 1995). SPRK shares six
of the eight consensus residues, and a fragment of SPRK containing the CRIB motif
has been demonstrated to associate with Cdc42 and Rac in filter binding assays
(Burbelo et al., 1995). SPRK and Cdc42 have been identified as upstream activators

of the c-Jun NHz-terminal kinase (INK). Although evidence exists that the small

GTPase Cdc42 as

 

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GTPase Cdc42 associates with SPRK and may be an upstream regulator of this
mixed-lineage kinase (Teramoto et al., 1996), the structural requirements and
mechanisms by which this small GTPase binds to SPRK and modulates its activity
are largely unknown. Accordingly the major aims of this thesis are: (1) to define the
structural requirements for activation of the protein kinase SPRK by the small
GTPase Cdc42, and (2) to investigate the role of subcellular targeting for SPRK
activation by Cdc42.

This thesis is essentially divided into five chapters. Chapter I includes a short
introduction and states the major aims and hypotheses. A review of the relevant past
and current literature in Chapter U will summarize our current understanding of
protein kinase regulation and of Rho-family GTPases, as well as of mitogen-
activated protein kinase (MAPK) signaling. The summary of MAPK signaling
mainly focuses on the INK pathway and its physiological importance. The last part
of the literature review will summarize the current knowledge about the mixed-
lineage kinase family.

Chapter 111 describes the research project which focuses on the regulation of
SPRK by Cdc42. This research project is designed to test the hypotheses that (1)
that the small GTPase Cdc42 binds to the protein kinase SPRK and increases its
catalytic activity, (2) that SPRK's CRIB motif is required for the Cdc42-.induced
activation of SPRK, (3) that the CRIB motif is necessary, but not sufficient to
mediate Cdc42-induced SPRK activation, and (4) that Cdc42-induced activation of

SPRK changes SPRK's in viva phosphorylation.

To further address the mechanism by which Cdc42 activates the protein
kinase SPRK, the effects of Cdc42 on SPRK's subcellular distribution was examined,
testing the hypotheses (1) that Cdc42 changes the subcellular distribution of SPRK
and (2) that plasma membrane targeting of SPRK is required for SPRK activation by
Cdc42. This research project is described in Chapter IV.

The final summary and the conclusion of this work are summarized in
Chapter V.

Understanding the structural requirements and molecular mechanism of
SPRK regulation by Cdc42 may provide insight into the regulation and function of
these two proteins. This knowledge may contribute to a better understanding of the
INK signaling cascade. Furthermore, these studies may shed light on how small

GTPases regulate the activity of other protein kinases.

 

 

11. Literature Review

1. Regulation of Protein Kinases

Protein kinases catalyze the transfer of the y-phosphate of ATP to their protein
substrates. This important physiological process is rendered reversible by. the action
of protein phosphatases. The first protein kinase discovered was glycogen
phosphorylase kinase in 1955, which activates glycogen phosphorylase (Fischer and
Krebs, 1955; Sutherland and Wosilait, 1955). At this point, it was believed that
phosphorylation of proteins was restricted to the amino acid residue serine and that
phosphorylation was characteristic for proteins involved in glycogen metabolism
This point of view was changed in 1968 with the discovery of the serine/threonine
kinase cyclic AMP-dependent kinase (PKA) (Walsh et al., 1968). In 1980 v-Src, the
gene product encoded by the Rous sarcoma virus src-gene was demonstrated to be a
tyrosine kinase (Hunter and Sefton, 1980). Phosphorylation of serine and threonine
residues by so called serine/threonine kinases and tyrosine phosphorylation by
tyrosine kinases are the most common modifications, although histidine kinases,
which phosphorylate histidines, have been identified in prokaryotes, fungi, molds,
and plants (Grebe and Stock, 1999; Pea and Saier, 1997). Additionally, there are so-
called dual specific kinases, which are able to phosphorylate both serine and
threonine, as well as tyrosine residues. Protein kinases represent one of the largest

protein superfamilies and it is estimated that the human genome may encode over

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2000 different protein kinases (Hunter, 1987b). In 1998, the crystal structures of the
catalytic domains of 12 protein kinases have been solved and all share a similar fold,
with conserved catalytic residues (Johnson et al., 1998), supporting the idea that their
catalytic mechanisms are similar. Understanding the regulation of a protein kinase
may provide important cues about the mechanism by which protein kinases, in
general, are regulated.

Protein kinases are involved in a variety of cellular processes ranging from
transcriptional activation, to muscle contraction, to long-term memory. In order to
control these processes properly it is clear that kinases themselves must be highly
regulated and controlled. The regulation of protein kinases is achieved by a number
of different mechanisms such as binding of an activator or release of an inhibitor,
posttranslational modification by phosphorylation or proteolysis, as well as

subcellular targeting.

1.1 Regulation of protein kinases by activators and inhibitors

A well-characterized mechanism of protein kinase regulation is by allosteric
ligand binding. Receptor tyrosine kinases, such as epidermal growth factor receptor,
platelet derived growth factor receptor, and the insulin receptor are activated upon
extracellular binding of epidermal growth factor, platelet-derived growth factor, and
insulin, respectively. Binding of the ligand dimerizes the receptor tyrosine kinase
and subsequently induces a conformational change. In response to the

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intracellular domain. The activation of the receptor triggers a variety of downstream
phosphorylation events and subsequently mediates changes in gene expression
(Carpenter, 1987; Fantl et al., 1993; Schlessinger and Ullrich, 1992; Ullrich and
Schlessinger, 1990).

Besides hormonal activation of receptor tyrosine kinases, protein kinases can be
activated by second messengers, such as the cyclic nucleotides cAMP and cGMP.
For instance, in the absence of cAMP, PKA is maintained in an inactive state as a
tetrameric holoenzyme consisting of two regulatory and two catalytic subunits
(Taylor et al., 1990). Upon binding of four CAMP molecules to the regulatory
subunit, the holoenzyme enzyme undergoes a conformational change and dissociates
in an R—subunit dimer and two active monomeric catalytic subunits (Taylor et al.,
1990). The R-subunits are considered physiological inhibitors of PKA since they
keep this kinase in an inactive state (Corbin et al., 1975; Hofmann et al., 1975;
Rosen et al., 1975).

Dimerization or oligomerization is a further mechanism of regulating protein
kinase activity. A well-established example of activation by dirnerization is the
activation of receptor tyrosine kinases. Upon binding of the ligand the receptor
dirnerizes and undergoes intermolecular autophosphorylation. In addition, many
intracellular protein kinases contain leucine zipper-like motifs that may mediate

oligomerization.

1.2 Regulation of protein kinases by phosphorylation

Many protein kinases are themselves regulated by phosphorylation. Activation
of the mitogen—activated protein kinase (MAPK) cascade involves sequential
phosphorylation, whereby a MAPK kinase kinase (MAPKKK) phosphorylates a
MAPK kinase (MAPKK), which in turn phosphorylates a MAPK For example, the
dual-specific kinase MKK4, an activator of cJun NHz-terminal kinase (INK),
requires phophorylation by a MAPKKK on Ser 254 and Thr 258 for activation (Yan
et al., 1994; Zheng and Guan, 1994).

The non-receptor tyrosine kinase Src is regulated by both activating and
inhibitory phosphorylation events (Hunter, 1987a). The autophosphorylation of
tyrosine 416, which resides within the activation loop of the kinase domain, has a
stimulatory effect on kinase activity (Ferracini and Brugge, 1990), whereas
phosphorylation of tyrosine 527 by c-Src kinase represses Src kinase activity (Nada
et al., 1991). Crystal structures of Src show that the SH2 domain of Src binds to its
own phosphorylated tail to exert autoinhibition (Gonfloni et al., 1999; Williams et

al., 1997; Xu et al., 1997).

1.3 Regulation by proteolytic cleavage

The onset of apoptosis is correlated with the proteolytic cleavage of nmny
proteins, including y-p21-activated kinase (PAK) by caspase CPP32 (caspase 3)
(Rudel and Bokoch, 1997). The cleavage by CPP32 produces two peptides, one

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catalytic domain (Walter et al., 1998). In contrast cleavage of y-PAK by caspase 3
results in a lO-fold stimulation (Walter et al., 1998). Upon caspase cleavage,
threonine 402 in the catalytic domain is autophosphorylated resulting in the
stimulation of the kinase activity (Walter et al., 1998). This threonine corresponds to
a regulatory phosphorylation site in the activation loop of many protein kinases as
determined by x-ray crystallography (Hanks and Quinn, 1991; Jeffrey et al., 1995;
Johnson et al., 1996; Knighton et al., 1991). It has been suggested that y-PAK
cleavage yields a constitutive active PAK, which induces cell death, whereas the
activation by Cdc42 allows a cycling of the kinase between an active and an inactive
form (Lee et al., 1997).

In addition to PAK, MEKKI, protein kinase N, and protein kinase C (PKC) can
also be activated by caspase 3 (Deak et al., 1998; Pongracz et al., 1999; Takahashi et
al., 1998). Pas-induced proteolytic cleavage of MEKKl leads to apoptosis (Deak et
al., 1998). It is believed that the cleavage renders a soluble kinase complex that
subsequently triggers apoptotic events. In contrast, uncleaved, activated MEKKI
renders an insoluble kinase complex, which may activate NF-xB and trigger a
survival response. This is an example of how the differential regulation of a kinase

may have opposite physiological outcomes.

1.4 Regulation by subcellular localization
Recently the regulation of protein kinases by subcellular targeting has received a

lot of attention. Compartmentalization may restrict a kinase to the vicinity of its

effector and result in the activation of the kinase or enhance its activity by increasing
accessibility to the substrate. Specific subcellular targeting may allow the
differential regulation of a kinase in one cell, enabling one enzyme to regulate
various physiological processes.

The serine/threonine kinase Raf, for example, requires membrane targeting for
full activation. In viva this takes place by binding to the GTP-bound form of Ras,
which recruits Raf to the plasma membrane. Appending a membrane-targeting
signal triggers Rafactivation in absence of Ras (Leevers et al., 1994; Stokoe et al.,
1994), suggesting that not Res-binding, but rather membrane-targeting is the key
activating step.

PKC is a well-studied example of a kinase whose activity depends upon proper
subcellular localization. The PKC protein kinase family consists of at least 11
family members (Newton, 1995). Most cells express more than one isoform, and all
of these isoforms have a very similar ligand-binding and substrate specificity.
Therefore proper subcellular localization is important to achieve specificity. PKCa,
for example is predominately found at the endoplasmic reticulum and at the cell
margin, PKCBII is associated with structures of the actin cytoskeleton, PKCy is
mainly targeted to the Golgi apparatus, whereas PKCe is associated with nuclear
membranes (Goodnight et al., 1995).

Multiple isozymes of PKA are expressed in mammalian cells and many
hormones use parallel pathways to activate PKA. Therefore a mechanism must exist

to ensure that the correct PKA isoform is activated at the right place at the right time

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(Harper et al., 1985). Appropriate subcellular localization is ensured by association
of PKA with so called, A-kinase anchoring proteins (AKAPs). For example, 75% of
type H PKA is found in association with AKAPs (Rubin, 1994). AKAPs are a
growing family of signaling molecules that target PKA to specific locations and
enhance substrate accessibility and specificity. Various AKAPs have been shown to
be targeted to centrosomes, endoplasmic reticulum, Golgi apparatus, microtubules,
mitochondria, membranes, nuclear matrix, and secretory granules (Coghlan et al.,
1994; Keryer et al., 1993; Lin et al., 1995b; McCartney et al., 1995; Ndubuka et al.,
1993).

In contrast to anchoring proteins which localize signaling molecules to the proper
subcellular localization, scaffold proteins bind an array of signaling molecules to
create a signaling complex. In yeast the same MAPKKK is used to elicit different
physiological effects depending upon the scaffold protein it is bound to. Two
MAPK scaffold proteins have been identified in Saccharamyces cervisiae. The
Ste5p scaffold protein coordinates the module, which induces mating in response to
pheromone stimulation (Choi et al., 1994; Kranz et al., 1994; Marcus et al., 1994;
Printen and Sprague, 1994), whereas the Pszp scaffold protein arranges the
signaling proteins, which induce glycerol synthesis in response to high osmolarity
(Posas and Saito, 1997). In both cases the first activated kinase is Stel 1p, but when
bound to different scaffolds it activates different downstream targets, and in turn
elicits different physiological outcomes (Fig. 1). In mammalian cells such scaffold

proteins have also been identified. INK-interacting protein (JIP)1 and JIP2 arrange

10

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modules of the JNK pathway (Whitmarsh et al., 1998; Yasuda et al., 1999). The
MEK-Partner (MP)1 scaffold complex coordinates the kinase MEK] and
extracellular signal-regulated kinase (ERK)1 (Schaeffer et al., 1998). There is also
evidence that the dual-specific kinase MKK4 organizes a signaling module
consisting of MEKKl, MKK4, and INK (Xia et al., 1998), and that ch-interacting
kinase (NIK), a Ste-related kinase, forms a complex with MEKKI, MKK4/7, and

INK (Su et al., 1997), similar to a Pszp-like complex (Fig.2).

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Fig. 1. MAPK scaffold complexes in Saccharonryces cervisiae. The mating
response is coordinated by the scaffold protein Ste5p, which binds the MAPKKK
Stel 1p, the MAPKK Ste7p, and the MAPK Fus3p. Increased glycerol synthesis in
response to increased osmolarity is controlled by the scaffold protein Pbs2p, which
acts as a scaffold protein and a MAPKK.

12

   

MEKK1 scaffold complex

 

MP1 scaffold complex

Fig. 2. Mammalian scaffold complexes. The scaffold proteins HP] and JIP2
coordinate components of the JNK signaling cascade in a SteSp-like complex.
MEKKI arranges a Pbs2p-like scaffold complex. The MP1 scaffold protein
coordinates kinases of the ERK signaling cascade.

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2. Small GTPases of the Rho-family

Small GTPases have been implicated in a variety of cellular processes, ranging
fiom intracellular signaling to synaptic transmission (Van Aelst and D'Souza-
Schorey, 1997). These small proteins, with molecular weights between 20 and 30
kDa, act as molecular switches, cycling between an inactive GDP-bound and an
active GTP-bound state (Fig. 3). The activity of small GTPases is determined by the
ratio of the GDP/GTP—bound state. This ratio is regulated by so-called guanine
nucleotide exchange factors (GEFs), which enhance the exchange of GDP to GTP,
and GTPase activating proteins (GAPS) (Boguski and McCormick, 1993). Since
small GTPases exert a very low intrinsic GTPase activity GAPS are required to
increase GTP hydrolysis and convert the GTPase into its inactive state (T rahey and
McCormick, 1987).

The Ras superfamily of small GTPases is divided into four different subfamilies
(Macara et al., 1996). The best-characterized family is the Ras family, which
consists of H-Ras, K-Ras, and N-Ras. The Ras family gained a lot of attention in the
early 1980s for its transforming activity. Indeed, in 10-20% of all human cancers
mutations in the ras-gene are found (Barbacid, 1987; B05, 1988).

The ARF and Rab families play important roles in the assembly and targeting of
vesicles to the appropriate cellular compartment. They are essential for the

recruitment of coatamer proteins and are important for proper docking and fusion of

vesicles (Macara et al., 1996).

14

The Ran family of small GTPases is associated with the nuclear import
machinery and is essential for nuclear import processes (Macara etal., 1996).

The Rho-family of small GTPases was first identified in yeast in the context of
cytoskeletal rearrangements. The mammalian Rho-family consists of at least 10
members, including Rho A, B, C, D, and E, Rac 1, 2, and E, and Cdc42 and TClO
(Van Aelst and D'Souza-Schorey, 1997). The members of the Rho-GTPase family
are key regulators linking surface receptors to the organization of the actin
cytoskeleton. Rho is implicated in the assembly of stress fibers and focal adhesion
(Ridley and Hall, 1992). Rac induces a meshwork of actin filaments at the cell
periphery to produce lamellipodia and membrane ruffles (Nobes er al., 1995; Ridley
et al., 1992). Cdc42 finally triggers actin rich surface protrusions, so called filopodia
(Kozma et al., 1995; Nobes et al., 1995). In Swiss 3T3 cells cross talk between the
Rho family members has been demonstrated (Chant and Stowers, 1995; Nobes and
Hall, 1995).

Besides their effects on the morphological changes of the cytoskeleton (Hall,
1998) Rho family members also have been implicated in apoptosis (Jimenez et al.,
1995), cell cycle progression (Olson et al., 1995), cellular transformation (Khosravi-
Far et al., 1995; Qiu et al., 1997; Qiu er al., 1995a; Qiu et al., 1995b; Tang et al.,
1999; Whitehead et al., 1998; Wu et al., 1998), and the regulation of MAPK
signaling pathways (Coso et al., 1995; Minden et al., 1995).

Cdc42 has been first identified in S. cervisiae as an important component in the

organization of the actin cytoskeleton during the cell cycle (Adams et al., 1990). In

15

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both yeast species, S. pambe and S. cervisiae, Cdc42 is essential for the viability of
the cell (Johnson and Pringle, 1990; Miller and Johnson, 1994). The mammalian
Cdc42, also known as 625K, was first purified from bovine brain (Waldo et al.,
1987) and human placental membranes (Evans et al., 1986; Polakis et al., 1989) and
shows a high degree of similarity to yeast Cdc42 (Johnson and Pringle, 1990; Polakis
et al., 1989). Studies of embryonic stem (ES) cells in which the gene encoding
Cdc42 is disrupted, show that in mammalian cells the small GTPase is not required
for cell viability, but confirmed its role in formation of the actin cytoskeleton, since
Cdc42-deficient cells show an abnormal cytokeletal arrangement (Chen et al., 2000).
In mammals Cdc42 appears to play a pivotal role in embryonic development.
Cdc42-deficient mice die very early during embryonic development. Histological
analysis of in utera embryos shows that the embryos are totally disorganized and
lack the primary ectoderm (Chen et al., 2000), supporting Cdc42's role in
cytoskeletal organization.

Cdc42 contains a so-called CAAX motif at the COOH-terminus, which is
required for geranylgeranylation of the small GTPase (Maltese and Sheridan, 1990).
This posttranslational modification allows the GTPase to associate with cellular
membranes, including the plasma membrane (Hart et al., 1990) and the Golgi
complex (Erickson et al., 1996). Interestingly, in yeast 3 Cdc42 variant which
cannot undergo prenylation, due to a change of the cysteine to a serine within the
CAAX motif, rescues the lethal phenotype induced by constitutively active Cdc42

(Ziman et al., 1991). Studies in yeast also show that Cdc42 is distributed

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differentially during the cell cycle and that proper localization is important for
polarized cell growth (Ziman et al., 1993). There is also evidence in mammalian
cells that Cdc42 may be required for the subcellular distribution of effector proteins.
In viva studies suggest that Cdc42 localizes the structural protein Wiskott Aldrich
Syndrome protein (WASP) to the plasma membrane, where a multiprotein complex
is formed, subsequently inducing actin polymerization and filopodium formation
(Castellano et al., 1999).

A very well studied downstream effector of Cdc42 is the serine/threonine kinase
PAK, which is activated by Rho-family GTPases (Manser et al., 1994). The
interaction between Cdc42 and PAK requires the so-called Cdc42/Rae interactive
binding (CRIB) motif (Burbelo et al., 1995), a short, and 14-16 amino acid residue
long motif. The structural requirements and the activation mechanism of PAK by
Cdc42 have been extensively studied (Gatti et al., 1999; Knaus et al., 1998; Manser
et al., 1997; Manser et al., 1994; Zenke et al., 1999; Zhao et al., 1998). Activation
of PAK by Cdc42 can be demonstrated in vitra using recombinant purified proteins
(Martin et al., 1995). This observation supports a stoichiometric model in which
Cdc42 directly increases PAK autophosphorylation activity, without the requirement
for additional effectors. In several studies it has been reported that membrane
localization of PAK may contribute to PAK activation (Daniels et al., 1998; Lu et
al., 1997a; Lu and Mayer, 1999). However, there is no direct evidence that the
interaction of Cdc42 with PAK is required for membrane translocation. In contrast a

variant of Ste20p, the PAK yeast homologue, which fails to bind Cdc42, shows

17

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cytoplasmic localization (Peter et al., 1996), suggesting that Cdc42 may be important
for targeting of Ste20p to emerging buds.

Besides the PAKs, Cdc42 interacts with various other kinases, including MEKKs
(Fanger et al., 1997; Gerwins et al., 1997) or the non-receptor tyrosine kinase
activated Cdc42HS-associated kinase (ACK) (Manser et al., 1993). Interestingly,
MEKKl (Fanger et al., 1997) and the ribosomal S6 kinase (Chou and Blenis, 1996)
bind to Cdc42 despite the fact that they lack a CRIB motif. In addition, deletion of
the CRIB motif in MEKK4 only partially diminishes binding of Cdc42 and Rac,
suggesting a CRIB-independent GTPase binding determinant. Thus, the role of
CRIB motifs and the mechanism by which small GTPases promote the activation of
protein kinase remains largely unexplained.

Cdc42 has been implicated in cell cycle progression, but its effect on this process
is still controversial. Olson and coworkers reported that microinjection of
constitutively active Cdc42 into Swiss-3 T3 cells increases bromodeoxyuridine
incorporation into DNA, whereas the dominant negative Cdc42 blocks the
incorporation (Olson et al., 1995), suggesting that Cdc42 may contribute to cell cycle
progression. In contrast, microinjection of wild type Cdc42 into synchronized NIH
3T3 cells caused cell cycle arrest, which was mediated by the p3 8/reactivating kinase
(p38/RK) pathway (Molnar et al., 1997). Besides its effects on the cell cycle,
activated variants of Cdc42 have transforming activity. There is evidence that Cdc42
is required for Ras-mediated transformation and that stable expression of

constitutively active Cdc42 in Rat] fibroblasts increases anchorage-independent

18

 

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growth in soft agar (Qiu et al., 1997). Injection of activated Cdc42 into nude mice
promotes tumor formation (Qiu et al., 1997). Taken together these data suggest that
Cdc42 (and Rac) indeed induce cellular transformation. Although Cdc42 appears to
regulate a variety of different cellular processes, including cell cycle progression,
cellular transformation, and invasiveness, these processes are all dependent on
cytoskeletal rearrangements.

A very fascinating field, which has recently drawn more attention, is the role of
Cdc42 in negative factor (NeD-dependent human immune deficiency virus (HIV)
replication. Nef of human and simian immune deficiency virus (HIV -1, HIV -2 and
SVI) is a myristylated plasma membrane-associated protein (Allan et al., 1987; Yu
and Felsted, 1992). Nef plays an important role in viral pathogenesis and is
responsible for high levels of viremia and for the progression of an HIV infection
towards acquired immune deficiency syndrome (AIDS) (Kestler et al., 1991). Nef
coimmunoprecipitates with the mammalian PAK-like serine/threonine kinase Nef-
associated kinase (NAK) (Nunn and Marsh, 1996; Sawai et al., 1994; Sawai et al.,
1995). It has been demonstrated that NAK kinase activity is increased upon
cotransfection with active Cdc42 and that dominant-negative Cdc42 blocks binding
of Nef to NAK and subsequent activation of the kinase. Moreover dominant-
negative Cdc42 and Rac decrease HIV-1 production (Lu et al., 1996). Thus these
data suggest a role for Cdc42 in the progression of an HIV infection towards AIDS.

Therefore understanding the regulation of Cdc42 and its effector proteins may shed

l9

further light on the progression of HIV infections and lead to the identification of
potential therapeutically useful targets.

Since Cdc42 regulates so many different proteins and thus, multiple
physiological processes, it is of great interest to learn more about the effector
proteins of this small GTPase. Understanding the Cdc42—mediated regulation of
proteins may provide important knowledge of how this small GTPase can exert so

many different effects and help to decipher the specific roles of the effector proteins.

20

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Fig. 3. Regulation of small GTPases. Small GTPase cycle between a GDP-bound
inactive, and a GTP-bound active form. Guanine nucleotide exchange factors
promote the exchange of GDP to GTP. GTPase-activating proteins (GAPS) enhance
the hydrolysis of GTP to GDP.

21

 

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3. The MAP Kinase Pathways

For proper development and survival, eukaryotic cells must be able to
communicate with their surroundings and to respond to various extracellular and
environmental stimuli. Exposure to external stimuli induces a variety of cellular
responses, such as proliferation, cell division, differentiation, or apoptosis. One
crucial question is how these external signals, which are sensed by cell surface
receptors, are processed within the cell to induce the changes in gene expression that
account for changes in cell phenotype and cell morphology. Some of the major
signal transduction pathways in eukaryotic cells are the protein kinase cascades,
which lead to the activation of so called mitogen-activated kinases (Cano and
Mahadevan, 1995; Marshall, 1994; Waskiewicz and Cooper, 1995). Although
different MAP kinase pathways are activated by different external stimuli they all
follow a common conserved module of activation (Fig. 4). The MAPK is activated
by phosphorylation on threonine and tyrosine residues within a TXY motif, by a
dual-specific MAPKK The MAPKKS are a highly conserved group of protein
kinases, which are activated by phosphorylation of serine and threonine residues by
MAPKKKS. MAPKKKS receive their signal from cell surface receptor through
various proteins, including small GTPases, protein kinases, and/or protein
phosphatases. In the different MAPK pathways the MAPKS, MAPKKS and
MAPKKKS are highly conserved within their catalytic domain, but can differ

significantly in their regulatory domains and substrate specificity.

22

 

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Fig. 4. MAPK pathway module. The core module of the mitogen-activated protein
kinase pathway is composed of three kinases that are sequentially activated by
phosphorylation.

23

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The first identified mammalian Signal transduction pathway was the ERK
cascade. Later a second MAP kinase pathway was discovered which induces the
activation of IN K. This pathway is also called stress-activated protein kinase
(SAPK) pathway, since its activation has been linked to various cellular stresses
(Derijard et al., 1994; Hibi et al., 1993; Kyriakis et al., 1994). Recently a third
MAPK the p38/RK pathway was identified (Han et al., 1994; Rouse et al., 1994). A
simplified overview of all three mammalian MAPK pathways is shown in Fig. 5.

The INK pathway is mainly known for its activation by cellular stresses, such as
osmotic stresses and UV-radiation (Derijard et al., 1994). Additionally the JNK
cascade is activated by cytokines such as tumor necrosis factor-a (TNF-ct) (Sluss et
al., 1994) and interleukin] (1L1) (Chaudhary and Avioli, 1998; Finch et al., 1997;
Guan et al., 1996). Furthermore, evidence exists that the JNK pathway can be
activated by growth factors (Bast et al., 1997).

Whereas the ERK pathway is activated by members of the Ras family of small
GTPases, small GTPases of the Rho-family, especially Racl, Rac2, and Cdc42 are
implicated in INK activation (Bagrodia et al., 1995a; Brown et al., 1996; C050 et al.,
1995; Hill et al., 1995; Minden et al., 1995; Zhang et al., 1995). Overexpression of
constitutively active Cdc42 in COS-7 cells, for example, increases in vitra INK
activity 5-10 fold, but Ins only little effect on ERK activity (Coso et al., 1995;
Teramoto et al., 1996). Similar results were observed in COS-1 (Bagrodia et al.,
1995a), HeLa, NIH-3T3, Ratl (Minden et al., 1995) and 293 cells (Teramoto et al.,

1996). Recent studies Showed that expression of dominant negative variants of

24

 

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Cdc42, Rae, and Ras block IN K1 activation, induced by hepatocyte growth factor,
TNF-a, or hypcrosmotic glucose (Auer et al., 1998). Interestingly, analysis of
cdc42-/- ES cells Showed that JNK and p38/RK activity induced by cellular stresses
is not impaired (Chen et al., 2000), suggesting that a Cdc42~independent mechanism
of JNK activation or that redundancy among Rho family members in terms of JNK
activation may exists.

MEKKS are MEKKKS that have been Shown to activate the ERK pathway
(Blank et al., 1996; Lange—Carter et al., 1993), but preferentially activate the JNK
pathway (Kyriakis et al., 1994; Minden et al., 1994; Yan et al., 1994). Four different
MEKKS have been cloned, MEKKl-4 (Blank et al., 1996; Gerwins et al., 1997;
Lange-Carter et al., 1993) among which MEKKl and 4 appear to be regulated by
Cdc42 and/or Rac.(Fanger et al., 1997). Kinase-inactive MEKK4 blocks INK
activation by constitutively activated Cdc42 and Rac (Gerwins et al., 1997), but no
direct activation of MEKK4 by these two small GTPases could be demonstrated yet.
In addition to Rho-GTPases, MEKKl also binds to the activated form of Ras
(Russell et al., 1995).

The protein kinase PAK has been shown to activate the JNK pathway
(Bagrodia et al., 1995a; Brown et al., 1996). Four different PAK isoforms, PAKl,
PAK2, PAK3 and PAK4 have been identified (Abo et al., 1998; Bagrodia et al.,
1995b; Brown et al., 1996; Jakobi et al., 1996; Manser et al., 1994) and PAKl-3

increase autophosphorylation in the presence of Cdc42 (Martin et al., 1995).

25

 

Although PAKs have been implicated in IN K activation, their physiological role in
the JNK pathway still remains unclear.

Members of the mixed lineage kinase family MLK2, SPRK/MLKB, and dual
leucine zipper-bearing kinase (DLK) have been identified as activators of the JNK
pathway. (Fan et al., 1996; Hirai et al., 1997; Nagata et al., 1998; Teramoto et al.,
1996). MLK2 and SPRK, both associate with the small GTPases Cdc42 and Rac in a
GTP-dependent manner (Burbelo et al., 1995; Nagata et al., 1998; Teramoto et al.,
1996) and coexpression of kinase-inactive SPRK with activated Cdc42 or Racl
blocks Cdc42-induced or Rae-induced IN K activation suggesting that SPRK/MLK3
acts downstream of Cdc42 and Rac (Teramoto et al., 1996). Furthermore it has been
demonstrated that hematopoietic protein kinase (HPK)1 associates with
SPRK/MLK3 and phosphorylates a kinase-inactive mutant of SPRK/MLK3. Thus
HPKI may be a potential activator of SPRK, although activation of SPRK/MLK3 by
HPKl has not been demonstrated (Kiefer er al., 1996). SPRK/MLK3 activates the
JNK pathway via the dual-specific kinases MKK4 (Rana et al., 1996) and MKK7
(Whitmarsh et al., 1998). SPRK/MLK3 activates MKK4 by phosphorylation of a
serine 254 and threonine 258 in the activation loop of the catalytic domain (Yan et
al., 1994; Zheng and Guan, 1994).

For activation INK has to be phosphorylated on threonine 183 and tyrosine
185 within the TXY-motif of the catalytic domain (Derijard et al., 1995). Two dual-
specific kinases, MKK4 (Derijard et al., 1995; Lin et al., 1995a) and MKK7

(Moriguchi et al., 1997; Tournier et al., 1997; Wu et al., 1997; Yao et al., 1997)

26

”I“

 

have been shown to phosphorylate INK. MKK7 has been found to associate with the
two scaffold proteins HP] and IIP2, which both mediate activation of the INK
pathway, presumably by assembling the individual signaling components, including
I-IPKl, DLK, MLK2, SPRK, MKK7, and INK into a multi-enzyme complex
(Whitmarsh et al., 1995; Yasuda etal., 1999).

Upon phosphorylation by a dual-specific kinase INK translocates into the
nucleus where it phosphorylates a variety of transcription factors. Originally INK
was identified by its ability to phosphorylate the transcription factor c-Iun on serine
63 and serine 73 (Derijard et al., 1994). These activating phosphorylations induce
transcriptional activity of c-Iun (Pulverer et al., 1991; Smeal et al., 1992; Smeal et
al., 1991). The INK family includes three different gene products, INK] (Derijard et
al., 1994), INK2 (Sluss et al., 1994) and INK3, which together due to alternative
splicing encode for 10 different INK isoforms (Gupta et al., 1996). INKl and INK2
are ubiquitously expressed (Kallunki et al., 1994), whereas INK3 is restricted to
neuronal cells (Gupta et al., 1996). The three INK isoforms differ in their ability to
phosphorylate c-Jun. INK2, for example, has a much higher affinity for c-Iun than
INKl. Due to the lower Km for c-Iun and higher me INK2 is estimated to be 25
times more efiicient in cJun phosphorylation than INKl (Kallunki et al., 1994).

The c-Iun transcription factor is involved in a variety of cellular events,
including proliferation, differentiation, and cellular transformation. c-Iun is a
member of the AP-l activator, which is composed of leucine zipper-bearing DNA

binding proteins including c-Iun, F05, and ATFZ (Sassone-Corsi et al., 1988). INK

27

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also phosphorylates and activates the transcription factor ATFZ, which is able to
dimerize with c-Iun and can induce transcription of c-Iun (Gupta et al., 1995;
Livingstone et al., 1995; van Dam et al., 1995). INK also regulates cFos expression,
which is mediated by the serum response factor (SRF) and the ternary complex
factor (T CF) (Treisman, 1992; Treisman, 1994). One well-characterized TCF is Elk-
1, which is phosphorylated by INK (Cavigelli et al., 1995; Gille et al., 1995;
Whitmarsh et al., 1995; Zinck et al., 1995). Interestingly, INK phosphorylates the
same activating sites as does ERK (Whitmarsh et al., 1995) suggesting an integration

of the ERK and the INK pathways.

28

GTPa

MAP!

“AF

MA

GTPases Rae Rae, Cdc42 Rae, Cdc42

 

 

 

 

PAK / HPK-1
/ 7 l?
l _. .
MAPKKK Raf MEKKt MLK2 SPRK MEKK4
DLK 3:- . , m;
MAPKK MEK 1, 2 MKK4 MKK3,
MKK7 MKKB
MAPK ERK JNK p38IRK

Fig. 5. Parallel mammalian MAPK pathways. Three MAPK pathways have been
identified in mammalian cells, the ERK, INK, and p3 8/RK pathway.

29

4. The physiological importance of the JNK pathway

The INK pathway has been mainly characterized as a MAPK cascade which
plays an important role in the induction of apoptosis in response to cellular stresses,
including UV and y-irradiation, protein synthesis inhibitors, osmotic stress, toxins,
ischemia/reperfusion injury in heart, ceramides, inflammatory cytokines, such as
TNF-a, or cytokine deprivation (Cuvillier et al., 1996; Verheij et al., 1996; Xia et
al., 1995; Zanke et al., 1996). Most studies showing the apoptotic effect were
performed in in vitro cell culture. Analysis of in vivo models finally demonstrated
that the INK pathway is not only a stress-activated pathway, but also an important
player in the inflammatory and humoral immune response and in developmental
processes. Drosophila melanogaster, as a genetic model, and genetically engineered
mice rendered much of the knowledge about the physiological function of the INK
signaling cascade. Following is a summary of knowledge obtained about the role of

INK signaling using Drosophila and knockout mice as a model system.

4.1 JNK signaling in Drosophila melanogaster

Many components of the mammalian INK signaling pathway are conserved in
mammals and insects (Glise et al., 1995; Holland et al., 1997; Lu et al., 1997b;
Moriguchi et al., 1997; Riesgo-Escovar et al., 1996; Sluss et al., 1996; Tournier et
al., 1997). The Drosophila INK homologue, also termed DINK or BSK, shares
about 80% sequence identity with the mammalian counterparts and contains the

INK-specific TPY regulatory motif (Riesgo-Escovar et al., 1996; Sluss et al., 1996).

30

 

Also a homologue of MKK7, the MAPKK, called DMKK7 or HEP (Holland et al.,
1997), and DPAK (Harden et al., 1996) have been cloned. In addition to protein
kinases of the INK pathway, the upstream activators, small GTPases of the Rho
family, termed D-Racl, D-Cdc42 (Luo et al., 1994) have been identified. Analogous
to the nnmmalian system DIN K phosphorylates Djun, the Drosophila homologue of
c-Iun, in vitro (Riesgo-Escovar et al., 1996; Sluss et al., 1996) and in viva (Hou et
al., 1997; Kockel et al., 1997; Riesgo-Escovar and Hafen, 1997). Studies in
Drosophila demonstrated that DINK is important for developmental processes such
as tissue polarity, dorsal closure, neural development, as well as the insect immune
response.

Dorsal closure occurs at mid-embryogenesis in Drosophila. It is a morphogenic
process, which requires a shape change of the epidermal cells and the movement of
these cells as a sheet, which surrounds the embryo (Martinez-Arias, 1993). Two
groups of proteins are required for dorsal closure, structural proteins that control cell
shape and signaling molecules that coordinate the morphogenic process. The two
signaling pathways, which contribute to dorsal closure, are the INK pathway
(Riesgo-Escovar et al., 1996; Sluss et al., 1996) and a transforming growth factor
(TGF)-B-like pathway (Glise and Noselli, 1997; Hou et al., 1997; Kockel et al.,
1997; Sluss and Davis, 1997). Loss of fimction mutations in hep, bsk and Djun
cause severe defects in dorsal closure (Glise et al., 1995; Glise and Noselli, 1997;
Hou et al., 1997; Kockel et al., 1997; Riesgo-Escovar and Hafen, 1997; Riesgo-

Escovar et al., 1996; Sluss and Davis, 1997; Sluss et al., 1996). In addition loss of

31

firmion m
332'. Pk}; (
31K‘ '5.

199"}. er.

r-y—q

filnction mutations of the upstream components of the INK pathway, Racl, Cdc42,
and PAK cause defective dorsal closure (Harden et al., 1996). Interestingly, mouse
MKK7 is able to rescue hep mutants when expressed in Drosophila (Holland et al.,
1997), emphasizing the conservation of this pathway among invertebrates and
vertebrates. Taken together theses results strongly suggest an important role for INK
during fly development.

It also has been proposed that the INK cascade is necessary in the insect immune
response. In order to fight microbial infection insects possess signal transduction
pathways (1p and Levine, 1994; Sluss et al., 1996). These signaling pathways, which
are conserved in fly and mammals, are involved in activation of the transcription
factors NF-KB and AP-l . Biochemical studies show that the Drosophila immune
response is initiated by bacterial lipopolysaccharides, which induce the activation of

the MAPKK HEP (Sluss et al., 1996).

4.2 The JNK Pathway in mammab

Although the investigation of INK activity in Drosophila rendered much
information about the developmental role of this pathway in the fruit fly the
generation of genetically engineered mice exerts great impact on the importance of
INK signaling in mammals.

Homozygous mice with a targeted disruption of the gene for the dual specific
kinase MKK4, also called SEKl, are embryonic lethal between embryonic day

(E)10.5 and E125 (Nishina et al., 1999; Yang et al., 1997a). The heterozygotes

32

 

show no phenotype compared with wild type mice (Ganiatsas etal., 1998; Nishina et
al., 1999; Yang et al., 1997a). ES cells, which lack the MKK4 gene, are no longer
able to activate the INK pathway in response to environmental stresses, such as
anisomycin treatment or heat shock demonstrating that MKK4 activates INK in viva.
Results from biochemical studies suggest that MKK4 activates both, INK and
p38/RK (Derijard et al., 1995; Lin et al., 1995a). Analysis of the ES cells with a loss
of fimction for MKK4, showed that only the activation of the INK pathway is
impaired and not activation of the p38/RK pathway, suggesting that MKK4 is a
specific INK activator in viva (Ganiatsas et al., 1998; Yang et al., 1997a). Analysis
of the phenotype of mkk4-l- mice showed that these animals have severe defects in
liver formation and hepatocyte development (Ganiatsas et al., 1998; Nishina et al.,
1999). The embryos still form the primordial liver anlage but the hepatocytes
undergo massive apoptosis and disappear, suggesting a role for MKK4 as an anti-
apoptotic survival signal during liver organogenesis (Ganiatsas et al., 1998; Nishina
er al., 1999). Interestingly mice lacking the transcription factor c-Iun also display a
similar defect in liver development and die between E13.5 and E14.5 (Hilberg et al.,
1993; Johnson et al., 1993). Taken together these results suggest that liver
development is dependent on c-Iun activation by the MKK4-INK signaling cascade.
Since the disruption of the MKK4 gene is embryonic lethal, it is not possible to
eXamine the physiological role of MKK4 in adult knockout animals. However, a
Complementation assay using ragZ-I- blastocytes rendered chimeric animals with

Mkk4-l- T and B cells (Nishina et al., 1997). In B cells the MKK4-INK signaling

33

 

l t .
C(‘A‘Ctdt 1)

i171"; R“ f.
me:

my.

Atl.

5 “‘93:

fig“?“_

‘ “L - xi)
' ‘

‘

h.
4n
..
3..
K‘“-\T>’:

cascade is activated in response to CD40 crosslinking (Berberich et al., 1996; Sakata
et al., 1995). INK activation also regulates 1L2 production in T cells (Su et al.,
1994). Animals with MKK4 null B cells show a partial block in B cell maturation,
but peripheral B cells show no defect demonstrating tlmt MKK4 may be important in
early B cell differentiation, but that it is not necessary for proliferation and Ig
secretion during the humoral immune response (N ishina et al., 1997). The chimeric
mice have smaller thymi, but normal numbers of T cells. This suggests that MKK4
is important for IL2-dependent T cell proliferation. Further analysis of these
chimeric mice showed that mkk4-l- mice failed to induce the expression of the death
suppressor Bel-XL in response to antigen receptor activation, suggesting that MKK4
transduces cellular survival signals during T cell stimulation (N ishina et al., 1998).

In contrast to MKK4 deficient mice, knockouts of the various INK isoforms are
viable (Dong et al., 1998; Yang et al., 1998; Yang et al., 1997b). The INKl and
INK2 isoforms are ubiquitously expressed in the body, whereas INK3 is mainly
restricted to the brain, with a lower expression in heart and testis (Gupta et al., 1996).

Analysis of jnkI-l- and jnkZ-l— mice showed that they have defects in T cell
differentiation (Dong et al., 1998). Two types of T lymphocytes cytotoxic T cells
and helper T cells (T it) exist. The TH cells stimulate the immune response of other
cells such as macrophages and B cells by secreting lymphokines, interleukins, and
cytokines. TH cells differentiate in two subclasses, TH] and TH2 cells, depending
upon the difl‘erentiation signal. Mice deficient for INKl produce a high level of THZ

cytokines and therefore differentiation into TH2 cells was enhanced (Dong et al.,

34

 

I -
.11 {on
diff-'31

.I'tc C

{.71

p.

1998) resulting in increased IL4 secretion. In jnk2-/- mice the differentiation into
TH] cells is also impaired, but this is due to a decrease in interferon (INF )y secretion,
which is required for differentiation into TH] cells (Sabapathy et al., 1999; Yang et
al., 1998). These results show that both INK] and INK2 are required for the proper
differentiation of T51 cells, but that they exert a different mechanism of regulation.
The My secretion, which requires INK, directly controls TH] proliferation and also
enhances the activation of cytotoxic T cells, whereas INK] controls IL4 secretion to
drive cells into TH] differentiation away from TH2 differentiation.

The compound knockout of INK] and INK2 is embryonic lethal between E1]
and E12, whereas compound knockouts of INK1/INK3 and INK2/INK3 are viable
and show no phenotype (Kuan et al., 1999). Mice deficient for INK] and INK2
have severe a dysregulation of apoptosis in the brain. Interestingly they show a
reduction of apoptosis in the lateral edges of the hindbrain prior to neural tube
closure and an increase of apoptosis in the forebrain with precarious degeneration,
suggesting that INK] and IN K2 have apoptotic as well as anti-apoptotic effects.
Furthermore this study shows that INK] and INK2 are absolutely required for proper
neural development but that their effects are redundant, since the single knockouts
show no neuro-developmental impairment.

Mice lacking the INK3 gene showed no abnormalities in the structural
organization and cellular composition of the brain, but they show a different
phenotype to wild type litterrnates when injected with kainic acid (Yang et al.,

1997b). Kainic acid mimics the effect of excitatory amino acids, which cause acute

35

 

membrane depolarization, seizures, and latent cellular toxicity. The cellular toxicity
induces apoptosis and plays an important role in the onset of neurodegenerative
diseases (Lipton and Rosenberg, 1994; Rothman and Olney, 1986). Kainic acid
causes seizures by direct stimulation of the glutamate receptor and indirectly by
increasing the release of excitatory amino acids firom nerve terminals (Ben-Ari,
1985; Ferkany et al., 1984). When treated with an epileptogenic dose of kainic acid
(30mg/kg body weight) wild type mice and heterozygous mice showed seizures of
progressive severity. The homozygous jnk-/- mice in contrast had much milder
symptoms and recovered faster after administration of kainic acid. The neural
damage caused by kainic acid is predominant in the hippocampus (Ben-Ari, 1985).
Analysis of hippocampi of systemically treated mice showed no signs of cell
destruction in the homozygous knockout animals, when compared to wild type or
heterozygous littermates. In knockout mice the phosphorylation of c-Iun and the
transcriptional activity of AP-l is decreased, suggesting that the protection against
kainic acid-induced symptoms is due to the blocking of the INK3 signaling cascade.
This effect is not seen in the INK] and INK2 knockout animals suggesting a very
Specific role for INK3 in mediating glutamate receptor signaling. Thus, INK3 may

be a potential target for the treatment of neurodegenerative diseases.

36

5. The Mixed-Lineage Kinase Family

The mixed-lineage kinase family of serine/threonine kinases consists of five
family members, MLK], MLK2, SPRK/MLK3, DLK, and leucine zipper-bearing
kinase (LZK). The three MLKs are very closely related, whereas DLK and LZK
compromise a more distant subfamily of the mixed-lineage kinases. This protein
kinase family is characterized by a kinase domain, which resembles in sequence both
tyrosine kinases and serine/threonine kinases but only serine/threonine kinase
activity has been demonstrated. The mixed-lineage kinases contain several potential
protein-protein interaction domains, including a leucine/isoleucine zipper and a
COOH-terminus, which is rich in serine, threonine, and proline. The bana fide
MLKs contain an NHz-terminal SH3 domain, whereas DLK and LZK do not. These
are the only reported serine/threonine kinases that contain an SH3 domain in the
absence of an SH2 domain.

All of the MLKs were identified by a PCR approach using degenerate
oligonucleotides corresponding to conserved regions in the catalytic domains of
protein kinases. The full-length clone of MLK1 has not been reported yet. MLK]
mRNA has been detected in epithelial cell lines of breast, colonic and esophageal
origin (Dorow et al., 1993) and shows differential mRN A expression in pancreatic B-
cell lines at different stages of maturation and embryonic pancreas development
(DeAizpurua et al., 1997). The protein kinase is primarily expressed in pancreatic

duct-like structures during mouse embryogenesis, whereas it is not detected in late

37

gestation or early adulthood suggesting a role of MLK1 in the early inductive phase
of pancreatic development.

According to Northern blot analysis MLK2 is rminly expressed in brain and
skeletal muscle, with a lower level of expression in the pancreas (Dorow et al.,
1993). Additionally, several tumor cell lines show high levels of MLK2 expression
(Katoh et al., 1995).

Overexpression of MLK2 in COS-1 cells increases INK activity 6 to 10-fold.
Only modest induction of p38/RK (LS-fold) and ERK (2 to 3-fold) (Hirai et al.,
1997; Nagata et al., 1998) was detected. INK activation is presumably mediated by
MKK4 and MKK7, since these two dual-specific kinases are activated upon
overexpression of MLK2 in COS-1 and human epithelial KB cells (Cuenda and
Dorow, 1998; Hirai et al., 1997; Hirai et al., 1998). Interestingly, it appears that
MKK7 is the preferred substrate since MLK2 overexpression in these cell types
resulted in a higher MKK7 than MKK4 activity (Cuenda and Dorow, 1998; Hirai et
al., 1998). The same result is obtained in in vitra kinase assays using a truncated,
recombinant MLK2 and purified GST-MKK4 or GST-MKK7 as a substrate (Hirai et
al., 1998). Evidence exists that transiently expressed MLK2 is able to activate
MKK3 and MKK6, the upstream activators of p38/RK (Cuenda and Dorow, 1998),
but the physiological importance of this activation remains unclear, since
overexpression of MLK2 does not elicit a prominent increase in p38/RK activity

(Cuenda and Dorow, 1998).

38

 

It

 

Examim:
pm‘maze d;
pTISTI‘A‘T) 1c:
precipitant
KIRK. RIF
L'arswn ca:
.2995! “OCT:

‘he vesicle 3r

Addmmgh

0]?“ dm

microtubuics
Nemesis. 1

 

 

Examination of the subcellular distribution of MLK2 in Swiss 3T3 showed a
punctuate distribution of microinjected MLK2 along microtubules, together with
phosphorylated INK (Nagata et al., 1998). Two-hybrid analysis and coimmuno-
precipitation experiments demonstrated tlmt MLK2 associates with KAP3 and
KIF3X. KIF3 is a member of the kinesin superfamily of motor proteins, which
transport cargo vesicles along microtubules towards the plus end (Brady and Sperry,
1995), whereas KAP3 is a cargo target molecule that serves as an adaptor between
the vesicle and the cargo molecule (Yamazaki et al., 1996). It appears that MLK2
can associate with both the motor protein itself and the cargo target molecule. This
may suggest that MLK2 may be involved in vesicular transport processes.
Additionally it has been reported that the SH3 domain of MLK2 can bind the
GTPase dynamin (Rasmussen et al., 1998). Dynarnin is a GTPase which binds to
microtubules (Cook et al., 1996) and which is involved in vesicle formation during
endocytosis, synaptic transmission, and secretion (Urrutia et al., 1997). Since the
study was performed only using the SH3 domain of MLK2 the specificity and
physiological significance of this interaction requires further investigation.

Studies of rat and mouse development suggest a role for MLK2 in testis
development. Whereas MLK2 expression is temporally and spatially restricted in
the rat testis, MKK4 is found at all stages and all cell types (Phelan et al., 1999)
suggesting that the INK pathway is employed in various cell types and has multiple
functions in rat testis, but that INK activation by MLK2 is temporally and spatially

restricted.

39

The mixed-lineage kinase MLK3 (Ing et al., 1994), also called SPRK (Gallo et
al., 1994) or protein tyrosine kinase-1 (PTK-l) (Ezoe et al., 1994), is a 95 kDa
protein consisting of 847 amino acid residues (Gallo et al., 1994). Despite the
similarity of SPRK's kinase domain to both, serine/threonine kinases and tyrosine
kinases only serine/threonine kinase activity has been demonstrated in vitra (Gallo et
al., 1994). SPRK contains an NHz-terminal glycine-rich region, an SH3 domain
followed by a catalytic domain, a leucine/isoleucine zipper motif, a CRIB motif, and
a proline/serine/threonine rich COOH-terminal region (Fig. 6).

SPRK mRN A expression is detected in various tissues, with lower expression in
heart and brain (Gallo et al., 1994). The MLK3 gene is localized to human
chromosome 11 ql3.1-l3.3 (Ing et al., 1994). Amplifications of this region have
been observed in several human malignancies, including melanonm, bladder, breast,
lung, esophagus, and head and neck carcinomas (Lammie and Peters, 1991).
Additionally a putative tumor suppressor gene has been mapped to 11q13 (Bale et
al., 1989; Larsson et al., 1988). Overexpression of SPRK induces cellular
transformation in NIH 3T3 fibroblasts, rendering them competent to grow in soft
agar (Hartkamp et al., 1999). Moreover, SPRK induces tumor formation in nude
mice (unpublished observation). However, very little is known about the
physiological fimction of this protein kinase in cellular transformation and its
potential role in human cancers.

Recently there is accumulating evidence that SPRK may be involved in T cell

receptor signaling. It has been demonstrated that overexpression of SPRK in T-cells

4o

 

induces
ILZ pro:
a ten ic;
subunit c
BCIIX It} i.
IO ID'CTGE:

323‘ m 9::

MEKKI a

 

induces INK-mediated TNF-a promotor activity (Hoffmeyer et al., 1999) as well as
1L2 promotor activity (Hoffmeyer et al., 1998). Upon overexpression in HeLa cells,
a cervical adenocarcinoma cell line, SPRK induces nuclear translocation of the p65
subunit of NF-ch, accompanied by an increase in NF-KB-dependent transcriptional
activity (Hehner et al., 2000). In vitra kinase assays demonstrate that SPRK is able
to increase Ich-kinase (IKK)a and IKKB activity, supporting the idea that SPRK
may mediate NF-xB-dependent transcription. However, one has to consider that
MEKK] and NTK can also act as IKKKs under these conditions, still leaving open
the identity of the physiological effector of this process. Electrophoretic mobility
shifts suggest that a combination of certain T cell activating stimuli decrease SPRK's
electrophoretic mobility in Iurkat cells supporting the idea that T cell activation
induces a change in SPRK phosphorylation. Upon overexpression of a kinase-
defective SPRK variant in Iurkat cells NF-ch-dependent transcription, which is
induced by these stimuli is blocked. The kinase-defective SPRK variant also inhibits
Cdc42 or Rac-induced NF-ch-dependent transcription. However, this effect may be
due to sequestration of the GTPases by the SPRK variant. Another study shows that
a kinase-defective SPRK variant blocks Vav-induced IL-2 transcription (Hehner et
al., 2000). These studies suggest a role for SPRK in T cell activation. Vav is a
guaninine-nucleotide exchange factor for the small GTPase Rac.

SPRK contains several domains predicted to mediate protein-protein interactions.
Two-hybrid analysis and coirnmunoprecipitation experiments, for example, showed

that HPKl binds to SPRK's SH3 domain (Kiefer et al., 1996). HPK] phosphorylates

41

kinase-inactive SPRK in an in vitra kinase assay (Kiefer et al., 1996), suggesting a
role for HPK] in SPRK regulation, although an effect of HPKl on SPRK activity has
not been demonstrated. Furthermore, kinase-defective SPRK variants and a non-
phosphorylatable MKK4 mutant block HPKl-induced INK activation placing SPRK
and MKK4 downstream of HPK] in the INK signaling cascade, at least upon
overexpression in COS-1 cells (Kiefer et al., 1996).

The leucine/isoleucine zipper is a potential motif for heterologous or/and
homologous oligomerization. Experiments using a SPRK variant bearing a point
mutation in the zipper motif indeed show that the zipper is required for SPRK
oligomerization. The study also shows that the zipper domain is important for basal
phosphorylation activity of SPRK and INK activation. Interestingly, the zipper is not
required for Cdc42-induced increase in SPRK autophosphorylation activity, but is
pivotal for phosphorylation of MKK4. This suggests that oligomerization by the
zipper is not required for SPRK activation per se but for interaction and
phosphorylation of MKK4 and subsequent INK activation (V acratsis and Gallo,
2000).

The CRIB motif is a short amino acid sequence that has been identified as the
binding motif for the GTPases Rae and Cdc42 (Burbelo et al., 1995). Filter binding
assays show that a CRIB motif-containing fragment of SPRK binds to GTP-bound
Rae and Cdc42, with a strong preference for activated Cdc42 (Burbelo et al., 1995).
The result of the filter-binding assay also could be recapitulated by a two-hybrid

analysis (Nagata et al., 1998), suggesting that the two proteins indeed associate in

42

 

3c33312..

 

viva. Upon coexpression of SPRK with Cdc42 or Rac in COS-7 cells SPRK
autophosphorylation is increased, suggesting a role for the small GTPases in SPRK
activation (Teramoto et al., 1996). SPRK contains only six of the eight conserved
residues of the consensus CRIB motif. Whether SPRK contains a fiinctional CRIB
motif, which mediates Cdc42 binding and perhaps Cdc42-induced, activation of
SPRK has not been determined.

SPRK has been identified as an upstream activator of the INK and p38/RK
pathways upon overexpression in COS-1 cells (Tibbles et al., 1996). However, no
activation of p38/RK could be demonstrated in 293 cells (Teramoto et al., 1996).
INK activation by SPRK is mediated by the dual specific kinases MKK4 (Rana et
al., 1996) and MKK7 (Whitmarsh et al., 1998). SPRK, MKK7, and INK bind to the
scaffold proteins ITP] (Whitmarsh et al., 1998) and IIP2 (Yasuda et al., 1999), which
subsequently induce INK activation. Stable expression of SPRK in NIH 3T3 cells
induces transformation and colony formation in soft agar (Hartkamp et al., 1999).
Interestingly, SPRK-expressing NIH 3T3 fibroblasts show increased MEK]
phophorylation, which is not accompanied by ERK activation. In contrast c-Iun is
highly phosphorylated in these cells, supporting the idea that SPRK upon
overexpression constitutively activates the INK pathway. This is also consistent
with our observation that Rat] fibroblasts, which stably express SPRK, have a 30-
40-fold increased INK activity compared to a vector control. Upon overexpression

of SPRK in HEK 293 cells an increase in ERK activation is detected (Hartkamp et

43

al., 1999), but whether there is a physiological role for MEK] and/or ERK activation
in NIH 3T3 cells is open for further investigations.

Although SPRK-induced INK activation has been demonstrated in a variety of
studies the upstream activators of SPRK still remain unclear. Since this protein
kinase contains several potential domains mediating protein-protein interaction, a
broad range of activators my exist. The small GTPase Cdc42 has been shown to
bind a CRIB-containing fi'agment of SPRK in a filter-binding assay (Burbelo et al.,
1995). Both, Cdc42 and SPRK have been identified as activators of the INK
pathway (Bagrodia et al., 1995a; Minden et al., 1995; Tibbles et al., 1996).
However, the effect of Cdc42 on SPRK's kinase activity still remains unclear. This
thesis work is mainly concerned with the structural requirements and molecular
mechanism of Cdc42-induced activation of SPRK. Understanding the mechanism by
which the small GTPase Cdc42 regulates and activates the protein kinase SPRK may
provide valuable infornntion about Cdc42-SPRK-induced INK activation. It may
provide more insight into the specific physiological role of SPRK and perhaps help
to identify the extracellular stimulus for SPRK activation. In addition, understanding
the molecular mechanism of SPRK regulation by Cdc42 may help to understand the

regulation of protein kinases by small GTPases in general.

 

 

Fig 6. I)
leucine ixIi]

high?!) and l

 

 

Fig. 6. Domain arrangement of SPRK. SPRK consists of an NHz-termina]

glycine-rich region, followed by an SH3 domain, a kinase domain and a
leucine/isoleucine zipper region. Adjacent to the CRIB motif SPRK contains a basic
region and a COOH-terminal serine/threonine/proline rich region.

45

 

[I]. C do]

viva: Re

 

 

III. Cdc42-Induced Activation of the Mixed-Lineage Kinase SPRK in
viva: Requirement of the Cdc42/Rae Interactive Binding Motif and

Changes in Phosphorylation

1. Abstract

SPRK/MLK-3 is a serine/threonine kinase tint upon overexpression in
mammalian cells activates the INK pathway. The mechanisms by which SPRK
activity is regulated are not well understood. The small Rho-family GTPases, Rae
and Cdc42, have been shown to bind and to modulate the activities of signaling
proteins, including SPRK, which contain Cdc42/Rae interactive binding motifs.
Coexpression of SPRK and activated Cdc42 increases SPRK’s activity. SPRK’s
CRIB-like motif contains six of the eight consensus residues. Using a site-directed
mutagenesis approach, we show that SPRK contains a fimctional CRIB motif that is
required for SPRK’s association with and activation by Cdc42. However,
experiments using a SPRK variant that lacks the COOH-terminal zipper region/basic
stretch suggest that this region may also contribute to Cdc42 binding. Unlike the
PAK family of protein kinases, we find that the activation of SPRK by Cdc42 cannot
be recapitulated in an in vitra system using purified, recombinant proteins.
Comparative phosphopeptide mapping demonstrates that coexpression of activated
Cdc42 with SPRK alters the in viva phosphorylation pattern of SPRK suggesting that

the mechanism by which Cdc42 increases SPRK’s catalytic activity involves a change

46

 

in {he in r
first dcrr
Pikrsp'mr}

mm

1 lntrod

 

 

in the in viva phosphorylation of SPRK. This is, to the best of our knowledge, the
first demonstrated example of a Cdc42-mediated change in the in viva
phosphorylation of a protein kinase. These studies suggest the requirement for an

additional component or the cellular environment for SPRK activation by Cdc42.

2. Introduction

The serine/threonine kinase SPRK is a member of the mixed-lineage kinase
Emily. SPRK contains various domains mediating protein-protein interaction,
including an NHz-terminal SH3 domain, a leucine/isoleucine zipper motif, a CRIB
motif, and a large COOH-terminal region that is rich in serine, threonine, and proline
residues (Fig. 7). The CRIB motif is a short amino acid sequence consisting of 14-16
amino acid residues, and has been identified as the minimal binding sequence for small
GTPases of the Rho-Emily (Burbelo et al., 1995). SPRK shares six of the eight
conserved amino acid residues within this motif. In filter binding assays it has been
demonstrated that SPRK associates with Rae and Cdc42, with a strong preference for
Cdc42 (Burbelo et al., 1995).

Cdc42 belongs to the Rho Emily of small GTPases. This protein Emily,
which includes Rho, Rae, and Cdc42, plays crucial roles in diverse cellular processes
(Hall, 1998; Van Aelst and D'Souza-Schorey, 1997), including cytoskeletal
rearrangements (Kozrna er al., 1995; Nobes and Hall, 1995; Ridley and Hall, 1992),

cell cycle progression (Olson et al., 1995), cellular transformation (Khosravi-Far et

47

 

01.. 1995; '
Tang e! u.’
(Bagrodia

Minden er .

his ac:

 

 

 

al., 1995; Qiu et al., 1997; Qiu et al., 1995a; Qiu et al., 1995b; Tang et al., 1997;
Tang et al., 1999; Whitehead et al., 1998; Wu et al., 1998), and nuclear signaling
(Bagrodia et al., 1995a; Brown et al., 1996; C050 et al., 1995; Hill et al., 1995;
Minden et al., 1995; Zhang et al., 1995). These proteins can also function as protein
kinase activators. One well-characterized target of Cdc42 and Rac is the
serine/tineonine kinase PAK (Bagrodia et al., 1995b; Manser et al., 1994; Martin et
al., 1995). The interaction between Cdc42 and the serine/threonine kinase PAK
requires the CRIB motif (Burbelo et al., 1995). The structural determinants required
for GTPase binding and the mechanism of activation of multiple PAK isoforms have
been extensively investigated (Gatti et al., 1999; Knaus et al., 1998; Manser et al.,
1997; Manser et al., 1994; Zenke et al., 1999; Zhao et al., 1998). CRIB-dependent
interaction of PAK with GTP-bound Rac/Cdc42 induces PAK autophosphorylation
and activation both in vitro and in viva.

Diverse proteins, including the tyrosine kinase, ACK (Manser et al., 1993),
and the non kinase, WASP (Aspenstrom et al., 1996; Kolluri et al., 1996), also
contain CRIB motifs, suggesting that mechanistically diverse regulatory pathways
may share this common structural motif. Likewise, not all protein kinases which
interactwitthc42andRacdo sothroughaCRIB motif. For instance, boththe 70
kDa ribosomal S6 kinase (Chou and Blenis, 1996) and MEKK] (Fanger et al., 1997),
which lack CRIB motifs, have been shown to interact with GTP-bound Cdc42 and

Rac. Furthermore, MEKK4 contains a modified CRIB motif whose deletion only

48

 

 

lik

10

Cd
Lin
Poi
inc;
C0:-
0P.

SP]

partially diminishes binding to Cdc42 and Rac, indicative of a CRIB-independent
GTPase binding determinant (Fanger eta1., 1997). Thus the role of CRIB motifs and
the mechanisms by which many protein kinases are activated by small GTPases remain
largely unexplored.

Whether activation by Cdc42 of the distantly related PAK and SPRK involves
mechanistically analogous processes is unknown. SPRK is Enctionally homologous
to Raf, and thus mechanistic aspects of Cdc42-SPRK and Ras-Raf activation may be
shared. Here we show that SPRK contains a Enctiona] CRIB motif that is absolutely
required for Cdc42 binding to and activation of SPRK. The zipper region and
adjacent basic sequences of SPRK may also contribute to Cdc42 binding. Binding of
Cdc42 to SPRK does not require SPRK catalytic activity. Interestingly, GTP-bound
Cdc42 has no effect on the activity of purified, catalytically active SPRK, suggesting
that an additional cellular component is required for kinase activation. These studies
point to an important distinction between PAK and SPRK in the mode of GTPase-
induced kinase activation. Comparative phosphopeptide mapping revealed that
coexpression of activated Cdc42 with SPRK alters the in viva phosphorylation sites
on SPRK. This change in serine/threonine phosphorylation correlates with increased
SPRK activity. These studies represent the first case where a Cdc42-mediated change
in the in viva phosphorylation sites of a protein kinase has been documented and

provide evidence for the involvement of in viva phosphorylation of SPRK for Cdc42-

49

 

induced a;

from the rr

3. Mater

3.] Constr

 

mutagEn.

 

induced activation. Thus, the Cdc42-mediated activation of SPRK is clearly distinct

fi‘om the mechanism previously described for Cdc42-induced activation of PAK.

3. Materials and Methods

3.1 Construction of mammalian expression vectors and mutagenesis

Construction of the cytomegalovirus-based expression vectors carrying the
cDNAs for wild type SPRK (pRKS-sprk), and for the kinase-defective variant of
SPRK (pRKS-sprkm‘“) has been described elsewhere (Gallo et al., 1994).
Expression plasmids encoding NIB-terminal Flag epitope-tagged wild type Cdc42
(pRKS-Nflag.cdc42) and the constitutively active variant (pRKS-Nflag.cdc42v‘2) of
Cdc42 were kindly provided by Avi Ashkenazi (Genentech, Inc., So. San Francisco,
CA).

Variants of SPRK containing point mutations in the CRIB motif were
constructed by a modified recombinant polymerase chain reaction (PCR) method
described in detail elsewhere (Higuchi, 1990). For each mutation two different PCRs
were performed, one containing a left mutagenesis primer and a left outside primer,
and one containing a right mutagenesis primer and a right outside primer. The left
and right outside primers used for all mutations were 5’-GATG-
AGTCATCTGAATCCAGG-3 ’ and 5’-CTGTGGCCTATGGCGTAGCTG-3’,
respectively. To obtain the three difierent CRIB mutants the following left and right

mutagenesis primers were used, respectively: (SPRKmsA), 5’-GGTGCTTGGCGT-

50

CGAGTGGCAT-3’ and 5’-ACTCGACGCCAAGCACCGCATC-3’; (SPRK"5°°A)'
5’-CTTCAAGGCCCGCATCACCGT-3’ and 5’-GGTGATGCGGGCCTTGAAG-
TCG-3’; (SPRKWW93A), 5’-GAAGTCGAGTGGCATGGCGGCACGCT-CG-3’
and 5’-CGAGCGTGCCGCCATGCCACTCGAC-3’. The presence of the desired
mutation was confirmed by DNA sequencing using the Sanger method, and the
absence of PCR-introduced errors was verified by automated sequencing.

Deletion of amino acids 430 through 486 of SPRK to yield SPRKAzip was
accomplished by digestion of the expression vector pRKS-sprk with BssH H, followed
by ligation with T4 DNA ligase. DNA modifying enzymes were purchased fi'om New

England Biolabs or Life Technologies, Inc..

3.2 Expression and purification of recombinant SPRK

An Nca I-Hind III fragment, containing the full length SPRK cDNA, was
subcloned fi'om pRKS-sprk into the pFastBac HTb baculovirus expression vector
(Life Technologies, Inc.) which contains a hexahistidine tag. Recombinant histidine-
tagged SPRK was expressed in Sf9 cells and purified by nickel affinity
chromatography according to the manufacturer's protocol. Fractions containing
histidine-tagged SPRK, as determined by SDS-PAGE followed by Coomassie Blue

staining, were pooled and concentrated using a Centriprep concentrator (Amicon).

51

 

3.3 Cell lines and transfections

Human fetal kidney 293 cells were maintained in Ham’s F12:low glucose
Dulbecco’s modified Eagle’s media (1:1) (Life Teclmologies, Inc.) supplemented with
8% fetal bovine serum (Life Technologies, Inc.), 2 mM glutamine, and
penicillin/streptomycin (Life Technologies, Inc.). Plasmids (5 pg each for 60 mm
dishes; 10 pg each for 100 mm dishes) were used to transfect 293 cells using the
calcium phosphate technique (Gorman, 1990). Cell monolayers were incubated with
the DNA precipitate for 4 h, then washed once with PBS (phosphate-bufi‘ered saline),

and cultured in the mdium described above. After 18 h cells were harvested.

3.4 Cell lysis and immunoprecipitation

Cells were washed with ice cold PBS and lysed for 5 min on ice by the addition
of 1 ml of lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCh, 2
mM EGTA, 1% Triton X—100, 10% glycerol, 10 mM NaF, 1 mM NaIP., 100 uM B-
glycerophosphate, 1 mM Na3VO4, 2 mM PMSF, and 0.15 U/ml aprotinin). The lysate
was clarified by centrifugation for 20 min at 14,000 rpm in an Eppendorf centrifuge at
4 °C. Rabbit polyclonal antiserum was raised against a peptide corresponding to the
COOH-terminal eight amino acids of SPRK and was purified by Protein A-Sepharose
chromatography. Antibodies against the proteins of interest were prebound to protein
A—agarose beads [SPRK antiserum (0.25 rig/u] slurry), M2 monoclonal antibody

(Kodak IBI) directed against the Flag epitope (0.45 rig/u] slurry) and INK C-17

52

antibody (Santa Cruz Biotechnology) (50 ng/ul slurry) as previously described (Gallo
er al., 1994). Clarified lysate (300 pl) was incubated with 20 u] of antibody-bound
Protein A-agarose for 90 min at 4 °C. Immunoprecipitates were washed with HNTG
buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton-X-100, 10% glycerol).
Immunoprecipitates used for kinase assays were washed three times with HNTG
buffer containing 1 M LiCl, three times with [MTG buffer, and twice with kinase
assay bufi‘er (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MnClz, 10 mM MgC12,

0.1 mM Na3VO4).

3.5 Gel electrophoresis and Western blot analysis

Lysates and immunoprecipitates of SPRK and Cdc42 were resolved by SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (Laemmli,
1970). Proteins were transferred to nitrocellulose and irnmunoblotted using either
SPRK antiserum (1 ug/ml) or M2 Flag monoclorml antibody (9 rig/ml), followed by
the appropriate horseradish peroxidase-conjugated secondary antibody (Life
Technologies, Inc.). Western blots were developed by chemiluminescence. Multiple
exposures of the Western blots were developed, and densitometry (NIH Image) of
unsaturated films was used to determine relative expression levels. Statistics were
compiled using an unpaired Student's t-test. A p—value smaller than 0.05 was

considered statistically significant.

53

3.6 In vitro kinase assays
Kimse assays were performed in 20 pl of kinase assay buffer containing 50 pM

ATP and 5 pCi [y-3ZP]-ATP (3000 Ci/mmol) (NEN Life Science Products). For the
SPRK kinase assay 10 pg of mixed histones (Boehringer Mannheim) was used as a
substrate and the reaction was carried out for 30 min at room temperature.
Independent experiments showed that the reaction was linear within this time range.
The reactions were terminated by the addition of an equal volume of 2x SDS sample
buffer (100 mM Tris (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromphenol blue, 100
mM DTT, 1% B—mercaptoethanol) containing 50 mM EDTA (pH 8.0).

For the kinase assays involving purified kinases, recombinant SPRK or PAK-2
(1 pg and 2 pg, respectively) was incubated in 50 pl of kinase assay buffer containing
4 pg of GST (glutathione S-transferase)-Cdc42 that had been preloaded with GTPyS
or GDP and assays were performed as above. GST-Cdc42 (19 pM) was preloaded
with GTPyS or GDP (4 mM) in buffer containing SOmM Tris (pH 7.5), 5 mM EDTA,
and 1 mM DTT. The mixture was incubated for 15 min at 30 °C. The nucleotide
loading reaction was quenched by the addition of 10 mM MgC12. Purified GST-Cdc42
was obtained from an E. cali overexpression system; and purified PAK-2, obtained
using the baculovirus expression system, was kindly provided by Dr. Arie Abo (Onyx
Pharmaceuticals) (Martin et al., 1995).

For the INK assays, 8 pg of GST-c-jun was used as the substrate, and the

reaction was carried out for 15 min at room temperature. The pGEX-c-jun (1-115)

54

vector was kindly provided by Dr. Ajay Rana (Massachusetts General Hospital,
Harvard Medical School, Boston, MA). GST-c-jun was expressed in XL-l Blue E.
cali and purified by glutathione Sepharose chromatography. Following the kinase
assay, proteins were separated by SDS-PAGE. Gels were rinsed in PBS, dried, and
incorporation of radioactivity into kinase, or substrates was determined by
Phosphorlmaging (Molecular Dynamics). To detect INK expression, proteins were
transferred fi'om an SDS polyacrylamide gel to a polyvinylidene difluoride (PVDF)

membrane and irnmunoblotted using the INK C-17 antibody (0.5 pg/ml).

3.7 In viva phosphopeptide mapping

After a 24 h transfection with pRKS-sprk in the presence or absence of pRKS-
Nflag.cdc42m, 293 cells were washed five times with phosphate-flee medium
(Dulbecco’s modified Eagle’s medium supplemented with 10% dialyzed fetal bovine
serum (Summit Biotechnology», and incubated at 37 °C for 2 h. The cells were then
incubated in phosphate-free medium containing 3 mCi/ml [”P] orthophosphate
(carrier free; NEN Life Science Products) for 4 h at 37 °C.

Cells were washed five times with ice cold PBS and then lysed in 1 ml of lysis
buffer. Lysates were clarified by centrifugation for 15 min at 14,000 rpm in an
Eppendorf centrifuge at 4 °C. SPRK was immunoprecipitated with SPRK antiserum
as described above. Immunoprecipitated proteins were resolved by SDS-PAGE and

transferred to a PVDF membrane. Radiolabeled bands that co-migrated with SPRK,

55

as judged by Western blotting, were excised fi'orn the PVDF membrane. After
washing three times with methanol and three times with water, the radioactive piece
of membrane was blocked with 1 ml of 0.5% polyvinylpyrrolidine-36O (Sigma)
containing 100 mM acetic acid for 30 min at 37 °C, and then washed five times with
water. Tryptic digestion was performed with 10 pg of sequencing grade trypsin
(Boehringer Mannheim) for 2 h in 200 pl of 50 mM NH4HC03, pH 8.3, at 37 °C. An
additional 10 pg of trypsin was added and the digestion mixture was incubated for an
additional 2 h at 37 °C. The membrane was then sonicated for 3 min in 300 pl of
water to remove additional tryptic peptides. The solution containing the released
tryptic peptides was concentrated in a SpeedVac (Savant Instruments).

The peptides were separated on cellulose thin layer chromatography (TLC)
plates (Kodak, 20 x 20 cm) by thin layer electrophoresis (TLE) in the first dimension
in pH 1.9 bufier (formic acid (88% w/v)/glacial acetic acid/water, 25:78:897, v/v/v) at
0°Cand1000Vfor30min,andseparatedintheseconddimensionbyTLCin
phosphochromatography buffer (n-butanol/pyridine/ glacial acetic acid/water,
15:10:3zl2, v/v/v/v). The radiolabeled phosphopeptides were visualized and

quantitated using a phosphorimager.

56

4. Results

4.1 Association of activated Cdc42 with SPRK does not require SPRK kinase

activity

Recently Hall and coworkers have shown that several proteins, including SPRK,
associate with GTP-bound Cdc42 and Rac in filter binding assays (Burbelo et al.,
1995). All of these proteins contain a 14-16 amino acid sequence that includes eight
consensus residues, which has been coined the CRIB motif. The CRIB motif of
SPRK contains six of the eight consensus amino acids (Fig. 8). In this study we
examined the structural requirements and mechanism of Cdc42 binding and activation
of SPRK.

SPRK and Flag epitope-tagged Cdc42 expression vectors were transiently
transfected in 293 cells. To mimic the GTP-bound state of Cdc42 we used a
constitutively active mutant of the GTPase, i.e.Cdc42V'2. Cellular lysates from 293
cells were immunoprecipitated with the Flag antibody and the presence of associated
SPRK was assessed by Western blot analysis with a SPRK antibody. In these co-
immunoprecipitation experiments (Fig. 9), the constitutively active form of Cdc42,
but not wild type Cdc42, associates with SPRK (first panel). We have not observed
association of SPRK with the dominant negative variant Cdc42N”, but, in our hands.
expression of this Cdc42 variant has been low. Expression levels of SPRK and Cdc42

in celluLar lysates were determined by Western blot analysis (Fig. 9, lower pane18).

57

These data indicate that SPRK preferentially associates with the GTP-bound form of
Cdc42.

SPRK can autophosphorylate in vitra (Gallo et al., 1994). Although SPRK’s
postulated site of interaction with Cdc42 is not within the catalytic domain, it is
plausible that either SPRK autophosphorylation or SPRK phosphorylation of another
interacting molecule might modulate the Cdc42-SPRK interaction. To examine
whether SPRK’s catalytic activity effects its ability to bind to Cdc42, the kinase-
defective SPRK variant (SPRKK'W‘) was tested for its ability to associate with
Cdc42m. The SPRKKIMA variant shows no autophosphorylation in an in vitra kinase
assay (Gallo et al., 1994).

Cotransfection and co-irmnunoprecipitation experiments with active and
inactive SPRK, and Cdc42m, demonstrate that constitutively active Cdc42 associates
equally well with wild type SPRK and the kinase—defective variant SPRKW" (Fig.
10, upper panel). This indicates that SPRK’s catalytic activity is not required for its
association with Cdc42. The retarded electrophoretic mobility of wild type SPRK, as
compared with that of inactive SPRK, suggests a more highly phosphorylated form of
SPRK that is likely due, at least in part, to autophosphorylation (Fig. 10, upper and
middle panels).

To determine whether association with Cdc42 effects SPRK’s catalytic
activity, an in vitra kinase assay for SPRK was developed. While its kinase domain

shares overall sequence similarity with both serine/threonine and tyrosine kinases,

58

SPRK shows high amino acid sequence identity with the serine/threonine kinase B-
Raf in a small region of subdomain VIb and VIII that is important for substrate
specificity and catalytic activity (Hanks et al., 1988). In addition, both Rafand SPRK
appear to function as MKKKs leading prirmrily to the activation of ERK and INK,
respectively. Since histones are commonly employed as exogenous substrates for Raf
in in vitra kinase assays (Kanakura et al., 1991; Tarnaki et al., 1992), a mixture of
histones was tested as an exogenous substrate for SPRK. SPRK, immunOprecipitated
from cellular lysates of transiently transfected 293 cells, exhibits basal
autophosphorylation as well as phosphorylation of the histones H3 and H4 (Fig. 11).
In contrast, the kinase-defective SPRK shows no autophosphorylation or histone
phosphorylation, indicating that the phosphorylation events observed in these assays
are attributable to SPRK, rather than to contaminating kinases.

W2
2

The expression of Cdc4 in transiently transfected 293 cells increases the
autophosphorylation activity as well as the substrate phosphorylation activity of
SPRK about 3- to 5-fold (Fig. 11). No background phosphorylation is observed with
the kinase-defective SPRK variant in the presence or absence of Cdc42v'2.
Interestingly, under these in vitra kinase assay conditions, we do not detect Cdc42

associated with SPRK by Western blotting.

59

4.2 SPRK’s CRIB motif is necessary for association with Cdc42 and for Cdc42

induced activation of SPRK

To test whether SPRK’s potential CRIB motif actually functions in the binding of
Cdc42 we took a site-directed mutagenesis approach. Three difierent SPRK variants
were generated by mutating conserved amino acids in the CRIB motif to alanine
residues: SPRKF‘98‘, SPRKHW, and Sl>RK"”"'S493A (Fig. 8). While we cannot
absolutely rule out the possibility that introduction of these mutations might alter
SPRK’s conformation, the expression levels of the SPRK CRIB mutants in transient
transfections of 293 cells are at least as high as that of wild type SPRK (Fig. 12),
suggesting that these variants are stable. The CRIB variants were coexpressed with
Cdc42V12 in 293 cells and the cellular lysates were subjected to co-
immunoprecipitation experiments. While wild type SPRK associates with Cdc42m,
none of the SPRK CRIB mutants detectably associates with the activated GTPase
(Fig. 12).

If Cdc42-induced activation of SPRK is mediated through its interaction with
SPRK’s CRIB motif, one would expect that the SPRK CRIB mutants should exhibit a
defect in Cdc42-induced activation. Accordingly, cells were transiently transfected
with cDNAs encoding wild type SPRK or SPRKWZA‘8493A, in the presence or absence
of Cdc42m. The activity of the immunoprecipitated SPRK or SPRK CRIB mutant
was measured in an in vitra kinase assay. In the absence of Cdc42m, both wild type

SPRK and the SPRK CRIB variant show similar levels of autophosphorylation and

60

substrate phosphorylation (Fig. 13, 14a and b). In the absence of Cdc42V”, the
differences in the activities of SPRK and the SPRK CRIB variants were not
statistically significant. This further supports the idea that the mutations in the CRIB
motif do not grossly perturb SPRK’s structure or inherent catalytic activity.
However, both autophosphorylation and substrate phosphorylation of the CRIB
variant is markedly lower (3-fold) than that of wild type SPRK, when each is co-
expressed with the activated GTPase (Fig. 13, 14a and b). The small increase in the
catalytic activity of the SPRK CRIB mutant when activated Cdc42 is coexpressed
my be due to Cdc42-activated endogenous SPRK. Alternatively there may be
residual binding of the CRIB variant to activated Cdc42 in viva, which we do not
detect in our in vitro co-immunoprecipitation assay. Taken together these results
demonstrate that SPRK does contain a functional CRIB motif, and that Cdc42-

induced activation of SPRK is mediated via association with this CRIB motif.

4.3 Effects of deleting the COOH-terminal portion of SPRK’s zipper domain

In WASP (Rudolph et al., 1998), PAK (Thompson et al., 1998; Zhao et al.,
1998) and ACK (Manser et al., 1993) the CRIB motif is necessary but not suflicient
for GTPase binding. Outside of the CRIB motif SPRK shares no sequence similarity
with the minimal GTPase binding domains that have been defined for these proteins.
Instead, SPRK contains two closely spaced leucine/isoleucine zipper motifs spanning

amino acids 400-462, NHz-terminal to the CRIB motif (Fig. 7). Considering the close

61

vicinity in linear sequence of the zippers and the CRIB motif we asked whether the
zipper motif might contribute to the binding of SPRK to Cdc42m. Accordingly, a
variant of SPRK, SPRKm’, which lacks amino acids 430-486, as shown in Fig. 7, was
constructed. This deletion removes the second half of the zipper region as well as 22
COOH-terminal amino acids, which include a short basic region, but leaves the entire
CRIB motif intact.

SPRK“ip is expressed in transiently transfected 293 cells at levels comparable
to that of wild type SPRK (Fig. 15). The ability of Cdc42v'2 to associate with
SPRKM" was tested in co-imrnunoprecipitation experiments with cellular lysates
harvested from transiently transfected 293 cells. The deletion of the second
zipper/basic stretch greatly diminishes the ability of SPRK to bind to Cdc42v'2,
despite the presence of the complete CRIB motif (Fig. 15). Based on these and our
previous results, both the CRIB motif, the second half of the zipper region and a
stretch of basic amino acids may contribute to Cdc42 binding. Alternatively, the
COOH-terminal zipper/basic stretch may not directly interact with Cdc42, but may be
required for the proper presentation and binding of the CRIB motif to the GTPase.

SPRK“? has approximately 70% ofthe basal autophosphorylation activity of
wild type SPRK (Fig. 16, 17a). However, in contrast to wild type SPRK, there is no
Cdc42-induced increase in autophosphorylation of SPRK”, consistent with the
finding that SPRKM" binds Cdc42VIZ only very weakly. In an in vitra kinase assay,

SPRK” expressed with or without mm"12 (Fig. 16, 17b) kicks the ability to

62

phosphorylate histones. To address whether the lack of histone phosphorylation
might be due to some unique feature of histones we performed the same experiment
with myelin basic protein as a substrate and obtained analogous results (data not
shown). These data suggest that the zipper domain/basic stretch may be

fimdamentally required for substrate pho sphorylation.

4.4 SPRKA" fails to activate JNK

SPRK has been identified as an upstream activator of the INK/SAPK pathway
(Rana et al., 1996; Teramoto et al., 1996; Tibbles et al., 1996). INK activity was
measured after immunoprecipitation of endogenous INK from cellular lysates of
transiently transfected 293 cells in an immune complex in vitra kinase assay using
GST-c—jun as the substrate. Overexpression of SPRK in 293 cells leads to a 5-fold
increase in INK activity over the basal activity in vector control-transfected cells (Fig.
18, 19). Transient expression of Cdc42V12 increases INK activity some 2- to 3-fold.
Although coexpression of SPRK and Cdc42VIZ increases SPRK catalytic activity, as
measured in an in vitra kinase assay, the observed SPRK-induced INK activation with
co-expressed Cdc42V12 over that of SPRK alone does not reach statistical
significance. A likely explanation for this finding is that the activity of overexpressed
SPRK alone is sufficient to maximally activate the endogenous INK in 293 cells.
SPRKAZ" is unable to phosphorylate an exogenous substrate in an in vitra kinase

assay, and, indeed, we have found tint it completely lacks the ability to activate the

63

INK pathway (Fig. 18, 19). Thus, it appears that the zipper motif is critical for

substrate phosphorylation by SPRK both in vitra and in viva.

4.5 Activated Cdc42 fails to activate SPRK in vilra

The small GTPases Rae and Cdc42 can stimulate the autophosphorylation activity
of the CRIB-containing serine/threonine kinase PAK2 in vitra (Martin et al., 1995).
To determine whether Cdc42-induced activation of SPRK can be recapitulated in a
completely in vitra system', hexahistidine NHz-terminal tagged SPRK was expressed
using the baculovirus system, and purified by metal-chelate chronntography’. GST-
Cdc42 was expressed in and purified fiom E. coli’. The purified SPRK is catalytically
active as judged by its basal autophosphorylation activity. GTPyS- or GDP-loaded
GST-Cdc42 was incubated with purified SPRK or PAK2 in an in vitra kinase assay
(Fig. 20b). While GTP-yS-loaded Cdc42 activates purified PAK2, it Eils to activate
purifed SPRK. Likewise, SPRK immunoprecipitated fi'om transfected 293 cells
carmot be activated in vitra by the addition of GTPyS-loaded Cdc42 (Fig. 20a).
These data support the requirement of a cellular context or coactivator for SPRK

activation by Cdc42.

4.6 Cdc42 alters the in viva phosphorylation pattern of SPRK
As described above, co-immunoprecipitation experiments and in vitra kinase

assays show that Cdc42“2 when coexpressed with SPRK can associate with SPRK

and modulate its catalytic activity. However, purified, activated Cdc42 cannot
stimulate the autophosphorylation of SPRK in vitra. In order to determine if the
presence of activated Cdc42 alters SPRK phosphorylation in viva, two-dimensional
phosphopeptide analysis of SPRK labeled in viva, in the absence and in the presence
of Cdc42m, was performed“.

The net incorporation of radiolabel into SPRK increased approximately three-
fold when SPRK was coexpressed with Cdc42m. Two-dimensional TLE/TLC
revealed that while the basic pattern of phosphopeptides from the two samples is
similar (Fig. 21a), there are notable differences. The major changes are observed in
the triangular cluster of phosphopeptides b, c, and d. Phosphopeptide a predominates
in both samples. Phosphopeptides b and c are detected in the triangular cluster of the
SPRK map, but are low in abundance relative to peptide 0. In the corresponding map
of SPRK that had been expressed in the presence of Cdc42m, however,
phosphopeptide b is not detected. Instead, phosphopeptide c is the prominent
phosphopeptide in the triangular cluster, with 70% of the radioactivity of
phosphopeptide a. (Fig. 21b). Furthermore, phosphopeptide (I, nearly undetectable in
the map of SPRK, appears at high levels in the map of SPRK expressed with
Cdc42m. For comparison, the level of another peptide (1:) relative to peptide 0 is
essentially constant in both maps. The labeling and mapping fiom three independent

experiments yielded the same results. These data indicate that the presence of

65

activated Cdc42 changes the in viva phosphorylation state of SPRK, which correlates

with an increase in SPRK catalytic activity.

66

Fig. 7. Schematic of SPRK. The numbers in the above diagram represent amino
acid number. The glycine-rich region (amino acids 1-42) is denoted by Gly. The
region containing the zipper motif and the polybasic stretch of amino acids includes
amino acids 400-486. The amino acid sequence corresponding to this region is
shown below with the basic stretch of amino acids printed in bold letters and the non-
aromatic hydrophobic residues predicted to occupy the d position in the zipper motifs
underlined. The sequence deleted in SPRKAzip (amino acids 430-486) is boxed.

67

 

 

5% r "trill 10444.waEJWSOSJImmggmmagammmmmi—mxEJBEJGO—mz;

 

 

 

 

 

 

 

 

 

 

 

 

0:-.. no 0.. . 23m >
a
ham «8 8m «aims So an .3 m2 9 F

 

68

 

 

Consensus stpxxxxrxnxxava
MLK2 m-rsxr. .xxr'xrixxrivca-487
WASP 233-IG'XP. .xxri'xnxxavcr-z51
ACK 510-:rsxraxxxxrl'xuxxuea-527
SPRK 492-stpxxxxrxnxx1vQ—sos
SPRKWW ALI l

SPRKWA a

SPRKHESOOA A

 

 

 

Fig. 8. Alignment of CRIB motifs and SPRK variants. The consensus sequence
for the CRIB motif as defined by Burbelo et a1. is shown aligned with the CRIB motif
of MLK2, WASP, ACK, and SPRK. Amino acid numbers are indicated to the left
and the right of each sequence. Amino acids occupying the consensus residues are
indicated by bold, with those that differ from the consensus italicized. Absent amino
acids are indicated by period The mutations in the engineered CRIB variants of
SPRK are shown with the conserved residue to alanine changes indicated by bold
letters.

69

SPRK: - - .. + + +
Cdc42: - V12 wr - v12 wr lmmunoblot

Flag IP . SPRK
I .5 .‘ SPRK

Flag-Cdc42

Fig. 9. Co-immunoprecipitation of SPRK with wild type (WT) Cdc42 or
Cdc42‘m. 293 cells were transfected with expression vectors containing the cDNA
indicated above. A minus sign indicates that a control empty vector was transfected.
Cdc42 was immunoprecipitated (IP) using the M2 antibody directed against the Flag
epitope appended to the NHz-terminus of Cdc42 and Cdc42m. The presence of
SPRK was determined by irnmunoblotting with a SPRK antibody (upper panel). The
middle and lower panels show SPRK and Cdc42 expression, respectively.

70

SPRK: WT K144A WTK144A

Flag IP - - SPRK
. ~~ SPRK
w Flag-Cdc42

Fig. 10. Co—immunoprecipitation of SPRKKI‘“ with activated Cdc42v". 293
cells were transfected with expression vectors containing the cDNA indicated above.
The upper panel shows the immunoprecipitation of wild type SPRK or SPRKK‘W‘
with activated Cdc42m. The presence of SPRK was determined using a SPRK
antibody. The middle and lower panel show SPRK and Cdc42 expression,

respectively.

71

SPRK: _ WT K144A WT K144A

 

Cdc42m= - .. - + + autoradiogram
I <— SPRK
" ‘— histonee
immunoblot
- ~~ SPRK
v Flag-Cdc42

Fig. 1]. In vitra kinase assay of SPRK and SPRK'm“ coexpressed with
Cdc42m. SPRK was immunoprecipitated from cellular lysates and subjected to an in
vitra kinase assay using histories as a substrate. The top panel shows an
autoradiogram with bands corresponding to SPRK autophophorylation and histone
phophorylation, indicated by arrows. The middle and lower panels show SPRK and
Cdc42 expression, respectively.

72

SPRK= -wrF H IWTF
Cdc42‘m:-. .++

Flag IF it ‘ '

' ”a i I‘“. SPRK

H I
+

immunoblot

SPRK

 

 

Flag-Cdc42

Fig. 12. Effects of mutations in the CRIB motif an association of SPRK with
Cdc42v". Co-irnmunoprecipitation experiments of SPRK or SPRK CRIB variants
with Cdc42m. The presence of bound SPRK or SPRK CRIB variant was assessed
by immunoblotting with a SPRK antibody as described previously (upper panel). The
middle and lower panels show SPRK and Cdc42 expression, respectively. WT
indicates wild type SPRK, F indicates SPRKFW, H indicates SPRKHSW, and I
indicates SPRKMQA'S493A.

73

SPRK: - wr |492A- - wr mm-
S493A S493A
Cdc42m: . _ _ + + + autoradiogram

4— SPRK

 

‘ ' ‘_ histories

Q ... -
lmmunoblot
it. than... In. spar

** w Flag-Cdc42

Fig. 13. Effects of mutations in the CRIB motif on SPRK in vitra kinase
activity. In vitro kinase assay of SPRK and SPRKWA'W“. The autoradiogram
(upper panel) shows SPRK autophosphorylation and histone phosphorylation
indicated by the arrows. The middle and lower panels show SPRK and Cdc42
expression, respectively.

74

auophoephoryletlen

C d N 0) ‘ U! a N
r a a r a l 4

Fold lncreaee In

 

h I
F

 

0 -e N U 1‘ (1| a N
zu_ A L_ # 4 4L A 1

Fold lncreeee In
hletone phoephoryleflon

Fig. 14. Quantitation of in vitro kinase assays of SPRK or SPRKmu‘w“. a,
SPRK autophosphorylation and b, histone phosphorylation which were
determined by in vitra kinase assays were quantified by PhosphorImaging and
normalized to relative SPRK expression levels. The mean :t SE. of three

independent experiments are shown.

75

SPRK: _ WT Azlp WT Azlp
Cdc42‘m: + 4-

Flag lP .

eaSPRK

— -
- .9er

t - Flag-Cdc42

immunoblot

   

Fig. 15. Effects of partial deletion of the zipper/basic stretch an association with
Cdc42v". Co-immunoprecipitation experiment of SPRK and SPRKM" with
Cdc42v'2. The presence of bound SPRK or SPRKM" was assessed by
irnmunoblotting with a SPRK antibody as described previously (top panel). The
middle and lower panels show SPRK expression, respectively.

76

SPRK: _ wr Azlp _ wr Azip

Cdc42”: - - - + + "’ autoradiogram

WP ‘. . “ELK ‘ <— SPRK
. . * hietonee
'4'" u ‘
immunoblot
~ ~ SPRK
m ' Flag-Cdc42

Fig. 16. Effects of partial deletion of the zipper/basic stretch on SPRK in vitro
kinase activity. In vitra kinase assay of SPRK and SPRKM" using histones as a
substrate. The top panel shows an autoradiogram with bands corresponding to
autophosphorylated SPRK and phosphorylated histories indicated by arrows. The
lower and middle panels show SPRK and Cdc42 expression, respectively.

77

 

 

‘
J

(.0

 

hleeene phoephoryleuon

Fold Inereaee In

 

Fig. 17. Quantitation of in vitro kinase assays of SPRK and SPRKMP. a,
SPRK autophosphorylation and b, histone phosphorylation which were
determined by in vitra kinase assays were quantified by PhosphorImaging and
normalized relative to SPRK expression levels. The mean i 8.13. of three

independent experiments are shown.

78

SPRK: _ wr Azip .. wr Azip

m.
66°42 - - - - + + + 'utondiognm
JNKlP V I ”(I' ’ A”:
m is”; _4—GST-e-jun

immunoblot

' “"91”". r.“‘!*"" ‘

JNKIP ”A“ + ”j. m...» 7w. pi JNK

”- ”Mm

Fig. 18. Effects of SPRKMp on JNK activity. Endogenous INK was
immunoprecipitated fi'om cellular lysates that had been transiently transfected with
cDNAs encoding the specified SPRK variants and/or Cdc42m. An in vitra kinase
assay for INK was performed using GST-c-jun as a substrate. The top panel shows
an autoradiogram with bands corresponding to phosphorylated GST-c-jun indicated
by an arrow. The second panel shows a INK irnmunoblot of the same
immunoprecipitated samples from the in vitra kinase assay. The third and bottom
panels show SPRK and Cdc42 expression, respectively.

79

Fold lnereaee In GST-e-jun

 

 

 

Fig. 19. Quantitation of JNK in vitra kinase assays. 293 cells were transiently
transfected with cDNAs encoding the specified SPRK variant and/or Cdc42. INK
activity was determined by GST-c-jun phosphorylation, which was quantified by
Phosphorlrnaging and normalized relative to SPRK expression levels. The mean t
SE. of three independent experiments are shown.

80

Cdc42: GDP GTPYS autoradiogram
‘ " a“ d—SPRK

immunoblot

”~— SPRK

Cdc42: GDP GTP-18 GDP GTPyS .
r, autoradlogram

W m d—SPRK

 

PAK 2 —> 'I‘

Fig. 20. In vitro kinase assay using purified Cdc42. a, SPRK was
immunoprecipitated fiom cellular lysates that had been transfected with cDNA
encoding SPRK and was incubated in an in vitra kinase assay in the presence of 4 pg
of GST-Cdc42 preloaded with either GDP or GTPyS. The top panel shows an
autoradiogram with bands corresponding to SPRK autophosphorylation indicated by
an arrow. The lower panel shows SPRK expression. b, purified SPRK and PAK2 (1
and 2 pg, respectively) were incubated in an in vitra kinase assay in the presence of 4
pg of GST-Cdc42 preloaded with either GDP or GTPyS. Shown is an autoradiogram
with bands corresponding to SPRK or PAK2 autophosphorylation.

81

Fig. 21. Phosphopeptide mapping of tryptic peptides derived from in viva
phosphorylated SPRK. a, two-dimensional phosphopeptide mapping of 32P-labeled
SPRK from 293 cells transfected with expression vectors for SPRK (top) or SPRK
and Cdc42V12 (bottom). SPRK was immunopurified fi'om cellular lysates, blotted
onto polyvinylidene difluoride membrane, and subjected to partial tryptic digestion.
Equal amounts of radioactivity, as determined by Cerenkov counting of the resultant
tryptic peptides , were analyzed by TLE in the first dimension and TLC in the second
dimension. The direction of electrophoresis and chromatography are indicated by
long arrows. Phosphopeptides were visualized by PhosphorImaging. The
phosphopeptides of interest are alphabetically labeled. Shown is a map representative
of three independent experiments. b, the percent radioactivity of the indicated
phosphopeptides compared with phosphopeptide a, calculated as: [(volume-
background)phosphopeptide]/[(volume-background)phosphopeptide a] x 100, using
Image Quant software (Molecular Dynamics).

82

 

 

 

 

 

 

 

' ' SPRK
x
a C b
C + d
w SPRK +
T ctle42V12
a .5; b
ehromato- . i- d
graph)! G A 4
electrophoresis _—P
b
% Radioactivity of Phosphopeptide a *
Peptides b c d x
SPRK 14 13 7 56

 

3:32:12 0 71 5o 44

 

 

 

 

 

 

 

83

5. Discussion

Small GTPases of the Ras superfamily have been shown to regulate protein
kinases. PAK has emerged as the paradigm CRIB-containing serine/threonine kinase
that is activated by GTP-bound Cdc42 and/or Rac. The PAKs play roles in diverse
processes, including apoptosis, modulation of actin cytoskeleton, gene transcription,
and cell cycle (Sells and Chernoff, 1997). SPRK is a member of the so-called mixed
lineage kinases. Except for the presence of a loosely conserved CRIB motif, SPRK
differs dramatically fiom the PAKs, both structurally and functionally. While the
mammalian PAK], 2, and 3 share 95% sequence similarity in their catalytic domains,
SPRK’s catalytic domain is just 20% similar to those of the mammalian PAKs. The
CRIB motif of the PAKs is found NHz-terrninal to the catalytic domain, whereas
SPRK’s CRIB motif is COOH-terminal to the catalytic domain. Flanking SPRK’s
catalytic domain is an NHz-terminal SH3 domain and a COOH-terminal leucine zipper
motif, both lacking in the PAKs. The only well-established function thus Er ascribed
to SPRK is as an MKKK in the activation of the INK pathway. Because the MLK3
are so different fi'om the PAK3, it is important to determine whether the structural
features of their binding to and the mechanisms of activation by Cdc42 and/or Rac
also differ fi'om that of the PAKs.

Whereas the three marmnalian PAK isoforms contain perfect consensus CRIB
motifs, as defined by Burbelo et al. (Burbelo et al., 1995), SPRK’s CRIB motif

contains only six of the eight consensus residues (Fig. 8). We show that mutations in

84

conserved residues of SPRK’s CRIB motif disrupt the ability of the Cdc42 to bind to
and activate SPRK, indicating that SPRK does indeed contain a functional CRIB
motif. WASP and ACK, two other proteins whose CRIB-dependent binding to Cdc42
has been well-established, also contain less than perfect CRIB motifs (Fig. 8), with
WASP (Aspenstrom et al., 1996; Kolluri et al., 1996), and ACK (Manser et al.,
1993) containing 7 of the 8 consensus residues, and 6 of the 8 consensus residues,
respectively. It may well be that the CRIB consensus motif is biased towards PAK,
due to the large number of PAK isoforms that have been identified.

SPRK and the closely-related MLK2 lack the second of the two conserved
histidine residues of the consensus CRIB motif (Fig. 8). We show here that mutation
of the first conserved histidine in SPRK to an alanine residue (1-1500A) disrupts
binding to Cdc42. The Ect that both SPRK (Burbelo et al., 1995) and MLK2
(Burbelo et al., 1995; Nagata et al., 1998) have been demonstrated to bind Cdc42
may indicate that the second of the two conserved histidines in the consensus CRIB
motif in other CRIB-containing proteins is not required for binding to Cdc42. Further
support for this notion is provided by the finding that the conserved Asp 38 in Cdc42
interacts primarily with the first of the two conserved histidine residues (11520) in
ACK (Matt at al., 1999). In addition, mutation of the first of the two conserved
histidine residues in N-WASP to aspartate (1-1208D) decreases the in vitra binding
aflinity for Cdc42 and Rac, as well as the activity of N-WASP in viva and in vitro

(Mild et al., 1998). The regions outside of the CRIB motifs of ACK and WASP

85

exhibit low sequence similarity, and, perhaps not surprisingly, low structural similarity
when bound to Cdc42 (Abdul-Manan et al., 1999; Mott et al., 1999). It is likely that
the GTPase binding domain of SPRK, with the exception of the CRIB motif, will
difl‘er structurally from those of both WASP and ACK.

Because of the proximity of SPRK’s zipper and CRIB motifs in linear
sequence, and because sequences flanking the CRIB motif in other proteins contribute
to Cdc42/Rae binding, we tested whether deletion of the COOH—terminal half of the
zipper motif effects Cdc42 binding. The binding of SPRK‘W (Fig. 7), which contains
an intact CRIB motif, to activated Cdc42 is reduced more than lO-fold, suggesting
that, in addition to SPRK’s CRIB motif, the zipper domain or the following short
basic region of SPRK may contribute to Cdc42 binding. Interestingly, the basal
autophosphorylation activity of SPRKM" is about 70% that ofwild type SPRK. This
may indicate some intrarnolecular autophosphorylation activity. Alternatively,
SPRK”? may homooligomerize and undergo intermolecular autophosphorylation.
Leung et al. (Leung and Lassam, 1998) recently reported a very large reduction in
GST-SPRK/MLK3 autophosphorylation activity in vitra upon deleting the entire
zipper region, but leaving the basic stretch intact.

Recent site-directed mutagenesis studies indicate that the serine/threonine
kinase PAK contains a basic stretch consisting of three contiguous lysine residues
upstream of the CRIB motif, whose charge is important for binding to Rae] and

Rac2, and whose presence is required for efficient PAK-1 activation by Rae], Rac2,

86

and Cdc42 (Knaus et al., 1998). SPRK contains four contiguous arginine residues
between the zipper domain and the CRIB motif. These basic amino acids are deleted
in the SPRK“ip variant, which exhibits greatly reduced binding to activated Cdc42.
Thus, it is plausible that the arginine tract in SPRK may contribute to Cdc42 binding
or activation of SPRK.

We show that SPRK and activated Cdc42 can be coirnrnunoprecipitated fiom
cellular lysates. However, under the conditions of our in vitra kinase assay, which
clearly show a Cdc42-induced increase in SPRK autophosphorylation and histone
phosphorylation, Cdc42 is not detected. Possibly once SPRK is activated by Cdc42,
SPRK lms a reduced affinity for the GTPase as has been demonstrated with PAK2
(Manser et al., 1994). Furthermore Panayiotis Vacratsis demonstrated that the sites of
in vitra autophosphorylation of SPRK expressed with and without Cdc42, and
isolated from nmmmalian cells, are essentially identical as judged by mapping of
tryptic phosphopeptides. The mechanism by which the highly conserved PAK Emily
members (PAKl, 2, and 3) are activated by Rac and Cdc42 has been well studied.
Increased autophosphorylation activity is observed upon incubation of purified
activated Cdc42 with purified PAK In contrast to PAK, purified, catalytically active
SPRK cannot be further activated in vitro by the addition of GTP-bound Cdc42.
These data are consistent with a catalytic model in which Cdc42 activates SPRK in
viva, but is not required to maintain SPRK in its activated state. We therefore decided

to assess whether Cdc42 induces differential phosphorylation of SPRK in viva.

87

Two-dimensional tryptic phosphopeptide mapping studies of in viva labeled
SPRK, expressed with or without constitutively active Cdc42, showed similar
phosphopeptide maps, with major differences observed in a triangular cluster of
phosphopeptides b, c, and d (Fig. 21a). In the map of SPRK alone, phosphopeptides b
and c are present, but at low levels. When activated Cdc42 is coexpressed with
SPRK, pho sphopeptide b is not detected and phosphopeptides c and d appear at high
levels. Since the change in in viva phosphorylation of SPRK with Cdc42 correlates
with increased SPRK activity, it is likely that phosphopeptides c and d contain
activating phosphorylation sites. Phosphopeptides c and d may be distinct
phosphopeptides. However, since their chromatographic mobilities are essentially
identical, it is also possible that phosphopeptides c and d result from differential
trypsin digestion. Phosphopeptides b and c lie on a diagonal which slopes toward the
anode, characteristic of peptides that are phosphoisomers. Upon phosphorylation, the
negative charge and polar nature of the added phosphate group reduces a peptide‘s
mobility in both the electrophoretic and chromatographic dimensions (Boyle et al.,
1991). Therefore, phosphopeptide c may differ from phosphopeptide b by the
addition of a phosphate group(s). This is consistent with the observation that when
activated Cdc42 is coexpressed with SPRK, phosphopeptide c emerges while
phosphopeptide b disappears.

Cdc42 may induce differential SPRK autophosphorylation in viva or,

alternatively, another SPRK-activating kinase may be responsible for the in viva

88

change in SPRK phosphorylation. Because Cdc42 is geranylgeranylated (Maltese and
Sheridan, 1990) and has been localized to cellular membranes (Backlund, 1993;
Erickson et al., 1996; Hart et al., 1990) as well as to cytoskeletal elements (Dash et
al., 1995; Hotta et al., 1996), it is possible that Cdc42 recruits SPRK to the vicinity
of an activating kinase. SPRK and the serine/threonine kinase Raf both appear to
function as MKKKs, activating the INK and ERK pathways, respectively. The idea
that Cdc42 may recruit SPRK to an activating kinase is reminiscent of the proposed
mechanism by which the small GTPase Ras activates Raf. Analogous to our findings
with Cdc42 and SPRK, the addition of purified, activated Ras to Raf in vitra is not
suficient for El] activation of Raf. However, appending a membrane targeting motif
to the COOH-terminus of Raf causes Raf translocation and activation in the absence
of activated Ras (Stokoe et al., 1994).

It has been recently shown that PAK3 phosphorylates Raf in viva and in vitra
leading to an increase in Raf activity (King et al., 1998), although it has yet to be
determined whether this event requires Raf translocation In yeast, the PAK homolog
STE20 functions as an MKKK in the activation of a yeast MAPK pathway. By
analogy, potential SPRK-activating kinases may include PAK-related kinases (Sells
and Chernoff, 1997) such as HPK-l. In coexpression studies HPK-l binds to SPRK's
SH3 domain and phosphorylates SPRK, but the effect on SPRK activity is not
reported (Kiefer et al., 1996). Interestingly, unlike PAK-1, -2, and -3, HPK-l lacks a

CRIB motif. Our finding that catalytically active SPRK cannot be further activated in

89

vitra by GTP-bound Cdc42 suggests that the GTPase activates SPRK differently than
the PAKs. Whereas the PAKs can be activated in vitra by interaction with
unprenylated GTP-bound Cdc42, the activation of SPRK by Cdc42 appears to require
prenylation of Cdc42, the cellular environment, or an as yet unidentified cellular
component. Our data support a model in which the in viva CRIB—dependent
interaction of SPRK and Cdc42 either allows SPRK to adopt a conformation that
leads to autophosphorylation or recruits SPRK to the vicinity of a serine/threonine
kinase that phosphorylates and activates SPRK Determination of the precise sites of
Cdc42-induced phosphorylation of SPRK, coupled with subcellular localization
studies, should shed finther light on the detailed mechanism of SPRK activation by

Cdc42.

 

' The in vitro kinase assay was performed by Panayiotis Vacratsis

2 Expression and purification of SPRK was done by Brian Qamirani

3 Expression and purification of GST-Cdc42 was performed by Hua Zhang

‘ Two-dimensional in viva phosphopeptide mapping was performed by Panayiotis Vacratsis

90

IV. Cdc42 Changes the Subcellular Distribution of the

Mixed-Lineage Kinase SPRK

1. Abstract

SPRK/MLK3 is a serine/threonine kinase that upon overexpression activates the
INK pathway. The small GTPase Cdc42 has been shown to associate with SPRK
and to increase its catalytic activity. Coexpression of SPRK with activated Cdc42
changes the in viva phosphorylation of the protein kinase. However, the Cdc42-
induced SPRK activation cannot be recapitulated in viira using recombinant, purified
proteins. Taken together these results suggest that SPRK activation by Cdc42
requires a cellular context.

In this study the effect of Cdc42 on the subcellular distribution of SPRK was
investigated. Upon overexpression in COS-7 cells, SPRK is predominantly found at
the Golgi apparatus. Coexpression of SPRK and Cdc42 in 293 cells induces a shift
of SPRK protein to a plasma membrane-enriched fraction, suggesting tlmt Cdc42
may indeed change SPRK's subcellular distribution. The Cdc42-induced change in
SPRK's localization requires prenylation, since a Cdc42 variant, which Eils to
undergo prenylation cannot induce the observed shift. Preliminary comparative two-
dirnensional phosphopeptide mapping suggests that subcellular targeting may be

required for Cdc42-induced activation of SPRK.

91

2. Introduction

Small GTPases act as molecular switches by cycling between two
interconvertible conformational states, a GTP-bound active form and a GDP-bound
inactive form. The activity of small GTPases is regulated by the ratio of GDP/GTP-
bound states. Guanine nucleotide exchange factors (GEFs) promote the exchange of
GDP and GTP, converting the GTPase to its activated state. GTPase activating
proteins (GAPS) enhance GTP hydrolysis, thus inactivating the GTPase (Boguski
and McCormick, 1993). GTPases regulate the activity of a variety of effector
molecules, including protein kinases.

A well-characterized target of the small Rho GTPases Rae and Cdc42 is the
protein kinase PAK. PAK was originally identified in rat brain as a serine/threonine
kinase that is activated by the small GTPases Rae and Cdc42 in a GTP-dependent
manner (Manser et al., 1994). The structural requirements and the mechanism of
PAK activation by these small GTPases have been vigorously investigated (Gatti et
al., 1999; Knaus et al., 1998; Manser et al., 1997; Manser et al., 1994; Zenke et al.,
1999; Zhao et al., 1998). The association of multiple PAK isoforms with Rae and
Cdc42 requires the CRIB motif of the protein kinase (Burbelo et al., 1995). The
CRIB motif is a 14-16 amino acid sequence containing eight conserved residues.
The direct interaction between PAK and Cdc42/Rae increases the
autophosphorylation activity of PAK, both in vitra and in viva.

Small GTPases can also change the subcellular distribution of protein kinases.
For example, the Ras-induced activation of the serine/threonine kinase Raf depends

on membrane targeting by activated, Ernesylated Ras. In contrast to PAK activation

92

by Cdc42, Raf activation by Ras cannot be recapitulated in vitra (Stokoe et al.,
1994). However, appending a membrane-targeting signal to Raf, which promotes
membrane association independent of Ras, induces Raf activation, suggesting that
not Ras binding per se, but rather membrane targeting, is the key-activating event
(Leevers et al., 1994; Stokoe et al., 1994).

By virtue of prenylation, small GTPases are able to associate with cellular
membranes and cytoskeletal elements (Dash et al., 1995; Erickson et al., 1996;
Hancock et al., 1991b; Hart et al., 1990; Hotta et al., 1996; Thissen et al., 1997).
Thus they have the potential to change the subcellular distribution of target proteins.
Prenylation is a posttranslational lipid modification, whereby a Ernesyl or
geranylgeranyl isoprenoid is covalently attached to the COOH-terminus of proteins.
Prenylation requires a so-called CAAX (cysteine-aliphatic-aliphatic-X) motif at the
extreme COOH-terminus of the protein. The prenyl group is attached by a thioether
linkage to the cysteine within the CAAX motif (Clarke, 1992).

Prenylation is a three-step process, which takes place in different
compartments of the cell. First the prenyl group is attached to the cysteine by
soluble prenyltransferases. Depending on the amino acid at the X position within the
CAAX motif, Ernesyltransferases or geranylgeranyltransferases catalyze this
process. The second step is the removal of the AAX residues by a prenyl-CAAX
protease. Finally the new COOH-terminus is methylated by a carboxyl
methyltransferases (Casey and Seabra, 1996). Whereas the prenyltransferases are
soluble (Casey and Seabra, 1996), the activities of the prenyl-CAAX proteases

(Hancock et al., 1991a) and the prenylcysteine carboxyl methyltransferase (Pillinger

93

et al., 1994) are associated with endomembranes. Recent evidence from studies
examining prenylation of various Ras isoforms supports the idea that the prenylated
CAAX motif serves as a target signal for association with the endOplasmic reticulum,
where the subsequent processing by proteolysis and methylation takes place (Choy et
al., 1999). The authors propose that after prenylation, the small GTPases use cellular
transport pathways to target to the plasma membrane.

The small GTPase Cdc42 is posttranslationally modified by
geranylgeranylation (Maltese and Sheridan, 1990), promoting membrane association.
Indeed, Cdc42 has been localized to cellular membranes, including the Golgi
apparatus (Erickson et al., 1996) and the plasma membrane (Hart et al., 1990), as
well as to cytoskeletal elements (Dash et al., 1995; Hotta et al., 1996).

SPRK contains a CRIB motif. The work described in this thesis and by Bock
et al., demonstrates that this CRIB motif is required for association of SPRK with
Cdc42 and for Cdc42-induced activation. Furthermore, the increase in activity is
accompanied by a change in the in viva phosphorylation of SPRK as determined by
two-dimensional phosphopeptide mapping, suggesting an activating phosphorylation
event. However, the Cdc42-induced activation of SPRK cannot be recapitulated in
vitra indicating the necessity for a cellular component in order to promote Cdc42-
induced SPRK activation. One intriguing possibility is that Cdc42 may induce a
change in localization of SPRK, for example by bringing SPRK into the vicinity of
an activating kinase. To investigate this possibility, studies were undertaken to

determine the effects of Cdc42 on SPRK subcellular localization and SPRK activity.

94

3. Materials and Methods

3.1 Construction of mammalian expression vectors and site-directed
mutagenesis

Construction of the cytomegalovirus-based vectors carrying the cDNAs for wild
type SPRK (pRK5 -sprk), for the kinase-defective variant (pRKS-sprkxm") and the
zipper mutant (pRKS-sprkw") has been described elsewhere (Beck et al., 2000;
Gallo et al., 1994). Expression plasmids encoding NHz-terminal Flag epitope-tagged
constitutively activated Cdc42 (pRK5-N-Flag.cdc42v'2) were kindly provided by
Avi Ashkermzi (Genentech, Inc.).

The Cdc42VIZ variant bearing an amino acid change of cysteine to serine in the
CAAX motif, Cdc42v'2'C'8ss was generated by site-directed mutagenesis using the
Quick Change Mutagenesis Kit (Stratagene). The mutagenesis primers used were
S’-GATGT‘TCATAGCAGCACAGATCTGCGGCTCTTCTTCG-3’ and 5’-CGAA-
GAAGAGCCGCAGATCTGTGCTGCTATGAACATC-3’. The expression vector
pRK5-Nflag.cdc42V12 was used as a template for the mutagenesis reaction. The
Cdc42 variant with a substitution of the polybasic amino acids in the COOH-
terminus to glutamines was generated by using the Quick Change Mutagenesis Kit
(Stratagene). To ensure that all four amino acids are changed to glutamine the
mutation was performed in two steps. For the first step and second step the following
mutagenesis primers were used, respectively: 5'-CCAGAACCGAAGAAGAGCC-
AGAGGTCTGTGCTGCTATGAAC-3' and 5'-GTTCATAGCAGCACAGACCT-
CTGGCTCTTCTTCGGTTCTGG-3'; 5'-GCCTCCAGAACCGCAGCAGAGCCA-

GCAGTCTGTGCTATGAACGCT-3' and 5 '-GTTCATAGCAGCACAGACTGC-

95

TGGCTCTGCTGCGGT'I‘CTGGAGGC-3'. The expression vector pRK5-

Nflag.ca’c42‘”2'c'388 was used as a template for the mutagenesis reaction.

3.2 Cell lines and transfections

Human fetal kidney 293 cells were maintained in Ham’s F 12:10w glucose
Dulbecco’s modified Eagle’s media (1:1) (Life Technologies, Inc.) supplemented
with 8% fetal bovine serum (Life Technologies, Inc.), 2 mM glutamine, and
penicillin/streptomycin (Life Technologies, Inc.). Plasmids (5 pg each for 60 mm
dishes; 10 pg each for 100 mm dishes) were used to transfect 293 cells using the
calcium phosphate technique [Goreman, 1990 #191]. Cell mono layers were
incubated with the DNA precipitate for 4 h, then rinsed once with PBS (phosphate-
buffered saline), cultured in the medium described above, and lmrvested for lysis
after 18 h.

COS-7 cells were maintained in high glucose Dulbecco’s modified Eagle’s
media (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life
Technologies, Inc.), 2 mM glutamine, and penicillin/streptomycin (Life
Technologies, Inc.). For transfection 7500 cells were plated on glass cover slips
(1.5, 22x22 mm) and transfected as described above. Cells were fixed for

microscopy 8 h post transfection.

3.3 Cell lysis, membrane fractionation, and immunoprecipitation
The crude membrane fi'actionation into 8100 and P100 fiactions was performed
as described previously (Stokoe et al., 1994). Briefly, transiently transfected 293

cells were rinsed twice with ice-cold PBS and lysed in 1 ml Buffer A (10 mM Tris

96

(pH 7.5), 35 mM NaF, 5 mM MgC12, 1 mM EGTA, 1 mM Na3VOa, 1 mM NaaPPi,
100 pM B—glycerophosphate, 2 mM PMSF and 0.15 U/ml aprotinin). After 10 min
on ice, the cells were homogenized with 30 strokes in a Dounce homogenizer.
Nuclei were pelleted by two centrifugation steps at 500 x g for 5 min at 4 °C. The
supernatant was ultracentrifuged at 100,000 x g for 1 h at 4 °C. The sedimented
P100 fi'action was rinsed twice with Buffer A and resuspended in Buffer A
containing 1% Nonidet P-40 (NP-40), resulting in the appearance of a particulate
solution. To obtain a plasma membrane-enriched fraction the experiment was
carried out as described previously with some modifications (Thissen and Casey,
1993). The cells were lysed in Buffer A, Dounce homogenized, and the nuclei
pelleted as described above. The supernatant was subjected to a centrifugation at
16,900 x g for 1 h at 4 °C to obtain a sedimented plasma membrane-emiched fraction
(Pl6.9). The P169 fi'action was rinsed twice with Buffer A and resuspended in
Buffer B (50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgC12, 2 mM EGTA,
1% Triton X-100, 10% glycerol, 10 mM NaF, 1 mM NaaPPi, 100 pM B-
glycerophosphate, 1 mM Na3VOa, 2 mM PMSF, and 0.15 U/ml aprotinin). The
supernatant ($16.9) was removed and subjected to an ultracentrifugation at 100,000
x g for 1 h at 4 °C. The sedimented fi‘action (PIOO) was rinsed twice with Buffer A
and the pellet was resuspended in Buffer B.

Rabbit polyclonal antiserum was raised against a peptide corresponding to
the C-terminal eight amino acids of SPRK and was purified by Protein A-Sepharose
chromatography. SPRK antiserum was prebound to protein A—agarose beads (0.25

pg/ p1 slurry) as previously described (Gallo et al., 1994). For immunoprecipitation

97

of SPRK 200-400 pl from a P16.9 fraction the resuspended P16.9 fiaction was
incubated with 20 pl of antibody-bound Protein A-agarose (Life Technologies, Inc.)
for 90 min at 4 °C. Immunoprecipitates were rinsed 3x with HNTG buffer (20 mM
HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton-X-100, 10% glycerol) containing 1 M
LiCl, 3x with HNTG buffer and twice with kinases buffer (50 mM Tris-HCl (pH
7.5), 100 mM NaCl, 1 mM MnClz, 10 mM MgC12, 0.1 mM Na3V04) and then
subjected to an in vitra immune complex kinase assay. Irnmunoprecipitation of
SPRK fiom total lysates, subjected to in viira kinase assays is described elsewhere
(Beck et al., 2000).

For co-irnmunoprecipitation experiments and in vitra kinase assays of SPRK
from total lysates, cells were lysed in 1 ml of Buffer B for 5 min on ice. The lysate
was clarified by centrifugation for 20 min at 14,000 rpm in an Eppendorf centrifuge
at 4 °C. The Flag epitope-tagged Cdc42 variants were immunoprecipitated fi'om the

clarified lysate (300 pl) with the M2 monoclonal antibody (Kodak IBI) directed

against the Flag epitope (0.45 pg/pl slurry).

3.4 Gel electrophoresis and Western blot analysis

Lysates and irnmunoprecipitates of SPRK and Cdc42 were resolved by SDS-
PAGE according to Laemmli (Laemmli, 1970). Proteins were transferred to
nitrocellulose and irnmunoblotted using either SPRK antiserum (1 pg/ml) or the M2
Flag monoclonal antibody (9 pg/ml), followed by the appropriate horseradish
peroxidase-conjugated secondary antibody (Life Technologies, Inc.). To determine

the distribution of SPRK and Cdc42 in the different membrane fiactions equal

98

amounts of protein were resolved by SDS-PAGE as described above. The protein
concentration of the various fractions was determined by Bradford analysis
(BioRad). Western blots were developed by chemiluminescence, and densitometry
(NIH Image) of unsaturated film was used to determine SPRK distribution in the

non-nuclear fiactions.

3.5 In vitra kinase assays and in viva phosphopeptide mapping

Kinase assays were performed as described previously (Bock et al., 2000), with
the exception that 10pg of purified GST-MKK4 was used as substrate. For non-
radioactive in vitra kinase assays the reaction was carried out in 20 pl of kinase
buffer containing 50 pM ATP and 10 pg GST-MKK4 for 30 min at room
temperature. The reaction was terminated by the addition of 2x SDS sample buffer
containing 50 mM EDTA (pH 8.0). To determine SPRK kinase activity 10 pl of the
reaction were separated by SDS-PAGE and transferred to nitrocellulose. The
Western blots were probed with an anti-phospho-MKK4 (New England Biolabs)
according to the manchturer’s protocol. To determine SPRK expression the same
blot was reprobed with SPRK polyclonal antiserum. GST-MKK4 was expressed
fiom the pGEX-vector in BL-21 E. cali and purified by glutathione Sepharose
aflinity chromatography as described previously (V acratsis and Gallo, 2000). Two-
dimensional in viva phosphopeptide mapping was performed as described previously

(Beck et al., 2000)

99

3.6 Immunofluorescence and microscopy

Transiently transfected COS-7 cells were fixed in 2% formaldehyde/PBS for 30
min at room temperature, rinsed 5x with PBS, and then permeabilized in PBS/0.2%
Triton-X-100 for 10 min at 37 °C. After three rinses with PBS, the samples were
blocked in PBS/5% BSA for 10 min at 37 °C. The cells were incubated with SPRK
antiserum (1:500) in PBS/0.5%BSA for l h at 37 °C. After three rinses cells were
incubated with SPRK antiserum (1:500) or the M2 monoclonal antibody (1:20) in
PBS/0.5%BSA for 1 h at 37 °C. Prior to incubation with the secondary antibody
cells were rinsed 3x with PBS. For single protein staining, the cells were incubated
with Alexa 488 goat anti-rabbit IgG (1:250) (Molecular Probes) or with the
FluoroLinltTM Cy3TM-labelled goat anti-mouse IgG (1:1000) (Amersham); for double
staining a dilution of 1:500 was used of each antibody. All incubations at 37 °C
were carried out in a humidified atmosphere. For Golgi apparatus staining, cells
were fixed as described above and incubated with 5 pM BODIPY TR ceramide
(Molecular Probes) in high glucose DMEM supplemented with 0.34 mg/ml BSA for
30 min at 4 °C. Cells were rinsed 5x with PBS containing 3.4 mg/ml BSA. For
double staining of SPRK and the Golgi apparatus cells were incubated in 1:750
Alexa 488 goat anti-rabbit IgG. After incubation with the secondary antibody and 5
rinses with PBS cells were incubated with 5 pM BODIPY TR ceramide in high
glucose DMEM supplemented with 0.34 mg/ml BSA for 30 min at 4 °C. Following
the incubation with ceramide the cells were rinsed 5x with PBS containing 3.4
mg/ml BSA. To examine cells stained with one fluorophore a Zeiss 210 confocal

laser scanning microscope was used. For the analysis of F luoroLinkTM Cy3m-

100

labelled and ceramide stained cells a helium neon laser with an excitation at 543 nm
was used. Staining with Alexa 488 was examined with an argon laser at 488 nm.
Doubly stained cells were examined with a Meridian INSIGHT microscope
(Genomic Solution, Lansing Division) at 488 nm and 514 nm. Images included in

this thesis are presented in color.

3.7 Protein markers

To analyze the subcellular fi'actions various cellular marker proteins were employed.
To determine the cytosolic fiaction a polyclonal rabbit antiserum against lactate
dehydrogenase (LDH) from pig muscle (~ 37 kDa) was used at a dilution of 1:500.
The LDH antiserum (anti-LDH) was kindly provided by Dr. John Wilson
(Department of Biochemistry, Michigan State University). The presence of
endoplasmic reticulum proteins was determined using a rabbit anti-ERp72 polyclonal
antibody (StressGen Biotechnologies Corp.) in a dilution 1:1000 according to the
manufacturer’s protocol. The presence of Golgi proteins was determined using the
monoclonal antibody M3A5 (Sigma-Aldrich), which recognizes the coatamer protein
B—COP, at a dilution of 1:50 according to the manufacturer’s protocol. To verify the
plasma membrane-enriched fi'action (Pl6.9) biotinylation of membrane proteins
using the EZ-Linkm Sulfa-NHS-LC-Biotin cross-linker (Pierce) was performed
(Shetty et al., 1993). The cell monolayer, grown on a 100 mm dish, was rinsed twice
with ice cold PBS and incubated in 1 ml of biotinylation buffer (120 mM NaCl, 30
mM NaHCO,, 5 mM KCl, (pH 8.5)), containing 0.1 mg/ml cross-linker for 30 min at

4 °C. Following biotinylation, cells were lysed in Buffer A and the membrane

101

fractionation was carried out as described above. Equal amounts of protein of the
S169 and P16.9 fiaction were separated by SDS-PAGE and tested for biotinylation
by Western blotting using horseradish peroxidase-conjugated strepavidin (Pierce) at
a dilution 1:500. The percentage of the protein amount in each fraction compared to
the total non-nuclear cellular protein in the single fractions was calculated as: [pg
protein fiaction y x 100]/(pg P16.9 protein + pg P100 protein + pg S100 protein). y

represents either the P16.9, P100, or either 8100.

4. Results

4.1 Analysis of cellular fractions

Initial studies of SPRK's subcellular distribution used a one-step crude
membrane fractionation method resulting in a soluble and membrane fraction. To
analyze the localization of SPRK in more detail, an additional centrifugation step
was introduced, resulting in a soluble 816.9 and a pelleted P16.9 fiaction. The 816.9
fi‘action was firrther separated, yielding 8100 and P100 fiactions. Fractions obtained
using the two-step method were analyzed by Western blot analysis employing
different marker proteins.

Lactose dehydrogenase (LDH) has been demonstrated to be present in the
cytosol (Vyakarnarn et al., 1998). As shown in Fig. 23a the soluble 8100 fiaction
contains LDH, whereas both pellet fiactions lack this enzyme, indicating that they
are not contaminated with cytosolic proteins.

To determine which fi'action contains plasma membrane proteins, a

biotinylation method was employed. Membrane proteins are translated at the rough

102

endoplasmic reticulum and posttranslationally modified in the endoplasmic
reticulum and the Golgi system before they are inserted into the plasma membrane.
Thus membrane proteins may have limited usefulness as marker proteins. Therefore
intact cells were treated with a biotin-conjugated membrane impermeable
crosslinking agent. Then a membrane fiactionation was performed to separate 816.9
and P16.9 fi'actions. After SDS-PAGE, the proteins were transferred to
nitrocellulose and the biotinylated proteins were visualized by chemiluminescence
using horseradish peroxidase-conjugated strepavidin. Over 95% of biotinylated
proteins are found in the P16.9 fi'action (Fig. 23b). Additionally the transmembrane
protein sodium potassium ATPase can be detected in the P16.9 fraction, but not in
the $16.9 fraction (personal communication, Dr. Julia V. Busik), validating that the
P16.9 fraction contains the majority of integral plasma membrane proteins.
Additionally the P16.9 fi'action contains endoplasmic reticulum as judged by the
presence of the endoplasmic reticulum protein (ERp)72,(Fig. 23a). ERp72 is a
member of the protein disulfide isomerase family and serves as a molecular
chaperone for newly translocated and glycosylated proteins.

Currently antibodies against human proteins, which reside within the Golgi
are not commercially available. Analysis of the obtained fi'actions with an antibody
directed against murine or-mannosidase II, a glycosylation enzyme within the Golgi
apparatus, was not successful since this antibody does not crossreact with the human
counterpart. Therefore a monoclonal antibody against B-Cop was used. B-COP is a
coatamer protein, which is found in the periphery of the Golgi system and on

vesicles scattered throughout the cytoplasm. As shown in Fig. 23 a, B-COP is

103

predominantly found in the 8100 fraction. The P100 fi'action is reported to contain
Golgi membranes (Thissen and Casey, 1993). It is likely that smaller Golgi-
associated vesicles, which are distributed within the cytosol, are found in the S100
fiaction whereas large Golgi stacks may pool in the P100 fi'action. A more reliable
method to determine the Golgi fraction may be provided by a galactosyl transferase
activity assay. Galactosyl transferase is an internal Golgi protein making it a better
marker for the Golgi complex. In addition to B-COP, ERp72 is detected in the $100
fiaction, indicating that the 8100 fiaction also contains endoplasmic reticulum. A

summary of the analysis of the fractions by marker proteins is given in Table 1.

4.2 Activated Cdc42 targets SPRK to cellular membranes

Transiently transfected 293 cells were used to test the effect of Cdc42 on
SPRK's subcellular distribution. A crude biochemical membrane fi'actionation,
employing one centrifirgation step at 100,000 x g was performed. Cellular lysates
were separated into a soluble and a membrane fi'action. As shown in Fig. 24,
overexpressed SPRK, in the absence of Cdc42, is distributed in both the soluble and
the membrane fiaction but, upon coexpression with activated Cdc42, nearly all
SPRK is found in the membrane fraction. From this biochemical data we conclude
that activated Cdc42 indeed affects SPRK's subcellular distribution, causing an

increased association of SPRK with cellular membranes.

104

4.3 Analysis of the subcellular distribution of SPRK by confocal microscopy

The biochemical fractionation shows that SPRK, overexpressed in 293 cells, is
distributed approximately equally between a soluble and a pelleted fraction. To
visually determine SPRK's distribution, COS-7 cells overexpressing SPRK were
analyzed by confocal microscopy. Recently it has been shown by confocal and
electron microscopy that the endogenous dual leucine zipper-bearing kinase (DLK)
associates with the cytoplasmic face of the Golgi apparatus in NIH 3T3 cells
(Douziech et al., 1999). Since DLK is related to SPRK and shares structural
similarity, SPRK may also localize to the Golgi apparatus. Because the Golgi
system has a diflerent appearance depending on the cell type, a crude visualization of
the Golgi apparatus, in COS-7 cells was performed using a ceramide analogue
coupled to the fluorophore Texas Red (Pagano et al., 1991). Cerarnides are
precursors for glycosphingolipids and sphingomyelin, which are synthesized in the
Golgi apparatus and hence can be employed to visualize the Golgi complex (Pagano
et al., 1991). The appearance of the Golgi apparatus in COS-7 cells is shown in Fig.
25a. Confocal microscopy using a polyclonal SPRK antiserum results in a
perinuclear staining and a Golgi-like staining (Fig. 25b). Additionally, SPRK shows
a difitlse distribution in the cytosol. Fig. 25c shows an overlay (yellow, right image)
of a cell stained for the Golgi apparatus (red, left image) and overexpressed SPRK
protein (green, middle image). The overlay suggests that SPRK indeed colocalizes
with the Golgi apparatus. The perinuclear staining may be indicative of some SPRK

association with the endoplasmic reticulum.

105

To analyze the effect of Cdc42V12 on SPRK distribution, COS-7 cells were
transfected with SPRK and Cdc42V12 and analyzed by irnmunofluorescence using
confocal microscopy. The Cdc42 distribution was determined using the M2
monoclonal antibody, which recognizes the Flag epitope, which is appended to the
NHz-terminus of Cdc42v’2. When using the same transfection conditions as in the
previous experiments, large vacuoles within the cells were observed. The cells
appeared to lose structural integrity and thus were impossible to analyze by confocal
microscopy. SPRK and Cdc42 have been shown to activate the INK pathway (Coso
et al., 1995; Minden et al., 1995; Tibbles et al., 1996), which has been implicated in
the induction of cellular death (Verheij et al., 1996; Xia et al., 1995; Zanke et al.,
1996). Therefore it is possible that overexpression of the two proteins may be toxic
to the cells and may promote the induction of cell death. This idea is also supported
by the morphological changes observed in 293 cells cotransfected with SPRK and
Cdc42V‘2. The 293 cells started rounding up 18 h post transfection and lifted fi'om
the culture dish approximately 48 h post transfection. This phenomena was not seen
when SPRK or Cdc42V12 were expressed alone.

To determine the earliest time point post transfection at which SPRK and
Cdc42V12 can be detected by Western blot analysis, a time course of the expression
of both proteins was analyzed. SPRK expression was already apparent 6 h post
transfection whereas Cdc42 expression was detected after 8 h (Fig. 26). Based on
this result the 8 h time point was chosen for further experiments to hopefirlly avoid
the gross morphological changes, which were observed when using longer

transfection times.

106

SPRK, expressed alone, is predominantly found in the Golgi apparatus, with a
diffuse distribution within the cytosol and some perinuclear localization (Fig. 25b).
Upon coexpression with constitutively active Cdc42, the majority of SPRK appears
at a Golgi-like structure and within the cytosol (Fig. 27a and b, H). The cytosolic
distribution appears more punctuate compared to SPRK alone. This may be
indicative of SPRK association with microtubules or other components of the
cytoskeleton. However, a possible association of SPRK with cytoskeletal elements
was not further investigated. A small amount of SPRK is also detected at the plasma
membrane, when activated Cdc42 is coexpressed. Analysis of Cdc42Vlz distribution
is very similar to that of SPRK with Cdc42 being found in wlmt appears to be the
Golgi apparatus, the cytosol, and maybe the plasma membrane (27 and b, I). The
overlay of SPRK and Cdc42V12 staining supports the idea that SPRK and activated
Cdc42 colocalize within COS-7 cells upon overexpression (Fig. 27a and b, 111).

Surprisingly, the small GTPase was also detected in the nucleus, independent of
the presence of SPRK (Fig. 27a and b, I and Fig. 283 and b). To exclude the
possibility that the nuclear staining results from non-specific binding of the M2
antibody or the secondary antibody, the subcellular distribution of NHz-terminal
F lag-tagged SPRK was determined using the M2 monoclonal antibody and the
SPRK antibody in parallel. No nuclear staining was detected when examining SPRK
distribution. To rule out that nuclear staining results fi'om the used concentrations of
antibody, lower concentrations of primary and secondary antibody were tested, again
resulting in nuclear staining. These data my suggest that under these experimental

conditions Cdc42 may be nuclear or may be associated with the nuclear envelope.

107

Due to geranylgeranylation, Cdc42 is able to associate with cellular membranes
(Erickson et al., 1996; Hart et al., 1990). Since the association of activated Cdc42
with the plasma membrane depends upon its prenylation, the translocation of SPRK
by activated Cdc42 might also depend on this posttranslational modification. To
investigate whether the prenylation of Cdc42 is required for Cdc42-induced
membrane targeting of SPRK a prenylation-defective variant of Cdc42 was
constructed. To prevent prenylation the cysteine residue within the CAAX motif of
Cdc42V12 was changed to a serine, resulting in the CAAX mutant Cdc42v12cms
(Fig. 22). The analogous mutation in yeast prevents prenylation and rescues the
lethal phenotype induced by constitutively active Cdc42V12 (Ziman et al., 1991).

To compare the subcellular distribution of Cdc42v'2 and Cdc42Vlzmss when
overexpressed in COS-7 cells, confocal microscopy was performed. The observed
plasma membrane staining with overexpressed Cdc42V12 (Fig. 28a and b) is largely
blunted with the Cdc42 CAAX variant (Fig. 28c and d), supporting the idea that
prenylation of Cdc42 contributes to membrane association of this small GTPase.
However, despite the lack of the prenyl group residual, Cdc42vncrsss is detected at
the plasma membrane.

SPRK alone is found predominately at the Golgi system and difiuse in the
cytosol (Fig. 25b). In contrast, overexpression of SPRK with activated Cdc42 results
mainly in a Golgi-like staining and a punctuate scattered distribution in the cytosol,
as well as some plasma membrane staining (27a and b, 11). When analyzing cells,
which coexpress SPRK and the Cdc42vrzcms mutant, SPRK distribution appears

very similar to the one seen with SPRK alone (Fig 29a and b; 11), with localization of

108

SPRK to a Golgi-like structure. Interestingly, Cdc42v'2'C1888 is also found at a
Golgi-like structure (Fig. 29a and b, I) where it colocalizes with SPRK (Fig. 29 a and
b, 11).

Taken together the data obtained by confocal microscopy suggest that activated
and prenylated Cdc42‘“2 changes the subcellular distribution of SPRK from the
Golgi system to a more punctuate distribution within the cytosol. This change in
distribution requires activated prenylated Cdc42. However, since the morphology of
cells coexpressing SPRK and Cdc42VIZ differs substantially from the phenotype
observed with SPRK alone, or SPRK coexpressed with the Cdc42 CAAX variant. it
is difficult to draw any hard conclusions. To further examine the effect on Cdc42 on
SPRK subcellular distribution, an additional modified biochemical fiactionation was

undertaken.

4.4 Analysis of SPRK distribution by biochemical fractionation

To complement confocal microscopy, the distribution of SPRK was further
examined by biochemical fractionation experiments. To obtain a plasma membrane-
enriched fiaction the one-step fractionation method was modified by introducing an
initial centrifirgation step at 16,900 x g, according to Thissen and Casey (Thissen and
Casey, 1993). This additional centrifugation yields a plasma membrane-enriched
fraction (Pl6.9). The soluble 816.9 fiaction was further separated into a soluble
(S100) and a pelleted (PlOO) fiaction by a second centrifirgation step at 100,000 x g.

It is estimated, based on four different fiactionation experiments, that approximately

109

25% of total non-nuclear cellular protein is found within the P16.9 fi'action, 8%
within the P100 fraction, and 67% within the $100 fraction (Table 2).

Analysis of the subcellular distribution of SPRK, when equal amounts of
protein are subjected to SDS-PAGE, showed that SPRK alone is distributed nearly
equally between the 816.9 and the P16.9 fiactions. Upon coexpression with
Cdc42“2 nearly all SPRK protein is found in the pelleted (Pl6.9) fiaction (Fig. 303,
top left panel), supporting the idea that activated Cdc42 may target SPRK to the
plasma membrane.

SPRK coexpressed with Cdc42v'2c188$ shows a similar distribution to that of
overexpressed SPRK alone, supporting the idea that Cdc42-induced SPRK
membrane targeting requires prenylation of Cdc42. Interestingly, when examining
the subcellular distribution of Cdc42“20888 it becomes clear that this mutant
associates to some extent with the plasma membrane-enriched fi'action, despite the
lack of the prenyl group (Fig. 30a, lower panel). Therefore, this mutant does not
yield a clean phenotype and thus, may target some SPRK protein to the plasma
membrane.

Hancock and coworkers showed tint, in addition to the CAAX motif, a so-
called polylysine stretch upstream of the CAAX-motif contributes to membrane
association of K-Ras (Hancock et al., 1990). Cdc42 also contains forn' basic amino
acids (two lysines and two arginines) near its CAAX motif (Fig.22), which may
contribute to membrane targeting. In yeast Cdc42“2 changing the basic residues
near the CAAX motif to glutamines results in a decrease in membrane association

(Davis et al., 1998). To test whether these four positively charged residues are

110

important for membrane association of human Cdc42 all four basic residues were
mutated to neutral ones in the Cdc42 CAAX mutant, resulting in a CAAX variant,
termed Cdc42v'2'cms'wR40 (Fig. 22). When tested in membrane fractionation
experiments, this Cdc42 variant shows no detectable association with the plasma
membrane-enriched P16.9 fraction (Fig. 30b, lower left panel), indicating that the
polybasic stretch in Cdc42, along with the CAAX-motif, participates in membrane
association of this small GTPase. It appears that both Cdc42 CAAX variants do not
influence the subcellular distribution of SPRK, when compared to SPRK expressed
alone, and are devoid of inducing the shift of SPRK to the P16.9 fiaction. This result
suggests that activated, prenylated Cdc42 is required for the observed change in
SPRK's subcellular distribution.

The distribution of overexpressed SPRK between the S169 and P16.9
fiaction is nearly equal when equivalent amounts of protein are analyzed (Fig. 30a
and b, top left panel). However, estimation of the fractional distribution of non-
nuclear SPRK reveals that approximately 27% of SPRK protein is found in the P16.9
fraction and 63% in the $16.9 fraction (Table 3). This distribution is dramatically
changed to 70% of SPRK protein in the P16.9 fiaetion when SPRK is coexpressed
with activated Cdc42. Coexpression of constitutively active Cdc42 does not
influence SPRK distribution in the P100 fraction, but in the 8100 fraction SPRK
protein is reduced fiom 62% to 18% as when compared to SPRK expressed alone
(Table 3). We conclude therefore that the Cdc42-induced redistribution of SPRK is
due to a shift of SPRK fi'om the 8100 to the P16.9 fraction, supporting the idea of the

Cdc42-mediated translocation of SPRK from the Golgi apparatus to the plasma

1]]

membrane. The percentage of SPRK protein found in the P100 fraction is not
influenced by Cdc42 (Table 3).

The small GTPase Rac also contains six contiguous basic amino acids NH;-
terminal to the CAAX motif. Changing these residues to neutral glutamine residues
results in the inability of Rac to bind PAK (Knaus et al., 1998). To rule out the
possibility that loss of membrane localization of SPRK is due to its inability to bind
to the Cdc42 CAAX variants, a co-irnmunoprecipitation experiment was performed.
The result of this experiment clearly shows that all Cdc42V12 variants associate with
SPRK to the same extent (Fig. 31, upper panel).

A portion of SPRK, upon overexpression, is found in the plasma membrane-
enriched fraction (Fig. 30a and b, top left panel). Overexpressed SPRK has high
autophosphorylation activity (Gallo et al., 1994). Therefore, it is conceivable that
this autophosphorylation activity is responsible for the presence of SPRK in the
plasma-membrane fraction. However, the subcellular distribution of SPRKKI’M, a
SPRK variant which lacks catalytic activity (Gallo et al., 1994), in presence or
absence of Cdc42v'2 revealed similar results as observed with wild type SPRK (Fig.
323) suggesting that membrane association is not dependent on SPRK's catalytic
activity.

SPRK contains a polyarginine tract of four contiguous arginines following
the zipper region. To test whether this polybasic stretch may mediate Cdc42-
independent plasma membrane association of SPRK the subcellular distribution of
the SPRK” variant was examined. This spinezip variant lacks the polybasic tract

as well as the second half of the zipper domain. SPRKAZ'I’ shows the same

112

distribution as wild type SPRK (Fig. 32b), suggesting that the polybasic stretch is not
required for Cdc42-independent membrane association of SPRK.

Because all of these experiments were performed with overexpressed SPRK
the observed subcellular distribution of SPRK may not reflect the distribution of
endogenous SPRK. To exclude the possibility that these results are influenced by
overexpression, the subcellular distribution of endogenous SPRK in the presence and
absence of activated Cdc42 was analyzed in 293 cells. A preliminary experiment
suggests that endogenous SPRK in the presence and absence of activated Cdc42
shows no striking difference in subcellular distribution compared to overexpressed

SPRK (Fig. 33).

4.5 Effect of membrane-targeting on SPRK's kinase activity

Activated Cdc42 increases the catalytic activity of SPRK and changes its in viva
phosphorylation. The Cdc42-induced activation cannot be recapitulated in vitra
(Bock et al., 2000), suggesting the requirement of a cellular component. Since the
results presented above show that Cdc42V12 is capable of localizing SPRK to a
plasma membrane-enriched fiaction, we wished to determine whether this membrane
targeting contributes to SPRK's activation. SPRK, when coexpressed with Cdc42vn,
has a slower electrophoretic mobility compared to SPRK alone or coexpressed with
Cdc42v'2‘C1838 (Fig. 34). Increased phosphorylation is often correlated with a
decrease in electrophoretic mobility. Thus, this mobility shift may suggest a change

in phosphorylation of SPRK that depends on prenylated activated Cdc42.

113

To test the hypothesis that membrane targeting by Cdc42 may be accompanied
by a change in in viva phosphorylation of SPRK two-dimensional comparative in
viva phosphopeptide mapping was performed'. Fig. 35 shows the in viva
phosphopeptide maps of SPRK expressed alone, or coexpressed with Cdc42V12 or
Cdc42V'2C'888. The basic pattern of the three phophopeptide maps is very similar.
The major changes are detected in the triangular cluster 0, b, and c. When SPRK is
expressed alone phosphopeptide a and b are detected in the triangular cluster. In the

2"”, phosphopeptide b is

corresponding map of SPRK, when coexpressed with Cdc4
not detected, whereas phosphopetides a and c are present at high levels. Analysis of
the preliminary phosphopeptide map of SPRK upon coexpression with Cdc42V'2'
Cms shows that phosphopeptide b is detected, but also a and c are highly abundant,
perhaps suggesting an intermediate phosphorylation phenotype. This may suggest
that membrane targeting is not required for the Cdc42-induced change in SPRK
phosphorylation. Alternatively, it is possible that this intermediate phenotype is
observed due to the fact that Cdc42v'2'c'8ss is detected within the plasma membrane-
enriched biochemical fiaction, and thus may target residual SPRK to the plasma
membrane, where in viva phosphorylation may take place. To more rigorously test if
membrane targeting of SPRK changes the in viva phosphorylation pattern of SPRK
this experiment should be performed employing the Cdc42‘mc'889"”1W2 variant,
which is devoid of membrane association.

To determine whether Cdc42-induced SPRK activation is dependent on
membrane targeting, total lysates of 293 cells were examined for their SPRK kinase

activity upon coexpression with Cdc42V12 or Cdc42v'zc'sss. Surprisingly, no

114

difference in kinase activity could be observed (Fig. 36) suggesting that Cdc42-
induced activation of SPRK under these experimental conditions may be
independent of membrane targeting. As shown in Fig. 30a the Cdc42 CAAX mutant
retains the ability to associate to some extent with the plasma membrane. This partial
membrane association is abolished in the c:dc42V"’-w”s"‘IR4Q variant (Fig. 30b).
Therefore the kinase activity of SPRK when coexpressed with Cdc42V12'C'885'm’Q
was measured. Upon coexpression of the Cdc42w2cms'wmQ variant with SPRK no
detectable difference in SPRK activity compared with Cdc42V12 in total cellular
lysates was found (Fig. 37).

To determine the kinase activity of membrane-associated SPRK, in vitra kinase
assays of SPRK in the P16.9 fiaction were performed. SPRK and either Cdc42"12 or
Cdc42V'2'Cm‘S'K’R40 was coexpressed in 293 cells. Following a membrane
fi'actionation, SPRK was immunoprecipitated from the P16.9 fiaction using SPRK
antiserum and the immunoprecipitates were subjected to an in vitra kinase assay.
The kinase assay employed in previous experiments using radiolabeled [y-32P]ATP
and histones as substrates were accompanied by high background radioactivity.
Therefore a non-radioactive kinase assay was developed using ATP and GST-MKK4
as substrates. Activation of MKK4 requires phosphorylation on serine 254 and
threonine 258 (Yan et al., 1994; Zheng and Guan, 1994). The activating
phosphorylation of MKK4 by SPRK was detected using an antibody, which
recognizes the phosphorylated form of threonine 258 of MKK4. Using this method
the background was dramatically reduced and SPRK substrate phosphorylation

activity could be determined (Fig. 38). In membrane fiactions SPRK coexpressed

115

with Cdc42V12 has a 3-fold higher kinase activity compared to SPRK alone, or SPRK
coexpressed with Cchvrzcrsssx/mo. The data shown in Fig. 38 is a representative
of three independent experiments. A preliminary experiment examining endogenous
SPRK in the P16.9 fiaetion for kinase activity in presence or absence or Cdc42V12
also shows an increase in SPRK kinase activity when activated, prenylated Cdc42 is
expressed (Fig. 39). As shown earlier, even without activated Cdc42, SPRK can be
detected in the immunoprecipitates of the P16.9, but as determined by in vitra kinase

assays this portion of SPRK is not active.

116

 

 

 

 

 

 

P16.9 P100 8100
LDH - - +
ERp72 + - +
B-COP - - +
Biotinylation + - -

 

 

 

 

117

Table 1. Analysis of subcellular fractions by marker proteins and biotinylation

 

 

Cdc42": PPEKKSRRCVLL
Cdc42v‘2‘c’m PPEKKSRRSVLL

ctlt:42‘""“""’°""m PPEQQSQQSVLL

 

 

 

Fig. 22. Alignment of various Cdc42 variants. The last 12 amino acid residues of
the COOH-terminus of the Cdc42 variants, which were used in this study, are shown.
The CAAX motifofczdc42V12 is in bold letters; amino acids, which were changed by
site-directed mutagenesis, are shown in bold and italicized letters.

118

816.9 P16.9

100
P16.9 S P
. LDH
can B-COP

 

Fig. 23. Analysis of subcellular fractions. a, the P16.9, $100, and P100 fiactions
were analysed using lactose dehydrogenase, ERp72, and B-COP as rmrker proteins.
Each lane contains equal amounts of protein. b, to determine the plasma membrane-
containing fi'action, the plasma membrane-bound proteins were cross-linked using a
biotinylated plasma membrane-impermeable crosslinker as described in "Materials
and Methods". After a membrane fi'actionation the proteins were separated by SDS-
PAGE and the biotinylated proteins were detected using horseradish peroxidase-
coupled strepavidin. Both lanes contain 0.3 pg of total protein.

119

SPRK: - + «l-
Cde42‘m: - - +
S a S A U y S M Immunoblot

. SPRK

 

“A

Fug4ana2

Fig. 24. Subcellular distribution of SPRK. Cells were transfected with expression
vectors containing the cDNA indicated above the figure. The minus sign indicates
the transfection of a control vector. S indicates the soluble fiaction and M the
membrane fiaction after a centrifugation at 100,000 x g. Transient transfections of
293 cells, membrane fractionation, and SDS-PAGE were performed as described in
"Materials and Methods". The upper panel shows SPRK distribution, the lower one
shows distribution of Cdc42v'2. Each lane contains equal amounts of protein. The
membrane fractionation shown is representative of five independent experiments.

120

   

Golgi SPRK

 

Golgi SPRK Overlay

Fig. 25. Subcellular localization of SPRK. COS-7 cells were plated onto glass
coverslips and transfected with an expression vector containing the SPRK cDNA.
SPRK subcellular distribution was determined by confocal irnmunofluorescence. a,
staining of the Golgi apparatus in COS-7 using a BODIPY-ceramide coupled to
Texas Red. b, localization of SPRK using an anti-SPRK antiserum as primary and
Alexa 488 goat anti-rabbit IgG as secondary antibody. c, cells were treated as
described under b and stained for both the Golgi apparatus (red, left panel) and
SPRK (green, middle panel). The right panel shows costaining of SPRK and the
Golgi apparatus (yellow).

12]

hours: 5 6 s 10 12 imam”,

. ‘7‘}
f ,e
.

 

 

FIag-Cde42

Fig. 26. Time course of SPRK and Cdc42Vlz expression. COS-7 cells were
transfected with expression vectors containing the cDNAs for SPRK and Cdc42V”.
Cells were harvested post transfection at the time points indicated above. The top
panel shows SPRK expression, the lower one expression of Cdc42V'2.

122

 

Flag-Cdc42": SPRK Overlay

Fig. 27. Subcellular distribution of coexpressed SPRK and Cdc42vu. COS-7
cells were plated onto glass coverslips, and transfected with expression vectors
encoding for SPRK or Cdc42V”. SPRK and Cdc42V12 subcellular distribution was
determined by confocal irnmunofluorescence. Both the a and b panels show cells
which coexpress SPRK and Cdc42V'2. (I), staining of Cdc42 (red) was performed
using the M2 monoclonal antibody as primary and the FluoroLinkTM Cy3TM-labelled
goat anti-mouse IgG as secondary antibody. (11) staining of SPRK protein (green)
was performed using an anti-SPRK antiserum as primary and the Alexa 488 goat
anti-rabbit IgG as secondary antibody. Arrowheads indicate areas where plasma
membrane staining is observed. (111), colocalization of SPRK and Cdc42V12
(yellow).

123

 

Fig. 28. Subcellular distribution of Cdc42Vlz and Cdc42V‘m“. cos-7 cells
were plated onto glass coverslips, and transfected with expression vectors encoding
for Cdc42V12 (a and b) or Cdc42v'2'cms (c and d). The subcellular distribution of
the two Cdc42 variants was determined by immunofluorescence using the M2
monoclonal antibody as primary and the FluoroLinkTM Cy3TM-labelled goat anti-
mouse IgG as secondary antibody. Arrowheads indicate areas of plasma membrane
staining.

124

 

Flag-Cdc42"“"“ SPRK Overlay

Fig. 29. Subcellular localization of coexpressed SPRK and Cdc42v'2‘cms. COS-
7 cells were plated onto glass coverslips, and transfected with expression vectors
encoding for SPRK and Cdc42v'2‘c‘m. SPRK and Cde42‘”w“s subcellular
distribution was determined by confocal immunofluorescence as described above.
The a and b panels show cells which coexpress SPRK and Cdc42V'2‘ms. (I),
staining of Cdc42v'2'c‘m (red); (11) staining of SPRK (green); (111), costaining of
SPRK and Cdc42V'2‘3ms (yellow).

125

- Ib‘“ - . Flag-Cdc42
16,900 x 9 100,000 x g
b
SPRK: + + + + + +
CIEIZ v11 - + 01W - + crass-mo
8 P 8 P 8 P 8 P 8 P 8 P Inlnunoblot
‘ SPRK
. e- r... M -Q a...“
' ._ it
~ Plug-06042
16.900 x a 100,000 rt 9

Fig. 30. SPRK's subcellular distribution upon coexpression with Cdc42v"
variants. 293 cells were transfected with expression vectors containing the cDNA
indicated above each figure. The membrane fiactionation was carried out as
described in "Materials and Methods". S indicates the soluble fiaction and P, the
pellet fiaction. The left hand panels contain soluble and pellet fiactions obtained
flour a centrifugation at 16,900 x g, the right hand panels contain soluble and pellet
fractions of a centrifugation at 100,000 x g. a, subcellular distribution of SPRK
when expressed alone or with either Cdc42V‘2, or Cdc42v'2'c'883. b, subcellular
distribution of SPRK when coexpressed with either Cdc42VIZ or Cdc42V‘M'835’WQ.
The reduced electrophoretic mobility of Cdc42V‘ZC'883'WQ may be due to the
substitution of positively charged residues to neutral residues. Equal amounts of
protein were loaded in corresponding experiments.

126

 

Fraction P16.9 P100 S100

 

 

% Protein 25 8 67

 

 

 

 

Table 2. Estimated distribution of total cellular non-nuclear protein among
biochemical fractions. The percentage of total cellular non-nuclear proteins found
in a given fiaction y was calculated as: [pg protein in fiaction y x 100]/(pg protein in
P16.9 fiaction + pg of protein in P100 fiaction + pg of protein in $100 fiaetion). y
represents either P16.9, P100, or 8100.

127

 

 

 

 

 

 

 

SPRK SPRK
(- Cdc42‘m) (+ Cdc42v”)
P16.9 27% 70%
816.9 (%P100 + %S]00) 63% 30%
P100 1 1% 12%
$100 62% 18%

 

 

 

Table 3. Estimated distribution of SPRK protein among biochemical fractions
in presence and absence of Cdc42v". The distribution of SPRK among the non-

nuclear biochemical fiactions was determined by densitometry (NIH Image) as
described in “Materials and Methods”, and corrected for the fiactional distribution of

total non-nuclear protein.

128

 

SPRK: - + + + +

m crass
Cdc42 = - - + crass KIR4Q
- . ‘ ‘ immunoblot

”'9", . - . SPRK

_-._..._.._._ .

’54;

one W
.O. Fla-scam

 

Fig. 31. Co-immunoprecipitation of SPRK and Cdc42 variants. Cdc42v‘2,
Cdc42V'2‘C'sss, or Cdc42 V‘“'”‘~‘*“"“Q was immunoprecipitated (IP) with the M2
antibody directed against the Flag epitope. The co-irnmunoprecipitation of SPRK by
the Cdc42Vlz variants was determined by immunoblotting with a SPRK antibody
(top panel). The middle panel and lower panel show Cdc42 expression, respectively.

129

SPRK'm“: + +
Cdc42m: _ +

 

 

 

 

S P

madam realm .~

. . .‘ .’c,_', i _

i‘ FSPRKW

q

Fig 32. Subcelluhr distribution of sprud‘mA and SPRKA". 293 cells were
transiently transfected with expression plasmids encoding a, SPRKK'W‘ and
Cdc42"12 or b, SPRK“. Cellular lysates were subjected to membrane fractionation.
The distribution of the SPRK variants was determined by Western blot analysis. P
indicates the pelleted 16.9 plasma membrane-emiched fraction and S the soluble
$16.9 fiaction. Each lane contains equal amounts of protein.

130

 

 

“7'. .. ,1 . It“ '_ -""M~"-~..fi‘

 

SIP 100,000 x 9

Fig. 33. Subcellular distribution of endogenous SPRK. 293 cells were transfected
with a control vector or an expression plasmid containing the Cdc42“2 cDNA. The
cellular lysates were subjected to membrane fractionation as described in "Materials
and Methods" and the SPRK distribution was determined by Western blot analysis. S
indicates the soluble fiaction, P the pellet fiaction of the indicated centrifugation
speed. Each lane contains equal amounts of protein.

131

SPRK: _ + + +
+ 01888

V12. - -
Cdc42 . __ .. . lmmunoblot

 

SPRK

 

Fig. 34. Electrophoretic mobility shift of SPRK. 293 cells were transiently
transfected with expression plasmids encoding the cDNAs indicated above. Cells
were lysed and the proteins were analyzed by Western blot analysis.

132

 

SPRK SPRK + SPRK +
Cduzv12ec1m Cdc42V12
. g ”h, . are.
a , W,
‘ E
b I b b
ehromato- .‘ ‘M .
graphy a c i ‘, C a“ c

 

 

 

 

 

 

electrophoresis —>

Fig. 35. Phosphopeptide mapping of in viva phosphorylated SPRK. Two-
dirnensional phosphopeptide mapping of 32P-labeled SPRK alone or coexpressed
with either Cdc42Vl2 or Cdc42V'2‘mS.
cellular lysates, proteins were separated by SDS-PAGE and blotted on
polyvinylidene difluoridemembrane, and subjected to a partial tryptic digest. The
resultant tryptic peptides were analyzed by TLE in the first and by TLC in the second

dimension. The elecreophoretic and the chromatographic directions are indicated by

SPRK was immunoprecipitated fi-om

arrows. The peptides of interest are indicated by italicized letters.

133

 

SPRK: - + + +
Cdc42”: - - + 01888 autoradiogram

*.., épg,.g;‘-giiig;i.jiig_, <F- esrauuel
SPRK4P i..'v-. 4i.-f;'g<'j~'§ f.

Immunoblot

I
_ - .. AL ‘- n
. _ ‘ -;ui. '. '. ' . p
_ r a . " ‘ ' ‘ .
ll." ‘ ., e _ ..
-r. -n— .. . - 7

SPRK

.. Fla-scam

Fig. 36. SPRK in vitro kinase activity upon coexpression with Cdc42"12 or
Cdcavrz-crsss. 293 cells were transfected with cDNAs indicated above. SPRK was
immunoprecipitated and subjected to a radioactive in vitro kinase assay using GST-
MKK4 as a substrate. The top panel shows an autoradiogram with bands
corresponding to phosphorylated GST-MKK4. The middle and lower panels show
SPRK and Cdc42 expression, respectively.

134

SPRK: - + + +

C1883-
Cdc42 "2: _ _ 4.
lmmunoblot

SPRKIP 3’ “a ”N“

SPRK IP ‘~ ~- SPRK

.- I ’ , , I. ~“7.l=rag-cde42

 

Fig. 37. SPRK in vitro kinase activity upon coexpression with Cdc42v" or
Cdc42V11'C‘m'm‘0. 293 cells were transfected with cDNAs indicated above.
SPRK was immunoprecipitated and subjected to an in vitro kinase assay using GST-
MKK4 as a substrate. MKK4 phosphorylation was examined using an anti-phospho
MKK4 antibody (upper panel). The middle and lower panels show SPRK and Cdc42
expression, respectively.

135

SPRK: - + + 4.

Cd 2,,,_ crass-
“ - '_ ' . 5“" {KIR4Q Immunoblot

P-MKKA

 

Fig. 38. Kinase activity of membrane-targeted SPRK. 293 cells were transiently
transfected with a vector containing the cDNA indicated above. Following a
membrane fiactionation SPRK was immunoprecipitated fi'om the P16.9 fiaction.
The SPRK kinase activity was analyzed using GST-MKK4 as a substrate. GST-
MKK4 phosphorylation was determined using an anti-phospho-MKK4 antibody.
The top panel shows phospho-MKK4. To determine SPRK expression the same blot
was reprobed with a SPRK antibody (lower panel).

136

vector + Cde42m Immunoblot

   

‘ it 4"- i’rkbm

Fig. 39. In vitro kinase assay of endogenous, membrane-targeted SPRK. 293
cells were transiently transfected with an expression vector encoding for Cdc42V12 or
a control plasmid. Cellular lysates were subjected to membrane fiactionation as
described in "Materials and Methods". SPRK was immunoprecipitated fiom the
P16.9 fiaction and subjected to an in vitro kinase assay employing GST-MKK4 as a
substrate. Phosphorylation of GST-MKK4 was determined using an anti-phospho-
MKK4 antibody (upper panel). To determine SPRK expression the same blot was
reprobed with a SPRK antibody (lower panel).

137

5. Discussion

The small GTPase Cdc42 increases the autophosphorylation and substrate
phosphorylation activity of the serine/threonine kinase SPRK (Beck et al., 2000).
However, Cdc42-induced activation of SPRK cannot be recapitulated in vitro using
recombinant proteins, suggesting that SPRK activation by Cdc42 requires the
cellular environment.

The small GTPase Cdc42, which is geranylgeranylated at the COOH-
terminus (Maltese and Sheridan, 1990), has been shown to associate with cellular
membranes, including the Golgi apparatus and the plasma membrane (Erickson et
al., 1996; Hart et al., 1990). Therefore, it was of interest to test the hypothesis if the
Cdc42-induced activation of SPRK requires membrane targeting of SPRK.

Subcellular localization by confocal microscopy shows that the majority of
SPRK colocalizes with the Golgi apparatus upon overexpression in COS-7 cells.
Coexpression of Cdc42 induces a shift of SPRK to a plasma membrane-enriched
biochemical fiaction. This P16.9 fiaction contains not only plasma membrane, but
also the endoplasmic reticulum. Data obtained by confocal microscopy support the
idea that Cdc42 changes SPRK's subcellular distribution resulting mainly in a
punctuate pattern within the cytosol and some plasma membrane association. This is
also supported by the finding that the non-prenylated Cdc42 variant is not able to
induce the observed shift, displaying a similar subcellular distribution as SPRK
alone. Costaining of SPRK and the endoplasmic reticulum has not been performed,
therefore it is not possible to draw any final conclusions on SPRK's targeting to the

endoplasmic reticulum. According to the literature the P100 fraction is supposed to

138

contain endomembranes (Thissen and Casey, 1993). Srmller and larger components
of the Golgi apparatus sediment differently. Therefore it may be feasible that the
$100 fi'action contains Golgi vesicles, whereas the P100 fiaction contains the large
Golgi stacks. An antibody directed against the coatamer protein B-COP was
employed as a Golgi marker, because it is commercially available. This protein is
found in the periphery of the Golgi complex and of a population of coatamers
scattered throughout the cyt0plasm. Therefore, B—COP may not be the most suitable
marker to examine the distribution of Golgi stacks in biochemical fiactionations,
since vesicles of Golgi origin may be present in the $100 fraction, and indeed B-COP
is detected in the S100 fiaction. A better analysis of the Golgi fiaction may require
the use of a protein, which resides within the Golgi complex. It was not possible to
use the anti-murine or-mannosidase H antibody, since the antibody does not
crossreact with the human homologue. A B-galactosyltransferase activity assay,
which is a commonly used and a sensitive method to determine Golgi fractions,
provide a better analysis of Golgi-containing fi'actions.

For many protein kinases it has been demonstrated that subcellular
distribution is important for the regulation of activity. Since Cdc42 changes SPRK's
subcellular distribution we wished to determine the effect of SPRK localization on
its kinase activity. Support for the idea that membrane translocation induces a
change in SPRK's phosphorylation comes from the electrophoretic mobility shift
which is observed when SPRK is coexpressed with constitutively active Cdc42.
However, preliminary comparative two-dimensional phosphopeptide mapping did

not show a dramatic change in the in viva phosphorylation pattern. However a

139

change of the phosphorylation pattern dependent on membrane translocation cannot
be excluded, because the Cdc42‘nm'38S variant has some residual plasma membrane-
binding activity. It may be worth while to repeat the phosphopeptide studies using
the Cdc42V‘2““"~""’IUQ variant, which is completely absent from the P16.9 fraction.

Examination of SPRK activity in total lysates upon coexpression with the
three Cdc42 variants showed no difference in autophosphorylation and substrate
phosphorylation. Interestingly, for K-Ras6Q, which exclusively localizes to the
cytosol (Stokoe et al., 1994) transforming activity has been reported when
overexpressed in NIH 3T3 cells (Cadwallader et al., 1994), suggesting that
overexpression may compensate for the impairment to associate with cellular
membranes and promote residual activity. Perhaps a similar phenomena is operating
here.

Further analysis of the plasma membrane-enriched fraction for SPRK kinase
activity shows a 3-fold higher kinase activity when SPRK is coexpressed with
constitutively active Cdc42 compared to SPRK alone, or to SPRK coexpressed with
Cdc42V12'C'888'm’Q. These results suggest that the physical presence of Cdc42 may
be required for SPRK activation rather then a specific subcellular localization. But
why is it not possible to recapitulate activation of SPRK by Cdc42 in vitro? It is
tempting to speculate that a third protein may be required to mediate Cdc42-induced
activation of SPRK and perhaps stabilize SPRK-Cdc42 interaction.

If membrane targeting is not required for the association of Cdc42 with
SPRK, membrane targeting of SPRK may increase SPRK's accessibility to activating

molecules. Small GTPases, for example, require guanine nucleotide exchange Ectors

140

(GEFs) to achieve their GTP-bound activated state. In yeast the activity of Cdc42 is
spatially and temporally regulated by the GEF Cdc24p, which localizes to the plasma
membrane (Michelitch and Chant, 1996). Also in mammalian cells GEFs regulate
the activity of small GTPases. One may envision that membrane targeting is
primarily required for Cdc42 activation by its GEF and not for SPRK activation per
se. Since constitutively active forms of Cdc42 were employed in this study the
requirement for Cdc42 activation was abrogated. To test whether the translocation
of Cdc42 to the plasma membrane is required for GTPase activity the experiments
should be repeated with non-prenylated Cdc42 variants, which exert wild type
GTPase activity.

Alternatively, membrane targeting of SPRK may increase the accessibility to
downstream targets and promote the formation of a signaling complex. Evidence for
Cdc42 as a membrane targeting molecule comes from a study investigating actin
polymerization and filopodium formation. This process requires the translocation of
WASP, a structural protein, by Cdc42 to the plasma membrane, which subsequently
induces the formation of a multiprotein complex (Castellano et al., 1999).

Although it is possible that the observed localization of SPRK to the Golgi
apparatus may be influenced by overexpression, preliminary analysis of the
subcellular distribution of endogenous SPRK shows that the distribution of SPRK is
similar to that seen with overexpressed SPRK. Various studies show that some
MAPKKK, such as SPRK, MLK2, and MEKKs, have high basal activity when
overexpressed. Recently it has been reported that microinjected MLK2 localizes to

microtubules in Swiss 3T3 cells (Nagata et al., 1998), whereas MEKKs localize to

141

the Golgi system (Fanger et al., 1997; Gerwins et al., 1997). To be kept away from
cellular substrates SPRK may be sequestered to the Golgi surface and then released
in response to an appropriate cellular signal. Recent studies in Drasaphila show that
components of the hedgehog signaling pathway are sequestered along microtubules
until they are released for activation (Robbins et al., 1997) . The Golgi apparatus
might have similar storage function.

The mixed-lineage kinase DLK has been localized to the cytoplasmic face of
the trans-Golgi in NIH 3T3 cells (Douziech et al., 1999). Since DLK localizes to the
Golgi system the authors speculate that this kinase may play a role in vesicular
transport during the secretory pathway. Further evidence for the role of rnixed-
lineage kinases in vesicular transport comes flour a study, which shows that
microinjected MLK2 in Swiss 3T3 cells colocalizes with INK], and INK2 at
punctuate structures along microtubules (Nagata et al., 1998). A yeast two-hybrid
experiment with a MLK2 and a SPRK variant revealed that both proteins interact
with KIF3, a member of the kinesin superfamily of motor proteins, and KAP3, a
kinectirr. Kinesins are motor proteins which transport cellular components, for
instance, vesicles, along microtubules towards the plus end (Brady and Sperry,
1995), hence from the Golgi apparatus in the direction of the plasma membrane.
Kinectins are the adaptors, which my regulate the binding of the cargo vesicles to
kinesins (Yamazaki et al., 1996). Furthermore, the MLK2-SH3 domain binds the
GTPase dynamin and cooperates with phospholipds to increase dynamin’s GTPase
activity (Rasmussen et al., 1998). Dynarnin is a GTPase, which has been

demonstrated to mediate vesicle formation during endocytosis, synaptic

142

transmission, and secretion (Urrutia et al., 1997). However, since this study was
only performed with the SH3 domin of MLK2 the physiological relevance of
MLK2 in the regulation of dynamin remains unclear. Also, Cdc42 has been shown
to associate in a two-hybrid assay with cytoskeletal elements (Dash er al., 1995) and
with kinectin (Hotta et al., 1996). Taken together these studies lead one to speculate
that mixed-lineage kinases in concert with GTPases are regulators of transport
processes along microtubules. Indeed, there is growing evidence that
phosphorylation is a way to regulate the activity of motor proteins. Two PKC Emily
members, for example, have been localized to the Golgi compartment and appear to
be important for regulating Golgi-related processes in NIH 3T3 cells, which my
include the regulation of the secretory pathway (Lehel et al., 1995; Prestle et al.,
1996).

Examination of SPRK distribution by confocal microscopy in COS-7 cells
when coexpressed with activated Cdc42 shows that less SPRK protein is present at
the Golgi apparatus as compared to when SPRK is overexpressed alone.
Furthermore SPRK showed a punctuate distribution within the cytosol when
coexpressed with activated Cdc42. Also a small amount of SPRK can be detected at
the plasma membrane. When analyzing the biochemical fiactions for SPRK
distribution upon coexpression with Cdc42V12 SPRK is present in the plasm
membrane-enriched fiaction to a much greater extent then can be accounted for by
plasm membrane staining of SPRK in COS-7 cells. A distinct punctuate
distribution of SPRK was observed in COS-7 cells. It is possible that the punctuate

structures represent some organized cytoskeletal elements. However, further

143

.-v

N

experiments are necessary to determine if the plasm membrane-enriched fi'action
contains cytoskeletal elements. Due to the observed staining pattern within the
cytosol it is tempting to speculate that the translocation takes place along
microtubules and that SPRK is transported along these cytoskeletal elements with
activated Cdc42 to its place of action. As mentioned above SPRK and Cdc42 have
been demonstrated to associate with components of the kinesin motor complex
(Hotta et al., 1996; Nagata et al., 1998). Additionally, a study which examined the
processing of Ras prenylation and Ras targeting within the cell showed that
farnesylated H-Ras and N-Ras associate first with the endoplasmtic reticulum and
the Golgi apparatus, before they are transported on the surEce of exocytotic vesicles
along microtubules to the plasm membrane (Choy et al., 1999). Also for Cdc42 one
my imagine a similar processing strategy, which my explain the association of the
small GTPase with the motor complex.

In this study we clearly demonstrate that SPRK largely associates with the
Golgi system and that upon coexpression with prenylated, activated Cdc42 the
protein kinase is shifted to a plasm membrane-enriched fiaction. Further
experiments will be necessary to determine if the plasm membrane-enriched
fiaction contains cytoskeletal elements. Whether SPRK is stored at the Golgi
apparatus and delivered to its site of action with or by Cdc42 along microtubules, or
if it plays a role in regulating vesicular transport per se will be open to further

investigations concerned with the physiological function of this protein kinase.

 

’ This expa'iment was performed by Panayiotis Vacratsis

144

V. Summary and Conclusions

The mjor objective of this thesis is to gain an understanding of how the smll
GTPase Cdc42 regulates the protein kinases SPRK The first part of this study was
focused on the structural requirements and mechanisms of Cdc42-induced SPRK
activation, whereas the second part was concerned with the role of Cdc42 in SPRK's
subcellular distribution and its effect on SPRK activity.

It was demonstrated that Cdc42 associates with SPRK in a GTP-dependent
mnner and that association does not require SPRK's kinase activity. A site-directed
mutagenesis approach was used to investigate the role of SPRK's CRIB motif on
SPRK-Cdc42 association and of Cdc42-induced activation of SPRK. The study
clearly demonstrates that SPRK contains a functional CRIB motif, which is required
for association with Cdc42 and Cdc42-induced activation. Additionally it was shown
that the zipper/basic stretch contributes to Cdc42 association of SPRK, confirming
that the CRIB motif is indeed necessary, but not suflicient, for the SPRK-Cdc42
interaction. Interestingly, the zipper/basic stretch not only contributes to Cdc42
binding, but it also plays an important role in transducing signals to downstream
targets that lead to the activation of INK Upon coexpression of SPRK with
activated Cdc42 in 293 cells an increase in SPRK activity was demonstrated in in
vitro kinase assays, even though Cdc42 cannot be detected in these kinase assays.
This result supports the idea of a catalytic model for SPRK activation by Cdc42, in

which the GTPase binds to SPRK and activates the kinase in viva, but is not required

145

to remin associated with SPRK to mintain the activated state. Comparative two-
dirnensional in viva phosphopeptide mpping clearly showed tint Cdc42 changes the
in viva phosphorylation pattern of SPRK, which is correlated with an increase in
kinase activity. Interestingly, an increase in kinase activity of SPRK cannot be
detected in an in vitro kimse assay using recombinant, purified proteins. Taken
together these results suggest that Cdc42-induced SPRK activation requires the
cellular environment.

To further analyze the mechanism by which Cdc42 activates SPRK, the effect of
the smll GTPase on the subcellular distribution of SPRK was examined. Upon
overexpression in COS-7 cells SPRK localizes predominantly to the Golgi apparatus.
Cdc42 changes SPRK's subcellular distribution by targeting the kinase presumbly to
the plasma membrane. This change in subcellular distribution of SPRK by Cdc42
requires membrane targeting of Cdc42, since a Cdc42 variant lacking the prenylation
signal fails to change SPRK subcellular distribution. In addition, coexpression with
prenylated, activated Cdc42 reduces the electrophoretic mobility of SPRK, whereas
the prenylation-defective mutant Eils to induce this mobility shift, perhaps indicating
a change in phosphorylation. However, the SPRK kinase activity in total cellular
lysates is the same whether SPRK was coexpressed with prenylated Cdc42V12 or the
non-prenylated Cdc42Vlz variants. Thus it was not possible to show that membrane

targeting is required for Cdc42-induced activation of SPRK.

146

Future Studies

Analysis of SPRK's subcellular distribution showed that Cdc42 targets SPRK to
a plasm membrane-enriched fi‘action. However, it was not possible to correlate the
translocation of SPRK by Cdc42 with an increase in SPRK activity. Comparative
two-dimensional in viva phosphopeptide mpping of SPRK when coexpressed with
Cdc42‘"“"188$ resulted in an intermediate phenotype allowing no conclusion about
the requirement of membrane targeting for a change in in viva phosphorylation of
SPRK. The Cdc42v'2‘C1888 variant retains residual membrane association as judged
by biochemical fractionations and confocal microscopy. Therefore it my be
feasible that the residual membrane association induces a partial activation of SPRK.
To more rigorously determine the effect of membrane targeting by Cdc42 on the in
viva phophorylation of SPRK in viva phosphopeptide mapping should be repeated
using the Cdc42V'2'C1888‘m’Q variant, which is devoid of membrane association.

Preliminary membrane fi'actionation experiments using 293 cells have
demonstrated that it is possible to examine the subcellular distribution of endogenous
SPRK and to determine its kinase activity. Examining the effect of Cdc42 on
endogenous SPRK my give a better reflection of the in viva situation than the
overexpression system, which was used in this study.

Smll GTPases are activated by GEFs. The yeast GEF of Cdc42, termed
Cdc24p, requires membrane targeting to promote guanine-nucleotide exchange and
to activate Cdc42. Therefore one my envision a scenario in which actually the

requirement for membrane translocation lies with Cdc42 and not with SPRK. In this

147

model translocation is not required for SPRK activation per se, but rather for
activation of Cdc42. Then upon activation, Cdc42 induces SPRK activation. The
experiments described in this thesis were done using constitutively active Cdc42
variants, abrogating the requirement for guanine-nucleotide excimnge. To test
whether membrane targeting is required for Cdc42 it my be useful to employ non-
prenylated Cdc42 variants with wild type GTPase activity.

To complete the biochemical fi'actionation studies, the content of the subcellular
fiactions need to be more precisely determined. The study clearly shows that the
P16.9 fiaction is the only subcellular fiaction containing integral plasma membrane
proteins. However, this fraction also contains endoplasmic reticulum. According to
the literature the contamination of a plasm membrane fiaction with endoplasmic
reticulum is very common and a clean plasm membrane fi‘action is not trivial to
obtain. Therefore colocalization of SPRK with the endoplasmic reticulum and the
plasm membrane, by confocal microscopy my be useful to differentiate between
these two cellular components. The content of the P100 fraction has definitely to be
determined, since SPRK protein is detected in this fraction. Based on the literature
the P100 fraction my contain Golgi membranes. Commercially available antibodies
against humn Golgi proteins are often directed against sru'Ece proteins of the Golgi
complex. Therefore these proteins are often found on budding Golgi vesicles. These
vesicles my fiactionate differently fiom the main Golgi stacks due to their different
density. To determine the presence of the Golgi membranes in the fractions 3

galactosyltransferase activity assay should be performed. Furthermore this study did

148

not examine if the plasm membrane-enriched fraction also contains cytoskeletal
elements. Since some evidence exists that SPRK my localize to cytoskeleton, in
particular to microtubules, it would be of great interest to determine if the plasm
membrane-enriched fiaction contains cytoskeletal elements.

The results obtained with the biochemical fractionation experiments are highly
reproducible and clearly indicate that prenylated activated Cdc42 changes the
subcellular location of SPRK However the colocalization experiments using
confocal microscopy were plagued with technical problems, which were certainly in
part due to the aberrant phenotype, which is induced upon overexpression of SPRK
and Cdc42. The development of cell lines with inducible Cdc42 expression my be a
usefirl way to monitor the subcellular localization of SPRK in the presence and
absence of Cdc42 by confocal microscopy avoiding the observed morphological
changes.

In conclusion this study provides important insights into the requirements and
mechanism of SPRK activation by Cdc42. Understanding the mechanism by which
the small GTPase Cdc42 regulates and activates the protein kinase SPRK my also
provide valuable knowledge about the role of SPRK and Cdc42 in INK activation.
Recent evidence supports the idea that SPRK my play a role in the immune
response and in cellular transformtion. Understanding the mechanism by which
SPRK is regulated my open the door to the development of specific inhibitors of
SPRK and/or mixed-lineage kinases in general, which my serve as useful

therapeutics.

149

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