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1198 chiRC/DatoDmpGS—pu

REGULATION OF CD45 TYROSINE
PHOSPHATASE ACTIVITY BY CASEIN KINASE 2

By

Wei Guo

A THESIS

Submitted to
Michigan State University
in partial fitlfillment of the requirements
for the Degree of

MASTER OF SCIENCE

Department of Microbiology

1 999

ABSTRACT
REGULATION OF CD45 TYROSINE
PHOSPHATASE ACTIVITY BY CASEIN KINASE 2
By

Wei Guo

CD45, a transmembrane protein tyrosine phosphatase (PTP), is critical in lymphocyte
activation and proliferation. Previous work in our lab established that casein kinase2
(CK2) is a major kinase that phosphorylates CD45 at the 19 amino acid acidic insert of
D2 domain. In this study, recombinant CD45 and its CK2 site mutants were expressed
and purified from E. coli and these proteins were used to examine the effects of
phosphorylation on its PTP activities. PTP activities of phosphorylated and non-
phosphorylated CD45 were compared using pNPP, Raytide, Myelin Basic Protein (MBP)
and recombinant Lck as substrates. It was found that phosphorylation of CD45 increased
its PTP activity toward myelin basic protein three fold and the increased activity was
reversed through dephosphorylation by PP2A, a serine phosphatase. Kinetic analysis
indicated that the increase of CD45 activity by phosphorylation was mainly due to an
increase of Vmax. However, no significant differences of PTP activity between
unphosphorylated and CK2-phosphorylated CD45 were observed using pNPP, Raytide
and recombinant Lck. In addition, our data showed that CD45 was able to differentially
dephosphorylate two Lck phosphorylation sites. Taken together, these data suggest that
CD45 has strong substrate specificity and phosphorylation of CD45 by CK2 may be one

of the mechanisms to regulate the PTP activity of CD45.

To my wife, Ying Wang
&
to my parents and sisters
Without their love and sustained support,

this could not be achieved.

ACKNOWLEDGEMENT

I am deeply grateful to my supervisor, Dr. Walter J. Esselman, for his inspiration and
his generous help through my graduate study. Working with him is not only a pleasant
and challenging but also a great experience that I will never forget in my life. Also I
would like to express my gratitude to my committee members: Dr. Ronald Patterson, Dr.
Richard Schwartz and Dr. Michael Garavito for their invaluable advice and their
understanding. I especially thank Dr. David DeWitt for his suggestions and technical
support in expression of recombinant Lck from insect cells. I thank Dr. Honggao Yan for
his helpful discussion in designing and interpreting of kinetics experiments. I also owe
my thanks to Dr. David McConnell for his support during my graduate career. I have
truly enjoyed working with my collogues, Lianzhu Liang and Lanlan Li, and thank them
for their friendship. Finally, I thank all members from Dr. Susan Conrad and Dr.

Catherine Gallo's lab. From the workshop with these labs, I have learned a lot in the area

of signal transduction and cell cycle.

TABLE OF CONTENTS

List of Tables ........................................................................................ vii
List of Figures ....................................................................................... viii
List of Abbreviations ................................................................................ ix
Chapter 1. Literature Review ........................................................................ 1
Introduction .................................................................................. 2
Protein Tyrosine Phosphatases ............................................................ 3
T Cell Antigen Receptor Signaling ..................................................... .8
CD45 and Its Biological Functions in Lymphocyte
Activation and Development ............................................................. 16
Conclusions ............................................................................... .22
Reference List ............................................................................. 24

Chapter 2. Regulation of CD45 Tyrosine Phosphatase Activity

by Casein Kinase 2 ......................................................................... 32
Introduction ............................................................................... .33
Materials and Methods .................................................................. .35
Cells Lines, Antibodies and Reagents ........................................ 35
Expression Vectors ............................................................ 35

Expression and Purification of Recombinant CD45, Lck, and
Src Kinase from E. Coli ....................................................... 35

Site-directed Mutagenesis and Construction of pFastBac-Lck(K/R). . .37
Transposition and Isolation of Recombinant Bacmid DNA .............. 37

Transfection of Sf21 Cells with Bacmid DNA and Screening
of Recombinant Baculovirus ................................................. 38

Infection and Purification of Recombinant Baculovirus Lck from
Sf21 Insect Cells ............................................................... 38

SfZl Insect Cells ............................................................... 38

Radioactive Labeling of Protein Substrates ................................. 39
PTP Assays ..................................................................... 4O
CK2 Phosphorylation of Hiss-cthD45 ..................................... 4O
FPLC Analysis .................................................................. 41
SDS-PAGE and Western Blot ................................................ 41
Kinetics Analysis ................................................................ 41
Protein Quantitative Analysis ................................................ 42
Results ..................................................................................... .43

Expression and Purification of CD45 and Its CK2 Site Mutants... .. ....43

CD45 Is Multiply Phosphorylated by CK2 ................................. 46
Kinetic Analysis of CD45 Phosphorylation by CK2 ...................... 46
Serine Phosphorylation of The Acidic Insert in The D2 Domain
Increases CD45 Activity Using MBP As Substrate ........................ 51
Effect of PP2A on CD45 Activity ........................................... 54
Kinetic Comparison of CD45 and Its CK2 Mutants ....................... 57
CD45 Differentially Dephosphorylates Lck Phosphorylation Sites. ....57
Discussion .................................................................................... 65
Reference.List .............................................................................. 70

vi

LIST OF TABLES

Table I. Kinetic parameters of the CK2 site-mutated
His6-cthD45 with 32P MBP as substrate ..................................................... 59

vii

LIST OF FIGURES

1. Schematic representation of CD45 cytoplasmic domain

and its phosphorylation sites .................................................................... 44
2. FPLC chromatography of three types of recombinant CD45 .............................. 45
3. SDS-PAGE analysis of purified recombinant CD45 proteins ............................. 47
4. Time course analysis of phosphorylation of CD45 by CK2 ............................... 48
5. Analytical F PLC separation of CK2-phosphorylated CD45 ............................... 49
6. Kinetic analysis of phosphorylation of CD45 by CK2 ..................................... 50
7. Comparison of PTP activities of recombinant wild type

D45 and CK2 site mutants using pNPP as substrate ........................................ 51
8. Comparison of PTP activity of recombinant wild type CD45

and CK2 site mutant CD45 using 32P-Raytide as substrate ................................. 53
9. Effect of phosphorylation of CD45 on PTP activity using 32P-MBP as substrate ...... 55
lO.Dephosphorylation of CD45 by PP2A: Effect on PTP activity ........................... 56
11.Kinetic comparison of wild type CD45, S/A and SE mutants

using 32P-MBP as substrate ..................................................................... 58
12.Selection of SB] clones expressing inactive Lck (I-Lck(K/R) ............................ 61

13.Comparison of autophosphorylation and Csk phosphorylation

of inactive Lck [I-Lck(K/R)] ................................................................... 62

l4.Comparison of PTP activity of mutant forms of CD45 with Csk

or autophosphorylated Lck as substrates ..................................................... 63

viii

LIST OF ABBREVIATIONS

PTP, protein tyrosine phosphatase; PTK, protein tyrosine kinase; TCR, T cell antigen
receptor; RPTPs, receptor-like protein tyrosine phosphatases; DSPs, dual-specificity
phosphatases; LMPs, low molecular weight (acid) phosphatases; MHC, major histo-
compatibility complex; ITAMs, immunoreceptor tyrosine-based activation motifs; SDS-
PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; Csk, c-Src kinase;
CK2, casein kinase 2; MBP, myelin basic protein; His6-cthD45, 6-His tagged form of

cytoplasmic domain of CD45; pNPP, p-nitrophenylphosphate.

Chapter 1.

Literature Review

Introduction

Immune system is an exquisite and complicated system functioning to protect
vertebrates from invasion of pathogens. This protection is carried out by two steps:
immune recognition and immune response. Recognition of self- and foreign antigens by
the immune system will trigger appropriate responses by a variety of molecules and cells.
Lymphocytes are one of the most important players in this process. Lymphocytes include
T and B lymphocytes, both of which originate from hematopoietic stem cells. Although
T and B cells have distinct functions in immune recognition and response, the molecular
signaling mechanisms employed by these two cells are similar in many respects. Due to
the crucial role of lymphocytes in the immune system, understanding lymphocyte
function at the molecular level has always been an important subject in immunology.

The response of lymphocytes to antigen stimulation requires the activation of
different signaling pathways. As noted in many other cell types, protein phosphorylation
and dephosphorylation is a general mechanism used in lymphocyte activation. The
importance of protein phosphorylation in control of cellular process was recognized as
early as the 1950's. Tyrosine phosphorylation was noticed after a protein tyrosine kinase
(PTK), Src kinase, had been purified. Since their catalytic domains are relatively
conserved, many protein tyrosine kinases have been identified by screening of cDNA
libraries at low stringency with kinase probes. To date, it has been well established that
protein tyrosine phosphorylation plays important roles in regulation of cellular growth,
differentiation, morphology and movement. In contrast, protein tyrosine
dephosphorylation was poorly understood until the first protein tyrosine phosphatase

(PTP) — PTPlB was characterized about ten years ago (Tonks et al., 1988a). In recent

years, advances in PTP studies have enriched our knowledge about how cells use delicate
phosphorylation/dephosphorylation mechanism to maintain their normal functions.
Lymphocyte signaling is a good example to promote the understanding of the dual
regulation by both PTKs and PTPs.

This review summarizes the functions of PTPs in the context of antigen
stimulated lymphocyte signaling in three parts. The first part provides general
information about the PTP family including the structure, catalytic action and activity
regulation. T cell antigen receptor (TCR) signaling will be discussed in the second part.
The third part focuses on how CD45, one of most important PTPs, acts as a key regulator
of lymphocyte activation and development. Evidence presented here strongly supports
the notion that PTPs are crucial for cellular response to extracellular stimuli as well as

having housekeeping functions.

Protein Tyrosine Phosphatases

PT P Family. It is estimated that there are as many as 500 human PTPs based on
genomic data (Neel and Tonks, 1997). To date, at least 75 PTPs have been identified.
These PTPs can be divided into four families: receptor-like PTPs (RPTPs), nonreceptor
PTPs, dual-specificity phosphatases (DSPs) and low molecular weight (acid)
phosphatases (LMPs). The RPTPs and nonreceptor PTPs are also referred to as "classical
PTPs". Every PTP has at least one catalytic domain, which is around 240 amino acids
with a unique signature motif ([IN]HCxAGxxR[S/T]). The invariant cysteine is
important for its enzymatic activity. Mutation of this residue eliminates all enzymatic

activity in PTPs (Streuli et al., 1989). Most RPTPs have two tandem catalytic domains

— a proximal domain (D1 domain) and a distal domain (D2 domain) based on their
relative position to the cytoplasmic membrane. It is generally believed that only the D1
domain has catalytic activity and the D2 domain exists in an inactive form. RPTPs have
a variety of extracellular domains and a short transmembrane region. Several structural
motifs such as fibronectin type-III, MAM, and "carbonic anhydrase" have been identified
in the extracellular domain of different RPTPs, which may relate to different
physiological functions. In contrast, all nonreceptor PTPs have only one catalytic domain
and no extracellular or transmembrane region. Some domains, such as 8H2, PEST and
Band 4.1, have been found in nonreceptor PTPs fused to either the amino- or carboxyl-
terminal of the PTP domain. These additional domains are believed to regulate
localization, enzymatic activity or protein-protein-protein interaction, and therefore serve
as regulatory domains. DSPs dephosphorylate phosphotyrosine and phosphothreonine in
a sequence specific way. MKP-l, PAC-l, cdc25A,B,C and VH-l are all members of this
family. LMPs have the lowest homology with other three families but they retain the
basic structure of the PTP catalytic domain. Little is known about this family.

Structural Features. Crystal structures of PTPs resolved in recent years provide
insights to the function of PTPs in signal transduction pathways. These structures
suggest that all classic PTPs and DSPs have similar three dimension architecture,
although overall sequence identity between them is only less than 5% (Barford et al.,
1994; Yuvaniyama et al., 1996). Basically, the catalytic domain has a (1+5 sandwich
structure. F our paralleled B-strands form a twisted sheet in the center with several

surrounding a helices. The residues of PTP signature motif are within a single loop

between B strand and on helix, and they form a pocket for incoming phosphotyrosine on

the substrate. At the base of the pocket is the catalytic cysteine (CyleS, numbered
according to PTPlB). Negatively charged Aspl 81 interacts directly with substrate
phosphate and undergoes a dramatic conformation change. The guanidinium group of
Arg221 facing the active site assists the phosphate binding. Tyr46 on a loop between or]
and [31 set the depth of the pocket and this depth determines the specificity of PTPs (Jia
et al., 1995). Compared with DSPs, tyrosine specific phosphatases have a deeper cleft in
which only phosphotyrosine moiety is long enough to reach the catalytic cysteine. The
domain structure of RPTPs is similar to the structure of PTPlB. A resolved structure
showed that RPTP exists as a symmetrical dimer. The helix-turn-helix structure from one
monomer acts as a wedge and inserts into the catalytic cleft of the other monomer. The
symmetrical insertion of the wedges from each monomer blocks the access of substrates
into catalytic clefts and thus results in a inactive enzyme. Two highly conserved acidic
residues within the wedge are thought to be critical to inhibit the enzymatic activity.
Therefore, the unique structure of RPTP implies that dimerization is a important
regulatory mechanism used by the RPTP family (Bilwes et al., 1996). Although the
overall structure of LMPs is totally different from the other three families, the structures
in the active site among these families are all similar suggesting that PTPs utilize a
common catalytic mechanism (Su et al., 1994; Zhang et al., 1994).

Enzymatic Reaction and Kinetics. The enzymatic mechanism for PTPs has been
extensively studied using a number of specific PTP inhibitors. The two-step reaction can

be represented by the equation shown below (Denu et al., 1996):

Kd TyFOH K(hydrolysis)

E+S —> ES EPO3 —> E+HP02

K(formation)

First, the unprotonated thiolate group of Cys215 attacks the phosphorus atom of the
substrate and releases dephosphorylated substrate. As a consequence, a thiol-phosphate
intermediate is formed. Then, this thiol-phosphate is hydrolyzed due to the nucleophilic
attack of activated water toward the phosphorus atom again. The second reaction is
generally considered as a rate-limiting step. Evidence from X-ray structures suggests that
both Arg221 and Asp181 are important for the catalysis. Protonated Asp181 as a general
acid binds with the phosphotyrosine substrate through hydrogen bond while the nitrogens
from Arg221 side chain and the amide groups from the active-site loop stabilize the
dianions of the phosphotyrosine. Once the phosphoenzyme immediate is formed,
Asp181 as a general base activates the water to facilitate hydrolysis. Dramatic movement
of Asp181 (about 8-12 A) has been noticed during the formation of phosphoenzyme
immediate. Asp181 to Ala mutant abolishes the catalytic activity and thus this mutant is
useful for substrate trapping.

Kinetics studies have showed that PTPs have high specific activities for artificial
substrates. However, free pTyr is a poor substrate (Km = 5 mM) demonstrating additional
interaction is important for the reaction. The specific activities of PTPlB using RCML
and MBP as substrates are up to 1000 fold higher than those of receptor-linked PTKs
(Tonks et al., 1988b). This implied that maintenance of low level of dephosphorylation
by PTPs might be necessary in control of normal cellular function. Although each PTP
has a preference for particular substrates, the Km of PTPs ranges within three orders of

magnitude.

Regulation of PTP Activity. The mechanism on the regulation of PTP activity is
not fully understood. Since some PTPs can be phosphorylated by serine/threonine and
tyrosine kinases, it is possible that PTP activities are regulated through phosphorylation.
Indeed, some studies indicated that phosphorylation of PTPs influence their activity.
However, these studies are inconsistent on the issue of whether phosphorylation up- or
down- regulates PTP activity. For example, the overall PTP activity increased when CV-
] kidney cells were treated with PKA/PKC activators or phosphatase inhibitors
(Brautigan and Pinault, 1991). Similarly, decreased CD45 activity induced by ionomycin
treatment correlated with decreased phosphorylation (Ostergaard and Trowbridge, 1991).
However, TPA treatment of human peripheral lymphocytes increased the
phosphorylation of CD45 but decreased its activity (Yamada et al., 1990). In addition,
several other reports showed that the in vitro phosphorylation of PTPs had no effect on
their activities (Pot et al., 1991; Stover et al., 1991; Tonks et al., 1990).

Based on the similarity of RPTPs with RPTKs, a regulation mechanism via ligand
binding has been speculated. To date, in spite of tremendous effort, few ligands have
been identified, and some ligands of RPTPs are not necessary to regulate enzymatic
activities of RPTPs. For example, no changes of the phosphatase activity and
downstream signaling have been detected after PTPB/Q bound with its ligand—contactin
(PELES et al., 1995). However, evidence derived from the crystal structure of PTPa
supports the hypothesis. EGF also can inhibit the T cell signaling mediated by EGF-
receptor/CD45 chimeras of ligand-mediated regulation through dimerization (Desai et al.,
1993; Majeti et al., 1998). The current model is that transducing of extracellular signals

involves a synergistic control of tyrosine phosphorylation, the activation of RPTKs and

inhibition of RPTPs by surface bound ligands (Neel and Tonks, 1997). Additional

evidence will be required to support this hypothesis.

T Cell Antigen Receptor Signaling

T cell activation is one of the important events during immune response against
pathogen and foreign substances. Activation also plays an important role during the
development of T cells in the thymus. Great progress has been made in the
understanding of the details of how this process occurs since the T cell antigen receptor
was identified in early 1980's. The study of T cell activation set a paradigm for signal
transduction, and yet many questions still remain to be answered. Overall, T cell
signaling includes four steps: 1) TCR binding to antigen-bound major histocompatibility
complex (MHC) -- this binding initiates the T cell signaling; 2) TCR-regulated protein
tyrosine kinases (PTKs) activation -- multiple tyrosine phosphorylation events result in
the activation of downstream signal pathways. 3) Cellular responses and related genes are
expressed. 4) TCR activation is terminated by negative regulators.

T Cell Antigen Receptors. The TCR complex consists of eight subunits derived
from six different polypeptide chains. These eight subunits are grouped into four hetero-
or homo- dimers: a/B, CD3y/CD33, CD3c/CD35 and TCRQIQ. While a/B dimers
function as a ligand-binding site, the other three dimers function in receptor assembly,
cell surface expression and signal transduction. Less than 5% of peripheral blood T cells
express 7/8 chains, instead of a/B, as an alternative ligand-binding site. An important
feature of the TCR is the presence of ten immunoreceptor tyrosine-based activation

motifs (ITAMs) in the cytoplasmic domains of CD3'Y/CD38 and CD3e/CD35. In

contrast, each TCRC/C; has three pairs of ITAMs in a tandem arrangement. The
consensus sequence on each ITAM has been identified as YxxL(x)5-ngxL (Qian and
Weiss, 1997). These ITAMs are phosphorylated upon TCR activation, and
phosphorylated tyrosines recruit SH2-containing proteins to TCR complex. Since one
TCR contains as many as ten ITAMs, these ITAMs are thought to function in amplifying
signals received by TCR (Weiss et al., 1994). Studies also suggest that the binding of
SH2-containing proteins with ITAMs is sequence specific, the specificity decides which
downstream pathways will be turned on under certain condition (Exley et al., 1994).
Thus, it is possible that T cells may be differentially activated via TCR-controlled
signaling. In addition, an intact ITAM is required for the association of TCRQ and CD38
with cytoskeleton (Caplan and Baniyash, 1995; Rozdzial et al., 1995).

TCR-regulated Protein Tyrosine Kinases (PT Ks). Tyrosine phosphorylation is an
important step for the initiation of downstream signaling pathways. Upon engagement of
TCR by antigen or anti-TCR antibodies, PTKs were activated and many proteins are
immediately phosphorylated by PTKs.. At least four families of PTKs including Src,
Csk, Tec and Syk families have been found in T cells (Qian and Weiss, 1997). Many of
these are positive regulators while some kinases such as Csk have negative effects on
TCR signaling. A few domain structures have been identified in these families. All four
families have SH2 domains, and three families have SH3 domain. The Tee family has
additional pleckstrin homology (PH) and Tec homology (TH) domains. These domains
are modules responsible for protein-protein and protein-lipid interaction (Pawson, 1995).
Extensive studies of these PTKs in recent years provide a lot of details about T cell

activation. Several important PTKs are briefly discussed as follows.

l) Lck and F yn. Both of these PTKs belong to Src kinase family and they have
similar structures: a myristylated glycine at the N-terminal, a kinase domain at its C-
terminal, a tandem SH3 and SH2 domain in the middle. The myristylated site facilitates
association with the plasma membrane. For Lck, this binding is related to its function in
TCR signaling (Veillette et al., 1989). There are two phosphorylated sites on Lck (Y505
and Y394). When Y505 is phosphorylated, the intramolecular interaction between Y505
and SH2 domain folds Lck into a loop and this conformation is inactive. Once this
conformation is formed, the autophosphorylation site Y394 is exposed and can be
accessed by other molecules. Studies showed that Lck is associated with CD4 or CD8
and this association is required for TCR mediated activation. In CD4'CD8' T cells, TCR
function was restored only by expression of CD4 with Lck binding site. CD4 mutants
without this binding site failed to restore normal TCR signaling (Glaichenhaus et al.,
1991).

Evidence suggests that Lck is critical in T cell activation. Lck negative cell lines
were unable to mobilize Ca2+ and to express activated antigens (Straus and Weiss, 1992).
Overexpression of Lck in murine T cells increased the level of tyrosine phosphorylation
and secretion of IL-2 (Abraham et al., 1991). Lck is also important for thymocyte
development. In Lck deficient mice, thymocyte numbers were dramatically decreased
(Molina et al., 1992) . While Lck was found to be associated with CD4 or CD8, co-

immunoprecipitation and immunofluorescence demonstrated that Fyn was associated
with TCR (; chain. Generally, Fyn has similar functions with Lck in TCR signaling, but

Fyn was not required for thymocyte development and proliferation in peripheral cells

10

(Appleby et al., 1992; Stein et al., 1992) . In addition, Fyn is not able to compensate for
Lck deficiency in Lck negative cells (Karnitz et al., 1992).

2) Syk and ZAP- 70. Syk and ZAP-70 play different roles in lymphocyte
activation and development although they have similar structures. Syk is important for B
cell antigen receptor signaling and B cell development. However, the T cell development
and TCR signaling were normal in Syk deficient mice (Cheng et al., 1995; Turner et al.,
1995) . In contrast, ZAP-70 was essential for TCR mediated pathways. Abnorrnally
expressed ZAP-70 in human cells resulted in severe combined immunodeficiency (Arpaia
et al., 1994; Chan et al., 1994; Elder et al., 1994). At least three tyrosine phosphorylation
sites, Y292, Y492 and Y493, have been identified within ZAP-70 (Watts et al., 1994).
While Y492 and Y493 locate inside the kinase domain, Y292 sits on the loop between
SH2 and kinase domain. Studies showed that Y493 is phosphorylated by a Src family
kinase and the phosphorylation further activates the kinase activity of ZAP-70. Also
phosphorylation of Y493 is required of the subsequent phosphorylation of Y492, Y292
and other sites, but how the sequential phosphorylation occurs is unclear. Y492F mutants
increase the kinase activity of ZAP-70, phosphorylation on Y492 probably turns down
the kinase activity. Unlike Y492 and Y493 mutants, Y292F had no effects on the kinase
activity and may only serve as a binding site for recruitment of other signal molecules

(Wange etal., 1995).

As its name indicated, ZAP-70 is a Q chain associated protein. The association of
ZAP-70 with Q chain upon TCR activation is important for initiating T cell signaling.
Two tandem SH2 domains on ZAP-70 bind two tyrosine phosphates on the ITAM of Q

chain. This high affinity binding facilitates the phosphorylation and activation of ZAP-

ll

70 by Src family kinases. Crystal structure of ITAM bound ZAP-70 showed that they
interact with each other in an anti-parallel way. In other words, the N-terminal SH2 of
ZAP-70 binds with C-terminal tyrosine phosphate on ITAM while the C-terminal SH2 of
ZAP-70 binds with N-terminal tyrosine phosphate on ITAM (Hatada et al., 1995). A
constitutive association between ZAP-70 and TCR (; chain was observed in murine
thymocytes and lymph node T cells. However, ZAP-70 in resting T cells was
unphosphorylated and' inactive until TCR was activated (vanOers et al., 1994). This
provides evidence that phosphorylation and activation of ZAP-70 is critical to TCR
signaling.

3) Csk and Itk. As mentioned above, Csk has negative effects on Src family
kinase. Phosphorylation on the C-terminal end of Lck and Fyn by Csk inactivated their
kinase activities. Itk is a member of Tec family kinases. Itk is a potential positive
regulator of TCR signaling since the number of thymocytes and CD4+ T cells was
abnormally low in Itk deficient mice (Liao and Littman, 1995). Further studies are
needed to elucidate the details.

Downstream Signaling Pathways. It is believed that antigen presentation to T
cells activates multiple pathways although some links to these pathways are still missing.
Activation of PTKs transduces signals in at least three parallel pathways: 1) PLCy
mediated lipid hydrolysis, Ca"+ mobilization and PKC activation. Ca2+ mobilization
results in the activation of Calcineurin, a serine/threonine phosphatase. Then NFAT is
dephosphorylated and activated by Calcineurin. 2) Ras/MAPK pathway. Activated
MAPK phosphorylates Elk, which then activates Fos transcription. 3) Rae/.HVK pathway.

Activated INK phosphorylates Jun, which combines with Fos to form AP-l. Once

12

transcription factors, such as NF AT, AP-l, NF «B and Oct-1, are activated, transcription
of cytokine genes like IL-2 will be stimulated. Thus, these activated pathways finally
result in T cell functional responses including cytokine secretion, proliferation and
cytolytic activity (Osman et al., 1996). Accumulated evidences suggest that ZAP-70 was
required for Ca2+ mobilization and activation of MAP kinase. ZAP-70 has been shown to
interact with many signaling molecules such as Lck, Abl, GAP, Vav, SHP-l, Cbl, LAT
and SLP-76 (Qian and Weiss, 1997; Peterson et al., 1998). Some of these signaling
proteins are substrates of ZAP-70. The interactions of ZAP-70 with downstream
pathways were partly, if not all, mediated by LAT. LAT is one of the adaptor proteins
specifically expressed in hematopoietic cells excluding B cells (Zhang et al., 1998). A
transmembrane domain was identified within LAT, and several tyrosines were found to
mediate the interactions of LAT with other SH2 containing proteins. Co-
irnmunoprecipitation showed those LAT associates with several signaling molecules

including Grb2, PI3K, PLCyl, SLP-76, Cbl and 803. This provided evidence that LAT

may couple Ca2+ mobilization by interacting with PLCy and couple Ras/MAPK pathway
by interacting with Grb2/Sos in T cells. In B cells, Shc instead of LAT recruits Grb2/Sos
to membrane and activates Ras/MAPK pathway. Besides LAT, other adaptor proteins
such as SLP-76, SLAP/Fyb and SKAP-55 also participate in the activation of Ras/MAPK
pathway. In addition, it has been known that SLP-76 can associate with Vav upon
stimulation (Michel et al., 1998; Wu et al., 1996). However, this pathway has not been
completely defined.

Negative Regulation of T Cell Activation. In order to maintain their normal

functions, T cells must shut down the activation by antigen presentation. To date, little is

13

known about how TCR signaling is terminated and how T cells keep a dynamic balance
between activation and inhibition. Studies suggest that the termination of T cell signaling
is controlled by multiple signal proteins including receptors, PTKs, PTPs and adaptor
proteins. Csk is the only PTK which has negative effects on TCR activation. Evidence
indicated that several receptors such as CD5, CTLA-4 and KIRs were involved in
negative regulation of T cell signaling. It has been known that the negative regulation by
CTLA-4 and KIRs is mediated by SHP-l or SHP-2 (Burshtyn and Long, 1997;
Marengere et al., 1996). However, the mediators of CD5 have not been found. In
addition, some adaptor proteins are suggested to be negative regulators. One of these
adaptors is Cbl which is able to associate with a variety of SH2 containing proteins such
as Vav, Crk, PI3K, Fyn and Syk family kinases (Peterson et al., 1998). There are two
hypotheses about the inhibitory mechanisms by Cbl. One is that the association of Cbl
with Syk family kinases blocks the recruitment of these kinases to activated receptors
(Ota and Samelson, 1997). The other is that Cbl can interact with CrkL/C3G complex
and result in the activation of Rapl , the activated Rapl competes with Ras for binding
Raf kinase, which results in the blockage of the Ras/MAPK pathway (Boussiotis et al.,
1997; Ichiba et al., 1997).

Extracellular Interactions and T cell Activation. Extensive investigation of the
signaling pathways in T cells have advanced our understanding of TCR activation. In
contrast, studies on how surface molecules of T cells initiate the signaling cascade are
just under way. Several questions have been raised about the interactions of TCR and
MHC complex such as steric hindrance, ligand concentration and affinity. l) Steric

hindrance. 30% of T cell surface is occupied by negatively charged glycoproteins.

14

These proteins have as long as 45 nm of extracellular domains. Thus TCRs are deeply
buried by these proteins (Cyster et al., 1991). To reach these TCRs, peptide-MHC
complexes must overcome not only electrical repulsion but also the steric hindrance. 2)
Low ligand concentration. It is estimated that the density of TCRs is about 200
molecules/um2 while the density of the peptide-MHC is only 0.1-1 molecules/um2 (Shaw
and Dustin, 1997). How can a single peptide-MHC activate up to 100 TCRs? 3) Low
aflinity. The dissociation constants between soluble TCR and peptide bound MHC range
from 10'4 to 10'7 M while the dissociation (constants between antigens and
immunoglobulins are less than 10'9 M. How can this weak interaction trigger a long
lasting activation event?

To answer these questions, a topological model for T cell activation has been
proposed (Shaw and Dustin, 1997). This model suggests that T cells must pass through
three checkpoints to reach maximal activation. The first checkpoint is integrin mediated
cell adhesion. Since the interaction between integrins is not affected by charge repulsion,
integrin mediated cell adhesion shortens the distance between two cells and thus peptide-
bound MHC can contact the buried TCRs. The second checkpoint is the formation of a
zone excluding PTPs, especially CD45. The exclusion of PTPs provides time for TCRs
initial tyrosine phosphorylation and Ca2+ mobilization. The third checkpoint is the
formation of contact cap. If the amount of TCR and peptide-MHC complex is sufficient,
a stable contact will be formed. In this model, time is thought to be a decisive factor. It
takes seconds for first step, minutes for second step, hours for third step. If a contact cap
can maintain several hours, a fully activated T cells is achieved. This model argues that

the rearrangement of proteins within a contact cap, rather than the engagement of TCR by

15

antigens, is critical to TCR activation. However, further evidence is needed to

substantiate this hypothesis.

CD45 and Its Biological Functions in Lymphocyte Activation and Development.

CD45 is one of the most abundant surface glycoproteins expressed exclusively in
all leukocytes. Due to its abundance and unique distribution, CD45 has been extensively
studied since mid 70's (Trowbridge and Thomas, 1994). After the identification of the
first PTP (PTPlB) in late 80's, CD45 was predicted to be a tyrosine phosphatase based on
sequence comparison (Charbonneau et al., 1988). CD453 PTP activity was quickly
confirmed (Tonks et al., 1988a). Further studies determined that CD45 is required for T
cell signaling since CD45 negative cell lines fail to respond to antigen-induced activation
(Pingel and Thomas, 1989). Although great advance has been made to understand the
biological functions of CD45 in hematopoietic cells, there are still a lot of questions
remained. For example, a ligand has not been found, the regulation of activity has not
been determined, and the function of the D2 domain has not been defined. Thus, the
further study of CD45 is important to fully understand lymphocyte development and
activation.

Structural Features. There are eight isoforms of CD45 (or, [3, y, 5, e, g, r], 0 and
t.) differentially expressed on the surface of leukocytes (Chang et al., 1989; Chang et al.,
1991). The multi-isoforms result from the alternate splicing of the exons from a single
gene. For example, mouse CD45 gene has 34 exons spanning 110 kb on the chromosome
1. Through alternate splicing, it produces a cDNA encoding 1291 amino acids for the

human protein (Streuli et al., 1987).

16

Like other RPTPs, CD45 contains three structural domains: An extracellular
domain (1-542 amino acids in human), a single transmembrane region (543-563 amino
acids) and cytoplasmic domain (564-1268 amino acids). There are two glycosylation
regions, O-linked glycosylation and N-linked glycosylation regions exist on the
extracellular domains. At the most N-terminal (1-177 amino acids), alternate exon use
generates eight isoforms. In addition, there are three cysteine rich fibronectin 111 like
repeats within the N-linked glycosylation region.

Role of CD45 in Lymphocyte Activation and Development. Accumulated
evidence has demonstrated the importance of CD45 in TCR-mediated signaling.
However, the function of CD45 in lymphocytes is not fully understood. Clearly, CD45
can act as a positive regulator of T cell signaling. The regulation of CD45 may vary
depending on different developmental stages of lymphocytes.

1) Regulation of PT K Activities. It has been established that CD45 functions to
regulate activities of PTKs such as Lek and F yn. This view is support by several studies.
First, Lck was hyperphosphorylated in CD45-deficient cell lines (Mustelin and Altman,
1990; Ostergaard et al., 1989). Second, CD45 has been suggested to be associated with
Lck through either direct or indirect interaction. The SH2 domain was critical for
Lek/CD45 interaction. One explanation of this interaction is that CD45 can be
phosphorylated by Csk, thus creating a binding site for SH2 domain on Lck (Autero et
al., 1994). However, in vitro studies showed that unphosphorylated recombinant CD45
bound to Lck suggesting a unconventional SH2 domain interaction (N g et al., 1996). An
indirect interaction mediated by CD45-AP was also suggested (McFarland and Thomas,

1995). Third, CD45 can dephosphorylate the negative regulatory tyrosine residues of

17

Lck and Fyn in vitro (Mustelin et al., 1989; Mustelin et al., 1992). Usually CD45 is
regarded as a positive regulator for Lck and Fyn. However, in CD45-deficient YAC-l
cells Lck and Fyn were hyperphosphorylated and their activities, instead of being
decreased, were also elevated (Burns et al., 1994). The observation implies that activities
of Lck and Fyn are not controlled by a single site, the phosphorylation of other site within
kinase domain could also contribute to the regulation. Although Lck and Fyn are similar,
their modulation by CD45 is structurally specific. While hyperphosphorylation of Lck in
CD45 deficient T cell lines increased 8-10 fold as compared to its wild type counterpart,
there was only 2-3 fold increase of hyperphosphorylation for Fyn in same cell line
(Hurley et al., 1993). Either deletion of unique domain of Lck or replacement of this
domain with corresponding sequence from Fyn dramatically decreased the
hyperphosphorylation of Lck suggesting differential regulation between these two
kinases by CD45 (Gervais and Veillette, 1995). Since CD45 has different isoforms and
these isoforms are differentially expressed in a variety of lymphocytes, Lck and Fyn may
also be differentially regulated by CD45 isoforms depending on developmental stage (see
below).

2) Differential Regulation of CD45 on Lymphocyte Development. The importance
of CD45 in lymphocyte development has been proven by studies from CD45 deficient
mice. Two kinds of CD45 deficient mice have been generated and their developmental
deficiencies on T cells and B cells have been investigated (Byth et al., 1996; Kishihara et
al., 1993). Since exon 6 is alternatively used in splicing process, CD45 deficient mice at
exon 6 were not complete nulls. In other words, CD45 isoforms without exon 6 were

normally expressed in these mice. In contrast, expression of CD45 in exon 9 targeted

18

mice was abnormal. Studies on these two deficient mice confirm that CD45 is required
for TCR, BCR signaling and lymphocyte development. Development of T cells can be
divided into two steps: 1) positive selection from CD4'CD8' double negative (DN) to
CD4+CD8Jr double positive (DP); and 2) negative selection from CD4+CD8+ double
positive to CD44r and CD8+ single positive (SP). Generally, deficiencies exhibited in
exon 6 and exon 9 targeted mice were similar. Both of them showed a lower number of
mature T cells. The DN to DP conversion decreased fivefold while the DP to SP
conversion decreased twofold. Thus CD45 was critical for each step of T cell
development, and it was more important in second step than in first step. It seems that
the function of CD45 was closely related to TCR signaling rather than other pathways
since only apoptosis through TCR was severely affected in these deficient mice. TCR
signaling of CD45 targeted mice was defective even in mature T cells from those
deficient mice. Similarly, T cells expressing certain CD45 isoforms in exon6 targeted
mice were unable to respond to antigen stimulation (Byth et al., 1996; Kishihara et al.,
1993). Either these T cells were intrinsically abnormal or different isoforms may have
distinct function. Compared with CD45 deficient mice, the impairment from DN to DP
in Lck deficient mice was more severe (Molina et al., 1992). It is possible that Lek is
regulated by CD45 in different stages or by different CD45 isoforms. Based on these
data, a threshold-regulating model was proposed (Neel, 1997). In this model, CD45 sets
the TCR signaling threshold by regulating Lck activity. Only strong signals beyond this
threshold can activate T cells in CD45 deficient mice and as a result those stimulated

cells are positively selected.

19

Although the number of B cells was normal, the development of B cells and BCR
signaling in CD45 deficient mice was affected. Unlike wild type mice in which most of
B cells are IgM'°IgDhi, CD45 deficient mice had increased number of IngIgDhi and
IthiIng° (Benatar et al., 1996). Like in T cells, CD45 may act as a positive regulator
for B cell selection, and the selection process is abnormal in CD45 deficient mice due to
the change of threshold set by CD45 (Cyster et al., 1996). BCR signaling stimulated by
IgM or IgD was also impaired in CD45 deficient mice while response to
lipopolysaccharrides and stimulation through CD40 in the same B cells was normal.
Upon antigen stimulation, deficient B cells lost their ability to proliferate. Although
intracellular Ca2+ mobilization was normal in these cells, extracellular Ca2+ influx is
abolished (Benatar et al., 1996; Byth et al., 1996). These data indicate that CD45 is
required for both lymphocyte development and proliferation in response to antigen
stimulation.

3) Function of CD45 Extracellular Domain in Cell Adhesion and TCR Signaling.
CD45 is a surface molecule with an extended and highly glycosylated extracellular
domain. However, the function of the extracellular domain in lymphocyte development
and proliferation are unknown. Accumulated evidence suggests that CD45 may interact
with other surface molecules and these interactions have impact on antigen mediated
signaling. The interaction of extracellular domains between CD45 and CD2 modulated
the signaling through CD2 stimulation (V erhagen et al., 1996). Also CD45 can formed
complex with CD4 and this association influenced antigen recognition. Interestingly,
only the CD45RO isoform, rather than CD45RABC, participated in this interaction. This

interaction may have also been related to the regulation of Lck activities (Leitenberg et

20

al., 1996). CD45 may participate in homotypic cell adhesion of lymphocytes.
Apparently, TCR signaling is required for T cell homotypic adhesion because only
activated T cells can aggregate. Furthermore, CD45 mediated homotypic adhesion
probably depends on its isoforms. Antibodies to some CD45 isoforms induced adhesion
while antibodies to other isoforms inhibited adhesion (Zapata et al., 1995). Cell adhesion
mediated by integrin was an important step for the initiation of TCR signaling. Some
evidence suggests that CD45 was also involved in this process. With the formation of
focal adhesions, multiple tyrosine phosphorylation events occur. Phosphorylation of
focal adhesion kinase (FAK) and paxillin was regulated by CD45 because these proteins
failed to be phosphorylated in CD45 deficient cells (Pao et al., 1997). Although these
data predict that CD45 is correlated with cell adhesion, details regarding this process are
still in question.

Regulation of CD45. Sufficient evidence supports the proposal that CD45 is a
regulator for lymphocyte activation, but how CD45 is regulated has not been defined.
Since CD45 is a receptor-like PTP, ligand binding is considered as a potential mechanism
for CD45 regulation. Nonetheless, the fact that a ligand for CD45 has not been found up
to date forces us search for other possible mechanisms. As mentioned before, it is
possible that CD45 is regulated by phosphorylation and dimerization (see regulation of
PTP activities). CD45 can be phosphorylated in vitro or in vivo by multiple
serine/threonine kinases such as PKC and casein kinase 2 and by tyrosine kinases like
Csk (Autero et al., 1994; Charbonneau and Tonks, 1992). However, the phosphorylation
of CD45 either increased or decreased its activity. One explanation for this inconsistency

is that CD45 is differentially regulated by multiple kinases. Although dimerization is a

21

tantalizing mechanism for CD45 regulation, EGF-CD45 chimera is not equivalent to
native CD45 molecules. It is difficult to confirm this hypothesis since the ligand of
CD45 is unknown. Another possibility for CD45 regulation is through protein-protein
interaction. CD45 can interact with a variety of molecules through either extra- or intra-
cellular domain interaction. Beside proteins mentioned earlier such as Lck, CD2 and
CD4, CD45 can also specifically associated with a glycoprotein (gp116) and a
cytoskeleton protein—fodrin (Arendt and Ostergaard, 1995; Okumura and Thomas,
1995). In addition, CD45 interacted with CD45AP through transmembrane domain
(Bruyns et al., 1995; Kitamura et al., 1995). It has been known that CD45AP co-
expressed with CD45 in T and B cells and a complex of CD45/CD45AP protected
CD45AP from degradation (Schraven et al., 1994). There is a putative WW domain
within the cytoplasmic domain of CD45AP. This WW domain can bind with proline-rich
sequences suggesting CD45AP may mediate the interaction of CD45 with a proline-rich
containing protein. Whether these proteins can regulate CD45 remains unknown due to

lack of details about their interactions with CD45.

Conclusions

Lymphocyte activation is an important event for immune recognition and immune
response. Numerous signaling molecules and multiple signaling cascades are involved in
this process. Integration of these signal pathways results in expression of specific genes
and ultimately cellular responses occur. Protein phosphorylation and dephosphorylation
are rapid chemical reactions and their fast nature predicts that these reactions are

extensively used in control of intracellular signals. Another advantage of

22

phosphorylation/dephosphorylation reactions is that they have capacity to amplify
transduced signals. Phosphorylation/dephosphorylation are not two absolutely opposite
actions in the process of signal transduction. Positive regulation of Src family kinase by
PTPs is a good example to indicate the importance of the cooperation between PTKs and
PTPs. As the first identified RPTP, CD45 is critical to the lymphocyte activation and
development. Great progress has been achieved to dissect the biological functions of
CD45 within the past few years. Many questions such as ligands of CD45, regulation of
PTP activities and the functions of D2 domain still remain. In addition, the differential
effects of CD45 isoforms in lymphocyte signaling are unclear. Without doubt, resolution
of these problems is a key step to deepen our understanding of how cells link and

equilibrate their intracellular signaling networks.

23

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Rozdzial, M.M., Malissen, B., And F inkel, TH. (1995). Tyrosine-Phosphorylated T-Cell
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Schraven, B., Schoenhaut, D., Bruyns, E., Koretzky, G., Eckerskom, C., Wallich, R.,
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a novel 32-kDa phosphoprotein that interacts with CD45 in human lymphocytes.
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Shaw, AS. and Dustin, ML. (1997). Making the T cell receptor go the distance: A
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Straus, D.B. And Weiss, A. (1992). Genetic-Evidence For The Involvement Of The Ick

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Streuli, M., Krueger, N.X., Tsai, A.Y., and Saito, H. (1989). A family of receptor-linked

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29

Tonks, N.K., Charbonneau, H., Diltz, C.D., Fischer, E.H., and Walsh, K.A. (1988a).
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30

Wu, J ., Motto, D.G., Koretzky, G.A., and Weiss, A. (1996). Vav and SLP-76 interact and
functionally cooperate in IL-2 gene activation. Immunity 4, 593-602.

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of CD45 and LFA-l. Blood 86, 1861-1872.

31

Chapter 2.

Regulation of CD45 Tyrosine Phosphatase Activity by Casein Kinase 2

32

Introduction

CD45 is a transmembrane protein tyrosine phosphatase (PTP) expressed on the
surface of nucleated hematopoietic cells. Because of its importance in lymphocyte
signaling, CD45 has been extensively investigated in recent years (Justement et al., 1994;
Kung and Thomas, 1997; Neel, 1997). There are at least eight different isoforms of
CD45, which distribute differentially on various lymphocyte types and in certain
lymphocyte developmental stages (Frearson and Alexander, 1996). CD45 isoforms result
from alternate usage of exons at the O-linked glycosylated region of its extracellular
domain (Chang et al., 1989; Chang et al., 1991). The function of extracellular domain of
CD45 in lymphocyte signaling remains unclear because chimeric proteins containing
only the cytoplasmic domain of CD45 were sufficient to restore T cell receptor activation
(Desai et al., 1993; Hovis et al., 1993; Volarevic et al., 1993). The cytoplasmic domain
of CD45 contains two tandem repeated PTP domains designated D1 and D2. The D1
domain is constitutively active and its catalytic activity is required for complementing T
cell receptor (TCR) signaling in CD45-deficient cell lines (Desai et al., 1993). The D2
domain is inactive. CD45 activates Src family kinase by dephosphorylating the
inhibitory pTyr near the C terminus in vitro and possibly in vivo. CD45 is a positive
regulator of TCR signal transduction. CD45 can also dephosphorylate several artificial
substrates in vitro. But it is unknown how the substrate specificity of CD45 is achieved
and how its PTP activity is regulated.

It has been shown that CD45 can be phosphorylated in vitro by a variety of
protein kinases, including protein kinase C, glycogen synthase kinase, casein kinase 2

(CK2), and Csk (Kung and Thomas, 1997). The relationship of CD45 phosphorylation to

33

its function in T cell signal transduction, however, remains unclear. Several studies on
the correlation of CD45 phosphorylation with its PTP activity have been reported but the
results are inconsistent (Autero et al., 1994; Ostergaard and Trowbridge, 1991; Stover
and Walsh, 1994). Previous work in our lab has shown that CK2 is a major kinase
responsible for phosphorylation of CD45 in vitro and in vivo. The phosphorylation sites
of CK2 has been identified in the 19 amino acid acidic region of the D2 domain of CD45.
In this study, the effects of CD45 phosphorylation on its PTP activity were examined
using recombinant CD45 and its CK2 site mutants. The data presented herein
demonstrate that phosphorylation of CD45 by CK2 increased CD45 activity three fold
using myelin basic protein as a substrate. In contrast, phosphorylation of CD45 by CK2
did not change the PTP activity toward other substrates such as pNPP, Raytide and
recombinant Lck. However, we found that CD45 could differentially dephosphorylate
two phosphorylation sites of Lck. The C-terminal inhibitory site of Lck was much more

easily dephosphorylated by CD45 than the autophosphorylation site.

34

Materials and Methods

Cell Lines, Antibodies and Regents. Sf21 insect cells were provided by Dr. D.
Dewitt (Michigan State University) and the cells were cultured in either TC-lOO (Sigma)
or HyQ medium (HyClone) with 1x Antibiotic-Antimyotic (Life Technologies).
Polyclonal rabbit anti-Lek serum was kindly provided by Dr. B. M. Sefton (Salk
Institute). Myelin basic protein (MBP) was purchased from Sigma and enhanced Raytide
was obtained from Calbiochem. Recombinant human Csk was a gift from Dr. P. A. Cole
(Rockefeller University).

Expression Vectors. pET3d-CD45, which expresses the cytoplasmic domain of
wild type murine CD45 with an amino terminal Hi86-tag, was provided by Dr. Pauline
Johnson (University of British Columbia). pET3d-CD45(S/A) and pET3d—CD45(S/E)
were generated by mutating serine at residues 965, 968, 969 and 973 in the acidic insert
of the D2 domain of CD45 into alanine or glutamate respectively (Wang et al., 1999).
pGEX-Src was obtained from Dr. J. E. Dixon (University of Michigan). Wild type
murine His6-tagged pFastBac-Lck was generously provided by Dr. B. M. Sefton (Salk
Institute) and pBluescript-Lck by Dr. R. Schwartz (Michigan State University). The
catalytically inactive form of Lek containing a Ly8273Arg mutation, designated pGEX-
Lck(K/R) and pFastBac-Lck(K/R), were prepared by mutagenesis as described below.

Expression and Purification of Recombinant CD45, Lck and Src Kinase from
E. Cali. Recombinant Hisé-tagged wild type and mutant CD45 were purified from E. coli
BL21(DE3) as described (Ng et al., 1995). Cultures were grown in LB containing 100
pg/ml ampicillin at 37°C until the optical density at 600 nm reached 0.6-0.8. IPTG

(Boehringer Mannheim) was added to a final concentration of 0.1 mM and cultures were

35

incubated at 30°C overnight. Then cells were collected by centrifugation and
resuspended in Buffer I [0.1% Triton X-100, 20 mM Tris-HCl (pH7.5), 150 mM NaCl,
20 mM imidazole 0.025% B-mercaptoethanol]. Cells were lysed by fast freezing in a dry

ice/ethanol bath followed by slow thawing at room temperature. DNase I (10 ug/ml)
(Boehringer Mannheim) was added to digest nuclear DNA. The cell debris was removed
by centrifugation at 10,000 x g for 10 min and the supernatant was filtered with a 0.22
pm low binding filter (Millipore). The protein sample was loaded onto a pre-equilibrated
Ni2+-NTA column (Qiagen) with Buffer 1. After washing with Buffer 1, proteins were
eluted from the column using Buffer II (1 M imidazole, pH7.2). A PD10 column
(Amersham Pharmacia Biotech) was used to remove the high concentration of imidazole
and proteins were eluted in Buffer III (0.1% Triton X-100, 20 mM Tris-HCl, pH8.0, 150
mM NaCl]. Finally, a Mono Q anion exchange column (Amersham Pharmacia Biotech)
was employed for the further purification of recombinant proteins.

Recombinant GST-Src was expressed in E. coli BL21(DE3) at lmM IPTG. E.
coli cells were lysed by fast freezing and slow thawing as described above and the
proteins were purified using GST-column according to the manufacturer's directions
(Amersham Pharmacia Biotech). GST-Lck(K/R) was expressed using a previously
described method with modification (Hlavac and Rouer, 1997). Cultures were induced
with 0.1 mM IPTG at room temperature for 24 h. After induction, cells were collected
and resuspended in Buffer IV (50 mM Tris-HCl, pH7.4, lmM EDTA, 100 mM NaCl,
10% glycerol and 1% NP-40) followed by sonication (Branson Sonofier 450, two 30-s
bursts at 50% duty cycle). The insoluble material was solubilized with 1% Sarcosyl and

diluted with Buffer 111 (1:10) and then centrifuged again to remove insoluble material.

36

The supernatant was loaded onto a glutathione-agarose affinity column (Amersham
Pharmacia Biotech) and eluted with 20 mM reduced glutathione (Sigma) in 50 mM Tris-
HCl (pH 8.0). Purified proteins were aliquoted and stored at -80°C.

Site-directed Mutagenesis and Construction of pFastBac-Lck(K/R). Point
mutation was carried out using QuickChange site-directed mutagenesis kit (Stratagene,
La Jolla, CA). Desired mutations (underlined) were incorporated into a pair of
oligonucleotide primers, each complementary to opposite strands of the parental DNA
template. The primers used for mutagenesis of lysine to arginine at residue273 were as
following: 5’-GGTGGCGGTGAQGAGTCTGAAACAAGGG-3’ and 5’-CCCTTGTTT-
CAGACTCQTCACCGCCACC-3’. The primers were extended by pfu DNA polymerase
during a short temperature cycling (95°C for 30 sec, 55°C for 1 min, 65°C for 13.5 min,
18 cycles) and the parental DNA template was then digested by Dpn I endonuclease.
Mutants were selected after the synthesized DNA was transformed into E. Coli XLl-Blue
and later verified by sequencing.

Transposition and Isolation of Recombinant Bacmid DNA. The mutated DNA
of pFastBac-Lck(K/R) from mini-preparation was transformed into DHlOBAC (Life
Technologies) for transposition into the bacmid. Transposition and isolation of bacmid
DNA were carried out according to the manufacturer's instruction (Life Technologies).
Briefly, transforrnants were plated on LB plate containing 50 ug/ml kanamycin, 7ug/ml
gentamicin, 10 ug/ml tetracycline 100 ug/ml X-gal and 40 ug/ml IPTG. The plate was
incubated at 37°C for at least 24 h and then white colonies containing the recombinant

bacmid were picked. Selected colonies were further confirmed for the presence of Lck

DNA by PCR analysis. The bacmid DNA was then isolated using a method specific for

37

large plasmids as follows: a bacterial colony was inoculated into 2ml of LB
supplemented with 50 ug/ml kanamycin, 7ug/ml gentamicin, 10 ug/ml tetracycline and
grown at 37°C to stationary phase. The culture was spun and resuspended in 0.3 ml of
Solution I [15 mM Tris-HCl (pH 8.0) 10 mM EDTA, 100 ug/ml RNase A]. 0.3 ml of
Solution 11 (0.2N NaOH, 1% SDS) was added and the lysate was incubated at room
temperature for 5 min followed by slowly adding 0.3 ml of 3 M potassium acetate (pH
5.5). After standing on ice for 5 to 10 min, the sample was centrifuged at 14,000 x g for
10 min. Transferred supernatant was precipitated by isopropanol and resuspended into
40 pl TE. A 0.5% agarose gel was used to analyze for the presence of high molecular
weight DNA.

Transfection of 8121 Cells with Bacmid DNA and Screening of Recombinant
Baculovirus. 9X105 Sf21 cells with a viability of more than 97% were seeded into 2ml
of TC-100 medium with antibiotics and incubated at 27°C for at least 1 h followed by
washing with TC-100 without antibiotics. A mixed suspension of bacmid DNA and
CellFECTIN (Life Technologies) in TC-lOO without antibiotics was prepared and
overlaid onto cells. After incubation at 27°C for 5b, the medium was replaced with TC-
100 medium containing antibiotics and incubated for 72 h. Recombinant baculovirus was
screened by either Western blot or Csk kinase assay.

Infection and Purification of Recombinant Baculovirus Lck from 8121 Insect
Cells. Sf21 insect cells were grown in 15 ml of T0100 with a 75 cm2 flask at 27°C for
several days and confluent cells were collected with 50 ml of mixed T0100 and HyQ
(1:1). The cell suspension was then transferred into a spinner and grown at 27°C with

gentle stirring. These cells were split with HyQ medium once a day until the desired

38

number of cells was achieved. Sf21 cells were infected with virus stock at a multiplicity
of infection (MOI) of 0.1. Infected cells were then harvested by centrifugation for 10
min at 1000 x g, and the cell pellet was resuspended in lysis buffer containing 50 mM
Tris-HCl (pH 8.5), 5 mM 2-mereaptoethanol, 100 mM KCl, 1 mM PMSF and 1% NP-40.
After a brief sonication, the cell debris was removed by centrifugation at 10,000 x g for
10 min and the supernatant was loaded onto pre-equilibrated Ni2+-NTA column as
described above. A PD-10 column was also used to remove high concentration of
imidazole. Purified Lek was aliquoted and stored at -80°C.

Radioactive Labeling of Protein Substrates. The 32P-labelling of MBP and
Raytide substrates was performed as described previously (Melkerson-Watson et al.,
1994). Briefly, 50 pg of enhanced Raytide (Calbiochem, San Diego, CA) or 250 pg of
MBP (Sigma) was labeled with 50 uCi of [y—32P]ATP (3000 Ci/mmol, Dupont NEN) by
incubation with 500 ng of recombinant Sre tyrosine kinase for 1 h at 30°C, in 160 pl of
reaction mixture containing 500 uM ATP, 10 mM MgC12, 16 mM HEPES (pH 7.5), 0.03
mM EDTA, 0.07% B-mercaptoethanol. Labeled 32P-Raytide or MBP was added to 100
pl of 5 mg/ml BSA and 70 ul of 50% cold TCA followed by centrifugation at 12,000 rpm
for 15 min. The pellet was then washed twice with 10% cold TCA and resuspended in
200 pl of 200 mM Tris-HCl (pH 8.0).

Recombinant Lek (200 ug) was labeled in 200 pl of reaction mixture containing
10 mM HEPES (pH7.4), 10 mM MnClz, 10 mM Mng 5 uM ATP and 50 uCi of [y-
32P]ATP (3000 Ci/mmol) in presence or absence of 100 ug of Csk at 30°C for 30 min.
Labeled Lek then was precipitated by adding 100 pl of 50% Ni2+-NTA bead slurry. The

slurry was incubated for 10 min at room temperature and the beads were washed twice

39

with Buffer I [0.1% Triton X-100, 20 mM Tris-HCl (pH7.5), 150 mM NaCl, 20 mM
imidazole 0.025% B-mercaptoethanol] and twice with 25 mM HEPES (pH7.5) 0.1% NP-
40. Finally, the beads were resuspended in 200 pl of 100 mM imidazole.

PTP Assays. The PTP assay using 32P-labelled MBP, Raytide and recombinant
Lek were carried out at 30°C and each assay contained 5 pl of 10x PTP buffer [250 mM
HEPES, (pH 7.3), 50 mM EDTA, 100 mM DTT], 35 pl H20, 5 pl of 32P-labeled
substrate and 5 pl of sample to be assayed. After incubation for various times, aliquots of
the reaction mixture were taken out and added to 0.75 ml acidic charcoal suspension [0.9
M HCl, 90 mM Na4PzO7, 2 mM NaH2P04, and 4% (w/v) active charcoal (Sigma)] to stop
the reaction. After centrifugation, the amount of released 32P in the supernatant was
measured in a scintillation counter.

PTP assay using pNPP as substrate was performed in a 96-well plate. Each well
contained 200 pl of solution containing 40 mM MES (pH 5.0), 1.6 mM DTT and 25 mM
pNPP. Reaction was initiated by adding 0.05-5 pg of purified protein and incubated at
30°C for 30 min. The process of reaction was monitored with a mierotiter plate reader

(Molecular Devices) at 410 nm and results were analyzed using SoftMaxPro (Version

2.1.0) software.

CK2 Phosphorylation of His6-cthD45. Wild type and mutant His5-cthD45
protein (1 pg) was either mock treated or treated with 0.04 mU recombinant CK2
(Boehringer Mannheim) at 30°C for 30 min in the presence of 20 mM Tris-HCl (pH 7.5),
5 mM MgC12, 1 mM DTT and 5 pM ATP. Treatment with PP2A treatment was
performed as follows. HISfi'CthD45 was phosphorylated as described above followed by

the addition of sufficient heparin (10 pg/ml) to inhibit CK2 without inhibiting CD45

40

(determined by titration of heparin with each enzyme). The phosphorylated CD45 was
then mock treated or treated with PP2A (Promega) for an additional 30 min at 30°C in 40
pl of reaction mixture containing 20 mM MgC12, 50 mM Tris-HCl (pH 8.5), 1 mM DTT,

1 pl of PP2A (0.5 units/pl)for. The mixture was then subjected to PTP assay as described
above.

FPLC Analysis. FPLC analysis was performed using a Mono Q anion exchange
column (Amersham Pharmacia Biotech) using Buffer A [0.1% Triton X-100, 20mM Tris-
HCl (pH8.0)] and Buffer B [0.1% Triton X-100, 20mM Tris-HCl (pH8.0), 2M NaCl] and
a NaCl gradient from 150 mM to 600 mM as described previously (Ng et al., 1995).
Eluted proteins were assayed by release of phosphate using pNPP as substrate.

SDS PAGE and Western Blot. 10% or 12% SDS polyaerylamide gel was ran at
130V for 1.5 h. Gel was stained with GELCODE blue stain reagent (Pierce).
Immunoblotting was performed on PVDF membrane with 1:1000 dilution of first
antibody followed by incubation with HRP-conjugated goat anti-rabbit secondary
antibody (BioRad) and visualization with chemiluminescence (Amersham Pharmacia
Biotech).

Kinetic Analysis. For the Hisé-cthD45 kinetics, specific activity was expressed
as nmol/min/mg protein and was plotted against substrate concentration. Kinetic
parameters were calculated by nonlinear curve fitting of the data to the Michaelis-Menten
equation using Origin software (Microcal Software). The kinetic parameters of CK2
(Boehringer Mannheim) with His6-cthD45 as substrate were determined with a CK2

assay using Whatrnan P81 phosphocellulose paper squares as described previously

(Tanks et al., 1990).

41

Protein Quantitative Analysis. Protein concentration was determined by the

Bio-Rad protein assay (Bio-Rad) using BSA (Sigma) as standard with a range of lpg to

Spg.

42

Results

Expression and Purification of CD45 and Its CK2 Site Mutants. Previous
work in our lab showed that CK2 is a major kinase responsible for the phosphorylation of
CD45 in viva and in vitro (Wang et al., 1999). Sequence analysis of CD45 indicated that
there are four consensus sequences, Ser-X-X-acidic group, residing in the l9-amino acids
acidic insert of the D2 domain and the consensus sequence is recognized by CK2
(Pearson And Kemp, 1991). Homology comparison showed that four serine residues
(Ser965, 968, 969 and 973) within the consensus sequences were highly conserved
among different species, and these four serines are potential CK2 phosphorylation sites
(Figure 1). To investigate the effect of phosphorylation by CK2 on the activity of CD45,
all these serines were mutated to either alanine, or all four to glutamate. Wild type and
mutant cytoplasmic domain CD45 (designated as His6-cthD45 wt, S/A or S/E
respectively) were expressed in E. coli BL21 (DE3). Expressed proteins were purified
using Ni2+-NTA column followed by Mono-Q anion exchange column chromatography
(Figure 2). All three proteins appeared as single peaks. The S/A mutant had an elution
profile identical to wild type CD45, both eluting at 240 mM NaCl (Figures 2A and 2B).
In contrast, the S/E mutant bound to Mono-Q more tightly and eluted at 340 mM (Figure
2C). The four additional negative charges introduced in the acidic insert enhanced the
interaction of CD45 with the matrix in the column and increased the binding affinity of
CD45. This observation suggested that the acidic insert was exposed on the surface of
native CD45 where it interacted with the matrix. Approximately 1 mg of recombinant

protein was obtained from 500 ml of bacterial culture. All three proteins appeared as one

43

A)

 

 

 

 

 

Phosphoryiaion sites
tm ”5 1200
H'—{ e '. ‘ 1;?4;;‘,‘gg;f;jj-$-?§-
817 1132 P PP
1204 1248
1248
B)

Ser 965 968,969 973
KESEPESD ESSDDDSDS Tsxrmns mouse
KESEAESD assnansos Tsxrruas rat
KESEHDSD ESSDEDSDS 133eran human
EE KEGEHDSE DSSDEDSDC SSRYINAS chicken
non mewsasnsan-snnsnn s'rxnnns shark

Bl-strand

acidic insert

Figure 1 Schematic representation of CD45 cytoplasmic domain and its
phosphorylation sites. (A) Two tandem catalytic domains are shown as gray
solid boxes and the 19 amino acid acidic insert is shown as the black box. Serine
and threonine phosphorylation sites are indicated underneath the structure. trn:
transmembrane region; C: invariant cysteine residue. (B) Comparison of
homologous sequences of CD45 acidic insert among different species. The region
of acidic insert is framed with an open box and four potential serine

phosphorylation sites are shaded. The starting position of Bl-strand is indicated
by an arrow.

44

 

 

 

Absarbance 280 nm

 

 

 

 

11111
010 2030

Fraction (ml)

Figure 2. FPLC chromatography of three types of recombinant CD45.
(A) Wild type CD45, (B) S/A and (C) S/E mutants were purified by Ni2+-NTA
and PD-lO column column chromatography and were loaded onto a Mono-Q
ion exchange column and eluted using a NaCl gradient from 150 to 600 mM.
PTP active fractions were determined by pNPP hydrolysis and are shaded
black. The full scale absorbance was 0.5.

45

major band of 95 kDa on 12% SDS-PAGE stained with Coomassie brillant blue (Figure
3).

CD45 Is Multiply Phosphorylated by CK2. To study the kinetics of
phosphorylation of CD45 by CK2, a time course analysis was performed using purified
wild type CD45 as a substrate (Figure 4). The results indicated that CD45 could be easily
phosphorylated by CK2. Phosphorylation of CD45 reached a plateau within less than 30
min. Based on the amount of incorporation of 32P into CD45, a stoichiometry of 2.5 mol
of phosphate/mol of protein was estimated suggesting that at least three serines on CD45
were phosphorylated by CK2 under the experimental conditions.

To confirm that CD45 was phosphorylated by CK2 at multiple sites, CK2 treated
CD45 wild type was subject to FPLC analysis using Mono-Q column (Figure 5). Free
ATP in the reaction mixture had a high absorbance at 280 nm but did not interfere with
the elution of CD45 since the mocked-treated CD45 eluted at same salt concentration as
purified wild type CD45 (compare Figures 5A and 2A). In contrast, CK2 phosphorylated
CD45 was retained by the column and eluted at 3 NaCl concentration of 340 mM to 360
mM. The phosphorylated CD45 eluted as two peaks probably resulting from differently
phosphorylated forms (Figure 5B). The fact that CK2 phosphorylated CD45 eluted near
the position of the S/E mutant supports the notion that the S/E mutant can mimic the
charge effect of phosphorylation. Taken together, the results indicate that there are at
least three sites (possibly four) on CD45 which can be phosphorylated by CK2 in vitro.

Kinetic Analysis of CD45 Phosphorylation by CK2. A kinetic analysis was
performed to further characterize the nature of CK2 phosphorylation of CD45 (Figure 6).

The Km was 0.51 pM and the Vmax was 35.5 nmol/min/mg with CD45 as a substrate of

46

Wt S/A S/E BSA

 

 

Figure 3. SDS-PAGE analysis of purified recombinant CD45 proteins.
A 5 pl sample of wild type (wt, lane 1-2), S/A (lane 34) and S/E (lane 5-6)
were loaded onto 12% SDS-PAGE gel followed by electrophoresis and
Commassie Blue staining. CD45 protein amounts were estimated by
comparison to 1 pg (lane7), 2 pg (lane 8) and 5 pg (lane 9) of BSA.

47

 

3000
2500 1 ‘

l

2000 ‘

1

pm

1
>

o 1500
1000

l

500 -A

 

 

 

0 20 4O 60

min

Figure 4. Time course analysis of phosphorylation of CD45 by CK2.
Recombinant wild type CD45 protein (1 pg) was mixed with CK2 (0.04 mU)
in a total volume of 50 pl under the conditions described in materials and

methods. An aliquot (5 pl) of the reaction was taken out at the indicated
times and used for measurement of 3'2P incorporation into CD45.

48

 

 

 

 

 

Absarbance 280 nm

 

 

 

 

 

 

0 10 20 30
Fraction (ml)

Figure 5. Analytical FPLC separation of CK2-phosphorylated CD45. (A)
Mock-treated and (B) CK2 treated recombinant wild type CD45 were loaded
onto Mono-Q ion exchange column and eluted using NaCl gradient from 150
mM to 600 mM as shown. PTP active fractions were determined by pNPP
hydrolysis are shaded black. An ATP peak before PTP active fractions is
shown by the arrow. The full scale absorbance was 0.02.

49

 

 

 

—\ 30-
3
‘~.
X
‘5”
2
'E 20—
a
e
.2.
ts
(U
:8 10- Vmax=35.46
a
m Km=051
é} .
I
0 j I ' I ' I ' I ' I ' I ' I
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

[CD45] 11M

Figure 6. Kinetic analysis of phosphorylation of CD45 by CK2. Specific
activity was determined by measuring 32P incorporation of recombinant wild
type CD45 from 0 to 3.5 mM. Kinetic parameters were generated by

nonlinear curve fitting of the data to the Michaelis-Menten equation using
Microcal Origin software.

50

CK2. These parameters were comparable to reports of CK2 kinetics with other protein
substrates. For example, the Km and Vmax of CK2 was 1.1 pM and 82.5 nmol/min/mg
using eIF-2 as substrate (Gonzatti-Haces and Traugh, 1982). With a Km in the sub-
micromolar range, CD45 is an excellent substrate for CK2 (Tuazon and Traugh, 1991).

Serine Phosphorylation of The Acidic Insert in The D2 Domain Increases
CD45 Activity Using MBP As Substrate. The high conservation of the CK2
phosphorylation sites in the acidic insert region of CD45 led us to hypothesize that
phosphorylation (or introduction of additional acidic residues) at this site would modulate
CD45 activity. To test the hypothesis, We evaluated the effect of CK2 phosphorylation
on CD45 activity using several artificial substrates--pNPP, Raytide and MBP. No
significant effects of phosphorylation on CD45 PTP activities were observed using pNPP
as a substrate at different concentrations (2.25 mM, 5.62 mM and 11.25 mM) (Figure 7).
Specific activities of CD45 toward 32P-raytide increased from 1.74 x 104 to 3.38 x 104
ng/min/mg upon comparison of unphosphorylated and CK2 phosphorylated CD45 wt.
Both phosphorylated and non-phosphorylated CD45 S/A mutant had similar specific
activities compared to unphosphorylated CD45wt (1.57 x 104 ng/min/mg). In contrast,
both forms of S/E mutants (phosphorylated and non-phosphorylated) demonstrated a
slight activity increase when compared with non-phosphorylated CD45 (from 1.74 x 104
to 2.33 x 104 ng/min/mg ) (Figure 8). We concluded that phosphorylation of CD45 by
CK2 has little effect on PTP activity toward pNPP or 32P-Raytide substrates.

Since pNPP is an analog of pTyr, and Raytide is a short peptide (2476 Da), we
attributed the unchanged activity after phosphorylation of CD45 to the small size of these

two substrates. In other words, the substrates may not be big enough to interact with the

51

 

 

 

E

g 14 - 2, - - w
8 12 - WTEI SA I SE I 1
e 10 l
g 8

e 6 - l
8 ‘ l
E 4 1
g 2 j l
E 0 n .7 ,_ l

2.25 5.62 11.25
[pNPP] mM

Figure 7. Comparison of PTP activities of recombinant wild type CD45
and CK2 site mutants using pNPP as substrate. The assays were performed
with three different concentrations of pNPP under the experimental conditions

described in materials and methods. 2.5 pg of enzyme was used in each assay.

52

80

 

[:1 no CK2 + CK2
60- 32P-Raytide

40-

20-

 

 

 

Specific activity (units/mg x 103)

wt ’ S/A' S/E

Figure 8. Comparison of PTP activity of recombinant wild type CD45
and CK2 site mutant CD45 using 32P-Raytide as substrate. CK2
phosphorylated (gray bar) and untreated (white bar) recombinant wild type
CD45 (wt), S/A and S/E mutants were incubated with 32P-Raytide as substrate
at 30°C for 10 minutes. PTP activities were determined by measurement of
the release of 32P after incubation.

acidic insert in the D2 domain of CD45. Therefore, potential regulatory effects of
phosphorylation on CD45 activity could not be observed using these substrates. To
examine this possibility, we compared the phosphorylated and non-phosphorylated forms
of CD45 wild type and the CK2 site mutants using a 18 kD protein — MBP (Figure 9).
Wild type CD45 activity was enhanced after CK2 phosphorylation by about 3-fold.
Neither the S/A mutant nor phosphorylated S/A increased the activity of CD45.
However, S/E mutant exhibited a three-fold activity increase compared to
unphosphorylated CD45 and there was no change in the activity of the S/E mutant after
phosphorylation with CK2. This result suggested that the contact between the acidic
insert in the D2 domain of CD45 and its substrates enhances the apparent regulation of
PTP activity and of CD45.

Effect of PP2A on CD45 Activity. In many cases the regulation of proteins by
phosphorylation acts like a molecular switch with a two-directional function. If
phosphorylation of CD45 up-regulates its PTP activity, then dephosphorylation of CD45
could possibly down-regulate its PTP activities. To determine the effect of de-
phosphorylation on the CD45 activity, PP2A, a serine phosphatase, was used to remove
the phosphate on CD45 after CK2 phosphorylation. As shown in Figure 10, CK2
increased the activity of CD45 and subsequent sequential dephosphorylation with PP2A
reversed the activation. Treatment of non-phosphorylated CD45 with PP2A did not alter
CD45 activity. In this experiment, a 10 pg/ml of heparin was added to inhibit CK2
activity before incubating with PP2A. The amount of heparin to be added was
determined by titration to achieve a balance in which the heparin inhibited the CK2

without inhibiting CD45 or PP2A (data not shown). Complete reversal had not been

54

 

Enocm i+CK2

 

 

 

Specific activity (units/mg x 103)

 

Figure 9. Effect of phosphorylation of CD45 on PTP activity using 32P-
MBP as substrate. CK2 phosphorylated (gray bar) and untreated (white bar)
recombinant wild type CD45 (wt) and the S/A and S/E mutants were incubated
at 30°C for 10 minutes with 32P-MBP. PTP activities were determined by
measurement of the release of 32F after incubation.

55

 

80

60-

 

 

Speeific activity (units/mg x 103)

X

r 0,
4° <2” 6' (30.1?
)8 quQ

Figure 10. Dephosphorylation of CD45 by PP2A: Effect on PTP
activity. Wild type CD45 was treated with CK2 and then incubated with
PP2A (+CK2, +PP2A) or without PP2A (+CK2) at 30°C. PTP activity was
measured using MBP as substrate. Wild type alone (wt) and wild type
treated with PP2A only (+PP2A) under same condition were used as
controls.

56

achieved during the short incubation possibly due to residual CK2 activity and the
presence of excessive ATP.

Kinetic Comparison of CD45 and Its CK2 Mutants. To further understand the
enzymatic mechanism of regulation of CD45 by phosphorylation, a kinetic analysis of
CD45 wild type and its CK2 mutants was carried out using MBP as a substrate. As
shown in Figure 11 and Table I, the S to A mutation (S/A) only slightly altered the basic
kinetic parameters of His6-cthD45 while the introduction of acidic residues, glutamate,
into the serine CK2 sites (S/E) resulted in a three fold increase in Vmax and a small
increase in Km.

CD45 Differentially Dephosphorylates Lck Phosphorylation Sites. Since Lek
has been suggested to be one of the physical substrates of CD45 in viva, we studied the
dephosphorylation of Lek by CD45. There are two regulatory phosphorylation sites on
Lek (Y505 and Y394 in human Lck). Y505 is phosphorylated by c-Src kinase (Csk) and
Y394 is the site of autophosphorylation. Both phosphorylation sites are believed to be
subject to dephosphorylation by CD45. To examine whether CD45 has distinct ability to
remove phosphate from these two sites, it was necessary to prepare the kinase-inactive
form of Lek (designated as I-Lck(K/R)) which abolished the autophosphorylation of Lek.
For this purpose, wild type murine Hisé-tagged pFastBac-Lck was mutated by replacing
lysine at position 273 with arginine. Mutated pFastBac-Lck(K/R) was then transposed
into bacmid. The bacmid DNA containing I-Lck(K/R) was transfected into Sf21 insect
cells and transfectants were screened by precipitating the cell lysate with Ni2+-NTA beads
followed by an evaluation of the ability of Csk to phosphorylate bead bound I-Lck(K/R)

(in vitro Csk kinase assay). Two positive transfectants (out of six) were selected as

57

 

 

 

 

_\ 80
§ . ‘ S/E
>< A
g1 60-
E
'E
3..
‘3‘ 40-
g . S/A
0 D
g 20_ o I wt
E). .
U)
0 I j I T I ' I ' I r
O 4 8 12
[MBP] mM

Figure 11. Kinetic comparison of wild type CD45, S/A and S/E mutants using
32P-MBP as substrate. The specific activity of each form was determined by
measuring 32F release from labeled substrate over the range of 0 to 12 mM.
Kinetic parameters were generated by nonlinear curve fitting the data to Michaelis-
Menten equation using Microcal Origin software.

58

Table 1. Kinetic parameters of the CK2 site-mutated His6-cthD45

 

 

 

 

with 32P MBP as substrate.
K... ' Vmax
CD45farm (pM) (pmol/min/mg)
wt 0.94 i 0.47b 24.2 1: 2.4
S/A° 1.18 i 0.42 33.3 i 2.7
3’5 1.95 i 0.29 85.1 :1: 3.5

 

a. Km and Vmax were calculated using nonlinear curve fitting with
Microcal Origin Software.

b. Average and standard deviation of three determinations.

c. S/A and S/E refer to the corresponding Ala and Glu mutations at

Ser residues 965, 968, 969 and 973.

59

shown in Figure 12. To confirm that the positive transfectants were inactive, one was
chosen to compare the effectiveness of autophosphorylation to that of phosphorylation by
Csk (Figure 13). When lowest amount of I-Lck(K/R) was present there was strong
phosphorylation by Csk with no detectable autophosphorylation (Figure 13, lane 1 and 2).
At this level the ratio of I-Lck(K/R) to Csk was 1/7 (w/w). When higher amount of I-
Lck(K/R) was present, a basal level of autophosphorylation was detected while the I-
Lck(K/R) was strongly phosphorylated by Csk. This result showed that mutated I-
Lck(K/R) had extremely low kinase activity.

The decreased kinase activity of I-Lck(K/R) also led to a higher expression in
insect cells. Usually, 400 pg of active Lek was obtained from 500 ml of insect cell
culture. In contrast, as high as 15 mg of I-Lck(K/R) was purified from 250 ml of culture.
The difference of yield between active and inactive form of Lek reflects the possible
toxicity in insect cells resulting from overexpression of active Lck.

To test whether phosphorylation of CD45 at CK2 sites had an impact on the
dephosphorylation of Lck, we treated the 32P labeled active and inactive forms of Lck
with the CK2 mutant forms of CD45 (S/A and S/E) (Figure 14). In this experiment no
significant difference in dephosphorylation of either form of Lck was detected between
wild type CD45 and the S/A or S/E mutants. That is, all the forms of CD45 had similar
PTP activity toward either the active or inactive form of Lek. This result implied that
phosphorylation status of CD45 in the acidic insert had no direct effect on functional
dephosphorylation of Lek. However, the enzymatic activity of CD45 toward the Csk-
phosphorylated, 32F I-Lck(K/R) was 400-fold greater than that for autophosphorylated 32P

Lek as substrate. This suggests a high substrate specificity of CD45 for the tail

60

I-Lek(K/R) expressing clones controls

 

.1 2... 3 .4 5 .6. + '

Lek

 

Figure 12. Selection of $121 clones expressing inactive Lck (I-Lck(K/R).
Clones of infected Sf21 cells (6 x105) were lysed with 1% NP-40 followed
by precipitation with Ni2+-NTA beads. In vitro kinase was performed by
incubation of the beads with 2.5 pg of Csk and [32P]yATP. The reaction
was analyzed using 10% SDS-PAGE followed by phosphorimage analysis.
Equal amount of Lek transfected cells (+) and uninfected Sf21cells (-) were
used as positive and negative controls.

61

I-Lck(K/R) 0.2pg lpg 2pg
Csk - + - + - +

Lek —>

 

Figure 13. Comparison of autophosphorylation and Csk phosphorylation
of inactive Lck [I-Lck(K/R)]. Different amounts of inactive Lek (0.2, 1 or 2
pg) were incubated [32P]yATP in presence or absence of Csk (1.5 pg) at 30°C
for 15 min. The reaction was terminated, analyzed by SDS-PAGE and
radioactivity was visualized by phosphorimage analysis.

62

250

 

 

 

 

 

Substrate

200 -

150 _ Csk phosphorylated
.i 32p I-Lck(K/R)
o:
E 100 _
g 50 .
E.
3. _
E
ii?
.9 B
"“5 1.5 -
8
(’3 Autophosphorylated

' * 32p Lck

 

 

 

Figure 14. Comparison of PTP activity of mutant forms of CD45 with Csk
or autophosphorylated Lck as substrates. (A) Recombinant wild type CD45
(1 ng) (wt) and the S/A and S/E mutants were incubated at 30°C for 10 minutes
with Csk-phosphorylated 32F I-Lck(K/R) (5 pg). The data represent the average
and SD of three determinations. (B) Recombinant wild type CD45 (100 ng) (wt)
and the S/A and S/E mutants were incubated with autophosphorylated 32P Lek (1
pg). One representative determination is shown. PTP activities were determined
by measurement of the release of 32P after incubation.

63

phosphorylated form of Lck and raises the possibility that CD45 differentially regulates
the two phosphorylation sites. Because Y505 is an inhibitory site for Lck kinase activity,
the preference of CD45 for this site supports the notion that CD45 is a positive regulator

for Lek.

Discussion

This study was designed to characterize the nature of phosphorylation of CD45 by
CK2 and to establish the relationship of phosphorylation of CD45 with its phosphatase
activity. The PTP activity of three forms of CD45 (wild type, S/A and S/E mutants) with
and without CK2 phosphorylation were compared using several different substrates. The
similarity in activities between S/E mutant and phosphorylated wild type CD45 showed
that S/E mutant was indeed a good analog to a phosphorylated form of protein. Both of
them have higher PTP activities toward MBP substrate than either unphosphorylated wild
type or S/A mutant suggesting that phosphorylation of CD45 by CK2 at the acidic insert
increases the phosphatase activity.

Protein phosphorylation is a universal regulatory mechanism used by cells to
respond to extracellular stimuli. Phosphorylation of CD45 by protein kinases had been
noticed for a long time, but little was known about how the phosphorylation of CD45
might regulate its biological function. The experiments of others suggested that CD45
activity either increased or decreased after cells were treated with PKC activators or
phosphatase inhibitors (Brautigan and Pinault, 1991; Ostergaard and Trowbridge, 1991;
Yamada et al., 1990). The discrepancy between these studies implicates that CD45 may
have various phosphorylation regulatory mechanisms depending on cell types, status or
conditions of stimuli. In other words, multiple phosphorylation sites may exist on CD45
that are differentially regulated by different protein kinases. Previous studies suggested
that the PTP activity of CD45 purified from lymphocytes was unchanged after
phosphorylation by CK2 (Tanks et al., 1990). A possible explanation is that the CD45

used in this study was purified from lymphocyte cells and was already highly

65

phosphorylated. This explanation can be supported by the results from our lab, which
showed that immunoprecipitated CD45 from Jurkat T cells could not be labeled easily by
exogenous CK2 (Wang et al., 1999).

Kinetic analysis shows that the increase of CD45 activity after phosphorylation by
CK2 is due to a large increase in its Vmax, instead of a decrease in its Km value. As
mentioned before, CD45 consists of two tandem PTP domains that are structurally
similar. The 19 amino acid acidic insert exists only in the D2 domain of CD45 but not in
D1 domain and not in other PTPs. Also, the acidic insert is highly conserved among
different species. The unique nature of this acidic insert suggests that it may be
functionally important for CD45.

Solution of the crystal structure of another receptor type PTP, RPTPa showed that
RPTPor may exist as an inhibited dimer in which the helix-turn-helix wedge from the N
terminal of each monomer blocks the mouth of catalytic cleft of the other monomer
(Bilwes et al., 1996). If CD45 has a similar overall structure with RPTPa, the 19 amino
acid acidic insert in D2 domain is potentially located in a loop at the N-terminal of [31
and this loop is close to the D2 catalytic cleft. Since the loop sticks out of the molecular
surface, phosphorylation of this loop may disrupt the inhibitory dimer structure and make
substrate interaction more accessible. As a consequence, the Vmax of CD45 might
increase. However, after CD45 was phosphorylated by CK2, the large increase of PTP
activity was only observed using MBP as a substrate while there were slight or no

increases of PTP activity using Raytide and pNPP. This result may suggest that CD45

has some substrate specificity.

66

In this study, we also developed the PTP assay using Lek as a substrate. Because
of the role of Lck in lymphocyte signaling, understanding the interaction of CD45 and
Lek is crucial to the dissection of the TCR signal pathways. For this purpose, we
attempted to express Lck using both E. coli and insect cell expression systems. It was
very difficult to purify active or inactive Lek from E. coli since the expressed Lck protein
formed inclusion bodies. Although detergents such as N-lauroylsarcosine dissolved the
inclusion bodies, the Lek was unable to bind to GST-column (data not shown). In
contrast, Lck protein expressed in insect cells was soluble. This observation suggested
that post-translational modifications unique to eukaryotic cells might be important to
maintain the correct protein conformation. In addition, the dramatic difference in yield
observed for expression of active and inactive Lck indicated the potential functional
effect of expression of this proto-oncogene in normal cells. Our data showed that
phosphorylation of CD45 at CK2 sites did not change the PTP activities toward Lek
substrate as it did for MBP. This result indicates that the regulation of CD45 PTP
activity might involve other mechanisms besides phosphorylation. Regulation of Lek by
CD45 might need the participation of other molecules. The fact that
coirnmunoprecipitation of CD45 with Lek has a low stoichiometry (N g et al., 1996)
implies that the interaction between Lek and CD45 is weak and not sufficient to hold
them in a correct position to allow detectable phosphorylation regulation of CD45.

In this study, CD45 was also shown to have a high substrate specificity toward
two different phosphorylation sites (Y505 and Y394) on the same Lek molecule.
According to resolved crystal structure of the inactive, Y505 phosphorylated Src kinase,

the phosphate on Y505 is buried inside of the SH2 domain of Lek, while phosphorylated

67

Y394 is on an exposed loop on the outside of Lck. However, our data show that CD45
dephosphorylated Lck preferentially at position of Y505 instead of Y394. Comparing the
enzyme activity of CD45 toward different 32P labeled substrates, Raytide, MBP and I-
Lck(K/R) seemed to serve as good substrates of CD45, while pNPP and
autophosphorylated Lek served as poor substrates of CD45. The substrate specificity of
CD45 strongly suggests that the environment in which the tyrosylphosphate resides, or
the steric conformation of the protein is a decisive factor in the PTP activity of CD45.

It has been known that mutation of these four serine sites to alanine abolished
greater than 95% of the ability of CK2 to phosphorylate CD45 (Wang et al., 1999). Here,
we demonstrate that there are multiple CK2 phosphorylation sites on CD45 based on the
stoichiometry of phosphorylation and on the fact that phosphorylated CD45 has similar
elution profile with S/E mutant. In addition, the FPLC elution profile indicated that CK2
treated CD45 had two elution peaks suggesting multiple forms of CD45 existed after
CK2 phosphorylation. However, the data presented here is unable to prove whether these
four phosphorylated serines contribute equally or preferentially to the increase of CD45
PTP activity. Mutation of each single site plus different site combination is needed to
answer this question. Analytic F PLC showed that the introduction of four negative
charges have significant effects on the interactions of CD45 with the Mono-Q column. It
may be that keeping the D2 insert acidic correlates with the function of CD45. However,
it is hard to understand why CK2 makes this region more acidic and more negatively
charged and why these four introduced phosphates significantly increases PTP activity.

CK2 is ubiquitously expressed in all cell types and it can phosphorylate a variety

of important signaling molecules, such as Jun, Myc, Myb, Rb and p53 (Litchfield and

68

Luscher, 1993; Litchfield et al., 1996; Tuazon and Traugh 1991). However, the fimction
of CK2 in signal transduction is still a puzzle. Recent studies showed that CK2 is
actively expressed in transformed and proliferating cells, and it can also act as an
oncogene in cooperation with Myc in transgenic mice (Seldin and Leder, 1994; Seldin
and Leder, 1995). Thus, CK2 is proposed to be a critical and essential serine/threonine
protein kinase in control of cell growth and differentiation.

Evidence from CD45 deficient mice suggested that regulation of Src family
kinase activity by CD45 varies in a development and cell type-dependent way. For
example, regulation of Lek activity by CD45 is less important in the early stages of
thymic development than in the late stages. These observations led to the suggestion that
CD45 may control lymphocyte signaling by setting different thresholds according to the
cellular environment and to the developmental stage of cells. If this is true, then
overexpression of CK2 should have an impact on the threshold set by CD45 since it can
up-regulate the PTP activity. Considering the high abundance and constitutively high
expressed activity of both CK2 and CD45, it is tantalizing to speculate that both CK2 and
CD45 act as “buffer molecules”, which are important in the maintenance of the basal

level of phosphorylation and in the adaptability of cells.

69

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