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301409 9158
ms ‘ LIBRARY
2.. Michigan State
University

 

 

This is to certify that the

dissertation entitled
CELL CYCLE-RELATED ACTIVITY OF CD45 AND
EXPRESSION OF A CD45-ASSOCIATED

SERINE-THREONINE KINASE
presentedby

Margaret Ellen Waldmann

has been accepted towards fulfillment
of the requirements for

P11- D- degree in W93:

 

/Mt 47/52: $71 1%ng '

Mafiipififesgn'

Date 14 March 1995

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

 

_¥' _____,

CELL CYCLE-RELATED ACTIVITY OF CD45 AND
EXPRESSION OF A CD45-ASSOCIATED SERINE-THREONINE KINASE

By

Margaret Ellen Waldmann

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology

1995

ABSTRACT

CELL CYCLE-RELATED ACTIVITY OF CD45 AND
EXPRESSION OF A CD45-ASSOCIATED SERINE-THREONINE KINASE

By

Margaret Ellen Waldmann

CD45 is an abundant cell-surface protein tyrosine phosphatase (PTPase) which
is essential for antigen activation of B and T lymphocytes. CD45 is also expressed
abundantly in many other hematopoietic cells indicating that it has additional
functions. In particular, the expression of CD45 in all hematopoietic cells except
erythrocytes and their immediate precursors suggests that CD45 may have a
fundamental role in cellular processes, such as cellular stasis or cell cycle regulation.
In the first study presented in this dissertation, the hypothesis that CD45 PTPase
activity varies during progression through the cell cycle was tested. Then, having
determined that the enzymatic activity of CD45 was elevated in mitosis, the second
study was undertaken to determine if accessory proteins associate with CD45 when it
exhibits peak PTPase activity late in the cell cycle. Centrifugal elutriation was used
in both studies to fractionate logag'thmicallyfgrowing asynchrpflnpusHCILL-Z. cellsjnto
cell cycle stage-enriched subpopulations. For this cytotoxic T cell line, CD45 PTPase
activity was elevated 2- to 5-fold in 62+M fractions when compared to its activity in
GI or 8 fractions. FACS and SMflgysesindicated that there was only a
minor increase in CD45 protein expression during progression through‘ thehcell’ cycle,
suggesting that increased CD45 PTPase activity was principally due to increased

.enzym§§9_agfiflw. -. The timing of this observation indicated that the peak PTPase

activity of CD45 occurred in mitosis. Treatment of cells with a chemical cross-
linking reagent prior to immunoprecipitation of CD45 indicated that 60 to 70 kD
proteins associated with CD45 in S and 62+M. The association of 60 to 70 kD
proteins with CD45 was confirmed by the analysis of CD45 immunoprecipitates from
cells lysed with the mild detergent digitonin. When CD45 immunoprecipitate
complexes from digitonin lysates were subjected to in vitro phosphorylation
treatments, it was determined that an active kinase was present in the complex and
that theactivityflpf the kinase from Gz+M—enriched subpopulations was elevated.
Rhosphqaminomd- analysis of kinase products demonstrated that theldnase was a
serinethreonine kinase. The elevation during mitosis of the activity of both CD45
and a CD45-associated kinase supports the hypothesis that CD45 has a role in

phosphorylation events during cell division.

This dissertation is dedicated to my family and the many educators who have helped

me attain this goal.

iv

ACKNOWLEDGEMENTS

I am greatly indebted to Dr. Walter Esselman for his guidance and support through
my years in his research group. I wish to express my gratitude to Drs. Kathryn
Brooks, Richard Schwartz, and Michael Zaroukian for their guidance and time. I am
also grateful to Drs. Lyla Melkerson-Watson, Hsu-Lang Chang, Louis King, Sue
Dagher, Laurie Iciek, and Jim Bretz and to Howard Beitenmiller and Mary Morrison

for their invaluable assistance.

TABLE OF CONTENTS

952nm

List of Tables ..................................

List of Figures ..................................

Chapter One: Literature Review ......................
Introduction ...............................
CD45 Gene and mRNA Transcripts ................
Altemate-Exon Usage .........................
Regulation of Splicing of the Alternate Exons ..........
CD45 PTPase Domain .........................
CD45 Function .............................
CD45 Interactions with Other Molecules .............
Regulation of the PTPase Activity of CD45 ............

References ...............................

Chapter Two: Elevation of Lymphocyte CD45 PTPase Activity during
Mitosis ..................................

vi

11

12

14

952nm

Materials and Methods ........................ 36
Results .................................. 41
Discussion ................................ 58
Acknowledgments ........................... 62
References ................................ 63

Chapter Three: Cell Cycle-Related Expression of a CD45-Associated

Serine—Threonine Kinase ....................... 67
Footnotes ................................ 68
Abstract ................................. 69
Introduction .............................. 70
Methods and Materials ........................ 73
Results .................................. 79
Discussion ................................ 100
Acknowledgments ........................... 105
References ................................ 106

vii

I

LIST OF TABLES

Chapter Two

Elutriation flow rates and recovery of CTLL-2 cells

viii

LIST OF FIGURES

Chapter One
CD45 structure ............................. 5
Chapter Two

FACS analysis of the DNA content of elutriated CTLL-2 cells 43

CTLL-2 CD45 PTPase activity during cell cycle progression 47

Cell cycle stage analysis of CTLL-2 elutriation fractions . . . . 49

CD45 fluorescence of elutriation fractions ............ 51

CTLL-2 expression of CD45 and CD8 through the cell cycle 54

Fluorescent microscopy images of elutriated CTLL-2 cell

populations .............................. 57
Chapter Three

FACS analysis of the DNA content of elutriated CTLL—2 cells 81

CD80: and CD45 immunoprecipitates from DTBP-treated CTLL-2
cells ................................... 84

FACS analysis of the DNA content of CTLL-2 elutriation
fractions analyzed in Fig. 4 ..................... 86

CD45 immunoprecipitates from DTBP-treated CTLL-2
elutriation fractions .......................... 88

In vitro phosphorylation of proteins coprecipitated with CD45 91

ix

FACS analysis of the DNA content of recultured CTLL-2
subpopulations .............................

In vitro phosphorylation of proteins coprecipitated with CD45

Phosphoamino acid analysis of in vitro phosphorylation products

96

99

CHAPTERONE

LITERATURE REVIEW

Introduction

The murine leukocyte common antigen (LCA, which is also known as T200,
B220, Ly-S, gplSO, and CD45) was first identified in 1975 by Trowbridge et al. (1)
as a group of high molecular weight species immunoprecipitated from T and B cell
lysates by rabbit anti-mouse lymphocyte serum and by Komura et al. (2) as a T
lymphocyte alloantigen system. Homologous proteins with highly conserved regions
(3) have been identified on human (4) and rat (5) leukocytes, and analogous LCA
have also been noted for sheep (6), chickens (7), cows, dogs, and sharks (8). CD45
was the first in a family of receptor-type protein tyrosine phosphatases (PTPases) to
be identified (9).

CD45 is an abundant transmembrane protein with large intra- and extracellular
regions (IO-12). Murine CD45 is expressed by all hematopoietic cells except
erythrocytes and their immediate precursors (13), but there is much cell lineage- and
activation state-related heterogeneity of structure in this family of glycoproteins
(reviewed in 3, 14). This heterogeneity occurs in the extracellular N—terminal region
due to variable use of at least three exons (15-19). The alternate use of 5’ exons
gives rise not only to different primary structures of CD45 proteins but also to
differences in glycosylation (14, 20). Because the primary structure of the

intracellular and transmembrane regions is not varied among isoforms (16, 18), it

3

appears that the intracellular signal transduction mechanism of this putative receptor
may be identical for the various cells expressing CD45, while the extracellular
binding of ligands could be cell-type specific.

Full-length murine CD45 is produced as a 1291 amino acid protein (18).
After the 23 residue leader sequence is removed, the extracellular region consists of
370-541 residues depending on exon usage, the transmembrane sequence of 22

residues, and the intracellular region of 705 amino acids (Fig. 1) ( 18).

CD45 Gene and mRNA Transcripts

The gene for murine CD45 is located on distal chromosome 1 in a large,
conserved linkage group, which is homologous to a group localized to chromosome
1q21-32 in the human genome (21). The CD45 locus is referred to as Ly—5, and there
are at three recognized alleles (21, 22). Ly-5 spans approximawa 120 kb and is
comprised of 34 exons (l9; nomenclature is from 23).

One of two alternate exons, denoted exons 1a and 1b, forms the 5’ end of
CD45 mRNA (19). Use of 1b is common in B cells, and in mRNA from T cells,
exon 1a is more abundant. There are also mRNA transcripts in T cells which are
exon-1a” and exon-lb‘, which may arise from an exon which is not yet identified.
Primer extension analysis has revealed that transcription may initiate at any one of a
number of sites at or near the 5’ end of exon 1a or 1b (19). Although there is
extremely high conservation of the nucleotide sequence 50 bp upstream of exon 1b

between the human and the mouse, the TATA and CAAT boxes which have been

Figure 1. CD45 structure. The overall structure of the receptor-type PTPase, CD45,
is shown depicting the extracellular domain, the transmembrane domain, and the
cytoplasmic domain. The extracellular domain contains the altemate-exon-encoded
sequences which generate the multiple isoforms. Glycosylation sites in the
extracellular domain are indicated. The location of a fibronectin-III-like (Fn-III—like)
repeat is shown. The cytoplasmic region contains two tandemly-repeated PTPase
domains along with a membrane proximal spacer, an inter-PTPase domain spacer, and
a C-terminal tail. Each cysteine essential for PTPase activity is depicted by C. The
position of a regulatory tyrosine residue phosphorylated by p50“" is indicated. The

numbers given in the figure refer to amino acid residue positions.

CD45 Protein Tyrosine Phosphatase

 

 

 

 

 

Alternate E O-linked glycosylation
exon ”59 ; . (many sites)
Extracellular .
1 - 541 CYS-n'ch % 2:3" N-linked glycosylation
(15 potential sites)
Trans— -
membrane [
542 - 563 "
PTPase active site I essential cys
Domain! / 817 and 1132
641 - 875 c
cytoplasmic a:
564 ‘ 1268 PTPase
Domain II
932 - 1190 3? + psocsk phosphorylation
T site

 

Figure I.

6

identified in some eukaryotic promoters are not found upstream of this exon or exon
la (19, 23). However, a number of TATA-like and CAAT-like sequences are located
upstream of each of these exons (19, 23).

Translation begins in the middle of exon 2 which encodes the 23 residues of
the signal sequence and is followed by an intron of about 50 kb (19). The seven
residues which are found at the N terminus of all CD45 isoforms are encoded by exon
3. While the use of exons 2 and 3 is invariant, mRNA species lacking any
combination of exons 4, 5, and 6 have been identified (17, 18, 24). Species also
lacking exons 7 and 8 have been found (25), and two other studies have also noted
mRNA species that cannot be explained by alternate use of exons 4, 5, and 6 (19,
24). Exon 16 encodes the transmembrane domain, and the termination of translation
and the 3’ untranslated region are encoded by exon 33 (19). There are two potential
polyadenylation sites in the 3’ untranslated region of exon 33, and it has been shown

that either one can be used (16).

Alternate-Emu Usage

Because cell type-specific patterns of altemate—exon usage are conserved
between human, mouse, and rat species, it is believed that CD45 isoform usage is
highly regulated (14, 26). While alternate-exon usage in nonlymphoid hematopoietic
cells has not been studied extensively (14), early studies of lymphoid cells led to the
generalizations that: 1) B cells express the highest molecular weight isoform of 220

kD which is produced when all of the exons are incorporated into CD45 mRNA, 2)

7
thymocytes express the smallest isoform of 180 kD which is encoded by mRNA

lacking exon 4, 5, and 6 sequences, and 3) T cells have complicated patterns of
altemate—exon usage and, thus, isoform expression (3). Subsequently, the former two
generalizations have been shown to be over—simplifications. Use of the polymerase
chain reaction technique has shown that all of the eight mRN A species which are
possible from the alternate use of exons 4, 5, and 6 can be found in a panel of B cell
tumor lines (24). Also, a number of studies have demonstrated that thymocyte
subsets can use alternate exons and that CD45 isoforms larger than 180 kD can be
found on the cell surface of thymocytes (25, 27-30).

In the study of cell typespecific expression of CD45 isoforms by T cell
subsets, anti-CD45 monoclonal antibodies (mAbs) with epitopes in the variably used
regions have been utilized (28, 29, 31): antibodies with specificities to epitopes
dependent on the expression of exon 4, also called exon A, are called CD45RA mAbs
(R denotes restricted epitope); those dependent on the expression of exon 6, i. e. B,
are CD45RB mAbs; those dependent on exon 7, i. e. C, are CD45RC mAbs; and
those which recognize the isoform produced from the mRNA lacking exons 4, 5, and
6 are called CD45R0 mAbs (R0 refers to restriction to the "0 exon” form). CD45R
is used to refer to any mAb with a restricted epitope, and antibodies which recognize
all CD45 isoforms are called CD45 or pan-CD45 mAbs. These terms are also used
to refer to the isoforms.

Rogers et al. (32) showed there is a high correlation of exon usage in CD45
mRNA in a cell line and the mAbs which react with CD45 on the cell surface. Of

the alternate exons, exon B is the most commonly used in humans and mice, but

8

mouse cells use alternate exons less often than their human counterparts (32). Among
the T helper subsets, altemate-exon use is more common in TH2 than in THl subsets
although there are numerous patterns of expression in each group (24, 28, 32-36).
CD8+ T cells use alternate exons more often and typically express higher molecular
weight isoforms than do CD4+ T cells (14, 25).

CD45R0 and CD45R have been considered to be markers for memory and
naive T cells, respectively (37-39). In the human system, CD45RA mAbs are
frequently used in memory studies while in the mouse system, in which alternate
exons are used less often, antibodies against epitopes from the most commonly-used
alternate exon, exon B, are typically used. Decreased use of exon B-dependent
epitopes by both CD4+ and CD8+ cells and a corresponding loss of 190 kD cell-
surface CD45 have been detected four days after in viva allostimulation (40). Also,
Lee et al. (41) determined that splenic T cells from newborn mice or mice raised in
aseptic environments are predominantly CD45RBhi. Increases in antigenic exposure
or age correlated with increases in the number of CD45RBl° T cells, and after long
term-in viva priming the majority of T cells were CD45RBl° (41). When either
CD45RBhi or CD45RB'° splenic T cells from primed donors were transferred into
nu/nu mice, recipients reconstituted with CD45RB'° cells exhibited an IgG response
while recipients reconstituted with CD45RBhi exhibited an IgM response (41). When
splenic CD4+ cells sorted into CD45RBhi and CD45RB'° populations were challenged
in vitro with antigen, from unprimed mice CD45RBhi cells proliferated well while

from mice previously primed with the antigen, CD45RB1° cells proliferated to a

9
greater degree (42). Hence, CD45RBhi cells appear to be naive T cells and a switch

to the CD45RB'° phenotype appears to occur after priming.

The same correlation of B-exon usage and age Lee and his co-workers (41)
reported was likewise observed by Ernst er al. (43). In the latter study, however,
although the CD45RB'° CD4+ T cells recovered from the spleens of older mice
appeared to produce more IL-4 than the cells from younger mice in response to in
vitro stimulation, the CD45RB'° CD4+ cells showed decreased proliferation when
compared to the cells from younger mice. Thus, in this study CD45RB'° cells did not
display the proliferative response typical of primed/ memory cells.

The switch from CD45R expression to CD45 R0 expression was considered to
be irreversible and unidirectional (44). The conversion of CD45R1°" to the
CD45RW+ phenotype, first observed in human and rat systems, has now also been
observed in mouse cells (36, 45-48). Thus, CD45RW+ and CD45R'°” phenotypes
may not be reliable markers for naive and memory cells (49-51). Instead, the
CD45R0+ CD45R' phenotype, which reflects the omission of the alternate exons from

mRNA, may be a marker for recently primed T cells.

Regulation of Splicing of the Alternate Exons

The regulation of splicing of the alternate exons was studied by Streuli and
Saito (52) using mini-LCA gene constructs encoding 5’ exons in an SV40
transcription unit. Cir-elements within or flanking the alternate exons 4 and 6 were

able to direct the tissue-specific splicing of the human gene constructs in murine T

10
(EL4) and pre-B (300—19) cell lines: in EL4 cells, each altemate-exon sequence was

omitted from cytoplasmic RNA and in 300-19 cells, it was included regardless of the
LCA exon flanking the alternate exon (52). A mini-gene construct with exon 5 did
not give a clear tissue-specific pattern of use, which may reflect its more common use
in T cells (32). When exon 9 was placed in the mini-gene construct instead of an
alternate exon, it was included in the mRNA of both cell types. The splicing pattern
typieal of thymocytes is seen in non-hematopoietic cell lines: murine NIH3T3 and L
cells spliced out the alternate exons in the production of mRNA from the mini-LCA
gene constructs; in human HeLa cells, though, a small amount of mRNA which
included exon 4 was detected (52). Several studies have determined that several
sequences within each alternate exon and in its nearby flanking sequences are required
to direct the tissue-specific splicing of the CD45 RNA (52-54). It is interesting to
note that the trans-acting factor which directs the alternate splicing of the exons is
apparently conserved between mice and humans because the factors produced by the
murine cells were able to faithfully splice the human gene constructs (52).

It was proposed that positive trans-acting factors produced by B cells direct
their use of the alternate exons based on the exon usage of T/B cell hybrids: the B
cell-splicing pattern was seen after stable hybrid lines were selected (54). However,
in the study of trans-acting factors, it is preferable to study the heterokaryons shortly
after fusion when the activities in one nucleus may (or may not) be altered by factors
clearly produced by the other nucleus, i. e. trans-acting factors (55). When murine T
cells and cells expressing the exon-usage pattern of human B cells were fused and the

products were analyzed within hours after the fusions for the presence of mRNA from

11

the human gene, increases in the splice products lacking alternate exons and decreases
in the products containing two or three of the alternate exons were noted (26).

Hence, T cells contain a regulatory signal which directs the splicing out of the
alternate exons. When murine B cells were fused with human T cells and likewise
analyzed, there was a slight, inconsistent increase in two-exon forms of human CD45
mRNA and no three-exon mRNA was detected. Thus, B cells do not appear to have
a positive regulatory signal to incorporate the alternate exons into mRNA. When
NIH3T3 cells were fused to human B cells, there was a shift to the production of
mRNA containing fewer alternate exons, which indicates that the negative regulatory
factor detected in T cells is also present in non-hematopoietic cells. The effect of
cycloheximide, a protein synthesis inhibitor, on the splicing patterns of normal human
thymocytes and peripheral T cells supported the theory that T cells express a negative
regulatory trans-acting factor which directs the removal of alternate exons from RNA

transcripts (26).

CD45PTPaseDomain

Mutational analysis of the murine cytoplasmic region expressed in vitro
indicated that the membrane proximal spacer and both PTPase domains must be
present for activity while the deletion of the cytoplasmic tail did not affect activity
(56). The deletion of the C terminus including 13 residues from the PTPase 11
domain, though, did abolish activity. Within the conserved domains of several

PTPases, there are several highly conserved features: a cysteine residue and its

12
surrounding 10-15 amino acids, a GXGXXG motif, and in PTPase 11 domains, a
region of about 20 acidic amino acids (56, 57). Since mutation of Cys317, the highly
conserved cysteine residue in domain I of murine CD45, but not of Cysmz, its
counterpart in domain 11, abolished PTPase activity, it was determined that the
PTPase 1 domain was responsible for the activity (56). Also, mutation of any of the
three glycine residues in the GXGXXG motif in PTPase I decreased activity (5-fold or
more) while only alteration of the first glycine in the motif in the second domain
affected activity (2-fold decrease). Deletion of the acidic region of PTPase H also
reduced activity (4-fold). Alteration of Tyr729 to phenylalanine abolished activity
while similar mutations of several other conserved tyrosine residues did not affect
activity (56). Analysis of human CD45 cytoplasmic sequences expressed in bacteria
determined that PTPase I is active by itself (57). The second PTPase domain of
CD45 has also been shown to become active in highly-specialized circumstances

involving phosphorylation of the cytoplasmic domain (58-60).

CD45 Function

Many of the early studies undertaken to determine the function of CD45 were
performed by observing the effects of anti-CD45 antibodies on cellular activities.
From determinations that such antibodies inhibit or enhance cytolytic functions of
antibody-dependent cell-mediated cytotoxicity, natural killer cells, and cytolytic T
cells (61-64), and proliferation and differentiation of B and T cells (63, 65-68), a role

for CD45 in a variety of signalling processes had been indicated.

13

Subsequently, two discoveries have entirely changed the methods used to study
CD45 function. The first revolution in CD45 experimentation occurred in 1988 when
Charbonneau and his colleagues proposed on the basis of amino acid sequence
similarity to a human placental enzyme that CD45 may be a PTPase (9). Shortly
thereafter, several groups confirmed that CD45 is indeed a PTPase (69-71). The
second discovery that changed the manner in which CD45 is studied was that CD45-
deficient lymphocytes have impaired immunological responses (72). This finding has
led to the extensive use of CD45' cell lines to study CD45 function.

CD45 is essential in both B and T cells to couple stimulation of , the antigen
receptor ,to secondmessenger_pathways. The earliest intracellular responses to
ligation of antigen receptors are tyrosine phosphorylation and phospholipase C
activation, which results in Ca2+ mobilization (73-76). In CI_)__4“5;cefll_i_iines~both
tyrosine phosphorylation and. Ca”? flux responses are diminished or“ absent (77-81).
Normal tyrosine phosphorylation and Ca2+ flux responses to” antigen receptor
stimulation. are restored to CD45' cells when the intracellular ”region of CD45. is
expressed. in the cells, even when the external CD45 region is, absent or replaced by
MHC class I or epidermal growth factor receptor extracellular sequences (79, 81-84).

~ln—‘NIJ- ‘w-IA...‘

CD45 amtgvtegulatc. the phosphorylation. and. activity ..9f_-§r9._€slll,ily

__.,_M

kinases; ”lymphocytes. Both p56"" and p590" are typically hyperphosphorylated in

. r ..‘_
”a _ .
“." "'r I.

5-“.

CD45' T cells, but the resulting effect on the protein tyrosine kinase (PTK) activity of
p56” and p59m is variable (71, 7o, 81, 85-89). Data derived from other
experimental approaches also provide evidence that CD45 can up- or down-regulate

p56"" and p590" PTK activity (71 , 90-92). Perhaps further studies based on the

l4
observation that CD45 regulates specific pools of p56” and p599" (89) will clarify

the seemingly contradictory data on the effect of CD45 on the activity of the src
family kinases.

In addition to its role in the activation of lymphocytes, CD45 appears to have
a role in mitosis. The PTPase activity of exponentially-growing cultures is higher
than that of stationary cultures (93, 94). Using centrifugal elutriation to obtain
subpopulations of asynchronous B and T cell cultures which were enriched for cells in
various stages of the cell cycle, it was determined that CD45 PTPase activity is
elevated 2- to 5-fold in Gz+M when compared to its activity during other stages of

the cell cycle.

CD45 Interactions with Other Molecules

The B lymphocyte adhesion molecule CD22 has been identified as a potential
ligand of CD45 (95). Although it has been determined that several human CD45
isoforms can interact with CD223 (95, 96), this interaction is not highly specific since
CD223 is a lectin which recognizes a number of glycoproteins on B and T cell
membranes with a-2,6-linked sialic acids (97, 98).

In studies using immunoprecipitation following chemical cross-linking or lysis
with the mild detergent digitonin, CD45 has been shown to associate with several
other molecules on lymphocyte cell surfaces including Thy-l, CD2, CD3, and CD26

(99-101). In addition, CD45 co-localization with capped CD4 (but not other T cell

15
surface molecules) has been observed (102). This CD4/CD45 interaction appeared to

be limited to low molecular weight isoforms of CD45 as CD45RB did not co-cap.

CD45 interacts with the cytoskeleton, and this interaction increases after
receptor patching and/or capping (103-105). In particular, the cytoskeletal element
fodrin appears to form a complex with CD45 (103, 104, 106).

In resting splenic murine B cells membrane IgM, p539“, and the antigen
receptor complex components MB-l and B29 have been coprecipitated with CD45
from digitonin lysates (107). In this study, many proteins were labelled in in vitro
phosphorylation reactions of CD45 immunoprecipitates from digitonin, CHAPS, and
Brij 35 lysates. Although the authors reported that the immunoprecipitates were
washed extensively before further analyses, they did not report the use of a high salt
solution in the washing process, a practice commonly used to reduce the presence of
nonspecifically bound material. Thus, care must be taken in the consideration of
these findings. Despite the lack of a high salt wash, however, the presence of the src
family kinase p531” in the CD45 complex was judged to be specific since other src-
related kinases which were expressed in the cells were not detected in the CD45
immunoprecipitates.

In the activation of T cells, CD45 may regulate the activity of both p56"" and
p590", two src family PTKs which are involved in the response to TCR/CD3
stimulation (71, 81, 85, 86, 88-92). Thus, an interaction of CD45 with each of these
PTKs can be inferred, and although binding of p56“ to CD45 has been observed,

similar data for p59" is yet to be presented.

16
The interaction of CD45 with p56“ appears to occur via the src homology

“my.” _

region _2_ (8.333???“ of the PTK and a phosphotyrosine residue on CD45 (60).
Although p56{‘5M some affinity for nonphosphorylated CD45, treatment of CD45
with 11,595.33 .3115, enhanced the association. In addition, the binding of p56"" to
CD45 was inhibited by the presence of recombinant SH2 domain.

Analyses of CD45 immunoprecipitates from digitonin or Brij 96 lysates of
human peripheral blood T lymphocytes and cultured human and murine T cells have
shown that CD45 associates with p56"" and a group of phosphoproteins of variable
molecular weights, referred to as pp29-34 or the lymphocyte phosphatase-associated
phosphoprotein (LPAP) (108-114). LPAP can act as a substrate for both p56“ and
CD45 in vitra (108). In vivo, LPAP is phosphorylated almost exclusively on serine,
and although it underwent change in electrophoretic mobility upon activation of cells,
no change in its phosphorylation could be detected (108, 109, 111, 112). It should be
noted the in viva phosphorylation analysis was performed without the use of the
potent PTPase inhibitor phenylarsine oxide, which was essential in the detection of
phosphotyrosine in CD45 (60, 115). The association of p56” with CD45 is not
mediated by CD4: even after CD4 was precleared from lysates, p56” still
coprecipitated with CD45 (112). Also, LPAP can bind to CD45 even in the absence
of p561“ (112). Interestingly, although when CD45 is immunoprecipitated from
digitonin lysates, p56” can be identified in the CD45 complex both by
immunoblotting and re-precipitation analyses (108, 111, 112), LPAP is
phosphorylated almost exclusively on serine after in vitra phosphorylation treatments

of the CD45 immunoprecipitates from Jurkat T cells (111). Thus, a serine-threonine

l7

kinase must also be present in the CD45 immunoprecipitates. It was determined that
the association of the serine-threonine kinase with the immunoprecipitate was specific
to CD45 because serine-threonine kinase activity was not found in mock CD45

immunoprecipitates from a CD45' clone (111).

Regulation of the PTPase Activity of CD45

CD45 PTPase activity is increased 7.5-fold after the binding of fodrin in vitro
(106). Thus, interaction with the cytoskeleton may play a role in regulating the
PTPase activity of CD45.

Dimerization may also regulate the activity of CD45. A chimeric form of
CD45, in which the external region was replaced by the external region of the
epidermal growth factor receptor, restored normal tyrosine phosphorylation and Ca2+
flux responses to a CD45' cell line. However, these responses were inactivated when
the chimeric CD45 was dimerized with the ligand epidermal growth factor (84).
Dimerization of CD45 may be a naturally-occurring phenomenon as dimeric forms of
CD45 have been recovered from the murine T cell line YAC-l (110).

Studies on the regulation of CD45 PTPase activity by phosphorylation indicate
that both serine and tyrosine phosphorylation are involved. Decreased PTPase
activity has been shown to correlate with decreased serine phosphorylation after Ca2 +
ionophore treatment of T cells (116). In contrast, increased serine phosphorylation of
CD45 was not associated with modulation of its enzymatic activity after either in vitra

treatment with casein kinase II and other serine-threonine kinases or after IL-2

18
stimulation of CTLL-2.4 cells (117, 118). The PTPase activity of CD45 can be

increased up to 8-fold in vitra by treatment with p50“", a PTK (60). In another
study, sequential in vitra phosphorylation of CD45 first on tyrosine with v-abl, then
on serine with casein kinase II was necessary to produce maximal enhancement of its
PTPase activity (59). Stover and Walsh (59) attributed the increased PTPase activity

they noted to activation of the second PTPase domain of CD45 .

10.

19

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Biochim. Biophys. Acta. 896:35.

Lin, J ., V. K. Brown, and L. B. Justement. 1992. Regulation of basal tyrosine
phosphorylation of the B cell antigen receptor complex by the protein tyrosine
phosphatase, CD45. J. Immunol. 149:3182.

Lokeshwar, V. B., and L. Y. Bourguignon. 1992. Tyrosine phosphatase
activity of lymphoma CD45 (GP180) is regulated by a direct interaction with
the cytoskeleton. J. Biol. Chem. 267:21551.

Brown, V. K., E. W. Ogle, A. L. Burkhardt, R. B. Rowley, J. B. Bolen, and
L. B. Justement. 1994. Multiple components of the B cell antigen receptor
complex associate with the protein tyrosine phosphatase, CD45. J. Biol.

Chem. 269:17238.

108.

109.

110.

111.

112.

113.

114.

115.

116.

117.

29

Schraven, B., H. Kirchgessner, B. Gaber, Y. Samstag, and S. Meuer. 1991.
A functional complex is formed in human T lymphocytes between the protein
tyrosine phosphatase CD45, the protein tyrosine kinase p56lck and pp32, a
possible common substrate. Eur. J. Immunol. 21:2469.

Schraven, B., A. Schirren, H. Kirchgessner, B. Siebert, and S. C. Meuer.
1992. Four CD45/P56lck-associated phosphorproteins (pp29-pp32) undergo
alterations in human T cell activation. Eur. J. Immunol. 22:1857.

Takeda, A., J. J. Wu, and A. L. Maizel. 1992. Evidence for monomeric and
dimeric forms of CD45 associated with a 30-kDa phosphorylated protein. J.
Biol. Chem. 267:16651.

Koretzky, G. A., M. Kohmetscher, and S. Ross. 1993. CD45-associated
kinase activity requires lck but not T cell receptor expression in the J urkat T
cell line. J. Biol. Chem. 268:8958.

Ross, S. E., B. Schraven, F. D. Goldman, J. Crabtree, and G. A. Koretzky.
1994. The association between CD45 and lck does not require CD4 or CD8

and is independent of T cell receptor stimulation. Biochem. Biophys. Res.
Commun. 198:88.

Takeda, A., A. L. Maize], K. Kitamura, T. Ohta, and S. Kimura. 1994.
Molecular cloning of the CD45-associated 30-kDa protein. J. Biol. Chem.
269:2357.

Schraven, B., D. Schoenhaut, E. Bruyns, G. Koretzky, C. Eckerskom, R.
Wallich, H. Kirchgessner, P. Sakorafas, B. Labkovsky, S. Ratnofsky, and S.
Meuer. 1994. LPAP, a novel 32-I(da phophoprotein that interacts with CD45
in human lymphocytes. J. Biol. Chem. 269(46):29102.

Stover, D. R., H. Charbonneau, N. l(. Tonks, and K. A. Walsh. 1991.
Protein-tyrosine-phosphatase CD45 is phosphorylated transiently on tyrosine
upon activation of Jurkat T cells. Proc. Natl. Acad. Sci. U. S. A. 88:7704.

Ostergaard, H. L. , and I. S. Trowbridge. 1991. Negative regulation of CD45
protein tyrosine phosphatase activity by ionomycin in T cells. Science.
253: 1423.

Tonks, N. K., C. D. Diltz, and E. H. Fischer. 1990. CD45, an integral
membrane protein tyrosine phosphatase. Characterization of enzyme activity.
J. Biol. Chem. 265:10674.

30

118. Valentine, M. A., M. B. Widmer, J. A. Ledbetter, F. Pinault, R. Voice, E.
A. Clark, B. Gallis, and D. L. Brautigan. 1991. Interleukin 2 stimulates serine
phosphorylation of CD45 in CTLL—2.4 cells. Eur. J. Immunol. 21:913.

CHAPTER TWO

ELEVATION OF LYNIPHOCYTE CD45 PTPASE ACTIVITY

DURING Ml'rosrsl

31

32

FOOTNOTES

1 This work was supported by NIH grant GM35774 and by a grant from the

Children’s Leukemia Foundation of Michigan.

2 Abbreviations used in this paper: PTPase, protein tyrosine phosphatase;
PTK, protein tyrosine kinase; PI, propidium iodide; DAPI, 4’,6—diamidino-2-

phenylindole; MPF, mitosis promoting factor.

33

ABSTRACT

CD45 is an abundant cell-surface protein tyrosine phosphatase (PTPase) of
hematopoietic cells which mediates specific tyrosine dephosphorylation in lymphocyte
antigen activation. The hypothesis that CD45 PTPase activity varies during
progression through the cell cycle was examined in this study. CD45 PTPase activity
was determined in the cytolytic T lymphocyte cell line CTLL-2 after fractionation by
counterflow centrifugal elutriation into cell cycle stage-enriched subpopulations. For
this cell line, CD45 PTPase activity was elevated 2- to 5-fold in G2+M fractions
compared to its PTPase activity in GI and S fractions. FACS and SDS-PAGE
analyses indicated that there was only a minor increase in CD45 protein expression
during progression through the cell cycle, suggesting that increased CD45 PTPase
activity was due to increased enzymatic activity. The timing of this observation
indicated that the peak PTPase activity of CD45 occurred in Gz-l-M or possibly in
cytokinesis. The elevation of CD45 PTPase activity in GZ+M supports the
hypothesis that CD45 has a role in phosphorylation events occurring during cell

division.

34

INTRODUCTION

The level of cellular tyrosine phosphorylation is a consequence of the opposing
actions of P'I'Pases2 and P’I‘Ks. Previous research has focused on the roles played by
PTKs in cellular regulation. However, since the identification of the PTPase family,
considerable effort has also been devoted to establishing the role of this class of
soluble and membrane-associated enzymes in the regulation of signal transduction and
cell cycle progression.

CD45 PTPase, a heterogeneous family of plasma membrane-associated
proteins, is ubiquitously and prominently expressed on the surface of all nucleated
hematopoietic cells (1). The intracellular region of CD45 consists of two tandemly-
repeated PTPase domains with enzyme activity residing primarily in the first domain
(2, 3). The level of CD45 expressed on the surface of a single lymphocyte can range
as high as 50,000 to 100,000 molecules (4), making it one of the major membrane
glycoproteins of the hematopoietic lineage. Deletion of CD45 severely impairs TCR-
and membrane IgM-mediated signal transduction (5-9). CD45 dephosphorylates src

6“", which are essential for facilitation of T cell activation

family kinases such as p5
(6, 7, 10, 11). Studies with CD45‘ cell lines show that CD45 is essential for coupling
antibody-mediated TCR stimulation to both the phosphatidyl inositol cascade and

tyrosine phosphorylation signalling pathways (7). The PTPase activity of T cell

35

CD45 has also been shown to correlate with serine or tyrosine phosphorylation of the
molecule (12, 13).

It has been proposed that the PTPase activity of CD45 is influenced by its
association with cytoskeletal elements (14, 15). CD45 contains fodrin/spectrin
binding domains and when fodrin or spectrin was added to purified CD45, its PTPase
activity increased up to 7.5 fold (14). Association of fodrin with other cytoskeletal
elements is believed to involve the phosphorylation state of these molecules. Further,
in B cells, cross-linking of CD45 has been shown to induce association of CD45 with
the cytoskeleton, resulting in capping of CD45 and increased tyrosine phosphorylation
of membrane lg-associated proteins (15). These reports suggest a regulatory role for
the CD45/cytoskeleton complex during lymphocyte activation and proliferation.

Although CD45 clearly has a specific role in lymphocyte signal transduction
and subsequent activation, the abundant expression of CD45 in many hematopoietic
cell types suggests that it may also play a fundamental role in other immunological or
cellular processes, such as maintenance of cellular stasis or in cell cycle regulation.
Using counterflow centrifugal elutriation and immunoanalysis, this study tested the
hypothesis that CD45 PTPase activity varies with cell cycle stage in a manner

consistent with its expected association with cytoskeletal elements.

36

MATERIALS AND METHODS

Cell culture

The cytolytic T cell line CTLL-2 was obtained from the American Type Culture
Collection (ATCC; Rockville, MD) and maintained at 37°C in a humidified
environment with 7.5% C02 in RPMI 1640 (GIBCO/BRL, Gaithersburg, MD)
containing 10% FBS (Biologos, Naperville, IL), 50 uM 2-ME (Sigma Chemical Co.,
St. Louis, MO), and 7 Wm] recombinant IL-2 (Cetus Corporation, Emeryville, CA)
(complete medium). Cells were maintained in a log exponential growth state (0.25 to
5.0 x 105 cells/ml). Cultures were typically harvested upon attaining a density of 1-4
x 105 cells/ml.

Counterflaw centrifilgal elutriation

Cells were separated using counterflow centrifugal elutriation carried out at 12°C and
2250 rpm in a JE-6B elutriation rotor in a 12-21M/E elutriation centrifuge system
(Beckrnan Instruments, Palo Alto, CA). Log phase cells were concentrated by
centrifugation at 1000 x g and resuspended at 4 x 10‘5 cells/ ml in complete medium.
Typically, 2-4 x 108 cells were loaded into the elutriation chamber with a peristaltic
pump at an initial flow rate of 4.5 ml/min with the elutriation buffer, Hank’s balanced
salt solution (GIBCO/BRL), pH 7.4, containing 1 mg/ml BSA (Sigma). After all of

the cells had entered the elutriation chamber, the system was equilibrated at 4.5

37

ml/min for 10-15 min. Elutriation fractions were obtained by increasing the flow rate
1-2 ml/min per fraction. Five minute fractions at each flow rate were collected in 50
ml conical tubes on ice and then pelleted and resuspended in minimal volumes for
analysis. The possibility that cells attached to the apparatus and bled out over time
was minimized by treating the elutriation chamber and tubing with silicone.
Immunoprecipitatian of CD45
Viable cell counts were obtained for concentrated cell fractions using trypan blue
exclusion. Cells were pelleted at 500 x g at 4°C for 5-10 min and were washed twice
with cold PBS (8 mM NazHPO4, 1 mM KH2P04, 137 mM NaCl, 3 mM KCl, pH
7.3). Following the final wash, cells were lysed by suspending the pellet in 1% (v/v)
Triton X-100 (Boehringer Mannheim, Indianapolis, IN); 0.5% (w/v) sodium
deoxycholate (Sigma); 0.1% (w/v) SDS (Bio-Rad Laboratories Inc., Melville, NY);
0.1 M NaCl; 10 mM phosphate buffer, pH 7.5; 0.01% NaN3 (l x 107 cells/m1 lysis
buffer). After 45 min on ice, the suspension was centrifuged at 12,000 x g for 15
min at 4°C. Postnuclear lysates were precleared with 25 [1.1 packed protein G-agarose
(Pharmacia, Piscataway, NJ, or Boehringer Mannheim) per 107 cells.

A 10 pl aliquot was removed for determining protein concentration ( l6).
CD45 and CD8 were immunoprecipitated from the remaining lysate by incubation
with anti-CD45 M1/9.3.4 mAb (ATCC) or with isotype-matched anti-CD801 53-6.72
(ATCC) for 60 min at 4°C, followed by addition of protein G-agarose with
continuous mixing for an additional 30 min at 4°C. Immunoprecipitates were washed
two times with 250 mM HEPES (Sigma), pH 7.3, and stored in 10X PTPase assay

buffer (250 mM HEPES, pH 7.3; 50 mM EDTA; 100 mM DTT) at -70°C.

38
Phosphatase assay

The PTPase activity of immunoprecipitated CD45 was determined using a
modification of established protocol (17). In brief, raytide (Oncogene Science,
Manhasset, NY) was labelled with [7-32P]-ATP (>6000 mCi/mmol; Dupont/New
England Nuclear, Boston, MA) using recombinant src tyrosine kinase (a gift of J.
Dixon, University of Michigan, Ann Arbor). The final substrate labelling reaction
contained 50 pg of raytide; 30 pM ATP (Sigma); 10 mM MgC12; 16 mM HEPES,
pH 7.5; 0.03 mM EDTA; 0.07% 2-ME; 400-800 ng src kinase; and 50 pCi ['y -32P]-
ATP. After incubation at 30°C for 40 min, the 32P-raytide was recovered by TCA
precipitation and dissolved in 200 pl 200 mM Tris (Sigma), pH 8. Each PTPase
assay contained up to 5 pl immunoprecipitated CD45, 5 pl 10X phosphatase assay
buffer (250 mM HEPES, pH 7.3; 50 mM EDTA; 100 mM DTI'), 5 pl 32P-raytide
(1-2 x 105 cpm) and H20 to 50 pl. Reactions were allowed to proceed for 5-20 min
at 37°C and then terminated by adding 750 pl of acidic charcoal suspension (0.9 M
BC], 90 mM Na4P207, 2 mM NaHzPO4, 4% [v/v] Norit A [Sigma]). After
cartrifugation for 3 min, 400 p1 of the supernatant were removed and the 32F
radioactivity of the sample was determined using liquid scintillation. The amount of
”Pi released was expressed as total cpm released per min per mg protein of the lysate
from which the CD45 was immunoprecipitated. A standard curve using CD45
immunoprecipitated from asynchronous cultures was used in each independent
experiment for establishing the linear range for the assay. CD45 PTPase activity was

measured in the linear range.

39

Immunofluorescent staining

For flow cytometry, cells were fixed in PBS/FBS/70% ethanol (0.2:O.2:l.2, v/v/v)
for 18-36 hrs at -20°C, washed twice with PBS containing 5% (v/v) FBS, and
immunolabelled on ice for 30 min with anti-CD45 mAb (Ml/9.3.4). The cells were
washed twice with PBS containing 5% (v/v) FBS and incubated with affinity-purified
FITC-conjugated goat anti-rat IgG (preabsorbed with murine IgG, Cappel
Laboratories, Durham, NC) for 30 min. The FITC mAb-labelled cells were washed
twice with ice-cold PBS and resuspended in P1 staining solution (PBS with 0.1%
Triton X-100, 0.1 mM EDTA, 0.05 mg/ml RNase A, and 50 pg/ml PI [Sigma]). For
fluorescent microscopy, cells were fixed in 2% formaldehyde in PBS and stained with
1% DAPI (Molecular Probes Inc., Eugene OR) in PBS.

Cell cycle analysis by FA CS

The cell cycle states of CTLL-2 elutriation fractions were determined by FACS
analyses of the DNA content of PI—stained, FI'l‘C-anti-CD45’r cell populations. PI
and FITC fluorescence were analyzed at 488 nm excitation on a Becton Dickinson
Vantage FACS (San Jose, CA). Pl emission was taken at 630 nm bandpass and FITC
fluorescence was read at 530 nm bandpass. Data were analyzed using PC-Lysys
version 1.0 software (Becton Dickinson, San Jose, CA). Mean FITC fluorescence
was determined following singlet discrimination based on P1 staining.

SDS-PAGE and silver staining

For each elutriation sample, the amount of CD45 or CD8 immunoprecipitate obtained
from 380 pg protein in the lysate was subjected to SDS-PAGE in a 4-15% gel for

approximately 1 hr at 125V ( 18). A variation of silver staining (19) was performed

40

as follows. Gels were first stained with Coomassie blue then destained. Gels were
equilibrated to H20 and treated with 5 pg DTT/ ml H20 for 30 min then with 1 mg
AgNO3/ml H20 for 30 min. The stain was developed with 3% (w/v) NaCO3, 0.05%

(v/v) HCHO, and development was stopped with 10% (v/v) CH3COOH.

41

RESULTS

The hypothesis of this study was that CD45 PTPase activity varied with cell
cycle stage. This hypothesis was based on the observation that CD45 PTPase activity
was influenced by culture density (data not shown). In addition, previous work with
chemical blocks suggested that there may be an increase in CD45 PTPase activity in
mitotic cells (data not shown). Therefore, further investigation utilizing counterflow
centrifugal elutriation was initiated to isolate subpopulations of cells representing each
of the phases of the cell cycle.

Counterflow centrifugal elutriation separates cells on the basis of velocity
sedimentation rates, permitting the study of cell cycle—dependent phenomena with
minimal perturbation of normal in vitro growth conditions (2023). Distinct
subpopulations from an asynchronously-proliferating p0pulation of cells were
separated, stained with P1 and FITC—anti-CD45, and analyzed by FACS. This
procedure resulted in effective isolation of subpopulations enriched for the various cell
cycle phases (Fig. 1). Debris and dead cells were recovered at less than 10 ml/min.
Early GI cells were concentrated in fractions obtained from flow rates of about 12

ml/min, late G] from 14-20 ml/min, S from 21-27 ml/min, early GZ+M from 28-31

ml/min, and late GZ+M from 32-37 ml/min. The percentage of cells recovered in

each fraction is shown in Table I. Elutriation flow rates varied slightly between

42

Figure I. FACS analysis of the DNA content of elutriated CTLL-Z cells. Two-color
FACS was performed on elutriation fractions from an exponentially-growing CFLL-2
culture. Cells from each sample were treated with FITC-anti—CD45 (Ml/9.3.4)
followed by P1 and analyzed by flow cytometry. PI fluorescence histograms of FITC-
anti-CD45+ cells are shown for (A) the asynchronous starting population of CTLL-2
cells and (B—P) elutriation fractions 8-25. Panel Q, noted with an asterisk,
corresponds to the maximal CD45 PTPase activity. The DNA staining patterns were
interpreted as follows: B—D, fiactions 8—10, GI phase; E-H, fractions 11-14, GIIS
phase; I-K, fractions 15-17, S phase; L-M, fiactions 18 and 19, S/62 phase; and N-S,
fractions 20-25, GZ+M phase. The flow rates and cell recoveries for each elutriation
fraction are shown in Table I. The cell cycle profiles are from a single experiment

and are representative of six independent experiments.

Table I. Elutriation flow rates and recovery of GILL—2 cells

 

 

Cell
Elutriation Flow Rate Percent Cycle
Fraction (ml/ min) Recovered Phase
8 12 1.0
9 14 10.2
10 15.5 7.7
11 17 7.2 GI
12 18 11.8
13 19.5 6.1
14 21 8.5
15 22.5 9.3
16 24.5 4.9 S
17 25.5 4.9
18 26.5 4.0
19 28.5 5.3
20 30 5.3
21 31 4.2 62+M
22 32 2.8
23‘it 34 4.1
24 35.5 1.2
25 37 1.4

 

aPeak of CD45 PTPase activity.

45

experiments because of limitations in the precision of the peristaltic pump. In the
asynchronous culture used in the elutriation analyzed in this paper, 43% of the cells
were in GI, 29% in S, and 28% in GZ+M (Fig. 1A).

The PTPase activity of CD45 immunoprecipitates was measured for the
elutriated fractions and, at flow rates from 34-36 ml/min, was consistently 2- to 5-
fold higher than in other fractions. A representative CD45 PTPase enzymatic profile
obtained from six independently-elutriated log-phase cultures showed recovery of
maximal CD45 PTPase activity as a peak (Fig. 2, fraction 23). Additionally, CD8
was immunoprecipitated as a control. Anti-CD8 immunoprecipitates of each
elutriation fraction were found to lack PTPase activity thus confirming that PTPase
activity did not nonspecifically associate with protein G/antibody complexes (Fig. 2).
A fairly high basal level of CD45 PTPase activity was observed in these cells.
Analysis of the DNA content of cells from these elutriation fractions by FACS
indicated that the peak of CD45 PTPase activity occurred in the 62+M population
(Fig. 3).

FACS analysis of FITC-anti-CD45-labelled cells from each elutriation fraction
showed that the increase in CD45 PTPase activity was not simply the result of an
increase in the amount of CD45 present in the cells of the 62+M fractions (Fig. 4).
The relative amount of CD45 was estimated by mean fluorescence measured by FACS
analysis (24). There was a relatively minor increase in mean green fluorescence of
elutriated cells during cell cycle progression. The method used for fixation of the
cells resulted in permeabilization of the plasma membrane and permitted effective

labelling of both cytoplasmic and membrane-associated CD45 protein. Cell size, as

46

Figure 2. CTLL-2 CD45 PTPase activity during cell cycle progression. The relative
PTPase activity of CD45 in each elutriation fraction (I) is expressed as the cpm of
the ”Pi released from 32P-raytide per min per mg protein in the lysate from which
CD45 was immunoprecipitated. The PTPase activities of the isotype-matched control
immunoprecipitates, i. e. CD8 immunoprecipitates, are also presented (A). Cell cycle

histograms for fractions 8-25 are given in Fig. 1 panels B-S, respectively.

PTPase ocfivfiy

47

01
o
l

 

 

 

¢/\ vvvvvvvvvvvvvvvvv
E A CD8 I CD45

x 4‘0 ’

D

~E‘ 30r

.E J

E 20 _

n.

N

n 10

E l

8'

V. O A—é—Q—é—A—Q—é—é—é—é—é—A—é—é—A—A—A—é-

 

‘IO 15 20 25

Elutriation fraction

Figure 2.

48

Figure 3. Cell cycle stage analysis of CTLL-2 elutriation fractions. The percent of
cells in each stage of the cell cycle, based on PI fluorescence depicted in Fig. l, is
presented for each elutriation fraction analyzed in Fig. 2. The fraction with the peak

CD45 PTPase activity is noted with an asterisk.

96 Cells

100

80'

60-

4o.

20'

49

 

 

v V T VVVVVVVVVV f ''''

 

Elutriation fraction

Figure 3.

 

50

Figure 4. CD45 fluorescence of elutriation fractions. CD45 expression of elutriated
populations was compared by analysis of the mean FITC-anti-CD45 fluorescence
exhibited by each elutriated fraction. This analysis is representative of six independent

experiments. Peak CD45 PTPase activity occurred in fraction 23, denoted by an

asterisk.

Mean fluorescence

100

80-

60-

40~

20

51

 

 

IIIIIIIIIIIIIIIIII

AAAAAAAAAAAAAAAAA

 

Elutriation fraction

Figure 4.

 

52

estimated by mean forward scatter, also increased proportionately, indicating that the
relative amount of CD45 did not increase more than would correspond to the size
increase of the cells (data not shown).

The amount of CD45 present in cells from each elutriation fraction was also
determined by SDS-PAGE analysis of CD45 immunoprecipitates. Immunoprecipitates
that were used for the PTPase assay were separated by SDS-PAGE and proteins were
visualized by silver staining (Fig. 5). This analysis demonstrated that CD45
immunoprecipitates contained only CD45 along with immunoglobulin heavy (about 50
kD) and light chains (about 23 kD) from the monoclonal antibody (Fig. 5A).
Importantly, no significant increases in CD45 protein levels were observed upon
comparison of elutriation fractions with basal levels of CD45 PTPase (Fig. 5A,
fractions up to 21) to those with elevated CD45 PTPase (Fig. 5A, fractions 22 and
23). For comparison, SDS-PAGE analysis of the PTPase-negative, anti-CD8
immunoprecipitates indicated the presence of CD80: along with immunoglobulin heavy
and light chains and only a few other contaminating proteins (Fig. SB).

These results confirm that the increase in CD45 PTPase activity in Gz-l-M
resulted primarily from an increase in relative PTPase activity rather than an increase
in protein expression. A consistent amount of CD45 was immunoprecipitated from
samples since all the conditions for immunoprecipitation were performed on a per cell
basis and each PTPase activity measurement was adjusted to the level of protein
present in the cell lysate.

Following fractionation by elutriation, cells were examined by staining with

DAPI to ascertain the mitotic nature of the cells in the fraction exhibiting elevated

53

Figure 5. CTLL-2 expression of CD45 and CD8 through the cell cycle. The
amounts of CD45 and CD8 proteins present in immunoprecipitates were determined
by silver staining of SDS-PAGE—separated proteins. (A) CD45 immunoprecipitates
from elutriation fractions showing CD45 (arrow) accompanied by immunoglobulin
heavy (about 50 kD) and light chains (about 23 kD). (B) Control immunoprecipitates
from elutriation fractions showing the position of CD80: (arrow) with immunoglobulin

heavy and light chains.

54

L‘ _ ‘7
files.

171819 20 2122 23 24 25

 

 

 

 

 

 

 

8 910111213141516

 

 

Figure 5.

55
CD45 PTPase activity. Cells recovered in early 6,, as determined by P1 analysis,

were small and exhibited a compact DNA staining pattern (Fig. 6A). The cells from
fractions in 62+M were large and exhibited bipolar DNA staining patterns with
numerous apparent mitotic figures (Fig. 6B). The nuclear staining patterns of cells
from the fractions with the highest CD45 PTPase activity were about 50% bipolar and
distinctly mitotic. In addition, a small population of cells with single-nucleus staining

patterns characteristic of 61 cells was observed. Taken together, these data indicated

that the cells with the highest level of CD45 PTPase activity were in 62+M.

56

Figure 6. Fluorescent microscopy images of elutriated CTLL-2 cell populations. The

DNA of elutriated cell populations was stained with DAPI and analyzed by
microscopy. (A) Representative cells from a GI phase-enriched fraction. (B)

Representative cells from the GZ+M fraction with the peak CD45 PTPase activity.

57

 

Figure 6.

58

DISCUSSION

Although CD45 clearly functions in antigen activation of B and T
lymphocytes, its abundance on other hematopoietic cells suggests that it may also play
a role in other basic immunological and cellular phenomena. Since it is believed that
CD45 interacts with cytoskeletal microfilaments via fodrin and because dividing cells
undergo extensive microfilament reorganization, we investigated the potential
modulation of CD45 PTPase activity in the progression through the cell cycle. CD45
PTPase activity was examined in CTLL-2 cells separated by centrifugal elutriation.
Cell cycle-dependent expression of CD45 PTPase activity reached a maximum in
Gz+M-enriched fraction. This fraction also contained cells with bipolar nuclear
staining patterns and some small Gl-type cells. The small cells in these fractions
were likely cells that had just completed cytokinesis during the time of separation in
the elutriator and during preparation of the cells for analysis. Once initiated,
cytokinesis is believed to proceed in a very short period of time. The possibility that
doublets were in the analyzed populations was eliminated by microscopic examination
of the elutriated subpopulations. Typically, doublets appeared after the flow rate
reached 36-40 ml/min and were not present in significant numbers in the
subpopulations analyzed for CD45 PTPase activity. These observations suggest that

peak CD45 PTPase activity occurred in GZ+M cells and may have occurred in cells

59
undergoing cytokinesis. The presence of elevated CD45 PTPase activity during

cytokinesis would be consistent with a role for CD45 in the regulation of
microfilament organization.

In other studies with 702 cells, it was determined that the increase in CD45
PTPase activity was placed among events occurring after anaphase by the observation
that the highest PTPase activity was found in elutriation fractions immediately
following the disappearance of the 62 kD cyclin B protein (data not shown). Cyclin B
accumulates within a cell as it proceeds through the cell cycle, associating with
p34c‘i‘2 kinase forming MPF or M-phase specific histone H1 kinase (25, 26). The
inactive MPF kinase complex attains a maximum level at prophase and becomes
activated via the dephosphorylation of p34““2 (27-29). Following activation, cells
enter metaphase and the cyclin B/p34‘d‘2 complex degrades by the completion of
anaphase (30). The observation that the peak of CD45 PTPase activity was apparent
only after cyclin B degradation supports the hypothesis that CD45 PTPase participates
in cytokinesis.

The results of analysis of cell cycle—dependent CD45 PTPase activity in the
CTLL-2 T cell line supports the hypothesis that CD45 plays a fundamental role in
mitosis. However, the CTLL-2 line consistently displayed a high basal level of CD45
PTPase activity throughout the cell cycle. The specificity of the observed CD45
PTPase activity in CTLL-2 cells was confirmed by the parallel precipitation with the
isotype-matched control antibody, anti-CD8. Even though CD8 was effectively

precipitated with this antibody, there was no PTPase activity associated with these

60

immunoprecipitates. The PTPase activity measured in these experiments was found to
be an accurate and reproducible measure of relative activity for each experiment.

In recent studies, CD45 was shown to contain a fodrin/spectrin binding domain
(14). Fodrin has been shown to mediate association of actin filaments with the
plasma membrane, leading to the proposal that it serves in membrane stabilization by
linking the cytoskeleton to the plasma membrane (31, 32). During cellular activation,
fodrin has been shown to serve as a kinase substrate, and this cytoskeletal protein is
isolated in close association with CD45 (14). Importantly, fodrin binding to CD45
enhanced CD45 PTPase activity 7.5 fold (14). Thus, the increase in CD45 PTPase
activity observed during late mitosis in this study may have resulted from binding to a
cytoskeletal element such as fodrin. This increased activity may cause the
dephosphorylation of fodrin or other microfilament proteins associated with
reorganization of actin microfilaments during cytokinesis.

Alternatively, elevation of CD45 PTPase activity could be due to modification
of phosphorylation of CD45 itself. Phosphorylation of tyrosine residues within CD45
has been detected after lectin or anti-CD3 activation of Jurkat cells (33) and after in
vitro treatment of CD45 with p50“" PTK (l3). Importantly, in the latter report, the
activity of CD45 increased several-fold after tyrosine phosphorylation by p50“" (13).
Serine residues on CD45 have also been shown to be phosphorylated in response to T
cell treatment with phorbol esters via protein kinase C (34), after in vitro treatment
with casein kinase 2 and other serine/threonine kinases (35), and after IL-2 treatment
of CTLL-2.4 cells (36). No modulation of CD45 PTPase activity was observed after

phosphorylation in the latter two reports. However, down-regulation of CD45

61
PTPase activity after addition of Ca2+ ionophores, i. e. ionomycin or A23187, to T

cells coincided with a decrease in serine phosphorylation (12). Thus, the increase in
CD45 activity observed in mitosis may be a consequence of serine/threonine or
tyrosine phosphorylation. Further experiments will be necessary to clarify this issue.

Recent investigation into the role of PTPases in the cell cycle has led to the
discovery that cdc25, a PTPase, was essential for activation of MPF and for allowing
entry into and progression through mitosis (28-30). In addition, over—expression of a
truncated form of another PTPase, TC.PTP, has been shown to result in cytokinetic
failure (37). These data support the potential involvement of tyrosine phosphorylation
in signalling events coordinating cell division.

These data and others support the concept that CD45 may have an important
role in the cellular mechanism regulating mitosis or cytokinesis. The observation of a
2- to 5-fold increase in CD45 PTPase activity during mitosis in CTLL-2 and 702 cell
lines suggests that phosphorylation of CD45 or association of CD45 with fodrin (or
fodrin-like molecules) during mitosis may result in the activation of a CD45 PTPase
domain. Further investigation into the phosphorylation state of CD45 and associated
proteins such as fodrin should provide information regarding the regulation of CD45

PTPase activity in mitosis.

62

ACKNOWLEDGMENTS

I thank Dr. L. Melkerson-Watson (Michigan State University, East Lansing,
MI) for her advice and assistance in this work, Dr. S. Conrad (Michigan State
University, East Lansing, M1) for use of the JE—68 elutriation system, Dr. L. King
(Michigan State University, East Lansing, MI) for technical support and assistance
with the FACS, and Dr. J. Dixon (University of Michigan, Ann Arbor, M1) for his

generous gift of src kinase.

63

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phosphorylation of the leukocyte common antigen CD45 in human T cells.
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Proc. Natl. Acad. Sci. U. S. A. 89:5422.

CHAPTER THREE

CELL CYCLE-RELATED EXPRESSION OF A CD45-ASSOCIATED

SERINE-THREONINE KINASEl

67

68

FOOTNOTES

1 This work was supported by NIH grant GM35774.

2 Abbreviations used in this paper: PTPase, protein tyrosine phosphatase;
PTK, protein tyrosine kinase; PAO, phenylarsine oxide; SH2, src homology
region 2; PI, propidium iodide; DTBP, dimethyl 3,3’-dithiobispropionimidate;
PVDF, polyvinylidene difluoride; ECL, Enhanced ChemiLuminescence; PHA,
phytohemagglutinin; LPAP, lymphocyte phosphatase-associated

phosphoprotein.

69

ABSTRACT

The protein tyrosine phosphatase (PTPase) CD45 is essential to couple antigen
receptor stimulation to second messenger signalling pathways in T and B cells. In
this role, CD45 appears to regulate src family kinases, and the use of mild detergents
has led to the identification of complexes of CD45 with p56"" in T cells and p53,” in
B cells. Although it has been hypothesized that CD45 also has a role in mitosis, the
accessory proteins in this role have not been identified. In this paper, we report that
a serine-threonine kinase is associated with CD45 throughout the cell cycle in CTLL-
2 cells. Centrifugal elutriation was used to obtain subpopulations of cells enriched for
the various stages of the cell cycle. Two approaches, chemical cross-linking and lysis
with a mild detergent, were used to identify accessory proteins which coprecipitate
with CD45. Proteins of 60 to 70 kD were found in CD45 immunoprecipitates arising
from both procedures. In in vitro phosphorylation reactions, these bands were
labelled on serine and, to a lesser extent, threonine residues. The in vitro
phosphorylation by the serine-threonine kinase in the CD45 immunoprecipitates was
elevated in late G2+ M-enriched samples, which correspond to the time in the cell

cycle when the PTPase activity of CD45 is elevated.

70

INTRODUCTION

The leukocyte common antigen, CD45, is a family of heterogeneous
transmembrane proteins which have been identified in numerous mammals, as well as
in chickens and a shark species (reviewed in l, 2). The extracellular region of CD45
varies as a result of cell lineage- and activation state-determined exon usage (1). In
contrast to the variation seen in the extracellular region of CD45, the intracellular
region of CD45 does not vary among leukocytes within an organism and is highly
conserved among mammals. The intracellular region of CD45 contains two active
PTPasez catalytic domains (3-6). CD45 is abundant on leukocyte membranes and
may constitute up to 10% of leukocyte surface glycoproteins (7, 8). Both the
abundance and conservation of CD45 in the hematopoietic system suggest that it is
involved in one or more basic roles critical to leukocyte function.

Indeed, CD45 is essential for activation of B and T lymphocytes through their
antigen and accessory receptors. CD45-deficient CD4+ and CD8+ T cell lines fail to
proliferate in response to TCR/CD3 stimulation, and, in addition, CD45-deficient
CD8+ cells have diminished capacity to produce cytokines and lyse cells after
stimulation (9, 10). CD45 deficiency uncouples TCR and CD2 stimulation from both
the tyrosine phosphorylation and phosphatidyl inositol second messenger pathways,

which are the initial responses detected after TCR or CD2 stimulation (1 1—14). CD45

71

expression is also required to couple the phosphatidyl inositol pathway to stimulation
through the membrane IgM antigen receptor on B cells (15). Normal tyrosine
phosphorylation and Ca2 + flux responses to antigen receptor stimulation are restored
to CD45' T and B cells when the intracellular region of CD45 is expressed in the
cells, even when the external CD45 region is absent or replaced by MHC class I or
epidermal growth factor receptor extracellular sequences (15—19).

In addition to its role in the activation of lymphocytes, CD45 appears to have
a role in mitosis (20). Using centrifugal elutriation to obtain subpopulations of
asynchronous B and T cell cultures which were enriched for cells in various stages of
the cell cycle, it was determined that CD45 PTPase activity is elevated 2— to 5-fold in
G2+M when compared to its activity during other stages of the cell cycle.

In the activation of T cells, CD45 may regulate the activity of both p56” and
p590", two src family PTKs which are involved in the response to TCR/CD3
stimulation (16, 21-28). Analyses of CD45 immunoprecipitates from digitonin or Brij
96 lysates of human peripheral blood T lymphocytes and cultured human and murine
T cells have shown that CD45 associates with p56“ and a group of phosphoproteins
of variable molecular weights, pp29-34 (29-33). Likewise, p53,”, a B cell PTK
implicated in the response to antigen, has been coprecipitated with CD45 from B cells
(34).

Phosphotyrosine—containing proteins of 55-62 kD have also been found in
CD45 immunoprecipitates from Triton X-100 lysates of human peripheral blood T
lymphocytes after treatment of the cells with PAO, a PTPase inhibitor (35). When

p56” was immunoprecipitated from PAC-treated, periodate-NaB3H4 surface-labelled

72
cells, tritiated cell-surface glycoproteins of 180-205 kD were found in the p56”

immunoprecipitates. Autero et al. (35) proposed that these data indicate that p56“
and tyrosine-phosphorylated CD45 form a stable complex in cells. In vitro studies
using purified CD45 and recombinant p561“ supported the theory that p56’“ binds to
tyrosine-phosphorylated CD45 via the SH2 domain of the PTK.

CD45 also interacts with cytoskeletal elements during its capping on B cells
and during capping of either CD45 or Thy-l on a thymocyte cell line (36-38).
Moreover, binding of fodrin to CD45 increases its PTPase activity 7.5-fold (39).

The present study was undertaken to determine what proteins interact with
CD45 in the progression through the cell cycle. In particular, we wished to
determine which proteins interact with CD45 when it reaches peak activity in the
GZ+M phases of the cell cycle. Centrifugal elutriation was used to obtain cell cycle
stage—enriched subpopulations of CTLL-2 cells, and proteins which coprecipitated

with CD45 were examined.

73

MATERIALS AND METHODS

Cell lines and antibodies

Cell lines were obtained from the American Type Culture Collection (Rockville,
MD). The cytolytic T cell line CTLL-2 was maintained at 37°C in a humidified
environment with 7.5% C02 in RPMI 1640 (GIBCO/BRL, Gaithersburg, MD) with
10% FBS (GIBCO/BRL), 15 mM HEPES (Boehringer Mannheim Biochemicals,
Indianapolis, IN), 50 pM 2-ME (Sigma Chemical Co., St. Louis, MO), and 7 U/ml
recombinant IL-2 (Cetus Corporation, Emeryville, CA) (complete medium). Anti-
CD45 mAb was prepared from the Ml/9.3.4 hybridoma line as were the control
mAbs: 33D], an anti-dendritic-cell receptor mAb, and $36.72, an anti-CD80: mAb.
Anti-fyn, anti-lck and anti-src polyclonal antibodies, which are specific for the
respective mouse as well as human proteins, were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA).

Counterflow centnfitgal elutriation

Approximately 4 x 108 exponentially-growing cells were loaded into the standard
elutriation chamber of a JE-6B rotor in a 12-2lM/E centrifuge (Beckman Instruments,
Palo Alto, CA) at 2250 RPM and 12°C with an initial buffer flow rate of 4.5 ml/min
produced by a Masterflex peristaltic pump (Cole Palmer Instruments, Chicago, IL).

Fractions were elutriated in Hank’s balanced salt solution (GIBCO/BRL) with 1

74

mg/ml bovine serum albumin (Sigma). Fractions were collected for 5 min at each
increment of flow rate, which was typically 1 to 2 ml/min.

In some experiments, cells collected aseptically during the elutriation were
resuspended at 5 x lo6 cells/ml in complete medium and recultured in standard
temperature and C02 conditions for 4 to 4.5 hrs, at which time the cells were
harvested and treated as were the samples collected directly from the elutriator.

Cell cycle analysis

For each sample, 1 x 106 cells were fixed by adding 1.2 ml ice-cold 70% ethanol to
cells suspended in 0.4 ml 50% PBS (8 mM NaQHPO4, 1 mM KHZPO4, 137 mM
NaCl, 3 mM KCl, pH 7.3), 50% FBS and storing the suspension for approximately
24 hr at -20'C. To stain the DNA of the fixed cells, the cells were washed twice in
PBS with 5% (v/v) FCS and suspended in P1 staining solution (PBS with 0.1% [v/v]
Triton X-100 [Boehringer Mannheim], 0.1 mM EDTA [Sigma], 50 pg/ml RNase A
[Boehringer Mannheim], and 50 pg/ ml PI [Sigma]) for 1 hr at room temperature.
Fluorescence of the singlet population was analyzed on a Becton Dickinson Vantage
FACS (San Jose, CA). Cell cycle analysis was performed using PC-Lysys version
1.0 software (Becton Dickinson).

Cross-linking and cytoskeletal preparations

Cells were washed twice with PBS and suspended at 6 x 105 cells/ml in pas, pH 8,
with 5 mM MgC12 and 1 mM Na3VO4 (Sigma), and with or without 3 mM DTBP
(Sigma) (40). The suspension was gently shaken every 5 min for 30 min at room
temperature. Cross-linking was quenched by the addition of Tris, pH 7 .4, to a final

concentration of 50 mM, and the cells were washed twice with PBS. Cells were

75
lysed at l x 108 cells/ml in a hypotonic, alkaline buffer (10 mM Tris, pH 8.9, 1 mM

EGTA, 1 mM PMSF) and rocked for 30 min at 4'C before the insoluble cytoskeletal
lattice was pelleted by centrifugation at 10,000 g for 10 min at 4°C (40). After the
supernatant was transferred to a new tube, its pH was neutralized by the addition of
20 pl 1 M Tris, pH 7.0, per ml. Proteins were extracted from the insoluble
cytoskeletal pellet for 30 min at 4 ‘ C on a rocker platform in Triton X-100 extraction
buffer (50 mM Tris, pH 7.4, 25 mM KCl, 5 mM MgC12, 0.5% Triton X-100, 1 mM
PMSF). The suspension was centrifuged at 10,000 g for 10 min at 4'C, and the
supernatant was transferred to a new tube. Proteins were further extracted from the
remaining insoluble pellet for 30 min at 4 ' C on the rocker platform in Triton X-
100/sarcosyl extraction buffer (50 mM Tris, pH 7.4, 25 mM KCl, 5 mM MgC12,
0.5% Triton X-100, 0.5% N-lauroylsarcosine [Sigma], 1 mM PMSF). After 10 min
centrifugation at 10,000 g and 4°C, the supernatant was transferred to a new tube. In
the extraction steps, 1 ml of Triton X-100 extraction buffer was used per 108 cells,
and 1 ml of Triton X-lOO/sarcosyl extraction buffer was used per 2 x 108 cells.
Bradford protein microassays were performed using 5 pl aliquots of the extracts (41).
For immunoprecipitation, each extract was divided into two aliquots, and
either CD45 or CD80: was immunoprecipitated from each aliquot for 1 hr at 4°C
using mAbs M1/9.3.4 or 53672, respectively, precoupled to protein G-agarose
(Pharmacia, Piscataway, NJ). Immunoprecipitate pellets were washed once with PBS

then 0.5 M LiCl, pH 7.4, and twice with 50 mM Tris, pH 8.

76

Coprecipitation of proteins with CD45 from digitonin lysates
A stock of 10% digitonin was prepared as described by Schraven et al. (29).
Digitonin (Sigma) was dissolved in boiling H20 for 2 min with stirring. After the
suspension was stored at room temperature for at least 3 days, the insoluble material
was removed by filtration through a 0.4 pm filter.

Cells were washed twice with PBS and lysed at 2.5 x lo7 cells/ml in digitonin
lysis buffer (20 mM Tris, pH 7.4, 140 mM NaCl, 1% digitonin, 1 mM PMSF, 1
pg/ml aprotinin, 0.7 pg/ml pepstatin A, 10 mM NaF, 1 mM Na3VO4, 10 mM
Na4PZO7). Postnuclear lysates were precleared with 100 p1 of packed protein G-
agarose and 100 pl of packed protein G-agarose precoupled to 33D1 per ml lysate.
Bradford protein assays were performed on 5 pl aliquots of the lysates.
Immunoprecipitation was done for 1 hr at 4'C using M1/9.3.4 mAb precoupled to
protein G-agarose. Immunoprecipitate pellets were washed once with digitonin lysis
buffer, twice with 0.5 M LiCl, pH 7.4, and twice with 50 mM Tris, pH 8. Pellets
were stored at —20 or 4‘C in an equal volume of 20 mM Tris, pH 7.4, 1 mM PMSF,
1 pg/ml aprotinin, 0.7 pg/ml pepstatin A, and 2 mM Na3VO4.
In vitro phosphorylation reactions
To normalize the amount of immunoprecipitate used in in vitro phosphorylation
reactions for a given elutriation, the average protein concentration for digitonin
lysates from the elutriation was calculated, and this was taken to be the protein
amount which is obtained from 1.25 x 105 cells, i. e. the number of cells expected to

be in each 5 pl aliquot used in the Bradford assay. For each sample, then, the

77

amount of immunoprecipitate which would have resulted from 1 x 10‘5 cells, based on
the average protein concentration, was used in the phosphorylation reaction.

In each 20 pl reaction, the amount of immunoprecipitate representing 1 x 106
cells was combined with 1 pCi [7-32P]-ATP (Dupont NEN, Boston, MA) in 20 mM
Tris, pH 7.4, 10 mM MnClz, 1 mM Na3VO4 and incubated at 30'C for 45 min. The
reaction was stopped by the addition of SDS-PAGE sample buffer and boiling for 2
min.
SDS-PA GE and Western blotting
For SDS-PAGE 4-15% gradient gels were used. For silver staining, gels were first
stained with Coomassie blue then destained. Gels were equilibrated to H20 and
treated with 5 pg/ml DTT in H20 for 30 min then with 1 mg/ml AgNO3 for 30 min.
The stain was developed with 3% (w/v) NazCO3, 0.05% (v/v) HCHO and developing
was stopped by the addition of 10% (v/v) CH3COOH (method modified from 42).

For Western blotting, gels were equilibrated to 25 mM Tris base, 192 mM
glycine, 20% (v/v) methanol and blotted onto PVDF membrane (Bio-Rad
Laboratories Inc., Melville, NY, or Millipore, Bedford, MA) for 30 min at 200
mAmps followed by 4.5 hrs at 400 mAmps, 4'C. To detect 32P-labelled
phosphorylation reaction products, blots were air dried and used to expose Hyperfilm
(Amersham, Arlington Heights, IL). For anti-fyn, anti-lck and anti-src probing of
Western blots, blots were blocked with 2 % (v/v) normal goat serum (GIBCO/BRL) in
TBS (10 mM Tris, pH 8, 150 mM NaCl) and probed with the primary antibody

diluted to 0.8 pg/ ml in TBST (TBS with 0.1% [v/v] Tween 20 [Bio-Rad]) followed

78
by goat anti-rabbit IgG-peroxidase (Boehringer Mannheim) in TBST. ECL reagents

(Amersham) and Hyperfilm were used to detect the bound antibodies.

Phosphoamino acid analysis

The protein bands for phosphoamino acid analysis were cut from the PVDF blots.
The blot pieces were wetted in methanol and equilibrated to H20 and then treated
with 5.7 N HCl at 100°C for 2 hr. The digest products were dried then dissolved in
5 or 10 pl phosphoamino acid standards (1 pg/pl each phosphoserine,
phosphothreonine, and phosphotyrosine). Samples were spotted onto thin layer
cellulose plates (Kodak, Rochester, NY) and electrophoresed at 500 to 1000 V in pH
2.5 buffer: 5.9% (v/v) acetic acid, 0.83% (v/v) formic acid, 0.33% (v/v) pyridine,
and 0.33 mM EDTA (43). Air-dried plates were sprayed with ninhydrin (Sigma) to
identify the positions of the phosphoamino acid standards, and the positions of the
32P-residues from the phosphorylation reaction products were determined by
autoradiography using Hyperfilm or by employing a Beta Scope 603 blot analyzer

(Betagen Corporation, Waltham, MA).

79

RESULTS

Centnfitgal elutriation of asynchronous cells produces fractions of cells enriched for
various stages of the cell cycle

In preparation for elutriation studies, CTLL-2 cells from unseparated, asynchronous
cultures were stained with P1 (a fluorescent DNA stain), and the fluorescence of the
cells was detected by flow cytometry. When DNA fluorescence was plotted as a
histogram, the predominant peak from the asynchronous cells represents cells which
are in G1 (Fig. 1A). As cells progress through S, DNA is duplicated leading to
increased PI fluorescence. Cells in GZ+M, having double the DNA material of cells
in G1, have approximately 2-fold higher PI fluorescence. Centrifugal elutriation was
then used to separate cells from the asynchronous culture into fractions enriched for
the various stages of the cell cycle with minimal perturbation to the cells (44, 45).
Flow cytometry of PI-stained CTLL-2 cells obtained from elutriation fractions
indicated that the fractions were sequentially enriched for the stages of the cell cycle
starting with G1 and progressing through S then GZ+M (Fig. l, B-Q).

Proteins of 60-70 kD are associated with CD45 in S and 62 +M stages of the cell
cycle

It has been shown that the activity of the PTPase CD45 is elevated during G2+M

(20). In order to identify proteins which interact with active CD45 in G2+M,

80

Figure I. FACS analysis of the DNA content of elutriated CTLL-2 cells.

Histograms of the PI fluorescence of cell populations are shown for (A) an
exponentially-growing asynchronous starting culture and (B-Q) its elutriation fractions
1-16. The DNA content based on P1 staining is indicated. (A) In asynchronous
cultures, the predominant peak represents cells in Gr? as cells progress through 8,
their DNA content and, thus, PI fluorescence increases; when cells are in G2+M,
their DNA content is double that of cells in G1, and hence their PI fluorescence is
approximately double that of G1 cells. The DNA staining patterns were interpreted as
follows: B-J, fractions 1-9, GI and S phases; K-M, fractions 10—12, S phase; and N-

Q, fractions 13-16, G2+M phases.

81

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82

cross-linking studies were undertaken. When asynchronous cells were treated with
the cross-linker DTBP and then cellular proteins were extracted sequentially with
hypotonic, alkaline buffer, Triton X-100 buffer, and Triton X-lOO/sarcosyl buffer,
CD45 was primarily found in the Triton X—100 extract while CD80: was found in the
hypotonic, alkaline extract (Fig. 2, lanes 1 and 6). CD45 was located in the Triton
X-100 extract even when the cross-linker was not used (Fig. 2, lane 5). Two protein
bands in the 50 to 60 kD range coprecipitated with CD8a: these may be forms of
p56”, which interacts with the cytoplasmic tail of CD80: (46, 47). Two proteins of
60 to 70 kD were precipitated along with CD45 from the Triton X-100 extract from
DTBP-treated cells (Fig. 2, lane 6).

When CD45 was immunoprecipitated from the Triton X-100 extracts from
fractions enriched for cells from the various stages of the cell cycle, i. e. from
elutriation fractions, protein bands in the 60 to 70 kD range were observed in CD45
immunoprecipitates from S through 62+M (Figs. 3 and 4A). The bands were
difficult to detect in gels run specifically for silver staining, but when gels were
stained after electroblotting, the bands could be readily seen above the
immunogloban heavy chain band (Fig. 4A). Other bands which appeared between
the immunoglobulin heavy and light chains were observed in many
immunoprecipitates, including those using other mAbs, and thus were not considered
to be specific to CD45 immunoprecipitates.

Western blot analysis showed that neither anti-fyn nor anti-lck antibodies
interacted with these bands, but the anti-src polyclonal antibody reacted with one or

two of the bands weakly (Fig 4B and data not shown). The bands seen as a result of

83

Figure 2. CD80: and CD45 immunoprecipitates from DTBP-treated CTLL-2 cells.
Samples of 5 x 106 asynchronous CTLL-2 cells were treated with the chemical cross-
linker DTBP (lanes 1, 3-4, 6—7 and 9) or, as a control, DTBP treatment was omitted
(lanes 2, 5, and 8). Proteins were then extracted from the cells initially with a
hypotonic, alkaline buffer (lanes 1-3), secondly with a Triton X-100 buffer (lanes 4-
6), and finally with a Triton X-lOO/sarcosyl buffer (lanes 7-9). Either CD80: (lanes
1, 4, and 7) or CD45 (lanes 2-3, 5—6, and 8-9) was then immunoprecipitated from the

extracts.

 

 

, _ TritonX—100
extract alkaline TrltonX-100 sarcosyl
2 3 4 5 6 7 8 9

       

203-

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CD8 CD45

Figure 2.

85

Figure 3. FACS analysis of the DNA content of CTLL-2 elutriation fractions
analyzed in Fig. 4. (A) Cell cycle profile of the asynchronous culture used in this
experiment. (B-F) DNA content histograms for the elutriation fractions 1-5 analyzed
further in Fig. 4. The DNA staining patterns were interpreted as follows: (B)
fiaction 1, G1 and S; (C and D) fractions 2 and 3, S; (E) fraction 4, S and 62; and

(F) fraction 5, GZ+M.

8

O\

 

lequlnu neg

Figure 3.

 

DNA content

87

Figure 4. CD45 immunoprecipitates from DTBP-treated CTLL—2 elutriation
fractions. Samples of 5 x 106 cells from the elutriation fractions analyzed in Fig. 3
were treated with DTBP and then lysed in hypotonic, alkaline buffer. Proteins were
extracted from the insoluble cytoskeletal matrix with Triton X-100 buffer, and CD45
was immunoprecipitated from this extract. Immunoprecipitates were subjected to
SDS-PAGE followed by Western blotting. Samples in lanes 1-5 are from elutriation
fractions 1-5, respectively, of Fig. 3 (panels B—F). (A) Silver-stained SDS-PAGE
gels. The coprecipitating 60 to 70 kD proteins were most readily seen in SDS-PAGE
gels after the majority of the proteins from the immunoprecipitates had been blotted
onto PVDF membrane. HC and LC: heavy and light chain, respectively, of
M1/9.3.4. (B) Anti-src probing of the Western blot of CD45 immunoprecipitates
from the elutriation fractions (lanes 1-5) and of a Western blot of whole cell lysate of

5 x 106 CTLL-2 cells (lane 6). The arrow indicates the position of p607“.

GZ+M

 

 

 

89

the anti-src probing of the elutriation fractions were located above the position of the
immunoglobulin heavy chain band.

Sixty to 70 kD proteins are labelled in in vitro phosphorylation reactions of CD45
immunoprecipitates

To determine if the 60 to 70 kD proteins could be precipitated with CD45 by a
second technique and to determine if any of the proteins was an active kinase, CD45
was precipitated from digitonin lysates. This technique has been used to show that
p56“ is associated with CD45 in T cells (29). CD45 immunoprecipitates
representing 1 x 106 cells were incubated with [7-32Pl-ATP in conditions in which src
family kinases can undergo autophosphorylation. To study the products from the in
vitro phosphorylation reactions, the reaction products were subjected to SDS-PAGE
followed by electroblotting.

Two 32P-labelled 60 to 70 kD proteins were found in the CD45
immunoprecipitates throughout the cell cycle (Figs. 1 and 5A). The 3‘fliP-labelling of
the larger band was greatly increased, and also the labelling of the smaller band to a
lesser extent, in the CD45 immunoprecipitate from the late G2+M fraction taken
directly from the elutriator in at least three experiments.

Labelling of the 60 to 70 kD proteins in in vitro phosphorylation reactions of CD45
immunoprecipitates from recultured, synchronized cells is elevated in the mitotic
fi'actions

Since the most highly-labelled fiaction was in late G2+M and this fraction was one of
the last elutriation fractions, we wished to further verify the cell cycle position of the

peak kinase activity by reculturing cells. This was performed by first obtaining

90

Figure 5. In vitro phosphorylation of proteins coprecipitated with CD45. CD45 was
immunoprecipitated from digitonin lysates of elutriation fractions, and the amount of
CD45 immunoprecipitate representing 1 x 10‘5 cells from each fraction was incubated
with [7-32P]-ATP in in vitro phosphorylation reactions and then subjected to SDS-
PAGE and Western blotting. Samples in lanes 1-16 are from elutriation fractions 1-
16, respectively, from Fig. 1 (panels B—Q). (A) In vitro-phosphorylated proteins were
visualized by autoradiography. (B) The Western blots were probed with anti-src

polyclonal antibody and the appropriate secondary antibody.

91

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92
synchronized cell populations by elutriation. The cells were then cultured for 4.25 hr

to allow them to progress through the cell cycle so that G2+M fractions were situated
between S and GI fractions. The final cell cycle analysis of the recultured samples is
shown in Fig. 6. Immunoprecipitation of CD45 from these recultured fractions
followed by in vitro phosphorylation treatment confirmed the coprecipitation of 60 to
70 kD proteins (Fig. 7). In addition, in CD45 immunoprecipitates from G2+M
fractions, the 60 to 70 kD bands were labelled more than those from other samples.
The 70 kD band reacts weakly with anti-src antibodies

These experiments demonstrated the presence of a kinase associated with CD45 in all
stages of the cell cycle. To identify this kinase, Western blot analysis was performed
with antisera to the src family PTKs, p60“, p590" and p561“.

When the blots of the in vitro phosphorylation products were probed with
antibodies, no binding of the anti-fyn polyclonal antibody was observed in any
immunoprecipitate, and anti-lck polyclonal antibody gave very weak signals in less
than 7% of the immunoprecipitates (data not shown). Probing of the blots with anti-
src polyclonal antibody, however, resulted in consistent yet weak labelling of a band
which corresponded with the higher molecular weight band labelled in the in vitro
phosphorylation reaction (Fig. 5B).

The active kinase coprecipitating with CD45 is a serine kinase

The specificity of the kinase associated with CD45 immunoprecipitates was
determined by phosphoamino acid analysis of the 32P—labelled products obtained from
the previous experiments. When the 60 to 70 kD 32P-labelled protein bands were

excised from blots and analyzed for phosphoamino acid content, approximately two

93

Figure 6. FACS analysis of the DNA content of recultured CTLL-2 subpopulations.
Samples of cells from elutriation fractions were recultured for 4.25 hrs prior to
analysis. (A-I) Cell cycle profiles of elutriation fractions 1—9 after reculturing. The
DNA staining patterns were interpreted as follows: (A-C) fiactions 1-3, S; (D)
fraction 4, S and G2; (E-G) fractions 5-7, M; (H-I) fractions 8 and 9, G1 and S (The
G2 peak is no longer decreasing so these cells do not appear to be progressing

through mitosis)

94

 

 

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requrnu "93

fi

 

Figure 6.

95

Figure 7. In vitro phosphorylation of proteins coprecipitated with CD45. CD45 was
immunoprecipitated from digitonin lysates of recultured CTLL-Z synchronized
subpopulations, and the amount of CD45 immunoprecipitate representing 1 x 106 cells
from each fraction was incubated with [7-32P]-ATP in in vitro phosphorylation
reactions and then subjected to SDS-PAGE and electroblotting. Phosphorylated
products were visualized by autoradiography. Samples in lanes 1-9 are from

recultured fractions 1-9, respectively, depicted in Fig. 6 (panels A-I).

8. 9

4'. ,
,VJ. .

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tree
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5 6 7
Figure7.

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97

thirds of the label was found in phosphoserine and one third in phosphothreonine
(Fig. 8 and data not shown). The phosphorylation pattern of the upper band was the
same as that of the lower band in each sample tested (data not shown). labelling of
phosphotyrosine was barely detectable in these samples. Thus we concluded that the
primary activity associated with CD45 immunoprecipitates was that of a serine

threonine kinase.

98

Figure 8. Phosphoamino acid analysis of in vitro phosphorylation products. 32P—
labelled bands were excised from the Western blots of kinase reaction products, and
the phosphoamino acids were released from the blot pieces by digestion with 5.7 N
HCl. One-dimensional electrophoresis on thin-layer cellulose plates was used to
resolve the phosphoamino acids. The 32P-labelled phosphoamino acids were
visualized with the Beta Scope analyzer. Lanes 1 and 2 show the phosphoamino acid
analysis of the lower band in lanes 4 and 5, respectively, of Fig. 7. Lane 3 shows
the analysis of a band from the fraction with peak kinase activity from an experiment
not depicted elsewhere in this paper. Lane 4 shows the analysis for the upper band
from lane 16 of Fig. 5. Lane 5 shows the position of the phosphotyrosine standard

while in lane 6 the positions of all three phosphoamino acid standards are shown.

P-Ser
P-Thr
P-Tyr

‘ P-SBI‘
P—Thr
P-Tyr

     

   

silt?

Figure 8.

100

DISCUSSION

The current study was undertaken to determine if accessory proteins associate
with CD45 when it exhibits peak PTPase activity in the GZ+M stages of the cell
cycle (20). Centrifugal elutriation of CTLL-2 T lymphocytes was used to obtain
subpopulations which were enriched for cells in each of the various stages of the cell
cycle. Two different approaches were used to assess the association of accessory
proteins with CD45: 1) chemical cross-linking of CD45 and putative accessory
proteins with DTBP followed by immunoprecipitation of CD45 and 2) lysis of cells
with the mild detergent digitonin followed by immunoprecipitation of CD45 and
analysis for the presence of kinase activity by use of an in vitro phosphorylation
assay. Using these approaches, 60 to 70 kD proteins were found throughout the cell
cycle in CD45 immunoprecipitates.

When the CD45 immunoprecipitates from digitonin lysates were incubated
with [7-32P]-ATP using conditions in which src family kinases undergo
autophosphorylation, the 60 to 70 kD proteins did indeed become phosphorylated.
The in vitro phosphorylation of these bands was higher in the CD45
immunoprecipitates from G2+M-enriched samples than from Gl or 8 samples.
Phosphoamino acid analysis of the labelled products determined that serine and

threonine residues were being phosphorylated in the in vitro reactions. These data

101

demonstrate that an active serine-threonine kinase is associated with CD45 in CTLL-2
cells throughout the cell cycle, and that its activity or its association with CD45 is
increased during G2+M.

When Western blots of the in vitro phosphorylation products were analyzed for
reactivity with anti-fyn, anti-lck, or anti-src antibodies, only the anti-src antibody
consistently, albeit weakly, reacted with a protein on the blots. The anti-fyn and anti-
lck antibodies did identify proteins of the appropriate molecular weights when blots of
whole cell lysates from 1 x 106 CTLL-2 cells were tested with the antibodies (data
not shown). Thus, the antibodies were capable of detecting their respective epitopes
on Western blots of CTLL-2 proteins.

In several previous studies of CD45 immunoprecipitates from digitonin lysates
of T cells, p56” has been found associated with CD45 (29, 32, 33). In the previous
studies, the CD45 immunoprecipitate from 1 to 5 x 107 cell equivalents was loaded
per lane on the Western blots used to detect p56“, amounts which were 10 to 50-fold
higher than the amounts used in the present study. When CD45 immunoprecipitates
from PHA-activated CTLL-2 cells were tested to determine the minimum cell number
necessary to detect p56’c" in the immunoprecipitates on Western blots, a signal was
obtained from 4 x 10‘5 cells, but not from fewer cells (data not shown). Thus, to
further test the CD45 immunoprecipitates from digitonin lysates of elutriation
fractions, Western blots of 5 x 106 cell equivalents were probed with the antibodies
for the src family kinases. The data obtained from these blots, however, did not
differ from those obtained from the in vitro phosphorylation product blots (data not

shown).

102

In previous studies, a group of proteins in the 29 to 34 kD range have been
identified in immunoprecipitates of CD45 when mild detergents, i. e. digitonin, Brij
58, or Brij 96, were used to lyse cells (29-33). Homologous genes encoding proteins
of about 30 kD which bind to CD45 have been cloned from both murine and human
genomes (48, 49). This protein has been named the lymphocyte phosphatase-
associated phosphoprotein (LPAP) (49). The multiple bands seen in CD45
immunoprecipitates from human T cells appear to arise from differential
phosphorylation of LPAP (49). In several of the previous studies involving in vitro
phosphorylation of CD45 immunoprecipitates, LPAP was the most predominant
labelled product (29, 30, 33). In contrast, almost no labelling of proteins in the 23 to
45 kD range was observed in our current study. There are several potential reasons
for this including: 1) CTLL—2 cells do not express as much LPAP as do the cell
types used in the other studies, 2) LPAP cannot be detected from 1 x 106 cells, or 3)
LPAP is present in the CD45 immunoprecipitates in the current study, but it is not
phosphorylated in the in vitro phosphorylation reactions. LPAP is in fact
phosphorylated in vivo on serine residues (29, 30, 32, 33). Phosphoamino acid
analysis of the 60 to 70 kD proteins which were labelled in the current study revealed
that the active kinase coprecipitating with CD45 is a serine-threonine kinase. Thus, it
is possible that in vivo phosphorylation of LPAP prevented subsequent
phosphorylation in in vitro reactions.

While one study reported the exclusive labelling of tyrosine residues in LPAP
in in vitro phosphorylation treatments of CD45 immunoprecipitates (29), a second

reported that both serine and, to a lesser extent, tyrosine were labelled (32).

103
Phosphoamino acid analyses of the 32P-labelled 50 to 60 kD proteins were not

reported in these studies although Schraven and his co-workers (29) noted that
autophosphorylation of p56”, which was readily observed in lck immunoprecipitates
from Triton X-100 lysates, was barely detected when lck was immunoprecipitated
from digitonin lysates. The in vitro labelling of both serine and tyrosine residues
suggests that multiple kinases may coprecipitate with CD45 .

In the current study, although some p60” appears to coprecipitate with CD45
from digitonin lysates of CTLL-2 cells, the predominant kinase activity in the
immunoprecipitates was clearly that of a serine-threonine kinase. The activity of the
serine-threonine kinase which coprecipitated with CD45 throughout the cell cycle was
increased in late G2+M-enriched fractions, a time in the cell cycle when the PTPase
activity of CD45 is elevated (20). In in vitro studies, Stover and Walsh (50)
determined that sequential phosphorylation of CD45 , first on tyrosine residues then on
serine residues, enhanced the PTPase activity of CD45 toward one artificial substrate
but not another. In addition, the activity of p607“ increases during mitosis coincident
with increases in threonine and possibly serine phosphorylation (51). Thus, it is
possible that the serine-threonine kinase which coprecipitates with CD45 could be
acting on CD45 itself or on other proteins associated with CD45 such as p60”.

Data on the in vivo phosphorylation state of CD45 would seem to support the
hypothesis that the serine-threonine kinase is acting on CD45. Early studies of the
phosphorylation of CD45 in vivo typically found serine residues were heavily
phosphorylated, and threonine and possibly tyrosine were phosphorylated weakly or

not at all (52-54). These studies were done without the use of the potent PTPase

104
inhibitor PAO, which allows the detection of phosphotyrosine in in vivo-labelled
CD45 (35, 55). In the present study, after in vitro phosphorylation treatments, 32P-
labelled protein bands in the appropriate range for CD45 were observed in a few
samples, but they were only weakly labelled (Figs. 5A and 7). Thus, the serine-
threonine kinase which coprecipitates with CD45 does not phosphorylate CD45, at
least in the conditions used in the present study.

The identity of the serine-threonine kinase responsible for the in vitro
phosphorylation observed in our studies is as yet unknown. One candidate kinase
being considered is Raf-l. Raf-l is a p70-74 serine-threonine kinase which is
activated after stimulation of T cells via the TCR/CD3 complex or a number of other
receptors (56-5 8). Raf-1 is capable of undergoing autophosphorylation in conditions
similar to those used in the in vitro phosphorylation reactions in the current study with
one notable difference: in vitro kinase treatments of immunoprecipitated Raf-l have
included the sulfhydryl-reducing compound DTT (59, 60). Interestingly, in a study
designed to determine the regulation of Raf-1 activity by tyrosine phosphorylation, it
was shown that in vitro treatment of Raf-1 with CD45 reduced the activity of this
serine-threonine kinase (61).

Future studies are planned to identify the serine-threonine kinase which
coprecipitates with CD45 from CTLL-2 cells. In addition, further studies will be
done to ascertain if p60” is indeed in the CD45 immunoprecipitates giving rise to the
weak signal which is detected after anti-src probing of the Western blots. Finally,

studies to determine who is acting upon whom in the CD45/p6OVC/serine-threonine

kinase complex are planned.

105

ACIC‘IOWLEDGMENTS

I thank Dr. S. Conrad (Michigan State University, East Lansing, M1) for use
of the JE-6B elutriation system and Dr. L. King (Michigan State University, East

Lansing, M1) for technical support and assistance with the FACS.

106

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