 |
INTRODUCTION |
The role of CD45 protein-tyrosine phosphatase
(PTP)1 in lymphocyte
signaling has been the subject of extensive investigation (1-4). CD45
is a transmembrane PTP of hematopoietic cells composed of 1268 total
amino acids with an external domain containing alternately used exons,
which leads to the lymphocyte-specific expression of at least eight
different isoforms (1, 5, 6). The cytoplasmic domain consists of 702 amino acids and contains two tandem repeated PTP domains designated D1
and D2 (1). The membrane-proximal PTP domain (D1) is constitutively
active, and the second PTP domain (D2) is considered to be inactive
(7). The catalytic activity of the D1 but not the D2 domain is required
for TCR signal transduction in CD45-deficient cell lines (8). The role
of CD45 in the antigen-specific activation of B and T cells has been
documented by demonstrating that T cells and B cells lacking CD45 fail
to respond to antigen stimulation (9, 10). This observation has been
confirmed in CD45 knockout mice in which the antigen signaling capacity of T and B cells was severely diminished and the transition of thymocytes to maturity was impaired (11, 12). CD45 is believed to
activate the Src family protein-tyrosine kinases by dephosphorylating the regulatory Tyr(P) near the C terminus of T cell receptor or B cell
receptor-associated Src family kinases (13-16). However, recently it
has become clear that the regulation of Src family kinases is likely to
be more complex since the discovery that the activating tyrosine
phosphorylation site in the kinase domain is also dephosphorylated by
CD45 (17). The importance of the CD45 PTP activity in the activation of
T cells has been demonstrated by showing that chimeric proteins
containing only the cytoplasmic domain of CD45 were capable of
restoring normal T cell receptor activation (18-20). Despite previous
research there is still much to be learned about the range of natural
substrates of CD45 as well as about the nature of other proteins that
may interact with CD45.
Phosphorylation of CD45 may play an essential role in the function of
CD45 and may regulate PTP activity, substrate specificity, subcellular
localization, and/or docking with other signaling molecules. Decreased
PTP activity of CD45 was found to correlate with decreased serine
phosphorylation after calcium ionophore treatment of T cells (21), and
serine residues on CD45 have been shown to be phosphorylated in
response to T cell treatment with phorbol esters (22) and after IL-2
treatment of CTLL-2.4 cells (23). Little or no modulation in CD45 PTP
activity was observed after phosphorylation in these reports. In other
studies, serine phosphorylation was observed after lectin treatment of T cells, and tyrosine phosphorylation of CD45 has been reported in
phenylarsine oxide-treated T cells (24, 25). CD45 was phosphorylated after in vitro treatment with casein kinase 2 (CK2) and
other serine/threonine kinases such as protein kinase C and glycogen synthase kinase (26). Increased CD45 PTP activity was found after
phosphorylation with p50csk tyrosine kinase (24) and after
sequential tyrosine phosphorylation by v-Abl kinase (using ATP
S)
followed by serine phosphorylation with CK2 (25).
Using two-dimensional TLC, HPLC, and matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS), we have identified several in vivo phosphorylation sites of CD45 (27). One
phosphorylation site identified in that study, Ser939, is
in a putative substrate-binding loop of the inactive D2 PTP domain.
Three other sites, Ser1204, Thr1246, and
Ser1248 are in the C-terminal tail of the molecule. Another
multiply phosphorylated region was tentatively localized to the
19-amino acid acidic insert in the D2 domain (27). The importance of CD45 phosphorylation sites to PTP activity or to T cell activation remains unknown. The present study was designed to evaluate the relationship of precise CD45 phosphorylation events to the functional role of CD45. In this study, we have identified CK2 as a lymphocyte kinase that targets CD45 and that is responsible for phosphorylation of
CD45 in the 19-amino acid acidic region of the D2 domain of CD45. This
region is a unique insert in the D2 domain that is not found in the
CD45 D1 domain or in any other PTP D1 or D2 domain. Investigation of
the relationship of the CK2 phosphorylation sites to the PTP activity
of CD45 showed that phosphorylation of the D2 acidic region by CK2
increased CD45 activity 3-fold toward phosphorylated myelin basic protein.
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EXPERIMENTAL PROCEDURES |
Cells and Cell Culture--
CTLL-2 (murine cytolytic T cell
line), DO-11.10 (murine T cell hybridoma) and 70Z/3.12 (murine pre-B
lymphocyte cell line) were obtained from ATCC (Rockville, MD). Jurkat
(clone E6-1) (human acute T cell leukemia cell line), and
CD45-deficient Jurkat clone (J45.01) were obtained from Dr. Gary
Koretzky (University of Iowa). The cells were cultured in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10%
heat-inactivated fetal bovine serum (Life Technologies),
streptomycin/penicillin (100 units/ml; Life Technologies), and 50 mM
-mercaptoethanol (Sigma). Recombinant IL-2 (Cetus
Corp.) was added to the CTLL-2 cultures at 7 units/ml. BW5147 (murine T
lymphoma), WEHI274.1 (murine monocyte), P815 (murine mastocytoma), and
NIH3T3 (murine fibroblast) were obtained from ATCC and were grown in
Dulbecco's modified Eagle's medium (Life Technologies) containing
10% heat-inactivated fetal bovine serum, streptomycin/penicillin (100 units/ml), and 50 mM 2-mercaptoethanol. Cells were
maintained in an exponential growth state (0.1-5.0 × 105 cells/ml), and cultures with viability greater than
97% were harvested for experimental use.
Site-directed Mutagenesis--
The bacterial expression vector
pET3D-His6CD45, which expresses the cytoplasmic
domain of murine CD45 with a His6 tag introduced to the
amino terminus, was kindly provided by Dr. Pauline Johnson (University
of British Columbia, Vancouver, Canada) (28). Multiple point mutations
in the acidic insert of the D2 domain of CD45 were made using the
QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).
Desired mutations (underlined) were incorporated into a pair of
oligonucleotide primers, each complementary to opposite strands of the
parental DNA template. The primers used for Ser to Ala (965, 968, 969, and 973) mutagenesis were as follows:
5'-GTGAGCCTGAAGCAGATGAGGCTGCAGATGATGACGCTGACTCAGAAG-3' and
5'-CTTCTGAGTCAGCGTCATCATCTGCAGCCTCATCTGCTTCAGGCTCAC-3'.
The primers used for Ser to Glu (965, 968, 969, and 973) mutagenesis were
5'-GTGAGCCTGAAGAAGATGAGGAGGAAGATGATGACGAGGACTCAGAAG-3'
and 5'-CTTCTGAGTCCTCGTCATCATCTTCCTCCTCATCTTCTTCAGGCTCAC-3'.
The primers were extended by Pfu DNA polymerase during a
short temperature cycling (95 °C for 30 s, 55 °C for 1 min,
65 °C for 13.5 min (2 min/kb of plasmid length), 18 cycles), and the
parental DNA template was then digested by DpnI
endonuclease. Mutants were selected after the synthesized DNA was
transformed into Escherichia coli XL1-Blue and later
verified by sequencing.
Purification of Recombinant CD45 Proteins and In-gel Kinase
Assay--
Recombinant wild type and mutant cytoplasmic domain CD45
(designated His6-cytCD45) was purified from E. coli BL21(DE3) as described (28). The size of the purified
proteins was determined on 10% SDS-polyacrylamide gel, and the
concentration was determined by the Bio-Rad protein assay. The in-gel
kinase assay was adapted from a previously described method (29).
Briefly, 10% polyacrylamide gel was cross-linked with 50 µg/ml of
His6-cytCD45 substrate, while the stacking gel was prepared
without the substrate. Cell lysates (1 × 106 cells)
or immunoprecipitates were loaded onto the gel for electrophoresis. The
gel was washed thoroughly with 20% 2-propanol to remove SDS, and the
protein kinases in the gel were then denatured with two incubations
with 6 M guanidine HCl (Life Technologies) and renatured with five changes of 0.04% Tween 40 (Sigma) at 4 °C. The gel was preincubated in the kinase assay buffer (40 mM HEPES, pH
8.0, 2 mM DTT, 0.1 mM EGTA, 5 mM
Mg(Ac)2, 0.2 mM Ca2+) for 30 min at
room temperature. The kinase reaction was started by incubating the gel
in the kinase assay buffer containing 5 µCi
[
-32P]ATP (3000 Ci/mmol, NEN Life Science Products)
for 1 h at 30 °C. After the reaction, the gel was washed four
to five times with 5% (w/v) trichloroacetic acid solution containing
1% sodium pyrophosphate, until the radioactivity of the solution
approached background. The gel was dried on 3MM Whatman paper and
subjected to PhosphorImager analysis (Molecular Dynamics Inc.,
Sunnyvale, CA).
Immunoprecipitation and Immunoblotting--
Cells were washed
twice with ice-cold phosphate-buffered saline (137 mM NaCl,
3 mM KCl, 8 mM Na2HPO4,
1 mM KH2PO4, pH 7.4) and then lysed
in an appropriate volume (5 × 107 cells/ml) of lysis
buffer (1% Nonidet P-40 (Pierce), 20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 0.23 units/ml aprotinin,
0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin A, 10 µg/ml DNase I, 1 µM okadaic acid, 6 mM sodium fluoride, 2 mM sodium orthovanadate, and 4 mM sodium
molybdate) for 30 min on ice. Cell nuclei were pelleted by
centrifugation at 12,000 rpm at 4 °C for 15 min, and the
supernatants were incubated with the antiserum to CK2
, CK2
', or
CK2
kindly provided by Dr. David Litchfield (University of Western
Ontario, London, Canada) (30) or with antibodies to CD45 (clone 9.4;
ATCC) for 2 h. GammaBind Plus Sepharose (Amersham Pharmacia
Biotech) was added, followed by rocking at 4 °C for 1 h. Immune
complexes were washed sequentially with 1% Nonidet P-40 lysis buffer,
phosphate-buffered saline (pH 7.4), 0.5 M LiCl (pH 7.4),
and 20 mM Tris (pH 7.4). Immunoblotting was performed on
polyvinylidene difluoride membrane with a mixture of anti-CK2
and
CK2
' (1:1000 dilution) (30) followed by incubation with horseradish
peroxidase-conjugated goat anti-rabbit secondary antibody (Bio-Rad) and
visualization with chemiluminescence (Amersham Pharmacia Biotech).
PTP Assay and Kinetic Analysis--
The PTP activity of CD45 was
determined as described previously (31) using 32P-labeled
myelin basic protein (MBP) and Raytide as substrates. Briefly, 50 µg
of Enhanced Raytide (Calbiochem) or 250 µg of MBP (Sigma) was labeled
with 50 µCi of [
-32P]ATP by incubation with 500 ng
of recombinant Src tyrosine kinase (obtained from Dr. J. Dixon,
University of Michigan) for 1 h at 30 °C in 160 µl of
reaction mixture containing 500 µM ATP, 10 mM
MgCl2, 16 mM HEPES, pH 7.5, 0.03 mM
EDTA, 0.07%
-mercaptoethanol. Labeled 32P-Raytide or
MBP was added to 100 µl of 5 mg/ml bovine serum albumin and 70 µl
of 50% cold trichloroacetic acid followed by centrifugation at 12,000 rpm for 15 min. The pellet was then washed twice with 10% cold
trichloroacetic acid and resuspended in 200 µl of 200 mM
Tris, pH 8.0. PTP assays were carried out at 30 °C, and each assay
contained 5 µl of 10× PTP buffer (250 mM HEPES, pH 7.3, 50 mM EDTA, 100 mM DTT), 35 µl of
H2O, 5 µl of 32P-labeled MBP, and 5 µl of
sample to be assayed. After incubation for various times, aliquots of
the reaction mixture were taken out and added to 0.75 ml of acidic
charcoal suspension (0.9 M HCl, 90 mM
Na4P2O7, 2 mM
NaH2PO4, and 4% (w/v) active charcoal (Sigma))
to stop the reaction. After centrifugation, the amount of released
32P in the supernatant was measured in a scintillation
counter. For the His6-cytCD45 kinetics, specific activity
is expressed as nmol/min/mg of protein and was plotted against
substrate concentration. Kinetic parameters were calculated by
nonlinear curve fitting of the data to the Michaelis-Menten equation
using Microcal Origin software (Microcal Software Inc., Northampton,
MA). The kinetics parameters of CK2 (Boehringer Mannheim) with
His6-cytCD45 as substrate were determined with a CK2 assay
using Whatman P81 phosphocellulose paper squares as described
previously (26).
CK2 Phosphorylation of His6-cytCD45--
Wild type
and mutant His6-cytCD45 protein (1 µg) was either
mock-treated or treated with 0.04 milliunits of recombinant CK2 (Boehringer Mannheim) at 30 °C for 30 min in the presence of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM DTT, and 5 µM ATP. PP2A treatment was
performed as follows. His6-cytCD45 was phosphorylated as
described above, followed by the addition of sufficient heparin (10 µg/ml) to inhibit CK2 without inhibiting CD45 (determined by
titration of heparin with each enzyme). The phosphorylated CD45 was
then mock-treated or incubated with PP2A (0.5 units; Promega) for an additional 30 min at 30 °C. The mixture was then subjected to PTP
assay as described above.
In Vitro Kinase Labeling of CD45 and PP2A
Treatment--
Immunoprecipitated CD45 from Jurkat T cells (1 × 107 cells for each sample) were either mock-treated or
treated with PP2A (Promega) for 1 h at 30 °C in 40 µl of
reaction mixture containing 20 mM MgCl2, 50 mM Tris, pH 8.5, 1 mM DTT, 1 µl of PP2A (0.5 units/µl). Treated immunoprecipitates were then washed with
phosphate-buffered saline (pH 7.4), 0.5 M LiCl (pH 7.4),
and 20 mM Tris (pH 7.4). The in vitro kinase
labeling by CK2 was performed at 30 °C. Each reaction contained 4 µl of 10× kinase buffer (200 mM Tris, pH 7.5, 50 mM MgCl2, 10 mM DTT), 20 µl of
immunoprecipitated CD45, 1 µl of recombinant CK2 (0.2 milliunits/µl; Boehringer Mannheim), 1 µl of 0.1 mM
ATP, 10 µCi of [
-32P]ATP (3000 Ci/mmol; NEN Life
Science Products), and H2O to 40 µl. The reaction was
incubated for 30 min and then terminated by the addition of SDS sample
buffer at 100 °C. Labeled CD45 immunoprecipitates were loaded onto
7.5% SDS-polyacrylamide gel, followed by electrophoresis and
PhosphorImager analysis.
FPLC Analysis--
FPLC analysis was performed using a Mono-Q
anion exchange column (Amersham Pharmacia Biotech) using buffer systems
described previously (28) and a NaCl gradient from 150 to 600 mM. Eluted proteins were assayed by the release of
phosphate using pNPP (Sigma) as substrate. CK2 phosphorylation of
His6-cytCD45 for FPLC analysis was performed as described above.
Trypsin Digestion and Mass Spectrometry--
CD45 trypsin
digestion and mass spectrometry were performed as described previously
(27). Briefly, SDS-PAGE-purified, 32P-labeled CD45 was
transferred to polyvinylidene difluoride membrane, excised, and
subjected to tryptic digestion with 10 µg of trypsin (Promega) at
37 °C. Tryptic peptides were recovered and subjected to HPLC
fractionation using a microbore reverse-phase HPLC system (Michrom
BioResources, Inc., CA) (27). The hydrophilic, multiply phosphorylated
HPLC fraction 4 (27) was subjected to MALDI-MS (Voyager Elite
time-of-flight; PerSeptive Biosystems, Framingham, MA) exactly as
described previously (27). A computer program, MSU MassMap (32), was
used to calculate the average masses of all possible peptide and
phosphopeptide fragments from CD45.
 |
RESULTS |
Identification of CD45 Kinase by In-gel Kinase Assay--
In-gel
kinase assays were used in an effort to identify kinases from T cells
with potential to phosphorylate CD45. The in-gel kinase assay was
performed by separating cytoplasmic lysates of CTLL-2 cells in SDS-PAGE
gels containing the His6-tagged recombinant cytoplasmic
domain of CD45 (His6-cytCD45) at a concentration of 50 µg/ml. The gel containing separated proteins was renatured, and
[
-32P]ATP was added to detect kinase activity (Fig.
1). Without in-gel substrate, several
radioactive bands between 40 and 120 kDa were present and most likely
represent the autophosphorylation of some CTLL-2 kinases. With CD45
in-gel substrate, the electrophoretically separated CTLL-2 lysates
exhibited enhanced labeling of two major bands at 40 and 45 kDa
(arrows) (Fig. 1A, lane 2),
which indicated phosphorylation of CD45 by a kinase doublet. Digestion
and phosphoamino acid analysis of both phosphorylated bands showed that
the proteins were exclusively phosphorylated on serine residues (not
shown). The enhanced labeling at 40 and 45 kDa was selective for CD45 because the incorporation into other substrates such as MBP at 50 µg/ml or even at 500 µg/ml only resulted in a basal level of phosphorylation at the same position (Fig. 1A,
lanes 3 and 4).

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Fig. 1.
In-gel kinase assay of T cell lysates.
In-gel kinase analyses of CTLL-2 lysates were performed without in-gel
substrate (lane 1), with His6-cytCD45
at 50 µg/ml of gel (lane 2) as substrate, and
with MBP at 50 µg/ml (lane 3) and at 500 µg/ml (lane 4) as substrate. The positions of
the radioactivity specifically associated with CD45 substrate are
indicated by arrows at 40 and 45 kDa. Protein size is
indicated by molecular mass markers in kDa. The positions of
radioactive bands were determined by PhosphorImager analysis throughout
this study.
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|
Distribution of CD45 In-gel Kinase Activity--
The CD45 in-gel
kinase activity was found to be broadly distributed among a panel of
murine cell lines, including T cell lines (CTLL-2, DO-11.10, BW5147), a
B cell line (70Z/3.12), myeloid cells (WEHI274.1, P815), a fibroblast
line (NIH3T3), and a human T cell line (Jurkat) (Fig.
2A). Each cell type exhibited
two bands resulting from a CD45-selective kinase at 40 and 45 kDa. This indicated that the CD45 kinase was ubiquitously expressed in cell lines
derived from various tissues. Candidate serine/threonine kinases in
this molecular weight range included MAP kinase and CK2. Examination of
the CD45 cytoplasmic domain sequence showed that CD45 had several
consensus sites for CK2 phosphorylation, while there were few conserved
serine or threonine residues that could serve as MAP kinase substrates.
Immunoblotting with a mixture of anti-CK2
and anti-CK2
' showed
that CK2 was widely expressed in these cell types and precisely
overlapped with the in-gel kinase activity (Fig. 2B). We
then addressed the question of the identity of the CD45 kinase by use
of CK2-specific inhibitors.

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Fig. 2.
Expression of CD45-targeted in-gel kinase
activity in various cell lines. A, lysates were
prepared from a panel of cell lines as indicated at the top
and subjected to electrophoretic separation with in-gel substrate
His6-cytCD45 (50 µg/ml of gel) and subsequent in-gel
kinase assay. A parallel separation was performed without added
substrate (bottom of A). B, equivalent
amounts of cell lysates from A were subjected to
electrophoresis and immunoblot analysis with antisera specific for
CK2 (45 kDa) and CK2 ' (40 kDa) as indicated by the
arrows.
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|
CD45 In-gel Kinase Activity Is Inhibited by CK2
Inhibitors--
In-gel kinase assays were performed with 50 µg/ml
His6-cytCD45 in the presence of known CK2 inhibitors
(33-35) to determine whether the observed CD45 in-gel kinase activity
was consistent with that of CK2 (Fig. 3).
The presence of CD45 in-gel kinase activity was almost completely
blocked by treatment with either 50 µg/ml heparin (Fig. 3,
lane 3), 200 µM GTP
(lane 4), or 2 mg/ml poly-GluTyr (4:1)
(lane 5). The activity of authentic recombinant CK2
was also almost completely abrogated by the inhibitors (Fig. 3,
lanes 7-9). Taken together, the size of the
in-gel kinase coupled with observed inhibition by specific CK2
inhibitors strongly suggested that the kinase responsible for
phosphorylation of CD45 in this assay was CK2.

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Fig. 3.
Inhibition of in-gel kinase activity with
inhibitors of CK2. CTLL-2 lysates (lanes 1-5) were
subjected to in-gel kinase assay with 50 µg/ml
His6-cytCD45 as substrate (lanes 2-5) in the
presence of inhibitors of CK2. CTLL-2 lysates were also analyzed with
no in-gel substrate (lane 1). Recombinant human CK2 was
also subjected to in-gel kinase assay as a control (lanes
6-9). The inhibitors used were heparin (lanes
3 and 7), GTP (lanes 4 and
8) and poly-Glu-Tyr (lanes 5 and
9). The positions of the CD45-targeted in-gel kinase bands
are indicated with arrows at 45 and 40 kDa. Protein size is
in kDa.
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Immunoprecipitation of the CD45-targeted In-gel Kinase with CK2
Antibodies--
To confirm the nature of the CD45 kinase, CK2
immunoprecipitates from cell lysates were subjected to in-gel kinase
assays with His6-cytCD45 as substrate (Fig.
4A). CK2 consists of
homotetrameric and heterotetrameric complexes
(
2
2,
'2
2,
and 
'
2) containing two catalytically active
-chains (45-kDa
-chains and 40-kDa
'-chains) and two
noncatalytic, regulatory
-chains (26 kDa each) (30). In-gel kinase
analysis of anti-CK2
immunoprecipitates resulted in prominent
labeling of CD45 substrate at 45 kDa (Fig. 4A,
lane 1). Analysis of CK2
' immunoprecipitates
resulted in prominent labeling at 40 kDa (Fig. 4A,
lane 2), and analysis of CK2
immunoprecipitates resulted in the visualization of radioactive bands
consistent in size with CK2
and CK2
' (Fig. 4A,
lane 3). Both
and
' are expected to exist
in CK2
,
', or
immunoprecipitates due to the existence of
heterotetramers as noted above. Precipitation with Sepharose G beads
alone did not result in the isolation of in-gel kinases (Fig.
4A, lane 4). Parallel samples
subjected to in-gel analysis without added CD45 protein did not result
in significant activity (Fig. 4A, lower
part). To further confirm that CK2 was the CD45 kinase in
CTLL-2 cells, specific CK2 antisera were used to deplete CK2 from
CTLL-2 lysates (Fig. 4B). After only one round of
immunoprecipitation, the 40-kDa CD45 in-gel kinase activity was
preferentially depleted using anti-CK2
' (Fig. 4B,
lane 3), while the CK2 activity was only slightly
depleted with anti-CK2
(Fig. 4B, lane
2) or anti-CK2
(Fig. 4B, lane
4). The relatively prominent labeling of the CK2
' in-gel
kinase band and immunodepletion by anti-CK2
' serum suggested that
CK2
' was the primary form of CK2 catalytic subunit that
phosphorylated CD45 in these cells.

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Fig. 4.
Immunoprecipitation of the CD45-targeted
kinase with anti-CK2 antiserum. A, CTLL-2 cell lysates
were subjected to immunoprecipitation with anti-CK2 (lane
1), anti-CK2 ' (lane 2), and
anti-CK2 (lane 3) and separated with a gel
containing His6-cytCD45 as substrate. Control
"precipitation" using beads alone is shown in lane
4. Immunoprecipitates were also analyzed without in-gel
substrate (bottom). The positions of CK2 and CK2 ' are
indicated with arrows at 45 and 40 kDa, respectively.
B, CK2 was depleted from CTLL-2 lysates with anti-CK2
(lane 2), anti-CK2 ' (lane
3), and anti-CK2 (lane 4), and the
depleted lysates were subjected to in-gel kinase assay using
His6-cytCD45 as substrate. The starting lysate was analyzed
in lane 1, and control depletion using beads
alone is shown in lane 5. Only one round of
depletion was performed.
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CD45 as a Substrate for CK2--
In order to further characterize
the nature of CK2 phosphorylation of CD45, a kinetic analysis was
performed. The Km was 0.51 µM, and the
Vmax was 35.5 nmol/min/mg with CD45 as a substrate of CK2. These parameters were comparable with reports of CK2
kinetics with other protein substrates (for example,
Km = 1.1 µM and
Vmax = 82.5 nmol/min/mg with eIF-2 (36)). With a
Km in the submicromolar range, we conclude that CD45 is an excellent substrate for CK2 (37).
Mutation of the CK2 Consensus Phosphorylation Sites Blocks
Phosphorylation by CD45-targeted In-gel Kinase Activity--
The
19-amino acid acidic insert of the D2 domain (Fig.
5, boxed) was compared for
different species and four highly conserved CK2 phosphorylation sites
consisting of the consensus sequence Ser-X-X-acidic group (38) were noted (Fig. 5,
shaded residues). All four CK2 consensus-site
serines at positions 965, 968, 969, and 973 were mutated to alanines in
His6-cytCD45 to preclude potential phosphorylation. The
mutated protein was then incorporated into an SDS-polyacrylamide gel at
50 µg/ml gel, and an in-gel kinase assay was performed using
immunoprecipitates of anti-CK2
, anti-CK2
', and anti-CK2
(Fig.
6A, lanes
1, 2, and 3, respectively). Control experiments used the same immunoprecipitates with wild type
His6-cytCD45 as substrate (Fig. 6B) and with no
substrate (Fig. 6C). The reduction of labeling with the
serine to alanine mutated form of His6-cytCD45 was
essentially complete, showing that these sites represented the major
CK2 phosphorylation sites in the CD45 cytoplasmic domain.

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Fig. 5.
CD45 sequence homology in the region of the
19-amino acid D2 insert. Comparison of the interspecies homology
of the CD45 D2 region containing the 19-amino acid acidic insert unique
to CD45. The insert is boxed, and the serine residues at
965, 968, 969, and 973 that are mutated in this study are
shaded. The position of the beginning of the 1-strand
(INAS) found in the family of PTP molecules is shown by the
arrow. The standard single-letter amino acid codes are used.
The accession numbers for the sequences are as follows: mouse, P06800;
rat, P04157; human, P08575; chicken, Z21960; and shark, U34750.
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Fig. 6.
In-gel kinase analysis of CK2
immunoprecipitates using mutant CD45 as substrate. Anti-CK2
(lane 1), anti-CK2 ' (lane
2), and anti-CK2 (lane 3)
immunoprecipitates from CTLL-2 cells were subjected to an in-gel assay
system containing the following substrates. A,
His6-cytCD45 with four Ser to Ala mutations at residues
965, 968, 969, and 973; B, wild-type
His6-cytCD45; C, no substrate. Control
"precipitation" using beads alone is shown in each lane
4. The size of SDS-PAGE markers is shown in kDa.
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Serine Phosphorylation of the Acidic Insert in the D2 Domain
Increases CD45 Activity--
The high conservation of the CK2
phosphorylation sites in the acidic insert region of CD45 led us to
hypothesize that phosphorylation (or introduction of additional acidic
residues) at this site would modulate CD45 activity. To test this
hypothesis, we compared the kinetics of the His6-cytCD45
mutant forms using 32P-MBP as a substrate (Fig.
7A). MBP was used because it is an excellent substrate for
CD45 (26) and because it was necessary to prepare it in sufficient
quantity to perform repeated kinetic analysis at substrate saturating
levels. The kinetics of wild type His6-cytCD45 are shown in
Table I and are comparable with previous
reports (26). Ser to Ala mutations only slightly altered the basic
kinetic parameters of His6-cytCD45, while the introduction of acidic residues (Glu) into the Ser CK2 sites resulted in a 3-fold
increase in Vmax and a small increase in
Km (Table I).
We then evaluated the effect of CK2 phosphorylation on CD45 activity at
single substrate concentrations (8 µM) as determined from
Fig. 7A. Wild type
His6-cytCD45 was phosphorylated with CK2 followed by
comparison of the PTP activity with the unphosphorylated form of the
protein. For 32P-MBP, wild type CD45 activity was enhanced
after CK2 phosphorylation by about 3-fold (Fig. 7B, wt;
gray bar). Mutation of the four consensus Ser
residues (965, 968, 969, and 973) to Ala (S/A) did not
increase the activity of CD45, and phosphorylation of the Ser to Ala
mutant did not exhibit an increase in activity (Fig. 7B,
gray bar). Mutation of the four Ser residues to
Glu (to mimic phosphorylation) resulted in a 3-fold activity increase
toward MBP (Fig. 7B, S/E, white
bar), and there was no change in the activity of the Ser to
Glu mutant after phosphorylation with CK2 (Fig. 7B,
S/E, gray bar). Interestingly, the
large increase in activity upon phosphorylation of the acidic domain
was only observed with MBP and not with two other small substrates, for
example 32P-Raytide (Fig. 7C) and pNPP (data not
shown). Only a small increase in activity toward
32P-Raytide was observed after phosphorylation of wild-type
His6-cytCD45 with CK2 (Fig. 7C, wt).
Further, neither Ser to Ala or Ser to Glu mutation nor CK2
phosphorylation had significant effect on the activity of CD45 with
Raytide as substrate (Fig. 7C, S/A and S/E).

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Fig. 7.
Effect of mutation and CK2 phosphorylation on
CD45 activity. A, kinetics of the
His6-cytCD45 mutant forms using 32P-MBP as
substrate. Specific activity of each CD45 CK2 site mutant
(Ser965, Ser968, Ser969, and
Ser973) Ser to Ala (S/A) and Ser to Glu
(S/E) was determined over the substrate concentration range.
The curves were calculated by nonlinear curve fitting of the data to
the Michaelis-Menten equation using Microcal Origin software.
B, PTP activity was determined using 32P-MBP for
His6-cytCD45 containing Ser to Ala (S/A) and Ser
to Glu (S/E) mutations and for wild type (wt)
CD45 (open bars). In parallel, wild-type and mutant CD45
proteins were phosphorylated with CK2 and ATP before PTP assay
(shaded bars). C, PTP activity using
32P-Raytide as substrate was determined for the
His6-cytCD45 forms as shown above. For B and
C, the average and S.D. of three separate experiments are
shown. D, comparison of PTP activity (with
32P-MBP as substrate) of CK2-phosphorylated, wild type
His6-cytCD45 (+CK2) with PTP activity after
sequential CK2 phosphorylation and subsequent dephosphorylation with
PP2A (+CK2 +PP2A). PP2A alone was added as a control
(+PP2A).
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We then determined the activity of CK2-phosphorylated CD45 after
removal of phosphate with PP2A (Fig. 7D). After the CK2
phosphorylation, we added heparin to inhibit CK2 activity and then
incubated with PP2A. The amount of heparin added (10 µg/ml) was
determined by titration to achieve a balance in which the heparin
inhibited the CK2 without inhibiting CD45 or PP2A (data not shown).
Using this protocol, we were able to show, in the same set of
experiments, that CK2 increased the activity of CD45 and that
subsequent dephosphorylation with PP2A reversed the activation.
Complete reversal may not have been achieved during the short
incubation, possibly due to residual CK2 activity and the presence of
excessive ATP.
In order to verify that treatment with CK2 resulted in the
phosphorylation of all the CD45 present, we subjected the CK2-treated, activated His6-cytCD45 from Fig. 7B to
analytical FPLC separation on a Mono-Q anion exchange column (Fig.
8). Mock-treated (ATP without added CK2)
and CK2-phosphorylated wild-type His6-cytCD45 were
separated using NaCl gradient elution from 150 to 600 mM. Wild type His6-cytCD45 eluted in about 14 ml at 240 mM NaCl (active fractions determined by pNPP hydrolysis are
shaded), and a large peak of ATP eluted at about 180 mM (about 10 ml) (Fig. 8A). Analysis of CK2
phosphorylated His6-cytCD45 indicated that the active
fractions were retained by the column and required from 340 to 360 mM NaCl for elution (about 19 ml) (Fig. 8B). The
phosphorylated CD45 appeared as two peaks probably resulting from
differently phosphorylated forms. One of these CD45 forms eluted at the
same salt concentration as the Ser to Glu mutant (340 mM
NaCl), which contains four additional negative charges out of 715 amino
acids (shown in Fig. 8E). The FPLC elution profiles of
wild-type and the Ser to Ala mutant are also shown for comparison (both
eluted at 240 mM NaCl) (Fig. 8, C and
D). Consideration of the FPLC data suggests that the two peaks represent CD45 forms containing different numbers of phosphates. In parallel experiments, the stoichiometry of phosphorylation of
His6-cytCD45 was estimated at 2.5 mol of phosphate/mol of
protein by quantitation of the incorporation of 32P of
known specific activity into CD45 (data not shown).

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Fig. 8.
Analytical FPLC separation of
CK2-phosphorylated CD45. His6-cytCD45 was subjected to
analytical FPLC separation on a Mono-Q anion exchange column using NaCl
gradient elution (fine line from 150 to 600 mM). A, elution profile of mock-treated
His6-cytCD45 containing ATP without added CK2 (CD45 eluted
at 240 mM NaCl). B, elution profile of
CK2-phosphorylated His6-cytCD45 (CD45 eluted at 340-360
mM NaCl). An absorbance scale of 0.01 was used, and PTP
active fractions were determined by pNPP hydrolysis (shaded
areas). ATP eluted at about 180 mM NaCl (peak
indicated by arrow). C-E, FPLC elution profile
of wild-type (wt), Ser to Ala (S/A), and Ser to
Glu (S/E) mutant forms of His6-cytCD45 separated
under conditions comparable with the other panels.
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CK2-targeted Sites in CD45 Are Phosphorylated in Vivo--
We
found that CK2 immunoprecipitates from Jurkat T cells had
constitutively high activity toward CD45, and the overall activity of
CK2 did not change after stimulation of Jurkat cells with anti-TCR, phorbol 12-myristate 13-acetate, or ionomycin (data not shown). This
result suggested that cytoplasmic CK2 maintained a high level of
phosphorylation of CD45 in the acidic insert. To address the question
of whether CD45 CK2 sites were phosphorylated in vivo, immunoaffinity-purified CD45 from Jurkat T cells was subjected to
in vitro kinase labeling by recombinant CK2
and
[
-32P]ATP (Fig. 9). It
was found that immunoprecipitated CD45 from Jurkat T cells could not be
labeled easily by exogenous CK2 (Fig. 9A, lane
1). Since phosphorylation at the CK2 sites could have blocked the addition of further phosphates, CD45 immunoprecipitates were pretreated with PP2A phosphatase before the kinase labeling. PP2A
treatment converted CD45 to a form that could be successfully phosphorylated with CK2 (Fig. 9A, lane
2). Immunoprecipitates from a CD45-deficient Jurkat clone
(J45.01) were used as controls (Fig. 9, lanes 3 and 4). Further evidence for the existence of in
vivo multiple phosphorylations at the CD45 CK2 sites was obtained by MALDI-MS analysis of tryptic peptides obtained from in vivo 32P-labeled CD45 (Fig. 9B). The hydrophilic
CD45 tryptic peptides from HPLC fraction 4 (27) were subjected to
MALDI-MS analysis exactly as described previously (27). Although these
hydrophilic peptides typically presented weak signals (not necessarily
reflective of abundance), the MALDI-MS analysis revealed a tryptic
peptide at (M + H)+ of 2542, which correlated with the CD45
tryptic peptide from the acidic insert with three phosphate residues
(predicted (M + H)+ of 2545; expected error of about
0.1%). Taken together, these results strongly support the notion that
the CD45 CK2 sites are multiply phosphorylated in vivo.

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Fig. 9.
CD45 is phosphorylated in
vivo. A, PhosphorImager analysis of
SDS-PAGE-separated CD45 immunoprecipitated from Jurkat cells that was
subjected to in vitro kinase phosphorylation with CK2 and
[ -32P]ATP. CD45 immunoprecipitate was mock-treated
(lane 1) or treated with PP2A before an in
vitro kinase reaction. Identical analysis of the
CD45 cell line, J45.01, was used as a control
(lanes 3 and 4). B,
MALDI-mass spectroscopic analysis of peptides obtained from a tryptic
digest of gel-purified CD45 after HPLC fractionation. The peak at (M + H)+ 2542 (noted by an arrow) correlates with the
calculated mass of the peptide shown, which contains the CD45 D2 acidic
insert with three phosphates.
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DISCUSSION |
In this report, we have positively identified the D2 acidic insert
as containing the sites for CK2 phosphorylation and have shown that
phosphorylation at those sites leads to a large increase in the
Vmax of CD45. This increase in
Vmax could lead to a major alteration of the
signaling capacity of CD45 in the initiation of antigen stimulation in
lymphocytes. Phosphorylation of CD45 at acidic domain CK2 sites
increased the PTP activity of CD45 about 3-fold using
32P-MBP as substrate. This activation was not apparent with
other substrates, suggesting substrate selectivity for phosphorylated CD45. A kinetic analysis of His6-cytCD45 found its
Vmax and Km to be in general
agreement with previously determined values (26, 39). By use of
analytical FPLC separation, we verified that all of the
His6-cytCD45 was phosphorylated after CK2 treatment as
indicated by increased retention on an anion exchange column (Fig.
8B). The fact that two FPLC peaks were observed after CK2 phosphorylation indicated the presence of multiple phosphorylated forms. Importantly, although there are other potential CK2 sites in
CD45, the Ser to Ala mutation of the D2 acidic insert blocked phosphorylation by CK2 and also increased activity after such treatment. The acidic insert contains four CK2 phosphorylation sites
that are conserved in all species examined, and mutation of these four
serine sites (965, 968, 969, and 973) abolished greater than 95% of
the ability of CK2 to phosphorylate CD45.
The increase in CD45 activity after phosphorylation is consistent with
a previous report in which decreased PTP activity of CD45 was
accompanied by a decrease in serine phosphorylation (21). In other
studies, PTP activity of CD45 was not modulated by CK2 phosphorylation,
possibly because the CD45 utilized in these studies was already highly
phosphorylated and therefore could not be further activated (25, 26).
The modulation of serine phosphorylation of CD45 has been demonstrated
under various stimuli (21-23). Some studies have failed to detect a
relationship between phosphorylation and PTP activity (22, 23), while
others have shown modulation of CD45 activity upon phosphorylation (21,
24, 25). The discrepancy between these studies may have stemmed from
the difficulty of isolating and assaying CD45 immediately after
stimulation and from the lack of precise knowledge of in
vivo phosphorylation states.
CK2 is a ubiquitous serine/threonine kinase that is expressed in
virtually all cell types (35, 40). CK2 has been reported to be highly
expressed in some transformed and proliferating cells, and, when
overexpressed in transgenic mice, the CK2 gene acts as an oncogene in
cooperation with myc (41, 42). CK2 exists as a tetramer
composed of two catalytic
-chains (
, 
', or
'
') and
two regulatory
-chains (40). The
-chain is almost identical among
various mammalian species, and the CK2
and CK2
' chains are highly
homologous to each other as well as highly conserved (40). CK2 is found
in both the nucleus and the cytoplasm, and it phosphorylates a number
of signaling proteins in both compartments (e.g. Jun, Myc,
Myb, Rb, and p53) (35) at consensus phosphorylation sites composed of
Ser/Thr-X-X-Glu/Asp or
Ser/Thr-X-X-acidic group (38). Although there has
been much work performed on the nature and activity of CK2, a
definitive role in signal transduction is still somewhat obscure
(40).
In this report, we showed that analysis of CK2 may be performed with an
in-gel kinase method using CD45 as a substrate. CD45 was an excellent
substrate for CK2 and became highly phosphorylated with only 50 µg of
substrate/ml of gel, while most other in-gel kinase methods have used
from 500 µg/ml to 1 mg/ml of substrate (43). The kinase that
phosphorylates CD45 was identified as CK2 by a combination of
immunoprecipitation, immunodepletion, specific inhibition, and mutation
of CK2 consensus sites. Our data demonstrated that while both the
-
and
'-chains of CK2 phosphorylate CD45, CK2
' is the most active
form on CD45. In addition, the relative ability of anti-CK2
' to
deplete CK2 from CTLL-2 lysates suggested that CK2
' was the
predominant form of CK2 that phosphorylated CD45 in these cells. In the
in-gel kinase assay, the CK2
chain was separated from the CK2
catalytic subunit and thus was not necessary for CD45 phosphorylation.
Immunoprecipitated CK2 in its native state (containing
-,
'-, and
-chains) also efficiently phosphorylated His6-cytCD45
(data not shown). Our results have extended previous reports that
predicted that CD45 would be a substrate of CK2 and that CK2 was able
to phosphorylate CD45 in vitro (25, 26, 44).
The insert that contains the CK2 phosphorylation sites is a conserved,
highly acidic sequence of 19 amino acids that exists only in the D2
domain of CD45 and not in other PTPs. Alignment of the D2 sequence of
CD45 to the x-ray crystal structure of other PTPs showed that the D2
acidic insert lies just N-terminal to the highly conserved YINAS
sequence that forms the
1-helix (45). This would place the acidic
insert of 19 amino acids in a loop near the opening of the inactive D2
catalytic cleft. Phosphorylation of this insert could interfere with
interdomain duplex formation postulated to involve the binding of the
N-terminal wedge of one PTP domain to the catalytic cleft of a second
PTP domain (7, 46, 47). This could make the catalytic site of the D1
domain more accessible to substrate and thus increase activity. It is also possible that the phosphorylation state could directly influence catalytic activity by affecting interactions between the D1 and D2
domains. The observation that CK2 phosphorylation increased the
activity of CD45 is consistent with either hypothesis.
Future work will focus on functional analysis of the 19-amino acid
acidic insert in vivo. It will be of great interest to find
out whether or not this unique insert serves as a docking site for
substrates or signaling molecules. Yet another question to be addressed
is why CD45 is endogenously phosphorylated to a high level in this
already very acidic region. Further investigation will be directed at
the physiological relevance of the insert and the phosphorylation
sites. It is expected that phosphorylation and/or dephosphorylation of
the acidic insert might play a role in the activation and/or
desensitization of the T cell receptor complex.