From the Department of Pathology, Roger Williams Medical Center-Boston University, Providence, Rhode Island 02908
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ABSTRACT |
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CD45-AP associates specifically with CD45, a
protein-tyrosine phosphatase essential for antigen receptor-mediated
signal transduction. CD45 modulates the activity of Src family
protein-tyrosine kinases involved at the onset of antigen
receptor-mediated signaling by dephosphorylating their regulatory
tyrosyl residues. We have shown that lymphocyte responses to antigen
receptor stimulation are impaired in CD45-AP-null mice. To
examine the possibility that CD45-AP coordinates the
interaction between CD45 and its substrates, we investigated the
associations of CD45-AP with several protein-tyrosine kinases.
Endogenous CD45-AP coimmunoprecipitated with Lck and ZAP-70 in both
CD45-positive T cells and their CD45-negative variants after
stimulation by antigen receptor ligation. Concomitantly, CD45
coimmunoprecipitated with Lck and ZAP-70 after T cell receptor-mediated stimulation of CD45-positive cells. Recombinant CD45-AP exhibited specific binding to Lck and ZAP-70 protein-tyrosine kinases, but not to
Fyn or Csk, in lysates of both CD45-positive and -negative T cells.
Specific interactions were demonstrated between the respective recombinant proteins as well. These results demonstrate that CD45-AP associates directly and selectively with Lck and ZAP-70 in response to
T cell receptor-mediated stimulation. The associations of CD45-AP with
Lck and ZAP-70 may mediate the functional interactions of these kinases
with CD45 during antigen receptor stimulation.
Protein-tyrosine phosphatase activity residing in the cytoplasmic
portion of CD45 (1) is essential for antigen receptor-mediated signal
transduction in lymphocytes (2). The T cell receptor (TCR)1 and its coreceptors
recruit Src family protein-tyrosine kinases (such as Lck and Fyn) and
Syk family protein-tyrosine kinases (such as ZAP-70) by forming
specific associations via their cytoplasmic segments (3-7). Tyrosine
phosphorylation of cellular proteins by these protein-tyrosine kinases
is one of the earliest events of TCR signaling (8-10). There is strong
evidence supporting the notion that CD45 activates Src family
protein-tyrosine kinases by dephosphorylating their down-regulatory
tyrosine residues (2, 11-14). On the other hand, considerable data
indicate that the effect of CD45 on antigen receptor-mediated signaling
can be inhibitory rather than stimulatory under certain circumstances.
For example, CD45 has been shown to specifically interact with and
dephosphorylate the tyrosine-phosphorylated Most of the CD45 in lymphocytes is associated with a predominantly
lymphocyte-specific protein, CD45-associated protein (CD45-AP), with an
estimated stoichiometry of 1 to 1 (18-20). Expression of CD45-AP is
limited to certain subsets of leukocytes, whereas CD45 is expressed in
all subsets of leukocytes. CD45-AP is expressed strongly in T, preB,
and B cells, very weakly in mast cells, and not at all in plasma cells
or cells of the monocyte/macrophage lineage (20). A human homologue of
CD45-AP, named LPAP, is also expressed in comparable subsets of
leukocytes (21, 22). Two forms of CD45-AP mRNA exist, and the
resulting two CD45-AP proteins are identical in their capacity for
specific binding to CD45 but employ different mechanisms for
endoplasmic reticulum membrane translocation (20). The potential
transmembrane segment of CD45-AP binds specifically to the
transmembrane segment of CD45, and this physical association does not
require the presence of other leukocyte-specific proteins, such as Lck,
Fyn, and ZAP-70 (23). Amino acid sequence analysis and protease
susceptibility analysis of CD45-AP indicate that only a short segment
at the amino terminus of CD45-AP protrudes extracellularly, whereas the
bulk of the protein is located intracellularly (23).
We have previously suggested that CD45-AP plays an adapter-like role
for CD45 (19). Evidence in support of this hypothesis was provided
recently by investigation of CD45-AP-null mice created by gene
targeting (24). CD45-AP-null mice do not exhibit any detectable
abnormality in lymphocyte differentiation in contrast to CD45-deficient
(25) or -null (26) mice that show a profound block in T cell
differentiation. However, similar to CD45-deficient or -null mice,
responses of CD45-AP-null mice to TCR-mediated or B cell
receptor-mediated stimuli are markedly reduced (24). Interestingly, the
interaction between CD45 and Lck is significantly decreased in
CD45-AP-null T cells, indicating that CD45-AP directly or indirectly
mediates the interaction of CD45 with Lck. Therefore, the present study
was carried out to examine the possibility that CD45-AP specifically
interacts with potential CD45 substrates. Specific interactions of
CD45-AP with Lck and ZAP-70, but not with Fyn or Csk, were detected in
both CD45-positive T cells and their CD45-negative variants in response
to TCR ligation. Moreover, a specific association of CD45-AP with Lck
and ZAP-70 was demonstrated by using the respective recombinant
proteins. In CD45-positive cells, TCR ligation resulted in association
of CD45 with Lck and ZAP-70. These results demonstrate that direct and
specific interactions of CD45-AP with Lck and ZAP-70 are triggered by
TCR ligation and support the notion that CD45-AP plays an adapter-like
role by coordinating the interaction between CD45 and specific
protein-tyrosine kinases.
Cell Culture--
The mouse T cell line YAC-1 (WT) and its
CD45-negative variant (N1) (27) were provided by Dr. J. Ashwell
(National Cancer Institute, Bethesda). The cells were cultured in
Iscove's modified Dulbecco's medium supplemented with 10% fetal calf
serum, 50 µM 2-mercaptoethanol, 2 mM
glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin.
Preparation of Cell Lysates--
YAC-1 or N1 cells, 40 × 106 cells/ml in Iscove's modified Dulbecco's medium
containing 2 mM sodium orthovanadate, were incubated first
with anti-CD3 Binding Assay of Recombinant CD45-AP with Cell
Lysates--
Recombinant full-length (F) and deleted (C) form of
CD45-AP fused to the carboxyl terminus of glutathione
S-transferase (GST) have been described previously (23). The
C form begins with amino acid position 47 and continues to the carboxyl
terminus of the CD45-AP cDNA. Approximately equimolar amounts of
full-length or C-form of recombinant CD45-AP proteins bound to
glutathione-Sepharose 4B beads were incubated at 37 °C for 1 h
with lysates of YAC-1 or N1 cells prepared as described above for
binding, followed by extensive washing to remove unbound material.
Total cell lysates and material bound to the beads were analyzed by
SDS-PAGE followed by Western blotting.
Immunoprecipitation--
Anti-CD45 antibody (29) cross-linked
with dimethyl pimelimidate (30) to protein G-Sepharose 4 beads
(Amersham Pharmacia Biotech) was used for anti-CD45
immunoprecipitation. Rabbit antiserum against CD45-AP (23) was used for
immunoprecipitation in combination with protein G-conjugated Sepharose
4 beads. Postnuclear supernatants of cell lysates prepared as described
above were precleared with protein G-Sepharose 4 and then used for
immunoprecipitation. Material bound to the beads was analyzed by
SDS-PAGE followed by Western blotting.
Western Blotting--
Western blotting was carried out with
antibodies against CD45-AP, CD45 (provided by Drs. J. Marth, University
of California, San Diego, La Jolla, CA, and I. Trowbridge, The Salk
Institute, San Diego, CA), Fyn (Oncogene Research Products and
Transduction Laboratories), Lck (Transduction Laboratories and Upstate
Biotechnology Incorporated), ZAP-70 (Transduction Laboratories), or Csk
(Transduction Laboratories) followed by horseradish
peroxidase-conjugated protein A, anti-mouse Ig antibody, or anti-rat Ig
antibody. The signals were detected with either the ECL Western
blotting system (Amersham Pharmacia Biotech) or Supersignal substrate
system (Pierce) and quantitated by densitometric analysis (Scan
Analysis version 2.56 by Biosoft).
Binding Assay of Recombinant CD45-AP with Recombinant Protein
Tyrosine Kinases--
The CD45-AP cDNA (19) was subcloned into the
pQE30 expression vector (Qiagen) downstream from six histidine residues
using the KpnI site. Mouse ZAP-70 cDNA (provided by Dr.
A. Weiss, University of California, San Francisco) (4) was excised by
XhoI and subcloned into the pQE32 expression vector (Qiagen)
downstream from six histidine residues at the BamHI sites by
using a BamHI linker. The respective histidine-tagged
recombinant proteins were produced in the JM109 strain of
Escherichia coli by
isopropylthio- Sucrose Gradient Ultracentrifugation--
Sucrose gradients
containing 0.4% BRIJ 96, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 0.1 mM EGTA, 2.5 mM thioglycolic acid, 0.2 mM phenylmethylsulfonyl fluoride, 10 mM sodium
fluoride, 1 mM sodium orthovanadate, 25 µM
phenylarsine oxide, and 10 mM sodium pyrophosphate were
prepared in a polyallomer tube (14 × 95 mm) from 5.6 ml each of
2.5 and 12.5% sucrose solutions over a 0.25-ml cushion of 40% sucrose
solution. Cell lysate (0.5 ml) obtained from 25 × 106
CD45-positive YAC-1 cells was applied to the top of the gradient. After
centrifugation at 36,000 rpm for 20 h at 4 °C in an SW40 swinging bucket rotor (L8-80 M Beckman ultracentrifuge),
the sucrose gradient was fractionated into 20 equal portions from the
bottom of the tube. A portion of each fraction was analyzed by SDS-PAGE followed by Western blotting.
Lck and ZAP-70 Interact with Recombinant CD45-AP Independently of
CD45--
The earliest stages of TCR signaling are mediated by
protein-tyrosine kinases such as Lck, Fyn, ZAP-70, and Syk (8-10). Lck and Fyn are most likely in vivo substrates of CD45, whereas
ZAP-70 may also be regulated directly or indirectly by CD45 (2, 11-14, 16). To examine the possibility that CD45-AP directs the interaction between CD45 and its substrates, the interactions of recombinant CD45-AP with several protein-tyrosine kinases were investigated.
Wild-type CD45-positive YAC-1 cells (WT) and their CD45-negative
variant cells (N1) (27) were stimulated by TCR cross-linking with
anti-CD3
For binding assays, either full-length recombinant CD45-AP (F) or the
bulk of its cytoplasmic portion (C) (Fig. 1B) bound to
glutathione-Sepharose 4B beads were incubated with the same cell
lysates. Material bound to the beads was analyzed by Western blotting
with the aforementioned antibodies. As shown in Fig. 1C,
binding of CD45 to recombinant CD45-AP remained unchanged with
stimulation. As expected from the fact that CD45 and CD45-AP interact
through their respective transmembrane segments (23), no CD45 bound to
the C form of CD45-AP. Lck and ZAP-70 specifically bound to recombinant
CD45-AP, and specificity of the binding was demonstrated by the fact
that Fyn, Csk (Fig. 1C), and other proteins such as Grb2 and
protein-tyrosine phosphatase-1C (not shown) did not bind to CD45-AP
under the same conditions. Interestingly, binding of Lck and ZAP-70 to
CD45-AP did not require the presence of CD45 since it occurred in
CD45-negative variant cells as well. Furthermore, the binding of Lck
and ZAP-70 to CD45-AP decreased for both the wild-type lymphocytes and
their CD45-negative variants when cells were stimulated by TCR ligation
although the total amounts of these protein-tyrosine kinases in the
cell lysates remained unchanged (see Fig. 1A). The greater
decrease of bound Lck in WT cells compared with that seen in N1 was not
a consistent observation as N1 cells exhibited greater decreases in
some experiments. Lck and ZAP-70 did not associate with the C form of
CD45-AP indicating that the transmembrane and/or the first five amino
acid residues of the cytoplasmic domain play critical roles for their
interactions. In addition, the binding of Lck and ZAP-70 to CD45-AP
occurred independently of the TCR coreceptor, CD4, since the cell lines used did not express CD4 on their cell surfaces (27).
The percentage of endogenous Lck and ZAP-70 that is capable of binding
to recombinant CD45-AP was then determined by using cell lysates
obtained from unstimulated CD45-positive YAC-1 cells (WT) and
CD45-negative N1 cells. The cell lysates were repeatedly subjected to
fresh aliquots of recombinant CD45-AP-conjugated beads, and the
material bound to each aliquot of beads was analyzed by Western
blotting with either anti-Lck or anti-ZAP-70 antibody (Fig.
1D). Approximately 2.1% of total Lck and 0.95% of total ZAP-70 present in WT cells bound to recombinant CD45-AP. On the other
hand, approximately 0.84% of total Lck and 0.66% of total ZAP-70
present in N1 cells bound to CD45-AP.
Lck and ZAP-70 Coimmunoprecipitate with CD45-AP after TCR-mediated
Stimulation Independently of CD45--
Next, we analyzed the effect of
anti-CD3
Interestingly, the binding of Lck and ZAP-70 to recombinant CD45-AP
decreased with stimulation (Fig. 1C), whereas association of
endogenous CD45-AP with these protein-tyrosine kinases increased (Fig.
2). This indicates that after 60 min stimulation, the protein-tyrosine kinase populations available for binding to CD45-AP have already bound
to endogenous CD45-AP and are not available for binding to recombinant
CD45-AP.
Specific Binding of CD45-AP to Lck and ZAP-70 Can Be Demonstrated
with the Respective Recombinant Proteins--
We have further
investigated the association of CD45-AP with Lck and ZAP-70 by binding
analysis employing recombinant forms of each protein. As shown in Fig.
3A, GST-tagged recombinant Lck protein bound to histidine-tagged recombinant CD45-AP that was conjugated to the nickel-nitrilotriacetate beads, and the binding was
prevented by adding increasing amounts of free GST-tagged CD45-AP as a
competitor for binding. An excess amount of control GST protein did not
compete for the binding. Likewise, as shown in Fig. 3B,
histidine-tagged recombinant ZAP-70 protein bound to GST-tagged
recombinant CD45-AP that was conjugated to the glutathione-Sepharose 4B
beads, and the binding was prevented by adding increasing amounts of
free histidine-tagged CD45-AP as a competitor for binding. An excess
amount of control histidine-tag protein did not compete for the
binding. The results demonstrate that both Lck and ZAP-70 recombinant
proteins bind specifically to recombinant CD45-AP and that the
interactions do not require other lymphocyte-specific proteins.
Lck and ZAP-70 Coimmunoprecipitate with CD45 after TCR-mediated
Stimulation--
Direct associations have been observed between CD45
and Lck (31), between CD45 and TCR
To determine the amounts of Lck and ZAP-70 that associate with CD45,
CD45-positive YAC-1 cells were stimulated for 60 min by TCR
cross-linking with anti-CD3 A Subpopulation of CD45-AP Is Not Bound to CD45--
In a previous
study, two-dimensional diagonal SDS-PAGE analysis of anti-CD45
immunoprecipitates obtained from YAC-1 cell lysates treated with a
chemical cross-linker revealed that approximately 70% of CD45 exists
as a complex with CD45-AP with a stoichiometry of 1 to 1 (18). To
determine whether a population of free CD45-AP exists in CD45-positive
cells, CD45 was depleted from lysates of CD45-positive YAC-1 cells by
repeated immunoprecipitation with anti-CD45 antibody-conjugated beads,
and the amount of CD45-AP in the lysates was determined (Fig.
5). After the exhaustive
immunoprecipitation, CD45 was barely detectable in lysates by Western
blotting as expected. On the other hand, approximately 24% of total
CD45-AP was estimated by densitometric analysis of the immunoblot to be
present in the lysate after CD45 depletion.
A similar estimate of the free form of CD45-AP was obtained by sucrose
gradient ultracentrifugation as well (Fig.
6). YAC-1 cell lysate was fractionated by
the ultracentrifugation, and each fraction was analyzed by Western
blotting with anti-CD45 and CD45-AP-antibodies. A majority of CD45-AP
cosedimented with CD45 as previously reported (18). However, a
subpopulation of CD45-AP sedimented at a much slower rate with a
distinct peak in fraction 18. The amount of CD45-AP present in
fractions 16-20 was estimated to be 26% of the total.
The present study demonstrates that CD45-AP specifically interacts
with Lck and ZAP-70 and that the interactions do not require CD45
(Figs. 1 and 2) or any other lymphocyte-specific proteins (Fig. 3).
Binding of Lck and ZAP-70 to exogenously added recombinant CD45-AP
decreases upon 60 min stimulation by TCR ligation (Fig. 1C),
whereas the amounts of these protein-tyrosine kinases
coimmunoprecipitating with endogenous cellular CD45-AP increase in both
CD45-positive YAC-1 cells and the CD45-negative variant N1 (Fig. 2).
The most plausible explanation for the reduced binding of recombinant
CD45-AP to Lck and ZAP-70 after TCR-mediated stimulation is that the
stimulation causes these protein-tyrosine kinases to associate with
endogenous CD45-AP, hence rendering them inaccessible to recombinant
CD45-AP. This is consistent with the fact that at a given time only a
small percentage of the total cellular protein-tyrosine kinases is able to interact with CD45-AP (Fig. 1D). The protein-tyrosine
kinase subpopulation that can interact with CD45 after stimulation is also small (Fig. 4B), and similar percentages of total Lck
and ZAP-70 have been reported in association with the TCR (8, 33). Therefore, the percentages of protein-tyrosine kinase subpopulations engaged in TCR signaling at a given time are consistent with the percentages of the protein-tyrosine kinase subpopulations capable of
associating with CD45-AP.
The absence of CD45 results in increased protein-tyrosine kinase
activity of Lck in N1 cells compared with CD45-positive YAC-1 cells
(34), probably due to the elevated level of tyrosine phosphorylation on
the positive regulatory site (17). As shown in Fig. 1D, the percentage of endogenous Lck that is capable of binding to recombinant CD45-AP is lower in N1 cells than in YAC-1 cells. However, no corresponding increase in binding to endogenous CD45-AP is observed in
N1 cells in the absence of stimulation (Fig. 2). It is therefore possible that the constitutively activated Lck in N1 cells is already
bound to various endogenous molecules and thus is less capable of
interacting with recombinant CD45-AP.
The mechanism that determines the availability of certain
protein-tyrosine kinase subpopulations for binding to CD45-AP in cells
is not known. Posttranslational modifications, interactions with other
molecules, and cellular localization may be required for
protein-tyrosine kinases to interact with CD45-AP. Likewise, it is
possible that endogenous CD45-AP acquires the ability to interact with
protein-tyrosine kinases only after certain
stimulation-dependent events that result in modification of
CD45-AP. Lck binds noncovalently to the cytoplasmic domain of the TCR
coreceptors, CD4 or CD8 (5, 7). Upon TCR ligation, these coreceptors
are thought to augment antigen-mediated signals by binding to major
histocompatibility complex molecules of target cells and bringing Lck
closer to the TCR. On the other hand, CD4 may also exert a negative
signal by sequestrating Lck from the TCR under certain circumstances
(35-37). The cell lines used in the present study, YAC-1 and N1 (27), express neither CD4 nor CD8. Therefore, it is possible that the absence
of CD4 and CD8 releases Lck from certain constraints and increases the
amount of Lck available for interacting with CD45 and CD45-AP in these
cell lines. On the other hand, it is clear that the presence of the
coreceptors does not prevent the interaction of Lck with CD45 since Lck
coimmunoprecipitates with CD45 in normal splenic T cells (24) as well
as in a CD4-positive T cell line (38). It is of interest to examine
whether transfection of CD4 to YAC-1 and N1 cells would alter the
interactions of Lck with CD45 and CD45-AP.
We have previously demonstrated that CD45-AP protein levels are much
lower in three out of four CD45-negative variant lymphoid cell lines,
including N1 cells, compared with their CD45-positive wild-type
parental lymphocytes (20). However, larger amounts of Lck and ZAP-70
coimmunoprecipitate with CD45-AP in N1 than in wild-type YAC-1 cells
after 60 min stimulation (Fig. 2). Therefore, it is possible that
CD45-AP/kinase dimers formed in N1 cells without CD45 are more
efficiently immunoprecipitated with anti-CD45-AP antibody than
CD45-AP/CD45/kinase trimers formed in wild-type cells. Alternatively,
CD45 and CD45-AP may not bind cooperatively to the protein-tyrosine
kinase to form ternary complexes, but instead the protein-tyrosine
kinase may be transferred from the CD45-AP·kinase complex to CD45 to
form a CD45/kinase complex in CD45-positive cells. In that case, fewer
CD45-AP/kinase complexes would accumulate in the presence of CD45.
Approximately 25% of total CD45-AP exists free of CD45 in
CD45-positive T cells (Figs. 5 and 6). On the other hand, our earlier
results indicate that approximately 30% of total CD45 is not bound to
CD45-AP (18). These significant subpopulations of CD45-AP and CD45 may
form binary complexes with protein-tyrosine kinases as a part of the CD45-mediated signaling process.
The C form of recombinant CD45-AP that encompasses the bulk of the
cytoplasmic domain except for the five amino acid residues adjacent to
the transmembrane segment failed to bind to Lck and ZAP-70 (Fig.
1C). Therefore, the transmembrane segment and/or the
membrane-proximal region of the cytoplasmic domain is critical for the
interactions. This region may be directly involved in the binding or,
alternatively, the cytoplasmic domain may not be able to form a correct
conformation without this region and thus fail to provide binding sites
for the protein-tyrosine kinases.
Fyn does not bind to recombinant CD45-AP but coimmunoprecipitates with
CD45 while Lck binds to recombinant CD45-AP and also coimmunoprecipitates with CD45 (Figs. 1 and 4). Thus CD45-AP appears to
be involved in the interaction between CD45 and Lck but not in the
interaction between CD45 and Fyn in YAC-1 cells. These findings are
consistent with the fact that Fyn and Lck have distinct characteristics
and roles in lymphocyte signal transduction. For example, Fyn has been
reported to be much less efficiently regulated than Lck by CD45
in vivo (39-41), and overexpression of Lck but not Fyn
leads to enhanced phosphorylation of ZAP-70 and Syk (42). Furthermore,
Fyn and Lck are required to different degrees at different stages of T
cell development (43-46). It is possible that CD45-AP plays a role in
a subset of CD45-mediated signal transduction pathways by mediating
interactions of CD45 with a selected group of potential substrates.
There is substantial evidence that Lck is a CD45 substrate in
vivo (2, 11, 13, 39-41). In addition, it has been shown that the
cytoplasmic portion of recombinant CD45 binds directly to recombinant
Lck (31). However, data from CD45-AP-null mice created by gene
targeting indicate that CD45-AP is required for an optimal CD45-Lck
interaction and antigen receptor-mediated signaling in lymphocytes
(24). T cells of CD45-AP-null mice exhibit markedly reduced
proliferative and functional responses to TCR-mediated stimuli.
Interestingly, the interaction between CD45 and Lck is significantly
diminished in these CD45-AP-null T cells. Moreover, the amount of Lck
associated with CD45 increases in wild-type cells after stimulation by
TCR ligation, whereas it remains at constantly lower levels in
CD45-AP-null T cells. Combined with the functional impairments
exhibited by CD45-AP-null T cells, the results indicate that CD45-AP is
required to coordinate the interaction of CD45 with Lck for appropriate
signaling. Further support for this notion comes from experiments in
which CD45 and Lck were coexpressed in fibroblasts (47). The failure of
CD45 to stably dephosphorylate kinase-active Lck in these experiments may well be due to the absence of CD45-AP expression in fibroblasts. It
would be interesting to determine whether cotransfection of a
CD45-AP-expressing construct would restore the ability of CD45 to
dephosphorylate Lck in this system.
It is thought that upon TCR-mediated stimulation, Src family kinases
such as Lck phosphorylate tyrosine residues of a signaling motif,
termed ITAM (immune receptor tyrosine-based activation motif), present
in the TCR
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
chain of the TCR (15).
Since the tyrosine-phosphorylated
chain recruits Syk family
protein-tyrosine kinases for TCR-mediated signal amplification (3, 4),
dephosphorylation of the
chain by CD45 will diminish the
stimulatory signal. In addition, it has been suggested that CD45
directly or indirectly inactivates ZAP-70 by dephosphorylating its key
tyrosyl residues (16). It has also been reported that CD45 can
down-regulate Lck by dephosphorylating its up-regulatory tyrosyl
residue in some cells (17). It remains unknown whether CD45 indeed
exercises both stimulatory and inhibitory effects on lymphocyte
signaling and, if so, how these opposing activities of CD45 are regulated.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
antibody (145-2C11) (28) on ice for 3 min. Following
the addition of anti-hamster IgG (12.5 µg/ml), cells were further
incubated either on ice for 60 min for negative controls or at 37 °C
for 5 or 60 min for stimulation. After a wash in cold Dulbecco's
phosphate-buffered saline, the cells were lysed at 20 × 106 cells/ml in 0.8% polyoxyethylene 10 oleyl ether (BRIJ
96) containing 50 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 1 mM MgCl2, 0.1 mM EGTA, 2.5 mM thioglycolic acid, 1 mM phenylmethylsulfonyl
fluoride, 10 mM sodium fluoride, 1 mM sodium
orthovanadate, 25 µM phenylarsine oxide, and 10 mM sodium pyrophosphate. Postnuclear supernatants of the
lysates were used for binding assays and immunoprecipitations as
described below.
-D-galactoside induction and purified by
binding to nickel-nitrilotriacetate resin (Qiagen). GST-tagged
recombinant Lck, produced by an expression construct (a mouse Lck
cDNA subcloned into pGEM-2T expression vector; provided by Dr. P. Johnson, University of British Columbia, Vancouver, Canada) (31), was
purified by binding to glutathione-Sepharose 4B beads. GST-tagged
recombinant CD45-AP has been described before (23). GST-tagged Lck
eluted from glutathione-Sepharose 4B beads was incubated with various
amounts of GST-tagged CD45-AP eluate at 37 °C for 30 min prior to
addition of histidine-tagged CD45-AP bound to nickel-nitrilotriacetate
beads and further incubation at 37 °C for 1 h for binding.
Likewise, histidine-tagged ZAP-70 eluted from nickel-nitrilotriacetate
beads was incubated with various amounts of histidine-tagged CD45-AP
eluate prior to addition of GST-tagged CD45-AP bound to
glutathione-Sepharose 4B beads and further incubation at 37 °C for
1 h for binding. Binding reactions were carried out in 0.4% BRIJ
96, 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM thioglycolic acid, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/ml ovalbumin.
GST or histidine tags without a fusion partner were isolated in the
same way as described above for fusion proteins and were used as
negative controls for competition experiments. Material bound to the
beads was analyzed by SDS-PAGE followed by Western blotting.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
antibody (28), and cell lysates were prepared at various
time points after stimulation. Whole cell lysates were analyzed by
Western blotting with anti-CD45, Fyn, Lck, ZAP-70, or Csk antibodies.
As shown in Fig. 1A, there is
no detectable CD45 in N1 cells. The absence of CD45 in N1 cells was
also confirmed by Northern hybridization (20), anti-CD45
immunoprecipitation combined with Western blotting, and flow cytometry
(data not shown). The wild-type and variant cells expressed comparable
amounts of Fyn, Lck, ZAP-70, and Csk, and the amounts remained
unchanged by stimulation. Syk is not expressed in YAC-1 cells (not
shown). Immunoblotting with anti-ZAP-70 antibody detected two bands
with different mobilities as has been reported previously by others (32). The structural difference between these two bands is not known.
However, the fast-migrating band indicated by the arrow appears to represent the subpopulation that interacts with CD45-AP and
CD45 as described below.
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Fig. 1.
Specific interaction of Lck and ZAP-70 with
recombinant CD45-AP. A, expression levels of CD45 and
protein-tyrosine kinases. Wild-type CD45-positive YAC-1 cells
(WT) and their CD45-negative variant cells (N1)
were stimulated by TCR cross-linking with anti-CD3 antibody for 0, 5, or 60 min. Cell lysates were analyzed by Western blotting with
anti-CD45, Fyn, Lck, ZAP-70, or Csk antibodies. B, the two
forms of GST-tagged recombinant CD45-AP: F (full-length;
amino acid positions 1-185 including the transmembrane segment
(TM) of amino acid positions 21-41) and C (amino
acid positions 47-185). C, binding of recombinant CD45-AP
to CD45 and protein-tyrosine kinases. The cell lysates described in
A were incubated with either the F or the C form of CD45-AP
bound to glutathione-Sepharose 4B beads. Material bound to the beads
was analyzed by Western blotting with the aforementioned antibodies.
D, percentages of Lck and ZAP-70 capable of binding to
CD45-AP. Cell lysates were prepared from unstimulated WT or N1 cells
(15 × 106) and were subjected repeatedly to fresh
aliquots of recombinant CD45-AP-conjugated beads. Material bound to
each aliquot of the beads (lanes 1-9) was analyzed by
Western blotting with either anti-Lck or anti-ZAP-70 antibody. The
total amount of Lck and ZAP-70 bound to recombinant CD45-AP was
estimated by densitometric analysis of the immunoblot. Percentages were
obtained by comparison to the total cellular amounts of Lck and ZAP-70
detected in lysates of 0.1 × 106 and 0.05 × 106 cells, respectively, determined by densitometric
analysis (shown in the left panels).
stimulation on associations between endogenous CD45-AP and
Lck or ZAP-70 in wild-type CD45-positive YAC-1 cells (WT) and their
CD45-negative variant cells (N1). Anti-CD45-AP immunoprecipitates were
prepared at various time points after stimulation, and material
coimmunoprecipitated with CD45-AP or with control beads was analyzed by
Western blotting with antibodies against Lck or ZAP-70 (Fig.
2). Lck and ZAP-70 coimmunoprecipitated
with CD45-AP after stimulation both in WT and N1 cells. In agreement
with the results of recombinant CD45-AP binding analysis (Fig.
1C), association of Lck or ZAP-70 with endogenous CD45-AP
did not require the presence of CD45 since it occurred in CD45-negative
variant cells as well. Larger amounts of Lck and ZAP-70
coimmunoprecipitated with CD45-AP in N1 than in WT cells despite the
greatly diminished presence of CD45-AP in N1 compared with WT cells
(20).
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Fig. 2.
Coimmunoprecipitation of Lck and ZAP-70 with
endogenous CD45-AP. Equal numbers of wild-type CD45-positive YAC-1
cells (WT) and their CD45-negative variant cells
(N1) were stimulated by TCR cross-linking with anti-CD3 antibody, and immunoprecipitates with anti-CD45-AP antibody were
prepared at various time points after stimulation. Material
coimmunoprecipitating with CD45-AP was analyzed by Western blotting
with antibodies against Lck or ZAP-70.
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Fig. 3.
Specific binding of CD45-AP to Lck and ZAP-70
using the respective recombinant proteins. A,
histidine-tagged recombinant CD45-AP bound to nickel-nitrilotriacetate
beads was incubated with a constant amount of GST-tagged recombinant
Lck protein in the presence or absence of increasing amounts of
GST-tagged recombinant CD45-AP or an excess of control GST protein. The
protein amount of control GST used corresponds approximately to the
highest amount of GST-tagged CD45-AP. Lck bound to the beads was
analyzed by Western blotting with anti-Lck antibody. B,
GST-tagged recombinant CD45-AP bound to glutathione-Sepharose 4B beads
was incubated with a constant amount of histidine-tagged recombinant
ZAP-70 protein in the presence or absence of increasing amounts of
histidine-tagged recombinant CD45-AP or an excess of histidine tag
control protein. The amount of control protein used corresponds
approximately to the highest amount of the histidine-tagged CD45-AP.
ZAP-70 bound to the beads was analyzed by Western blotting with
anti-ZAP-70 antibody.
chain (15), between TCR
chain and ZAP-70 (3, 4), and between Lck and ZAP-70 (32). To determine
the effect of stimulation on these associations, wild-type CD45-positive YAC-1 cells were stimulated by TCR cross-linking with
anti-CD3
antibody, and anti-CD45 immunoprecipitates were prepared
at various time points after stimulation. Material coimmunoprecipitated with CD45 was then analyzed by Western blotting with antibodies against
CD45, CD45-AP, Fyn, Lck, or ZAP-70 (Fig.
4). In agreement with the results
obtained by recombinant CD45-AP binding analysis (Fig. 1C),
the amount of CD45-AP coimmunoprecipitating with CD45 did not change
after stimulation. Fyn and Lck coimmunoprecipitated with CD45 after 60 min stimulation, whereas a small amount of ZAP-70 coimmunoprecipitated
with CD45 without stimulation and the amount increased after a short
period of stimulation. These results indicate that TCR-associated
protein-tyrosine kinases bind to CD45 in response to TCR-mediated
stimulation, whereas the interaction between CD45-AP and CD45 remains
unchanged.
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Fig. 4.
Coimmunoprecipitation of Lck and ZAP-70 with
cellular CD45. A, wild-type CD45-positive YAC-1 cells
were stimulated by TCR cross-linking with anti-CD3 antibody, and
immunoprecipitates with anti-CD45 antibody were prepared at various
time points after stimulation. Material coimmunoprecipitating with CD45
was analyzed by Western blotting with antibodies against CD45, CD45-AP,
Fyn, Lck, or ZAP-70. B, YAC-1 cells were stimulated for 60 min by TCR cross-linking with anti-CD3
antibody, and the lysate
derived from 20 × 106 cells was subjected to repeated
immunoprecipitation with anti-CD45-conjugated beads. The
immunoprecipitates (lanes 1-8) were then analyzed by
Western blotting with either anti-Lck or anti-ZAP-70 antibody. The
amounts of protein-tyrosine kinases associated with CD45 were estimated
by densitometric analysis of the immunoblot. The total cellular amounts
of Lck and ZAP-70 detected in lysates obtained from 0.25 × 106 and 0.1 × 106 cells, respectively,
served as references for obtaining percentages by the densitometric
analysis (shown in the left panels).
antibody, and a lysate derived from
20 × 106 cells was subjected to repeated
immunoprecipitation with anti-CD45-conjugated beads. The
immunoprecipitates were then analyzed by Western blotting with either
anti-Lck or anti-ZAP-70 antibody, and the amounts of the
protein-tyrosine kinases associated with CD45 were estimated as a
percentage of the total cellular amount present in lysates before
immunoprecipitation. As shown in Fig. 4B, approximately 4.2% of total Lck and 2.5% of total ZAP-70 coimmunoprecipitated with CD45.
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Fig. 5.
The presence of CD45-AP in CD45-depleted cell
lysate. CD45 was depleted from a lysate of CD45-positive YAC-1
cells by repeated immunoprecipitation with anti-CD45
antibody-conjugated beads. The cell lysates before and after the
exhaustive immunoprecipitation were analyzed by Western blotting with
antibodies against CD45 and CD45-AP. An estimate of CD45-AP present
after CD45 depletion was obtained by densitometric analysis of the
immunoblot.
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Fig. 6.
A subpopulation of CD45-AP that does not
cosediment with CD45 in sucrose gradient ultracentrifugation.
CD45-positive YAC-1 cell lysate was fractionated by sucrose gradient
ultracentrifugation in 2.5-12.5% sucrose with a cushion of 40%
sucrose. After centrifugation, the sucrose gradients were fractionated
into 20 equal fractions starting from the bottom of the tube, and each
fraction was analyzed by Western blotting with anti-CD45 and CD45-AP
antibodies. The amount of CD45-AP that did not cosediment with CD45
(fractions 16-20) was estimated by densitometric analysis
of the immunoblot.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
chain and CD3 peptides (48). The tyrosine-phosphorylated
chain recruits ZAP-70 by binding through the SH2 domains (3, 4).
The TCR-associated ZAP-70 then becomes tyrosine-phosphorylated and
activated by Lck leading to phosphorylation of other cellular
substrates and amplification of the signal (49, 50). Thus, Lck and
ZAP-70 are thought to work in synergy to transduce TCR-mediated
stimulatory signals. A direct interaction of Lck with ZAP-70 has been
reported (32). However, the interaction of ZAP-70 with CD45-AP probably
does not proceed via Lck since recombinant ZAP-70 protein directly
binds to recombinant CD45-AP (Fig. 3). The specific interaction of
ZAP-70 with CD45-AP and the coimmunoprecipitation of ZAP-70 with CD45
(Fig. 4) demonstrated in the present study support the possibility that
ZAP-70 is also a CD45 substrate. Phosphorylation on Tyr-492 and -493 of
ZAP-70 leads to down- and up-regulation of its protein-tyrosine kinase activity, respectively, whereas phosphorylation of Tyr-292, the major
autophosphorylation site, is thought to down-regulate ZAP-70 function
without affecting the kinase activity (51, 52). It is possible that the
CD45 protein-tyrosine phosphatase activates ZAP-70 by dephosphorylating
one or both of its down-regulatory tyrosyl residues. As described
above, responses to TCR-mediated stimuli are diminished in
CD45-AP-knock-out mice. These data suggest that CD45-AP coordinates the
interaction between ZAP-70 and CD45 during TCR signal transduction.
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ACKNOWLEDGEMENTS |
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We thank J. Stoeckler and N. Yaseen for useful comments on the paper, and J. Ashwell, P. Johnson, J. Marth, I. Trowbridge, and A. Weiss for generously providing reagents.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM 48188 (to A. T.) and the Department of Pathology Research and Development Fund (to A. L. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: First Dept. of Internal Medicine, Sapporo Medical
University School of Medicine, Sapporo, 060 Japan.
§ Present address: Dept. of Orthopedics, Goryokaku Hospital, Goryokaku-cho 38-3, Hakodate, 040 Japan.
¶ To whom correspondence should be addressed: Dept. of Pathology, Roger Williams Medical Center-Boston University, 825 Chalkstone Ave., Providence, RI 02908. Tel.: 401-456-6557; Fax: 401-456-6569; E-mail: Akiko_Takeda{at}brown.edu.
The abbreviations used are: TCR, T cell receptor; BRIJ 96, polyoxyethylene 10 oleyl ether; CD45-AP, CD45-associated protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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