Interactions of CD45-associated Protein with the Antigen
Receptor Signaling Machinery in T-lymphocytes*
André
Veilletteabcdef,
David
Soussouabg,
Sylvain
Latourah,
Dominique
Davidsona, and
François G.
Gervaisabi
From the a McGill Cancer Centre and the Departments of
b Biochemistry, c Medicine, and
d Oncology, McGill University, Montréal,
Québec H3G 1Y6, and the e Departments of
Medicine and Oncology, Montreal General Hospital, Montréal,
Québec H3G 1A4, Canada
 |
ABSTRACT |
CD45 is a transmembrane protein tyrosine
phosphatase playing an essential role during T-cell activation. This
function relates to the ability of CD45 to regulate
p56lck, a cytoplasmic protein tyrosine kinase
necessary for T-cell antigen receptor (TCR) signaling. Previous studies
have demonstrated that CD45 is constitutively associated in
T-lymphocytes with a transmembrane molecule termed CD45-AP (or
lymphocyte phosphatase-associated phosphoprotein). Even though the
exact role of this polypeptide is unclear, recent analyses of mice
lacking CD45-AP have indicated that its expression is also required for
optimal T-cell activation. Herein, we wished to understand better the
function of CD45-AP. The results of our studies showed that in T-cells,
CD45-AP is part of a multimolecular complex that includes not only
CD45, but also TCR, the CD4 and CD8 coreceptors, and
p56lck. The association of CD45-AP with TCR,
CD4, and CD8 seemed to occur via the shared ability of these molecules
to bind CD45. However, binding of CD45-AP to
p56lck could take place in the absence of other
lymphoid-specific components, suggesting that it can be direct.
Structure-function analyses demonstrated that such an interaction was
mediated by an acidic segment in the cytoplasmic region of CD45-AP and
by the kinase domain of p56lck. Interestingly,
the ability of CD45-AP to interact with Lck in the absence of other
lymphoid-specific molecules was proportional to the degree of catalytic
activation of p56lck. Together, these findings
suggest that CD45-AP is an adaptor molecule involved in orchestrating
interactions among components of the antigen receptor signaling
machinery. Moreover, they raise the possibility that one of the
functions of CD45-AP is to recognize activated Lck molecules and bring
them into the vicinity of CD45.
 |
INTRODUCTION |
Activation of T-lymphocytes by antigen is initiated by protein
tyrosine phosphorylation (1-4). Although the T-cell antigen receptor
(TCR)1 and the associated CD3
and
subunits are devoid of intrinsic protein tyrosine kinase
activity, they can recruit two classes of cytoplasmic protein tyrosine
kinases to mediate this response, the Src family and the Syk/Zap-70
family. The Src-related enzymes Lck and FynT initiate TCR-mediated
signals by phosphorylating a signaling motif in the cytoplasmic
domain of CD3 and
termed ITAM (for immunoreceptor tyrosine-based
activation motif). Following this phosphorylation, the Syk/Zap-70
family kinases are activated through binding of their tandem Src
homology 2 (SH2) domains to doubly phosphorylated ITAMs. Together with
Src family kinases, Zap-70 and Syk are responsible for subsequent
tyrosine phosphorylation of several signal transduction molecules
including phospholipase C-
1, Cbl, Vav, Slp-76, and Lat.
CD45 is a 180-220-kDa transmembrane protein tyrosine phosphatase
expressed on all nucleated hemopoietic cells (5, 6). In T-cells it
constitutes ~10% of all cell surface glycoproteins. Previous studies
have shown that CD45 is necessary for T-cell activation because of its
ability to promote constitutive dephosphorylation of the inhibitory
carboxyl-terminal tyrosine of p56lck, tyrosine
505 (7-9). This dephosphorylation is necessary for Lck to become
activated and phosphorylate the TCR complex upon antigen stimulation.
There is also an indication that CD45 is able to dephosphorylate other
substrates, including the positive regulatory site of Lck (tyrosine
394) and the
subunit of TCR (10-12). Although the precise
mechanism by which CD45 specifically targets these substrates is not
established, it is noteworthy that small amounts of
p56lck, but not FynT, can be found to
coimmunoprecipitate with CD45 in mild detergent lysates of T-cells
(13-15). Furthermore, CD45 has been shown to colocalize on the cell
with several other molecules, including the TCR complex, CD4, CD2, and
Thy-1 (16-21). Thus, it is possible that the specificity of CD45
toward its substrates is dictated in part by its relative distribution
on the cell surface.
Previous studies have demonstrated that the majority of CD45 molecules
in T-cells is associated with a 30-kDa polypeptide termed
CD45-associated protein (CD45-AP) or lymphocyte phosphatase-associated phosphoprotein (hereafter named CD45-AP) (22-24). Through molecular cloning, CD45-AP was shown to be a novel transmembrane protein with a
short extracellular domain of ~9 amino acids, a single transmembrane
segment, and a cytoplasmic region of ~145 residues (see Fig.
8A) (23, 24). Although CD45-AP has no clear homology with
any other known polypeptide, one group pointed out that it may possess
a WW domain in its intracytoplasmic region (25). CD45-AP is expressed
on T-cells, B-cells, and some myeloid cells and is absent in
non-hematological cell types (23, 26, 27). Structure-function analyses
and experiments with recombinant proteins have revealed that the
interaction between CD45 and CD45-AP is direct and that it occurs via
their respective transmembrane segments (25, 28, 29). CD45-AP was also
reported to associate directly with Lck in transfected HeLa cells (25);
however, the affinity of this association was apparently low because it
could be detected only through in vitro kinase reactions and
not by Western blotting.
The importance of CD45-AP in normal cellular physiology was addressed
recently by the creation of CD45-AP-deficient mice through homologous
recombination in embryonic stem cells (30). Although T-cell development
was not noticeably affected in these mice, thymocytes and splenic
T-cells exhibited a decrease in proliferation in response to TCR
stimulation. CD45-AP-deficient T-cells were also less efficient at
mediating effector functions such as cytolysis. Although a precise
biochemical explanation for these defects was not established, it is
noteworthy that CD45-AP-deficient T-cells also demonstrated a reduction
in CD45 expression and in the amount of p56lck
that can be coimmunoprecipitated with CD45.
In this paper, we wish to explore further the role of CD45-AP in
T-lymphocytes. The results of our experiments showed that CD45-AP is
part of a complex that contains not only CD45 but also the antigen
receptor, the CD4 and CD8 coreceptors, and
p56lck. They also demonstrated clearly that an
interaction between CD45-AP and Lck can occur in the absence of other
lymphoid-specific molecules and that this association is proportional
to the degree of enzymatic activation of
p56lck.
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MATERIALS AND METHODS |
Cells--
Thymocytes were extracted from 5-6-week-old Balb/c
mice. BI-141 is an antigen-specific mouse T-cell line (31). It
expresses TCR, CD3, and CD45 but lacks CD4 and CD8. Because BI-141
contains very low amounts of endogenous p56lck,
a variant transfected with a wild-type lck cDNA was used
for some of the experiments (32). Monoclonal cell lines expressing either Tac-
or Tac-
were generated by
transfection.2 YAC-1 and the
CD45-negative variant YAC-N1 were described previously (33) and were
kindly provided by Dr. Jonathan Ashwell, National Institutes of Health.
LSTRA is a lymphoid cell line expressing CD45 but lacking TCR, CD4, and
CD8. It expresses markedly elevated levels of Lck as a result of
retroviral promoter insertion (34, 35). All lymphoid cells were
propagated in RPMI 1640 medium supplemented with 10% fetal bovine
serum, glutamine, and antibiotics. Whenever appropriate, the
aminoglycoside G418 (0.6 mg/ml) was also added to the growth medium.
COS-1 cells were grown as detailed elsewhere (36).
cDNAs and Transfections--
A cDNA encoding mouse
CD45-AP was cloned by polymerase chain reaction from mouse thymus RNA.
Variants in which the sequences coding for the carboxyl-terminal region
of CD45-AP were truncated were also generated by polymerase chain
reaction. In the process, a Myc-derived epitope was inserted at their
carboxyl terminus. cDNAs coding for wild-type,
U,
SH3,
SH2, Y394F, Y505F, G2A/Y505F,
U/Y505F, K273R/Y505F, and
Y394F/Y505F Lck were reported previously (32, 37-41). An
lck cDNA lacking the sequences encoding the kinase domain (
K lck) was produced by digesting the wild-type
cDNA with NcoI, blunting the resulting DNA fragment, and
ligating with a linker containing a stop codon. This deletion removed
the kinase and negative regulatory domains of
p56lck. All constructs were verified by
sequencing to ensure that no unwanted mutation had been introduced
(data not shown). For transfection in COS-1 cells, the various
cDNAs were cloned in the multiple cloning site of pXM139, which
contains the SV40 origin of replication. Transfections in COS-1 cells
were performed as described elsewhere (36), using the DEAE-dextran
method and a fixed total amount of DNA (8 µg).
Antibodies--
A polyclonal antiserum directed against CD45-AP
was produced in rabbits using a fusion protein encompassing the
cytoplasmic domain of CD45-AP as an immunogen. Rabbit antisera directed
against Lck, Fyn, Csk, Chk, Zap-70, Syk, Cbl, Vav, and Shc were
described previously (36, 42-48). Monoclonal antibodies (MAbs)
reacting with CD45 (M1.89.18.7), TCR (F23.1 and H57), CD3 (145-2C11
for immunoprecipitation; HMT-3 for immunoblotting),
(H146), Thy-1 (G7), CD4 (GK1.5), CD8 (2.43), Tac (7G7), and Myc (9E10) were reported elsewhere.
Immunoprecipitations and Immunoblots--
After washing in
phosphate-buffered saline, cells were lysed in Brij-97-containing
buffer (50 mM Tris, pH 8.0, 1% Brij-97, 2 mM
EDTA) supplemented with 10 µg/ml each of the protease inhibitors leupeptin, aprotinin, N-tosyl-L-phenylalanine
chloromethyl ketone, N-p-tosyl-L-lysine chloromethyl
ketone, and phenylmethylsulfonyl fluoride, as well as the phosphatase
inhibitors sodium fluoride (50 mM) and sodium orthovanadate
(1 mM). For COS-1 cells, cells were lysed in TNE buffer (50 mM Tris, pH 8.0, 1% Nonidet P-40, 2 mM EDTA),
supplemented with the same inhibitors. Polypeptides were recovered by
immunoprecipitation from equivalent amounts of total cellular proteins
using the indicated antibodies, precoupled to protein A or protein
G-Sepharose (Amersham Pharmacia Biotech). In some cases, immune
complexes were collected with Staphylococcus aureus protein
A (Pansorbin; Calbiochem), coupled, if indicated, to rabbit anti-mouse
immunoglobulin (Ig) G. Immunoprecipitates were washed three times with
lysis buffer containing 1 mM sodium orthovanadate. Proteins
were then eluted in sample buffer, boiled, electrophoresed in 8%
(Lck), 10% (CD45-AP and
) or 12% (
) SDS-polyacrylamide gels,
and transferred onto Immobilon membranes (Millipore) for immunoblotting. Immunoblots were performed according to a protocol described previously (42). After incubation with
125I-protein A (Amersham Pharmacia Biotech) or
125I-goat anti-mouse IgG (ICN), immunoreactive products
were detected by autoradiography and quantitated with a PhosphorImager
(BAS 2000; Fuji).
 |
RESULTS |
Coimmunoprecipitation of CD45-AP with the Antigen Receptor Complex
in T-lymphocytes--
To gain more understanding of the function of
CD45-AP in T-lymphocytes, its ability to interact with molecules other
than CD45 was evaluated. Normal ex vivo mouse thymocytes
were lysed in Brij-97-containing buffer, and various polypeptides were
immunoprecipitated from postnuclear cell lysates using the indicated
antibodies. After separation in 10% SDS-polyacrylamide gels, the
presence of CD45-AP in these immunoprecipitates was determined by
immunoblotting with a rabbit antiserum directed against the cytoplasmic
domain of CD45-AP (Fig. 1A).
As reported earlier (23, 24), large amounts of CD45-AP were found to
coimmunoprecipitate with CD45 (lane 3).Unexpectedly, though,
we observed that appreciable quantities of CD45-AP were also present in
immunoprecipitates of Lck (lanes 4 and 5), CD4
(lane 8), CD8 (lane 9), and TCR-associated CD3
(lane 10). In contrast, no CD45-AP was detected in FynT
immunoprecipitates (lanes 6 and 7) or in
immunoprecipitates obtained with normal rabbit serum (lane
11). It is notable that CD45-AP migrated as three distinct species
in these gels, in agreement with earlier reports (23). These variations
in the apparent molecular mass of CD45-AP are apparently caused by
serine phosphorylation (23).

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Fig. 1.
Association of CD45-AP with signaling
molecules in mouse thymocytes. Mouse thymocytes were lysed in
Brij-97-containing buffer, and the presence of CD45-AP in various
immunoprecipitates was assessed by immunoblotting with anti-CD45-AP
antibodies. For panel A, 100 µg of cellular proteins was
used for immunoprecipitation in lanes 2 and 3;
400 µg was used in the other lanes. 100 µg of cellular
lysates was resolved in lane 1. For panel B, 500 µg of cellular lysates was utilized in all lanes. The
positions of prestained molecular mass markers are shown on the
right; those of the heavy chain of immunoglobulin
(Ig(H)) and CD45-AP are indicated on the left.
NRS, normal rabbit serum. Exposures: panel
A, 9 h; panel B, 4 h.
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To address the specificity of these associations, the ability to
CD45-AP to coimmunoprecipitate with a series of other signal transduction molecules expressed in thymocytes was assessed (Fig. 1B). Contrary to p56lck (lane
1) and the TCR-associated
chain (lane 10), no
CD45-AP was associated with the protein tyrosine kinases Csk
(lane 3) and Chk (lane 4), the adaptor molecules
Cbl (lane 7) and Shc (lane 9), and the guanine
nucleotide exchange factor Vav (lane 8). However, a small
quantity was detected in Zap-70 (lane 5) and Syk (lane 6) immunoprecipitates. Because Syk/Zap-70 family kinases are known to be constitutively associated with TCR in thymocytes (49), it is
plausible that these interactions were mediated via the TCR.
We also wanted to test whether components of the TCR complex could be
found in immunoprecipitates of CD45-AP (Fig.
2). Therefore, thymocyte lysates were
immunoprecipitated with antibodies directed against either CD45-AP or
CD45, and the presence of the
subunit of TCR in these
immunoprecipitates was revealed by immunoblotting with anti-
MAb
H146. This experiment demonstrated that significant amounts of
were
immunoprecipitated both with CD45-AP (lane 1) and with CD45
(lane 2) but not when normal rabbit serum (lane 4) or an irrelevant MAb antibody (anti-Tac MAb 7G7; data not
shown) was used as immunoprecipitating reagent. As expected, large
quantities of
were also detectable in anti-CD3 immunoprecipitates
(lane 3). In combination, these data revealed that CD45-AP
could be coimmunoprecipitated not only with CD45 but also with several elements of the antigen receptor complex in thymocytes, including TCR,
CD3
, CD4, and CD8, as well as with
p56lck.

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Fig. 2.
Association of the chain of TCR with CD45 and CD45-AP in mouse thymocytes.
Procedures were as in Fig. 1 except that immunoprecipitates were
immunoblotted with anti- MAb H146. 500 µg of protein was used in
each immunoprecipitation. The migrations of prestained molecular weight
markers are indicated on the right; those of the heavy chain
of Ig (Ig(H)) and are shown on the left.
NRS, normal rabbit serum. Exposure was for 24 h.
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The Interaction between CD45-AP and the TCR Complex Requires the
Presence of CD45--
To understand the mechanism(s) of these
interactions, we first investigated whether they required the presence
of CD4 and CD8 by performing similar analyses with the CD4-negative,
CD8-negative mouse T-cell line BI-141 (Fig.
3). As was the case in thymocytes (Fig.
1), significant amounts of CD45-AP were present in immunoprecipitates of CD45 (Fig. 3, lane 2), CD3 (lane 6), TCR
(lanes 7 and 8), and
(lane 9) in
BI-141 cells. Smaller quantities were also detected in anti-Lck
immunoprecipitates (lane 3). However, there was no CD45-AP
associated with FynT (lane 4) or the
glycosylphosphatidylinositol-linked molecule Thy-1 (lane
10), even though both molecules are expressed abundantly in BI-141
cells (data not shown). Moreover, no CD45-AP was recovered when an
antibody against CD4 (lane 5) or normal rabbit serum
(lane 11) was used for immunoprecipitation. Thus, the
interaction of CD45-AP with TCR (and Lck) could take place in the
absence of CD4 and CD8.

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Fig. 3.
Association of CD45-AP with signaling
molecules in BI-141 T-cells. The antigen-specific mouse T-cell
line BI-141 was lysed in Brij-97-containing buffer, and various
immunoprecipitates were probed by immunoblotting with anti-CD45-AP
antibodies. For immunoprecipitation, 100 µg of cellular proteins was
used in lanes 1 and 2; 500 µg was used in the
other lanes. The positions of prestained molecular mass
markers are shown on the right; those of the heavy chain of
immunoglobulin (Ig(H)) and CD45-AP are indicated on the
left. NRS, normal rabbit serum. Exposure was for
7 h.
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Next, we wished to determine whether the CD45-AP-TCR interaction was
direct. Because CD45-AP contains only a few extracellular residues and
its transmembrane region is already involved in binding to CD45, we
were especially interested in testing the possibility that its
intracytoplasmic region may bind the intracellular sequences of CD3
and/or
. To this end, subclones of BI-141 bearing chimeras in which
the cytoplasmic sequences of either
or the
subunit of CD3 were
fused to the extracellular and transmembrane regions of Tac were
studied (Fig. 4). As in control BI-141
cells (Fig. 4A, lanes 2 and 3),
substantial amounts of CD45-AP were immunoprecipitated with CD45
(lanes 7 and 12) and CD3 (lanes 8 and
13) in these derivatives. In contrast, though, none was
associated with Tac-
(lane 9) or Tac-
(lane
14), despite the fact that both chimeras were expressed at the
cell surface (data not shown) and were present in amounts comparable or
superior to those of the endogenous TCR-associated chains (Fig.
4B; data not shown). Similar results were obtained when
individual chains of CD3
were transfected with CD45-AP in COS-1
cells (data not shown). Hence, these results implied that the
interaction between CD45-AP and TCR was not mediated by the cytoplasmic
domain of either
or
. Unfortunately, similar experiments could
not be performed with chimeras encompassing the cytoplasmic domain of
the
or
subunit of CD3 because fusions were internalized rapidly
and not expressed stably at the cell surface (data not shown).

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Fig. 4.
Association of CD45-AP with
CD3 and chimeras in
BI-141 T-cells. The ability of Tac- and Tac- to associate
with CD45-AP in BI-141 T-cells was ascertained as described for Fig. 1.
60 µg of cellular protein was used for immunoprecipitation of CD45
and CD45-AP; 300 µg was used for all other immunoprecipitations.
Panel A, anti-CD45-AP immunoblot. The positions of molecular
mass markers are indicated on the right; those of the heavy
chain of immunoglobulin (Ig(H)) and CD45-AP are shown on the
left. Exposure was for 8.5 h. Panel B,
anti- immunoblot. The presence of CD3 was detected by
immunoblotting with an antibody directed against the cytoplasmic domain
of (MAb HMT-3). The migrations of prestained molecular mass markers
are indicated on the right; those of Tac- and are
shown on the left. Exposure was for 16 h.
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Studies of splicing isoforms of CD45 have indicated that CD45 and the
TCR complex can interact by way of their extracellular domains (20,
50). On this basis, the possibility that the CD45-AP-TCR interaction
was mediated by CD45 was ascertained by studying T-cells lacking CD45
expression (Fig. 5). CD45-positive YAC-1
T-cells, as well as a variant lacking CD45 (YAC-N1), were lysed and
processed for immunoprecipitation as outlined above. In keeping with
our earlier observations (Figs. 1 and 3), CD45-AP was present in
immunoprecipitates of CD3 and
from CD45-positive YAC-1 cells (Fig.
5, lanes 3 and 4); however, it was not observed when similar immunoprecipitates were obtained from the CD45-negative variant (lanes 8 and 9). This difference was not
the result of variations in TCR expression because both cell lines
expressed comparable levels of TCR at the cell surface (data not
shown). However, it is noteworthy that the abundance of CD45-AP in
YAC-N1 (lane 6) was reduced ~5-fold compared with that of
its CD45-positive counterpart (lane 1). A similar decrease
in CD45-AP expression has been reported in other CD45-negative T-cells
(23, 27, 51). Therefore, it is conceivable that the ability to detect CD45-AP·TCR complexes in YAC-N1 was hampered by the lowered abundance of CD45-AP. Nonetheless, because the amount of CD45-AP remaining in
these cells was still significant (~20% of control), we believe that
this result is consistent with the idea that CD45 mediates the
CD45-AP-TCR interaction. Similar findings were made using thymocytes
from CD45-deficient mice (data not shown).

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Fig. 5.
Role of CD45 in the CD45-AP-TCR
interaction. The ability of CD45-AP to coimmunoprecipitate with
TCR in CD45-positive and CD45-negative variants of YAC-1 T-cells was
examined as outlined in the legend of Fig. 1. 1 mg of protein was used
in each immunoprecipitation. The migrations of prestained molecular
weight markers are indicated on the right; those of the
heavy chain of Ig (Ig(H)) and CD45-AP are shown on the
left. NRS, normal rabbit serum. Exposure was for
21 h.
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Association of CD45-AP with Lck Can Occur Independently of CD45,
CD4, CD8, and TCR--
Previously it was reported that small
quantities of Lck could be observed in immunoprecipitates of the CD45
phosphatase obtained from mild detergent lysates of T-cells (13, 14).
Although the basis for this association was not determined, it was
shown to happen in the absence of CD4 and CD8 expression (15). Further experiments with recombinant proteins suggested that this interaction was possibly direct, as a result of an association between the cytoplasmic portion of CD45 and the unique and SH2 domains of p56lck (52). Nevertheless, recent analyses of
CD45-AP-deficient T-cells suggested that the association between CD45
and Lck may be facilitated by CD45-AP, especially in TCR-stimulated
cells (30). Further credence for this view was provided by the
observation that CD45-AP and Lck can associate weakly in HeLa cells, in
the absence of CD45 (25).
To examine further the idea that CD45-AP may interact directly with
p56lck, we first studied their capacity to be
coimmunoprecipitated in LSTRA, a lymphoid cell line lacking not only
CD4 and CD8, but also TCR, while expressing markedly elevated levels of
Lck (Fig. 6A) (34, 35). This
analysis showed that significant quantities of CD45-AP existed in
anti-p56lck immunoprecipitates (lane
3) in LSTRA cells. As expected, CD45-AP was also associated with
CD45 (lane 2) in these cells, but it was absent in
immunoprecipitates obtained with preimmune serum (lane 4).
Therefore, CD45-AP and Lck could interact in lymphoid cells in the
absence of TCR, CD4, and CD8 (this figure and Fig. 3).

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Fig. 6.
Association of CD45-AP with Lck can occur
independently of other lymphoid-specific components. Panel
A, association of CD45-AP with p56lck in
LSTRA cells. 100 µg of cellular proteins was used for
immunoprecipitation in lanes 1 and 2; 500 µg
was used in the other lanes. The positions of prestained
molecular mass markers are indicated on the right; those of
the immunoglobulin heavy chain (Ig(H)) and CD45-AP are shown
on the left. Exposure was for 7 h. Panel B,
association of CD45-AP with p56lck in COS-1
cells. COS-1 cells were transiently transfected with the indicated
cDNAs. After lysis in Nonidet P-40-containing buffer, the ability
of CD45-AP to associate with Lck was determined as indicated under
"Materials and Methods." 250 µg of cellular proteins was used in
each immunoprecipitation. The migrations of prestained molecular weight
markers are shown on the right; those of CD45-AP and Lck are
indicated on the left. Exposures were for 16 h.
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Second, the capacity of CD45-AP to associate with Lck was characterized
in COS-1 cells, a non-lymphoid cell line lacking CD4, CD8, TCR, and
CD45 (Fig. 6B). Cells were transiently transfected by the
DEAE-dextran method with a mouse cd45-ap cDNA, in the
absence or presence of a wild-type mouse lck cDNA. After
60 h, cells were lysed in Nonidet P-40-containing buffer, and the
capacity of CD45-AP and p56lck to interact was
evaluated by immunoblotting Lck immunoprecipitates with anti-CD45-AP
antibodies (Fig. 6B, top panel). Although no CD45-AP was present in Lck immunoprecipitates obtained from cells lacking Lck (lane 2), detectable amounts were present in
those generated from Lck-containing cells (lane 4).
Likewise, appreciable quantities of p56lck were
noted in CD45-AP immunoprecipitates produced from Lck-expressing cells
(second panel from top, lane 4),
whereas little or none was found in cells devoid of CD45-AP (lane
3). These differences were not caused by inadequate expression of
CD45-AP or Lck, as documented by parallel immunoblots of total cell
lysates with anti-CD45-AP (third panel from top)
or anti-Lck (bottom panel) sera. In agreement with the
findings of Cahir McFarland et al. (25), these results
indicated that the association between CD45-AP and Lck could occur in
the absence of any other lymphoid-specific components.
Structural Requirements for CD45-AP·Lck Complex Formation in
Non-lymphoid Cells--
To explore the CD45-AP-Lck interaction in the
absence of other lymphoid-specific molecules, its structural
requirements were identified using the COS-1 cell system. The roles of
the various domains of p56lck were initially
examined, using a series of previously described Lck mutants (37, 38,
41, 53). Cells were transfected with cDNAs encoding these mutants
in the absence or presence of wild-type CD45-AP. After lysis, the
association between the two molecules was revealed by immunoblotting of
CD45-AP immunoprecipitates with an anti-Lck serum (Fig.
7B, top panel).
This assay demonstrated that, compared with wild-type Lck (lane
9), Lck polypeptides carrying a deletion of the SH3 domain (
SH3
Lck; lane 11) or SH2 region (
SH2 Lck; lane
12), or a mutation of the inhibitory carboxyl-terminal tyrosine,
tyrosine 505 (Y505F Lck; lane 14) exhibited a ~5-6-fold greater efficiency at binding CD45-AP. Interestingly, these three mutations have all been shown to cause constitutive activation of
p56lck (41, 53-55). In contrast, variants
lacking the unique domain (
U Lck; lane 10) or carrying a
mutation of the site of positive regulation, tyrosine 394 (Y394F;
lane 13) had binding capacities similar to that of wild-type
p56lck (lane 9). No significant
immunoprecipitation of any of these polypeptides occurred in the
absence of CD45-AP (lanes 3-8).

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Fig. 7.
Structure-function analyses of
p56lck. Panel A, structure
of Lck. The positions of the unique SH3, SH2, and kinase domains, as
well as the sites of myristoylation (glycine 2; G2), ATP
binding (lysine 273; K273), autophosphorylation (tyrosine
394; Y394), and negative regulation (tyrosine 505;
Y505) are indicated. Panel B, effects of single
mutations on the ability of Lck to associate with CD45-AP. COS-1 cells
were transfected with the indicated cDNAs. After lysis in Nonidet
P-40-containing buffer, the ability of wild-type CD45-AP to associate
with various Lck mutants was studied. 300 µg of cellular protein was
used for immunoprecipitation. The positions of Lck and CD45-AP are
indicated on the left. Exposures: top, 5 h;
middle, 5 h; bottom, 16 h. Panel
C, effects of supplementary mutations on the ability of Y505F Lck
to associate with CD45-AP. The procedures were as in panel B
except that all cells were transfected with the cd45-ap
cDNA. For immunoprecipitation, 250 µg of cellular protein was
used. The migrations of Lck and CD45-AP are shown on the
left. Exposures were for 3.5 h.
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To examine further the possibility that CD45-AP recognized activated
Lck molecules preferentially, the impact of supplementary mutations on
the capacity of Y505F Lck to associate with CD45-AP was assessed (Fig.
7C). This experiment revealed that derivatives of Y505F Lck
carrying a mutation of the site of myristoylation (G2A/Y505F Lck;
lane 3), ATP binding (K273R/Y505F Lck; lane 5), or positive regulation (Y394F/Y505F Lck; lane 6) had a
reduced capacity to bind to CD45-AP compared with Y505F Lck (lane
2). Their binding efficiency was comparable to that of wild-type
Lck (lane 1). In contrast, a mutant lacking the unique
domain (
U/Y505F Lck; lane 4) behaved like Y505F Lck
(lane 2). Once again, the ability of CD45-AP to bind Lck
correlated perfectly well with enzymatic activation of
p56lck (37, 41). Because the unique SH3 and SH2
domains of Lck were dispensable for the association with CD45-AP (Fig.
7B), these data suggested that the binding was mediated by
the Lck catalytic domain. To test this idea more directly, we evaluated
the capacity of an Lck variant lacking the kinase domain to bind
CD45-AP. As expected, this mutant (
K Lck; Fig. 7C,
lane 7) was incapable of interacting with CD45-AP.
Finally, we determined which region in CD45-AP was involved in binding
Lck (Fig. 8). For this purpose, various
carboxyl-terminal truncations of CD45-AP were created by polymerase
chain reaction (Fig. 8A). The first mutant (
C1 CD45-AP)
lacked 38 amino acids at the carboxyl terminus of the protein. The
second (
C2 CD45-AP) was missing 98 residues, including a region rich
in aspartic acid and glutamic acid ("acidic" region). Finally, the
third carried a truncation of 128 amino acids at the carboxyl terminus,
including part of the potential WW domain. For adequate detection of
these polypeptides, they were also engineered to possess a Myc-derived epitope at their carboxyl terminus.

View larger version (23K):
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|
Fig. 8.
Structure-function analyses of CD45-AP.
Panel A, structure of CD45-AP. The positions of the
extracellular (EC), transmembrane (TM),
intracytoplasmic (IC), WW-like, and acidic regions of
CD45-AP are indicated. The locations of the carboxyl-terminal
boundaries of the truncations are also depicted. Panel B,
association of CD45-AP mutants with Y505F Lck. COS-1 cells were
transfected with the indicated cDNAs. After lysis in Nonidet
P-40-containing buffer, the ability of various CD45-AP mutants to
associate with Y505F Lck was examined. 400 µg of cellular protein was
used for immunoprecipitation. The positions of Lck and CD45-AP-Myc are
indicated on the left. Exposures: top, 36 h;
middle, 36 h; bottom, 16 h.
|
|
When tested in the COS-1 system (Fig. 8B),
C1 CD45-AP
(top panel, lane 4) was able to associate with
Y505F Lck in a manner analogous to wild-type CD45-AP (lane
3). Although the association of this variant with Lck may appear
greater than that of wild-type CD45-AP, it should be noted that the
mutant protein accumulated in greater amounts in COS-1 cells
(bottom panel). Contrary to
C1 CD45-AP, however, the
C2 CD45-AP (lane 5) and
C3 CD45-AP (lane 6)
mutants were incapable of associating with
p56lck even though they were also expressed
adequately in COS-1 cells (bottom panel). It is worth
pointing out that, unlike wild-type CD45-AP, all truncated proteins
migrated as a single species in these gels. This finding suggested that
the sites of phosphorylation responsible for the variations in the
electrophoretic mobility of CD45-AP (23) were positioned within the
last 38 residues of the protein. Taken together, these results
indicated that the CD45-AP-Lck association was mediated by an
acidic-rich region in the cytoplasmic domain of CD45-AP and by the
catalytic domain of p56lck.
 |
DISCUSSION |
In this paper, we have examined the ability of CD45-AP to interact
with various constituents of the antigen receptor signaling machinery
in T-lymphocytes. We found that in mouse thymocytes, CD45-AP
coimmunoprecipitated not only with CD45 but also with the antigen
receptor complex (which incorporates TCR, CD3,
, CD4, and CD8 in
these cells) and the protein tyrosine kinase
p56lck. These interactions were specific, as
CD45-AP was not associated with Thy-1, FynT, Csk, Chk, Cbl, Vav, and
Shc. Further studies showed that the association between CD45-AP and
TCR occurred independently of the presence of CD4 and CD8 but that it
required the expression of CD45. It was also determined that this
interaction was not mediated by the cytoplasmic region of the
TCR-associated CD3
and
chains. In other experiments, we
established that binding of CD45-AP to p56lck
could occur in cells lacking CD45, CD4, CD8, TCR, and other
lymphoid-specific components, suggesting that it can be direct.
Structure-function analyses revealed that such an interaction was
assured by the cytoplasmic domain of CD45-AP and the catalytic region
of Lck. Moreover, they revealed that CD45-AP was more apt to associate with enzymatically activated Lck molecules than with wild-type or
inactive Lck polypeptides.
CD45-AP was discovered on the basis of its capacity to associate with
CD45, via its transmembrane domain (25, 28, 29, 51). In addition to
confirming this association, our results revealed that CD45-AP could be
coimmunoprecipitated with the antigen receptor complex in Brij-97
lysates of T-cells. Although the precise basis for this interaction
needs to be established, several observations suggest that it was
mediated by CD45. First, we observed that the cytoplasmic domain of at
least two of the components of the TCR complex, CD3
and
, was
unable to engage in an association with CD45-AP, suggesting that the
extracellular and/or transmembrane segments of the TCR complex were
responsible for the interaction. Because CD45-AP contains little
extracellular sequences, and its transmembrane domain is already bound
to CD45, it thus appears unlikely that CD45-AP made direct contact with
the TCR complex. Second, we noted that the CD45-AP-TCR interaction was
absent in cells lacking CD45. Even though the levels of CD45-AP were
reduced in CD45-negative YAC-1 cells, the inability of the remaining
CD45-AP (20% of control) to coimmunoprecipitate with TCR would be
consistent with the notion that CD45 was bridging the interaction
between these two molecules. This idea is also in keeping with the
previous observations that CD45 can interact with the TCR complex by
way of its extracellular domain (20, 50), whereas it associates with
CD45-AP via its transmembrane domain (25, 28, 29, 51).
These findings indicate that CD45-AP is part of a multimolecular
complex that includes TCR as well as the CD4 and CD8 coreceptors in
addition to CD45 (Fig. 9). Obviously, the
function of CD45-AP in this complex remains to be established.
Nevertheless, it is provocative to note that the absence of CD45-AP in
mice was accompanied by a reduction in CD45 expression at the cell
surface (30). Thus, CD45-AP may be a "chaperone" for one or more of
these molecules. In agreement with this notion, it has been reported
that CD45-AP associates with CD45 shortly after its synthesis (51).
Although an analysis of the levels of TCR, CD4, and CD8 in
CD45-AP-deficient mice has yet to be reported, CD45-AP may also
provide, albeit indirectly, a similar function for TCR, CD4, and CD8.
It is also conceivable that CD45-AP participates in organizing the
membrane microdomains containing the components of the antigen receptor signaling machinery. Such an organization may be important for optimal
generation of TCR-triggered signals and may explain the diminished
antigen receptor-induced responses in CD45-AP-deficient mice (30).
Clearly, future studies are warranted to test these various
possibilities.
The mechanisms underlying the association between CD45-AP and Lck in
T-cells are apparently complex. In part, this interaction is probably
indirect, as a consequence of the capacity of
p56lck to associate with CD4 and CD8 through its
unique region (42, 56) and possibly with CD45 via its unique and SH2
domains (52). Nonetheless, our transient expression experiments in
COS-1 cells, and those in HeLa cells reported by others (25), showed
that an association between CD45-AP and Lck could take place in the absence of other lymphoid-specific components. Notably, however, the
association of CD45-AP with wild-type or inactive Lck molecules was
rather weak in these systems. It was much more efficient with activated
Lck variants such as Y505F Lck,
SH3 Lck, and
SH2 Lck. Therefore,
the capacity of CD45-AP to bind Lck in the absence of other lymphoid
molecules seems to be dictated by the degree of enzymatic activation of
Lck. Presumably, CD45-AP recognizes the "active" conformation of
the Lck kinase domain. Along these lines, it is worth noting that this
association was mediated by an acidic-rich region in the cytoplasmic
domain of CD45-AP. Interestingly, an acidic domain in the
chain of
the interleukin-2 receptor has also been shown to bind the kinase
domain of p56lck (57), raising the possibility
that the catalytic region of p56lck may
recognize certain acidic motifs.
In light of these results, we propose the following scheme for the
interaction between CD45-AP and p56lck (Fig. 9).
The ability of CD4, CD8, and, in all likelihood, CD45 to bind Lck
brings the kinase in the vicinity of CD45-AP in unstimulated T-cells.
Once activated by engagement of TCR, CD4, or CD8, Lck molecules become
more susceptible to bind CD45-AP. This binding may help deliver
activated Lck polypeptides to CD45. Given the capacity of CD45 to
dephosphorylate the positive regulatory site of Lck (tyrosine 394) and
cause its inactivation (11, 12), this could be part of a negative
feedback mechanism aimed at limiting TCR signaling. Such a concept was
also suggested by the earlier findings that TCR stimulation resulted in
an enhancement of the extent of association of CD45 with Lck and that
such an increase was absent in T-cells lacking CD45-AP (30).
Alternatively, CD45-AP may bring activated
p56lck into closer proximity to TCR, thereby
facilitating ITAM phosphorylation and amplifying TCR signaling.
Additional analyses of T-cells from CD45-AP-deficient mice should help
distinguish between these two opposing scenarios. Finally, it is
plausible that CD45-AP has a direct impact on the kinase activity
and/or substrate specificity of Lck via steric hindrance or
conformational modification. However, this notion seems less likely
because we found that CD45-AP expression alone had little effect on the
activity of Y505F Lck in transfected fibroblasts or COS-1
cells.3
In summary, the results presented in this manuscript show that CD45-AP
is part of a multimolecular complex that includes the TCR complex, CD4,
CD8, CD45, and p56lck. These associations are
mediated at least in part through the ability of CD45-AP to associate
with CD45 via its transmembrane domain and with activated Lck molecules
via its cytoplasmic region. Even though additional studies are
necessary to determine the precise role of CD45-AP in this complex, our
data are consistent with the notion that it is an adaptor or
chaperone-like molecule coordinating interactions among components of
the antigen receptor signaling machinery.
 |
ACKNOWLEDGEMENTS |
We thank Jonathan Ashwell and Larry Samelson
for gifts of reagents and Eric Henry for technical help.
 |
FOOTNOTES |
*
This work was supported in part by grants from the Medical
Research Council of Canada and the National Cancer Institute of Canada
(to A. V.).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.
f
Scientist of the Medical Research Council of
Canada. To whom correspondence should be addressed: Rm. 715, McIntyre
Medical Sciences Bldg., McGill University, 3655 Drummond St.,
Montréal H3G 1Y6, Canada. Tel.: 514-398-8936; Fax: 514-398-4438;
E-mail: andrev{at}med.mcgill.ca.
g
Supported in part by a studentship from Canderel.
h
Recipient of a fellowship from the Medical
Research Council of Canada.
i
Holder of a Steve Fonyo studentship from the
National Cancer Institute of Canada.
2
S. Latour, E. Henry, and A. Veillette,
unpublished results.
3
F. G. Gervais and A. Veillette, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
TCR, T-cell antigen
receptor;
ITAM, immunoreceptor tyrosine-based activation motif;
CD45-AP, CD45-associated protein;
MAb, monoclonal antibody.
 |
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