(Received for publication, August 24, 1995; and in revised form, October 20, 1995)
From the
p56 is a potential in vivo substrate for the tyrosine-specific phosphatase, CD45. In this
study, recombinant purified p56
was found to
specifically associate with recombinant CD45 cytoplasmic domain
protein, but not to the cytoplasmic domain of another related tyrosine
phosphatase, receptor protein-tyrosine phosphatase
. Under
equilibrium binding conditions, the binding was saturable and occurred
at a 1:1 molar stoichiometry. A fusion protein containing only the
amino-terminal region of p56
(residues
34-150) also bound to recombinant CD45, and further analysis of
this region indicated that glutathione S-transferase fusion
proteins of the unique amino-terminal region and the SH2 domain, but
not the SH3 domain of p56
, bound to recombinant
CD45. The SH2 domain protein bound with a higher affinity than the
amino-terminal region, but both were able to compete for the binding of
p56
to CD45, and when added together worked
synergistically to compete for p56
binding. The
SH2 domain interaction with CD45 was specific as glutathione S-transferase-SH2 fusion proteins from p85
subunit of
phosphatidylinositol 3-kinase and SHC did not bind to CD45. In
addition, this interaction occurred in the absence of any detectable
tyrosine phosphorylation on CD45, suggesting a nonconventional SH2
domain interaction.
p56 and CD45 (see (1) for
review) are both required to generate an effective T cell antigen
receptor (TCR)(
)-mediated signal, being required for the
earliest detectable event to occur upon TCR stimulation, the tyrosine
phosphorylation of cellular proteins(2, 3) .
p56
is a member of the Src family of tyrosine
kinases and is thought to be regulated by tyrosine phosphorylation at
the autophosphorylation and negative regulatory sites. Tyrosine
phosphorylation at the carboxyl-terminal negative regulatory site is
believed to result in an intramolecular interaction with its SH2 domain
which results in an inactive or inaccessible kinase(4) . In the
presence of CD45 this negative regulatory tyrosine residue in
p56
is
dephosphorylated(5, 6, 7, 8, 9) ,
and p56
is then thought to be able to
participate in TCR-mediated signal transduction
events(10, 11, 12) . Exactly how
dephosphorylation of p56
results in effective
TCR-mediated signal transduction remains unclear as recent data
indicate that this dephosphorylation event may not necessarily lead to
increased kinase activity(13) . Dephosphorylation of these
kinases may also result in the release of the intramolecular binding of
the carboxyl-terminal tyrosine to the SH2 domain, which would then
allow the unoccupied SH2 domain to bind to other
tyrosine-phosphorylated proteins. Hence CD45 may act to regulate the
SH2 domain interactions of p56
, which itself
could be crucial in mediating a TCR induced signal. Consistent with
such a model is the demonstration that the SH2 domain of
p56
also plays an active role in T cell
activation(14) .
CD45 is a major lymphocyte glycoprotein and
a transmembrane two domain tyrosine-specific phosphatase (reviewed in (15) ). CD45 can dephosphorylate several protein substrates and
phosphorylated peptides in
vitro(6, 7, 16, 17) , yet only
p56 and another Src family kinase,
p59
, have clearly been identified as potential in vivo substrates(5, 8, 9) . Both
p56
and, to a lesser extent, p59
were found to be hyperphosphorylated in the absence of CD45,
whereas the tyrosine phosphorylation state of p60
was reported to be unaffected (9) . How the
substrate specificity of CD45 is achieved in vivo is not
known, although substrate accessibility may be a factor. There is some
evidence indicating that p56
may be associated
with CD45 in T cells(18, 19, 20) .
p56
and proteins of approximately 30 kDa can be
co-precipitated with CD45 using a mild detergent to lyse the
cells(18, 21, 22) . CD4 and CD8 are strongly
associated with p56
(reviewed in (1) )
and have been reported to associate with
CD45(23, 24) , yet the interaction between
p56
and CD45 was shown to occur independently of
the expression of CD4, CD8, and the TCR(25) . Hence in T cells,
CD45 may associate directly with p56
or
indirectly via the 30-kDa proteins.
Recently, an interaction between
p56 and tyrosine-phosphorylated CD45 was
observed in phenylarsine oxide-treated T cells(26) . In
addition, tyrosine phosphorylation of CD45 in vitro by
p50
resulted in the binding of
p56
to CD45. This binding was thought to be
mediated by the SH2 domain of p56
as the
interaction was prevented by the addition of excess recombinant
p56
SH2 domain(26) .
To further
investigate the potential interaction of p56 and
CD45, we chose to analyze this association using purified, recombinant
forms of p56
and the cytoplasmic domain of CD45.
Recombinant CD45 cytoplasmic domain protein (34) was
covalently coupled to CNBr-activated Sepharose CL-4B, and approximately
2.5 µg of the immobilized protein was incubated with approximately
200 ng of recombinant p56(32) at concentrations
of 700 and 90 nM, respectively. Under the binding conditions
used (the presence of phosphatase inhibitors, the absence of ATP and
divalent cations, and with CD45 covalently coupled to beads) neither
CD45 nor p56
was enzymatically active. After a 2-h
incubation at 4 °C, the CD45-conjugated beads were washed three
times in RIPA buffer, and any remaining proteins were subjected to
SDS-PAGE, transferred to PVDF membrane, and probed by Western blotting
for the presence of p56
. The results are shown in Fig. 1and illustrate that p56
bound to
CD45-coupled beads (lane 2), but did not bind to an equivalent
amount of Sepharose beads alone (lane 1) or to equivalent
amounts of Sepharose beads covalently coupled to two irrelevant
antibody proteins (lanes 4 and 5). Furthermore,
p56
did not associate with a recombinant GST fusion
protein of the cytoplasmic domain of a related protein-tyrosine
phosphatase, RPTP
, which had been immobilized on
glutathione-Sepharose beads (Fig. 1, lane 3). It was
verified by Coomassie Blue staining of recombinant CD45 and RPTP
proteins that similar amounts of protein had been taken and coupled to
beads (data not shown). In addition, immobilized CD45 did not associate
with a recombinant GST protein under similar binding conditions, and
likewise, p56
did not bind to another immobilized
recombinant protein with a 6-histidine tag (endoglucanase, CenA) (37) (data not shown), further indicating the specificity of
the interaction.
Figure 1:
Binding of recombinant p56 to the cytoplasmic domain of CD45. Approximately 200 ng of
p56
was incubated with 2.5 µg of various
proteins coupled to beads at 4 °C for 2 h in 40 µl of binding
buffer. After washing in RIPA buffer, the entire sample was subjected
to SDS-PAGE, transferred to PVDF membrane, and bound p56
was detected by immunoblot analysis using
anti-p56
antisera. Lane C is a control
containing 25 ng of recombinant p56
. Lanes
1-5 show the amount of recombinant p56
remaining bound to Sepharose CL-4B beads alone (lane
1), recombinant CD45 cytoplasmic domain conjugated to Sepharose
beads (lane 2), recombinant RPTP
cytoplasmic domain GST
fusion protein coupled to glutathione-Sepharose beads (lane
3), I3/2 antibody conjugated to Sepharose beads (lane 4),
Ly 5.2 antibody conjugated to Sepharose beads (lane
5).
To determine the stoichiometry of the interaction,
increasing amounts of P-labeled p56
were
incubated with immobilized CD45. Under equilibrium binding conditions
the specific binding of p56
to the cytoplasmic domain of
CD45 was saturable (see Fig. 2). It was determined from
double-reciprocal plot analysis of seven experiments that 1.1 ±
0.2 mol of p56
bound to 1 mol of the cytoplasmic domain
of CD45 under saturating binding conditions. Attempts were made to
determine the affinity of this interaction, but Scatchard analysis
generated a nonlinear plot which suggested a complex interaction from
which the affinity constant could not be readily determined.
Figure 2:
Graph showing amount of radioactively
labeled p56 bound to immobilized cytoplasmic
domain of CD45 at equilibrium. A, graph of total amount of
radioactive p56
(ng) bound to 300 ng of
immobilized cytoplasmic domain of CD45 after incubation in increasing
concentrations (nanomolar) of radioactively labeled p56
(
).
is nonspecifically bound radioactive
p56
(nanograms), and
is the amount of
radioactive p56
that specifically bound.
Specifically bound radioactive p56
was
calculated from ((total radioactivity added - amount free)
- nonspecifically bound
radioactivity).
It was
observed that when antisera raised against amino acid residues
34-150 of p56 was added together with recombinant
p56
, significant inhibition of the association occurred
(data not shown). To confirm whether this region of p56
could mediate the binding to cytoplasmic CD45, approximately 200
ng of a purified TrpE fusion protein containing residues 34-150
of p56
was incubated with immobilized recombinant CD45
cytoplasmic domain. This TrpE-p56
fusion protein has a
predicted molecular mass of 51 kDa and specifically bound to the
recombinant cytoplasmic domain of CD45 immobilized on Sepharose beads (Fig. 3). To further localize which specific region of the
TrpE-p56
fusion protein was mediating this interaction,
the immobilized CD45 cytoplasmic domain was incubated with 2.0 µg
of purified GST fusion proteins containing the unique amino-terminal
region of p56
, the SH3 domain of p56
, or
the SH2 domain of p56
. From the Western blot probed with
anti-GST antisera in Fig. 4A, it can be seen that GST
fusion proteins containing the unique amino-terminal region and SH2
domain of p56
, but not the SH3 domain of p56
or GST alone, specifically bound to the immobilized cytoplasmic
domain of CD45. The GST-p56
unique amino-terminal region
and SH2 fusion proteins did not associate with an immobilized
irrelevant protein, an anti-CD45 monoclonal antibody. Since a similar
detection system was employed to identify both the unique
amino-terminal and SH2-GST fusion proteins, the results indicated that
a greater amount of the SH2-GST fusion protein bound to recombinant
CD45, implying that the SH2 domain had a higher affinity for CD45 than
the unique amino-terminal region. It was verified by Coomassie Blue
staining that similar amounts of recombinant GST fusion proteins were
incubated with recombinant CD45 cytoplasmic domain-coupled Sepharose
beads (Fig. 4B). Thus, two distinct regions of
p56
can bind to the cytoplasmic domain of CD45.
Figure 3:
Binding of the TrpE- p56 fusion protein to the cytoplasmic domain of CD45.
Approximately 200 ng of p56
(lanes 1 and 2) or 200 ng of TrpE-p56
fusion protein containing amino acids 34-150 of
p56
(lanes 3 and 4) were
incubated with immobilized recombinant CD45 cytoplasmic domain protein (lanes 1 and 3) or with an irrelevant protein
(monoclonal antibody) coupled to beads (lanes 2 and 4). Recombinant p56
and
TrpE-p56
fusion proteins remaining bound to the
immobilized proteins were detected by immunoblot analysis using
p56
antisera specific for the amino-terminal
region of p56
.
Figure 4:
Binding of recombinant GST fusion proteins
to the cytoplasmic domain of CD45. A, Western blot analysis of
GST fusion proteins remaining bound to the immobilized cytoplasmic
domain of CD45 (odd-numbered lanes) or to the control I3/2
antibody (even-numbered lanes). GST fusion proteins were
detected using GST antisera. 2.0 µg of the GST fusion proteins were
added to 2.5 µg of immobilized CD45 cytoplasmic domain and
incubated for 2 h at 4 °C in 40 µl of binding buffer. The beads
were washed three times in RIPA buffer and subjected to SDS-PAGE. Lanes 1 and 2, GST protein alone; lanes 3 and 4, GST p56 unique
amino-terminal region; lanes 5 and 6, GST
p56
SH3; lanes 7 and 8, GST
p56
SH2. B, SDS-PAGE analysis of the
purity of GST fusion proteins used in the binding assay, stained with
Coomassie Blue. Lane 1, GST protein alone; lane 2,
GST p56
unique amino-terminal region; lane
3, GST p56
SH3; and lane 4, GST
p56
SH2.
Competition binding assays were performed to determine the role of
these two regions in the binding of intact p56 to CD45.
Both the unique amino-terminal region and the SH2-GST fusion protein
could compete for the binding of p56
to CD45 (Fig. 5). The SH2 domain was approximately 5-fold more efficient
at competing with p56
for binding to CD45 than the unique
amino-terminal region. This supports the earlier observation that the
SH2 domain of p56
bound with a higher affinity to CD45.
Interestingly, when the two domains were used together, they acted
synergistically to compete with p56
for binding to CD45,
but were still not as efficient as the intact p56
protein.
Figure 5:
Using GST fusion proteins to compete for
the binding of p56 to the cytoplasmic domain of
CD45. 300 ng of immobilized recombinant cytoplasmic domain of CD45 was
mixed with
P-labeled p56
for 8 h,
washed, and then incubated with various concentrations of competing GST
fusion protein (
, GST-p56
;
, GST-unique
amino-terminal region;
, GST-SH3 domain;
, GST-SH2 domain;
, GST unique amino-terminal region and GST-SH2 domain) for 12 h.
The immobilized CD45 was centrifuged, the supernatant removed, and the
remaining
P-labeled p56
bound to
CD45 determined.
The specificity of the interaction with the SH2
domain of p56 was further evaluated by determining if
other GST-SH2 domain fusion proteins would bind to the immobilized
recombinant CD45 cytoplasmic domain. It was found that neither the
carboxyl-terminal SH2 domain from the p85
subunit of
phosphatidylinositol 3-kinase nor the SH2 domain of SHC bound to
recombinant cytoplasmic CD45 protein (Fig. 6).
Figure 6:
Binding of recombinant GST SH2 domain
fusion proteins to the cytoplasmic domain of CD45. A, Western
blot analysis of GST fusion proteins remaining bound to the immobilized
cytoplasmic domain of CD45 (odd-numbered lanes) or to the
control I3/2 antibody (even-numbered lanes). GST fusion
proteins were detected using GST antisera. 2.0 µg of the GST fusion
proteins were added to 2.5 µg of immobilized CD45 cytoplasmic
domain and incubated for 2 h at 4 °C in 40 µl of binding
buffer. The beads were washed three times in RIPA buffer and subjected
to SDS-PAGE. Lanes 1 and 2, GST p56 SH3; lanes 3 and 4, GST p56
SH2; lanes 5 and 6, GST p85
SH2; lanes 7 and 8, GST SHC SH2; lanes 9 and 10, GST protein alone. B, SDS-PAGE analysis of GST
fusion proteins used in the binding assay and stained with Coomassie
Blue. Lane 1, GST p56
SH3; lane
2, GST p56
SH2; lane 3, GST
p85
SH2; lane 4, GST SHC SH2; and lane 5, GST
protein alone.
Protein-protein interactions involving SH2 domains have previously
been shown to be mediated by the binding of the SH2 domain to a region
of the protein containing a phosphorylated tyrosine residue (reviewed
in (38) ). In this situation, the possibility that the SH2
domain of p56 was binding to a phosphorylated tyrosine
residue in CD45 was considered unlikely for two reasons. First,
recombinant CD45 had been generated and purified from E. coli,
which is not known to contain any tyrosine kinase activity, and second,
no tyrosine kinase or ATP was present in the incubation of recombinant
cytoplasmic CD45 with the SH2-GST fusion proteins, which themselves do
not possess tyrosine kinase activity. However, in order to evaluate
this possibility experimentally, 2.5 µg of recombinant CD45 was
incubated with approximately 200 ng of p56
or 2.0 µg
of GST-p56
SH2 domain fusion protein in the binding
buffer for 2 h at 4 °C. These proteins and aliquots of purified
proteins that had not been incubated were separated by SDS-PAGE and
then either transferred to PVDF membrane and Western blotted with 4G10,
a monoclonal antibody specific for phosphotyrosine residues (Fig. 7A) or stained with Coomassie Blue (Fig. 7B). As can be seen in Fig. 7A (lanes 1 and 2) approximately 200 ng of
p56
gave a strong signal with the anti-phosphotyrosine
antibody. Even 20 ng of p56
(lanes 6 and 7) provided a strong signal indicating that recombinant
p56
, produced using a baculovirus expression system, was
tyrosine-phosphorylated. However, even with 100 times the amount
required to give a signal for p56
, no bands at 95 kDa
representing the cytoplasmic domain of CD45 (lanes 2, 3, 4,
and 6) were observed using the anti-phosphotyrosine antibody.
These data demonstrate that neither CD45 nor the GST-p56
SH2 domain proteins were detectably tyrosine-phosphorylated and
that the tyrosine phosphorylation state of p56
and CD45
did not change during the course of the binding assay, indicating that
under these conditions both p56
and CD45 were not
catalytically active. Thus, the SH2 domain of p56
interacts with the cytoplasmic domain of CD45 by a mechanism that
does not depend on the tyrosine phosphorylation of CD45.
Figure 7:
Tyrosine phosphorylation state of
recombinant p56 and CD45 cytoplasmic domain
proteins. A, Western blot of soluble recombinant proteins
probed with the 4G10, an anti-phosphotyrosine monoclonal antibody.
Proteins were incubated in 40 µl of binding buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.025%
-mercaptoethanol,
containing protease and phosphatase inhibitors) for 2 h at 4 °C. Lane 1, approximately 200 ng of p56
; lane 2, approximately 200 ng of p56
,
and 2.5 µg of cytoplasmic CD45; lane 3, 2.5 µg of
cytoplasmic CD45; lane 4, 2.0 µg of GST p56
SH2 domain and 2.5 µg of cytoplasmic CD45; lane
5, 2.0 µg of GST p56
SH2 domain; lane 6, 20 ng of p56
and 2.5 µg of
cytoplasmic CD45; and lane 7, 20 ng of
p56
. B, SDS-PAGE analysis of
recombinant proteins detected by Coomassie Blue staining. Lanes
1-5 are identical to those in A.
This work demonstrates that recombinant p56 can
specifically associate in vitro with the recombinant
cytoplasmic domain of CD45. The specificity of this interaction has
been defined on the basis of the following criteria: 1) the interaction
did not occur with a structurally related tyrosine phosphatase,
RPTP
; 2) the binding was saturable and occurred at a stoichiometry
of 1:1; 3) the binding could be localized to distinct protein domains
of p56
, and 4) the association was still detectable after
washing in the presence of 0.1% SDS, 1% Nonidet P-40, 0.5% sodium
deoxycholate, and 150 mM NaCl (RIPA buffer).
The
cytoplasmic region of RPTP has a similar predicted domain
structure to CD45, having two tandemly repeated phosphatase domains
separated by a unique spacer region and flanked by unique membrane
proximal and carboxyl-terminal sequences. The PTP domain 1 and domain 2
of CD45 share 47 and 33% sequence identity with PTP domain 1 and domain
2 of RPTP
, respectively(27, 33) . The observation
that p56
associates with CD45 but not with RPTP
implies a specific interaction, but at present the significance of this
interaction is not known. The in vitro interaction also
occurred in the absence of PTP inhibitors ruling out the possibility
that these inhibitors were mediating the interaction between
p56
and CD45 (data not shown). In addition, p56
also bound to an immobilized recombinant CD45 protein in which
the catalytic cysteine (C817) had been replaced with a serine,
indicating that a phosphocysteine intermediate was not responsible for
the observed interaction (data not shown). From the binding assay, only
a small percentage of p56
(
3-4%) bound to
recombinant CD45, yet under equilibrium binding conditions and using
saturating amounts of p56
, a 1:1 interaction was
indicated. These differences may reflect the fact that in the initial
binding experiments, the amount bound was observed after washing three
times in stringent conditions (RIPA buffer).
In an attempt to
determine the affinity of the interaction, Scatchard analysis of the
equilibrium binding data was performed. However, a nonlinear plot was
obtained, indicating that it was not a simple single binding site
interaction (data not shown). Interpretation of the plot as two
distinct binding sites with different affinities was also precluded as
the interaction at saturation resulted in a 1:1 stoichiometry. This
indicated that the criteria for Scatchard analysis was not met and thus
could not be used to determine the affinity of the interaction.
Additional data using GST-fusion proteins indicated that two distinct
regions of p56, the unique amino-terminal region and the
SH2 domain, could specifically bind to recombinant CD45. Competition
analysis showed that the SH2 domain was approximately 5-fold more
effective than the unique amino-terminal region in competing with
intact p56
. Interestingly, together these two domains
were able to compete with p56
more effectively than
either domain alone, suggesting a synergistic effect. This strongly
implies that both regions are involved in mediating the interaction
between p56
and CD45. However, together, these two
domains were still not as efficient as the intact p56
in
these competition assays, suggesting that either these two domains need
to be in a certain configuration for optimum binding or that additional
regions within the COOH-terminal region of p56
are
involved in binding. Since CD45 can dephosphorylate Tyr
and Tyr
residues, both of which are located in the
COOH-terminal region, an interaction with this region must occur in
order for CD45 to dephosphorylate these residues. Whether this region
can interact stably with the cytoplasmic domain of CD45 in the absence
of these two regulatory regions or whether these regulatory regions
interact with CD45 to facilitate the dephosphorylation of p56
remains to be determined and is currently under investigation.
It was also shown that the association between p56 or
the GST-p56
SH2 fusion protein with the cytoplasmic
domain of CD45 did not require the tyrosine phosphorylation of CD45.
This is in contrast to previous studies by Autero et al.(26) where an interaction between p56
and
CD45 was observed when CD45 was tyrosine-phosphorylated in vitro by p50
. It is possible that p56
may
bind to both non-tyrosine-phosphorylated and phosphorylated forms of
CD45. In support of this notion, another Src family kinase,
p59
, has been reported to associate with both
non-tyrosine-phosphorylated and phosphorylated forms of Ig
.
Increased binding of p59
was observed upon tyrosine
phosphorylation of Ig
, which resulted in an increase in the kinase
activity of p59
(39) .
Tyrosine
phosphorylation-independent interactions with SH2 domains have been
reported for three other tyrosine kinases; ABL, p59, and
p60
(40, 41) . In these examples, the SH2
domains were thought to bind to phosphoserine/threonine residues. It
remains to be determined whether the interaction between the
p56
SH2 domain and the cytoplasmic domain of CD45
requires serine/threonine phosphorylation. At present, this interaction
has been observed with the SH2 domain of p56
but not with
two other SH2 domains derived from two other signaling proteins, the
p85
subunit of phosphatidylinositol 3-kinase and SHC.
Although
we have demonstrated a direct interaction between p56 and
the cytoplasmic domain of CD45 in vitro, it still remains to
be established whether a direct interaction also occurs in vivo and whether such an interaction is regulated by the association of
other proteins such as CD45AP. In T cells, p56
can be
detected in immunoprecipitates of CD45 along with the presence of a
30-kDa protein (18, 25) and can be co-precipitated
with CD45 under conditions where CD45 is not detectably tyrosine
phosphorylated. (
)Since p56
has not been
co-precipitated in the absence of the 30-kDa protein (CD45AP or LPAP),
it is not known whether p56
can directly associate with
CD45 in T cells. In contrast, p30 has been shown to co-precipitate with
CD45 in the absence of p56
(20) , and recently,
the transmembrane region of CD45 has been shown to be required for the
association of p30(42) . However, as only a small percentage of
p56
is co-precipitated with CD45,
the
physiological significance of this association remains to be
established. In the in vitro assay conditions used to observe
the binding of p56
to the cytoplasmic domain of CD45,
neither enzyme was catalytically active and p56
, but not
CD45, was detectably tyrosine-phosphorylated. It is possible that these in vitro conditions favor or stabilize the interaction between
these proteins whereas conditions in the cell may favor a more
transient association.
Data from CD45 negative and positive cell
lines have indicated that the absence of CD45 has a larger effect on
the phosphorylation state of p56 than p59
and that the phosphorylation state of p60
is
unaffected(9) . Since all these kinases are closely related,
particularly at the dephosphorylation site, it is not clear why some
members of this family appear to be preferred substrates in
vivo. Recent data using a p56
/p59
chimera has shown that the unique amino-terminal region of
p56
, but not that of p59
, was required for
the dephosphorylation of Tyr
in p56
,
suggesting that the unique amino-terminal region of p56
may influence substrate specificity(43) . In this paper
we have demonstrated that both the unique amino-terminal region and SH2
domain of p56
can bind to the cytoplasmic domain of CD45 in vitro, and thus, it is tempting to speculate that an
interaction between these noncatalytic domains of p56
and
CD45 may act to facilitate the dephosphorylation of p56
.