(Received for publication, June 7, 1995; and in revised form, February 16, 1996)
From the
A number of protein-tyrosine kinases have been shown to be
important in T cell activation. One such kinase, Lck, has been
demonstrated genetically to be essential for T cell receptor (TcR)
signaling, and the SH2 and SH3 (src homology 2 and 3) domains
of Lck have been shown to be indispensable for T cell activation. We
have sought substrates with which the SH2,3 domain would interact
following T cell activation, using fusion proteins containing the Lck
SH2 and SH3 domains linked to glutathione S-transferase. We
demonstrate that the SH2,3 region interacts specifically and directly
with numerous tyrosine-phosphorylated molecules following TcR
cross-linking, including constitutively with mitogen-activated protein
kinase (MAPK)/extracellular-regulated kinase and inducibly with the
chain of the TcR. The interaction with
MAPK/extracellular-regulated kinase was via the SH3 domain. The
interaction with the tyrosine-phosphorylated
chain, while
phosphotyrosine-dependent, required both the SH3 and SH2 domains. These
interactions were specific as molecules known to be
tyrosine-phosphorylated following TcR cross-linking, phospholipase
C-
1 and Fyn, were not bound. Thus, we suggest that during TcR
signaling, Lck interacts with numerous molecules, including MAPK and
TcR-
, via its SH2,3 domain. The interaction with MAPK would place
Lck in a position to be involved in the complex resulting in the
activation of MAPK. In addition, the binding of Lck to the
tyrosine-phosphorylated
chain of the TcR would serve to
strengthen the interaction of the associated CD4 and the TcR complex,
leading to increased avidity for the antigen-major histocompatibility
protein complex.
The antigen-specific T cell receptor serves to activate T cells
and imparts antigen specificity. A number of investigators have
demonstrated that following cross-linking of the TcR, ()a
number of molecules become tyrosine-phosphorylated, including the
activation of protein-tyrosine kinases such as p59
and the
chain-associated kinase ZAP-70. These
activated kinases presumably then act on other substrates that result
in the tyrosine phosphorylation of a number of other molecules,
including the
chain of the TcR and the
chains of CD3, CD5,
CD6, phospholipase C-
1, MAPK, and Vav, and the activation of Ras
(reviewed (1) ). While the protein-tyrosine kinase Lck has been
shown genetically to be essential for signaling via the
TcR(2) , whether this kinase actually becomes activated has
been controversial, with some reports of it becoming activated
following TcR cross-linking (3, 4) and some reports
of no activation(1, 5) .
The protein-tyrosine kinases Fyn, ZAP-70, and Lck all possess SH2 domains, examples of which have been demonstrated to bind to tyrosine-phosphorylated molecules in the context of surrounding amino acids(6) . In addition, both Fyn and Lck have SH3 domains, examples of which have been shown to bind to proline-rich regions in a number of intracellular molecules, and both have the organization of these modules in the following order: N-terminal unique region, SH3 domain, SH2 domain. Recent work has shown that the kinase domain of Lck seems to be dispensable for mature T cell activation, at least in the context of activating T cell hybridomas in an antigen-specific manner with CD4 as a co-receptor(7, 8) . The SH2 and SH3 domains, however, were found to be indispensable for the activation of these hybridomas, suggesting an important role for these domains of Lck(8) . This suggests that molecules that interact with these domains of Lck may play important roles in T cell activation. Along these lines, several groups have recently demonstrated that the SH3 domain of Lck interacts with the lipid kinase phosphatidylinositol 3-kinase(9, 10) .
The substrate specificity of some
SH2-containing tyrosine kinases seems to reside in the specificity of
their SH2 domains(11) . To investigate the role of Lck in T
cell activation and as a first step in finding substrates for this
protein-tyrosine kinase, it would be of interest to determine the
spectrum of molecules that would bind to the SH3 and SH2 domains of
Lck, some of which would probably include substrates of this
protein-tyrosine kinase. We demonstrate here that the SH2,3 domain of
Lck binds the serine/threonine kinase MAPK and the
tyrosine-phosphorylated chains of the TcR. The binding of MAPK
was via the SH3 domain of Lck and probably involved an indirect
interaction. While the tyrosine-phosphorylated
chain did not bind
the SH3 domain, it also did not bind the isolated SH2 domain, and
binding required both the SH3 and SH2 domains, although this binding
was phosphotyrosine-dependent. This supports the contention that the
kinase specificity of these SH2-containing kinases is regulated by the
specificities of their SH2 domains as the
chain has been reported
to be a substrate of Lck(1, 11) .
The protein-tyrosine kinase Lck has been shown to be
essential for TcR signaling(2) , and the SH2 and SH3 domains
were deemed to be essential in this process(8) . We reasoned
that part of this may represent the binding of the Lck SH2 and/or SH3
domain to important effector molecules required for the efficient
activation of the T cell. To this end, a SH2,3lckgst fusion protein was
generated and used as a probe for molecules binding to the SH2,3 domain
of Lck. These fusion proteins were used as probes using lysates of the
Jurkat T cell line E6-1 following cross-linking of the TcR. As
previously reported, cross-linking of the TcR complex resulted in
tyrosine phosphorylation of a number of proteins (Fig. 1a). A subset of these proteins bound to the
SH2,3lckgst fusion protein (Fig. 1, b and c),
with none interacting with GST alone (see also Fig. 5b, lane 3) ()or when Jurkat cells were stimulated with
pervanadate.
Two prominent tyrosine-phosphorylated
molecules were observed (arrowheads in Fig. 1, b and c), one of 42 kDa and one of
20 kDa. The one at
42 kDa had a molecular mass similar to that of the serine/threonine
kinase MAPK(16) . Cross-linking the TcR results in the tyrosine
phosphorylation and activation of MAPK(1) , suggesting that
this protein may be MAPK. We therefore tested if MAPK would bind to the
SH2,3lckgst fusion protein. The CD3 complex of the TcR was
cross-linked, and the resultant lysates were precipitated with the
SH2,3lckgst fusion protein. The resultant proteins were then probed for
the presence of MAPK. Fig. 2demonstrates that MAPK binds the
SH2,3 domain in both resting and TcR-cross-linked cell lysates. The
lysates were also denatured to determine if the SH2,3 domain of Lck
could interact directly with MAPK. Fig. 2demonstrates that the
amount of MAPK bound to the SH2,3 domain after denaturation of the
lysates was less than that seen when the lysates were in the native
form. It is possible that the reproducible residual binding that we
observed is due to incomplete denaturation. The results observed do,
however, clearly demonstrate reduced binding of MAPK to the Lck SH2,3
domain under denaturing conditions and support an indirect interaction.
MAPK bound in a manner that was independent of TcR stimulation, binding
both before and after TcR cross-linking. This binding was also largely
phosphotyrosine-independent, as illustrated in Fig. 3, where
phenyl phosphate, a phosphotyrosine mimic, competed very poorly to
elute the bound MAPK since only minimal MAPK was recovered in the
eluate. Other tyrosine-phosphorylated proteins, including ZAP-70, were,
however, eluted using this treatment with phenyl phosphate, as seen in
anti-phosphotyrosine immunoblots of these membranes.
In
addition, a similar amount of MAPK equivalent to that seen in the input
was recovered on the beads following phenyl phosphate treatment.
To demonstrate that the MAPK bound to the Lck SH2,3 domain
represented functional MAPK, Jurkat cells were stimulated with anti-CD3
antibodies for the indicated time periods, and the SH2,3-binding
proteins were isolated. These precipitates were then tested for MAPK
activity as measured by the ability to phosphorylate a synthetic MAPK
substrate as described under ``Experimental Procedures.'' Fig. 4a shows that the fusion protein bound MAPK activity.
This activity increased in parallel with the increases in
phosphotyrosine in the 42-kDa protein (see arrowhead in Fig. 1b). (Note that the time courses in the two
experiments are different. In Fig. 1b, the peak of
tyrosine phosphorylation (45 s) is earlier than the peak of
SH2,3lckgst-precipitated MAPK activity seen in Fig. 4a (2 min). While we have seen that the peaks of tyrosine
phosphorylation and MAPK activity vary within 30-45 s in
different experiments, they have not coincided and may reflect
different pools of MAPK being activated with different kinetics.) The
proteins bound were also blotted onto PVDF membrane and probed for MAPK (Fig. 4b). Thus, Fig. 1b, Fig. 2, Fig. 3, and Fig. 4b show that the
fusion protein bound MAPK constitutively, and the increase in activity
seen in Fig. 4a largely represents activation of MAPK
as a consequence of TcR cross-linking. The binding of MAPK to the SH2,3
domain of Lck therefore seems to be constitutive, with only its
activity regulated by the TcR. There is a small increase in binding of
MAPK to the SH2,3 domain of Lck upon tyrosine phosphorylation; however,
this amount is small compared with the total binding (Fig. 3,
compare amounts in lanes 3 and 4 with those in lanes 1 and 2; also seen in Fig. 4). The
constitutive binding of MAPK is characteristic of proteins that bind to
SH3 domains(6) . Indeed, MAPK bound to a fusion protein
consisting of only the SH3 domain of Lck, but not to GST (Fig. 5) or to the SH2 domains of Lck and Src (Fig. 6).
MAPK was also not bound by the SH2,3 domain of Csk or by the
full-length Grb2-GST and Nck-GST fusion proteins (Fig. 6). Thus,
the data indicate that MAPK can bind constitutively to the SH3 domain
of Lck.
Figure 1:
Binding of tyrosine-phosphorylated
molecules to the SH2,3 domain of Lck. Jurkat cells were stimulated for
the indicated periods of time using anti-CD3 antibodies, and either
total cell protein (a) or SH2,3lckgst fusion protein
precipitates (b and c) were separated by PAGE,
blotted onto PVDF membrane, and probed with anti-phosphotyrosine
antibodies (pY). In b, the arrowhead points to the 42-kDa MAPK. In c, the arrowhead points to the
chain of the TcR. c is from 30 kDa to
the dye front. CD3X, cross-linked CD3; Ip,
immunoprecipitate.
Figure 5:
The SH3 domain of Lck binds MAPK, but not
the chain of the TcR. Jurkat cells were either stimulated or not
with anti-CD3 antibodies, and the lysates were precipitated with an
SH3lckgst fusion protein (lanes 1 and 2) or with GST
alone (lane 3). Lane 4 is the whole cell lysate of
Jurkat E6-1 cells. The bound proteins were separated by SDS-PAGE,
blotted, and probed for MAPK (a) or the
chain of the TcR (b). The bracket indicates MAPK in a, and
the arrow indicates the
chain in b. CD3X, cross-linked CD3; Ip,
immunoprecipitate.
Figure 2: MAPK can bind constitutively to the SH2,3lckgst fusion protein. Jurkat cells were stimulated with anti-CD3 antibodies for 2 min, and half of the lysates were boiled (lanes 3 and 4) or not (lanes 1 and 2). The lysates were then precipitated with the SH2,3lckgst fusion protein. Lanes 1 and 3 are control cells; lanes 2 and 4 are CD3-stimulated cells. The precipitates were then blotted for MAPK. The isoforms of MAPK are bracketed. CD3X, cross-linked CD3; Ip, immunoprecipitate.
Figure 3: MAPK binds to the SH2,3lckgst fusion protein in a phosphotyrosine-independent manner. Jurkat cells were stimulated with anti-CD3 antibodies for 2 min, and the cells were lysed and precipitated with the SH2,3 fusion protein. The precipitates were washed and split in half; one-half was incubated with phenyl phosphate; and the supernatant was collected. The other half was not treated. The proteins from the supernatant (lanes 3 and 4) and the untreated beads (lanes 1 and 2) were separated by 10% SDS-PAGE, blotted onto PVDF membrane, and probed for MAPK. Lanes 1 and 3 are control cells; lanes 2 and 4 are CD3-stimulated cells. The bracket indicates MAPK isoforms. CD3X, cross-linked CD3; Ip, immunoprecipitate.
Figure 4: Bound MAPK is activated following TcR cross-linking. a, Jurkat cells were stimulated with anti-CD3 antibodies for the indicated time periods, and the lysates were precipitated with the SH2,3lckgst fusion protein. The precipitates were then assayed for MAPK activity on a synthetic MAPK substrate. b, the precipitates assayed in a were blotted for MAPK protein. The three isoforms of MAPK are bracketed. CD3X, cross-linked CD3; IP, immunoprecipitate.
Figure 6: Other SH2 and SH3 domains do not bind MAPK. Jurkat lysates were incubated with the indicated GST fusion proteins, and the precipitates were separated by 10% SDS-PAGE. The gel was blotted onto PVDF membranes and probed with an antibody to MAPK. Lane 1, glutathione-agarose beads alone; lane 2, Lck SH2 domain; lane 3, Src SH3 domain; lane 4, Abl SH3 domain; lane 5, Src SH2 domain; lane 6, Grb2; lane 7, Nck; lane 8, Csk SH2,3 domain; lane 9, whole cell (W/C) lysate of Jurkat cells. The band seen in lane 2 is a nonspecific band seen with secondary reagents alone. The MAPK isoforms are bracketed. Ip, immunoprecipitate.
It is shown in Fig. 1c that a
tyrosine-phosphorylated protein with a molecular mass similar to that
of the chain of the TcR was precipitated by the SH2,3lckgst
fusion protein following TcR cross-linking. This protein could be
recognized by antibodies to the
chain of the TcR, suggesting that
the binding is inducible.
To investigate this further,
precipitates of the fusion protein either before or after TcR
cross-linking were blotted with antibodies to the
chain of the
TcR. Fig. 7shows that the
chain can be detected only
after TcR cross-linking, and GST alone does not bind the
chain
(data not shown and Fig. 5b). This suggests that
tyrosine phosphorylation of the
chain could serve as a signal for
the binding of the SH2,3 domain of Lck. This is unlike the results seen
with MAPK, where the binding was constitutive and via the SH3 domain.
The binding of the
chain to the SH2,3 domain of Lck is largely
phosphotyrosine-dependent as a phosphotyrosine mimic, phenyl phosphate,
can compete for the binding and can elute the
chain from the
domains. In addition to the
chain, we also detected a protein of
70 kDa, which represents the
chain-associated kinase ZAP-70
(Fig. 8a) (very little
immunoreactivity was left on the beads following elution with phenyl
phosphate
). Since the
chain becomes associated with
ZAP-70 following TcR cross-linking(1) , one would expect to see
this molecule in precipitates containing the
chain. In accord
with the phosphotyrosine-dependent binding of the
chain to the
SH2,3 domain, the SH3 domain of Lck does not bind the
tyrosine-phosphorylated
chain (Fig. 5b).
Figure 7:
Regulation of binding of with
SH2,3lckgst by the TcR. Jurkat cells were stimulated with anti-CD3
antibodies, and the lysates were precipitated with the fusion protein.
The gel was then blotted for
using antisera to the
chain of
the TcR. Lane 1 is control lysates; lane 2 is
CD3-stimulated lysates. The arrow denotes the position of the
chain. CD3X, cross-linked CD3; Ip,
immunoprecipitate.
Figure 8:
The SH2,3 domain of Lck binds to the
chain in a phosphotyrosine-dependent manner. Jurkat cells were
stimulated or not with anti-CD3 antibodies, and the lysates were
precipitated with the SH2,3 domain fusion protein. The precipitated
proteins were incubated with phenyl phosphate, and the supernatants
were collected and separated by SDS-PAGE, blotted, and probed for
anti-phosphotyrosine antibodies (
pY) (a) and the
chain (b). CD3X, cross-linked CD3; Ip,
immunoprecipitate.
To
determine if the binding to the tyrosine-phosphorylated chain was
determined by the SH2 domain of Lck, similar experiments were performed
using the isolated SH2 domain of Lck. Fig. 9(a and b) demonstrates that the SH2 domain of Lck does not bind to
the tyrosine-phosphorylated
chain of the TcR, although it does
bind to a tyrosine-phosphorylated protein of
70 kDa
(which represents ZAP-70)(17) . The SH2,3 domain of Lck,
on the other hand, does bind to the tyrosine-phosphorylated
chain. Thus, the SH2 domain alone is insufficient for binding to the
tyrosine-phosphorylated
chain, the binding of which requires the
SH3 domain along with the SH2 domain as the binding is
phosphotyrosine-dependent. This binding was direct as it was also
observed to the same level under denaturing conditions.
The
SH2 domain of Src, however, does bind to the tyrosine-phosphorylated
chain (Fig. 9, a and b), suggesting that
the Src SH2 domain has higher affinity for the tyrosine-phosphorylated
chain. The SH2,3 domains of Csk and Emt/ITK (Fig. 9b) did not bind to the tyrosine-phosphorylated
chain. Fusion proteins of full-length Grb2 and Nck, molecules
with both SH2 and SH3 domains, both bound to the
tyrosine-phosphorylated
chain. While the SH2 domain of Grb2 has
been reported previously not to bind the tyrosine-phosphorylated
chain(18) , we have used full-length Grb2, and the presence of
the SH3 domains may increase the binding affinity for the
tyrosine-phosphorylated
chain, similar to our findings with Lck.
Thus, the data with the Lck fusion proteins fit well with the recently
derived crystal structure of Lck and a tyrosine-phosphorylated peptide
modeled after the C-terminal tail of Lck, suggesting that this
phosphopeptide fits in the groove between the SH3 and SH2
domains(19) . Therefore, the SH3 domain may be important in
determining binding to some tyrosine-phosphorylated proteins, as
demonstrated here for binding to the tyrosine-phosphorylated
chain.
Figure 9:
The SH2 and SH3 domains of Lck are
required for binding of the phosphorylated chain. Jurkat cells
were either not stimulated(-) or were stimulated for 2 min with
anti-CD3 antibody and rabbit anti-mouse (+). The cells were lysed
and precleared with GST-agarose beads. Proteins were then precipitated
with the GST fusion proteins indicated. The precipitates were washed
and separated by SDS-PAGE, blotted onto PVDF membrane, and probed with
an antibody to the
chain. The arrows indicate the
chain. a: lanes 1 and 2, Lck SH2 domain; lanes 3 and 4, Grb2; lanes 5 and 6,
Nck; lanes 7 and 8, Src SH2 domain. b: lanes 1 and 2, Src SH2 domain; lanes 3 and 4, Csk SH2,3 domain; lanes 5 and 6, Lck
SH2,3 domain: lanes 7 and 8, Lck SH2 domain; lanes 9 and 10, Lck SH3 domain; lane 11,
Grb2; lane 12, Src SH3 domain; lane 13, Emt PH
(pleckstrin homology) SH2,3 domain. -, control unstimulated
cells; +, CD3-stimulated cells. CD3X, cross-linked CD3; Ip, immunoprecipitate.
Since a number of molecules become tyrosine-phosphorylated
following TcR cross-linking, the SH2,3 domain of Lck may bind to these
molecules nondiscriminately. Arguing against this is the fact that only
a subset of the molecules that become tyrosine-phosphorylated following
TcR cross-linking are bound by the fusion protein. In
addition, Fyn, phospholipase C-
1, and Ras GTPase-activating
protein could not be detected in the precipitates before or after TcR
cross-linking.
To determine if these interactions occur in vivo, we stimulated Jurkat cells with pervanadate.
Immunoprecipitates were formed using antibodies to the chain, and in vitro kinase reactions were performed. The precipitates
were then washed and boiled to release the precipitated proteins. The
resultant proteins were re-immunoprecipitated using antibodies against
either Lck or the anti-
chain. Fig. 10a shows that Lck
can be re-immunoprecipitated from the anti-
precipitates. In
similar experiments in which cells were stimulated with anti-CD3
antibodies, anti-Lck immunoprecipitates were probed with anti-MAPK
antibodies (Fig. 10b), demonstrating that MAPK is present
both before and after stimulation. Finally, when Jurkat cells were
stimulated with anti-CD3 antibodies and Lck was immunoprecipitated, an
increase in MAPK activity against a MAPK substrate could be detected (Fig. 10c).
Figure 10:
Binding of the chain and MAPK to
Lck in vivo. Jurkat cells were stimulated with pervanadate for
15 min and lysed, and either the
chain (a) or Lck (b) was immunoprecipitated. Following an in vitro kinase reaction, the precipitates were boiled and reprecipitated
with the indicated antibodies. a, shown is the anti-
chain followed by anti-Lck antibodies (lanes 1 and 2)
or anti-
antibodies (lanes 3 and 4). The upper unlabeled arrow points to a nonspecific band, and the lower arrow points to Lck. b, cells were stimulated
with anti-CD3 antibodies (lane 2) or not (lane 1),
and the Lck precipitates were probed with anti-MAPK antibodies. Lane 3 is total cell protein from rat fibroblasts. Lanes 1 and 2 are longer exposures than lane 3. The arrow points to MAPK. c, Jurkat cells were stimulated
with anti-CD3 antibodies for 2 min, and Lck was immunoprecipitated and
analyzed for the presence of MAPK using a MAPK kinase-specific
substrate in kinase reactions. The open bar represents resting
cells, and the solid bar represents TcR-stimulated cells. Ip., immunoprecipitate; Ip. ab, immunoprecipitated
antibody.
The TcR signals given to T cells result in the tyrosine
phosphorylation of numerous molecules. Some of these molecules are
activated as a consequence of this phosphorylation. Another consequence
of tyrosine phosphorylation can be relocation within the cell, such as
from the cytoplasm to the membrane, or association with other
molecules. We suggest that some of the molecules that are
tyrosine-phosphorylated as a consequence of TcR signaling bind to the
SH2,3 domain of Lck and localize themselves to this kinase. Lck would
then be able to act on these molecules. Peri et al.(21) and Amrein et al.(20) have recently
reported that a GST fusion protein of the SH2 domain of Lck can
interact with numerous tyrosine-phosphorylated molecules in T
lymphocytes following CD3 cross-linking and in fibroblasts,
respectively. We demonstrated herein that the SH2,3 domain of Lck can
interact with both MAPK and the chain of the TcR, with the
interaction with MAPK occurring in a phosphotyrosine-independent manner
via the SH3 domain and the interaction with the
chain occurring
in a phosphotyrosine-dependent manner via the SH2 and SH3 domains.
Furthermore, we demonstrated that the pool of MAPK that can associate
with the SH3 domain of Lck does so both before as well as after TcR
cross-linking. Of interest are findings that Lck may be associated with
the kinase Raf1 (22) or Raf1-related kinases(23) . The
Raf1 kinase is upstream of MAPK (24) and may be found in
complexes with MAPK/extracellular-regulated kinase and
Ras(25) . As MAPK/extracellular-regulated kinase can interact
with MAPK, one may expect that MAPK also associates with Lck. Finally,
Lck is a substrate for activated MAPK, with MAPK phosphorylating Lck on
Ser
following TcR and B cell receptor
cross-linking(26) . Recently, Chou and Hanafusa (27) reported a serine/threonine kinase Nck-associated kinase
that interacts with the SH3 domain of Nck. This kinase was different
from the known MAPKs. In addition, Weng et al.(28) have reported a serine/threonine kinase different
from MAPK that interacts with the Src SH3 domain. These results add
credence to the results obtained in this report that serine/threonine
kinases can interact with SH3 domains.
We also demonstrated that the
tyrosine-phosphorylated chain only associates with the SH2 domain
of Lck when the SH3 domain is present. The fact that the binding is
inducible following TcR cross-linking, when the
chain becomes
tyrosine-phosphorylated, suggests that the binding is via a
phosphotyrosine moiety. This was confirmed by the phenyl phosphate
competition experiments. Thus, the binding of the SH2,3 domain of Lck
to the tyrosine-phosphorylated
chain is unlike the binding of the
SH2 domain of Lck to the ZAP-70 tyrosine kinase (17) . This
result also rules out an indirect binding effect since we can detect
binding of the SH2 domain only to ZAP-70, but not to the
tyrosine-phosphorylated
chain.
These results fit the
crystallographic model of Lck by Eck et al.(19) : the
two domains (SH2 and SH3) complexed with the Lck C-terminal
phosphopeptide, with the peptide bound in a phosphotyrosine-dependent
manner, fitted in a groove running between the two domains.
The
association of the tyrosine-phosphorylated chain with Lck would
seem to follow as
is a substrate for Lck (1) and becomes
tyrosine-phosphorylated both after activation of Lck as well as after
cross-linking of the TcR(29) . Thus, one can envision a
scenario in which Lck tyrosine-phosphorylates the
chain and then
associates with it, leading to the localization of kinase activity at
the signaling complex. Since phospholipase C-
1 has been shown to
associate with the TcR complex (30) as well as with Lck, in
this case via the SH2 domain of phospholipase C-
1(31) ,
this would place Lck in an optimal position for its action. This
localization of tyrosine kinases may serve to increase the potency of
activation of the T cell. This is underscored in T cell lines that lack
the
chain. Normally, cross-linking of the CD4-Lck complex with
the TcR complex increases the potency of activation; however, in these
cell lines lacking the
chain, the increase in activation when
CD4-Lck is co-cross-linked with the TcR complex does not
occur(32) . Recently, Thome et al.(33) reported co-immunoprecipitation of
with Lck,
although they proposed that this association occurred via the SH2
domain of Lck binding to ZAP-70 as they did not detect the
chain
using the isolated Lck SH2 domain, similar to the results reported
here. It is, however, likely that in the full-length protein, the SH3
domain cooperates with the SH2 domain in binding to the
chain, as
seen in our studies and as supported by crystallographic
analysis(19) .
The binding of the SH2,3 domain of Lck to the
chain of the TcR suggests that binding of this domain of Lck can
serve not only to bind and localize itself to intracellular enzymes and
substrates, but also to heighten the interaction of the associated CD4
with the TcR complex by binding to the tyrosine-phosphorylated
chain. The reported interactions of CD4 with the TcR (32) may
be a result of such an interaction. In addition, this interaction has
been postulated to increase following cross-linking of the TcR or
contact of the TcR with antigen plus the major histocompatibility
complex. This would then serve to strengthen the avidity of the TcR and
its associated co-receptor CD4 for the antigen-major histocompatibility
protein complex. There is some support for this hypothesis as CD4
mutants that cannot bind Lck do not interact with the TcR, and these
cells have diminished responses to antigen(34) . The SH2,3
domain of Lck can thus serve a kinase-independent function in T cell
activation by binding multiple tyrosine-phosphorylated molecules,
including MAPK and the tyrosine-phosphorylated
chain. In
conclusion, there are obviously additional substrates that bind to Lck,
and by identifying them, we may be able to discern the full role of Lck
in signal transduction via the TcR and activation of the T cell.