(Received for publication, July 5, 1995; and in revised form, January 9, 1996)
From the
Tyrosine kinase activity is required for signal transduction
through the T cell antigen receptor (TCR). The Src family tyrosine
kinase Lck appears to play a key role in the initiation of TCR
signaling events. We have investigated the role of the
phosphotyrosine-binding Src homology-2 (SH2), domain of Lck in TCR
signaling. Lck containing a mutation in the phosphotyrosine binding
pocket of the SH2 domain was expressed in an Lck-deficient cell line.
We found that, in contrast to wild-type Lck, the SH2 domain mutant was
unable to restore even the earliest TCR-mediated signaling events. To
investigate the role of the Lck SH2 domain, we examined the association
of tyrosine phosphoproteins with Lck. The predominant associated
phosphoprotein was the ZAP-70 tyrosine kinase, which has also been
implicated in the initiation of TCR signaling. In addition, the
subunit of the T cell receptor was found to weakly associate with Lck.
Further analysis indicated that the SH2 domain of Lck can directly
recognize both ZAP-70 and
in immunoprecipitates from
TCR-stimulated cells. Our findings demonstrate that the SH2 domain of
Lck is essential for the initiation of signaling events following TCR
stimulation probably as a result of its ability to mediate an
interaction between Lck and the ZAP-70 tyrosine kinase and/or the
subunit of the T cell receptor.
Stimulation of the T cell antigen receptor (TCR) ()initiates intracellular signaling events that lead to T
cell differentiation and proliferation. Several pathways have been
implicated in this activation process, notably the induction of
tyrosine phosphorylation and the phosphatidylinositol and Ras signaling
pathways(1, 2) . Cross-linking of the TCR results in
the recruitment and apparent activation of protein-tyrosine kinases,
which then leads to the phosphorylation of downstream substrates. The
increase in tyrosine phosphorylation of cellular proteins is a crucial
event in TCR signaling(3) . Tyrosine phosphorylation of
phospholipase C-
1 is increased following TCR
stimulation(4, 5) , which leads to an induction of
enzymatic activity and an increase in the second messengers of the
phosphatidylinositol pathway. Activation of the Ras pathway also
requires protein-tyrosine kinase activity (6) . Ras may become
activated through phosphotyrosine-dependent recruitment of a guanine
nucleotide exchange factor to the membrane (7) or through the
tyrosine phosphorylation and activation of Vav(8) .
At least
two distinct types of protein-tyrosine kinases have been implicated in
TCR signal transduction. The ZAP-70 tyrosine kinase, homologous to Syk,
associates with the tyrosine-phosphorylated cytoplasmic domains of the
TCR subunits following receptor stimulation (9, 10) .
Patients with a rare form of severe combined immunodeficiency have been
identified whose T cells fail to express ZAP-70. These patients exhibit
a defect in the development of CD8+ T cells, and their peripheral
CD4+ T cells fail to respond to stimulation through the TCR (11, 12, 13) . In addition to ZAP-70, the Src
family of tyrosine kinases has been implicated in the initiation of
signaling through the TCR. Expression of the activated forms of either
the Fyn or Lck tyrosine kinases leads to enhanced tyrosine
phosphorylation following TCR
stimulation(14, 15, 16) . Fyn can be found
associated with the TCR(17) , and mature thymocytes that are
homozygous for null alleles of Fyn show defects in TCR-mediated signal
transduction(18, 19) . Mice that lack a functional Lyk
gene show a severe defect in thymocyte development(20) . This
may be related to a deficit in TCR function since thymocytes that carry
null alleles at the TCR locus are blocked at the same stage of
development(21) . Direct evidence for the role of Lck in TCR
signaling has come from the analysis of cell lines that are deficient
in Lck function(22, 23) . These mutant cell lines fail
to respond to stimulation through the TCR. Previously we have shown
that signaling events are blocked at the earliest stages following TCR
stimulation in an Lck kinase-deficient derivative of the Jurkat cell
line(22) . Subsequent work using a COS cell reconstitution
system has indicated that one function of Lck in the initiation of TCR
signaling is to mediate the association of ZAP-70 with the receptor
through the phosphorylation of the cytoplasmic domains of the
TCR(24) .
To further examine the action of Lck in the initiation of TCR signaling events, we have investigated the role of the Lck Src homology 2 (SH2) domain. SH2 domains can mediate protein interactions by virtue of their ability to bind phosphotyrosine residues, and they are present in a large number of signal-transducing proteins(25) . The SH2 domains of the Src family kinases are thought to participate in the control of kinase activity through an intramolecular interaction with a carboxyl-terminal phosphotyrosine residue(26) . Consistent with this, deletion of the Lck SH2 domain enhances its oncogenic potential in fibroblasts(27) . However, such a deletion also inhibits the ability of an activated form of Lck to increase tyrosine phosphorylation in T cells, suggesting that the SH2 domain may also have a positive role in Lck function(28) . We have taken advantage of the Lck-deficient cell line JCaM1 to examine how the phosphotyrosine binding function of the SH2 domain of Lck might contribute to the initiation of TCR signal transduction.
To study the function of the Lck SH2 domain in TCR signal transduction we took advantage of the Lck kinase-deficient cell line JCaM1. This mutant, which was derived from Jurkat, is defective in its response to stimulation of the TCR unless wild-type Lck is re-expressed by transfection(22) . To determine the importance of the SH2 domain in Lck function, we transfected JCaM1 with an IL-2-luciferase reporter plasmid and either wild-type Lck or Lck containing an arginine to lysine substitution at position 154 (LckR154K). The arginine at this position is crucial for SH2 domain function since it is responsible for making direct contact with the substrate phosphotyrosine residue(35, 36) . As shown in Fig. 1, stimulation of the TCR on JCaM1 leads to an induction of luciferase expression following transfection with wild-type Lck but not with the LckR154K mutant. This finding indicates that the SH2 domain function of Lck is essential at some point in the TCR signaling pathway that leads to IL-2 gene expression.
Figure 1: Induction of IL-2-luciferase activity in JCaM1 transfected with Lck+ or LckR154K. JCaM1/TAg cells were transfected with an IL-2-luciferase reporter plasmid and pBJ-neo-Lck+ or pBJ-neo-lckR154K, or without an Lck plasmid (mock). 48 h after transfection cells were left unstimulated or were stimulated with phorbol 12-myristate 13-acetate alone (solid bars), or with phorbol 12-myristate 13-acetate and anti-TCR antibody C305 (open bars). 6 h after stimulation, cells were lysed and luciferase activity was measured. The data are presented as the ratio of luciferase activity in cell extracts from stimulated and unstimulated cultures. In parallel experiments, immunoblotting cell lysates from transfectants demonstrated that equivalent levels of Lck+ and LckR154K were expressed. The data are representative of three experiments.
To understand how the SH2 domain of Lck contributes to TCR signaling, we undertook a biochemical analysis of JCaM1 transfectants expressing LckR154K to determine at what step the signaling pathway was blocked. Cell lines stably expressing LckR154K were obtained by electroporation of JCaM1. However, the level of Lck expression achieved was only 10-20% of levels normally found in Jurkat. Thus, a JCaM1 transfectant expressing similar levels of wild-type Lck was obtained as a matched control for the LckR154K transfectant (Fig. 2). One of the early events that occurs following TCR stimulation is an increase in intracellular calcium levels due to the induction of the phosphatidylinositol pathway. Analysis of calcium levels using the fluorescent calcium-binding dye indo-1 indicated that receptor stimulation was unable to elicit a significant rise in intracellular calcium in JCaM1 transfected with LckR154K (Fig. 3B). This result is in contrast with the rise in intracellular calcium that is observed following stimulation of the TCR on JCaM1 transfected with wild-type Lck (Fig. 3A and (22) ). Thus, the Lck SH2 domain is required for function of the TCR signaling pathway prior to the release of calcium from intracellular compartments.
Figure 2: Lck levels in lysates of Jurkat, JCaM1/Lck+, and JCaM1/LckR154K. Nonidet P-40 lysates of equivalent cell numbers were prepared from the indicated cell lines and analyzed on an 8% SDS-polyacrylamide gel, blotted onto nitrocellulose, and probed with anti-Lck antibody. The lower molecular weight band present in the JCaM1 transfectants is also present in parental JCaM1 lysates and represents the protein product of mis-spliced Lck mRNA expressed in JCaM1(22) .
Figure 3: Intracellular calcium levels in JCaM1/Lck+ (A), JCaM/LckR154K (B), and JCaM1 (C) following TCR stimulation. Calcium levels were measured at 37 °C in a spectrofluorimeter using the calcium-binding dye indo-1 described under ``Materials and Methods.'' Cells were stimulated with saturating amounts of anti-TCR antibody C305 as indicated. All three cell lines demonstrated a large increase in intracellular calcium in response to treatment with ionomycin.
To determine the contribution of Lck SH2
domain function to the earliest signaling events, we examined induction
of tyrosine phosphorylation of cellular proteins following receptor
stimulation of the LckR154K transfectant. Lysates from unstimulated or
stimulated transfectants were resolved on SDS-polyacrylamide gels and
blotted with anti-phosphotyrosine antibody (Fig. 4). The
LckR154K transfectant showed only a weak induction of tyrosine
phosphoproteins compared with the matched wild-type control. This
finding suggests that the Lck SH2 domain plays an important role in the
initiation of the protein-tyrosine kinase pathway following TCR
stimulation. To define this role in more detail we examined the
tyrosine phosphorylation of the chain of the TCR and the ZAP-70
tyrosine kinase. Tyrosine phosphorylation of these proteins appears be
required for their role in the initiation of receptor
signaling(37, 38, 39) . Anti-phosphotyrosine
immunoblots of
or ZAP-70 immunoprecipitates from the
transfectants showed that tyrosine phosphorylation of these signaling
molecules induced by TCR stimulation was substantially reduced in the
LckR154K transfectant (Fig. 5, A and B). These
results show that the SH2 domain of Lck is required for the induction
and/or maintenance of normal levels of
and ZAP-70 tyrosine
phosphorylation following TCR stimulation.
Figure 4: Induction of tyrosine phosphoproteins in JCaM1/Lck+ and JCaM1/LckR154K following anti-TCR stimulation. Nonidet P-40 lysates were prepared from the indicated cell lines; samples were either unstimulated or stimulated for 2 min at 37 °C with the anti-TCR antibody C305. After resolution on 10% SDS-polyacrylamide gels, proteins were transferred to nitrocellulose and probed with anti-phosphotyrosine antibody.
Figure 5:
Induction of TCR and ZAP-70 tyrosine
phosphorylation following TCR stimulation in JCaM1/Lck+ and
JCaM1/LckR154K. A, was immunoprecipitated from Nonidet
P-40 lysates prepared from unstimulated or anti-TCR-stimulated cells.
Immunoprecipitates were analyzed on 11.5% SDS-polyacrylamide gels,
transferred to nitrocellulose, and probed with anti-phosphotyrosine
antibody. Stripping and reprobing the blot with anti-
antibody
demonstrated that equivalent levels of
were immunoprecipitated
from both cell lines. B, induction of ZAP-70 tyrosine
phosphorylation following TCR stimulation in JCaM1/Lck+ and
JCaM1/LckR154K. ZAP-70 was immunoprecipitated from Nonidet P-40 lysates
prepared from unstimulated or anti-TCR-stimulated cells.
Immunoprecipitates were analyzed on SDS-polyacrylamide gels,
transferred to nitrocellulose, and probed with an anti-phosphotyrosine
antibody. Parallel experiments demonstrated that the JCaM1/Lck+
and JCaM1/LckR154K cell lines express equivalent levels of ZAP-70 (data
not shown).
Since SH2 domains can
mediate protein interactions through the binding of phosphotyrosine
residues it is likely that the essential role of the Lck SH2 domain in
TCR signal transduction involves the association of Lck with a tyrosine
phosphoprotein. To identify potential substrates of the Lck SH2 domain,
we examined Lck immunoprecipitates from unstimulated and stimulated
cell lysates of the Jurkat T cell line with anti-phosphotyrosine
antibody. As shown in Fig. 6A, lane 4, several tyrosine
phosphoproteins were observed in Lck immunoprecipitates from cells
stimulated through the TCR. Most prominent among the tyrosine
phosphoproteins was Lck itself, both the 56- and 60-kDa forms. We have
confirmed the induced tyrosine phosphorylation of Lck by in vivo labeling studies (data not shown), and similar findings have been
previously reported(40) . In addition to Lck we observed a
70-kDa tyrosine phosphoprotein. This phosphoprotein co-migrated with
phospho-ZAP-70 present in immunoprecipitates from stimulated cells (Fig. 6A, lane 5). Although the Fyn tyrosine
kinase was also tyrosine-phosphorylated following TCR stimulation,
there was no detectable association of the 70-kDa tyrosine
phosphoprotein (Fig. 6A, lane 2). We confirmed
the identity of the 70-kDa tyrosine phosphoprotein by directly
immunoblotting Lck immunoprecipitates with anti-ZAP-70 antibody (Fig. 6B). In addition to ZAP-70 we have also
reproducibly observed a low level of a 20-23-kDa tyrosine
phosphoprotein in Lck immunoprecipitates from stimulated cells. This
phosphoprotein is at least in part the
subunit of the TCR since
it co-migrates with phospho-
present in ZAP-70 immunoprecipitates
and can be immunoprecipitated with anti-
antiserum following
solubilization of in vitro phosphorylated Lck
immunoprecipitates (data not shown).
Figure 6: Association of ZAP-70 with Lck. A, anti-phosphotyrosine blot of Fyn, Lck, or ZAP-70 immunoprecipitates from unstimulated or stimulated Jurkat cells. The Fyn, Lck, or ZAP-70 kinases were immunoprecipitated from Nonidet P-40 lysates of unstimulated or anti-TCR-stimulated Jurkat cells with anti-peptide antisera, resolved on SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with anti-phosphotyrosine antibody. B, ZAP-70 blot of Lck immunoprecipitates from unstimulated or stimulated Jurkat cells. Immunoprecipitates of Lck from Nonidet P-40 lysates of unstimulated or anti-TCR-stimulated cells were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose, and blotted with anti-ZAP-70 antibody. The bands at 50-55 kDa are immunoglobulin heavy chain from the immunoprecipitating antisera.
The presence of ZAP-70 in Lck
immunoprecipitates could be the result of an interaction between the
Lck SH2 domain and tyrosine-phosphorylated ZAP-70. To investigate this
we constructed glutathione S-transferase fusion proteins using
either the wild-type or the R154K mutant form of the Lck SH2 domain.
These fusion proteins were then incubated with lysates from
unstimulated or stimulated Jurkat cells, and the presence of ZAP-70 in
the precipitates was determined by immunoblotting (Fig. 7).
These results show that the Lck SH2 domain can associate with ZAP-70
from stimulated cells. The R154K mutation disrupted the interaction
between the Lck SH2 domain and ZAP-70 (Fig. 7, lanes 3 and 4) as well as the interaction between the Lck SH2
domain and other tyrosine phosphoproteins (data not shown). Since other
proteins are present in the GST-Lck(SH2) precipitates the interaction
between the fusion protein and ZAP-70 could be indirect. To address
this possibility we probed blots of ZAP-70 and immunoprecipitates
using a GST-Lck SH2 domain fusion protein (Fig. 8). These
studies showed that the fusion protein was capable of directly
interacting with both ZAP-70 and
. This interaction was enhanced
by TCR stimulation, suggesting that it was mediated by the increased
tyrosine phosphorylation of ZAP-70 and
following receptor
stimulation. These results indicate that the interaction of Lck with
ZAP-70 and
can be mediated by a direct interaction between the
Lck SH2 domain and phosphotyrosine residues on ZAP-70 and
.
Figure 7: Association of ZAP-70 with wild-type or R154K GST-Lck(SH2) fusion protein. Lysates from unstimulated or anti-TCR-stimulated Jurkat cells were incubated with 1 µg of GST-Lck+(SH2) or GST-LckR154K(SH2) fusion protein. The associated proteins were then analyzed on SDS-polyacrylamide gels, immunoblotted with anti-ZAP-70 antibody, and developed using a chemiluminescence detection system. The immunoblot was exposed for 1 min. Extended exposure of the immunoblot revealed bands corresponding to ZAP-70 in the GST-LckR154K(SH2) fusion protein precipitates. However, this association may be nonspecific, as it was unaltered by TCR stimulation.
Figure 8:
GST-Lck(SH2) fusion protein blot of ZAP-70
or TCR immunoprecipitates from unstimulated or
anti-TCR-stimulated Jurkat cells. ZAP-70 and
immunoprecipitates
were prepared from Nonidet P-40 lysates of unstimulated or
anti-TCR-stimulated cells. Immunoprecipitates were resolved on
SDS-polyacrylamide gels, transferred to nitrocellulose, and probed
first with purified GST-Lck(SH2) fusion protein, and then with anti-GST
antibody as described under ``Materials and
Methods.''
Our analysis indicates that the SH2 domain of Lck plays a
crucial role in TCR signal transduction. Expression of an Lck cDNA
containing an arginine to lysine substitution within the SH2 domain
(LckR154K) was unable to restore signaling function to the
Lck-deficient cell line JCaM1. The failure to restore signaling
function to JCaM1 occurred at the earliest steps in the signal
transduction pathway. Unlike the wild-type Lck transfectant, TCR
stimulation of JCaM1 transfected with LckR154K did not increase the
level of intracellular calcium or result in a significant induction of
tyrosine phosphoproteins. More detailed analysis showed that two of the
most proximal signaling events, tyrosine phosphorylation of the TCR
chain as well as tyrosine phosphorylation and recruitment of the
ZAP-70 tyrosine kinase, were substantially reduced in the LckR154K
transfectant. These results indicate that the function of the Lck SH2
domain is crucial for initiating signal transduction events following
TCR stimulation.
Crystallographic analysis of the Lck SH2 domain complexed with a phosphopeptide indicates that the arginine residue at position 154 makes direct contact with the phosphotyrosine residue of the peptide (35) . Substitution of the analogous arginine residue in c-Abl with lysine eliminates binding to phosphotyrosine(36) . Additionally, we have found that this mutation eliminates the ability of the Lck SH2 domain to associate with tyrosine phosphoproteins (data not shown). The fact that the LckR154K mutant is unable to function in the TCR signaling pathway suggests that the interaction of Lck with a tyrosine phosphoprotein is crucial for initiation of TCR-mediated signaling. Anti-phosphotyrosine blots of Lck immunoprecipitates revealed that a 70-kDa tyrosine phosphoprotein associated with Lck immediately following receptor stimulation. Direct blotting of Lck immunoprecipitates demonstrated that this protein was the ZAP-70 tyrosine kinase. To investigate whether the interaction of the two kinases was mediated by SH2-phosphotyrosine interaction we blotted ZAP-70 immunoprecipitates with a purified GST-LckSH2 domain fusion protein. These blots demonstrated that the association of Lck and ZAP-70 following TCR stimulation could be the result of a direct interaction between phosphotyrosine residues on ZAP-70 and the LckSH2 domain. We found that purified GST-LckSH2 domain fusion protein could associate with ZAP-70 in lysates from stimulated cells and confirmed that this interaction was disrupted by the R154K mutation. Similar biochemical results have been reported by Duplay et al.(41) .
Anti-phosphotyrosine blots of Lck
immunoprecipitates also revealed the weak association of a tyrosine
phosphoprotein that co-migrated with the TCR chain present in
ZAP-70 immunoprecipitates. Lck immunoprecipitates labeled by in
vitro phosphorylation with [
P]ATP do
contain
, as demonstrated by reprecipitation (data not shown).
Blotting of
immunoprecipitates with purified GST-LckSH2 fusion
protein further demonstrated a potential direct interaction between Lck
and the
chain. Lck has been found to associate with
in
natural killer cells in the context of the Fc
RIIIA(42) ;
however, it has been difficult to demonstrate such an association in T
cells. The presence of only a low level of
in Lck
immunoprecipitates may reflect a very unstable association of the two
molecules, and it may also reflect an indirect association in vivo since
has been shown to bind ZAP-70
tightly(24, 43) . It is likely that
serves as a
substrate for Lck in T cells, and the two molecules may remain
associated following phosphorylation of
via the SH2 domain of
Lck, but this interaction may be displaced by the stronger binding of
the dual SH2 domains of ZAP-70 to tyrosine-phosphorylated
(44) .
It appears likely that the interaction between
Lck and ZAP-70 mediated by the SH2 domain is crucial in the initiation
of TCR signaling. Co-expression of Lck and ZAP-70 in Sf9 cells leads to
the phosphorylation of ZAP-70 on the same sites as observed in
activated T cells and also results in an increase in ZAP-70 kinase
activity(39) . Cross-linking of the TCR leads to the
Lck-dependent phosphorylation of the TCR and CD3 cytoplasmic
domains as well as the recruitment of ZAP-70. Presumably, the
co-localization of Lck and ZAP-70 could lead to a direct interaction of
the two kinases, mediated by recognition of phosphotyrosine residues on
ZAP-70 by the Lck SH2 domain. Once bound, Lck may further phosphorylate
and activate ZAP-70, thereby leading to the phosphorylation and
activation of downstream signaling molecules. It is unclear whether Lck
binds ZAP-70 at a site of autophosphorylation or at a site previously
phosphorylated by Lck (or another kinase). This question could be
addressed in part by analyzing the ability of Lck to interact with a
kinase-deficient mutant of ZAP-70.
An alternative explanation for
the role of the Lck SH2 domain in TCR signaling is that it is required
not only for a direct interaction between Lck and ZAP-70, but also for
the phosphorylation of the TCR cytoplasmic domains, which is required
for the initial recruitment of ZAP-70 to the TCR. This is consistent
with the observation that phosphorylation is reduced in JCaM1
transfected with the LckR154K mutant. Recent studies on the
phosphorylation of the carboxyl-terminal tail of RNA polymerase II, or
p130, by the Abl tyrosine kinase has indicated that SH2 domain function
is required for complete phosphorylation of the
substrate(45, 46) . Presumably the SH2 domain allows
the kinase to remain in association with the substrate, thereby
promoting phosphorylation of multiple tyrosine residues. This model is
consistent with the observation that substrate specificity of tyrosine
kinases correlates with the specificity of their SH2
domains(47) . However, unlike the Abl kinase, mutation of the
Lck SH2 domain does not significantly reduce the ability of Lck to
phosphorylate the cytoplasmic domain of
in vitro (data
not shown). This may not be surprising in light of the fact that only
one of six tyrosines in the
cytoplasmic domain is found in the
preferred context for Lck SH2 binding, YEEI(48) .
Alternatively, the reduction in
phosphorylation observed with the
SH2 domain mutant may be due to reduced protection of phospho-
from dephosphorylation by tyrosine phosphatases. Phospho-
could be
protected through a direct interaction with Lck, as discussed above. In
addition, ZAP-70 has been shown to protect phospho-
from
dephosphorylation in COS cells(24) . The protection of
phospho-
may be enhanced by the SH2 domain-mediated interaction
between Lck and ZAP-70.
In previous work a functional interaction
between Lck and ZAP-70 was observed when both kinases were transfected
into COS cells(24) . Co-expression of the kinases led to
enhanced tyrosine phosphorylation of cellular proteins and the
association of ZAP-70 with the tyrosine-phosphorylated cytoplasmic
domain of the TCR chain. In contrast to our findings with the
JCaM1 transfectants, in COS cells Lck with the R154K mutation
functioned as well as wild-type Lck. The different requirements for
LckSH2 domain function probably reflect a difference in the level of
expression between the two experimental systems. The high level
expression of transfected cDNA clones in COS cells may have overcome
the requirement for the LckSH2 domain to mediate a specific interaction
between the two kinases or between Lck and the
cytoplasmic
domain.
In summary, our results indicate that the phosphotyrosine
binding function of the Lck SH2 domain is crucial for the role of Lck
in T cell receptor signal transduction. Lck carrying a mutation in the
phosphotyrosine binding pocket of the SH2 domain is unable to restore
even the earliest signaling events to an Lck-deficient cell line. The
major tyrosine phosphoprotein that associates with Lck is the ZAP-70
tyrosine kinase. TCR chain also associates with Lck but
apparently much more weakly than ZAP-70. Further experiments showed
that the Lck SH2 domain could mediate a direct interaction between Lck
and ZAP-70, as well as between Lck and
. Since the Lck SH2 domain
mutant is defective in signaling function, it is likely that the
interaction between Lck and ZAP-70 and/or possibly
, is essential
for the initiation of TCR signal transduction.