(Received for publication, May 15, 1995; and in revised form, November 16, 1995)
From the
The COOH-terminal Src kinase (Csk) is responsible for the
phosphorylation of the conserved, negative regulatory,
carboxyl-terminal tyrosine of most of the Src family protein tyrosine
kinases. Up to now, no stable binding of Csk to Src kinases has been
detected. We therefore decided to analyze this interaction using two
systems which allow detection of transient interaction. We produced and
purified recombinant proteins in the glutathione S-transferase
prokaryotic expression system. First, using real-time biospecific
interaction analysis (BIAcore), we detected in vitro a specific interaction between Csk and one of its substrates Lck,
a lymphocyte-specific member of the Src family. This interaction
requires the autophosphorylation of Lck on tyrosine 394 (the
phosphorylation of which is correlated with an increase of the kinase
activity) and involves a functional Csk SH2 domain. Second, using the
yeast two-hybrid system, we confirmed in vivo the physical
interaction between Csk and Lck. Furthermore, in vitro we
showed that autophosphorylation of Lck on tyrosine 394 enhances the
phosphorylation of Lck by Csk on the negative regulatory site, tyrosine
505, suggesting that activated Lck serves preferentially as substrate
for Csk. These findings might explain the mechanism(s) by which Csk
interacts with most of Src kinases to down-regulate their kinase
activity.
The protein tyrosine kinases (PTKs) ()of the Src
family are nonreceptor kinases which are involved in cell proliferation
and differentiation. This family comprises nine proto-oncogenes:
c-src, c-yes, fyn, lyn, lck, blk, hck, c-fgr, yrk(1) . The kinase activity of Src PTKs is regulated
by phosphorylation of two highly conserved tyrosine (Tyr) residues. One
of these residues, located in the catalytic domain, is involved in
positive regulation of the kinase activity upon
autophosphorylation(2) . Contrary to their viral counterparts,
the products of the cellular genes have their activity repressed by
phosphorylation of their COOH-terminal Tyr (3) which probably
generates a binding domain for their own Src homology 2 (SH2) domain,
thereby placing the molecule in an inactive
conformation(4, 5) . A widely expressed PTK isolated
initially by Okada and Nakagawa in 1988, Csk, has been shown in
vitro to phosphorylate the negative regulatory Tyr of
c-Src(6, 7) , Lyn, Fyn(8) , Lck(9) ,
and c-Fgr (10) .
Csk is related to Src PTKs but does not contain a myristylation site, or the conserved autophosphorylated Tyr and the regulatory COOH-terminal Tyr(6, 11) . Thus, the mechanism by which Csk function is regulated is not known. Similar to Src kinases, Csk contains one SH3 and one SH2 domain, which both are found in a number of catalytic and non catalytic signal transduction molecules and mediate protein-protein interactions(12) . In Src kinases, the SH2 domain is considered to be involved in the intramolecular suppression of the kinase activity (4, 5) and in the binding to sequence specific tyrosine phosphorylated proteins(13, 14) , whereas the SH3 domain is involved in the recruitment of substrates containing proline-rich sequences(15) .
Several systems provide evidence for the involvement of Csk in the regulation of cell activation through down-regulation of Src PTKs. Thus in vivo, (i) overexpression of Csk in v-Crk/c-Src transformed fibroblast cells causes reversion to normal phenotype(16) , (ii) overexpression of Csk in a T cell line inhibits TcR-induced tyrosine protein phosphorylation and lymphokine production(17) , (iii) the kinase activity of Lyn is constitutively activated in Csk-negative B-cell clones(18) , and (iv) coexpression of Csk counteracts cell death caused by expression of c-Src in Schizosaccharomyces pombe(19) and Saccharomyces cerevisiae(20) . Furthermore, Csk-deficient mouse embryos are developmentally arrested at the somite stage, and the kinase activity of several members of the Src family is greatly enhanced in these embryos(21, 22) .
Although Src is a substrate
of Csk, no stable binding of Csk to c-Src or v-Src has been
detected(11, 23) . Therefore, we postulated that the
binding of Src PTKs to Csk must be transient with a rapid off-rate, and
decided to study the interaction between Csk and one of its substrate
Lck, a Src PTK involved in T cell signaling(24) , using
real-time biospecific interaction analysis
(BIAcore)(25) . Toward this end, we produced
recombinant Csk and Lck proteins in bacteria using the glutathione S-transferase (GST) expression system (26) . We
demonstrated that Csk interacts physically with Lck through its SH2
domain and that this interaction requires the phosphorylation of
Tyr
of Lck. We confirmed the interaction between Csk and
Lck in vivo using the yeast two-hybrid system which also
allows to detect transient interaction(27) . Furthermore,
through in vitro phosphorylation assays, we demonstrated that
Lck phosphorylation on Tyr
by Csk is enhanced by
autophosphorylation of Lck on Tyr
.
We have previously shown that
with the exception of the inactive mutant (GST-Lck.K273E), all other
GST-Lck proteins were tyrosine phosphorylated in vivo in
bacteria due to autophosphorylation. We have previously determined that
Tyr was phosphorylated in the GST-Lck.WT and
GST-Lck.Y505F mutant, and Tyr
was phosphorylated in the
GST-Lck.WT and GST-Lck.Y394F mutant(28) . Therefore, in our
binding and phosphorylation experiments, these GST-Lck proteins were
used without prior in vitro phosphorylation. On the contrary,
the purified c-Fgr was not tyrosine-phosphorylated in vivo. It
has been shown that c-Fgr autophosphorylates in vitro on the
conserved autophosphorylation site, Tyr
(10) .
Thus, for our binding studies, we used c-Fgr either nonphosphorylated
or after in vitro autophosphorylation.
Figure 1: Binding studies of recombinant GST-Lck and purified c-Fgr to immobilized Csk. Purified Csk was immobilized within the flow-cell matrix and each protein was injected for 10 min. Arrows indicate the degree of binding, in relative RU, taken just at the end of the injection. Regeneration after each binding experiment was performed by continuous buffer flow for 20 min. A, purified GST-Lck proteins were injected at a concentration of 1 µM. B, Purified GST-Lck.Y505F and purified c-Fgr, either prephosphorylated in vitro with cold ATP (+ATP) or not (-ATP), were injected at 500 nM.
In a search for modified sequences surrounding the autophosphorylated Tyr of Src family PTKs, we noticed that c-Fgr is the only Src PTK to have a different motif at the autophosphorylation site (Tyr-Asn-Pro-Cys instead of Tyr-Thr-Ala-Arg)(41) . Therefore we tested the interaction of purified c-Fgr with immobilized Csk (Fig. 1B). We did not detect any binding of c-Fgr to Csk either nonphosphorylated (30 RU) or after in vitro autophosphorylation (32 RU), whereas, on the same Csk surface, a strong binding of GST-Lck.Y505F was measured (750 RU). This suggests that the conserved sequence at the autophosphorylation site is required for Lck interaction with Csk.
To determine which part of Csk was involved in the interaction with
Lck, we injected GST-Lck.Y505F either alone or preincubated with
different subdomains of Csk over a Csk immobilized surface. The results
are summarized in Table 1. Complete inhibition of the binding was
observed when GST-Lck.Y505F was preincubated either with full-length
Csk (purified without GST) or with GST-Csk SH3/SH2 fusion protein.
However, the recombinant GST-Csk SH3/SH2.S108C protein with the
point-mutation in the SH2 domain which abolishes its binding to
phosphotyrosine proteins(30) , did not prevent Lck interaction
with Csk. Furthermore the binding was not affected by preincubation of
GST-Lck.Y505F with GST-Csk SH3 fusion protein. These results suggest
that Csk interacts with Lck via its SH2 domain. Surprisingly, the
GST-Csk SH2 fusion protein was unable to inhibit this interaction. By in vitro binding assays, we have observed that Csk SH2 domain
alone was unable to bind phosphotyrosine proteins, whereas Csk SH3/SH2
domains clearly displayed such an
interaction()(30) . These results suggest that the
SH3 domain of Csk participates in the interaction, presumably by
allowing the SH2 domain to have the required steric conformation.
Figure 2:
Kinetic analysis of recombinant GST-Lck.WT
and GST-Lck.Y505F interaction with immobilized Csk. Purified GST-Lck.WT
and GST-Lck.Y505F proteins were used at various concentrations (conc.) ranging from 62.5 nM to 1000 nM and
allowed to interact with immobilized Csk for 10 min (sensorgrams A and B, respectively). The association phase was analyzed
in a dR/dt versus R plot for each GST-Lck concentration. The
slope values obtained are plotted against GST-Lck.WT and GST-Lck.Y505F
concentrations (C and D, respectively). The
dissociation phase was followed in continuous buffer flow during 200 s
after the end of the injection and was analyzed in a ln (R/R
) versus time plot for GST-Lck.WT and GST-Lck.Y505F (E and F, respectively). Equilibrium values were determined by
fitting the steady state binding values (Req) to the binding
equation by Scatchard analyses, giving rise to a R
/GST-Lck concentration versus R
plot for GST-Lck.WT and GST-Lck.Y505F (G and H,
respectively).
Figure 3:
Interaction of Csk and Lck proteins in the
yeast two-hybrid system. The L40 reporter strain was transformed with
the indicated plasmids. Growth in the absence of histidine indicates
the interaction between hybrid proteins. L40
pLex-Ras/pGAD-Raf was used as a positive control. Each
patch represents an independent
transformant.
Figure 4:
Analysis of recombinant GST-Lck.WT
phosphorylation by Csk. A, in vitro phosphorylation
of Lck. 100 nM GST-Lck.WT was incubated in vitro with (lane 2) or without (lane 1) 100 nM of Csk
in presence of [-
P]ATP and analyzed on 10%
SDS-PAGE followed by autoradiography. B, tryptic
phosphopeptide mapping. In vitro phosphorylated GST-Lck.WT by
Csk (as described in A) was excised from dried acrylamide gels
and digested with trypsin (lane 1). 10 µg of Lck-Y505
synthetic tryptic peptide were phosphorylated with 40 nM of
Csk in presence of [
-
P]ATP (lane
2). Tryptic phosphopeptides were analyzed by running on 40%
polyacrylamide gel followed by autoradiography. C,
phosphorylation of Lck mutants. 100 nM of GST-Lck.WT (lane
1), GST-Lck.K273E (lane 2) or GST-Lck.Y394F (lane
3) was incubated in vitro with 100 nM of Csk in
presence of [
-
P]ATP and analyzed on 10%
SDS-PAGE followed by autoradiography. D, inhibition of Lck
phosphorylation. 100 nM of GST-Lck.WT was incubated in
vitro with 100 nM of Csk and
[
-
P]ATP, alone or in presence of various
concentrations of phosphorylated (P) and nonphosphorylated
Lck-Y394 peptide. The reaction was resolved by SDS-PAGE and the
radioactivity of labeled proteins was counted by Cerenkov radiation.
Results are expressed as a percent inhibition of GST-Lck.WT
phosphorylation and the values represent an average of three separate
experiments.
Using two systems which allow to detect transient
interaction, the in vitro real-time interaction analysis
(BIAcore) and the in vivo yeast two-hybrid
system, we have shown that Csk interacts physically with Lck. Through
BIAcore
experiments, we noticed the absence of detectable
binding of Lck.K273E and Lck.Y394F to Csk, and the inhibition of
Lck-Csk interaction by pre-incubation of Lck with Csk SH3/SH2 domains,
which is not observed with a mutant of Csk SH3/SH2 protein (S108C)
unable to bind phosphotyrosine proteins. This strongly suggests that
the autophosphorylated Tyr
of Lck interacts with the SH2
domain of Csk. Furthermore, using a Y505F mutant of Lck we observed an
increase in the rate of association but no change in the rate of
dissociation compared to wild-type Lck. On the one hand, the fact that
the Lck.Y505F mutant which corresponds to an hyperactive form due to
the absence of the negative regulatory site, binds to Csk with a higher
rate of association, suggests that the binding of Csk to Lck is
enhanced by an increase of the kinase activity of Lck through a yet
unknown mechanism. On the other hand, the fact that we did not observe
any change in the dissociation rate of the Lck.Y505F mutant which is
not phosphorylated by Csk, suggests that the release of Lck after
interaction with Csk is not due to phosphorylation by Csk, but might be
due to the transience of the interaction. Thus, the off-rate might be
too rapid to detect a stable complex between Src PTKs and Csk by
conventional binding assays(11, 23) . Finally, through in vitro phosphorylation experiments, we have also
demonstrated that Lck phosphorylated on Tyr
is more
efficiently phosphorylated on Tyr
by Csk, suggesting a
functional interaction between Lck and Csk involving the phosphorylated
Tyr
of Lck. Similarly, another group has observed that
Csk phosphorylates cellular proteins, including Lck, in a
phosphotyrosine dependent manner, suggesting that previous substrate
tyrosine phosphorylation may be critical for the substrate selection of
Csk. (
)Interestingly, we have shown by immunofluorescence
microscopy that upon T cell activation the cytoplasmic Csk (revealed
with fluorescent tagged monoclonal anti-Csk antibody) translocates to
plasma membrane where most of Lck is localized.
This is
compatible with the hypothesis that after T cell stimulation, membrane
bound Lck is activated, phosphorylated on Tyr
, which
creates a temporary binding site for Csk. We propose that the binding
of Csk SH2 domain to the autophosphorylated Tyr of the Src kinases
facilitates phosphorylation of the COOH-terminal Tyr by Csk thereby
repressing the kinase activity. This mechanism may be a general feature
for down modulation of the kinase activity of most Src PTKs.
We
observed that both the SH3 and the SH2 domains of Csk are required for
the binding to phosphorylated Tyr of Lck, suggesting that
the SH3 domain participates in the interaction. Such a role has been
reported by several groups (19, 20, 44) who
found that the SH3 domain of c-Src was required for the intramolecular
binding of the phosphorylated COOH-terminal Tyr to the SH2 domain.
Furthermore, Panchamoorthy et al.(45) have recently
shown that the presence of the adjacent SH3 domain increases the
affinity of Fyn SH2 domain for phosphotyrosyl peptide motifs. It
remains to determine the mechanism by which SH3 domains are involved in
phosphotyrosine-SH2 domains interactions. The recent crystal structure
of Lck SH3-SH2 fragment suggests that both the SH3 and the SH2 domains
participate to form dimer and that the phosphorylated COOH-terminal Tyr
binds at the intermolecular SH3/SH2 contact(46) .
The absence of Csk binding to autophosphorylated c-Fgr, the only Src PTK with a different sequence in the autophosphorylation site, is further evidence for the specificity of Csk SH2 domain interaction with amino acids surrounding the autophosphorylated Tyr. It has been shown that the selectivity of SH2 domains for specific tyrosine phosphorylated sequences is provided by the 3 amino acids immediately carboxyl-terminal of the phosphotyrosine(13, 14) . The finding that c-Fgr does not bind to Csk is consistent with the fact that once autophosphorylated, c-Fgr is no more susceptible to down-regulation by Csk phosphorylation. Indeed, previous autophosphorylation of c-Fgr does not affect its phosphorylation by Csk and even though autophosphorylated c-Fgr is still phosphorylated by Csk, this phosphorylation does not lead to down-regulation of c-Fgr. The authors propose that autophosphorylation of c-Fgr could induce an intermolecular interaction between the autophosphorylated Tyr and the SH2 domain, resulting in an active homodimeric form(10) .
An
interaction between Csk SH2 domain and the autophosphorylated Tyr of
Src PTKs has been postulated by Songyang et al.(14) who have shown that the sequence motif Tyr(P),
Thr/Ala, Lys/Arg, Met/Ile/Val/Arg which is found within the
autophosphorylation site of the Src kinases (with the exception of
c-Fgr), has high affinity for the SH2 domain of Csk. Furthermore, a
functional and physical interaction of Fyn and Csk has been reported
and a mutant of Fyn that is highly autophosphorylated on Tyrin vivo was shown to form a more stable complex with Csk
than with wild-type Fyn(47) .
The regulation of Csk-Src PTKs
interaction proposed in this report does not explain the fact that an
inactive form of Src expressed in mouse embryo fibroblasts lacking
endogenous Src, is still phosphorylated on Tyr(48) and a Lck.Y394F mutant transfected in NIH3T3 cells
is still phosphorylated on Tyr
(5) . However, in
fibroblasts these phosphorylations might be due either to
autophosphorylation, as previously reported for Src in yeast cells (49) and for Lck in bacteria(28) , or to
phosphorylation by other members of the Csk family. It has been shown
that in cell lines established from embryos lacking Csk, the endogenous
c-Src is still phosphorylated on Tyr
(21) ,
suggesting that other kinases may phosphorylate this site in
vivo. Several groups have recently described cDNAs encoding a
second Csk-related protein tyrosine kinase: termed either matk(50) , hyl(51) , ctk(52) , ntk(53) , or lsk(54) . These kinases might have different
affinities for Src PTKs and might be responsible for the regulation of
those PTKs which do not bind to Csk. In vitro Ctk/Ntk is
capable of phosphorylating Lck at its negative regulatory site (52, 53) and might also phosphorylate the Lck.Y394F
mutant. In the same way, Matk can phosphorylate the COOH-terminal Tyr
of c-Src (55) and might also phosphorylate the Src.Y416F. Thus,
it is likely that these Csk-related kinases are capable of
phosphorylating the COOH-terminal Tyr of Src PTKs in the absence of the
autophosphorylated Tyr.
Little is known about the regulation of Csk
itself. It has been shown that Csk SH3 an SH2 domains are both required
for the suppression of c-Src kinase activity (30) and for its
negative impact on T-cell activation(56) , suggesting that Csk
kinase activity is regulated through these domains. Colocalization of
Csk with activated Src to podosomes has been observed, and this
requires both the SH3 and SH2 domains of Csk but not its kinase
activity, suggesting that these domains are both necessary to target
Csk to places where Src is active (23) . It has been proposed
that Csk delocalization from the cytoplasm to the plasma membrane upon
Src activation, is due to an interaction between Csk SH2 domain and the
tyrosine phosphorylated Ras GTPase activating-associated p62
protein(57) . It has also been proposed that the binding of Csk
SH2 domain to tyrosine phosphorylated cytoskeleton proteins, paxillin
and pp125, may promote the accessibility of Csk to c-Src
localized at certain subcellular regions (30, 58) .
However, the cytosolic non myristylated form of c-Src can be fully
phosphorylated at Tyr
(59) , suggesting that Csk
can phosphorylate c-Src in cytoplasm in the absence of any interaction
with others proteins. All these findings strongly suggest that Csk
function is regulated through protein-protein interactions involving
its SH2 and/or SH3 domains. These domains may be required to target
cytosolic Csk to the plasma membrane or cytoskeletal structure, where
the known cellular substrates for Csk are localized. This could occur
either through an interaction of Csk SH2 domain with tyrosine
phosphorylated Src PTKs substrates, or, as we propose, through an
interaction of Csk SH2 domain with autophosphorylated Src PTKs. In both
case, this interaction occurs only after Src PTKs activation.
In
sum, we showed that (i) the interaction of Csk with Lck requires the
phosphorylated Tyr of Lck, the phosphorylation of which
is correlated with its kinase activity; (ii) the hyperactive form of
Lck (Y505F) has much higher affinity for Csk than the wild-type Lck,
and (iii) Lck phosphorylated on Tyr
is more efficiently
phosphorylated by Csk. Altogether these results suggest that activated
and autophosphorylated Lck interacts preferentially with Csk. We
propose that the modification of Lck accessibility upon activation may
be a mechanism for the control of Csk-Lck interaction. Therefore, one
might assume that the control of Csk function occurs through its
substrate accessibility rather than through an intrinsic mechanism.