(Received for publication, November 16, 1995)
From the
p130 is a major tyrosine-phosphorylated protein
that tightly binds v-Crk in v-crk-transformed cells and v-Src
in v-src-transformed cells. The ``substrate domain''
of p130
contains 15 possible Src homology (SH) 2-binding
motifs, most of which conform to the binding motif for the Crk SH2
domain. Another region near its C terminus contains possible binding
motifs for the Src SH2 domain and proline-rich sequences that are
candidates for SH3-binding sites.
Using GST fusion proteins, we
revealed that both SH2 and SH3 domains of Src bind p130,
whereas v-Crk binds p130
through its SH2 domain. We
located the binding site of p130
for the Src SH3 domain
at the sequence RPLPSPP in the region near its C terminus. Mutations
within this sequence or at Tyr
of p130
caused a significant reduction in the association of p130
with Src, and no association was detected when both of them were
deleted. The kinase activity in v-Crk-transformed cells was also
associated with p130
through this region. On the other
hand, the deletion of the substrate domain abolished the binding with
v-Crk. The association through the C-terminal region of p130
with Src kinase may facilitate effective hyperphosphorylation of
tyrosine residues in the substrate domain of p130
,
resulting in the binding of SH2-containing molecules to
p130
.
Src family cytoplasmic tyrosine kinases, including Src, Fyn, and
Lyn, possess conserved, noncatalytic, and regulatory domains named Src
homology 2 (SH2) ()and Src homology 3 (SH3) domains. SH2
domains are composed of
100 amino acids and specifically interact
with sequences containing
phosphotyrosine(1, 2, 3) . SH3 domains are
composed of
50 amino acids and interact with proline-rich amino
acid sequences(1, 2, 3, 4) . These
SH2 and SH3 domains exert their functions, e.g. regulating
their own kinase activity(5, 6, 7) ,
connecting other signaling molecules to tyrosine
kinases(8, 9, 10) , and locating the proteins
to the site of cytoskeleton (11) , by inter- or intramolecular
association with specific polypeptide sequences.
The SH2 and SH3
domains are also found in many signaling molecules other than Src
family tyrosine kinases. Among them, proteins called ``adapter
proteins'' such as Crk(2) , Nck(12) , and
Grb2/Ash/Sem-5 (13, 14, 15) have only SH2
and SH3 domains and no catalytic domain. v-Crk, a transforming
oncoprotein encoded by avian sarcoma viruses, is a fusion protein of
viral gag protein and the SH2 and SH3 domains derived from
c-Crk, a cellular counterpart of v-Crk(2) . v-Crk has an
oncogenic potential and induces tyrosine phosphorylation of several
proteins when expressed in fibroblasts, although the mechanism of
phosphorylation is unknown. The most prominent tyrosine-phosphorylated
protein among them is p130, which forms a tight complex with
v-Crk(2, 16, 17) . We recently cloned the
cDNA for p130 and named its product as p130 (
Crk-associated substrate)(18, 19) . On the other hand,
activated forms of Src tyrosine kinase, e.g. v-Src and
527F-c-Src, are known to induce tyrosine phosphorylation of several
cellular proteins(20, 21) . One of the major
tyrosine-phosphorylated proteins, p130, appears in v-Src-transformed
cells and is known to bind
Src(20, 22, 23, 24) . Using the
peptidase mapping analysis, we demonstrated that the p130 associated
with v-Src is identical to p130
(18) . Although
the phosphorylation of p130
was shown to closely
correlate with the transformation in NIH 3T3 cells(18) , the
mechanism of phosphorylation of p130
is unclear.
The
primary structure of p130 reveals that it has a
proline-rich region and several tyrosine residues near its C terminus (18) , and that these motifs fairly well conform to the
consensus binding sequences for the SH2 and SH3 domains of Src.
Moreover, it has 15 YXXP motifs following its own SH3
domain(18) . Many of these YXXP motifs are estimated
to be ideal substrates for cytoplasmic tyrosine kinases (25) and are very close to the consensus binding motif for the
Crk SH2 domain(26) . In addition to these domains, this
molecule has several possible binding motifs for SH3 domains, and it is
thus suggested that p130
may act as a ``docking
protein'' in intracellular signal transduction. Thus far, the SH2
domains of Src and Crk were known to associate with
p130
(21, 24, 27, 28) .
As for the interaction between p130 and the Src SH3 domain, there was a
suggestion that the SH3 domain might be involved in the
association(24, 29) , although the direct binding
through SH3 domain is not proved.
To clarify the role of
p130 in the oncogenic signal transduction by v-Crk or
v-Src, we investigated the manner of association between p130
and v-Src or v-Crk. In this report, we demonstrate that both SH2
and SH3 domains of Src associate with the C-terminal region of
p130
and determined the exact binding sites of
p130
to Src. We further show that v-Crk binds to the
substrate domain of p130
and that the C-terminal
Src-binding region of p130
is also associated with the
kinase activity in v-Crk-transformed cells.
A eukaryotic GST (glutathione S-transferase)
fusion expression vector, pEBG and pEBG-p130 (36) are generous
gifts from B. J. Mayer. Mutations of the p130 moiety,
described above, were also introduced into this vector.
To make GST-SrcSH2, the XhoI-MluI fragment of D3 mutant of chicken c-src(6) was blunt-ended with Klenow fragment of Escherichia coli and was cloned into the SmaI site of pGEX-1. For GST-CrkSH2 fusion protein, the SfiI-Eco81I fragment of v-crk was blunt-ended with the Klenow fragment of E. coli and ligated to the SmaI site of pGEX-1. To generate GST-SrcSH3 protein and GST-CrkSH3 protein, oligonucleotides flanking SH3 domains of c-Src (residues 81-140) and v-Crk (residues 357-440) and introducing restriction sites were used for polymerase chian reaction, and the products were cloned into BamHI-EcoRI sites of pGEX-1 and pGEX-2T, respectively. GST-Grb2/AshSH2 is a kind gift from T. Takenawa.
For immunoprecipitations, 250
µl of 3Y1 cell lysates or 100 µl of COS-1 cell lysates
(approximately 4 µg/µl) were mixed with 5 µl of anti-Cas2
and incubated for 1 h at 4 °C. Samples were rotated with protein
A-Sepharose (Sigma) for 1 h at 4 °C, and then the beads were washed
four times with 1% Triton buffer and boiled in 1 sample buffer.
Western blotting was performed as described (37) using
anti-Cas2 (1:2500), 4G10 (5 µg IgG/ml) as first antibodies and
detected by ProtoBlot Western AP system (Promega). In some cases,
Western blotting was performed using anti-Cas2 (1:50,000), 4G10 (0.5
µg/ml IgG), or mAb 327 (1:5,000) as first antibodies and detected
by ECL Western blotting analysis system (Amersham Corp.).
Silver staining for checking the expression levels was performed as described(38) .
Figure 1:
Association of
p130 with SH2 or SH3 domains of Src and Crk in
vitro. Cell lysates of 3Y1-vec (lanes 1, 4, 7, 10, 13, 16, and 19),
SR-3Y1 (lanes 2, 5, 8, 11, 14, 17, and 20), 3Y1-Crk (lanes 3, 6, 9, 12, 15, 18, and 21) were precipitated with anti-Cas2 (lanes 1, 2, and 3), GST-SrcSH2 (lanes 4, 5,
and 6), GST-SrcSH3 (lanes 7, 8, and 9), GST-CrkSH2 (lanes 13, 14, and 15), GST-CrkSH3 (lanes 16, 17, and 18), and as a negative control GST (lanes 10, 11, and 12) and GST-Grb2/AshSH2(lanes 19, 20, and 21). Immunoblotting was performed using
anti-Cas2.
Both GST-SrcSH2
and GST-CrkSH2 bound to the C form of p130 but not to the
A or B form (Fig. 1, lanes 4-6 and 16-18). GST-SrcSH3 bound to the A, B, and C forms of
p130
(Fig. 1, lanes 7-9). We could
not detect the binding of GST-CrkSH3 to p130
(Fig. 1, lanes 19-21). Negative controls,
GST and GST-Grb2/AshSH2, did not bind p130
(Fig. 1, lanes 10-12 and 13-15). From these results, we concluded that the SH2
domains of Src and Crk are associated with p130
in a
phosphorylation-dependent manner and that the Src SH3 domain can be
associated with p130
in a phosphorylation-independent
manner.
Figure 2:
A schematic diagram representing the various p130 constructs used. A, p130
constructs for
eukaryotic expression vectors. These mutant constructs were introduced
to pSSR
vector. P1 and P2 indicate two
proline-rich regions that can be considered to match the consensus
sequence for the SH3-binding site. Eukaryotic GST fusion protein
expression vectors were constructed by exchanging the p130 moiety of
pEBG-p130 (36) with these mutants. B, GST-p130
constructs for bacterial expression
system.
Figure 3:
Binding of GST-SrcSH3 to wild-type and
mutant forms of p130 expressed in COS-1 cells. The
migrations (in thousands) of molecular weight standards are indicated
on the left. A, anti-Cas2 immunoblot. Approximately
500 µg of cellular lysates from COS-1 cells transiently transfected
with the expression vectors for p130
mutants were
immunoprecipitated with anti-Cas2 (lanes 1, 3, 5, 7, 9, and 11) or recombinant
GST-SrcSH3 immobilized on glutathione-Sepharose beads (lanes
2, 4, 6, 8, 10, and 12). The transfected constructs are mock (lanes 1 and 2), wild-type p130
(lanes 3 and 4),
SH3 (lanes 5 and 6),
P1 (lanes 7 and 8),
SD (lanes 9 and 10), and
SB (lanes 11 and 12). B, anti-Cas2 immunoblot. Cellular lysates from COS-1 cells
were immunoprecipitated with anti-Cas2 (lanes 13, 15,
and 19), or recombinant GST-SrcSH3 (lanes 14, 16, and 20), recombinant GST-W119ASrcSH3 (lane
17), or GST (lane 18) immobilized on
glutathione-Sepharose beads. The transfected constructs were mock (lanes 13 and 14), wild-type p130
(lanes 15-18), and RPLP* (lanes 19 and 20).
The deleted region in SB
mutant contains the sequence RPLPSPPKF, corresponding to residues
733-741. This sequence is close to the class I consensus
sequence, RXLPPLPR
(
represents a hydrophobic
residue) for the Src SH3 domain(39, 40) . A mutant
RPLP* (Fig. 2A), in which the RPLPSPPKF sequence was
converted to RSLGSPPKF, was expressed in COS-1 cells. GST-SrcSH3 fusion
protein failed to bind this mutant (Fig. 3B, lanes
19 and 20). Thus, we located the binding site of the Src
SH3 domain to the RPLPSPPKF sequence of p130
.
Furthermore, the W119A mutant of c-Src(6) , which has an
alanine residue instead of the tryptophan residue well conserved in
various SH3 domains, failed to bind the wild-type p130
(Fig. 3B, lane 17), confirming that this
tryptophan is essential to the binding of the Src SH3 domain with
p130
.
Figure 4:
Binding of GST-SrcSH3 or GST-W119ASrcSH3
to bacterially expressed GST-p130 constructs. The
position of GST-SrcSH3 and GST-W119ASrcSH3 is indicated on the left. Bacterially expressed GST-p130
constructs
were purified with glutathione-Sepharose beads and eluted using
glutathione. The eluted solution was dialyzed in boric acid buffer.
Purified GST-SrcSH3 or GST-W119ASrcSH3 was reacted with these proteins
immobilized on CNBr-activated Sepharose beads, washed four times with
radioimmune precipitation buffer, and resuspended in 1% SDS solution.
Samples were separated with SDS-PAGE and visualized by Coomassie Blue
staining. The immobilized proteins were GST-SDa (lane 1),
GST-SDb (lane 2), GST-SB (lanes 3 and 5),
and GST-SB-RPLP* (lane 4). The reacted proteins were
GST-SrcSH3 (lanes 1-4) and GST-W119ASrcSH3 (lane
5). One-fourth of mixed GST-SrcSH3 (lane 6), or
GST-W119ASrcSH3 (lane 7) fusion proteins were also
electrophoresed.
Figure 5:
Association between 527F-c-Src and
p130 mutants co-expressed in COS-1 cells. COS-1 cells
were transiently transfected with pSSR
-527F-c-Src plus
pSSR
-wild-type p130
or its mutants. The transfected
p130
constructs were
SD (lane 1),
SB (lane 2), wild-type p130
(lane 3),
RPLP* (lane 4), Y751F (lane 5), Y762F (lane
6), and Y751F Y762F (lane 7). After 72 h, cell lysates
were collected and, 100 µl (approximately 500 µg) of lysates
were immunoprecipitated with anti-Cas2. Immunoblotting was performed
using mAb 327 (A), anti-Cas2 (C), 4G10 (D).
Total lysates were electrophoresed and immunoblotted by mAb 327 (B) to confirm that the expression levels of 527F-c-Src were
comparable.
To elucidate the interaction through the SH2 domain, another
series of mutants, of which tyrosine residues were converted to
phenylalanine residues, were constructed and co-expressed in COS-1
cells with 527F-c-Src. The mutation of Tyr of
p130
to Phe reduced the binding ability to 527F-c-Src to
less than one-third of that of the wild type (Fig. 5A, lane 6), but deletion of the substrate domain and the mutation
of Tyr
to Phe did not reduce the binding ability at all (Fig. 5A, lane 5). These results suggest that
Tyr
also contributes to the binding with 527F-c-Src.
Since SH2 domains are considered to bind phosphorylated tyrosine
residues, Tyr
should be the binding site for the Src SH2
domain in vivo.
Figure 6:
v-Crk binds the substrate domain of
p130. COS-1 cells were transiently transfected with pEBG (lane 1), pEBG-p130 (lane2), or pEBG-
SD (lane 3) plus pSSR
-v-Crk (lanes 1, 2,
and 3) or pSSR
-antisense-v-Crk (lane 4). Cell
lysates were collected, and 500 µg of lysates were precipitated
with glutathione-Sepharose beads. A, immunoblotting were
performed with
Hcrk. C, Coomassie Blue staining. B, 50 µg of total cell lysates were immunoblotted with
Hcrk.
Figure 7:
C-terminal region of p130 is
also associated with kinase activity in Crk-transformed cells. A, 3T3-Crk cells were transfected eukaryotic GST fusion
expression vectors carrying wild-type p130
(lanes 2 and 7), RPLP* (lanes 3 and 8), Y762F (lanes 4 and 9),
SB (lanes 5 and 10) and vector alone (lanes 1 and 6).
Lysates were precipitated with glutathione-Sepharose beads and washed,
and in vitro kinase reaction was performed without adding
substrates (lanes 1-5) or with poly-Glu-Tyr (lanes
6-10). Proteins were separated with SDS-PAGE and
autoradiographed. B, silver staining of the same lysates
precipitated with glutathione-Sepharose beads. Lane 1,
wild-type p130
; lane 2, RPLP*; lane 3,
Y762F; lane 4,
SB. The positions of GST-wild-type
p130
and GST-
SB are
indicated.
p130 is a phosphoprotein that has
characteristic, clustered, and repeated (I/V/L)YXXP motifs (Table 1) in its ``substrate domain'' (18) and
is supposed to be an ideal substrate for tyrosine kinases including Src
family kinases and Abl(25) . Furthermore, these motifs conform
very well to the consensus binding sequence for the Crk SH2 domain.
p130
has also a proline-rich region and several tyrosine
residues near its C terminus, suggesting that this region could provide
the binding sites for the SH2 and SH3 domains. Here we report that both
SH2 and SH3 domains of Src tyrosine kinase bind to the C-terminal
region of p130
, whereas the v-Crk binds to the substrate
domain through the SH2 domain.
We revealed that Tyr of
p130
is one of the binding sites for Src. As SH2 domains
are thought to interact with phosphorylated tyrosine, Tyr
is estimated to be the binding site for the Src SH2 domain. The
sequence around Tyr
(Table 1) is similar to the
consensus sequence for the Src SH2 domain determined by the
phosphopeptide library (26) and has a hydrophobic amino acid,
valine, at the +3 position. A phosphorylated tyrosine and a
hydrophobic amino acid residue at the +3 position are considered
to be important for the binding between SH2 domains and
tyrosine-containing peptides(41) .
The binding sequence for
the Src SH3 domain is a RPLPSPPKF sequence corresponding with amino
acid residues 733-741 of p130. The association
between purified GST-fusion proteins suggests that the interaction
between the Src SH3 domain and p130
is direct and does
not require any intermediate proteins. The RPLPSPP sequence of
p130
matches the Class I consensus sequence for the Src
SH3 domain (Table 2) determined by a biased random peptide
library(39, 40) . The known ligands for the Src SH3
domain are shown in Table 2(8, 42, 43, 44) ,
including the RPLPSPPKF sequence of p130
.
So far,
three substrates for Src family kinase,
AFAP-110(11, 24) , Sam68(9, 10) , and
GAP-associated p62 (29, 45) were reported to interact
with Src family kinase through both SH2 and SH3 domains. In these
cases, mutants of Src family kinases in the SH3 domains could not
tyrosine-phosphorylate these
substrates(9, 24, 45) . Therefore, the
interaction through SH3 domains is assumed to be involved in the
substrate recognition by these kinases. In the case of
p130, although a mutant in the SH3 domain of Src, which
had an impaired SH3 binding ability, could tyrosine-phosphorylate p130,
the phosphorylation level was reported to be low(24) . This
fact suggests that the Src SH3 domain plays a role in phosphorylating
p130
. In this report, we show that the Src SH3 domain
binds p130
. Furthermore, in the co-expression system of
COS-1 cells, the mutant that destroyed only the RPLPSPP sequence showed
a certain level of phosphorylation; however, the mobility on SDS-PAGE
had changed. Although it is possible that the mutation itself caused
this change of mobility, the mobility shift may be the result of a low
level of phosphorylation. We also revealed that the tyrosine kinase
activity for poly-Glu-Tyr and p130
itself was associated
with the RPLPSPPKF sequence of p130
in 3T3-v-Crk cells.
These results suggest that the binding through the SH3 domain would be
important in tyrosine-phosphorylating p130
.
In our
model, the binding through the Src SH3 domain is thought to have a role
in substrate recognition before tyrosine phosphorylation, and the Src
SH2 domain reinforces the binding after tyrosine phosphorylation. As a
result, the two-site binding interaction creates a strong association
between Src and p130. This tight association may cause
the effective hyperphosphorylation of p130
by Src
tyrosine kinase. In the association between c-Src and
p130
, p130
might open the ``closed
form'' of c-Src by binding to the regulatory domain and
up-regulate the kinase activity of c-Src. This possibility still
remains under investigation now. Tyrosine phosphorylation of
p130
should allow the recruitment of
SH2-domain-containing signaling molecules such as c-Crk (46) or
Nck (47) and their associated proteins. This might enable
these proteins to be tyrosine-phosphorylated by Src or to interact with
the molecules associated with the SH3 domain of p130
.
Thus, p130
may serve as a ``docking protein''
linking Src to downstream signaling molecules. To clarify the role of
p130
in transformation, we are now searching the
molecules that comprise the complex with p130
.
Although we revealed that the W119A mutant of the Src SH3 domain
fails to bind p130, the W119A mutant of 527F-c-Src had a
full transforming activity compared with parental
527F-c-Src(6, 7) . At present, we have no evidence to
tell whether the binding of p130
to the Src SH3 domain
controls the kinase activity of Src positively or negatively. There are
several proteins that bind the Src SH3 domains other than
p130
. The cooperative roles of these molecules on the
regulation of the transforming activity of Src oncoprotein should be
elucidated.
In the transformation by v-Crk, the tyrosine kinase that
phosphorylates p130 is not known. In this report, we
reveal that most of the tyrosine kinase activity in v-Crk-transformed
cells is also associated with the Src-binding C-terminal region of
p130
, especially the RPLPSPP sequence. This suggests that
the binding of some kinase(s) to this sequence through the SH3 domain
like Src kinase plays a role in phosphorylating p130
.
Formerly, it was reported that v-Crk caused the elevation of the
co-overexpressed c-Src kinase activity in 3Y1 cells (48) . In
our previous reports, co-overexpression of c-Src in v-Crk-transformed
NIH 3T3 cells raised the tyrosine phosphorylation of
p130
(18) , and p130
could be a
substrate for c-Src in vitro(19) , suggesting that
c-Src might be the kinase to phosphorylate p130
. We
showed here that the p130
-associated tyrosine kinase
activity in v-Crk-transformed cells is mostly associated with the
Src-binding region of p130
, whereas v-Crk binds the
substrate domain of p130
. Therefore, the kinase activity
that we see here is more likely to be caused by some other kinases that
bind the Src-binding region of p130
rather than by
v-Crk-associated kinases such as
Abl(36, 49, 50) . However, our data cannot
exclude the possibility that some tyrosine kinase that does not form a
stable complex with p130
phosphorylates p130
in 3T3-v-Crk cells. We are now searching for the kinase that
tyrosine-phosphorylates p130
in v-Crk-transformed cells.
Furthermore, the mechanism by which v-Crk activates the tyrosine kinase
and the pathway through which the phosphorylation of p130
is involved in the cell transformation should be elucidated.
Our results give an insight into the mechanism of signal
transduction in v-Src- and v-Crk-transformed cells and also provide
information on the physiological role of unphosphorylated p130 as a partner of tyrosine kinase.