Possible v-Crk-induced transformation through activation of Src kinases.

p47gag-crk (v-Crk) encoded by avian sarcoma virus CT10, causes an elevation of tyrosine phosphorylation of several cellular proteins. The lack of a protein-tyrosine kinase domain in v-Crk suggests its co-operation with cellular protein-tyrosine kinase activity. We have shown that suppression of a certain fraction of c-Src activity by Csk may require the binding of Csk to tyrosine-phosphorylated paxillin. In this study, we detected co-immunoprecipitation of tyrosine-phosphorylated paxillin with v-Crk in CT10-transformed chicken embryo fibroblasts (CEF), and demonstrated that v-Crk binding to paxillin can inhibit Csk binding to paxillin. A phosphotyrosine peptide, which can inhibit v-Crk binding to paxillin, did not inhibit Csk binding to paxillin, suggesting that v-Crk and Csk bind to different tyrosine-phosphorylated sites in paxillin. We also found that the kinase activity of the endogenous c-Src in CEF is elevated severalfold after CT10-transformation. We therefore suggest that the competitive binding of overexpressed v-Crk affects an efficient interaction of Csk with tyrosine-phosphorylated paxillin in CT10-transformed CEF. This would result in a failure in the suppression of the kinase activities of a population of c-Src and other Src family protein-tyrosine kinases as well, and these kinases may then contribute to the phosphorylation of cellular proteins in CT10-transformed CEF.

A viral oncogene product, p47 gag-crk (v-Crk), 1 encoded by avian sarcoma virus CT10 (1), consists of viral gag sequences fused to the src homology 2 and 3 (SH2 and -3) sequences of cellular crk (1,2). Although v-Crk shows no intrinsic proteintyrosine kinase activity, an increase in protein tyrosine phosphorylation of several proteins (130, 125, 110, and 70 -75 kDa) accompanies cellular transformation of chicken embryo fibroblasts (CEF) by the CT10 virus (1,3). The SH2 domain of v-Crk shows a broad range of binding to various tyrosine-phosphorylated proteins (4,5). Deletion mutations within the SH2 domain of v-Crk abolish the transforming capacity of the protein, and such mutants fail to increase cellular protein tyrosine phosphorylation (6,7). The binding of the v-Crk SH2 domain to tyrosine-phosphorylated proteins has also been demonstrated to protect them from dephosphorylation by protein-tyrosine phosphatase activities (8,9). Thus, highly expressed v-Crk in transformed cells seems to cooperate with or activate some cellular protein-tyrosine kinase activities (5), but these proteintyrosine kinases have not been identified.
Unlike in chicken cells, overexpression of v-Crk in rat cells does not cause efficient cell transformation (10). However, by overexpressing proteins via cDNA transfection, we previously showed that the kinase activity of c-Src can cooperate with v-Crk to increase cellular protein tyrosine phosphorylation and cause morphological transformation in rat 3Y1 cells (10). The kinase activity of the c-Src overexpressed with v-Crk was elevated 3-4-fold above basal levels, which may correspond to an activation of 10 -20% of the total c-Src. The activation of c-Src kinase activity by v-Crk is indirect because no significant stable complex between c-Src and v-Crk was detected. This transformation, as well as the kinase activity of c-Src, can be suppressed to basal levels by overexpression of Csk in rat 3Y1 cells (10), which has been shown to regulate the kinase activities of c-Src and several other Src family protein-tyrosine kinases by phosphorylation of their C-terminal tyrosine residues (11)(12)(13)(14). These results suggest that c-Src function may be related to the function of v-Crk and may play a role in v-Crk transformation.
Csk has an SH2 domain (12,15,16). In normal rat or chicken fibroblasts, we found that the major tyrosine-phosphorylated protein that binds to the Csk SH2 domain is a focal adhesion protein, paxillin (17)(18)(19). A minor fraction of Csk seems to associate with Focal Adhesion Kinase, pp125 FAK (19 -21). It is conceivable that the anchoring of Csk to these proteins is required for its suppression of the activity of a certain fraction of c-Src as well as other Src family protein-tyrosine kinases, which may function at focal adhesion plaques (19).
In this study, we show that the activity of endogenous c-Src kinase is activated severalfold in CT10-transformed CEF. We also demonstrate that binding of v-Crk to paxillin (9) can inhibit the binding of Csk to paxillin, although the primary binding sites in paxillin for these proteins seem to differ. The binding of v-Crk to paxillin phosphorylated by Src proteintyrosine kinase in vivo as well as in vitro but not by Csk was effectively inhibited by a peptide that specifically inhibited the binding of v-Crk to paxillin in v-Crk-transformed CEF. Therefore, we suggest that c-Src may participate in the phosphorylation of cellular proteins such as paxillin in CT10-transformed CEF. v-Crk binding to paxillin may account for the partial activation of c-Src in v-Crk-transformed cells.
Preparation of Glutathione S-Transferase (GST)-Fusion Proteins-GST-Csk SH3/2 proteins containing both of the SH3 and SH2 domains of chicken Csk, its mutants within the SH2 domain, R106K and S108C, were prepared as described previously (19). GST-v-Crk protein containing the Gag-v-Crk fragment was prepared as described previously (9).
Immunoprecipitation and Immunoblotting Analysis-Paxillin was immunoprecipitated from cell lysates prepared in RIPA buffer using an anti-paxillin antibody (Zymed) coupled to protein G-Sepharose (Pharmacia Biotech Inc.). v-Crk was immunoprecipitated from cell lysates prepared in 1% Nonidet P-40 buffer using an anti-Gag antibody (3C2) (23) coupled to protein G-Sepharose. Csk was immunoprecipitated using protein A-purified anti-Csk polyclonal antibodies (gift from M. Okada and K. Tobe). For the precipitation using GST-fusion proteins, 250 g of cell lysates prepared in 1% Nonidet P-40 buffer were incubated with 5 g of GST-fusion proteins coupled to glutathione-Sepharose (Pharmacia Biotech Inc.) in a total volume of 300 l for 30 min at 4°C, unless otherwise indicated. After being washed 4 times with respective buffer, the samples were boiled in Laemmli's SDS sample buffer and separated by SDS-polyacrylamide gel electrophoresis (PAGE) (8% gel). After electrophoresis, the proteins were transferred to membrane filters (Immobilon P, Millipore), blocked with Tris-buffered saline containing 0.1% Tween 20 (Sigma) and 5% BSA (radioimmunoassay grade, Sigma), and probed with appropriate antibodies as described previously (10,19). The antibodies retained on filter membranes were then detected by peroxidase-conjugated secondary antibody and visualized by enzyme-linked chemiluminescence method according to the manufacture's instruction (ECL, Amersham Corp.). Anti-phosphotyrosine polyclonal antibodies were prepared against tyrosine-phosphorylated v-Abl proteins as described previously (24).
In Vitro Kinase Assay-c-Src protein was immunoprecipitated from cell lysates prepared in RIPA buffer using an anti-Src antibody (Ab327) (26), coupled to protein A-Sepharose. After being washed 4 times with RIPA buffer and once with kinase buffer without divalent cations, aliquots of the samples were separated on SDS-PAGE (8% gel) and subjected to immunoblot analysis using Ab327 to quantify the relative amounts of c-Src protein in each sample. Then the samples, containing similar amounts of c-Src, were subjected to in vitro kinase assay with 10 M ATP and 10 Ci of [␥-32 P]ATP (3000 Ci/mmol, Amersham Corp.) in 50 l of kinase buffer (20 mM Tris-HCl (pH 7.4), 5 mM MgCl 2 , 0.1% Nonidet P-40, 0.1 mM Na 3 VO 4 ) with 2 g of acid-denatured enolase (Sigma) as a exogenous substrate, and incubated for 10 min at 30°C. The samples were run on SDS-PAGE (8% gel); the gel was then dried and exposed to film. The same amount of the samples used for in vitro kinase assay were again separated on SDS-PAGE (8% gel) and subjected to immunoblot analysis using Ab327 to quantitate the levels of c-Src protein. Specific activity was calculated by dividing each activity by the respective amount of c-Src protein quantitated by immunoblotting as described previously (19).
Phosphopeptide Library Screening and Rational Design of Phos-phopeptides-Affinity purification of phosphopeptides specific for GST-Csk SH3/2 has been described previously (27). For the peptide synthesis, an Fmoc-based strategy for sequential peptide synthesis was used in combination with standard side chain-protecting groups as described previously (28,29), and N- ) was used to incorporate phosphotyrosine (29). Peptides were purified by preparative reversed-phase high pressure liquid chromatography (28), and purified products were analyzed by amino acid composition. Sequences used in this study were DNEpYTARNGAK (c-Src 416), PVSpYADMRTGI (IRS-1 1010), and DpYDAPA (Crk 1) (9, 30). (10), c-Src kinase activity may also be up-regulated in v-Crk-transformed chicken cells. Therefore, we examined the level of the kinase activity of endogenous c-Src in CT10-transformed CEF. With equivalent amounts of cell lysates, we noticed that the levels of c-Src protein immunoprecipitated with Ab327 from CT10transformed CEF were often slightly lower than those from normal CEF. Thus we adjusted to use same amounts of c-Src protein in these in vitro kinase assay. As shown in Fig. 1, about 4-fold activation of the specific activity of c-Src kinase was detected in CT10-transformed cells using enolase as an exogenous substrate, as compared with that in normal CEF. The degree of the activation varied with each preparation of primary culture of CEF, but with five different preparations of CEF, we observed an average of 3-4-fold activation of the specific activity of the endogenous c-Src after transformation by the CT10 virus (data not shown). In 3Y1 cells, the specific activity of endogenous c-Src kinase was also activated to a similar level when cells were transformed by expression of v-Crk at high levels by cDNA transfection (data not shown). Again the protein levels of endogenous c-Src were significantly reduced (about 1.5-2-fold) in these cells (data not shown).

Activation of the Specific Activity of Endogenous c-Src Kinase in CT10-Transformed Cells-Because c-Src activation was involved in v-Crk transformation of rat 3Y1 cells
v-Crk Binds to Paxillin in CT10-transformed CEF As Well As Paxillin Phosphorylated by c-Src or by Csk-Tyrosine phosphorylation of paxillin is highly elevated in v-Src-transformed cells (31). Elevation in the tyrosine phosphorylation of paxillin was also observed in CT10-transformed CEF (Fig. 2, A and D). Tyrosine phosphorylation of pp125 FAK has also been shown to be increased 2-3-fold in CT10-transformed CEF as compared with normal CEF (3) (also see Fig. 2A). To examine the binding of v-Crk protein to these tyrosine-phosphorylated proteins, we immunoprecipitated v-Crk protein with the 3C2 monoclonal antibody, which recognizes the Gag portion of the v-Crk protein. As shown in Fig. 2, paxillin was co-immunoprecipitated with v-Crk in CT10-transformed CEF (Fig. 2), as has been shown using the GST fusion form of v-Crk SH2 domain in vitro (9). On the other hand, co-immunoprecipitation of pp125 FAK and v-Crk was not detected under these conditions (Fig. 2C). The GST fusion form of the Csk SH3 and SH2 (Csk SH3/2) domains binds to tyrosine-phosphorylated forms of paxillin and pp125 FAK in CT10-transformed CEF cell lysates, as shown previously with normal CEF cell lysates (19). Mutations in the SH2 domain of Csk such as R106K and S108C (19) reduced its binding to these proteins, suggesting that binding was primarily mediated by the SH2 domain.
We have shown that both c-Src and Csk can phosphorylate paxillin on tyrosine residues generating slow migrating isoforms of paxillin (19). 2 Both of these kinases phosphorylate paxillin at multiple sites, but they generate isoforms of paxillin that migrate differently on SDS-PAGE 2 (also see Fig. 3). As shown in Fig. 3, GST fusion forms of v-Crk and Csk SH3/2 were able to bind to both forms of these slow migrating paxillin phosphorylated in vitro either by Csk or by c-Src. Tyrosine phosphorylation of slow migrating paxillin was confirmed by immunoblotting using polyclonal anti-phosphotyrosine antibodies (data not shown).
v-Crk Binding to Paxillin Inhibits Csk Binding to Paxillin-It has been shown that even subnanomolar concentrations of the v-Crk SH2 domain fused to GST can bind to tyrosine-phosphorylated paxillin in cell lysates of CT10-transformed CEF (9). Under similar conditions, we assessed an apparent binding affinity of Csk SH3/2 to paxillin from CT10-transformed CEF. A titration experiment showed that half maximal binding was observed at around 10 -20 nM of the GST-Csk SH3/2 protein (Fig. 4A), an affinity that is severalfold weaker than that of v-Crk binding to paxillin.
We next examined the possible competition of binding of Csk and v-Crk toward paxillin. Cell lysates from CT10-transformed CEF were preincubated with either purified recombinant v-Crk or purified recombinant Csk, and then the binding of cellular tyrosine-phosphorylated proteins to GST-Csk SH3/2 was analyzed. As shown in Fig. 5A, in the presence of almost equimolar concentrations of recombinant Csk (5 g, 50-kDa) and GST-Csk SH3/2 (4 g, 46-kDa), the binding of GST-Csk SH3/2 to paxillin was reduced to half (55% reduction quantitated with a densitometer). On the other hand, substantial inhibition (88% reduction quantitated with a densitometer) of GST-Csk SH3/2 binding to paxillin was observed when it was preincubated with equimolar concentrations of recombinant v-Crk (5 g, 47 kDa). The binding of GST-Csk SH3/2 to pp125 FAK was also reduced to about half in the presence of 5 g of recombinant Csk, whereas only a slight reduction in the binding of Csk SH3/2 to pp125 FAK was observed by recombinant v-Crk and analyzed for paxillin binding with GST-Csk SH3/2 by immunoblotting using an anti-paxillin antibody. Glutathione-Sepharose beads were added to give a similar amount of beads during incubation in each sample. B and C, cell lysates prepared in 1% Nonidet P-40 buffer from CEF or CT10-transformed CEF, along with authentic recombinant v-Crk (45) or recombinant Csk (19), were separated on SDS-PAGE and subjected to immunoblotting analysis using an anti-v-Crk polyclonal antibodies (5) or anti-Csk polyclonal antibodies (19). To verify whether the concentration of the v-Crk protein in CT10-transformed CEF is high enough to inhibit the binding of Csk to tyrosine-phosphorylated paxillin, we measured the cellular levels of v-Crk and Csk. In 50 g of lysate of transformed CEF prepared with 1% Nonidet P-40 buffer, the level of the v-Crk protein was found to be approximately 100 -300 ng, whereas the amount of Csk was approximately 1-3 ng (Fig. 4, B and C). Therefore, the molar concentration of v-Crk appears to be almost 100 times that of Csk in CT10-transformed CEF.

FIG. 2. Tyrosine phosphorylation of paxillin in CT10-transformed CEF and binding of endogenous v-Crk or GST-Csk
We then tried to assess whether this type of inhibition by v-Crk was taken place in CT10-transformed cells. Csk has been shown to be co-precipitated clearly with paxillin when Csk was overexpressed (19). With endogenous Csk, however, it was relatively difficult to show such a high level of co-precipitation of paxillin (data not shown). Therefore, we used cell lysates prepared at high protein concentrations, and purified anti-Csk antibodies to avoid nonspecific binding of paxillin to rabbit serum. As shown in Fig. 5C, a higher level of paxillin was detected in a Csk immunoprecipitant from CEF than that from CT10-transformed CEF.
Difference in the Binding Specificity between Csk and v-Crk toward Paxillin-By analysis of specific binding of each SH2domain to a degenerate phosphopeptide library, a consensus amino acid sequence of pY(T/A)XX was identified for optimal Csk SH2 binding, whereas a pYXXP sequence for the v-Crk SH2 domain was determined (27,30). Using phosphopeptides containing these sequences, we examined difference in the binding of the SH2 domains of Csk and v-Crk toward paxillin in cell lysates prepared from CT10-transformed CEF. Preincubation of GST-v-Crk with 500 M of the Crk binding peptide, DpYDAPA, almost completely inhibited its binding to paxillin (70 -75-kDa), but only slightly inhibited its binding to proteins of 110 and 130-kDa in size, as reported previously (9) (Fig. 6A). On the other hand, the same peptide was totally ineffective in inhibition of the binding of GST-Csk SH3/2 to tyrosine-phosphorylated proteins, including paxillin (Fig. 6A). Two phosphopeptides containing pY(T/A)XX sequences, PVSpYADM-RTGI and DNEpYTARGAK, significantly reduced the GST-Csk SH3/2 binding to pp125 FAK (Fig. 6A). Only a marginal inhibition in the binding of the GST-Csk SH3/2 to paxillin was observed with these two peptides (Fig. 6A). These two peptides were totally ineffective in inhibition of the v-Crk binding to tyrosine phosphorylated proteins, including paxillin (Fig. 6A).
Similar experiments were also performed using cell lysates prepared from v-Src-transformed CEF. Again, the peptide with the pYXXP motif inhibited the binding of GST-v-Crk to paxillin very effectively (Fig. 6B). The two peptides with pY(T/A)XX motifs partially reduced the GST-Csk SH3/2 binding to proteins of 110 -130-kDa, while little inhibition was observed in its binding to paxillin (Fig. 6). The PVSpYADMRTGI peptide seemed to be slightly more effective than the DNEpYTARGAK peptide in their inhibition of GST-Csk SH3/2 to these high FIG. 5. Competition of v-Crk and Csk in binding to paxillin. A and B, each 200 g of cell lysate from CT10-transformed CEF was preincubated with respective amounts of purified recombinant v-Crk or purified recombinant Csk for 30 min at 4°C and then incubated with 5 g of GST-Csk SH3/2 and analyzed for proteins binding to GST-Csk SH3/2 by immunoblotting analysis using an anti-paxillin antibody (A) and anti-Fak polyclonal antibody (B). C, co-immunoprecipitation of paxillin with Csk in CEF and CT10-transformed CEF. Each 4 mg of cell lysate (8 mg/ml) from CEF or from CT10-transformed CEF was subjected to immunoprecipitation (IP) using anti-Csk polyclonal antibody (␣ Csk). Paxillin precipitated with an anti-paxillin antibody (␣ Pax), and precipitation using rabbit IgG (IgG) were also included. Proteins precipitated with Sepharose beads were analyzed by immunoblotting using an anti-paxillin antibody .  Fig. 3 was added (80 l) and further incubated for 30 min at 4°C. After being washed, the samples were separated on SDS-PAGE and analyzed for proteins associated with each GST-fusion protein by immunoblotting analysis using anti-phosphotyrosine polyclonal antibody, anti-Fak polyclonal antibody, or an anti-paxillin antibody, as indicated. Thick bands that appeared at around 65 kDa in A and B in the anti-phosphotyrosine blot are due to an artifact as described previously (19). For C, 1 mg/ml acetylated BSA was added to each sample during the incubation. Phosphopeptides used were DNEpYTARGAK (lane 2), PVSpYADMRTGI (lane 3) and DpYDAPA (lane 4). Control included no addition of a phosphopeptide (lane 1). molecular weight proteins (Fig. 6B).
The pYXXP Peptide Inhibits v-Crk Binding to Paxillin Phosphorylated by c-Src in Vitro-Paxillin seems to have multiple tyrosine phosphorylation sites. 2 The experiment described above suggested that the kinase(s), which are involved in the tyrosine phosphorylation of paxillin in CT10-transformed CEF, may have a substrate specificity similar to v-Src. To obtain a clue to the nature of the protein-tyrosine kinase(s) phosphorylating paxillin in CT10-transformed cells, we examined the ability of these peptides to inhibit the binding of v-Crk to paxillin phosphorylated in vitro. As shown in Fig. 6C, the peptide with the pYXXP motif was very effective in inhibition of GST-v-Crk binding to paxillin phosphorylated by c-Src. However, it exhibited a partial inhibition in the case of GST-v-Crk binding to paxillin phosphorylated by Csk. Two other kinds of peptides with pY(T/A)XX motifs were totally ineffective in the inhibition of v-Crk binding to these in vitro phosphorylated paxillin isoforms. None of these phosphopeptides significantly inhibited Csk SH3/2 binding to paxillin phosphorylated in vitro by either kinase (Fig. 6C).

DISCUSSION
In this report, we showed that both v-Crk and Csk bind to tyrosine-phosphorylated paxillin, at primarily different sites from each other as assessed by the phosphopeptide inhibition assay. However, we demonstrated that v-Crk binding has a potential to block the binding of Csk to paxillin phosphorylated in CT10-transformed cells. No significant physical complex formation between Csk and v-Crk was observed (10). Therefore, this binding competition seems to be due to steric hindrance. The 100-fold higher cellular concentration of v-Crk over Csk in CT10-transformed CEF, as well as the stronger affinity of v-Crk for paxillin than that of Csk for paxillin suggests that a significant inhibition of binding of Csk to paxillin can take place in CT10-transformed CEF.
We also showed that the specific activity of c-Src kinase in CT10-transformed CEF is elevated severalfold over that in normal cells. Immunohistochemical staining of c-Src protein had revealed that the majority of c-Src seems to localize at cell-to-cell adhesion junctions, juxtanuclear regions, and centrosomes (32). However, an increasing number of studies have suggested that c-Src localizes to and may have a function at focal adhesions; an activated form of c-Src has been shown to bind to pp125 FAK via its SH2 domain (33,34) and is also able to bind to paxillin through its SH3 domain (35). We have indicated that Csk binds to focal adhesion proteins to suppress c-Src kinase activity (19). Our model is consistent with the observation that Csk colocalizes with activated c-Src to cellular structures resembling podosomes (36). Furthermore, the kinase activity of a certain fraction of cellular c-Src is regulated by cell to substratum adhesion (19). Failure to find c-Src at focal adhesion plaques by immunostaining analysis (32) may be due to the fact that only a small fraction of c-Src can localize to focal adhesion plaques (19). Or, only when activated, c-Src can stay stably at focal adhesion plaques through the binding of its SH2 domains with several focal adhesion proteins including pp125 FAK (33,34). Because v-Crk overexpressed in CT10transformed cells can block the binding of Csk to paxillin, it is likely that the negative regulation of c-Src by Csk through Csk's binding to paxillin can be reduced and thus results in activation of the kinase activity of a certain fraction of c-Src. This mechanism may also explain why only a small fraction of cellular c-Src appears to be activated in CT10-transformed CEF (Fig. 1) as well as in rat 3Y1 cells overexpressing both c-Src and v-Crk as we have shown previously (10). Among Src family protein-tyrosine kinases, Fyn also has been shown to bind to pp125 FAK , with relatively stronger affinity than that of Src (33). Thus, it would be interesting to examine whether other Src-family kinases such as Fyn whose activities could be negatively regulated by Csk, are also activated in a similar way in CT10-transformed cells.
Our study described here also suggests that at least one of the protein-tyrosine kinases involved in the increased phosphorylation of paxillin in CT10-transformed CEF is c-Src. Paxillin is highly phosphorylated in v-Src transformed cells (31). We showed that the same phosphotyrosine-peptide can block the binding of v-Crk to paxillin either from CT10-transformed CEF or from v-Src-transformed CEF. Furthermore, the same peptide blocks v-Crk binding to paxillin phosphorylated by c-Src in vitro, whereas it exhibits a partial blockage in v-Crk binding to Csk-phosphorylated paxillin. Consistent with our model, it is worthy to note a recent demonstration that depletion of c-Src kinase in csk-negative cells caused lowered tyrosine phosphorylation of paxillin (37).
In addition to paxillin, other proteins whose tyrosine phosphorylation is increased in v-Src transformed CEF, such as pp125 FAK and 110-and 130-kDa proteins, are also highly tyrosine-phosphorylated in CT10-transformed CEF (1, 3). Consistent with the partial activation of the c-Src kinase, the level of overall kinase activities in CT10-transformed cells seems to be much lower than that of the v-Src kinase in v-Src-transformed cells, where an amount of v-Src protein comparable with that of endogenous c-Src is expressed (5). 3 In addition to the activation of the kinase activity of a small fraction of c-Src, the binding of v-Crk to tyrosine-phosphorylated forms of paxillin, pp125 FAK and 110-and 130-kDa proteins with high affinity, would protect them from cellular tyrosine phosphatase activities (8,9) and thereby contribute to the steady state accumulation of tyrosine phosphorylation in these proteins in CT10-transformed CEF. Recently, several proteins including c-Abl protein-tyrosine kinase (38,39), and C3G with a guanine nucleotide-releasing property (40), have been shown to bind to the SH3 domain of Crk. How much of these Crk-binding proteins contribute to the protein-tyrosine phosphorylation as well as cell transformation in CT10-transformed cells remains to be studied.
Although pp125 FAK did not co-precipitate with endogenous v-Crk (Fig. 2), it was recovered in a protein complex bound to GST-v-Crk (Fig. 6A). This difference is probably due to the higher concentration of GST-v-Crk than endogenous v-Crk used in our experiments. However, since paxillin itself can associate with pp125 FAK (41), our analysis cannot distinguish whether v-Crk directly binds to pp125 FAK .
The cell-to-substratum adherence seems to be greatly altered in CT10-transformed CEF; cells become fusiform in morphology and acquire an ability to grow in soft agar (1). It is interesting to note that several types of v-Src mutants whose kinase activities are weaker than that of wild-type v-Src cause transformation with a fusiform morphology rather than a rounded-up one as caused by wild-type v-Src (42)(43)(44). The v-Crkinduced activation of c-Src kinase activity at focal adhesion plaques may contribute to the loss of the tight adherence of cells to the extracellular matrix and vice versa, as we have discussed previously (19). The binding of v-Crk to paxillin may affect a function of paxillin other than its function as a mediator for the interaction between the Csk and Src kinases. v-Crk binding to paxillin may also block the phosphorylation of paxillin by Csk, which takes place at an early stage of cell to substratum adhesion, 2 thus affecting the normal function of the focal adhesion plaques.