CAS-SD tyrosine phosphorylation is an important event in integrin control of cell behavior, but understanding the mechanism by which this is achieved is complicated by the dual interactions CAS makes with the tyrosine kinases FAK and Src and by the direct binding and regulatory interactions made between FAK and Src. As a consequence of this complexity, five plausible models have been proposed for CAS-SD phosphorylation by FAK or Src (Fig. ). In this study, we used biochemical and structure/function approaches to test these models. Our results indicate that CAS-SD tyrosine phosphorylation is most efficiently achieved by a FAK/Src cooperative mechanism whereby CAS bound via its SH3 domain to FAK becomes phosphorylated by Src bound to the FAK pTyr-397 site (model E). FAK therefore plays a major role as a docking protein that acts to bring Src to its substrate CAS. Our results also support a FAK-independent mechanism of CAS-SD phosphorylation by Src resulting from direct recruitment of Src to the CAS-SBD (model A), although this mechanism appears to account for substantially less CAS phosphorylation than occurs through Src bound to FAK. Our findings that FAK exhibits very weak catalytic activity toward CAS, relative to Src, and does not appear to uniquely phosphorylate CAS tyrosines, including those in the SBD, downplay other FAK/Src cooperative models involving direct phosphorylation of CAS by FAK.
Model E, along with models B to D, predict that FAK and Src act cooperatively to promote CAS-SD phosphorylation. Accordingly, we found that expression of both kinases in COS-7 cells results in substantially higher levels of cellular CAS phosphorylation and CAS-associated kinase activity recovered in immunoprecipitates than is achieved when either kinase is expressed alone (Fig. and ). Models E and D, but not B and C, also predict that FAK's role in promoting CAS phosphorylation is dependent on its ability to directly interact with both Src and CAS, and this prediction is supported by our findings that FAK mutants unable to bind either Src (F397) or CAS (A712/A715/A873/A876 [mPR]) impaired FAK enhancement of CAS tyrosine phosphorylation (Fig. ) as well as did the CAS ΔSH3 mutant which cannot bind FAK (Fig. ). In support of model D, suggested by past findings that the FAK F576/F577 mutation results in diminished FAK activity (12
), we found that Src expression enhances CAS phosphorylation by FAK immunoprecipitates when assayed in the presence of a Src inhibitor (Fig. ). However, other results strongly support model E over model D. Thus, the vast majority of kinase activity toward CAS recovered in FAK immunoprecipitates is due to coprecipitating Src rather than FAK itself (Fig. ), and FAK's own kinase activity is not critical for FAK-promoted CAS phosphorylation in COS-7 cells (Fig. ). Initial support for model E came from the observation that expression of a CD2-FAK chimeric protein caused elevated CAS phosphotyrosine levels that were aberrantly maintained when cells were held in suspension, while the F397 and R454 mutants of CD2-FAK were unable to achieve this response (64
). Also consistent with model E are findings of enhanced adhesion-dependent CAS phosphotyrosine induced by expression of WT-FAK, but not by F397- or mPR-FAK mutants (14
). A mechanism similar to model E may also regulate tyrosine phosphorylation of paxillin (54
In addition to the FAK/Src cooperative mechanism of model E, our results also support the FAK-independent mechanism depicted in model A, whereby CAS-SD phosphorylation is due to Src recruited directly to CAS via its SH3 and SH2 domain interactions with the SBD. Thus, even in the absence of FAK, we found that expression of Src in COS-7 cells promoted some CAS tyrosine phosphorylation—measured to be ~10 to 20% of that induced when FAK is coexpressed with Src (Fig. ). Consistent with model A, this activity is retained for the CAS ΔSH3 mutant but is reduced in the CAS-SBD mutants, mPR and F668/F670 (Fig. ). The loss of activity in the CAS-F668/F670 mutant is consistent with the notion that Src, once initially bound by its SH3 domain, phosphorylates the Tyr668/670 site to further stabilize its interaction by SH2 binding. Other studies supporting model A have shown that CAS-SBD mutations disrupt Src/CAS coimmunoprecipitation and reduce CAS tyrosine phosphorylation (40
) and that the Src-SH3/CAS-SBD interaction is sufficient to recruit and activate Src to phosphorylate CAS-SD (11
). Also, recovery of CAS-associated activity from Crk-transformed cells was found to be greatly reduced by mutation of the SH3-binding site in the CAS-SBD (40
). Thus, model A appears to be an alternative mechanism for CAS-SD phosphorylation and could be the major mechanism under conditions where FAK is not associated with CAS or is not highly phosphorylated at the Tyr-397 site.
In our studies we primarily used the n-Src neuronal isoform, which contains a short insert in the SH3 domain. It could be argued that this insert may impair the binding affinity for the CAS-SBD relative to the nonneuronal c-Src SH3 domain, as indeed appears to be the case for certain other SH3 ligands (50
). If the affinity of the n-Src SH3 domain for the CAS-SBD site is considerably less than that of nonneuronal c-Src, then the relative contribution made by c-Src bound directly to CAS could be substantially greater than is observed for n-Src. However, when we directly compared the ability of n-Src versus c-Src to promote CAS tyrosine phosphorylation we found that c-Src behaves essentially the same as n-Src in this regard. Thus, both Src isoforms were able to promote CAS tyrosine phosphorylation to only a low level in the absence of FAK and, for both, the coexpression of FAK led to a dramatic enhancement of CAS phosphotyrosine levels (Fig. ). Thus the argument that FAK plays an important role by recruiting Src to phosphorylate CAS need not be restricted to neuronal cell types where n-Src is expressed.
Several results from the study indicated that model B, in which FAK phosphorylates the CAS-SBD tyrosines 668 and 670 to promote Src binding, is unlikely to make a significant contribution to CAS-SD phosphorylation. Most notable was the finding that the CAS 668 and 670 tyrosines are not required for FAK/Src-enhanced CAS phosphorylation (Fig. ). Also, phosphopeptide mapping did not provide evidence for this site being among the major sites phosphorylated by FAK (Fig. ), and FAK kinase activity was found to be dispensible for FAK-enhanced CAS phosphorylation while the Src-binding Tyr-397 site was required (Fig. ). Model B stems from observations showing that FAK stimulates CAS-SD phosphorylation following coexpression of the two proteins in COS-1 cells, while FAK appeared capable only of directly phosphorylating the Tyr-668 and 670 site (59
). The same study found that the ability of FAK to promote CAS-SD phosphorylation required CAS Tyr-668 and 670 and FAK kinase activity but was independent of FAK Tyr-397, which is in contrast to our own observations. We cannot explain this discrepency, but we note that the earlier study (59
) analyzed the effects on CAS phosphorylation of expressing only FAK with CAS. Given the intricate nature of the interactions between CAS, FAK, and Src and the capacity for mutual catalytic activation by FAK and Src, a full assessment of the mechanism of CAS phosphorylation should consider the combined action of both kinases. Indeed, we found FAK expression alone resulted in very little CAS tyrosine phosphorylation compared to when both FAK and Src were expressed. While it is conceivable that the model B mechanism could account for the minimal CAS phosphorylation induced when FAK alone is expressed, the much greater capacity for CAS-SD phosphorylation by Src bound to FAK indicate that model B is unlikely to play a significant role in CAS signaling.
Our results are also inconsistent with model C, whereby Src acts as a bridge to recruit FAK to phosphorylate CAS. According to model C, the ability of the Src SH3 domain to bind the CAS-SBD is critical for FAK/Src-promoted CAS-SD phosphorylation. Yet it is clear from our analysis of CAS mutants (Fig. ) that this is not the case. The ability of FAK and Src to cooperatively promote CAS tyrosine phosphorylation is little affected by the mPR mutation in the CAS-SBD. Rather, it is the CAS ΔSH3 mutation that abolishes this phosphorylation response, indicating the importance of a direct CAS-FAK interaction, as does the finding that the FAK-mPR mutation, which disrupts the FAK-CAS interaction (22
), eliminates FAK-promoted CAS tyrosine phosphorylation in the presence of Src (Fig. ). Model C also predicts a major role for the kinase activity of FAK, rather than Src activity, but FAK kinase activity is not required for FAK to promote CAS phosphorylation (Fig. ). Also, we have observed that FAK does not efficiently promote cellular CAS tyrosine phosphorylation when coexpressed with KD-Src (data not shown). Model C stemmed from the observation that expression in Src null cells of a truncated Src protein that lacks its kinase domain but includes both SH3 and SH2 domains is able to enhance cellular CAS phosphotyrosine levels, coupled with data showing that immunoprecipitates of FAK from Src null cells still are able to phosphorylate CAS with high efficiency (57
). While the mechanism by which the truncated Src promotes CAS tyrosine phosphorylation remains uncertain, coprecipitating SFKs, i.e., Fyn and Yes, could have accounted for the bulk of the CAS kinase activity observed in the FAK immunoprecipitates from Src null cells. Even if full-length Src could act as a bridge to bring FAK to CAS, it seems unlikely that the meager FAK catalytic activity toward CAS, relative to that of Src, would critically impact CAS-SD phosphorylation and downstream signaling events.
The complexity of the CAS tryptic phosphopeptide maps (four or five major spots and several additional minor spots) indicate that CAS is phosphorylated at multiple tyrosine sites by the FAK/Src complex. These sites appear to all lie in or very near the SD region, as indicated by the essentially identical appearance of the maps for full-length mouse CAS and the GST-CAS-SD fusion protein (Fig. ). It will be of future interest to determine which YXXP tyrosines account for the observed phosphopeptides and if other tyrosines in the region are phosphoacceptor sites. It was somewhat surprising that our maps did not provide evidence for a major phosphopeptide representing the Tyr668/Tyr670 site. Previous studies have shown that mutation of the Tyr-668 equivalent in rat CAS can reduce the interaction between CAS and Src following their coexpression in COS-1 cells (40
) and that the C-terminal region of CAS containing Y668/670 is efficiently phosphorylated by a baculoviral Src preparation, enhancing its ability to bind Src in a blot overlay assay (10
). In light of these studies, we do not discount the possibility that the Tyr-668/670 site was phosphorylated in our in vitro kinase reactions but was insufficiently labeled to give rise to a major phosphopeptide spot.
While our studies indicated that CAS-SD tyrosine phosphorylation by FAK and Src is primarily achieved by the mechanism represented by model E, with model A having a secondary role when FAK is present, the relative importance of these and other mechanisms could be different for other complexes containing members of the FAK, Src, and CAS families. Since FAK and the FAK-related kinase PYK2 appear to have differences in substrate specificity (34
), it is possible that PYK2 could be a more efficient CAS kinase than FAK such that models B to D could be more important. In addition, there are substantial differences in the SBDs of CAS and the two CAS-related proteins HEF1/CAS-L (32
) and Efs/Sin (1
), which could impact on the SD phosphorylation mechanism. For example, the binding site for the Src SH3 domain is not conserved in HEF1/CAS-L. Thus, the relative importance of the various possible mechanisms for SD phosphorylation of CAS family members by SFKs and FAK/PYK2 kinases will need to be examined on an individual basis.