We have here shown that endogenously expressed CAP can be Tyr phosphorylated in Hep3B hepatocytes, whereas previous findings from others have demonstrated that in these cells, CAP can be coimmunoprecipitated with c-Abl kinase [4
]. Although other members of the SoHo protein family have been shown to be phosphorylated by c-Abl [20
], our report is the first demonstration of Tyr phosphorylation of CAP by any kinase. CAP has been shown to play a role in membrane receptor signal transduction, e.g. insulin and neurotrophic factor signaling [10
]. We were not able to detect phosphorylated CAP in insulin treated Hep3B cells, whereas vanadate stimulation resulted in robust Tyr phosphorylation of CAP. This might indicate that the phosphorylation of CAP during signaling is regulated by rapid dephosphorylation by phosphatases, and thus phosphorylation can only be detected upon inhibition of phosphatase activity by vanadate. It will be of interest to determine if CAP is phosphorylated in other cell types during signaling processes, and if so, which kinases and phosphatases mediate the phosphorylation and dephosphorylation.
The third SH3 domain of CAP (SH3-C) has previously been shown to bind to c-Abl in vitro [4
]. According to our results, the delta-SH3 mutant of CAP missing all three SH3 domains was neither capable of coprecipitating c-Abl nor phosphorylated by it. However, we observed only a slightly reduced coprecipitation of c-Abl with the mutants in which either SH3-C or all three SH3 domains of CAP were mutated. As the Trp substitutions render SH3 domains incapable of binding their poly-Pro containing recognition signals, these results would suggest that although the binding site of Abl in CAP is located in the C-terminal part containing the SH3 domains, there have to be determinants other than the SH3 domains, e.g. the linker regions in between, which are important for the interaction of Abl with CAP. Interestingly, the linker region between SH3-B and -C contains a putative Pro-rich sequence (PQQP) which might function as a binding site for the SH3 domain of c-Abl, thus facilitating a dual interaction mediated by SH3 domains/Pro-rich regions in both proteins. A similar binding mode has also been implicated between vinexin and c-Abl [20
]. However, mutation of the PQQP motif into AEEA did not significantly reduce the phosphorylation of CAP in the presence of intact SH3 domains (data not shown). Although this would rather speak against the role of the PQQP motif in the binding of Abl, an interaction mediated by more than one determinant would also be supported by the fact that the constitutively active Abl-PP mutant coprecipitated with CAP more than c-Abl. In Abl-PP, two Pro residues in the linker region between the SH2 and kinase domain have been mutated so that the protein acquires an open conformation [25
] and might be able to bind more efficiently to its substrate, CAP. On the other hand, since phosphorylation of CAP was readily observed in cells coexpressing WT Abl and CAP, this might imply that binding of CAP to c-Abl could even increase the activity of the kinase which is under tight regulation in normal cells (see [26
] for review). However, further experiments will be required to clarify the exact binding mechanism between CAP and Abl.
We have here identified Tyr360 in CAP as a major phosphorylation site by c-Abl, although Tyr 632 also might contribute since its substitution in combination with the Y360F mutation reduced the phosphorylation of CAP to a very low level. Both Tyr conform to the consensus c-Abl phosphorylation motif. Interestingly, in mouse vinexin, the major phosphorylation site of c-Abl is provided by Y127 with some possible contribution by Y269 and Y394 [20
]. However, different from Y360 in CAP, Tyr127 of vinexin does not seem to reside in a consensus phosphorylation motif for c-Abl.
CAP localizes to focal adhesions, where it can bind vinculin, FAK, paxillin and filamin C and to actin stress fibers, which is probably, at least to some degree, mediated by different sets of binding partners [1
]. In fibroblasts, overexpression of CAP has been described to slow down the spreading of cells onto extracellular matrix [8
]. However, according to our data, CAP does neither inhibit nor enhance the spreading of epithelial cells onto fibronectin. This could be explained by the differences in the modes of regulation of focal adhesions and cell spreading between different cell types or by the presence of binding partners of CAP in fibroblasts that result in inhibitory effects. Although c-Abl is an important regulator of cytoskeletal remodeling and many of its substrates are known to participate in the coordination of actin-dependent processes, we could not observe any effect on cell spreading when Y360, the major c-Abl phoshorylation site of CAP, or Y632 were mutated to Phe. Both mutants were also localized in both focal adhesions and stress fibers in a very similar way to the WT CAP protein. The Y632F mutant also exhibited some nuclear localization which is at present difficult to explain. Although a nuclear form of CAP, termed R85Fl [3
], has been described, the isoform used here does not contain the nuclear localization signal shown to mediate the nuclear transport of R85Fl. Thus, the nuclear translocation of Y632F mutant might be mediated by its interaction with another protein that is capable of being transported into the nucleus, as has been shown the case with teneurin-1 and CAP [29
Since substitution of the major Abl phosphorylation sites in CAP resulted in normal spreading of the cells, phosphorylation of CAP by c-Abl does not seem to play a major role in this process. However, the mutation of Y326 slowed down the spreading, indicating that phosphorylation of this Tyr by another kinase might be important for the regulation of CAP function during cell spreading. Indeed, we could show that Y326 is phosphorylated by c-Src, another non-receptor Tyr kinase that regulates many actin dependent cellular processes, including focal adhesion assembly and spreading [30
]. If phosphorylation of Y326 by c-Src indeed plays a role in the regulation of cell spreading by CAP still needs to be clarified in more detail in future studies.
As with Abl, the binding and phosphorylation of CAP by Src was severely compromized by deletion of the C-terminal SH3 region but not by mutations in the critical Trp residued in the SH3 domains of CAP. Thus, also in the case of Src, the linker regions between the SH3 domains and the Pro rich sequences might mediate the interaction between the kinase and its substrate. The main phosphorylation sites Y326 and Y360 do not reside in the SH3 region but more N-terminally to it, and thus the reduced phosphorylation of ΔSH3 CAP by Src and Abl is unlikely to be simply due to deletion of phosphorylatable Tyr residues. In addition, mutation of six tyrosines within the SH3 domain region of CAP (Y527, Y587, Y592, Y632, Y678 and Y638) which all fit well to the consensus phosphorylation sequence of Src did not affect the phosphorylation of CAP by c-Src (our unpublished data). Thus, the SH3 region is more likely to be important for the substrate-kinase interaction rather than providing substrate tyrosines.
Substitution of Y360, the major Abl phosphorylation site, alone only moderately impaired the phosphorylation of CAP by Src. However, the double mutant Y326F/Y360F showed a severely impaired phosphorylation by both Src and Abl, implicating that both kinases might actually cooperate to achieve the full degree of phosphorylation of CAP. It could be speculated that Src plays only an indirect role in the phosphorylation of CAP by facilitating Abl activation and thus increasing Abl-mediated CAP phosphorylation. However, our in vitro phosphorylation assay with purified proteins showed that CAP is a direct substrate of Src. Furthermore, CAP interacted with Src in coimmunoprecipitation experiments, and Y326 fits well to the consensus phosphorylation sequence of c-Src. Thus, it appears that both c-Src and c-Abl are capable of directly phosphorylating tyrosines 326 and 360 in CAP, but each show a preference to a different Tyr. However, inhibition of Src activity in cells expressing c-Abl resulted in slightly reduced phoshorylation of CAP. Thus, both kinases appear to contribute to the phosphorylation and thereby generate binding sites for SH2-containing proteins which would link CAP downstream towards processes resulting in actin remodeling. The details of the interplay between Src and Abl in the phosphorylation of CAP should be clarified in future studies in order to understand the role of phosphorylation in the regulation of the function of CAP.