Identification and characterization of novel focal adhesion kinase substrates
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Focal Adhesion Kinase (FAK) regulates many parameters of normal embryonic development, such as vascular formation, and of cancer progression, especially those involved in metastatic cell motility and invasiveness. FAK interacts with Src to initiate signals controlling cell proliferation, survival, adhesion and motility. The FAK/Src complex phosphorylates many substrates, however although there is evidence that FAK's kinase is required for many oncogenic functions, little is known about its specific or shared substrates. Moreover, the development of FAK kinase inhibitors that exhibit anti-tumor activity underlines the need to comprehensively identify FAK-specific substrates. We attempted to identify FAK specific substrates using two approaches: an in situ/kinase screen using protein or tyrosine-containing peptide microarrays, and mass spectrometry of cell lysate substrates phosphorylated by FAK. Several substrates were validated, including vitronectin, protein tyrosine phosphatase non-receptor type 5, programmed cell death 6, and I-kappa B kinase alpha. Furthermore, we found that the preferred FAK substrate motif is D-E-D-pY-E-V-P. A truncated version of p38-regulated and activated kinase (PRAK), identified initially as a FAK substrate, was phosphorylated in cells and in vitro as its full-length protein by Src. PRAK plays roles in cell growth, survival and motility, and has been implicated in cancer progression. The tyrosine phosphorylation of PRAK was induced by cell adhesion to fibronectin, during which a small portion of PRAK relocalizes to FAK- and Src-containing focal adhesions. Phosphorylation of Y188 and Y216 by Src appears to drive the relocalization of PRAK. Active Src induced the p38-independent activation of PRAK, suggesting that tyrosine phosphorylation of PRAK modulates its ability to affect cell migration and adhesion by enhancing its kinase activity. In sum, we have identified several novel substrates of FAK and have demonstrated that the tyrosine phosphorylation of one FAK/Src substrate, PRAK, appears to mediate aspects of FAK/Src-regulated adhesion and cell motility.