Focal adhesion kinase (FAK) was first described in 1992 as a novel nonreceptor protein-tyrosine kinase localized prominently within focal adhesions, suggesting a signaling role in regulating cell behavior resulting from integrin interaction with the extracellular matrix. Subsequent studies over the past decade have established functional roles for FAK as a positive regulator of both cell motility and cell survival, while providing considerable insight into signaling mechanisms involved. FAK signaling results from its ability to become highly phosphorylated in response to integrin-mediated adhesion on Tyr-397, permitting interactions with a number of different signaling effectors containing Src homology 2 (SH2) domains. Src-family kinases recruited to the Tyr-397 site phosphorylate two FAK-interacting proteins, Crk-associated substrate (CAS) and paxillin, which results ultimately in regulation of Rho-family GTPases contributing to cell motility. CAS phosphorylation, as well as phosphatidylinositol 3-kinase (PI3K) activation resulting from its binding to the FAK Tyr-397 site, have been implicated as downstream FAK signaling events that confer a resistance to apoptosis. This article reviews these and other aspects of FAK signaling and function.

Tyrosine phosphorylation of CAS (Crk-associated substrate, p130Cas ) has been implicated as a key signaling step in integrin control of normal cellular behaviors, including motility, proliferation, and survival. Aberrant CAS tyrosine phosphorylation may contribute to cell transformation by certain oncoproteins, including v-Crk and v-Src, and to tumor growth and metastasis. The CAS substrate domain (SD) contains 15 Tyr-X-X-Pro motifs, which are thought to represent the major tyrosine phosphorylation sites and to function by recruiting downstream signaling effectors, including c-Crk and Nck. CAS makes multiple interactions, direct and indirect, with the tyrosine kinases Src and focal adhesion kinase (FAK), and as a result of this complexity, several plausible models have been proposed for the mechanism of CAS-SD phosphorylation. The objective of this study was to provide experimental tests of these models in order to determine the most likely mechanism(s) of CAS-SD tyrosine phosphorylation by FAK and Src. In vitro kinase assays indicated that FAK has a very poor capacity to phosphorylate CAS-SD, relative to Src. However, FAK expression along with Src was found to be important for achieving high levels of CAS tyrosine phosphorylation in COS-7 cells, as well as recovery of CAS-associated Src activity toward the SD. Structure-functional studies for both FAK and CAS further indicated that FAK plays a major role in regulating CAS-SD phosphorylation by acting as a docking or scaffolding protein to recruit Src to phosphorylate CAS, while a secondary FAK-independent mechanism involves Src directly bound to the CAS Src-binding domain (SBD). Our results do not support models in which FAK either phosphorylates CAS-SD directly or phosphorylates CAS-SBD to promote Src binding to this site. CAS (Crk-associated substate, p130Cas ) was first recognized as a tyrosine-phosphorylated protein in cells transformed by v-Crk or v-Src (27, 38, 52) and later characterized as a docking protein containing multiple protein-protein interaction domains, including a Src-homology 3 (SH3) domain at the N terminus, a Src-binding domain (SBD) near the C terminus, and a large interior substrate domain (SD) (40,47,52). The CAS SH3 domain may function as a molecular switch regulating CAS tyrosine phosphorylation since it interacts with tyrosine kinases focal adhesion kinase (FAK) (22,47,48) and the FAK-related kinase PYK2 (also known as CAK␤, RAFTK, and CADTK) (3, 42) and also with tyrosine phosphatases PTP-1B (35) and PTP-PEST (18). The SBD represents a second site of CAS interaction with tyrosine kinases and consists of a proline-rich motif, RPLPSPP (amino acid residues 639 to 645 in mouse CAS), that can interact with the SH3 domains of Src-family kinases (SFKs) and a nearby tyrosine phosphorylation site (Tyr-668 and/or Tyr-670) that can promote an interaction with the Src-homology 2 (SH2) domain of SFKs (37, 40). CAS-SD, the major region of tyrosine phosphorylation, is characterized by 15 tyrosines present in Tyr-X-X-Pro (YXXP) motifs. When phospho...

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Crk-associated substrate (CAS, p130Cas) is a major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. We recently reported that reexpression of CAS in CAS-deficient mouse embryo fibroblasts transformed by oncogenic Src promoted an invasive phenotype associated with enhanced cell migration through Matrigel, organization of actin into large podosome ring and belt structures, activation of matrix metalloproteinase-2, and elevated tyrosine phosphorylation of the focal adhesion proteins FAK and paxillin. We have now extended these studies to examine the mechanism by which CAS achieves these changes and to evaluate the potential role for CAS in promoting in vivo tumor growth and metastasis. Whereas the presence or absence of CAS did not alter the primary growth of subcutaneous-injected Src-transformed mouse embryo fibroblasts, CAS expression was required to promote lung metastasis following removal of the primary tumor. The substrate domain YxxP tyrosines, the major sites of CAS phosphorylation by Src that mediate interactions with Crk, were found to be critical for promoting both invasive and metastatic properties of the cells. The ability of CAS to promote Matrigel invasion, formation of large podosome structures, and tyrosine phosphorylation of Src substrates, including FAK, paxillin, and cortactin, was also strictly dependent on the YxxP tyrosines. In contrast, matrix metalloproteinase-2 activation was most dependent on the CAS SH3 domain, whereas the substrate domain YxxP sites also contributed to this property. Thus multiple CAS-mediated signaling events are implicated in promoting invasive and metastatic properties of Src-transformed cells. (Mol Cancer Res 2005;3(6):307 -15)

Liver microsomes are widely used to study xenobiotic metabolism in Vitro, and covalent binding to microsomal proteins serves as a surrogate marker for toxicity mediated by reactive metabolites. We have applied liquid chromatography-tandem mass spectrometry (LC-MS-MS) to identify protein targets of the biotin-tagged model electrophiles 1-biotinamido-4-(4′-[maleimidoethylcyclohexane]-carboxamido)-butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) in human liver microsomes. The biotin-tagged peptides resulting from in-gel tryptic digestion were enriched by biotin-avidin chromatography and LC-MS-MS was used to identify 376 microsomal cysteine thiol targets of BMCC and IAB in 263 proteins. Protein adduction was selective and reproducible, and only 90 specific cysteine sites in 70 proteins (approximately 25% of the total) were adducted by both electrophiles. Differences in adduction selectivity correlated with different biological effects of the compounds, as IAB-but not BMCC-induced ER stress in HEK293 cells. Targeted LC-MS-MS analysis of microsomal glutathione-S-transferase cysteine 50, a target of both IAB and BMCC, detected time-dependent adduction by the reactive acetaminophen metabolite N-acetyl-p-benzoquinoneimine during microsomal incubations. The results indicate that electrophiles selectively adduct microsomal proteins, but display differing target selectivities that correlate with differences in toxicity. Analysis of selected microsomal protein adduction reactions thus could provide a more specific indication of potential toxicity than bulk covalent binding of radiolabeled compounds.