Induced Focal Adhesion Kinase (FAK) Expression in FAK-Null Cells Enhances Cell Spreading and Migration Requiring Both Auto- and Activation Loop Phosphorylation Sites and Inhibits Adhesion-Dependent Tyrosine Phosphorylation of Pyk2

Abstract

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAKnull mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130 Cas , were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAKrelated kinase Pyk2/CAK␤/RAFTK/CadTK. FAK (focal adhesion kinase) is a widely expressed nonreceptor protein tyrosine kinase found in focal adhesions of cultured cells (23,61). FAK becomes activated by tyrosine phosphorylation in response to integrin clustering achieved by cell adhesion or antibody cross-linking (5,19,23,34,40). FAK Tyr-397 is an autophosphorylation site and a high-affinity binding site for Src homology 2 (SH2) domains of Src family kinases, including c- Src and Fyn (48,62,80). This interaction could contribute both to the recruitment of Src family kinases to sites of cell adhesion and to their catalytic activation through C-terminal tail displacement. Other adhesion-regulated sites of FAK phosphorylation are tyrosines 407, 576, 577, 861, and 925 (7, 8, 65). These tyrosines do not appear to be autophosphorylation sites but are efficiently phosphorylated by c-Src in vitro and elevated in Src-transformed cells (7,8,66). Tyr-397 can also be phosphorylated by c-Src (7); hence, it is not strictly an autophosphorylation site. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and mutation of these residues reduces FAK catalytic activity (7, 42). The potential for reciprocal activation of FAK and Src family kinases suggests a mechanism for signal amplification following an initial integrin-induced FAK autophosphorylation event. Other sites of FAK tyrosine phosphorylation are likely to participate in downstream signaling through recruitment of additional SH2-containing proteins. Indeed, phosphoryl...

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…This observation is not altogether novel: studies have shown that cells overexpressing FAK mutants incapable of binding paxillin exhibit the same phenotype [123]. Since FAK-null cells have also been documented to have more persistent adhesions and a reduced rate of migration when compared to reconstituted cells, we would suggest that with respect to adhesion dynamics, an inability of FAK to bind paxillin, is similar to a FAK-null phenotype [165]. Alternatively, the observation that the S250 phospho-isoform is present along peripheral actin stress fibres but absent from adhesions would suggest that this FAK-paxillin interaction takes place outside of the focal adhesion.…”

Intrinsic focal adhesion kinase activity controls orthotopic breast carcinoma metastasis via the regulation of urokinase plasminogen activator expression in a syngeneic tumor model

“…As strategies using antisense oligonucleotides (Xu et al, 1996;Hauck et al, 2001) or RNA interference (RNAi) (Duxbury et al, 2003;Han et al, 2004) can reduce FAK expression in tumor cells, these methods have not yet been tested for specificity via FAK reconstitution and rescue assays. Only in RNAi-treated normal fibroblasts (Tilghman et al, 2005) and in reconstituted FAK-null (Owen et al, 1999;Sieg et al, 1999) or v-Src-transformed FAK-null fibroblasts (Hsia et al, 2003;Wu et al, 2005) have FAK re-expression and tyrosine phosphorylation been linked to the promotion of cell motility and/or invasion.…”

Differential FAK phosphorylation at Ser-910, Ser-843 and Tyr-397 induced by angiotensin II, LPA and EGF in intestinal epithelial cells

“…[6]. Subsequent Src-mediated phosphorylation of FAK at Tyr-576, Tyr-577, Tyr-861 and Try-925, is important for the maximal activation of FAK and phosphorylation at Tyr-397 [4,9,10]. This model of FAK activation and Tyr-397 phosphorylation is thought to be of central importance for FAKdependent signaling.…”

“…This model of FAK activation and Tyr-397 phosphorylation is thought to be of central importance for FAKdependent signaling. The biological importance of FAK-mediated signal transduction is underscored by the fact that this tyrosine kinase plays a fundamental role in embryonic development [11,12] and in the control of cell migration [9,[12][13][14][15][16][17][18][19], cell cycle progression [20] and apoptosis [21][22][23][24]. Furthermore, there is increasing evidence linking overexpression of FAK to the invasive properties of cancer cells [25][26][27][28][29][30][31].…”