Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration

The focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways, We show that expression of the FAK-related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak(-/-) embryos (FAK(-)) compared with cells from fak(+/+) embryos (FAK(+)). Pyk2 was localized to perinuclear regions in both FAK(+) and FAK(-) cells, Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK(-) but not FAK(+) cells. Increased Pyk2 tyrosine phosphorylation paralleled the timecourse of Grb2 binding to Shc and activation of ERK2 in FAK(-) cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK(-) cells. However, Pyk2 associated with active Src-family PTKs after FN but not poly-L-lysine replating of the FAK(-) cells, Overexpression of both wild-type (WT) and kinase-inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN-stimulated c-Src in vitro kinase activity in FAK(-) cells, but only WT Pyk2 overexpression enhanced FN-stimulated activation of co-transfected ERK2, Interestingly, Pyk2 overexpression only weakly augmented FAK(-) cell migration to FN whereas transient FAK expression promoted FAK(-) cell migration to FN efficiently compared with FAK(+) cells, Significantly, repression of endogenous Src-family PTK activity by p50(csk) overexpression inhibited FN-stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK(-) but not in FAK(+) cells, These studies show that Pyk2 and Src-family PTKs combine to promote FN-stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK(-) cell migration defects.