Abstract

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 time-course 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.