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

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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 p50csk 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.