Targeting Pyk2 to beta 1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cells

Focal adhesion kinase-null (FAK(-/-)) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpres sion. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is exp ressed in FAK(-/-) cells, yet it exhibits a perinuclear distribution and do es not functionally substitute for FAK, Chimeric Pyk2/FAK proteins were cre ated and expressed in FAK-/- cells to determine the impact of Pyk2 localiza tion to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chime ra was perinuclear distributed, stable expression of a Pyk2 chimera with th e FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-recons tituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to p romote cell motility, Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a beta1-integrin-containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/F AK-CT S-1034 reconstituted FAK(-/-) cells, exhibit elevated FN-stimulated e xtracellular signal-regulated kinase 2 (ERK2) and c-Jun NH2-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhan ced both the extent and duration of FN-stimulated ERK2 activity which was n ecessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation a s well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH2-terminal and kinase domains of P yk2 can functionally substitute for FAK in promoting FN-stimulated signalin g and motility events when localized to P-integrin-containing focal contact sites via interactions mediated by the FAK-CT domain.