N-WASP-directed actin polymerization activates p130Cas phosphorylation and lamellipodium spreading

Abstract

Tyrosine phosphorylation of the substrate domain of Cas (CasSD) correlates with increased cell migration in healthy and diseased cells. Here we address the mechanism leading to CasSD phosphorylation in the context of fibronectin-induced early spreading of fibroblasts. We previously demonstrated that mechanical stretching of CasSD exposes phosphorylation sites for Src family kinases (SFKs). Surprisingly, phosphorylation of CasSD was independent of myosin contractile activity, but dependent on actin polymerization. Further, we found that CasSD phosphorylation in early cell spreading required: (1) integrin anchorage and integrin-mediated SFK activation, (2) association of Cas with focal adhesion kinase (FAK) and (3) N-WASP actin assembly activity. These findings and analyses of Cas domain interactions indicate that Cas N-terminus associates with FAK/N-WASP complex at the cell's protrusive edge and that Cas C-terminus associates with immobilized integrin-SFK cluster. Thus, extension of the leading edge by actin polymerization could stretch Cas in early cell spreading, priming it for phosphorylation.