Inhibition of cell spreading by expression of the C-terminal domain of focal adhesion kinase (FAK) is rescued by coexpression of Src or catalytically inactive FAK: A role for paxillin tyrosine phosphorylation

pp125(FAK) is tyrosine kinase that appears to regulate the assembly of focal adhesions and thereby promotes cell spreading on the extracellular matrix. In some cells, the C terminus of pp125(FAK) is expressed as a separate protein, pp41/43(FRNK). We have previously shown that overexpression of pp41/43(FRNK) inhibits tyrosine phosphorylation of pp125(FAK) and paxillin and, in addition, delays cell spreading and focal adhesion assembly. Thus, pp41/43(FRNK) functions as a negative inhibitor of adhesion signaling and provides a tool to dissect the mechanism by which pp125(FAK) promotes cell spreading. We report here that the inhibitory effects of pp41/43(FRNK) expression can be rescued by the co-overexpression of wild-type pp125(FAK) and partially rescued by catalytically inactive variants of pp125(FAK). However, coexpression of an autophosphorylation site mutant of pp125(FAK), Which fails to bind the SH2 domain of pp60(c-Src), or a mutant that fails to bind paxillin did not promote cell spreading. In contrast, expression of pp41/43(FRNK) and pp60(c-Src) reconstituted cell spreading and tyrosine phosphorylation of paxillin but did so without inducing tyrosine phosphorylation of pp125(FAK). These data provide additional support for a model whereby pp125(FAK) acts as a ''switchable adaptor'' that recruits pp60(c-Src) to phosphorylate paxillin, promoting cell spreading. In addition, these data point to tyrosine phosphorylation of paxillin as being a critical step in focal adhesion assembly.