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

Abstract

pp125^FAK is a 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.