Integrin-dependent PLC-1 phosphorylation mediates fibronectin-dependent adhesion

Although integrin engagement initiates signaling events such as focal-adhesion kinase (FAK) and Src kinase activation, the role of phosphoinositide turnover in cell adhesion is less clear. To assess PLC-1 function in this process, Plcg1^-/- fibroblasts (Null) were compared with the same fibroblasts in which PLC-1 was re-expressed (Null+). Following plating on fibronectin, Null cells displayed a significantly impaired rate of adhesion compared with Null+ cells. This defect was detected at low concentrations of fibronectin; at high fibronectin concentrations, the Null and Null+ cells displayed equivalent adhesion characteristics. The differences were not due to PLC-1-dependent changes in integrin subunit expression, nor was integrin receptor clustering impaired with the absence of PLC-1. Experiments with site-specific antibodies and PLC-1 mutants showed that fibronectin selectively increased phosphorylation of Tyr783 and that mutagenesis of this residue, but not Tyr771 or Tyr1253, abrogated fibronectin-dependent adhesion. The SH2 domains of PLC-1 were also required for maximal adhesion on fibronectin. Adhesion to fibronectin induced PLC-1 tyrosine phosphorylation that was inhibited by a Src-kinase inhibitor, but not an epidermal-growth-factor-receptor kinase inhibitor. Moreover, in cells null for Src family members, but not in cells null for FAK family members, integrin-dependent PLC-1 tyrosine phosphorylation was greatly reduced. Finally, the data demonstrated that PLC-1 co-immunoprecipitated with Src following fibronectin-induced integrin activation, and this association did not depend on FAK expression.