IDENTIFICATION OF INTEGRIN-STIMULATED SITES OF SERINE PHOSPHORYLATIONIN FRNK, THE SEPARATELY EXPRESSED C-TERMINAL DOMAIN OF FOCAL ADHESIONKINASE - A POTENTIAL ROLE FOR PROTEIN-KINASE-A

Focal adhesion kinase (pp125(FAK)) is a protein tyrosine kinase that i s localized to focal adhesions in many cell types and which undergoes tyrosine phosphorylation after integrin binding to extracellular matri x, In some cells the C-terminal non-catalytic domain of pp125(FAK) is expressed as a separate protein referred to as FRNK (FAK-related, non- kinase). We have previously shown that overexpression of FRNK inhibits tyrosine phosphorylation of pp125(FAK) and its substrates as well as inhibiting cell spreading on fibronectin. In this report we identify S er(148) and Ser(151) as residues in FRNK that are phosphorylated after tyrosine phosphorylation of pp125(FAK) and in response to integrin bi nding to fibronectin. Tyrosine phosphorylation of pp125(FAK) appears t o be an early event after integrin occupancy, and serine phosphorylati on of FRNK occurs significantly later. Treatment of fibroblasts with a series of protein kinase A inhibitors delayed serine phosphorylation of FRNK as well as cell spreading on fibronectin and tyrosine phosphor ylation of pp125(FAK). However, these PKA inhibitors are unlikely to d elay cell spreading simply by preventing serine phosphorylation of FRN K, as overexpression of FRNK containing mutations of Ser(148) and Ser( 151) either singly or jointly to either alanine or glutamate residues did not significantly alter the ability of FRNK to act as an inhibitor of pp125(FAK).