Identification of integrin-stimulated sites of serine phosphorylation in FRNK, the separately expressed C-terminal domain of focal adhesion kinase: a potential role for protein kinase A

Focal adhesion kinase (pp125^FAK) is a protein tyrosine kinase that is localized to focal adhesions in many cell types and which undergoes tyrosine phosphorylation after integrin binding to extracellular matrix. 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 Ser¹⁴⁸ and Ser¹⁵¹ as residues in FRNK that are phosphorylated after tyrosine phosphorylation of pp125^FAK and in response to integrin binding to fibronectin. Tyrosine phosphorylation of pp125^FAK appears to be an early event after integrin occupancy, and serine phosphorylation 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 phosphorylation of pp125^FAK. However, these PKA inhibitors are unlikely to delay cell spreading simply by preventing serine phosphorylation of FRNK, as overexpression of FRNK containing mutations of Ser¹⁴⁸ and Ser¹⁵¹ 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.