Activation of G(s alpha) by the epidermal growth factor receptor involves phosphorylation

Previous studies from our laboratory have shown that epidermal growth factor (EGF) stimulates cAMP accumulation in the heart via a process involving G(s alpha) and the EGF receptor (EGFR) protein tyrosine kinase activity (Nair, B. G., Parikh, B., Milligan, G., and Patel, T. B. (1990) J. Biol. Chem. 265, 21317-21322; Nair, B. G., and Patel, T. B. (1993) Biochem. Pharmacol. 46, 1239-1245). Therefore, studies were performed to investigate the hypothesis that the EGFR protein tyrosine kinase phosphorylates G(s alpha) and activates this protein. Employing purified EGFR and G(s alpha), we have demonstrated that the EGFR kinase phosphorylates G(s alpha) in a time-dependent manner with a stoichiometry of 2 mol of phosphate incorporated/mol of G(s alpha). As determined by phosphoamino acid analysis, the phosphorylation of G(s alpha) by the EGFR kinase was exclusively on tyrosine residues. Interestingly, GDP and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) inhibited the phosphorylation of G(s alpha) without altering EGFR autophosphorylation, However, G protein beta gamma subunits protected against GDP- and GTP gamma S-mediated inhibition of phosphorylation of G(s alpha). In functional studies, phospho-G(s alpha) demonstrated a greater GTPase activity and also a greater capacity to bind GTP gamma S as compared to the nonphosphorylated G(s alpha). Moreover, the phospho-G(s alpha) augmented adenylyl cyclase activity in S49 cyc(-) cell membranes to a greater extent than its nonphosphorylated counterpart. Therefore, we conclude that phosphorylation of G(s alpha) on tyrosine residues by the EGFR kinase activates this G protein and increases its ability to stimulate adenylyl cyclase.