Activation of Gsalpha 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 Gsalpha 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 Gsalpha and activates this protein. Employing purified EGFR and Gsalpha, we have demonstrated that the EGFR kinase phosphorylates Gsalpha in a time-dependent manner with a stoichiometry of 2 mol of phosphate incorporated/mol of Gsalpha. As determined by phosphoamino acid analysis, the phosphorylation of Gsalpha by the EGFR kinase was exclusively on tyrosine residues. Interestingly, GDP and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) inhibited the phosphorylation of Gsalpha without altering EGFR autophosphorylation. However, G protein betagamma subunits protected against GDP- and GTPgammaS-mediated inhibition of phosphorylation of Gsalpha. In functional studies, phospho-Gsalpha demonstrated a greater GTPase activity and also a greater capacity to bind GTPgammaS as compared to the nonphosphorylated Gsalpha. Moreover, the phospho-Gsalpha augmented adenylyl cyclase activity in S49 cyc- cell membranes to a greater extent than its nonphosphorylated counterpart. Therefore, we conclude that phosphorylation of Gsalpha on tyrosine residues by the EGFR kinase activates this G protein and increases its ability to stimulate adenylyl cyclase.
Poppleton
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Department of Pharmacology, the Center for Health Sciences, University of Tennessee, Memphis, Tennessee 38163, USA.
Sun
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Fulgham
D
D
Bertics
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P
Patel
T B
TB
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D018076:DNA, Complementary; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation
D018076:DNA, Complementary; D066246:ErbB Receptors; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases