G-PROTEIN BETA-GAMMA-SUBUNITS STIMULATE PHOSPHORYLATION OF SHC ADAPTER PROTEIN

The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive G(i)-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21(ras) activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21(ras) activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to G(i) in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximate to 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa, The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21(ras). Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K, These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21(ras) activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors,