G protein βγ subunits stimulate phosphorylation of Shc adapter protein

Abstract

The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive G(i) coupled receptors is known to involve the βγ subunits of heterotrimeric G proteins (Gβγ), p21(ras) activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of Gβγ- stimulated p21(ras) activation, Gβγ-mediated tyrosine phosphorylation was examined by overexpressing Gβγ or α2-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 ≃50 kDa (designated as p52) in Gβγ-transfected cells or in α2-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 Gβγ- or α2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of β-adrenergic receptor kinase (a Gβγ-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 Gβγ- 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.