An essential role for src tyrosine kinase/shc adapter protein complexes in gβy subunit-mediated mitogen-activated protein kinase activation

Several G protein-coupled recepiors which interact with pertussis toxin-sensitive heterotrimenc G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gβysubunit-mediated increases in tyrosine phosphorylation of the She adapter protein, GRB2/She complex formation and recruitment of Ras guanine nucleotide exchange factor activity. We investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src, in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or transient expression of Gβy subunits in COS-7 cells. In vitro kinase assays of She immunoprecipitates following LPA stimulation demonstrated rapid, (ransient recruitment of tyrosine kinase activity into She immune complexes. Recruitment of lyrosine kinase aclivity was pertussis toxin-sensitive and mimicked by cellular expression of Gβy subunits. Immunoblots for coprecipitated proteins in she immunoprecipitates revealed a transient association of She and c-Src following LPA stimulation which coincided with increases in She-associated tyrosine kinase activity and she tyrosine phosphorylation. LPA stimulation or expression of Gβy subuntis resulted in c-Src activation as assessed by increased c-Src autophosphorylation. Overexpression of wild type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in She immunoprecipitates, increased She tyrosine phosphorylation and Shc/GRB2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gβy suhumts or expression of e-Src was sensitive to dominant negatives of mSos, Ras and Raf Coexpression of Csk, which inactivates Src family kinases by phosphoryfating the regulatory C-terminai lyrosine residue, inhibited LPA stimulation of She [yrosine phosphorylalion, Shc/GRB2 complex formation and MAP kinase activation. These data suggest that Gβy subunit-mediated formation of Shc/e-Sre complexes and cSrc kinase activation are eariy events m Ras-dependent activation of MAP kmasc via pertussis toxin sensitive G protein-coupled receptors.

Duke Scholars

Author Robert J. Lefkowitz Medicine, Cardiology