ROLE OF C-SRC TYROSINE KINASE IN G PROTEIN-COUPLED RECEPTOR-MEDIATED AND G-BETA-GAMMA-SUBUNIT-MEDIATED ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASES

Several G protein-coupled receptors that interact with pertussis toxin -sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases, The mechanism involves G b eta gamma subunit-mediated increases in tyrosine phosphorylation of th e She adapter protein, Shc-Grb2 complex formation, and recruitment of Pas guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activa tion of the MAP kinase pathway via endogenous G protein-coupled lysoph osphatidic acid (LPA) receptors or by transient expression of G beta g amma subunits in COS-7 cells. In vitro kinase assays of She immunoprec ipitates following LPA stimulation demonstrated rapid, transient recru itment of tyrosine kinase activity into She immune complexes, Recruitm ent of tyrosine kinase activity was pertussis toxin sensitive and mimi cked by cellular expression of G beta gamma subunits. Immunoblots for coprecipitated proteins in She immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincid ed with increases in Shc-associated tyrosine kinase activity and She t yrosine phosphorylation. LPA stimulation or expression of G beta gamma subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation, Overexpression of wild-type or constitutively ac tive mutant c-Src, but not kinase inactive mutant c-Src, lead to incre ased 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 G be ta gamma subunits, or expression of c-Src was sensitive to dominant ne gatives of mSos, Pas, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosi ne residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc-Grb2 complex formation, and MAP kinase activation, These data sug gest that G beta gamma subunit-mediated formation of Shc-c-Src complex es and c-Src kinase activation are early events in Ras-dependent activ ation of MAP kinase via pertussis toxin-sensitive G protein coupled re ceptors.