Touhara, KHawes, BEvan Biesen, TLefkowitz, RJ2013-09-101995-09-260027-8424https://hdl.handle.net/10161/7837The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 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 p21ras. Worthmannin, 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 p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.Adaptor Proteins, Signal TransducingAdaptor Proteins, Vesicular TransportAndrostadienesAnimalsBenzoquinonesCalcium-Calmodulin-Dependent Protein KinasesCell LineCercopithecus aethiopsEnzyme ActivationEnzyme InhibitorsGTP-Binding ProteinsGenisteinHumansIsoflavonesKidneyKineticsLactams, MacrocyclicMacromolecular SubstancesPertussis ToxinPhosphatesPhosphatidylinositol 3-KinasesPhosphorylationPhosphotransferases (Alcohol Group Acceptor)Protein-Tyrosine KinasesProteinsProto-Oncogene Proteins p21(ras)QuinonesReceptors, Adrenergic, alpha-2Recombinant ProteinsRifabutinShc Signaling Adaptor ProteinsSrc Homology 2 Domain-Containing, Transforming Protein 1Tetradecanoylphorbol AcetateTransfectionVirulence Factors, BordetellaJournal article