G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.
The 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.
Touhara
K
K
Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
Hawes
B E
BE
van Biesen
T
T
Lefkowitz
R J
RJ
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016227:Benzoquinones; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D019833:Genistein; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology; D007668:Kidney; D007700:Kinetics; D047029:Lactams, Macrocyclic; D046911:Macromolecular Substances; D037342:Pertussis Toxin; D010710:Phosphates / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011506:Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology; D018341:Receptors, Adrenergic, alpha-2 / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017828:Rifabutin / Q000031:analogs & derivatives; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000077191:Wortmannin
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000730:Androstadienes; D016227:Benzoquinones; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D019204:GTP-Binding Proteins; D019833:Genistein; D007529:Isoflavones; D047029:Lactams, Macrocyclic; D046911:Macromolecular Substances; D037342:Pertussis Toxin; D010710:Phosphates; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; D011506:Proteins; D016283:Proto-Oncogene Proteins p21(ras); D011809:Quinones; D018341:Receptors, Adrenergic, alpha-2; D011994:Recombinant Proteins; D017828:Rifabutin; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013755:Tetradecanoylphorbol Acetate; D010566:Virulence Factors, Bordetella; D000077191:Wortmannin; C528024:SHC1 protein, human; C020754:herbimycin; C501009:HRAS protein, human