G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.
Abstract
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.
Type
Journal articleSubject
Adaptor Proteins, Signal TransducingAdaptor Proteins, Vesicular Transport
Androstadienes
Animals
Benzoquinones
Calcium-Calmodulin-Dependent Protein Kinases
Cell Line
Cercopithecus aethiops
Enzyme Activation
Enzyme Inhibitors
GTP-Binding Proteins
Genistein
Humans
Isoflavones
Kidney
Kinetics
Lactams, Macrocyclic
Macromolecular Substances
Pertussis Toxin
Phosphates
Phosphatidylinositol 3-Kinases
Phosphorylation
Phosphotransferases (Alcohol Group Acceptor)
Protein-Tyrosine Kinases
Proteins
Proto-Oncogene Proteins p21(ras)
Quinones
Receptors, Adrenergic, alpha-2
Recombinant Proteins
Rifabutin
Shc Signaling Adaptor Proteins
Src Homology 2 Domain-Containing, Transforming Protein 1
Tetradecanoylphorbol Acetate
Transfection
Virulence Factors, Bordetella
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Show full item recordScholars@Duke
Robert J. Lefkowitz
The Chancellor's Distinguished Professor of Medicine
Dr. Lefkowitz’s memoir, A Funny Thing Happened on the Way to Stockholm, recounts his
early career as a cardiologist and his transition to biochemistry, which led to his
Nobel Prize win.
Robert J. Lefkowitz, M.D. is Chancellor’s Distinguished Professor of Medicine and
Professor of Biochemistry and Chemistry at the Duke University Medical Center. He
has bee

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