Dual Regulation of Akt/Protein Kinase B by Heterotrimeric G Protein Subunits

While positive regulation of c-Akt (also known as protein kinase B) by receptor tyrosine kinases is well documented, compounds acting through G protein-coupled receptors can also activate Akt and its downstream targets. We therefore explored the role of G protein subunits in the regulation of Akt in...

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Bibliographic Details
Published in:The Journal of biological chemistry 2000-12, Vol.275 (49), p.38870-38876
Main Authors: Bommakanti, R K, Vinayak, S, Simonds, W F
Format: Article
Language:English
Subjects:
Animals
Cell Line
Chlorocebus aethiops
Chromones - pharmacology
COS Cells
Enzyme Inhibitors - pharmacology
GTP-Binding Protein alpha Subunits, Gi-Go - metabolism
GTP-Binding Protein alpha Subunits, Gq-G11
Heterotrimeric GTP-Binding Proteins - metabolism
Humans
Isoproterenol - pharmacology
Morpholines - pharmacology
Protein Subunits
Protein-Serine-Threonine Kinases
Protein-Tyrosine Kinases - metabolism
Proto-Oncogene Proteins - metabolism
Proto-Oncogene Proteins c-akt
Receptors, Adrenergic, beta-2 - drug effects
Receptors, Adrenergic, beta-2 - physiology
Receptors, Muscarinic - drug effects
Receptors, Muscarinic - physiology
Recombinant Proteins - metabolism
Substrate Specificity
Transfection
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Summary:While positive regulation of c-Akt (also known as protein kinase B) by receptor tyrosine kinases is well documented, compounds acting through G protein-coupled receptors can also activate Akt and its downstream targets. We therefore explored the role of G protein subunits in the regulation of Akt in cultured mammalian cells. In HEK-293 and COS-7 cells transiently transfected with β 2 -adrenergic or m2 muscarinic receptors, respectively, treatment with agonist-induced phosphorylation of Akt at serine 473 as evidenced by phosphoserine-specific immunoblots. This effect was blocked by the phosphatidylinositol-3-OH kinase inhibitor LY294002 and wild-type Gα i1 , and was not duplicated by co-transfection of the constitutively active Gα s -Q227L or Gα i -Q204L mutant. Co-transfection of Gβ 1 , Gβ 2 but not Gβ 5 together with Gγ 2 activated the kinase when assayed in vitro following immunoprecipitation of the epitope-tagged enzyme. In contrast, constitutively activated G protein subunits representing the four Gα subfamilies were found unable to activate Akt in either cell line. The latter results are in disagreement with a report by Murga et al. (Murga, C., Laguinge, L., Wetzker, R., Cuadrado, A., and Gutkind, J. S. (1998) J. Biol. Chem . 273, 19080–19085) that described activation of Akt in response to mutationally activated Gα q and Gα i transfection in COS cells. To the contrary, in our experiments Gα q -Q209L inhibited Akt activation resulting from βγ or mutationally activated H-Ras co-transfection in these cells. In HEK-293 cells Gα q -Q209L transfection inhibited insulin-like growth factor-1 activation of epitope-tagged Akt. In m1 muscarinic receptor transfected HEK-293 cells, carbachol inhibited insulin-like growth factor-1 stimulated phosphorylation at Ser 473 of endogenous Akt in an atropine-reversible fashion. We conclude that G proteins can regulate Akt by two distinct and potentially opposing mechanisms: activation by Gβγ heterodimers in a phosphatidylinositol-3-OH kinase-dependent fashion, and inhibition mediated by Gα q . This work identifies Akt as a novel point of convergence between disparate signaling pathways.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M007403200