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 cultured mammalian cells,In HEK-293 and COS-7 cells transiently transfected with beta (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 G alpha (il), and was not duplicated by co-transfection of the constitutively active G alpha (s)-Q227L or G alpha (i)-Q204L mutant, Co-transfection of G beta (1), G beta (2) but not G beta (5) together with G gamma (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 G alpha 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 G alpha (q) and G alpha (i) transfection in COS cells. To the contrary, in our experiments G alpha (q)-Q209L inhibited Akt activation resulting from beta gamma or mutationally activated H-Ras co-transfection in these cells. In HEK-293 cells G alpha (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. me conclude that G proteins can regulate Akt by two distinct and potentially opposing mechanisms: activation by G beta gamma heterodimers in a phosphatidylinositol-3-OH kinase-dependent fashion, and inhibition mediated by G alpha (q). This work identifies Akt as a novel point of convergence between disparate signaling pathways.