Regulation of phosphoinositide 3-kinaseγ protein kinase activityin vitro and in COS-7 cells

Abstract

Phosphoinositide 3-kinases (PI3K) are a family of signaling enzymes with dual specificity. While their lipid kinase function has been extensively studied, there is only limited knowledge about the regulation of their protein kinase activity and its physiological function. Here we report that the protein kinase activity of the PI3K species γ expresses a biphasic response to the Gβγ subunits of heterotrimeric G proteins in vitro. A slight stimulatory effect of Gβγ on PI3Kγ autophosphorylation and on trans-phosphorylation of the protein kinase MEK-1 has been observed in the low nanomolar range. Higher Gβγ concentrations inhibit, rather than stimulate the protein kinase activity. The regulatory effects of Gβγ on PI3Kγ activities have been further investigated in COS-7 cells. Recently we demonstrated that overexpression of PI3Kγ induces mitogen-activated protein kinase (MAPK) activation, probably related to PI3Kγ protein kinase activity. Now we show that PI3Kγ-induced MAPK activation can be further stimulated by coexpression of low amounts of Gβγ, while augmentation of Gβγ expression induced abolition of MAPK stimulation. In contrast to MAPK stimulation, PI3Kγ-dependent protein kinase B (PKB) activity revealed sensitivity to higher Gβγ expression in COS-7 cells, most likely due to the induction of PI3Kγ lipid kinase activity. Expression of PI3KγFRAP, a lipid kinase inactive mutant, induced MAPK activity in a similar manner as the wild-type enzyme, but did not affect PKB. These data are consistent with the hypothesis that varying activation states of Gβγ subunits and the related G protein-coupled receptors differentially affect the complex cellular signaling events induced by PI3Kγ.