Gβγ stimulates phosphoinositide 3-kinase-γ by direct interaction with two domains of the catalytic p110 subunit

Class I phosphoinositide 3-kinases (PI3Ks) regulate important cellular processes such as mitogenesis, apoptosis, and cytoskeletal functions. They include PI3K alpha, -beta, and -delta isoforms coupled to receptor tyrosine kinases and a PI3K gamma isoform activated by receptor-stimulated G proteins. This study examines the direct interaction of purified recombinant PI3K gamma catalytic subunit (p110 gamma) and G beta gamma complexes. When phosphatidylinositol was used as a substrate, G beta gamma stimulated p110 gamma lipid kinase activity more than 60-fold (EC50, similar to 20 nM). Stimulation was inhibited by G alpha(o)-GDP or wortmannin in a concentration-dependent fashion. Stoichiometric binding of a monoclonal antibody to the putative pleckstrin homology domain of p110 gamma did not affect G beta gamma-mediated enzymatic stimulation, whereas incubation of G beta gamma with a synthetic peptide resembling a predicted G beta gamma effector domain of type 2 adenylyl cyclase selectively inhibited activation of p110 gamma. G beta gamma complexes bound to N- as well as C-terminal deletion mutants of p110 gamma. Correspondingly, these enzymatically inactive N- and C-terminal mutants inhibited G beta gamma-mediated activation of wild type p110 gamma. Our data suggest that (i) p110 gamma directly interacts with G beta gamma, (ii) the pleckstrin homology domain is not the only region important for G beta gamma-mediated activation of the lipid kinase, and (iii) G beta gamma binds to at least two contact sites of p110 gamma, one of which is close to or within the catalytic core of the enzyme.