Gβγ Stimulates Phosphoinositide 3-Kinase-γ by Direct Interaction with Two Domains of the Catalytic p110 Subunit

Abstract

Class I phosphoinositide 3-kinases (PI3Ks) regulate important cellular processes such as mitogenesis, apoptosis, and cytoskeletal functions. They include PI3K␣, -␤, and -␦ isoforms coupled to receptor tyrosine kinases and a PI3K␥ isoform activated by receptor-stimulated G proteins. This study examines the direct interaction of purified recombinant PI3K␥ catalytic subunit (p110␥) and G␤␥ complexes. When phosphatidylinositol was used as a substrate, G␤␥ stimulated p110␥ lipid kinase activity more than 60-fold (EC 50 , ϳ20 nM). Stimulation was inhibited by G␣ o -GDP or wortmannin in a concentration-dependent fashion. Stoichiometric binding of a monoclonal antibody to the putative pleckstrin homology domain of p110␥ did not affect G␤␥-mediated enzymatic stimulation, whereas incubation of G␤␥ with a synthetic peptide resembling a predicted G␤␥ effector domain of type 2 adenylyl cyclase selectively inhibited activation of p110␥. G␤␥ complexes bound to N-as well as C-terminal deletion mutants of p110␥. Correspondingly, these enzymatically inactive N-and C-terminal mutants inhibited G␤␥-mediated activation of wild type p110␥. Our data suggest that (i) p110␥ directly interacts with G␤␥, (ii) the pleckstrin homology domain is not the only region important for G␤␥-mediated activation of the lipid kinase, and (iii) G␤␥ binds to at least two contact sites of p110␥, one of which is close to or within the catalytic core of the enzyme.

Heterotrimeric G-protein βγ-dimers in growth and differentiation

“…However, a deletion analysis of p110g-GST fusion protein appears to indicate that Gbg-dimer binds to multiple regions of the p110g-subunit. Interactions of multiple regions of p110g-subunit with Gbg-dimers is further supported by the observation that both N-and C-terminal fragments of p110g inhibit Gbg-dimer stimulated PI3Kg activity (Leopoldt et al, 1998). Involvement of the p101-subunit in binding to Gbg-dimer, is supported by a deletion analysis of p101 which indicates that Gbg-stimulated activity requires the N-terminus of p101 and correlates with the tightness of binding of p101 to p110g (Krugmann et al, 1999).…”

“…Direct binding of Gbg-dimer to p110g-subunit is evidenced by co-puri®cation of Gbg-dimers with a p110g-GST fusion protein (Leopoldt et al, 1998). However, a deletion analysis of p110g-GST fusion protein appears to indicate that Gbg-dimer binds to multiple regions of the p110g-subunit.…”

Cellular function of p70S6K: A role in regulating cell motility

“…2 For G-protein-coupled receptor systems, the signalling for PI 3-kinase activation is less clear and several mechanisms have been suggested. A number of reports have identified a p110γ catalytic subunit, which is activated through binding either directly to the βγ subunits of activated trimeric G-proteins 3 or through interaction with a p101 adaptor protein. 4,5 Other studies have suggested alternative mechanisms involving G-protein-mediated activation of receptor tyrosine kinase activity 6 or through the p110β isoform, which can also be activated directly by G-protein βγ subunits.…”

The Gβγ-Src signaling pathway regulates TNF-induced necroptosis via control of necrosome translocation

“…Previous studies have shown that cAMP, PI3K, GIRK channel, calcium and Src pathways can act downstream of the Gβγ complex [20][21][22][23][24]51]. Among these pathways, we found that the cAMP pathway is not involved in TNF-induced necroptosis in L929 cells ( Figure 4A).…”

“…The first evidence indicating that Gβγ functions not only as a scaffold but also as a signal transducer/activator came from the study of the activation of muscarinic-gated potassium channels in chicken embryonic atrial cells [19]. More recently, Gβγ has also been revealed to participate in the activation of a variety of signaling pathways including the cAMP/PKA, PI3K, calcium, Src (Rous sarcoma oncogene) kinase and GIRK pathways [20][21][22][23][24]. Gβγ dimer within the heterotrimeric G protein complex is composed of Gβ and Gγ subunits.…”