Ras is an indispensable coregulator of the class I _B phosphoinositide 3-kinase p87/p110γ

Abstract

Class IB phosphoinositide 3-kinase ␥ (PI3K␥) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3K␥ consists of a catalytic p110␥ and a regulatory p87 PIKAP (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein ␤␥ (G␤␥)-mediated PI3K␥ regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110␥ activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3K␥-complemented bone marrow-derived mast cells (BMMCs) from p110␥ -/-mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein-coupled receptor (GPCR)-induced PI3K␥ activation. Although p101 strongly interacted with G␤␥, thereby mediating PI3K␥ membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing G␤␥-dependent membrane recruitment of p87/p110␥ by direct interaction with p110␥, suggesting the indispensability of Ras for activation of p87/p110␥. Consequently, interference with Ras signaling indeed selectively blocked p87/p110␥, but not p101/ p110␥, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110␥ activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3K␥ that could open up new possibilities for therapeutic intervention.confocal life cell imaging ͉ G protein ͉ receptor signaling ͉ mast cells

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein–coupled receptors (GPCRs)

“…This is in sharp contrast to the 552 DD-p110γ/p101 complex ( Fig. 2A), suggesting that p87 makes much less contribution to Gβγ interaction compared with p101, which is consistent with previous reports (22,45,46). (28,36,45,47).…”

Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

“…The direct stimulation of p110␥ lipid kinase activity by Ras was found only modest in one study (1309), but oncogenic Ras mutants potently stimulated p110␥ both in vitro and in transfected cells in another (1184). Importantly, the G␤␥ sensitivity of PI3K␥ stimulation is greatly enhanced by the p101 regulatory subunit in vitro or in cells (833,1465), yet p101 does not change the Ras sensitivity of the kinase. Accordingly, a mutant p110␥ with impaired Ras binding still responds to G␤␥, and this response is still enhanced by p101 (1184,1497).…”

“…In contrast, p84/p110␥ complexes are not sensitized to G␤␥ stimulation. Instead, the p84/p110␥ complex requires Ras binding for membrane recruitment and activation by GPCRs (833). Therefore, it appears that p110␥ complexed to the two distinct adaptors is regulated in a clearly distinct manner and participates in distinct cellular responses.…”

Angiotensin II Stimulates Thick Ascending Limb NO Production via AT2 Receptors and Akt1-dependent Nitric-oxide Synthase 3 (NOS3) Activation

“…Recent reports suggest that the G␤␥ subunit of G proteins can directly interact with certain PI3K isoforms promoting their translocation to the plasma membrane, a prerequisite for PI3K activation. Depending on the isoform, this process may or may not require participation of Ras-GTP (40). The mechanism whereby AT 2 activation leads to increased PI3K activity in the TAL needs to be further explored.…”