Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein-coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme's activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gβγ heterodimers, we mapped these regulatory interactions using hydrogen-deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gβγ heterodimers, leading to PI3Kγ activation. Mutating Gβγ-interaction sites of either p110γ or p101 ablates G-protein-coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen-deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gβγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gβγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gβγ heterodimers; and the β-adrenergic receptor kinase.

Synergistic activation by heterotrimeric guanine nucleotide–binding protein (G protein)-coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110β from other class IA phosphoinositide 3-kinases (PI3Ks). Activation of p110β is specifically implicated in various physiological and pathophysiological processes, such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110β-dependent functions, we identified the site in p110β that binds to the Gβγ subunit of G proteins. Mutation of this site eliminated Gβγ-dependent activation of PI3Kβ (a dimer of p110β and the p85 regulatory subunit) in vitro and in cells, without affecting basal activity or phosphotyrosine peptide–mediated activation. Disrupting the p110β-Gβγ interaction by mutation or with a cell-permeable peptide inhibitor blocked the transforming capacity of PI3Kβ in fibroblasts, and reduced proliferation, chemotaxis, and invasiveness of PTEN-null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3Kβ could provide a therapeutic approach for tumors that depend on p110β for growth and metastasis.

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

Background: p87 and p101 represent non-catalytic subunits of class I B PI3K␥. Results: Expression and activity of PI3K␥ is modified differently by p87 and p101 in vitro and in living cells. Conclusion: Non-catalytic subunits of PI3K␥ represent two different regulators in the absence of G␤␥ or Ras. Significance: p87 and p101 determine diversity within class I B PI3K␥ and allow integration in distinct PI3K␥ signaling pathways.

G-protein-regulated PI3Kγ (phosphoinositide 3-kinase γ) plays a crucial role in inflammatory and allergic processes. PI3Kγ, a dimeric protein formed by the non-catalytic p101 and catalytic p110γ subunits, is stimulated by receptor-released Gβγ complexes. We have demonstrated previously that Gβγ stimulates both monomeric p110γ and dimeric p110γ/p101 lipid kinase activity in vitro. In order to identify the Gβ residues responsible for the Gβγ-PI3Kγ interaction, we examined Gβ1 mutants for their ability to stimulate lipid and protein kinase activities and to recruit PI3Kγ to lipid vesicles. Our findings revealed different interaction profiles of Gβ residues interacting with p110γ or p110γ/p101. Moreover, p101 was able to rescue the stimulatory activity of Gβ1 mutants incapable of modulating monomeric p110γ. In addition to the known adaptor function of p101, in the present paper we show a novel regulatory role of p101 in the activation of PI3Kγ.

The PI3-kinase pathway is commonly activated in tumors, most often by loss of PTEN lipid phosphatase activity or the amplification or mutation of p110α. Oncogenic mutants have commonly been found in p110α, but rarely in any of the other catalytic subunits of class I PI3-kinases. We here characterize a p110β helical domain mutation, E633K, first identified in a Her2-positive breast cancer. The mutation increases basal p110β activity, but does not affect activation of p85/p110β dimers by phosphopeptides or Gβγ. Expression of the mutant causes increases in Akt and S6K1 activation, transformation, chemotaxis, proliferation and survival in low serum. E633 is conserved among class I PI3 Ks, and its mutation in p110β is also activating. Interestingly, the E633K mutant occurs near a region that interacts with membranes in activated PI 3-kinases, and its mutation abrogates the requirement for an intact Ras-binding domain in p110β-mediated transformation. We propose that the E633K mutant activates p110β by enhancing its basal association with membranes. This study presents the first analysis of an activating oncogenic mutation of p110β.

Class IB phosphoinositide 3-kinases (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled-receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody (mAb(A)p110γ) to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ-interface, hydrogen-deuterium exchange coupled to mass spectrometry measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gβγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gβγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ Gβγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gβγ-stimulated lipid kinase activity of p87-p110γ 30 times more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gβγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs.

Phosphoinositide 3-kinases (PI 3-kinases) are regulated by a diverse range of upstream activators, including receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and small GTPases from the Ras, Rho and Rab families. For the Class IA PI 3-kinase PI3Kβ, two mechanisms for GPCR-mediated regulation have been described: direct binding of Gβγ subunits to the C2-helical domain linker of p110β, and Dock180/Elmo1-mediated activation of Rac1, which binds to the Ras-Binding Domain of p110β. We now show that the integration of these dual pathways is unexpectedly complex. In breast cancer cells, expression of constitutively activated Rac1 (CA-Rac1) along with either GPCR stimulation or expression of Gβγ led to an additive PI3Kβ-dependent activation of Akt. Whereas CA-Rac1-mediated activation of Akt was blocked in cells expressing a mutated PI3Kβ that cannot bind Gβγ, Gβγ and GPCR-mediated activation of Akt was preserved when Rac1 binding to PI3Kβ was blocked. Surprisingly, PI3Kβ-dependent CA-Rac1 signaling to Akt was still seen in cells expressing a mutant p110β that cannot bind Rac1. Instead of directly binding to PI3Kβ, CA-Rac1 acts by enhancing Gβγ coupling to PI3Kβ, as CA-Rac1-mediated Akt activation was blocked by inhibitors of Gβγ. Cells expressing CA-Rac1 exhibited a robust induction of macropinocytosis, and inhibitors of macropinocytosis blocked the activation of Akt by CA-Rac1 or lysophosphatidic acid. Our data suggest that Rac1 can potentiate the activation of PI3Kβ by GPCRs through an indirect mechanism, by driving the formation of macropinosomes that serve as signaling platforms for Gβγ coupling to PI3Kβ.