PLEKHS1 drives PI3Ks and remodels pathway homeostasis in PTEN-null prostate.

The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP3/PI(3,4)P2-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP3 signaling, and supports tumor progression.

Gßγ is a direct regulator of endogenous p101/p110γ and p84/p110γ PI3Kγ complexes in mouse neutrophils.

The PI3Kγ isoform is activated by Gi-coupled GPCRs in myeloid cells, but the extent to which the two endogenous complexes of PI3Kγ, p101/p110γ and p84/p110γ, receive direct regulation through Gßγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gßγ binding to p110γ (RK552DD) or to p101 (VVKR777AAAA) and investigated the effects of these mutations in primary neutrophils and in mouse models of neutrophilic inflammation. Loss of Gßγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gßγ binding to p101 caused more variable effects, depending on both the agonist and cellular response, with the biggest reductions seen in PIP3 production by primary neutrophils in response to LTB4 and MIP-2 and in the migration of neutrophils during thioglycolate-induced peritonitis or MIP2-induced ear pouch inflammation. We also observed that p101VVKR777AAAA neutrophils showed enhanced p84-dependent ROS responses to fMLP and C5a, suggesting that competition may exist between p101/p110γ and p84/p110γ for Gßγ subunits downstream of GPCR activation. GPCRs did not activate p110γ in neutrophils from mice lacking both the p101 and p84 regulatory subunits, indicating that RAS binding to p110γ is insufficient to support GPCR activation in this cell type. These findings define a direct role for Gßγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs.

Frontline Science: TNF-α and GM-CSF1 priming augments the role of SOS1/2 in driving activation of Ras, PI3K-γ, and neutrophil proinflammatory responses.

Circulating neutrophils are, by necessity, quiescent and relatively unresponsive to acute stimuli. In regions of inflammation, mediators can prime neutrophils to react to acute stimuli with stronger proinflammatory, pathogen-killing responses. In neutrophils G protein-coupled receptor (GPCR)-driven proinflammatory responses, such as reactive oxygen species (ROS) formation and accumulation of the key intracellular messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3 ), are highly dependent on PI3K-γ, a Ras-GTP, and Gßγ coincidence detector. In unprimed cells, the major GPCR-triggered activator of Ras is the Ras guanine nucleotide exchange factor (GEF), Ras guanine nucleotide releasing protein 4 (RasGRP4). Although priming is known to increase GPCR-PIP3 signaling, the mechanisms underlying this augmentation remain unclear. We used genetically modified mice to address the role of the 2 RasGEFs, RasGRP4 and son of sevenless (SOS)1/2, in neutrophil priming. We found that following GM-CSF/TNFα priming, RasGRP4 had only a minor role in the enhanced responses. In contrast, SOS1/2 acquired a substantial role in ROS formation, PIP3 accumulation, and ERK activation in primed cells. These results suggest that SOS1/2 signaling plays a key role in determining the responsiveness of neutrophils in regions of inflammation.

Localizing the lipid products of PI3Kγ in neutrophils.

Class I phosphoinositide 3-kinases (PI3Ks) are important regulators of neutrophil migration in response to a range of chemoattractants. Their primary lipid products PtdIns(3,4,5)P3 and PtdIns(3,4)P2 preferentially accumulate near to the leading edge of migrating cells and are thought to act as an important cue organizing molecular and morphological polarization. We have investigated the distribution and accumulation of these lipids independently in mouse neutrophils using eGFP-PH reportersand electron microscopy (EM). We found that authentic mouse neutrophils rapidly polarized their Class I PI3K signalling, as read-out by eGFP-PH reporters, both at the up-gradient leading edge in response to local stimulation with fMLP as well as spontaneously and randomly in response to uniform stimulation. EM studies revealed these events occurred at the plasma membrane, were dominated by accumulation of PtdIns(3,4,5)P3, but not PtdIns(3,4)P2, and were dependent on PI3Kγ and its upstream activation by both Ras and Gßγs.

GPCR activation of Ras and PI3Kc in neutrophils depends on PLCb2/b3 and the RasGEF RasGRP4.

The molecular mechanisms by which receptors regulate the Ras Binding Domains of the PIP3-generating, class I PI3Ks remain poorly understood, despite their importance in a range of biological settings, including tumorigenesis, activation of neutrophils by pro-inflammatory mediators, chemotaxis of Dictyostelium and cell growth in Drosophila. We provide evidence that G protein-coupled receptors (GPCRs) can stimulate PLCb2/b3 and diacylglycerol- dependent activation of the RasGEF, RasGRP4 in neutrophils. The genetic loss of RasGRP4 phenocopies knock-in of a Ras-insensitive version of PI3Kc in its effects on PI3Kc-dependent PIP3 accumulation, PKB activation, chemokinesis and reactive oxygen species (ROS) formation. These results establish a new mechanism by which GPCRs can stimulate Ras, and the broadly important principle that PLCs can control activation of class I PI3Ks.

Use of the GRP1 PH domain as a tool to measure the relative levels of PtdIns(3,4,5)P3 through a protein-lipid overlay approach.

We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] and its application to measure, in neutrophils, the activation of phosphoinositide 3-kinase (PI3K). This protein-lipid overlay-based assay allowed us to confirm and extend the observations, first, that N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P(3) levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P(3) accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kgamma (p110gamma(DASAA/DASAA)) is substantially dependent on the Ras binding domain of PI3Kgamma.

Gbetagammas and the Ras binding domain of p110gamma are both important regulators of PI(3)Kgamma signalling in neutrophils.

Through their ability to regulate production of the key lipid messenger PtdIns(3,4,5)P(3), the class I phosphatidylinositol-3-OH kinases (PI(3)Ks) support many critical cell responses. They, in turn, can be regulated by cell-surface receptors through signals acting on either their adaptor subunits (for example, through phosphotyrosine or Gbetagammas) or their catalytic subunits (for example, through GTP-Ras). The relative significance of these controlling inputs is undefined in vivo. Here, we have studied the roles of Gbetagammas, the adaptor p101, Ras and the Ras binding domain (RBD) in the control of the class I PI(3)K, PI(3)Kgamma, in mouse neutrophils. Loss of p101 leads to major reductions in the accumulation of PtdIns(3,4,5)P(3), activation of protein kinase B (PKB) and in migration towards G-protein activating ligands in vitro, and to an aseptically inflamed peritoneum in vivo. Loss of sensitivity of PI(3)Kgamma to Ras unexpectedly caused similar reductions, but additionally caused a substantial loss in production of reactive oxygen species (ROS). We conclude that Gbetagammas, p101 and the Ras-RBD interaction all have important roles in the regulation of PI(3)Kgamma in vivo and that they can simultaneously, but differentially, control distinct PI(3)Kgamma effectors.