Characterization of metal ion-induced [3H]inositol hexakisphosphate binding to rat cerebellar membranes

The binding of [3H]inositol hexakisphosphate ([3H] InsP6) to rat cerebellar membranes has been characterized with the objective of establishing the role, if any, of a membrane protein receptor. In the presence of EDTA, we have previously identified an InsP6-binding site with a capacity of approximately 20 pmol/mg protein (Hawkins, P. T., Reynolds, D. J. M., Poyner, D. R., and Hanley, M. R. (1990) Biochem. Biophys. Res. Commun. 167, 819-827). However, in the presence of 1 mM Mg2+, the capacity of [3H]InsP6 binding to membranes was increased approximately 9-fold. This enhancing effect of Mg2+ was reversed by addition of 10 microM of several cation chelators, suggesting that the increased binding required trace quantities of other metal cations. This is supported by experiments where it was possible to saturate binding by addition of excess membranes, despite not significantly depleting radioligand, pointing to removal of some other factor. Removal of endogenous cations from the binding assay by pretreatment with chelex resin also prevents the Mg(2+)-induced potentiation. Consideration of the specificity of the chelators able to abolish this potentiation suggested involvement of Fe3+ or Al3+. Both these ions (but not several others) were able to increase [3H]InsP6 binding to chelex-pretreated membranes at concentrations of 1 microM. It is possible to demonstrate synergy between Fe3+ and Mg2+ under these conditions. We propose that [3H]InsP6 may interact with membranes through non-protein recognition possibly via phospholipids, in a manner dependent upon trace metals. The implications of this for InsP6 biology are considered

Phosphoproteomic Analyses of Interleukin 2 Signaling Reveal Integrated JAK Kinase-Dependent and -Independent Networks in CD8+ T Cells

SummaryInterleukin-2 (IL-2) is a fundamental cytokine that controls proliferation and differentiation of T cells. Here, we used high-resolution mass spectrometry to generate a comprehensive and detailed map of IL-2 protein phosphorylations in cytotoxic T cells (CTL). The data revealed that Janus kinases (JAKs) couple IL-2 receptors to the coordinated phosphorylation of transcription factors, regulators of chromatin, mRNA translation, GTPases, vesicle trafficking, and the actin and microtubule cytoskeleton. We identified an IL-2-JAK-independent SRC family Tyr-kinase-controlled signaling network that regulates ∼10% of the CTL phosphoproteome, the production of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), and the activity of the serine/threonine kinase AKT. These data reveal a signaling framework wherein IL-2-JAK-controlled pathways coordinate with IL-2-independent networks of kinase activity and provide a resource toward the further understanding of the networks of protein phosphorylation that program CTL fate

Neutrophils from p40phox−/− mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing

The generation of reactive oxygen species (ROS) by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex plays a critical role in the antimicrobial functions of the phagocytic cells of the immune system. The catalytic core of this oxidase consists of a complex between gp91phox, p22phox, p47phox, p67phox, p40phox, and rac-2. Mutations in each of the phox components, except p40phox, have been described in cases of chronic granulomatous disease (CGD), defining their essential role in oxidase function. We sought to establish the role of p40phox by investigating the NADPH oxidase responses of neutrophils isolated from p40phox−/− mice. In the absence of p40phox, the expression of p67phox is reduced by ∼55% and oxidase responses to tumor necrosis factor α/fibrinogen, immunoglobulin G latex beads, Staphylococcus aureus, formyl-methionyl-leucyl-phenylalanine, and zymosan were reduced by ∼97, 85, 84, 75, and 30%, respectively. The defect in ROS production by p40phox−/− neutrophils in response to S. aureus translated into a severe, CGD-like defect in the killing of this organism both in vitro and in vivo, defining p40phox as an essential component in bacterial killing

In B cells, phosphatidylinositol 5-phosphate 4-kinase-α synthesizes PI(4,5)P2 to impact mTORC2 and Akt signaling.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases with physiological functions that are incompletely understood, not the least because genetic deletion and cell transfection have led to contradictory data. Here, we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4Kα was removed, either stably by genetic deletion or transiently (within 1 h) by tagging the endogenous protein genomically with the auxin degron. In both cases, removal impacted Akt phosphorylation, and by leaving one PI5P4Kα allele present but mutating it to be kinase-dead or have PI4P 5-kinase activity, we show that all of the effects on Akt phosphorylation were dependent on the ability of PI5P4Kα to synthesize phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] rather than to remove PI5P. Although stable removal of PI5P4Kα resulted in a pronounced decrease in Akt phosphorylation at Thr308 and Ser473, in part because of reduced plasma membrane PIP3, its acute removal led to an increase in Akt phosphorylation only at Ser473. This process invokes activation primarily of mammalian target of rapamycin complex 2 (mTORC2), which was confirmed by increased phosphorylation of other mTORC2 substrates. These findings establish PI5P4Kα as a kinase that synthesizes a physiologically relevant pool of PI(4,5)P2 and as a regulator of mTORC2, and show a phenomenon similar to the "butterfly effect" described for phosphatidylinositol 3-kinase Iα [Hart JR, et al. (2015) Proc Natl Acad Sci USA 112(4):1131-1136], whereby through apparently the same underlying mechanism, the removal of a protein's activity from a cell can have widely divergent effects depending on the time course of that removal.S.J.B. was supported by an A.J. Clark Studentship from the British Pharmacological Society, A.D. by Sidney Sussex College, the Cambridge Overseas Trust and the Säid Foundation, and J.H.C by the MRC (Grant RG64071).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Proceedings of the National Academy of Sciences (PNAS)

Phosphoinositide 3-OH Kinase Regulates Integrin-Dependent Processes in Neutrophils by Signaling through Its Effector ARAP3

ARAP3, a GTPase activating protein for Rho and Arf family GTPases, is one of many phosphoinositide 3-OH kinase (PI3K) effectors. In this study, we investigate the regulatory input of PI3K upstream of ARAP3 by analyzing neutrophils from an ARAP3 pleckstrin homology (PH) domain point mutation knock-in mouse (R302, 303A), in which ARAP3 is uncoupled from activation by PI3K. ARAP3 PH domain point mutant neutrophils are characterized by disturbed responses linked to stimulation by either integrin ligands or immobilized immune complexes. These cells exhibit increased β2 integrin inside-out signaling (binding affinity and avidity), and our work suggests the disturbed responses to immobilized immune complexes are secondary to this. In vitro, neutrophil chemotaxis is affected in the mutant. In vivo, ARAP3 PH domain point mutant bone marrow chimeras exhibit reduced neutrophil recruitment to the peritoneum on induction of sterile peritonitis and also reduced inflammation in a model for rheumatoid arthritis. The current work suggests a dramatic regulatory input of PI3K into the regulation of β2 integrin activity, and processes dependent on this, by signaling through its effector ARAP3. Copyright © 2012 by The American Association of Immunologists, Inc

Compensation between CSF1R+ macrophages and Foxp3+ Treg cells drives resistance to tumor immunotherapy.

Redundancy and compensation provide robustness to biological systems but may contribute to therapy resistance. Both tumor-associated macrophages (TAMs) and Foxp3+ regulatory T (Treg) cells promote tumor progression by limiting antitumor immunity. Here we show that genetic ablation of CSF1 in colorectal cancer cells reduces the influx of immunosuppressive CSF1R+ TAMs within tumors. This reduction in CSF1-dependent TAMs resulted in increased CD8+ T cell attack on tumors, but its effect on tumor growth was limited by a compensatory increase in Foxp3+ Treg cells. Similarly, disruption of Treg cell activity through their experimental ablation produced moderate effects on tumor growth and was associated with elevated numbers of CSF1R+ TAMs. Importantly, codepletion of CSF1R+ TAMs and Foxp3+ Treg cells resulted in an increased influx of CD8+ T cells, augmentation of their function, and a synergistic reduction in tumor growth. Further, inhibition of Treg cell activity either through systemic pharmacological blockade of PI3Kδ, or its genetic inactivation within Foxp3+ Treg cells, sensitized previously unresponsive solid tumors to CSF1R+ TAM depletion and enhanced the effect of CSF1R blockade. These findings identify CSF1R+ TAMs and PI3Kδ-driven Foxp3+ Treg cells as the dominant compensatory cellular components of the immunosuppressive tumor microenvironment, with implications for the design of combinatorial immunotherapies.D. Gyori was funded by a research grant from Karus Therapeutics. E.L. Lim was supported by a Yousef Jameel Scholarship (Cambridge Trust). R. Roychoudhuri and K. Okkenhaug received institute support from Biotechnology and Biological Sciences Research Council (BBSRC) (BBS/E/B/000 -C0407, -C0409, -C0427 and -C0428). K. Okkenhaug was also supported by Wellcome Trust grant 095198/Z/10/Z. L.R. Stephens and P.T. Hawkins were supported by and institute grant from the BBSRC (BB/J004456/1). R. Roychoudhuri is supported by the Wellcome Trust/Royal Society (Grant 105663/Z/14/Z), the UK Biotechnology and Biological Sciences Research Council (Grant BB/N007794/1), and Cancer Research UK (Grant C52623/A22597)

Regulation of PTEN Inhibition by the Pleckstrin Homology Domain of P-REX2 During Insulin Signaling and Glucose Homeostasis

Insulin activation of phosphoinositide 3-kinase (PI3K) signaling regulates glucose homeostasis through the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3). The dual-specificity phosphatase and tensin homolog deleted on chromosome 10 (PTEN) blocks PI3K signaling by dephosphorylating PIP3, and is inhibited through its interaction with phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2 (P-REX2). The mechanism of inhibition and its physiological significance are not known. Here, we report that P-REX2 interacts with PTEN via two interfaces. The pleckstrin homology (PH) domain of P-REX2 inhibits PTEN by interacting with the catalytic region of PTEN, and the inositol polyphosphate 4-phosphatase domain of P-REX2 provides high-affinity binding to the postsynaptic density-95/Discs large/zona occludens-1-binding domain of PTEN. P-REX2 inhibition of PTEN requires C-terminal phosphorylation of PTEN to release the P-REX2 PH domain from its neighboring diffuse B-cell lymphoma homology domain. Consistent with its function as a PTEN inhibitor, deletion of Prex2 in fibroblasts and mice results in increased Pten activity and decreased insulin signaling in liver and adipose tissue. Prex2 deletion also leads to reduced glucose uptake and insulin resistance. In human adipose tissue, P-REX2 protein expression is decreased and PTEN activity is increased in insulin-resistant human subjects. Taken together, these results indicate a functional role for P-REX2 PH-domain-mediated inhibition of PTEN in regulating insulin sensitivity and glucose homeostasis and suggest that loss of P-REX2 expression may cause insulin resistance

The Parkinson's disease gene PINK1 activates Akt via PINK1 kinase-dependent regulation of the phospholipid PI(3,4,5)P3

Akt signalling is central to cell survival, metabolism, protein and lipid homeostasis, and is impaired in Parkinson's disease (PD). Akt activation is reduced in the brain in PD, and by many PD-causing genes, including PINK1 This study investigated the mechanisms by which PINK1 regulates Akt signalling. Our results reveal for the first time that PINK1 constitutively activates Akt in a PINK1-kinase dependent manner in the absence of growth factors, and enhances Akt activation in normal growth medium. In PINK1-modified MEFs, agonist-induced Akt signalling failed in the absence of PINK1, due to PINK1 kinase-dependent increases in PI(3,4,5)P3 at both plasma membrane and Golgi being significantly impaired. In the absence of PINK1, PI(3,4,5)P3 levels did not increase in the Golgi, and there was significant Golgi fragmentation, a recognised characteristic of PD neuropathology. PINK1 kinase activity protected the Golgi from fragmentation in an Akt-dependent fashion. This study demonstrates a new role for PINK1 as a primary upstream activator of Akt via PINK1 kinase-dependent regulation of its primary activator PI(3,4,5)P3, providing novel mechanistic information on how loss of PINK1 impairs Akt signalling in PD

Functional Redundancy of Class I Phosphoinositide 3-Kinase (PI3K) Isoforms in Signaling Growth Factor-Mediated Human Neutrophil Survival

We have investigated the contribution of individual phosphoinositide 3-kinase (PI3K) Class I isoforms to the regulation of neutrophil survival using (i) a panel of commercially available small molecule isoform-selective PI3K Class I inhibitors, (ii) novel inhibitors, which target single or multiple Class I isoforms (PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ), and (iii) transgenic mice lacking functional PI3K isoforms (p110δKOγKO or p110γKO). Our data suggest that there is considerable functional redundancy amongst Class I PI3Ks (both Class IA and Class IB) with regard to GM-CSF-mediated suppression of neutrophil apoptosis. Hence pharmacological inhibition of any 3 or more PI3K isoforms was required to block the GM-CSF survival response in human neutrophils, with inhibition of individual or any two isoforms having little or no effect. Likewise, isolated blood neutrophils derived from double knockout PI3K p110δKOγKO mice underwent normal time-dependent constitutive apoptosis and displayed identical GM-CSF mediated survival to wild type cells, but were sensitized to pharmacological inhibition of the remaining PI3K isoforms. Surprisingly, the pro-survival neutrophil phenotype observed in patients with an acute exacerbation of chronic obstructive pulmonary disease (COPD) was resilient to inactivation of the PI3K pathway

PI3Kδ Forms Distinct Multiprotein Complexes at the TCR Signalosome in Naïve and Differentiated CD4 + T Cells

Phosphoinositide 3-kinases (PI3Ks) play a central role in adaptive immunity by transducing signals from the T cell antigen receptor (TCR) via production of PIP3. PI3Kδ is a heterodimer composed of a p110δ catalytic subunit associated with a p85α or p85β regulatory subunit and is preferentially engaged by the TCR upon T cell activation. The molecular mechanisms leading to PI3Kδ recruitment and activation at the TCR signalosome remain unclear. In this study, we have used quantitative mass spectrometry, biochemical approaches and CRISPR-Cas9 gene editing to uncover the p110δ interactome in primary CD4+ T cells. Moreover, we have determined how the PI3Kδ interactome changes upon the differentiation of small naïve T cells into T cell blasts expanded in the presence of IL-2. Our interactomic analyses identified multiple constitutive and inducible PI3Kδ-interacting proteins, some of which were common to naïve and previously-activated T cells. Our data reveals that PI3Kδ rapidly interacts with as many as seven adaptor proteins upon TCR engagement, including the Gab-family proteins, GAB2 and GAB3, a CD5-CBL signalosome and the transmembrane proteins ICOS and TRIM. Our results also suggest that PI3Kδ pre-forms complexes with the adaptors SH3KBP1 and CRKL in resting cells that could facilitate the localization and activation of p110δ at the plasma membrane by forming ternary complexes during early TCR signalling. Furthermore, we identify interactions that were not previously known to occur in CD4+ T cells, involving BCAP, GAB3, IQGAP3 and JAML. We used CRISPR-Cas9-mediated gene knockout in primary T cells to confirm that BCAP is a positive regulator of PI3K-AKT signalling in CD4+ T cell blasts. Overall, our results provide evidence for a large protein network that regulates the recruitment and activation of PI3Kδ in T cells. Finally, this work shows how the PI3Kδ interactome is remodeled as CD4+ T cells differentiate from naïve T cells to activated T cell blasts. These activated T cells upregulate additional PI3Kδ adaptor proteins, including BCAP, GAB2, IQGAP3 and ICOS. This rewiring of TCR-PI3K signalling that occurs upon T cell differentiation may serve to reduce the threshold of activation and diversify the inputs for the PI3K pathway in effector T cells