Publications by authors named "Phillip T Hawkins"

66 Publications

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

Sci Signal 2020 11 3;13(656). Epub 2020 Nov 3.

Signalling Programme, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK.

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 Gβγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gβγ 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 Gβγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gβγ binding to p101 caused more variable effects, depending on both the agonist and cellular response, with the biggest reductions seen in PIP 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 p101 neutrophils showed enhanced p84-dependent ROS responses to MLP and C5a, suggesting that competition may exist between p101/p110γ and p84/p110γ for Gβγ 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 Gβγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs.
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http://dx.doi.org/10.1126/scisignal.aaz4003DOI Listing
November 2020

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

J Cell Sci 2019 10 22;132(20). Epub 2019 Oct 22.

School of Biochemistry and Cell Biology, Biosciences Institute, University College Cork, Cork City T12 YT20, Ireland

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 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)P at both plasma membrane and Golgi being significantly impaired. In the absence of PINK1, PI(3,4,5)P 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)P, providing novel mechanistic information on how loss of PINK1 impairs Akt signalling in PD.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.233221DOI Listing
October 2019

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.

J Leukoc Biol 2019 10 5;106(4):815-822. Epub 2019 Feb 5.

Inositide Laboratory, The Babraham Institute, Cambridge, UK.

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 (PIP ), are highly dependent on PI3K-γ, a Ras-GTP, and Gβγ 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-PIP 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, PIP 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.
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http://dx.doi.org/10.1002/JLB.2HI0918-359RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6977543PMC
October 2019

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

JCI Insight 2018 06 7;3(11). Epub 2018 Jun 7.

Signalling ISP, Babraham Institute, Babraham Research Campus, Cambridge, Cambridgeshire, United Kingdom.

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.
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http://dx.doi.org/10.1172/jci.insight.120631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124419PMC
June 2018

In-depth PtdIns(3,4,5)P signalosome analysis identifies DAPP1 as a negative regulator of GPVI-driven platelet function.

Blood Adv 2017 Jun;1(14):918-932

School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom.

The class I phosphoinositide 3-kinase (PI3K) isoforms play important roles in platelet priming, activation, and stable thrombus formation. Class I PI3Ks predominantly regulate cell function through their catalytic product, the signaling phospholipid phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P], which coordinates the localization and/or activity of a diverse range of binding proteins. Notably, the complete repertoire of these class I PI3K effectors in platelets remains unknown, limiting mechanistic understanding of class I PI3K-mediated control of platelet function. We measured robust agonist-driven PtdIns (3,4,5)P generation in human platelets by lipidomic mass spectrometry (MS), and then used affinity-capture coupled to high-resolution proteomic MS to identify the targets of PtdIns (3,4,5)P in these cells. We reveal for the first time a diverse platelet PtdIns(3,4,5)P interactome, including kinases, signaling adaptors, and regulators of small GTPases, many of which are previously uncharacterized in this cell type. Of these, we show dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1) to be regulated by Src-family kinases and PI3K, while platelets from DAPP1-deficient mice display enhanced thrombus formation on collagen in vitro. This was associated with enhanced platelet α/δ granule secretion and αβ integrin activation downstream of the collagen receptor glycoprotein VI. Thus, we present the first comprehensive analysis of the PtdIns(3,4,5)P signalosome of human platelets and identify DAPP1 as a novel negative regulator of platelet function. This work provides important new insights into how class I PI3Ks shape platelet function.
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http://dx.doi.org/10.1182/bloodadvances.2017005173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5726495PMC
June 2017

PTEN Regulates PI(3,4)P Signaling Downstream of Class I PI3K.

Mol Cell 2017 Nov 19;68(3):566-580.e10. Epub 2017 Oct 19.

Signalling Programme, Babraham Institute, Cambridge, UK. Electronic address:

The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P. PI(3,4,5)P can be dephosphorylated by 3- or 5-phosphatases, the latter producing PI(3,4)P. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4)P 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4)P phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P 4-phosphatase, leads to synergistic accumulation of PI(3,4)P, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4)P in the phenotype caused by loss-of-function mutations or deletions in PTEN.
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http://dx.doi.org/10.1016/j.molcel.2017.09.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678281PMC
November 2017

Class (I) Phosphoinositide 3-Kinases in the Tumor Microenvironment.

Cancers (Basel) 2017 Mar 4;9(3). Epub 2017 Mar 4.

The Babraham Institute, Cambridge CB22 3AT, UK.

Phosphoinositide 3-kinases (PI3Ks) are a diverse family of enzymes which regulate various critical biological processes, such as cell proliferation and survival. Class (I) PI3Ks (PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ) mediate the phosphorylation of the inositol ring at position D3 leading to the generation of PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 can be dephosphorylated by several phosphatases, of which the best known is the 3-phosphatase PTEN (phosphatase and tensin homolog). The Class (I) PI3K pathway is frequently disrupted in human cancers where mutations are associated with increased PI3K-activity or loss of PTEN functionality within the tumor cells. However, the role of PI3Ks in the tumor stroma is less well understood. Recent evidence suggests that the white blood cell-selective PI3Kγ and PI3Kδ isoforms have an important role in regulating the immune-suppressive, tumor-associated myeloid cell and regulatory T cell subsets, respectively, and as a consequence are also critical for solid tumor growth. Moreover, PI3Kα is implicated in the direct regulation of tumor angiogenesis, and dysregulation of the PI3K pathway in stromal fibroblasts can also contribute to cancer progression. Therefore, pharmacological inhibition of the Class (I) PI3K family in the tumor microenvironment can be a highly attractive anti-cancer strategy and isoform-selective PI3K inhibitors may act as potent cancer immunotherapeutic and anti-angiogenic agents.
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http://dx.doi.org/10.3390/cancers9030024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5366819PMC
March 2017

cAMP Signaling of Adenylate Cyclase Toxin Blocks the Oxidative Burst of Neutrophils through Epac-Mediated Inhibition of Phospholipase C Activity.

J Immunol 2017 02 30;198(3):1285-1296. Epub 2016 Dec 30.

Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology of the CAS, v.v.i., 142 20 Prague, Czech Republic; and

The adenylate cyclase toxin-hemolysin (CyaA) plays a key role in immune evasion and virulence of the whooping cough agent Bordetella pertussis. CyaA penetrates the complement receptor 3-expressing phagocytes and ablates their bactericidal capacities by catalyzing unregulated conversion of cytosolic ATP to the key second messenger molecule cAMP. We show that signaling of CyaA-generated cAMP blocks the oxidative burst capacity of neutrophils by two converging mechanisms. One involves cAMP/protein kinase A-mediated activation of the Src homology region 2 domain-containing phosphatase-1 (SHP-1) and limits the activation of MAPK ERK and p38 that are required for assembly of the NADPH oxidase complex. In parallel, activation of the exchange protein directly activated by cAMP (Epac) provokes inhibition of the phospholipase C by an as yet unknown mechanism. Indeed, selective activation of Epac by the cell-permeable analog 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate counteracted the direct activation of phospholipase C by 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide. Hence, by inhibiting production of the protein kinase C-activating lipid, diacylglycerol, cAMP/Epac signaling blocks the bottleneck step of the converging pathways of oxidative burst triggering. Manipulation of neutrophil membrane composition by CyaA-produced signaling of cAMP thus enables B. pertussis to evade the key innate host defense mechanism of reactive oxygen species-mediated killing of bacteria by neutrophils.
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http://dx.doi.org/10.4049/jimmunol.1601309DOI Listing
February 2017

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

Proc Natl Acad Sci U S A 2016 09 6;113(38):10571-6. Epub 2016 Sep 6.

Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom;

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035885PMC
http://dx.doi.org/10.1073/pnas.1522478113DOI Listing
September 2016

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

Immunity 2016 09 23;45(3):685-700. Epub 2016 Aug 23.

Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK. Electronic address:

Interleukin-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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040828PMC
http://dx.doi.org/10.1016/j.immuni.2016.07.022DOI Listing
September 2016

Coincident signals from GPCRs and receptor tyrosine kinases are uniquely transduced by PI3Kβ in myeloid cells.

Sci Signal 2016 08 16;9(441):ra82. Epub 2016 Aug 16.

Inositide Laboratory, Babraham Institute, Babraham Research Campus, Babraham, Cambridge CB223AT, UK.

Class I phosphoinositide 3-kinases (PI3Ks) catalyze production of the lipid messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), which plays a central role in a complex signaling network regulating cell growth, survival, and movement. This network is overactivated in cancer and inflammation, and there is interest in determining the PI3K catalytic subunit (p110α, p110β, p110γ, or p110δ) that should be targeted in different therapeutic contexts. Previous studies have defined unique regulatory inputs for p110β, including direct interaction with Gβγ subunits, Rac, and Rab5. We generated mice with knock-in mutations of p110β that selectively blocked the interaction with Gβγ and investigated its contribution to the PI3K isoform dependency of receptor tyrosine kinase (RTK) and G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) responses in primary macrophages and neutrophils. We discovered a unique role for p110β in supporting synergistic PIP3 formation in response to the coactivation of macrophages by macrophage colony-stimulating factor (M-CSF) and the complement protein C5a. In contrast, we found partially redundant roles for p110α, p110β, and p110δ downstream of M-CSF alone and a nonredundant role for p110γ downstream of C5a alone. This role for p110β completely depended on direct interaction with Gβγ, suggesting that p110β transduces GPCR signals in the context of coincident activation by an RTK. The p110β-Gβγ interaction was also required for neutrophils to generate reactive oxygen species in response to the Fcγ receptor-dependent recognition of immune complexes and for their β2 integrin-mediated adhesion to fibrinogen or poly-RGD+, directly implicating heterotrimeric G proteins in these two responses.
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http://dx.doi.org/10.1126/scisignal.aae0453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5417692PMC
August 2016

Emerging evidence of signalling roles for PI(3,4)P2 in Class I and II PI3K-regulated pathways.

Biochem Soc Trans 2016 Feb;44(1):307-14

Signalling Programme, Babraham Institute, Cambridge CB22 3AT, U.K.

There are eight members of the phosphoinositide family of phospholipids in eukaryotes; PI, PI3P, PI4P, PI5P, PI(4,5)P2, PI(3,4)P2, PI(3,5)P2 and PI(3,4,5)P3. Receptor activation of Class I PI3Ks stimulates the phosphorylation of PI(4,5)P2 to form PI(3,4,5)P3. PI(3,4,5)P3 is an important messenger molecule that is part of a complex signalling network controlling cell growth and division. PI(3,4,5)P3 can be dephosphorylated by both 3- and 5-phosphatases, producing PI(4,5)P2 and PI(3,4)P2, respectively. There is now strong evidence that PI(3,4)P2 generated by this route does not merely represent another pathway for removal of PI(3,4,5)P3, but can act as a signalling molecule in its own right, regulating macropinocytosis, fast endophilin-mediated endocytosis (FEME), membrane ruffling, lamellipodia and invadopodia. PI(3,4)P2 can also be synthesized directly from PI4P by Class II PI3Ks and this is important for the maturation of clathrin-coated pits [clathrin-mediated endocytosis (CME)] and signalling in early endosomes. Thus PI(3,4)P2 is emerging as an important signalling molecule involved in the coordination of several specific membrane and cytoskeletal responses. Further, its inappropriate accumulation contributes to pathology caused by mutations in genes encoding enzymes responsible for its degradation, e.g. Inpp4B.
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http://dx.doi.org/10.1042/BST20150248DOI Listing
February 2016

Investigating the effect of arachidonate supplementation on the phosphoinositide content of MCF10a breast epithelial cells.

Adv Biol Regul 2016 09 14;62:18-24. Epub 2015 Nov 14.

Inositide Laboratory and Babraham Institute, Babraham Research Campus, Babraham, Cambridge, CB223AT, UK. Electronic address:

Phosphoinositides in primary mammalian tissue are highly enriched in a stearoyl/arachidonyl (C38:4) diacylgycerol backbone. However, mammalian cells grown in culture typically contain more diverse molecular species of phosphoinositides, characterised by a reduction in arachidonyl content in the sn-2 position. We have analysed the phosphoinositide species in MCF10a cells grown in culture by mass spectrometry. Under either serum or serum starved conditions the most abundant species of PI, PIP, PIP and PIP had masses which corresponded to C36:2, C38:4, C38:3, C38:2 and C36:1 diacylglycerol backbones and the relative proportions of each molecular species were broadly similar between each phosphoinositide class (approx. 50%, 25%, 10%, 10% and 10% respectively, for the species listed above). Supplementing the culture medium with BSA-loaded arachidonic acid promoted a rapid increase in the proportion of the C38:4 species in all phosphoinositide classes (from approx. 25%-60% of total species within 24 h), but the total amount of all combined species for each class remained remarkably constant. Stimulation of cells, cultured in either normal or arachidonate-enriched conditions, with 2 ng/ml EGF for 90 s caused substantial activation of Class I PI3K and accumulation of PIP. Despite the increased proportion of C38:4 PIP under the arachidonate-supplemented conditions, the total amount of all combined PIP species accumulating in response to EGF was the same, with or without arachidonate supplementation; there were however small but significant preferences for the conversion of some PIP species to PIP, with the polyunsaturated C38:4 and C38:3 species being more favoured over other species. These results suggest the enzymes which interconvert phosphoinositides are able to act on several different molecular species and homoeostatic mechanisms are in place to deliver similar phosphoinositide pool sizes under quite different conditions of arachidonate availability. They also suggest enzymes regulating PIP levels downstream of growth factor stimulation (i.e. PI3Ks and PIP-phosphatases) show some acyl selectivity and further work should be directed at assessing whether different acyl species of PIP exhibit differing signalling potential.
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http://dx.doi.org/10.1016/j.jbior.2015.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061326PMC
September 2016

Localizing the lipid products of PI3Kγ in neutrophils.

Adv Biol Regul 2016 Jan 10;60:36-45. Epub 2015 Nov 10.

The Signalling Department, The Babraham Institute, The Babraham Research Campus, Cambridge, CB22 3AT, United Kingdom. Electronic address:

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 Gβγs.
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http://dx.doi.org/10.1016/j.jbior.2015.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4739120PMC
January 2016

The cytotoxic T cell proteome and its shaping by the kinase mTOR.

Nat Immunol 2016 Jan 9;17(1):104-12. Epub 2015 Nov 9.

Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dow Street, Dundee, UK.

We used high-resolution mass spectrometry to map the cytotoxic T lymphocyte (CTL) proteome and the effect of the metabolic checkpoint kinase mTORC1 on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes, and mTORC1 selectively repressed and promoted expression of a subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for negative control of the production of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) by mTORC1 in CTLs. mTORC1 repressed PtdIns(3,4,5)P3 production and determined the requirement for mTORC2 in activation of the kinase Akt. Our unbiased proteomic analysis thus provides comprehensive understanding of CTL identity and the control of CTL function by mTORC1.
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http://dx.doi.org/10.1038/ni.3314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4685757PMC
January 2016

The regulatory subunits of PI3Kγ control distinct neutrophil responses.

Sci Signal 2015 Jan 20;8(360):ra8. Epub 2015 Jan 20.

Signalling Department, Babraham Institute, Babraham Research Campus, Cambridge CB22 4AT, UK.

Neutrophils, which migrate toward inflamed sites and kill pathogens by producing reactive oxygen species (ROS), are important in the defense against bacterial and fungal pathogens, but their inappropriate regulation causes various chronic inflammatory diseases. Phosphoinositide 3-kinase γ (PI3Kγ) functions downstream of proinflammatory G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) in neutrophils and is a therapeutic target. In neutrophils, PI3Kγ consists of a p110γ catalytic subunit, which is activated by the guanosine triphosphatase Ras, and either a p84 or p101 regulatory subunit. Loss or inhibition of p110γ or expression of a Ras-insensitive variant p110γ (p110γ(DASAA/DASAA)) impairs PIP3 production, Akt phosphorylation, migration, and ROS formation in response to GPCR activation. The p101 subunit binds to, and mediates PI3Kγ activation by, G protein βγ subunits, and p101(-/-) neutrophils have a similar phenotype to that of p110γ(-/-) neutrophils, except that ROS responses are normal. We found that p84(-/-) neutrophils displayed reduced GPCR-stimulated PIP3 and Akt signaling, which was indistinguishable from that of p101(-/-) neutrophils. However, p84(-/-) neutrophils produced less ROS and exhibited normal migration in response to GPCR stimulation. These data suggest that p84-containing PI3Kγ controls GPCR-dependent ROS production. Thus, the PI3Kγ regulatory subunits enable PI3Kγ to mediate distinct neutrophil responses, which may occur by targeting PIP3 signaling into spatially distinct domains.
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http://dx.doi.org/10.1126/scisignal.2005564DOI Listing
January 2015

Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion.

EMBO Mol Med 2015 Feb;7(2):127-39

Department of Medicine, Cambridge Institute for Medical Research University of Cambridge, Cambridge, UK Cambridge Centre for Lung Infection, Papworth Hospital, Cambridge, UK

Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell-based screening of FDA-approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug-resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR-independent pathway controlled by cellular depletion of myo-inositol. While strain-specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug-resistant mycobacterial infection.
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http://dx.doi.org/10.15252/emmm.201404137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4328644PMC
February 2015

Dictyostelium uses ether-linked inositol phospholipids for intracellular signalling.

EMBO J 2014 Oct 1;33(19):2188-200. Epub 2014 Sep 1.

Signalling Programme Babraham Research Campus, Cambridge, UK

Inositol phospholipids are critical regulators of membrane biology throughout eukaryotes. The general principle by which they perform these roles is conserved across species and involves binding of differentially phosphorylated inositol head groups to specific protein domains. This interaction serves to both recruit and regulate the activity of several different classes of protein which act on membrane surfaces. In mammalian cells, these phosphorylated inositol head groups are predominantly borne by a C38:4 diacylglycerol backbone. We show here that the inositol phospholipids of Dictyostelium are different, being highly enriched in an unusual C34:1e lipid backbone, 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), in which the sn-1 position contains an ether-linked C16:0 chain; they are thus plasmanylinositols. These plasmanylinositols respond acutely to stimulation of cells with chemoattractants, and their levels are regulated by PIPKs, PI3Ks and PTEN. In mammals and now in Dictyostelium, the hydrocarbon chains of inositol phospholipids are a highly selected subset of those available to other phospholipids, suggesting that different molecular selectors are at play in these organisms but serve a common, evolutionarily conserved purpose.
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http://dx.doi.org/10.15252/embj.201488677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282506PMC
October 2014

The hexosamine biosynthesis pathway and O-GlcNAcylation maintain insulin-stimulated PI3K-PKB phosphorylation and tumour cell growth after short-term glucose deprivation.

FEBS J 2014 Aug 14;281(16):3591-608. Epub 2014 Jul 14.

Inositide Laboratory, Cancer Research UK Manchester Institute, University of Manchester, UK.

Glucose provides an essential nutrient source that supports glycolysis and the hexosamine biosynthesis pathway (HBP) to maintain tumour cell growth and survival. Here we investigated if short-term glucose deprivation specifically modulates the phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) cell survival pathway. Insulin-stimulated PKB activation was strongly abrogated in the absence of extracellular glucose as a consequence of the loss of insulin-stimulated PI3K activation and short-term glucose deprivation inhibited subsequent tumour cell growth. Loss of insulin-stimulated PKB signalling and cell growth was rescued by extracellular glucosamine and increased flux through the HBP. Disruption of O-GlcNAc transferase activity, a terminal step in the HBP, implicated O-GlcNAcylation in PKB signalling and cell growth. Glycogenolysis is known to support cell survival during glucose deprivation, and in A549 lung cancer cells its inhibition attenuates PKB activation which is rescued by increased flux through the HBP. Our studies show that rerouting of glycolytic metabolites to the HBP under glucose-restricted conditions maintains PI3K/PKB signalling enabling cell survival and proliferation.
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http://dx.doi.org/10.1111/febs.12879DOI Listing
August 2014

The phosphoinositide 3-kinase isoform PI3Kβ regulates osteoclast-mediated bone resorption in humans and mice.

Arthritis Rheumatol 2014 Aug;66(8):2210-21

Semmelweis University School of Medicine, and MTA-SE "Lendület" Inflammation Physiology Research Group of the Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.

Objective: While phosphoinositide 3-kinases (PI3Ks) are involved in various intracellular signal transduction processes, the specific functions of the different PI3K isoforms are poorly understood. We have previously shown that the PI3Kβ isoform is required for arthritis development in the K/BxN serum-transfer model. Since osteoclasts play a critical role in pathologic bone loss during inflammatory arthritis and other diseases, we undertook this study to test the role of PI3Kβ in osteoclast development and function using a combined genetic and pharmacologic approach.

Methods: The role of PI3Kβ in primary human and murine osteoclast cultures was tested with the PI3Kβ-selective inhibitor TGX221 and by using PI3Kβ(-/-) mice. The trabecular bone architecture of PI3Kβ(-/-) mice was evaluated using micro-computed tomography and histomorphometric analyses.

Results: The expression of PI3Kβ was strongly and specifically up-regulated during in vitro osteoclast differentiation. In vitro development of large multinucleated osteoclasts from human or murine progenitors and their resorption capacity were strongly reduced by the PI3Kβ inhibitor TGX221 or by the genetic deficiency of PI3Kβ. This was likely due to defective cytoskeletal reorganization and vesicular trafficking, since PI3Kβ(-/-) mouse multinucleated cells failed to form actin rings and retained intracellular acidic vesicles and cathepsin K. In contrast, osteoclast-specific gene expression and the survival and apoptosis of osteoclasts were not affected. PI3Kβ(-/-) mice had significantly increased trabecular bone volume and showed abnormal osteoclast morphology with defective resorption pit formation.

Conclusion: PI3Kβ plays an important role in osteoclast development and function and is required for in vivo bone homeostasis.
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http://dx.doi.org/10.1002/art.38660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4314683PMC
August 2014

P-Rex1 directly activates RhoG to regulate GPCR-driven Rac signalling and actin polarity in neutrophils.

J Cell Sci 2014 Jun 21;127(Pt 11):2589-600. Epub 2014 Mar 21.

Inositide laboratory, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK

G-protein-coupled receptors (GPCRs) regulate the organisation of the actin cytoskeleton by activating the Rac subfamily of small GTPases. The guanine-nucleotide-exchange factor (GEF) P-Rex1 is engaged downstream of GPCRs and phosphoinositide 3-kinase (PI3K) in many cell types, and promotes tumorigenic signalling and metastasis in breast cancer and melanoma, respectively. Although P-Rex1-dependent functions have been attributed to its GEF activity towards Rac1, we show that P-Rex1 also acts as a GEF for the Rac-related GTPase RhoG, both in vitro and in GPCR-stimulated primary mouse neutrophils. Furthermore, loss of either P-Rex1 or RhoG caused equivalent reductions in GPCR-driven Rac activation and Rac-dependent NADPH oxidase activity, suggesting they both function upstream of Rac in this system. Loss of RhoG also impaired GPCR-driven recruitment of the Rac GEF DOCK2, and F-actin, to the leading edge of migrating neutrophils. Taken together, our results reveal a new signalling hierarchy in which P-Rex1, acting as a GEF for RhoG, regulates Rac-dependent functions indirectly through RhoG-dependent recruitment of DOCK2. These findings thus have broad implications for our understanding of GPCR signalling to Rho GTPases and the actin cytoskeleton.
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http://dx.doi.org/10.1242/jcs.153049DOI Listing
June 2014

Regulation of PTEN inhibition by the pleckstrin homology domain of P-REX2 during insulin signaling and glucose homeostasis.

Proc Natl Acad Sci U S A 2014 Jan 23;111(1):155-60. Epub 2013 Dec 23.

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029.

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.
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http://dx.doi.org/10.1073/pnas.1213773111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890808PMC
January 2014

Signaling via class IA Phosphoinositide 3-kinases (PI3K) in human, breast-derived cell lines.

PLoS One 2013 4;8(10):e75045. Epub 2013 Oct 4.

The Babraham Institute, Babraham, Cambridge, United Kingdom.

We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ) and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/-) cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN(-/-) MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110β function in the absence of PTEN.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075045PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790768PMC
July 2014

A new approach to measuring phosphoinositides in cells by mass spectrometry.

Adv Biol Regul 2014 Jan 17;54:131-41. Epub 2013 Sep 17.

Babraham Institute, Babraham Research Campus, Cambridge, UK. Electronic address:

The phosphoinositide family of phospholipids, defined here as PtdIns, PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, play pivotal roles in organising the location and activity of many different proteins acting on biological membranes, including those involved in vesicle and protein trafficking through the endolysosomal system and receptor signal transduction at the plasma membrane. Accurate measurement of the cellular levels of these lipids, particularly the more highly phosphorylated species, is hampered by their high polarity and low cellular concentrations. Recently, much progress has been made in using mass spectrometry to measure many different lipid classes in parallel, an approach generally referred to as 'lipidomics'. Unfortunately, the acidic nature of highly phosphorylated phosphoinositides makes them difficult to measure using these methods, because they yield low levels of useful ions; this is particularly the case with PtdIns(3,4,5)P3. We have solved some of these problems by methylating the phosphate groups of these lipids with TMS-diazomethane and describe a simple, integrated approach to measuring PtdIns, PtdInsP, PtdInsP2 and PtdInsP3 classes of lipids, in parallel with other phospholipid species, in cell and tissue extracts. This methodology is sensitive, accurate and robust, and also yields fatty-acyl compositions, suggesting it can be used to further our understanding of both the normal and pathophysiological roles of these important lipids.
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http://dx.doi.org/10.1016/j.jbior.2013.09.001DOI Listing
January 2014

3D time series analysis of cell shape using Laplacian approaches.

BMC Bioinformatics 2013 Oct 4;14:296. Epub 2013 Oct 4.

Warwick Systems Biology Centre, University of Warwick, Coventry CV4 7AL, UK.

Background: Fundamental cellular processes such as cell movement, division or food uptake critically depend on cells being able to change shape. Fast acquisition of three-dimensional image time series has now become possible, but we lack efficient tools for analysing shape deformations in order to understand the real three-dimensional nature of shape changes.

Results: We present a framework for 3D+time cell shape analysis. The main contribution is three-fold: First, we develop a fast, automatic random walker method for cell segmentation. Second, a novel topology fixing method is proposed to fix segmented binary volumes without spherical topology. Third, we show that algorithms used for each individual step of the analysis pipeline (cell segmentation, topology fixing, spherical parameterization, and shape representation) are closely related to the Laplacian operator. The framework is applied to the shape analysis of neutrophil cells.

Conclusions: The method we propose for cell segmentation is faster than the traditional random walker method or the level set method, and performs better on 3D time-series of neutrophil cells, which are comparatively noisy as stacks have to be acquired fast enough to account for cell motion. Our method for topology fixing outperforms the tools provided by SPHARM-MAT and SPHARM-PDM in terms of their successful fixing rates. The different tasks in the presented pipeline for 3D+time shape analysis of cells can be solved using Laplacian approaches, opening the possibility of eventually combining individual steps in order to speed up computations.
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http://dx.doi.org/10.1186/1471-2105-14-296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3871028PMC
October 2013

Two distinct functions for PI3-kinases in macropinocytosis.

J Cell Sci 2013 Sep 10;126(Pt 18):4296-307. Epub 2013 Jul 10.

Cardiovascular Research Institute and Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA.

Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins.
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http://dx.doi.org/10.1242/jcs.134015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772393PMC
September 2013

Lysophosphatidylinositol-acyltransferase-1 (LPIAT1) is required to maintain physiological levels of PtdIns and PtdInsP(2) in the mouse.

PLoS One 2013 5;8(3):e58425. Epub 2013 Mar 5.

Inositide Laboratory, Babraham Institute, Babraham Research Campus, Cambridge, United Kingdom.

We disrupted the gene encoding lysophosphatidylinositol-acyltransferase-1 (LPIAT1) in the mouse with the aim of understanding its role in determining cellular phosphoinositide content. LPIAT1(-/-) mice were born at lower than Mendelian ratios and exhibited a severe developmental brain defect. We compared the phospholipid content of livers and brains from LPIAT1(-/-) and LPIAT1(+/+) littermates by LC-ESI/MS. In accord with previous studies, the most abundant molecular species of each phosphoinositide class (PtdIns, PtdInsP, PtdInsP2 and PtdInsP3) possessed a C38∶4 complement of fatty-acyl esters (C18∶0 and C20∶4 are usually assigned to the sn-1 and sn-2 positions, respectively). LPIAT1(-/-) liver and brain contained relatively less of the C38∶4 species of PtdIns, PtdInsP and PtdInsP2 (dropping from 95-97% to 75-85% of the total species measured for each lipid class) and relatively more of the less abundant species (PtdInsP3 less abundant species were below our quantification levels). The increases in the less abundant PtdIns and PtdInsP2 species did not compensate for the loss in C38∶4 species, resulting in a 26-44% reduction in total PtdIns and PtdInsP2 levels in both brain and liver. LPIAT1(-/-) brain and liver also contained increased levels of C18∶0 lyso-PtdIns (300% and 525% respectively) indicating a defect in the reacylation of this molecule. LPIAT1(-/-) brain additionally contained significantly reduced C38∶4 PC and PE levels (by 47% and 55% respectively), possibly contributing to the phenotype in this organ. The levels of all other molecular species of PC, PE, PS and PA measured in the brain and liver were very similar between LPIAT1(-/-) and LPIAT1(+/+) samples. These results suggest LPIAT1 activity plays a non-redundant role in maintaining physiological levels of PtdIns within an active deacylation/reacylation cycle in mouse tissues. They also suggest that this pathway must act in concert with other, as yet unidentified, mechanisms to achieve the enrichment observed in C38∶4 molecular species of phosphoinositides.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058425PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589398PMC
December 2013

Activation of the neutrophil NADPH oxidase by Aspergillus fumigatus.

Ann N Y Acad Sci 2012 Dec;1273:68-73

MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.

Upon infection of the respiratory system with the fungus Aspergillus fumigatus various leukoctytes, in particular neutrophils, are recruited to the lung to mount an immune response. Neutrophils respond by both phagocytosing conidia and mediating extracellular killing of germinated, invasive hyphae. Of paramount importance to an appropriate immune response is the neutrophil NADPH oxidase enzyme, which mediates the production of various reactive oxygen species (ROS). This is evidenced by the acute sensitivity of both oxidase-deficient humans and mice to invasive aspergillosis. Herein we briefly review the mechanisms and functions of oxidase activation and discuss our recent work identifying at least some of the important players in hyphal-induced oxidase activation and neutrophil function. Among these we define the phosphoinositide 3-kinase enzyme and the regulatory protein Vav to be of critical importance and allude to a kinase-independent role for Syk.
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http://dx.doi.org/10.1111/j.1749-6632.2012.06821.xDOI Listing
December 2012

Phosphoinositide 3-OH kinase regulates integrin-dependent processes in neutrophils by signaling through its effector ARAP3.

J Immunol 2013 Jan 23;190(1):381-91. Epub 2012 Nov 23.

The Inositide Laboratory, The Babraham Institute, Cambridge CB22 3AT, United Kingdom.

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.
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http://dx.doi.org/10.4049/jimmunol.1201330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3672969PMC
January 2013

Functional redundancy of class I phosphoinositide 3-kinase (PI3K) isoforms in signaling growth factor-mediated human neutrophil survival.

PLoS One 2012 24;7(9):e45933. Epub 2012 Sep 24.

Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge, United Kingdom.

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 (p110δ(KO)γ(KO) or p110γ(KO)). 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 p110δ(KO)γ(KO) 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045933PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3454369PMC
March 2013