Life Sciences Research for Lifelong Health

Publications phillip-hawkins

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Two distinct functions for PI3-kinases in macropinocytosis.
O Hoeller, P Bolourani, J Clark, LR Stephens, PT Hawkins, OD Weiner, G Weeks, RR Kay

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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Journal of cell science, 126, Pt 18, 4296-307, 2013

PMID: 23843627
DOI: 10.1242/jcs.134015

Open Access

Lysophosphatidylinositol-acyltransferase-1 (LPIAT1) is required to maintain physiological levels of PtdIns and PtdInsP(2) in the mouse.
KE Anderson, A Kielkowska, TN Durrant, V Juvin, J Clark, LR Stephens, PT Hawkins

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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PloS one, 8, 3, e58425, 2013

PMID: 23472195
DOI: 10.1371/journal.pone.0058425

Open Access

Activation of the neutrophil NADPH oxidase by Aspergillus fumigatus.
KB Boyle, LR Stephens, PT Hawkins

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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Annals of the New York Academy of Sciences, 1273, , 68-73, 2012

PMID: 23230839
DOI: 10.1111/j.1749-6632.2012.06821.x

Phosphoinositide 3-OH kinase regulates integrin-dependent processes in neutrophils by signaling through its effector ARAP3.
L Gambardella, KE Anderson, Z Jakus, M Kovács, S Voigt, PT Hawkins, L Stephens, A Mócsai, S Vermeren

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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Journal of immunology (Baltimore, Md. : 1950), 190, 1, 381-91, 2013

PMID: 23180820
DOI: 10.4049/jimmunol.1201330

Open Access

Functional redundancy of class I phosphoinositide 3-kinase (PI3K) isoforms in signaling growth factor-mediated human neutrophil survival.
JK Juss, RP Hayhoe, CE Owen, I Bruce, SR Walmsley, AS Cowburn, S Kulkarni, KB Boyle, L Stephens, PT Hawkins, ER Chilvers, AM Condliffe

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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PloS one, 7, 9, e45933, 2012

PMID: 23029326
DOI: 10.1371/journal.pone.0045933

Open Access

PI3K signalling: the path to discovery and understanding.
B Vanhaesebroeck, L Stephens, P Hawkins

Over the past two decades, our understanding of phospoinositide 3-kinases (PI3Ks) has progressed from the identification of an enzymatic activity associated with growth factors, GPCRs and certain oncogene products to a disease target in cancer and inflammation, with PI3K inhibitors currently in clinical trials. Elucidation of PI3K-dependent networks led to the discovery of the phosphoinositide-binding PH, PX and FYVE domains as conduits of intracellular lipid signalling, the determination of the molecular function of the tumour suppressor PTEN and the identification of AKT and mTOR protein kinases as key regulators of cell growth. Here we look back at the main discoveries that shaped the PI3K field.

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Nature reviews. Molecular cell biology, 13, 3, 195-203, 2012

PMID: 22358332
DOI: 10.1038/nrm3290

SCFAs induce mouse neutrophil chemotaxis through the GPR43 receptor.
MA Vinolo, GJ Ferguson, S Kulkarni, G Damoulakis, K Anderson, M Bohlooly-Y, L Stephens, PT Hawkins, R Curi

Short chain fatty acids (SCFAs) have recently attracted attention as potential mediators of the effects of gut microbiota on intestinal inflammation. Some of these effects have been suggested to occur through the direct actions of SCFAs on the GPR43 receptor in neutrophils, though the precise role of this receptor in neutrophil activation is still unclear. We show that mouse bone marrow derived neutrophils (BMNs) can chemotax effectively through polycarbonate filters towards a source of acetate, propionate or butyrate. Moreover, we show that BMNs move with good speed and directionality towards a source of propionate in an EZ-Taxiscan chamber coated with fibrinogen. These effects of SCFAs were mimicked by low concentrations of the synthetic GPR43 agonist phenylacetamide-1 and were abolished in GPR43(-/-) BMNs. SCFAs and phenylacetamide-1 also elicited GPR43-dependent activation of PKB, p38 and ERK and these responses were sensitive to pertussis toxin, indicating a role for Gi proteins. Phenylacetamide-1 also elicited rapid and transient activation of Rac1/2 GTPases and phosphorylation of ribosomal protein S6. Genetic and pharmacological intervention identified important roles for PI3Kγ, Rac2, p38 and ERK, but not mTOR, in GPR43-dependent chemotaxis. These results identify GPR43 as a bona fide chemotactic receptor for neutrophils in vitro and start to define important elements in its signal transduction pathways.

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PloS one, 6, 6, e21205, 2011

PMID: 21698257
DOI: 10.1371/journal.pone.0021205

Open Access

The GTPase-activating protein ARAP3 regulates chemotaxis and adhesion-dependent processes in neutrophils.
L Gambardella, KE Anderson, C Nussbaum, A Segonds-Pichon, T Margarido, L Norton, T Ludwig, M Sperandio, PT Hawkins, L Stephens, S Vermeren

Neutrophils form a vital part of the innate immune response, but at the same time their inappropriate activation contributes to autoimmune diseases. Many molecular components are involved in fine-tuning neutrophil function. We report here the first characterization of the role of ARAP3, a PI3K and Rap-regulated GTPase-activating protein for RhoA and Arf6 in murine neutrophils. We show that neutrophils lacking ARAP3 are preactivated in vitro and in vivo, exhibiting increased β2 integrin affinity and avidity. ARAP3-deficient neutrophils are hyperresponsive in several adhesion-dependent situations in vitro, including the formation of reactive oxygen species, adhesion, spreading, and granule release. ARAP3-deficient cells adhere more firmly under flow conditions in vitro and to the vessel wall in vivo. Finally, loss of ARAP3 interferes with integrin-dependent neutrophil chemotaxis. The results of the present study suggest an important function of ARAP3 downstream of Rap. By modulating β2 integrin activity, ARAP3 guards neutrophils in their quiescent state unless activated.

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Blood, 118, 4, 1087-98, 2011

PMID: 21490342
DOI: 10.1182/blood-2010-10-312959

Open Access

PI3Kβ plays a critical role in neutrophil activation by immune complexes.
S Kulkarni, C Sitaru, Z Jakus, KE Anderson, G Damoulakis, K Davidson, M Hirose, J Juss, D Oxley, TA Chessa, F Ramadani, H Guillou, A Segonds-Pichon, A Fritsch, GE Jarvis, K Okkenhaug, R Ludwig, D Zillikens, A Mocsai, B Vanhaesebroeck, LR Stephens, PT Hawkins

Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the β isoform of phosphoinositide 3-kinase (PI3Kβ) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kβ alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kβ and PI3Kδ, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3Kβ by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B₄. Coincident activation by a tyrosine kinase-coupled receptor (FcγR) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the β isoform of PI3K. PI3Kβ-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kβ and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kβ as a potential therapeutic target in inflammatory disease.

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Science signaling, 4, 168, ra23, 2011

PMID: 21487106
DOI: 10.1126/scisignal.2001617

Open Access

Structure of lipid kinase p110β/p85β elucidates an unusual SH2-domain-mediated inhibitory mechanism.
X Zhang, O Vadas, O Perisic, KE Anderson, J Clark, PT Hawkins, LR Stephens, RL Williams

Phosphoinositide 3-kinases (PI3Ks) are essential for cell growth, migration, and survival. The structure of a p110β/p85β complex identifies an inhibitory function for the C-terminal SH2 domain (cSH2) of the p85 regulatory subunit. Mutagenesis of a cSH2 contact residue activates downstream signaling in cells. This inhibitory contact ties up the C-terminal region of the p110β catalytic subunit, which is essential for lipid kinase activity. In vitro, p110β basal activity is tightly restrained by contacts with three p85 domains: the cSH2, nSH2, and iSH2. RTK phosphopeptides relieve inhibition by nSH2 and cSH2 using completely different mechanisms. The binding site for the RTK's pYXXM motif is exposed on the cSH2, requiring an extended RTK motif to reach and disrupt the inhibitory contact with p110β. This contrasts with the nSH2 where the pY-binding site itself forms the inhibitory contact. This establishes an unusual mechanism by which p85 SH2 domains contribute to RTK signaling specificities.

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Molecular cell, 41, 5, 567-78, 2011

PMID: 21362552
DOI: 10.1016/j.molcel.2011.01.026

Open Access

Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry.
J Clark, KE Anderson, V Juvin, TS Smith, F Karpe, MJ Wakelam, LR Stephens, PT Hawkins

Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P(3)). PtdIns(3,4,5)P(3) regulates major aspects of cellular behavior, and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P(3) have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based on phosphate methylation coupled to high-performance liquid chromatography-mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P(3) and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP(2) are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P(3) in unstimulated mouse and human cells (≥10(5)) or tissues (≥0.1 mg) and their increase upon appropriate stimulation.

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Nature methods, 8, 3, 267-72, 2011

PMID: 21278744
DOI: 10.1038/nmeth.1564

Open Access

Class IA phosphoinositide 3-kinase β and δ regulate neutrophil oxidase activation in response to Aspergillus fumigatus hyphae.
KB Boyle, D Gyori, A Sindrilaru, K Scharffetter-Kochanek, PR Taylor, A Mócsai, LR Stephens, PT Hawkins

An effective immune response to the ubiquitous fungus Aspergillus fumigatus is dependent upon production of reactive oxygen species (ROS) by the NADPH oxidase. This is evidenced by the acute sensitivity of oxidase-deficient humans and mice to invasive aspergillosis. Neutrophils are recruited to the lungs shortly postinfection and respond by phagocytosing conidia and mediating extracellular killing of germinated hyphae in a ROS-dependent manner. However, the signaling mechanisms regulating the generation of ROS in response to hyphae are poorly understood. PI3Ks are important regulators of numerous cellular processes, with much recent work describing unique roles for the different class I PI3K isoforms. We showed by live-cell imaging that the lipid products of class I PI3Ks accumulated at the hyphal-bound neutrophil plasma membrane. Further, we used pharmacological and genetic approaches to demonstrate essential, but overlapping, roles for PI3Kβ and PI3Kδ in the ROS and spreading responses of murine neutrophils to Aspergillus hyphae. Hyphal-induced ROS responses were substantially inhibited by deletion of the common β2-integrin subunit CD18, with only a minor, redundant role for Dectin-1. However, addition of soluble algal glucans plus the genetic deletion of CD18 were required to significantly inhibit activation of the PI3K-effector protein kinase B. Hyphal ROS responses were also totally dependent on the presence of Syk, but not its ITAM-containing adaptor proteins FcRγ or DAP12, and the Vav family of Rac-guanine nucleotide exchange factors. These results start to define the signaling network controlling neutrophil ROS responses to A. fumigatus hyphae.

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Journal of immunology (Baltimore, Md. : 1950), 186, 5, 2978-89, 2011

PMID: 21257963
DOI: 10.4049/jimmunol.1002268

Open Access

Signalling via class IA PI3Ks.
L Stephens, P Hawkins

Advances in enzyme regulation, 51, 1, 27-36, 2011

PMID: 21035483
DOI: 10.1016/j.advenzreg.2010.09.007

Phosphorylation of threonine 154 in p40phox is an important physiological signal for activation of the neutrophil NADPH oxidase.
TA Chessa, KE Anderson, Y Hu, Q Xu, O Rausch, LR Stephens, PT Hawkins

The neutrophil nicotinamide adenine dinucleotide phosphate-oxidase is a multisubunit enzyme (comprising gp91(phox), p22(phox), p67(phox), p40(phox), p47(phox), and Rac) that plays a vital role in microbial killing. The recent discovery of a chronic granulomatous disease patient who expresses a mutant p40(phox) subunit, together with the development of mouse models of p40(phox) function, indicate phosphatidylinositol 3-phosphate binding to the PX domain of p40(phox) is an important signal for oxidase activation. However, the presence of other conserved residues and domains in p40(phox) suggest further regulatory roles for this protein. To test this, we introduced wild-type and mutated versions of p40(phox) into fully differentiated mouse neutrophils by retroviral transduction of p40(phox)(-/-) bone marrow progenitors and repopulation of the bone marrow compartment in radiation chimaeras. Phosphorylation of p40(phox) on threonine 154, but not serine 315, was required for full oxidase activation in response to formylated bacterial peptide fMLP, serum-opsonized S aureus, and immunoglobulin-opsonized sheep red blood cells. A functional SH3 domain was not required for oxidase activation, and deletion of the entire domain resulted in enhanced oxidase responses. Phosphorylation of threonine 154 in response to S aureus was mediated by protein kinase Cδ and was required for full translocation of p47(phox) to phagosomes. These results define an important new element in the physiological activation of the oxidase.

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Blood, 116, 26, 6027-36, 2010

PMID: 20861461
DOI: 10.1182/blood-2010-08-300889

Open Access

PtdIns3P and Rac direct the assembly of the NADPH oxidase on a novel, pre-phagosomal compartment during FcR-mediated phagocytosis in primary mouse neutrophils.
KE Anderson, TA Chessa, K Davidson, RB Henderson, S Walker, T Tolmachova, K Grys, O Rausch, MC Seabra, VL Tybulewicz, LR Stephens, PT Hawkins

The generation of reactive oxygen species (ROS) by the nicotinamide adenine dinucleotide phosphate oxidase is an important mechanism by which neutrophils kill pathogens. The oxidase is composed of a membrane-bound cytochrome and 4 soluble proteins (p67(phox), p40(phox), p47(phox), and GTP-Rac). These components form an active complex at the correct time and subcellular location through a series of incompletely understood mutual interactions, regulated, in part, by GTP/GDP exchange on Rac, protein phosphorylation, and binding to lipid messengers. We have used a variety of assays to follow the spatiotemporal assembly of the oxidase in genetically engineered primary mouse neutrophils, during phagocytosis of both serum- and immunoglobulin G-opsonized targets. The oxidase assembles directly on serum-Staphylococcus aureus-containing phagosomes within seconds of phagosome formation; this process is only partially dependent (∼ 30%) on PtdIns3P binding to p40(phox), but totally dependent on Rac1/2 binding to p67(phox). In contrast, in response to immunoglobulin G-targets, the oxidase first assembles on a tubulovesicular compartment that develops at sites of granule fusion to the base of the emerging phagosome; oxidase assembly and activation is highly dependent on both PtdIns3P-p40(phox) and Rac2-p67(phox) interactions and delivery to the phagosome is regulated by Rab27a. These results define a novel pathway for oxidase assembly downstream of FcR-activation.

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Blood, 116, 23, 4978-89, 2010

PMID: 20813901
DOI: 10.1182/blood-2010-03-275602

Open Access

Synthesis and biological evaluation of phosphatidylinositol phosphate affinity probes.
SJ Conway, J Gardiner, SJ Grove, MK Johns, ZY Lim, GF Painter, DE Robinson, C Schieber, JW Thuring, LS Wong, MX Yin, AW Burgess, B Catimel, PT Hawkins, NT Ktistakis, LR Stephens, AB Holmes

The synthesis of the complete family of phosphatidylinositol phosphate analogues (PIPs) from five key core intermediates A-E is described. These core compounds were obtained from myo-inositol orthoformate 1 via regioselective DIBAL-H and trimethylaluminium-mediated cleavages and a resolution-protection process using camphor acetals 10. Coupling of cores A-E with phosphoramidites 34 and 38, derived from the requisite protected lipid side chains, afforded the fully-protected PIPs. Removal of the remaining protecting groups was achieved via hydrogenolysis using palladium black or palladium hydroxide on carbon in the presence of sodium bicarbonate to afford the complete family of dipalmitoyl- and amino-PIP analogues 42, 45, 50, 51, 58, 59, 67, 68, 76, 77, 82, 83, 92, 93, 99 and 100. Investigations using affinity probes incorporating these compounds have identified novel proteins involved in the PI3K intracellular signalling network and have allowed a comprehensive proteomic analysis of phosphoinositide interacting proteins.

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Organic & biomolecular chemistry, 8, 1, 66-76, 2010

PMID: 20024134
DOI: 10.1039/b913399b

CD18-dependent activation of the neutrophil NADPH oxidase during phagocytosis of Escherichia coli or Staphylococcus aureus is regulated by class III but not class I or II PI3Ks.
KE Anderson, KB Boyle, K Davidson, TA Chessa, S Kulkarni, GE Jarvis, A Sindrilaru, K Scharffetter-Kochanek, O Rausch, LR Stephens, PT Hawkins

Phagocytosis and activation of the NADPH oxidase are important mechanisms by which neutrophils and macrophages engulf and kill microbial pathogens. We investigated the role of PI3K signaling pathways in the regulation of the oxidase during phagocytosis of Staphylococcus aureus and Escherichia coli by mouse and human neutrophils, a mouse macrophage-like cell line and a human myeloid-like cell line. Phagocytosis of these bacteria was promoted by serum, independent of serum-derived antibodies, and effectively abolished in mouse neutrophils lacking the beta(2)-integrin common chain, CD18. A combination of PI3K isoform-selective inhibitors, mouse knock-outs, and RNA-interference indicated CD18-dependent activation of the oxidase was independent of class I and II PI3Ks, but substantially dependent on the single class III isoform (Vps34). Class III PI3K was responsible for the synthesis of PtdIns(3)P on phagosomes containing either bacteria. The use of mouse neutrophils carrying an appropriate knock-in mutation indicated that PtdIns(3)P binding to the PX domain of their p40(phox) oxidase subunit is important for oxidase activation in response to both S aureus and E coli. This interaction does not, however, account for all the PI3K sensitivity of these responses, particularly the oxidase response to E coli, suggesting that additional mechanisms for PtdIns(3)P-regulation of the oxidase must exist.

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Blood, 112, 13, 5202-11, 2008

PMID: 18755982
DOI: 10.1182/blood-2008-04-149450

Open Access

Moving towards a better understanding of chemotaxis.
L Stephens, L Milne, P Hawkins

Eukaryotic cells are thought to move across supporting surfaces through a combination of coordinated processes: polarisation; extension of dynamic protrusions from a leading edge; adhesion-associated stabilisation of some protrusions; centripetal pulling against those leading adhesions; and de-adhesion at the rear. Gradients of extracellular ligands can be detected by cells and then used to guide them either towards the source (in the case of a chemoattractant) or away from the source (in the case of a chemorepellent)--such migration is termed chemotaxis. Recent work suggests that chemotaxis probably emerges from the ability of cells to spatially encode extracellular gradients of ligands, a process for which phosphoinositide 3'-kinase (PI3K) signals alone are insufficient, and to use that vectorial information to bias movement by enhancing the survival, and not the formation, of the protrusions that experience the greatest stimulation.

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Current biology : CB, 18, 11, R485-94, 2008

PMID: 18522824
DOI: 10.1016/j.cub.2008.04.048

Open Access

PI3K class IB pathway in neutrophils.
S Andrews, L Stephens, P Hawkins

Activation of G(i)-coupled receptors in neutrophils stimulates class IB phosphoinositide 3-kinase (PI3K) (also known as PI3Kgamma) through the combined actions of Gbetagamma subunits and the small guanosine triphosphatase (GTPase) Ras, resulting in the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] in the plasma membrane. In most cases, the effectors of this pathway possess a pleckstrin homology (PH) domain that mediates the interaction with and regulation by these two lipid messengers. These direct effectors sit within a complex regulatory network that includes several other signaling pathways and that is responsible for the control of important neutrophil functions, including adhesion, chemotaxis, secretion, and the "respiratory burst" [activation of the nicotinamide adenosine diphosphate (NADPH) oxidase]. Although the molecular details that link the direct effectors of class IB PI3K to these complex cell responses are still largely unknown, these responses involve complex regulation of small GTPases of the Rac, Rho, and Arf families.

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Science's STKE : signal transduction knowledge environment, 2007, 407, cm3, 2007

PMID: 17925574
DOI: 10.1126/stke.4072007cm3

PI3K class IB pathway.
Andrews S, Stephens LR, Hawkins PT

Class I phosphoinositide 3-kinases (PI3Ks) are well-established signal transduction enzymes that play an important role in the mechanisms by which a wide variety of cell surface receptors control several cellular functions, including cellular growth, division, survival, and movement. Class IB PI3K (also known as PI3Kgamma) allows fast-acting, heterotrimeric GTP-binding protein-coupled receptors to access this pathway. Activation of class IB PI3K results in the rapid synthesis of phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and its dephosphorylation product, PI(3,4)P2, in the plasma membrane. These two lipid messengers bind to multiple, pleckstrin homology (PH) domain-containing effectors, which together regulate a complex signaling web downstream of receptor activation. This pathway regulates the activity of protein kinases and small guanosine triphosphatases that control cellular movement, adhesion, contraction, and secretion. Most of the ligands that have been established to activate class IB PI3K are involved in coordinating the body's response to injury and infection through the regulation of multiple cell types in the immune system and vascular lining. Mice lacking the catalytic subunit of class IB PI3K are remarkably resistant to the development of several inflammatory pathologies in mouse models of human inflammatory disease. These results suggest small molecule inhibitors of class IB PI3K may represent a novel class of therapeutic agents that may complement existing anti-inflammatory treatments.

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Science's STKE : signal transduction knowledge environment, 2007, 1525-8882, cm2, 2007

PMID: 17925573

Open Access

PI3Kgamma is a key regulator of inflammatory responses and cardiovascular homeostasis.
PT Hawkins, LR Stephens

Class I phosphoinositide 3-kinase (PI3K) signaling pathways regulate several important cellular functions, including cellular growth, division, survival, and movement. Class IB PI3K (also known as PI3Kgamma) links heterotrimeric GTP-binding protein-coupled receptors to these pathways. Activation of class IB PI3K results in the rapid synthesis of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] and its dephosphorylation product PtdIns(3,4)P2 in the plasma membrane. These two lipid messengers bind to pleckstrin homology domain-containing effectors that regulate a complex signaling web downstream of receptor activation. Characteristic features of this pathway are the regulation of protein kinases and the regulation of small guanosine triphosphatases that control cellular movement, adhesion, contraction, and secretion. Most of the ligands that activate class IB PI3K are involved in coordinating the body's response to injury and infection, and recent studies suggest that small molecule inhibitors of this enzyme may represent a novel class of anti-inflammatory therapeutic agents.

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Science (New York, N.Y.), 318, 5847, 64-6, 2007

PMID: 17916723
DOI: 10.1126/science.1145420

The role of PI3Ks in the regulation of the neutrophil NADPH oxidase.
PT Hawkins, K Davidson, LR Stephens

The NADPH oxidase complex of neutrophils and macrophages is an important weapon used by these cells to kill microbial pathogens. The regulation of this enzyme complex is necessarily complicated by the diverse receptor types that are needed to trigger its activation and also the tight control that is required to deliver this activation at the appropriate time and place. As such, several signalling pathways have been established to regulate the NADPH oxidase downstream of cell surface receptors. Central amongst these are PI3K- (phosphoinositide 3-kinase)-dependent pathways, blockade of which severely limits activation of the oxidase to several soluble and particulate stimuli. The precise roles of the phosphoinositide products of PI3K activity in regulating NADPH oxidase assembly and activation are still unclear, but there is emerging evidence that they play a key role via regulation of guanine nucleotide exchange on Rac, a key component in the oxidase complex. There is also very strong evidence that the PI3K products PtdIns(3,4)P2 and PtdIns3P can bind directly to the PX (Phox homology) domains of the core oxidase components p47phox and p40phox respectively. However, the significance of these interactions in terms of membrane localization or allosteric consequences for the oxidase complex remains to be established.

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Biochemical Society symposium, , 74, 59-67, 2007

PMID: 17233580
DOI: 10.1042/BSS0740059

Structural determinants of LL5beta subcellular localisation and association with filamin C.
V Paranavitane, LR Stephens, PT Hawkins

PI3K signalling pathways link cell surface receptors to the control of several intracellular functions including cell growth, survival and movement. Filamins are important regulators of cortical actin structure and function. LL5beta is a filamin binding protein that is an effector of the PI3K signalling pathway. We define an N-terminal region of LL5beta that is responsible for binding to the C-terminus of filamins. Under conditions of very low PI3K activity, we show that this region, together with an additional domain of the protein, is responsible for localising the complex to punctate structures that are also decorated by L-FILIP (a protein previously characterised to bind filamin and accelerate its destruction). Under conditions of significant PI3K activity, PtdIns(3,4,5)P(3) binding to the C-terminal PH domain in LL5beta prevents localisation to these structures. These observations start to define the basis for PI3K regulation of filamin through LL5beta.

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Cellular signalling, 19, 4, 817-24, 2007

PMID: 17174070
DOI: 10.1016/j.cellsig.2006.10.007

PI(3)Kgamma has an important context-dependent role in neutrophil chemokinesis.
GJ Ferguson, L Milne, S Kulkarni, T Sasaki, S Walker, S Andrews, T Crabbe, P Finan, G Jones, S Jackson, M Camps, C Rommel, M Wymann, E Hirsch, P Hawkins, L Stephens

The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells, such as mammalian phagocytes, allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) accumulate in a PI(3)Kgamma-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradient-sensing and gradient-biased movement. PI(3)Kgamma activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kgamma activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kgamma is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrin-based adhesion and the accumulation of polymerized (F)-actin at the leading-edge.

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Nature cell biology, 9, 1, 86-91, 2007

PMID: 17173040
DOI: 10.1038/ncb1517

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.
H Guillou, C Lécureuil, KE Anderson, S Suire, GJ Ferguson, CD Ellson, A Gray, N Divecha, PT Hawkins, LR Stephens

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.

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Journal of lipid research, 48, 3, 726-32, 2007

PMID: 17130283
DOI: 10.1194/jlr.D600038-JLR200

Open Access