Keith Davidson

Signalling through Class I PI3Ks in mammalian cells

It is now accepted that activation of Class I PI3Ks (phosphoinositide 3-kinases) is one of the most important signal transduction pathways used by cell-surface receptors to control intracellular events. The receptors which access this pathway include those that recognize growth factors, hormones, antigens and inflammatory stimuli, and the cellular events known to be regulated include cell growth, survival, proliferation and movement. We have learnt a great deal about the family of Class I PI3K enzymes themselves and the structural adaptations which allow a variety of cell-surface receptors to regulate their activity. Class I PI3Ks synthesize the phospholipid PtdIns(3,4,5)P3 in the membranes in which they are activated, and it is now accepted that PtdIns(3,4,5)P3 and its dephosphorylation product PtdIns(3,4)P2 are messenger molecules which regulate the localization and function of multiple effectors by binding to their specific PH (pleckstrin homology) domains. The number of direct PtdIns(3,4,5)P3/PtdIns(3,4)P2 effectors which exist, even within a single cell, creates an extremely complex signalling web downstream of PI3K activation. Some key players are beginning to emerge, however, linking PI3K activity to specific cellular responses. These include small GTPases for the Rho and Arf families which regulate the cytoskeletal and membrane rearrangements required for cell movement, and PKB (protein kinase B), which has important regulatory inputs into the regulation of cell-cycle progression and survival. The importance of the PI3K signalling pathway in regulating the balance of decisions in cell growth, proliferation and survival is clear from the prevalence of oncogenes (e.g. PI3Kalpha) and tumour suppressors [e.g. the PtdIns(3,4,5)P3 3-phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome 10)] found in this pathway. The recent availability of transgenic mouse models with engineered defects in Class I PI3K signalling pathways, and the development of PI3K isoform-selective inhibitors by both academic and pharmaceutical research has highlighted the importance of specific isoforms of PI3K in whole-animal physiology and pathology, e.g. PI3Kalpha in growth and metabolic regulation, PI3Kbeta in thrombosis, and PI3Kdelta and PI3Kgamma in inflammation and asthma. Thus the Class I PI3K signalling pathway is emerging as an exciting new area for the development of novel therapeutics.

PtdIns(3)P Regulates the Neutrophil Oxidase Complex by Binding to the PX Domain of p40phox

The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b558 (gp91phox and p22phox) and three soluble factors (GTP–Rac, p47phox and p67phox (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40phox has subsequently been discovered as a binding partner for p67phox (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation but through an unknown mechanism. We show that the addition of p40phox to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns(3)P to the PX domain of p40phox. These results offer new insights into the roles for PI(3)Ks and p40phox in ROS formation and define a cellular ligand for the orphan PX domain.

Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.

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

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

p84, a New Gβγ-Activated Regulatory Subunit of the Type IB Phosphoinositide 3-Kinase p110γ

Summary A variety of genetic and inhibitor studies have shown that phosphoinositide 3-kinase γ (PI3Kγ) plays an essential role in a number of physiological responses, including neutrophil chemotaxis, mast cell degranulation, and cardiac function [1–6]. PI3Kγ is currently thought to be composed of a p110γ catalytic subunit and a single regulatory subunit, p101. The binding of p110γ to p101 dramatically increases the activation of the complex by Gβγ subunits and, hence, is thought to be critical for the coupling of PI3Kγ to G protein coupled receptors [7–9]. Here, we characterize a new regulatory subunit for PI3Kγ. p84 is present in human, mouse, chicken, frog, and fugu genomes and is located beside the p101 locus. It is broadly expressed in cells of the murine immune system. Both recombinant and endogenous p84 bind p110γ specifically and with high affinity. Binding of p84 to p110γ substantially increases the ability of Gβγ to stimulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5) P 3 ) production both in vitro and in vivo. However, the p84/p110γ heterodimer is approximately 4-fold less sensitive to Gβγs than p101/p110γ. Endogenous murine p84 expression is substantially reduced in the absence of p110γ expression. We conclude that p110γ has two potential regulatory subunits in vivo, p84 and p101.

Gβγs and the Ras binding domain of p110γ are both important regulators of PI3Kγ signalling in neutrophils

Through their ability to regulate production of the key lipid messenger PtdIns(3,4,5)P3, the class I phosphatidylinositol-3-OH kinases (PI(3)Ks) support many critical cell responses. They, in turn, can be regulated by cell-surface receptors through signals acting on either their adaptor subunits (for example, through phosphotyrosine or Gβγs) or their catalytic subunits (for example, through GTP-Ras). The relative significance of these controlling inputs is undefined in vivo. Here, we have studied the roles of Gβγs, the adaptor p101, Ras and the Ras binding domain (RBD) in the control of the class I PI(3)K, PI(3)Kγ, in mouse neutrophils. Loss of p101 leads to major reductions in the accumulation of PtdIns(3,4,5)P3, activation of protein kinase B (PKB) and in migration towards G-protein activating ligands in vitro, and to an aseptically inflamed peritoneum in vivo. Loss of sensitivity of PI(3)Kγ to Ras unexpectedly caused similar reductions, but additionally caused a substantial loss in production of reactive oxygen species (ROS). We conclude that Gβγs, p101 and the Ras–RBD interaction all have important roles in the regulation of PI(3)Kγ in vivo and that they can simultaneously, but differentially, control distinct PI(3)Kγ effectors.

A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanase and α-galactosidase

Abstract Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase A (XylA) from Neocallimastix patriciarum in combination with mannanase and α-galactosidase. Mannanase A (ManA) from Pseudomonas fluorescens subsp. cellulosa and ManA from Clostridium thermocellum, both family 26 glycosyl hydrolases, are structurally diverse and exhibit different pH and temperature optima. Although neither mannanase was effective in pretreating softwood pulp alone, both enzymes were able to enhance the production of reducing sugar and the reduction of single-stage bleached κ number when used with the xylanase. Sequential incubations with XylA and P. fluorescens ManA produced the largest final κ number reduction in comparison to control pretreated pulp. The release of galactose from softwood pulp by α-galactosidase A (AgaA) from P. fluorescens was enhanced by the presence of ManA from the same microorganism, and a single pretreatment with these enzymes, in combination with XylA, gave the most effective κ number reduction using a single incubation. Results indicated that mixtures of hemicellulase activities can be chosen to enhance pulp bleachability.

PtdIns3P binding to the PX domain of p40phox is a physiological signal in NADPH oxidase activation

The production of reactive oxygen species by the NADPH oxidase complex of phagocytes plays a critical role in our defence against bacterial and fungal infections. The PX domains of two oxidase components, p47phox and p40phox, are known to bind phosphoinositide products of PI3Ks but the physiological roles of these interactions are unclear. We have created mice which carry an R58A mutation in the PX domain of their p40phox gene, which selectively prevents binding to PtdIns3P. p40phoxR58A/R58A embryos do not develop normally but p40phoxR58A/− mice are viable and neutrophils from these animals exhibit significantly reduced oxidase responses compared to those from their p40phox+/− siblings (e.g. 60% reduced in response to phagocytosis of Staphylococcus aureus). Wortmannin inhibition of the S. aureus oxidase response correlates with inhibition of phagosomal PtdIns3P accumulation and overlaps with the reduction in this response caused by the R58A mutation, suggesting PI3K regulation of this response is substantially dependent on PtdIns3P‐binding to p40phox. p40phoxR58A/− mice are significantly compromised in their ability to kill S. aureus in vivo, defining the physiological importance of this interaction.

PI3Kβ Plays a Critical Role in Neutrophil Activation by Immune Complexes

The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory diseases. The Integrating Isoform The class I phosphoinositide 3-kinases (PI3Ks) are implicated in processes such as growth factor signaling and inflammation. PI3Kγ is activated by G protein–coupled receptors (GPCRs), whereas PI3Kα and PI3Kδ are activated by protein tyrosine kinase–coupled receptors. PI3Kβ is unusual in that it appears to respond to signals from both types of receptors, depending on the cellular context. Kulkarni et al. investigated the responses of mouse neutrophils to immune complexes of antibody and antigen, which trigger chronic inflammation in conditions such as autoimmune arthritis. Genetic and pharmacological evidence suggested that immune complexes stimulated PI3Kβ in a process involving activation of FcγR, a tyrosine kinase–coupled low-affinity antibody receptor, and autocrine signaling by a proinflammatory lipid (LTB4) through its GPCR. Mice deficient in PI3Kβ fared better than did controls in models of arthritis and inflammatory skin disease. These data implicate PI3Kβ in the integration of signals from tyrosine kinase–coupled receptors and GPCRs—and suggest that this isoform may be an effective therapeutic target in inflammatory diseases. 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.

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

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.