Chris D. Ellson

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.

The Crystal Structure of the Px Domain from P40Phox Bound to Phosphatidylinositol 3-Phosphate

More than 50 human proteins with a wide range of functions have a 120 residue phosphoinositide binding module known as the PX domain. The 1.7 A X-ray crystal structure of the PX domain from the p40(phox) subunit of NADPH oxidase bound to PtdIns(3)P shows that the PX domain embraces the 3-phosphate on one side of a water-filled, positively charged pocket and reveals how 3-phosphoinositide specificity is achieved. A chronic granulomatous disease (CGD)-associated mutation in the p47(phox) PX domain that abrogates PtdIns(3)P binding maps to a conserved Arg that does not directly interact with the phosphoinositide but instead appears to stabilize a critical lipid binding loop. The SH3 domain present in the full-length protein does not affect soluble PtdIns(3)P binding to the p40(phox) PX domain.

Roles of PI3Ks in leukocyte chemotaxis and phagocytosis

Through selective disruption of phosphoinositide 3-kinase (PI3K) activity and the use of green fluorescent protein tagged derivatives of domains capable of specifically binding the lipid products of PI3Ks in vivo, it has been shown that this family of signalling enzymes have vital and distinct roles in chemotaxis, phagocytosis and phagosome maturation in leukocytes.

Phosphatidylinositol 3-phosphate is generated in phagosomal membranes.

Phagocytic cells such as neutrophils and macrophages engulf and destroy invading microorganisms. After internalization, material captured within the phagosomal membrane is destroyed by a complex process of coordinated delivery of digestive enzymes and reactive oxygen species. Several endosomal, lysosomal, and oxidase components expected to participate in these events have recently been shown to bind PtdIns3P, suggesting that this lipid may play a role in this process. We used live, digital fluorescence imaging of RAW 264.7 cells stably expressing either a PtdIns3P binding GFP-PX domain or a GFP-FYVE domain to visualize changes in the levels and subcellular localization of PtdIns3P during phagocytic uptake of IgG-opsonized zymosan particles. Very similar results were obtained using both PtdIns3P probes. The basal distribution of each PtdIns3P probe was partially cytosolic and partially localized to EEA-1-positive endosomal structures. Within about 2-3 min of zymosan attachment and concomitant with the closure of the phagosomal membrane, GFP-positive vesicles moved toward and attached to a localized area of the phagosome. A dramatic, transient accumulation of GFP probe around the entire phagosome rapidly ensued, accompanied by a transient drop in cytosolic GFP fluorescence. The magnitude and timing of this rise in PtdIns3P clearly suggest that it is an ideal candidate for controlling the early stages of phagosomal maturation.

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.

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.

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.

A novel pathway of cellular phosphatidylinositol(3,4,5)-trisphosphate synthesis is regulated by oxidative stress

BACKGROUND Phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is a key second messenger found ubiquitously in higher eukaryotic cells. The activation of Class I phosphoinositide 3-kinases and the subsequent production of PtdIns(3,4,5)P(3) is an important cell signaling event that has been causally linked to the activation of a variety of downstream cellular processes, such as cell migration and proliferation. Although numerous proteins regulating a variety of biological pathways have been shown to bind PtdIns(3,4,5)P(3), there are no data to demonstrate multiple mechanisms for PtdIns(3,4,5)P(3) synthesis in vivo. RESULTS In this study, we demonstrate an alternative pathway for the in vivo production of PtdIns(3,4,5)P(3) mediated by the action of murine Type Ialpha phosphatidylinositol 4-phosphate 5-kinase (Type Ialpha PIPkinase), an enzyme best characterized as regulating cellular PtdIns(4,5)P(2) levels. Analysis of this novel pathway of PtdIns(3,4,5)P(3) synthesis in cellular membranes leads us to conclude that in vivo, Type Ialpha PIPkinase also acts as a PtdIns(3,4)P(2) 5-kinase. We demonstrate for the first time that cells actually contain an endogenous PtdIns(3,4)P(2) 5-kinase, and that during oxidative stress, this enzyme is responsible for PtdIns(3,4,5)P(3) synthesis. Furthermore, we demonstrate that by upregulating the H(2)O(2)-induced PtdIns(3,4,5)P(3) levels using overexpression studies, the endogenous PtdIns(3,4)P(2) 5-kinase is likely to be Type Ialpha PIPkinase. CONCLUSIONS We describe for the first time a novel in vivo activity for Type Ialpha PIPkinase, and a novel pathway for the in vivo synthesis of functional PtdIns(3,4,5)P(3), a key lipid second messenger regulating a number of diverse cellular processes.

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

We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] 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)P3 levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P3 accumulation in neutrophils isolated from mice carrying a Ras-insensitive (‘DASAA’) knock-in of PI3Kγ (p110γDASAA/DASAA) is substantially dependent on the Ras binding domain of PI3Kγ.