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PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40phox

Nature Cell Biology volume 3, pages 679–682 (2001)Cite this article

Abstract

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 formation4,5 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 domain6 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.

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Figure 1: Dependence of ROS formation activated by PtdIns(3,4,5)P3 and PtdIns(3)P on purified components.
Figure 2: PtdIns(3)P activates a relipidated oxidase complex in the presence of p40phox.
Figure 3: PtdIns(3)P binds to the PX domain of p40phox.

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Acknowledgements

We thank A. Hall for GST–p67phox and GST–47phox bacterial expression plasmids, C. Erneux for SHIP-1 cDNA, and F. A. Norris for the inositol polyphosphate 4-phosphatase cDNA. We also acknowledge the Isaac Newton Trust for a contribution towards the Biacore 3000 machine. K.E.A. is a Beit Memorial Fellow, and P.T.H. is a BBSRC Advanced Research Fellow. This work was supported by the BBSRC, by the MRC (studentship to C.D.E.) and by a National Cancer Institute core grant (to P.T.).

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Authors and Affiliations

  1. The Inositide Laboratory, The Babraham Institute, Babraham, CB2 4AT, Cambridge, UK

    Chris D. Ellson, Stéphanie Gobert-Gosse, Karen E Anderson, Keith Davidson, John Coadwell, Phill T. Hawkins & Len R. Stephens

  2. Centre de Génétique Moléculaire et Cellulaire, Université Claude Bernard Lyon, 43 Boulevard du 11 Novembre 1918, Villeurbanne, 69622, France

    Stéphanie Gobert-Gosse

  3. Memorial Sloan-Kettering Cancer Center, New York, 10021, New York, USA

    Hediye Erdjument-Bromage & Paul Tempst

  4. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK

    Jan W. Thuring, Matthew A. Cooper, Ze-Yi Lim & Andrew B. Holmes

  5. Department of Biochemistry and Molecular Biology, University College London, London, WC1E 6BT, UK

    Piers R. J. Gaffney

  6. Division of Respiratory Medicine, Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge, CB2 2QQ, UK

    Edwin R. Chilvers

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Correspondence to Len R. Stephens.

Supplementary information

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Method S1 Purification and assaying of cytosolic factors from pig neutrophil cytosol. (PDF 57 kb)

Method S2 Assay of phosphoinositide phosphatase activities of SHIP-1 and Inositol polyphosphate 4-phosphatase mutants

Figure S1 Purification of three factors allowing PtdIns(3,4,5)P3 to stimulate ROS production.

Figure S2 SHIP-1 and Inositol polyphosphate 4-phosphatase lipid phosphatase activity are required for PtdIns(3,4,5)P3 stimulated ROS formation.

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Ellson, C., Gobert-Gosse, S., Anderson, K. et al. PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40phox. Nat Cell Biol 3, 679–682 (2001). https://doi.org/10.1038/35083076

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