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 b 558 (gp91 phox and p22 phox ) and three soluble factors (GTP–Rac, p47 phox and p67 phox (refs 1 , 2 ). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40 phox has subsequently been discovered as a binding partner for p67 phox (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 4 , 5 but through an unknown mechanism. We show that the addition of p40 phox 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 6 of p40 phox . These results offer new insights into the roles for PI(3)Ks and p40 phox in ROS formation and define a cellular ligand for the orphan PX domain.