FcγR-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

Abstract

The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b₅₅₈ and cytosolic p67^phox, p47^phox, and p40^phox subunits that undergo membrane translocation upon cellular activation. The function of p40^phox, which binds p67^phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40^phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40^phox in FcγR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcγR-induced phagocytosis. YFP-tagged p67^phox and p40^phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67^phox and p47^phox accumulation on nascent and internalized phagosomes did not require p40^phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40^phox PI3P-binding domain or wortmannin. Translocation of p40^phox to nascent phagosomes required binding to p67^phox but not PI3P, although the loss of PI3P binding reduced p40^phox retention after phagosome internalization. We conclude that p40^phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization.