The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b 558 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 Fc␥R-induced oxidase activation, we used immunofluorescence and real-time imaging of Fc␥R-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 Fc␥R-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization. (Blood.
2008;112:3867-3877)
IntroductionPhagocytic leukocytes are the front-line cellular defense against microbial attack, and are mobilized rapidly to the sites of infection where they ingest and kill opsonized microorganisms. The NADPH oxidase complex plays a central role in this process, as its assembly and activation on phagosomal membranes generate superoxide, the precursor of potent microbicidal oxidants. The importance of this enzyme is demonstrated by genetic defects in the NADPH oxidase complex that cause chronic granulomatous disease (CGD), characterized by recurrent severe and potentially lethal bacterial and fungal infections. 1 The NADPH oxidase includes the membrane-integrated flavocytochrome b, composed of gp91 phox and p22 phox , and the cytosolic components p47 phox , p67 phox , p40 phox , and Rac, a Rho-family GTPase, which translocate to flavocytochrome b upon cellular stimulation to activate superoxide production. [2][3][4] Segregation of regulatory components to the cytosol in resting cells facilitates the temporal and spatial regulation of NADPH oxidase activity. The p67 phox subunit is a Rac-GTP effector 2-4 containing a domain that activates electron transport through the flavocytochrome. 5 In resting cells, p67 phox is associated with p40 phox via complementary PB1 (phagocyte oxidase and Bem1p) motifs present in each protein. 2,[6][7][8] p67 phox is also linked to p47 phox via a high-affinity interaction involving an SH3 domain and a proline-rich region, respectively, in the C-termini of...