p47(phox) PX domain of NADPH oxidase targets cell membrane via moesin-mediated association with the actin cytoskeleton

Activation of phagocytic NADPH oxidase requires association of its cytosolic subunits with the membrane-bound flavocytochrome. Extensive phosphorylation of the p47(phox) subunit of NADPH oxidase marks the initiation of this activation process. The p47(phox) subunit then translocates to the plasma membrane, bringing the p67(phox) subunit to cytochrome b558 to form the active NADPH oxidase complex. However, the detailed mechanism for targeting the p47(phox) subunit to the cell membrane during activation still remains unclear. Here, we show that the p47(phox) PX domain is responsible for translocating the p47(phox) subunit to the plasma membrane for subsequent activation of NADPH oxidase. We also demonstrate that translocation of the p47(phox) PX domain to the plasma membrane is not due to interactions with phospholipids but rather to association with the actin cytoskeleton. This association is mediated by direct interaction between the p47(phox) PX domain and moesin.     

p47phox Phox Homology Domain Regulates Plasma Membrane but Not Phagosome Neutrophil NADPH Oxidase Activation

The assembly of cytosolic subunits p47^phox, p67^phox, and p40^phox with flavocytochrome b₅₅₈ at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47^phox subunit plays a critical role in oxidase assembly. Recent studies showed that the p47^phox Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47^phox PX domain in neutrophils is unclear, we investigated its role using p47^phox knock-out (KO) mouse neutrophils to express human p47^phox and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47^phox proteins were expressed at levels similar to endogenous murine p47^phox, with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47^phox rescued p47^phox KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47^phox with Arg⁹⁰ substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47^phox Arg⁹⁰ mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47^phox PX domain for neutrophil NADPH oxidase activation.     

Binding of the PX domain of p47(phox) to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction

p47(phox) is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X-ray structure of the p47(phox) PX domain revealed two distinct basic pockets on the membrane-binding surface, each occupied by a sulfate. These two pockets have different specificities: one preferentially binds phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and is analogous to the phophatidylinositol 3-phosphate (PtdIns3P)-binding pocket of p40(phox), while the other binds anionic phospholipids such as phosphatidic acid (PtdOH) or phosphatidylserine. The preference of this second site for PtdOH may be related to previously observed activation of NADPH oxidase by PtdOH. Simultaneous occupancy of the two phospholipid-binding pockets radically increases membrane affinity. Strikingly, measurements for full-length p47(phox) show that membrane interaction by the PX domain is masked by an intramolecular association with the C-terminal SH3 domain (C-SH3). Either a site-specific mutation in C-SH3 (W263R) or a mimic of the phosphorylated form of p47(phox) [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater affinity than the isolated PX domain.