Phosphatidylinositol 3-Phosphate-dependent and -independent Functions of p40phox in Activation of the Neutrophil NADPH Oxidase
Bissonnette, Sarah A. | Glazier, Christina M. | Stewart, Mary Q. | Brown, Glenn E. | Ellson, Chris D. | Yaffe, Michael B.
In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40phox, p47phox, and p67phox, and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40phox remains less well defined than those of p47phox and p67phox. We investigated the biological role of p40phox in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40phox using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67phox levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40phox reduces both the maximal rate and total amount of ROS produced without altering the KM value of the oxidase for NADPH. Using a series of mutants, p47PX-p40phox chimeras, and deletion constructs, we found that the p40phox PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67phox requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40phox, however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40phox can increase oxidase activity ~2.5-fold above that of wild-type p40phox but maintain the requirement for PX and SH3 domain function. We present a model where p40phox translocates p67phox to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.Show more [+] Less [-]