Phosphatidylinositol 3-Phosphate-dependent and -independent Functions of p40 super(phox) in Activation of the Neutrophil NADPH Oxidase

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, p40 super(phox), p47 super(phox), and p67 super(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40 super(phox) remains less well defined than those of p47 super(phox) and p67 super(phox). We investigated the biological role of p40 super(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40 super(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67 super(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40 super(phox) reduces both the maximal rate and total amount of ROS produced without altering the K sub(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40 super(phox) chimeras, and deletion constructs, we found that the p40 super(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67 super(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40 super(phox), 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 p40 super(phox) can increase oxidase activity similar to 2.5-fold above that of wild-type p40 super(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40 super(phox) translocates p67 super(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.