Roles for Proline-Rich Regions of p47phoxand p67phoxin the Phagocyte NADPH Oxidase Activationin Vitro

Abstract

The cytosolic proteins p47phoxand p67phox,each containing two SH3 domains, are required for activation of the superoxide-producing phagocyte NADPH oxidase in a cell-free system with human neutrophil membrane and the small GTPase Rac. Here we focus on roles of proline-rich regions (PRRs) that reside in p47phoxand p67phox.Deletion of the p47phoxPRR, to which the C-terminal SH3 domain of p67phoxbinds, results in three-fold decreased activation of the enzyme in the cell-free system with the full-length p67phox,suggesting a modulatory role of the p47phoxPRR. The modulation is likely mediated via the C-terminal region of p67phox,since the p47phoxmutant protein fully activates the oxidase in combination with the N-terminus of p67phox.Neither deletion of the p67phoxPRR nor substitutions for prolines in the region affects the ability to support superoxide production under the cell-free conditions, indicating that the PRR of p67phoxhas no primary function in the oxidase activation.