Rac1 Disrupts p67phox/p40phox Binding: A Novel Role for Rac in NADPH Oxidase Activation

Phagocytic cells possess a tightly regulated multicomponent enzyme complex, the NADPH oxidase, which produces superoxide, a reactive oxygen molecule that is an essential component of host defense against infection. Upon stimulation, a functional NADPH oxidase is assembled when the cytosolic proteins, Rac, p67 phox, p47 phox, and possibly p40 phox, associate with the gp91 phox and p22 phox transmembrane proteins. Rac is a GTPase that in the GTP-bound state binds p67 phox to activate NADPH oxidase. The function of p40 phox is not known; it is believed to have a regulatory function in sequestering p67 phox and p47 phox in a cytosolic complex. We investigated binding interactions between p40 phox, p67 phox, and Rac and found that Rac1-GTP displaced p67 phox bound to p40 phox. In contrast, Cdc42, a GTPase homologous to Rac, did not displace p67 phox from p40 phox. A synthetic peptide corresponding to p67 phox amino acids 170–199, a region identified previously as a Rac binding domain, significantly reduced the ability of Rac1-GTP to disrupt p67 phox /p40 phox binding. We hypothesize that Rac-GTP binds the p67 phox N-terminal domain encompassing amino acids 170–199 that transmits a conformational change which causes p40 phox to dissociate from its binding site in the p67 phox C-terminus.