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, p67phox, p47phox, and possibly p40phox, associate with the gp91phox and p22phox transmembrane proteins. Rac is a GTPase that in the GTP-bound state binds p67phox to activate NADPH oxidase. The function of p40phox is not known; it is believed to have a regulatory function in sequestering p67phox and p47phox in a cytosolic complex. We investigated binding interactions between p40phox, p67phox, and Rac and found that Rac1-GTP displaced p67phox bound to p40phox. In contrast, Cdc42, a GTPase homologous to Rac, did not displace p67phox from p40phox. A synthetic peptide corresponding to p67phox amino acids 170-199, a region identified previously as a Rac binding domain, significantly reduced the ability of Rac1-GTP to disrupt p67phox/p40phox binding. We hypothesize that Rac-GTP binds the p67phox N-terminal domain encompassing amino acids 170-199 that transmits a conformational change which causes p40phox to dissociate from its binding site in the p67phox C-terminus.