Role of the small GTPase Rac in p22phox-dependent NADPH oxidases

The superoxide-producing phagocyte NADPH oxidase gp91phox/Nox2 and the non-phagocytic oxidases Nox1 and Nox3 each form a complex in the membrane with p22phox, which provides both stabilization and a docking site for organizer proteins. The p22phox-complexed Nox2 and Nox1 are dormant on their own, and their activation requires soluble supportive proteins such as a Nox organizer (p47phox or Noxo1) and a Nox activator (p67phox or Noxa1). The small GTPase Rac directly binds to the activators, and thus plays an essential role in the Nox2-based oxidase containing p47phox and p67phox or a positive role in Nox1 activity supported by Noxo1 and Noxa1. Although Nox3 complexed with p22phox constitutively produce superoxide, the production can be enhanced by supportive proteins. Here we compare the roles of Rac in these p22phox-dependent oxidases using the organizer and activator in different combinations. Expression of constitutively active Rac1(Q61L) is essential for activation of the Nox2- or Nox1-based oxidase containing the organizer p47phox and either p67phox or Noxa1. When these oxidases use Noxo1 as an organizer instead of p47phox, they produce a small but significant amount of superoxide without expression of Rac1(Q61L), although the production is enhanced by Rac1(Q61L). Thus p47phox is likely related to strict dependence on Rac. The Nox3-based oxidase has a similar tendency in the change of the dependence: Rac plays a positive role in Nox3 activation in the presence of p47phox and either p67phox or Noxa1, whereas Rac fails to upregulate Nox3 activity when p47phox is replaced with Noxo1. We also demonstrate that, in the Nox3-based oxidase containing solely p67phox as supportive protein, expression of Rac1(Q61L) enhances not only superoxide production but also membrane translocation of p67phox. Since the enhancements are not observed with a mutant p67phox defective in binding to Rac, this GTPase appear to directly recruit p67phox to the membrane.