Role of the small GTPase Rac in p22^phox-dependent NADPH oxidases

The superoxide-producing phagocyte NADPH oxidase gp91^phox/Nox2 and the non-phagocytic oxidases Nox1 and Nox3 each form a complex in the membrane with p22^phox, which provides both stabilization and a docking site for organizer proteins. The p22^phox-complexed Nox2 and Nox1 are dormant on their own, and their activation requires soluble supportive proteins such as a Nox organizer (p47^phox or Noxo1) and a Nox activator (p67^phox or Noxa1). The small GTPase Rac directly binds to the activators, and thus plays an essential role in the Nox2-based oxidase containing p47^phox and p67^phox or a positive role in Nox1 activity supported by Noxo1 and Noxa1. Although Nox3 complexed with p22^phox constitutively produce superoxide, the production can be enhanced by supportive proteins. Here we compare the roles of Rac in these p22^phox-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 p47^phox and either p67^phox or Noxa1. When these oxidases use Noxo1 as an organizer instead of p47^phox, they produce a small but significant amount of superoxide without expression of Rac1(Q61L), although the production is enhanced by Rac1(Q61L). Thus p47^phox 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 p47^phox and either p67^phox or Noxa1, whereas Rac fails to upregulate Nox3 activity when p47^phox is replaced with Noxo1. We also demonstrate that, in the Nox3-based oxidase containing solely p67^phox as supportive protein, expression of Rac1(Q61L) enhances not only superoxide production but also membrane translocation of p67^phox. Since the enhancements are not observed with a mutant p67^phox defective in binding to Rac, this GTPase appear to directly recruit p67^phox to the membrane.