Involvement of Rac1 in activation of multicomponent Nox1- and Nox3-based NADPH oxidases

Several Nox family NADPH oxidases function as multicomponent enzyme systems. We explored determinants of assembly of the multicomponent oxidases Nox1 and Nox3 and examined the involvement of Rac1 in their regulation. Both enzymes are supported by p47(phox) and p67(phox) or homologous regulators called Noxo1 and Noxal, although Nox3 is less dependent on these cofactors for activity. Plasma membrane targeting of Noxal depends on Noxo1, through tail-to-tail interactions between these proteins. Noxal can support Nox1 without Noxo1, when targeted to the plasma membrane by fusing membrane-binding sequences from Rac1 (amino acids 183 to 192) to the C terminus of Noxal. However, membrane targeting of Noxal is not sufficient for activation of Nox1. Both the Noxo1-independent and -dependent Nox1 systems involve Rac1, since they are affected by Rac1 mutants or Noxal mutants defective in Rac binding or short interfering RNA-mediated Rac1 silencing. Nox1 or Nox3 expression promotes p22(phox) transport to the plasma membrane, and both oxidases are inhibited by mutations in the p22(phox) binding sites (SH3 domains) of the Nox organizers (p47(phox) or Noxo1). Regulation of Nox3 by Rac1 was also evident from the effects of mutant Rac1 or mutant Nox3 activators (p67(phox) or Noxa1) or Rac1 silencing. In the absence of Nox organizers, the Nox activators (p67(phox) or Noxa1) colocalize with Rac1 within ruffling membranes, independently of their ability to bind Rac1. Thus, Rac1 regulates both oxidases through the Nox activators, although it does not appear to direct the subcellular localization of these activators.