Nox1-dependent Reactive Oxygen Generation Is Regulated

, is activated by Rac1. When Nox1 is co-expressed alongwith its regulatory subunits NOXO1 and NOXA1, significant ROSgeneration is seen. Herein, co-expression of constitutively activeRac1(G12V), but not wild-type Rac1, resulted in marked furtherstimulation of activity. Decreased Rac1 expression using smallinterfering RNA reduced Nox1-dependent ROS. CDC42(G12V)failed to increase activity, and small interfering RNA directedagainstCDC42failedtodecreaseactivity,pointingtospecificityforRac.TPRdomainmutantsofNOXA1thatinterferewithRac1bind-ing were ineffective in supporting Nox1-dependent ROS generation.Immunoprecipitation experiments demonstrated a complex contain-ing Rac1(G12V), NOXO1, NOXA1, and Nox1. CDC42(G12V) couldnotsubstituteforRac1(G12V)insuchacomplex.Nox1formedacom-plexwithRac1(G12V)thatwasindependentofNOXA1andNOXO1,consistent with direct binding of Rac1(G12V) to Nox1. Rac1(G12V)interactionwithNOXA1wasenhancedbyNox1andNOXO1,suggest-ingcooperativebinding.AmodelispresentedcomparingactivationbyregulatorysubunitsofNox1