RAS EFFECTOR-HOMOLOG REGION ON RAC REGULATES PROTEIN ASSOCIATIONS IN THE NEUTROPHIL RESPIRATORY BURST OXIDASE COMPLEX

Rac, a small molecular weight GTPase in the Ras superfamily, participa tes in the activation of the multicomponent superoxide-generating NADP H oxidase of human neutrophils. Rac is 30% identical to Ras overall, b ut is 75% identical within the sequence corresponding to the effector region of Ras, which regulates mitogenesis through interactions with t he protein kinase Raf1. We investigated the role of this region in Rac 1 using site-directed mutagenesis. In a cell-free semirecombinant NADP H oxidase system, mutants in the 26, 33, 38, and 45 amino acids showed 20-110-fold reduced binding to the oxidase complex as judged by EC(50 ) values and reduced (44-80%) maximal activities in superoxide generat ion. Only the GTP gamma S-bound form associated, since the GDP-bound f orm of Rac neither activated alone nor competed with GTP gamma S-Rac. EC(50) values for neither p47-phox nor p67-phox were affected when mut ant Racs were used in place of Rac. Data indicate direct binding of th e Rac effector region to one or more components of the respiratory bur st oxidase, Results indicate a general role for conserved effector-equ ivalent regions in small GTPases in the regulation of protein-protein interactions.