Ras effector-homologue region on Rac regulates protein associations in the neutrophil respiratory burst oxidase complex.
Rac, a small molecular weight GTPase in the Ras superfamily, participates in the activation of the multicomponent superoxide-generating NADPH oxidase of human neutrophils. Rac is 30% identical to Ras overall, but is 75% identical within the sequence corresponding to the effector region of Ras, which regulates mitogenesis through interactions with the protein kinase Raf1. We investigated the role of this region in Rac1 using site-directed mutagenesis. In a cell-free semirecombinant NADPH 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 EC50 values and reduced (44-80%) maximal activities in superoxide generation. Only the GTP gamma S-bound form associated, since the GDP-bound form of Rac neither activated alone nor competed with GTP gamma S-Rac. EC50 values for neither p47-phox nor p67-phox were affected when mutant Racs were used in place of Rac. Data indicate direct binding of the Rac effector region to one or more components of the respiratory burst oxidase. Results indicate a general role for conserved effector-equivalent regions in small GTPases in the regulation of protein-protein interactions.
Freeman
J L
JL
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322.
Kreck
M L
ML
Uhlinger
D J
DJ
Lambeth
J D
JD
D000595:Amino Acid Sequence; D019204:GTP-Binding Proteins / Q000235:genetics*; D011905:Genes, ras; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology; D016897:Respiratory Burst; D013481:Superoxides / Q000378:metabolism; D020744:rac GTP-Binding Proteins
D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins