title: Identification of a conserved Rac-binding site on NADPH oxidases supports a direct GTPase regulatory mechanism.
abstract: The NADPH oxidases (Noxs) are a family of superoxide-generating enzymes implicated in a variety of biological processes. Full activity of Nox1, -2, and -3 requires the action of a Rac GTPase. A direct regulatory interaction of Rac with Nox2 has been proposed as part of a two-step mechanism for regulating electron transfer during superoxide formation. Using truncation analysis of Rac binding to the cytoplasmic tail of Nox2, along with peptides derived from this region in cell-free assays, we identify a Rac interaction site within amino acids 419-430 of Nox2. This region is required for binding Rac2 but not p47(phox) or p67(phox) cytosolic regulatory factors. A cell-permeant version of the peptide encompassing amino acids 419-430 specifically inhibits NADPH oxidase activation in intact human neutrophils. Mutational analysis of the putative Rac-binding site revealed specific residues, particularly Lys-421, Tyr-425, and Lys-426, individually required for Rac-dependent NADPH oxidase activity that are conserved in the Rac-regulated Nox1, Nox2, and Nox3 enzymes but not in Nox4 or Nox5. Mutation of the conserved residues in the Rac-binding site of Nox1 also result in the loss of Rac-dependent activity. Our data identify a functional Rac interaction site conserved in Rac-dependent Noxs and support a direct regulatory interaction of Rac GTPases to promote activation of these NADPH oxidases.
authors.affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Yu-Ya
authors.initials: YY
authors.lastname: Kao
authors.forename: Davide
authors.initials: D
authors.lastname: Gianni
authors.forename: Benjamin
authors.initials: B
authors.lastname: Bohl
authors.forename: Ross M
authors.initials: RM
authors.lastname: Taylor
authors.forename: Gary M
authors.initials: GM
authors.lastname: Bokoch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019255:NADPH Oxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D016415:Sequence Alignment; D020744:rac GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D019255:NADPH Oxidases; D020744:rac GTP-Binding Proteins