title: Dual role of Rac in the assembly of NADPH oxidase, tethering to the membrane and activation of p67phox: a study based on mutagenesis of p67phox-Rac1 chimeras.
abstract: NADPH oxidase activation involves the assembly of membrane-localized cytochrome b559 with the cytosolic components p47phox, p67phox, and the small GTPase Rac. Assembly is mimicked by a cell-free system consisting of membranes and cytosolic components, activated by an anionic amphiphile. We reported that a chimeric construct, consisting of residues 1-212 of p67phox and full-length Rac1, activates the oxidase in vitro in an amphiphile-dependent manner, and when prenylated, in the absence of amphiphile and p47phox. We subjected chimera p67phox-(1-212)-Rac1 to mutational analysis and found that: 1) replacement of a single basic residue at the C terminus of the Rac1 moiety by glutamine is sufficient for loss of activity by the non-prenylated chimera; replacement of all six basic residues by glutamines is required for loss of activity by the prenylated chimera. 2) A V204A mutation in the activation domain of the p67phox moiety leads to a reduction in activity. 3) Mutating residues, known to participate in the interaction between free p67phox and Rac1, in the p67phox-(R102E) or Rac1 (A27K, G30S) moieties of the chimera, leads to a marked decrease in activity, indicating a requirement for intrachimeric bonds, in addition to the engineered fusion. 4) Chimeras, inactive because of mutations A27K or G30S in the Rac1 moiety, are reactivated by supplementation with exogenous Rac1-GTP but not with exogenous p67phox. This demonstrates that Rac has a dual role in the assembly of NADPH oxidase. One is to tether p67phox to the membrane; the other is to induce an "activating" conformational change in p67phox.
authors.affiliation: Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research and the Ela Kodesz Institute of Host Defense against Infectious Diseases, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Rive
authors.initials: R
authors.lastname: Sarfstein
authors.forename: Yara
authors.initials: Y
authors.lastname: Gorzalczany
authors.forename: Ariel
authors.initials: A
authors.lastname: Mizrahi
authors.forename: Yevgeny
authors.initials: Y
authors.lastname: Berdichevsky
authors.forename: Shahar
authors.initials: S
authors.lastname: Molshanski-Mor
authors.forename: Carolyn
authors.initials: C
authors.lastname: Weinbaum
authors.forename: Miriam
authors.initials: M
authors.lastname: Hirshberg
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Dagher
authors.forename: Edgar
authors.initials: E
authors.lastname: Pick
mesh_terms: D000818:Animals; D002474:Cell-Free System; D004252:DNA Mutational Analysis; D004789:Enzyme Activation / Q000235:genetics; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C079789:neutrophil cytosol factor 67K; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein