title: A prenylated p67phox-Rac1 chimera elicits NADPH-dependent superoxide production by phagocyte membranes in the absence of an activator and of p47phox: conversion of a pagan NADPH oxidase to monotheism.
abstract: Activation of the superoxide-generating NADPH oxidase of phagocytes is the result of the assembly of a membrane-localized flavocytochrome (cytochrome b(559)) with the cytosolic components p47(phox), p67(phox), and the small GTPase Rac. Activation can be reproduced in an in vitro system in which cytochrome b(559)-containing membranes are mixed with cytosolic components in the presence of an anionic amphiphile. We proposed that the essential event in activation is the interaction between p67(phox) and cytochrome b(559) and that Rac and p47(phox) serve as carriers for p67(phox) to the membrane. When prenylated, Rac can fulfill the carrier function by itself, supporting oxidase activation by p67(phox) in the absence of p47(phox) and amphiphile. We now show that a single chimeric protein, consisting of residues 1-212 of p67(phox) and full-length Rac1 (residues 1-192), prenylated in vitro and exchanged to GTP, becomes a potent oxidase activator in the absence of any other component or stimulus. Oxidase activation by prenylated chimera p67(phox) (1-212)-Rac1 (1-192) is accompanied by its spontaneous association with membranes. Prenylated chimeras p67(phox) (1-212)-Rac1 (178-192) and p67(phox) (1-212)-Rac1 (189-192), containing specific C-terminal regions of Rac1, are inactive; the activity of the first but not of the second chimera can be rescued by supplementation with exogenous nonprenylated Rac1-GTP. An analysis of prenylated p67(phox)-Rac1 chimeras suggests that the basic requirements for oxidase activation are: (i) a "two signals" membrane-localizing motif present in Rac, comprising the prenyl group and a C-terminal polybasic sequence and (ii) an intrachimeric or extrachimeric protein-protein interaction between p67(phox) and Rac1, causing a conformational change in the "activation domain" in p67(phox).
authors.affiliation: Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Yara
authors.initials: Y
authors.lastname: Gorzalczany
authors.forename: Nathalie
authors.initials: N
authors.lastname: Alloul
authors.forename: Natalia
authors.initials: N
authors.lastname: Sigal
authors.forename: Carolyn
authors.initials: C
authors.lastname: Weinbaum
authors.forename: Edgar
authors.initials: E
authors.lastname: Pick
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D006168:Guinea Pigs; D009249:NADP / Q000378:metabolism*; D010586:Phagocytes / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D017368:Protein Prenylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D009249:NADP; D020830:rac1 GTP-Binding Protein