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

被引:109
作者
Sarfstein, R
Gorzalczany, Y
Mizrahi, A
Berdichevsky, Y
Molshanski-Mor, S
Weinbaum, C
Hirshberg, M
Dagher, MC
Pick, E [1 ]
机构
[1] Tel Aviv Univ, Sackler Sch Med, Dept Human Microbiol, Julius Friedrich Cohnheim Minerva Ctr Phagocyte R, IL-69978 Tel Aviv, Israel
[2] Tel Aviv Univ, Sackler Sch Med, Ela Kodesz Inst Host Def Infect Dis, IL-69978 Tel Aviv, Israel
[3] Duke Univ, Med Ctr, Dept Pharmacol & Canc Biol, Durham, NC 27710 USA
[4] Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England
[5] CEA Grenoble, Lab DBMS BBSI, F-38054 Grenoble 9, France
关键词
D O I
10.1074/jbc.M312394200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
NADPH oxidase activation involves the assembly of membrane-localized cytochrome b(559) with the cytosolic components p47(phox), p67(phox), 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 p67(phox) and full-lengthRac1, activates the oxidase in vitro in an amphiphile-dependent manner, and when prenylated, in the absence of amphiphile and p47phox. We subjected chimera p67(phox)-(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 p67(phox) moiety leads to a reduction in activity. 3) Mutating residues, known to participate in the interaction between free p67(phox) and Rac1, in the p67(phox)( 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 p67(phox). This demonstrates that Rac has a dual role in the assembly of NADPH oxidase. One is to tether p67(phox) to the membrane; the other is to induce an "activating" conformational change in p67(phox).
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收藏
页码:16007 / 16016
页数:10
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