Dual Role of Rac in the Assembly of NADPH Oxidase, Tethering to the Membrane and Activation of p67
| Autor: | Carolyn Weinbaum, Yara Gorzalczany, Ariel Mizrahi, Miriam Hirshberg, Shahar Molshanski-Mor, Rive Sarfstein, Edgar Pick, Marie-Claire Dagher, Yevgeny Berdichevsky |
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| Rok vydání: | 2004 |
| Předmět: |
0303 health sciences
Conformational change Oxidase test NADPH oxidase C-terminus 030302 biochemistry & molecular biology Cytochrome b559 RAC1 Cell Biology Biology Biochemistry 03 medical and health sciences Chimera (genetics) Biophysics biology.protein Moiety Molecular Biology 030304 developmental biology |
| Zdroj: | Journal of Biological Chemistry. 279:16007-16016 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m312394200 |
| Popis: | 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 anamphiphile-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. |
| Databáze: | OpenAIRE |
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