Redox regulation of human Rac1 stability by the proteasome in human aortic endothelial cells.
Rac1 has been shown to activate a NADPH oxidase complex producing superoxide anions in a variety of mammalian cell types. We evaluated the impact of Rac1-induced reactive oxygen species production on the turnover of Rac1 itself in human aortic endothelial cells. The concentration of a constitutively active mutant of Rac1 (Rac1(V12)) was increased by treatment of the cells with diphenylene iodinium (DPI), an inhibitor of the NADPH oxidase. Such an effect was not observed for the dominant negative form of Rac1 (Rac1(N17)). We showed a decrease in proteolytic degradation of Rac1(V12) in the presence of DPI, and showed that short term treatment with H(2)O(2) reverses the effect of DPI. We found that proteasome inhibitors (lactacystin and MG132) increased Rac1(V12) protein level. In support of this finding, we have identified in the primary sequence of Rac1 a potential destruction box domain, which is known to be a signal for protein degradation mediated by the ubiquitin/proteasome system. We show that Rac1(V12) is ubiquitinated before degradation. By contrast Rac1(N17) induces an accumulation of the ubiquitinated form of Rac1. These results suggest that Rac1 activation of NADPH oxidase is necessary for the proteolytic degradation of Rac1 itself.
Kovacic
H N
HN
UMR CNRS 6032, Faculté de Pharmacie, Université de la Méditerranée, 13385 Marseille, France. kovacic@pharmacie.univ-mrs.fr
Irani
K
K
Goldschmidt-Clermont
P J
PJ
D000975:Antioxidants / Q000494:pharmacology; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D009097:Multienzyme Complexes / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D046988:Proteasome Endopeptidase Complex; D017382:Reactive Oxygen Species; D025801:Ubiquitin / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D000975:Antioxidants; D017931:DNA Primers; D009097:Multienzyme Complexes; D016877:Oxidants; D017382:Reactive Oxygen Species; D025801:Ubiquitin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D020830:rac1 GTP-Binding Protein