Redox regulation of Rac1 by thiol oxidation

被引:30
作者
Hobbs, G. Aaron [1 ]
Mitchell, Lauren E. [1 ]
Arrington, Megan E. [1 ,2 ]
Gunawardena, Harsha P. [1 ,3 ]
DeCristo, Molly J. [4 ,5 ]
Loeser, Richard F. [6 ,7 ]
Chen, Xian [1 ,3 ]
Cox, Adrienne D. [5 ,8 ,9 ]
Campbell, Sharon L. [1 ,8 ]
机构
[1] Univ N Carolina, Dept Biochem & Biophys, Chapel Hill, NC 27599 USA
[2] Univ N Carolina, Dept Chem, Chapel Hill, NC 27599 USA
[3] Univ N Carolina, Program Mol Biol & Biotechnol, Chapel Hill, NC 27599 USA
[4] Univ N Carolina, Dept Biol, Chapel Hill, NC 27599 USA
[5] Univ N Carolina, Dept Pharmacol, Chapel Hill, NC 27599 USA
[6] Univ N Carolina, Dept Med, Chapel Hill, NC 27599 USA
[7] Univ N Carolina, Thurston Arthrit Res Ctr, Chapel Hill, NC 27599 USA
[8] Univ N Carolina, Lineberger Comprehens Canc Ctr, Chapel Hill, NC 27599 USA
[9] Univ N Carolina, Dept Radiat Oncol, Chapel Hill, NC 27599 USA
基金
美国国家卫生研究院;
关键词
Rac1; GTPases; Reactive oxygen species; Reactive nitrogen species; Glutathiolation; Cysteine oxidation; Free radicals; GUANINE-NUCLEOTIDE EXCHANGE; ACTIN STRESS FIBERS; NF-KAPPA-B; NITRIC-OXIDE; REACTIVE OXYGEN; RHO-GTPASES; NADPH OXIDASE; S-GLUTATHIOLATION; PROTEIN; RAS;
D O I
10.1016/j.freeradbiomed.2014.09.027
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
070307 [化学生物学]; 071010 [生物化学与分子生物学];
摘要
The Rac1 GTPase is an essential and ubiquitous protein that signals through numerous pathways to control critical cellular processes, including cell growth, morphology, and motility. Rac1 deletion is embryonic lethal, and its dysregulation or mutation can promote cancer, arthritis, cardiovascular disease, and neurological disorders. Rac1 activity is highly regulated by modulatory proteins and posttranslational modifications. Whereas much attention has been devoted to guanine nucleotide exchange factors that act on Rac1 to promote GTP loading and Rac1 activation, cellular oxidants may also regulate Rac1 activation by promoting guanine nucleotide exchange. Herein, we show that Rac1 contains a redox-sensitive cysteine (Cys(18)) that can be selectively oxidized at physiological pH because of its lowered pKa. Consistent with these observations, we show that Rac1 is glutathiolated in primary chondrocytes. Oxidation of Cys(18) by glutathione greatly perturbs Rac1 guanine nucleotide binding and promotes nucleotide exchange. As aspartate substitutions have been previously used to mimic cysteine oxidation, we characterized the biochemical properties of Rac1(C18D). We also evaluated Rac1(C18S) as a redox-insensitive variant and found that it retains structural and biochemical properties similar to those of Rac1(WT) but is resistant to thiol oxidation. In addition, Rac1(C18D), but not Rac1(C18S), shows greatly enhanced nucleotide exchange, similar to that observed for Rac1 oxidation by glutathione. We employed Rac1(C18D) in cell-based studies to assess whether this fast-cycling variant, which mimics Rac1 oxidation by glutathione, affects Rac1 activity and function. Expression of Rac1(C18D) in Swiss 3T3 cells showed greatly enhanced GTP-bound Rac1 relative to Rac1(WT) and the redox-insensitive Rac1(C18S) variant. Moreover, expression of Rac1C18D in HEK-293T cells greatly promoted lamellipodia formation. Our results suggest that Rac1 oxidation at Cys(18) is a novel posttranslational modification that upregulates Rac1 activity. (C) 2014 Elsevier Inc. All rights reserved.
引用
收藏
页码:237 / 250
页数:14
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