Redox regulation of Rac1 by thiol oxidation.

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 p K a . 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 Rac1 C18D 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. [ABSTRACT FROM AUTHOR]