Redox regulation of Rac1 by thiol oxidation

Abstract

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 (Cys18) 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 Cys18 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 Rac1C18D. We also evaluated Rac1C18S as a redox-insensitive variant and found that it retains structural and biochemical properties similar to those of Rac1WT but is resistant to thiol oxidation. In addition, Rac1C18D, but not Rac1C18S, shows greatly enhanced nucleotide exchange, similar to that observed for Rac1 oxidation by glutathione. We employed Rac1C18D in cell-based studies to assess whether this fast-cycling variant, which mimics Rac1 oxidation by glutathione, affects Rac1 activity and function. Expression of Rac1C18D in Swiss 3T3 cells showed greatly enhanced GTP-bound Rac1 relative to Rac1WT and the redox-insensitive Rac1C18S variant. Moreover, expression of Rac1C18D in HEK-293T cells greatly promoted lamellipodia formation. Our results suggest that Rac1 oxidation at Cys18 is a novel posttranslational modification that upregulates Rac1 activity.

Metabolic rewiring in melanoma

“…The inability of antioxidants to induce migratory and invasive phenotypes in the presence of BSO indicates that glutathione biosynthesis, not just GSSG reduction, is necessary for the effect. Changes in redox status also reportedly alter nucleotide binding by the RHO family of GTP-ases 45, 46, 79 . GTP loading of RHOA but not RAC1 increases consistently following treatment with antioxidants like NAC and the vitamin E analog Trolox.…”

Antioxidants can increase melanoma metastasis in mice

“…S4 and table S1). RHO family proteins RHOA and RAC1 mediate cytoskeletal changes during cell migration and invasion, and recent studies indicate that they are regulated by the cell's redox status through direct modification of residues involved in nucleotide binding (16)(17)(18). We therefore determined whether antioxidants affect the activation of RHOA and RAC1.…”

Redox regulation of the actin cytoskeleton and its role in the vascular system

“…Such a scenario is the case for Rac1, which has been shown to be activated by ROS/RNS both in vitro [138, 140] and in cellulo [139–142]. Early work by Heo and colleages [138] showed that the dramatic increase in Rac1 nucleotide dissociation induced by O 2 •− in vitro has no effect on a Rac1 C18S mutant, suggesting that activation of Rac1 is due to direct oxidation at Cys18.…”