title: 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 (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 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.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260.
authors.forename: G Aaron
authors.initials: GA
authors.lastname: Hobbs
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Mitchell
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260; Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290.
authors.forename: Megan E
authors.initials: ME
authors.lastname: Arrington
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260; Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Harsha P
authors.initials: HP
authors.lastname: Gunawardena
authors.affiliation: Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280; Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599-7365.
authors.forename: Molly J
authors.initials: MJ
authors.lastname: DeCristo
authors.affiliation: Department of Medicine and the Thurston Arthritis Research Center, University of North Carolina, Chapel Hill, NC 27599-7280.
authors.forename: Richard F
authors.initials: RF
authors.lastname: Loeser
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260; Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Xian
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599-7365; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295; Department of Radiation Oncology, University of North Carolina, Chapel Hill, NC 27599-7512.
authors.forename: Adrienne D
authors.initials: AD
authors.lastname: Cox
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295. Electronic address: campbesl@med.unc.edu.
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Campbell
mesh_terms: D000595:Amino Acid Sequence; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D005978:Glutathione / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D055550:Protein Stability; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
chemical_list: C107953:RAC1 protein, human; D013438:Sulfhydryl Compounds; D020830:rac1 GTP-Binding Protein; D005978:Glutathione
keywords: Cysteine oxidation; Free radicals; Glutathiolation; Rac1 GTPases; Reactive nitrogen species; Reactive oxygen species