Mechanism of p21Ras S-nitrosylation and kinetics of nitric oxide-mediated guanine nucleotide exchange.
Nitric oxide (NO), a highly reactive redox molecule, can react with protein thiols and protein metal centers to regulate a multitude of physiological processes. NO has been shown to promote guanine nucleotide exchange on the critical cellular signaling protein p21Ras (Ras) by S-nitrosylation of a redox-active thiol group (Cys(118)). This increases cellular Ras-GTP levels in vivo, leading to activation of downstream signaling pathways. Yet the process by which this occurs is not clear. Although several feasible mechanisms for protein S-nitrosylation with NO and NO donating have been proposed, results obtained from our studies suggest that Ras can be S-nitrosylated by direct reaction of Cys(118) with nitrogen dioxide (*NO(2)), a reaction product of NO with O(2), via a Ras thiyl-radical intermediate (Ras-S*). Results from our studies also indicate that Ras Cys(118) can be S-nitrosylated by direct reaction of Cys(118) with a glutathionyl radical (GS*), a reaction product derived from homolytic cleavage of S-nitrosoglutathione (GSNO). Moreover, we present evidence that reaction of GS* with Ras generates a Ras-S* intermediate during GSNO-mediated Ras S-nitrosylation. The Ras-S(*) radical intermediate formed from reaction of the Ras thiol with either *NO(2) or GS*, in turn, reacts with NO to complete Ras S-nitrosylation. NO and GSNO modulate Ras activity by promoting guanine nucleotide dissociation from Ras. Our results suggest that formation of the Ras radical intermediate, Ras-S*, may perturb interactions between Ras and its guanine nucleotide substrate, resulting in enhancement of guanine nucleotide dissociation from Ras.
Heo
Jongyun
J
Department of Biochemistry and Biophysics, University of North Carolina, 530 Mary Ellen Jones Building, Chapel Hill, North Carolina 27599-7260, USA.
Campbell
Sharon L
SL
D006150:Guanine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000737:chemistry / Q000378:metabolism; D009603:Nitroso Compounds / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000378:metabolism*; D026422:S-Nitrosoglutathione / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
D006150:Guanine Nucleotides; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D011506:Proteins; D013438:Sulfhydryl Compounds; D006153:Guanosine Diphosphate; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D006160:Guanosine Triphosphate; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)