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Mechanism of p21Ras S-Nitrosylation and Kinetics of Nitric Oxide-Mediated Guanine Nucleotide Exchange

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journal contribution
posted on 02.03.2004, 00:00 by Jongyun Heo, Sharon L. Campbell
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 (Cys118). 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 Cys118 with nitrogen dioxide (NO2), a reaction product of NO with O2, via a Ras thiyl-radical intermediate (Ras-S). Results from our studies also indicate that Ras Cys118 can be S-nitrosylated by direct reaction of Cys118 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 NO2 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.