Superoxide anion radical modulates the activity of Ras and Ras-related GTPases by a radical-based mechanism similar to that of nitric oxide

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. The activity of Ras is up-regulated by cellular agents, including both protein ( guanine nucleotide exchange factors) and redox-active agents ( nitric oxide ( NO) and superoxide anion radical (O-2(.-))). We have recently elucidated the mechanism by which NO promotes guanine nucleotide dissociation of redox-active NKCD motif-containing Ras and Ras-related GTPases. In this study, we show that guanine nucleotide dissociation is enhanced upon exposure of the redox-active GTPases, Ras and Rap1A, to O-2(.-) and provide evidence for the efficient guanine nucleotide reassociation in the presence of the radical quenching agent ascorbate to complete guanine nucleotide exchange. In vivo, guanine nucleotide reassociation is necessary to populate Ras in its biologically active GTP-bound form after the dissociation of GDP. We further show that treatment of the redox-active GTPases with O-2(.-) releases GDP in the form of an unstable oxygenated GDP adduct, putatively assigned as 5-oxo-GDP. 5-Oxo-GDP was not produced from either the C118S or the F28L Ras variants upon the treatment of O-2(.-), supporting the involvement of residues Cys118 and Phe(28) in O-2(.-)-mediated Ras guanine nucleotide dissociation. These results indicate that the mechanism of O2.- mediated Ras guanine nucleotide dissociation is similar to that of NO/O-2-mediated Ras guanine nucleotide dissociation.