Ras regulation by reactive oxygen and nitrogen species

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. We have previously shown that both NO/O-2 (via nitrogen dioxide, (NO2)-N-.) and superoxide radical anion (O-2(.-)) promote Ras guanine nucleotide dissociation. We now show that hydrogen peroxide in the presence of transition metals (i.e., H2O2/transition metals) and peroxynitrite also trigger radical-based Ras guanine nucleotide dissociation. The primary redox-active reaction species derived from H2O2/transition metals and peroxynitrite is O-2(.-) and (NO2)-N-., respectively. A small fraction of hydroxyl radical (OH.) is also present in both. We also show that both carbonate radical (CO3.-) and (NO2)-N-., derived from the mixture of peroxynitrite and bicarbonate, facilitate Ras guanine nucleotide dissociation. We further demonstrate that NO/O-2 and O-2(.-) promote Ras GDP exchange with GTP in the presence of a radical-quenching agent, ascorbate, or NO, and generation of Ras-GTP promotes high-affinity binding of the Ras-binding domain of Raf-1, a downstream effector of Ras. S-Nitrosylated Ras (Ras-SNO) can be formed when NO serves as a radical-quenching agent, and hydroxyl radical but not (NO2)-N-. or O-2(.-) can further react with Ras-SNO to modulate Ras activity in vitro. However, given the lack of redox specificity associated with the high redox potential of OH., it is unclear whether this reaction occurs under physiological conditions.