Biochemical And Tumorigenic Effects Of Redox Modification Of Ras-G12c By Nitric Oxide

The Ras family of small GTPases cycle between an inactive, GDP-bound state and an active, GTP-bound state. When bound to GTP, Ras engages and activates a number of effectors that mediate proliferative and survival signals. Ras is mutated in over 30% of human cancers, usually at codons 12, 13, or 61, to remain in this active, GTP-bound state, which promotes tumorigenesis. One of these oncogenic mutations that commonly occurs in lung cancer is G12C. Recently, it was shown that alkylating agents that react with the thiol functional group of this mutant amino acid can inactivate oncogenic Ras^G12C.

Given that Cys¹² of Ras^G12C is accessible to thiol alkylating agents and forms interactions within the electrostatic phosphoryl-binding loop of Ras, we postulated that Cys¹² may possess an altered pKa, potentially allowing this residue to be modified by NO and other cellular oxidants.

We conducted several biochemical analyses to determine whether nitrosylation of Ras^G12C alters its activity and structure in vitro. We also determined the biological effects of increasing NO production on the tumorigenic growth of cells transformed by Ras^G12C.

We found that Cys¹² has a depressed pKa of 7.4, which increases the susceptibility of the thiol to modification by oxidation or nitrosylation at physiological pH. We also found that coexpressing active eNOS^S1177D and Ras^G12C accelerated tumorigenic growth of human and murine cell line xenografts.

Modification of Cys¹² in mutant, oncogenic Ras^G12C may promote its tumorigenic activity.