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 RasG12C.



Given that Cys12of RasG12Cis accessible to thiol alkylating agents and forms interactions within the electrostatic phosphoryl-binding loop of Ras, we postulated that Cys12may 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 RasG12Calters its activity and structure in vitro. We also determined the biological effects of increasing NO production on the tumorigenic growth of cells transformed by RasG12C.



We found that Cys12has 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 eNOSS1177Dand RasG12Caccelerated tumorigenic growth of human and murine cell line xenografts.



Modification of Cys12in mutant, oncogenic RasG12Cmay promote its tumorigenic activity.
Duke University Medical Center, Durham, USA.
Matthew
M
Crowe
D000818:Animals; D045744:Cell Line, Tumor; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D009363:Neoplasm Proteins; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D018631:ras Proteins; D003545:Cysteine