Abstract IA18: Activation of RAS by post-translational modification: Ubiquitination and thiol oxidation

Ras proteins are critical regulators of multiple pathways involved in cellular growth control. Activating mutations in Ras genes are found in 33% of human cancers, with Ras well recognized as the most commonly mutated oncogene in human cancer. Mutations in Ras associated with cancer promote cellular activation by populating the active state of Ras, most frequently by impairing the action of proteins that downregulate its activity. We have investigated novel mechanisms of Ras activation by posttranslational modification. One such modification is monoubiquitination. While polyubiquitination of proteins leads to protein degradation, reversible covalent modification by monoubiquitination can lead to alterations in trafficking and subcellular localization. It has recently been shown that K-Ras monoubiquitination, upregulates its activity by populating the active GTP-bound state, enhances interactions with certain downstream effectors and promotes Ras-mediated tumorigenesis [1]. These findings represent a paradigm shift in our understanding of the role monoubiquitination may play in protein regulation as well as new mechanisms for regulation of Ras activity. To determine the mechanism by which ubiquitination activates Ras, we conducted NMR, modeling, biochemical and biological studies of monoubiquitinated H-Ras and K-Ras. We find that monoubiquitination of K- and H-Ras occurs at distinct sites, both of which can upregulate Ras activity, but by different mechanisms [2-4]. We will also present our recent work on an oncogenic variant prevalent in lung cancer, K-Ras G12C. We find that this activating mutation introduces a redox active cysteine that renders Ras G12C sensitive to thiol oxidation. We will present recent findings on how thiol oxidation at this site modulates Ras activity. 1. Ubiquitination of K-Ras enhances activation and facilitates binding to select downstream effectors. Sasaki AT, Carracedo A, Locasale JW, Anastasiou D, Takeuchi K, Kahoud ER, Haviv S, Asara JM, Pandolfi PP, Cantley LC. Sci Signal. 2011 4(163):ra13. 2. Site-Specific Monoubiquitination Activates Ras by Impeding GTPase Activating Protein Function. Baker R, Lewis SM, Wilkerson EM, Sasaki AT, Cantley LC, Kuhlman B, Dohlman H and Campbell, SL. Nat Struct Mol Biol. 2013 Jan;20(1):46-52. 3. Differences in the regulation of k-ras and h-ras isoforms by monoubiquitination.Baker R, Wilkerson EM, Sumita K, Isom DG, Sasaki AT, Dohlman HG, Campbell SL.J Biol Chem. 2013 Dec 27;288(52):36856-62. 4.Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function. Hobbs GA, Gunawardena HP, Baker R, Campbell SL. Small GTPases. 2013 Sep 12;4(3). Citation Format: Rachael Baker, Aaron Hobbs, Minh Huynh, Atsuo Sasaki, Henrik Dohlman, Sharon L. Campbell. Activation of RAS by post-translational modification: Ubiquitination and thiol oxidation. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA18. doi: 10.1158/1557-3125.RASONC14-IA18