Developing a Chemically-Controlled RAS Toolset

Project Abstract RAS GTPase operates as a molecular switch toggling between GDP-bound (inactive) and GTP-bound forms (active), and orchestrates dynamic cellular processes such as proliferation, migration, survival, and T-cell development.1,2 Despite its binary nature, RAS exhibits sophisticated and dynamic signaling behavior influenced by cellular context and subcellular localization. The lack of a precise understanding of the roles of spatiotemporal compartmentalization in RAS hinders our understanding of fundamental signaling mechanisms and limits our ability to develop targeted therapies for RAS-driven cancers. To address this, we will leverage a chemical genetic tool called Chemically-Inducible Activator of RAS (CIAR) that allows the rapid and dose-dependent activation of wild-type RAS with bio-orthogonal small molecules.8-10 Aim 1 describes efforts to use transmembrane-tethered versions of NS3a-CIAR to dissect the impact of differential membrane localization on wild-type RAS activation and downstream signaling. This Aim also describes the development of chemical tools for quantitatively measuring the transmembrane localization of proteins and using a novel tool for probing RAS-GTP in its native cellular context. Aim 2 aims to investigate the role of oligomerization of signaling proteins at membranes. An engineered protein toolkit will be used to control the oligomeric state and study its impact on Ras-mediated signaling. Finally, Aim 3 explores efforts to dissect how the intracellular signaling environment affects the kinetics of magnitude of RAS activation and downstream signaling. This Aim also explores the development of a number of chemical genetic tools for enabling intracellular protein display at membranes. Overall, the studies described herein will offer new and important mechanistic insight into RAS signaling.