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.

Grant Number: 5F31GM155953-02
NIH Institute/Center: NIH
Principal Investigator: Fernando Banales Mejia