Chemical Genetic Analysis of RAS Signaling

摘要

Abstract The GTPase RAS functions as a molecular “on/off” switch, existing both in GDP-bound (inactive) and GTP- bound forms (active). Despite functioning as a simple binary switch, RAS is capable of directing complex and diverse cellular processes, including proliferation, migration, survival, and T-cell development. Recent work suggests that the ability of RAS to play complex, often conflicting roles in diverse processes results from differences in cellular context and and/or subcellular localization of its signaling. We have developed a novel chemical genetic tool–called Chemically-Inducible Activator of RAS (CIAR)–to study the dynamics of the signaling networks that are mediated by RAS activity. CIAR allows rapid and dose-dependent activation of RAS signaling with a cell permeable small molecule. With CIAR, we propose to use targeted, quantitative phosphoproteomics and transcriptomics to study the fundamental dynamic behavior of RAS-driven signaling. Subcellularly-localized versions of CIAR will be used to determine the effects of localized RAS activation on the dynamics of RAS-mediated signaling. We will also delineate the interplay between WT RAS and oncogenic RAS signaling and the contribution of WT RAS-mediated signaling to direct inhibitors of oncogenic RAS mutants. Finally, we will develop and use a chemically-controlled toolset for the rapid activation of signaling enzyme oligomers, which will be used to dissect the role that oligomerization state plays in RAS signaling dynamics.