Chemical Genetic Analysis of RAS Signaling

摘要 GTP酶RAS是一种分子“开/关”开关，存在于GDP结合型(非活性)和GTP- 装订表单(活动)。尽管RAS起着简单的二元开关的作用，但它能够引导复杂的和 不同的细胞过程，包括增殖、迁移、生存和T细胞发展。近期工作 这表明，RAS在不同的过程中扮演复杂的、往往是相互冲突的角色的能力源于 细胞背景和/或其信号的亚细胞定位的差异。我们开发了一部小说 化学遗传学工具--称为RAS的化学诱导激活剂(CIAR)--用于研究 由RAS活动介导的信号网络。Ciar允许快速且依赖于剂量的激活 RAS信号与细胞通透性小分子。对于Ciar，我们建议使用有针对性的、量化的 磷酸蛋白质组学和转录组学，以研究RAS驱动的信号的基本动态行为。 Ciar的亚细胞本地化版本将用于确定本地化RAS激活对 RAS介导的信号传递动力学。我们还将描述WT RAS和致癌基因之间的相互作用 RAS信号及WT-RAS介导的信号对直接抑制致癌RAS的作用 变种人。最后，我们将开发和使用一个化学控制的工具包，用于快速激活信号 酶低聚物，将用于剖析齐聚状态在RAS信号转导中所起的作用 动力学。

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.