Chemical Genetic Analysis of RAS Signaling

摘要 GT-RAS作为分子"开/关"开关发挥作用，存在于GDP结合（无活性）和GTP结合（无活性）中。 绑定表单（活动）。尽管作为一个简单的二进制开关，RAS是能够指导复杂的， 不同的细胞过程，包括增殖、迁移、存活和T细胞发育。最近的工作 表明RAS在不同过程中扮演复杂的、经常相互冲突的角色的能力来自于 细胞环境和/或其信号传导的亚细胞定位的差异。我们已经开发出一种新颖 化学遗传工具-称为RAS的化学诱导激活剂（CIAR）-研究RAS的动力学。 RAS活性介导的信号网络。CIAR允许快速和剂量依赖性激活 RAS信号转导与细胞可渗透的小分子。通过CIAR，我们建议使用有针对性的，定量的 磷酸蛋白质组学和转录组学研究RAS驱动的信号传导的基本动态行为。 将使用亚细胞定位的CIAR版本来确定局部RAS激活对细胞凋亡的影响。 RAS介导的信号传导的动力学。我们还将描述WT RAS和致癌性RAS之间的相互作用。 RAS信号传导和WT RAS介导的信号传导对致癌RAS直接抑制剂的贡献 变种人最后，我们将开发和使用一个化学控制的工具集，用于快速激活信号 酶寡聚体，这将用于剖析的作用，寡聚状态发挥RAS信号 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.