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来研究RAS驱动的信号传导的基本动态行为。 亚细胞定位版本的CIAR也将用于确定局部RAS激活的影响， RAS介导的信号动力学。最后，我们将探讨积极和消极的反馈， RAS驱动的信号通路通过选择性增强或抑制下游信号组分。

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 also be used to determine the effects of localized RAS activation on the dynamics of RAS-mediated signaling. Finally, we will explore positive and negative feedback within RAS-driven signaling pathways by selectively enhancing or inhibiting downstream signaling components.