Molecular mechanisms of ZAK- and GCN2-mediated signaling in response to ribotoxic stress

项目总结/摘要 细胞识别和响应外在和内在扰动的能力对于它们的生长至关重要。 生存最近的研究表明，翻译核糖体的状态是不断监测， 监测因素，以确定翻译困扰的迹象。例如，细胞暴露在紫外线下 辐射和活性氧导致转录组范围的RNA损伤和细胞周期的延长停滞。 受损信使RNA编码序列中的核糖体引发核糖体碰撞。最近我们 实验室已经表明，促分裂原活化蛋白激酶ZAK，和一般对照 非去阻遏蛋白2（GCN 2）激酶在碰撞的核糖体上被激活，以触发核糖毒性蛋白的激活， 综合应激反应程序（RSR和ISR）分别。 我的建议的目标是了解细胞如何安装测量的ZAK和GCN 2介导的 依赖于核糖体碰撞的普遍性来决定细胞命运的反应。初步 实验中，我进行了定量的时间分辨磷酸蛋白质组学，以确定时间顺序 在响应核糖毒性应激的细胞中激活信号级联。由此产生的数据提供了一个 前所未有的高分辨率的信号事件发生在响应核糖体碰撞的图谱，并将 进一步探讨了两个拟议的目标。在目标1中，我将确定ZAK是如何响应核糖体激活的。 碰撞并通过激活p38和JNK信号级联调节细胞命运结果。我也会 测试ZAK的负反馈回路如何使细胞对核糖毒性应激产生可测量的反应。 这些研究将采用生物化学、定量蛋白质组学和活细胞成像方法。在目标2中，我 将确定GCN 2如何调节ISR通路，以及JNK和mTOR活性以响应核糖毒性应激 通过遗传和化学干扰GCN 2的激酶活性。最后，我将测试辅助因子和调节因子 GCN 2的表达为它在核糖体碰撞时的激活设定了阈值。在一起，成功 完成这些互补的方法将提供基本的机械的见解，如何RSR 和ISR途径由ZAK和GCN 2协调以调节核糖毒性应激期间的细胞命运决定。 为此，建议的实验将使我能够开发新的技能和技术， 定量蛋白质组学和活细胞成像方法，这将使我能够充分区分我的 我的导师绿色博士的研究项目。我相信我的合作者团队 已经组装和伴随的培训计划，将有助于我的成长和发展， 独立研究项目并为终身教职做准备

Project Summary / Abstract The ability of cells to recognize and respond to extrinsic and intrinsic perturbations is critical for their survival. Recent studies have shown that the status of translating ribosomes is constantly monitored by surveillance factors to determine signs of translational distress. For example, the exposure of cells to ultraviolet radiation and reactive oxygen species causes transcriptome-wide RNA damage and prolonged stalling of ribosomes within the coding sequence of damaged messenger RNAs triggers ribosomal collisions. Recently, our laboratory has shown that the mitogen-activated protein kinase kinase kinase ZAK, and the general control nonderepressible 2 (GCN2) kinase are activated on collided ribosomes to trigger activation of the Ribotoxic and Integrated Stress Response programs (RSR and ISR) respectively. The goal of my proposal is to understand how the cell mounts a measured ZAK- and GCN2-mediated response that depends on the pervasiveness of ribosome collisions to determine cell fate. In preliminary experiments, I performed quantitative time-resolved phosphoproteomics to determine the temporal order of activation of signaling cascades in cells responding to ribotoxic stress. The data thus generated is providing an unprecedented high-resolution atlas of signaling events occurring in response to ribosome collisions and will be further explored in two proposed aims. In Aim 1, I will determine how ZAK is activated in response to ribosomal collisions and regulates cell fate outcomes through activation of the p38 and JNK signaling cascades. I will also test how a negative-feedback loop for ZAK enables cells to mount a measured response to ribotoxic stress. These studies will employ biochemical, quantitative proteomics, and live cell imaging approaches. In Aim 2, I will determine how GCN2 regulates the ISR pathway, and JNK and mTOR activity in response to ribotoxic stress by genetically and chemically perturbing GCN2’s kinase activity. Finally, I will test how co-factors and regulators of GCN2 set the threshold for its activation in response to ribosomal collisions. Together, the successful completion of these complementary approaches will provide foundational mechanistic insights into how the RSR and ISR pathways are coordinated by ZAK and GCN2 to regulate cell fate decisions during ribotoxic stress. Towards this, the experiments proposed will enable me to develop new skills and technologies in quantitative proteomics and live-cell imaging approaches, which will allow me to substantially differentiate my own research program from that of my mentor Dr. Green. I am confident that the team of collaborators that I have assembled and the accompanying training plan, will be instrumental in the growth and development of my independent research program and prepare me for a tenure-track position.