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

点击翻译按钮获取中文摘要

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.