Molecular mechanisms of ZAK- and GCN2-mediated signaling in response to ribotoxic stress - PROJECT SUMMARY (See instructions): Cellular fitness depends on the ability to sense and respond to proteotoxic stress. Recent studies, including my own, have shown that the status of translating ribosomes is monitored by surveillance factors to determine signs of translational distress. My previous work established that ribosomal collisions, which are triggered by mRNA-damaging insults such as UV radiation, activate the stress-responsive kinases ZAK and GCN2, which initiate the Ribotoxic (RSR) and Integrated Stress Response (ISR) pathways, respectively. I propose that ribosomal collisions occurring on mRNAs exemplify a broader phenomenon known as ribosomal crowding. In this model, cells directly sense alterations in ribosome concentration and translational flux, which activate stress signaling pathways that respond to changes in translation dynamics. My central hypothesis is that ZAK and GCN2 have evolved as molecular sensors of ribosomal crowding, enabling cells to recognize and respond to translational stress. The goal of my proposal is to determine how (1) ribosomal crowding serves as a cue for translational stress sensors, and (2) how the Ribotoxic and Integrated Stress Response pathways are activated by ZAK and GCN2 to regulate cellular life and death decisions. In Aim 1 , I will define the molecular mechanisms of ZAK activation, determining (1) how ribosome crowding relieves its autoinhibition, and (2) how various co-factors and its phosphorylation state regulates its activity on and off the ribosome. Addressing this knowledge gap is critical to understand how ZAK activation thresholds vary across cell types with distinct translational loads, and to identify ZAK-centric vulnerabilities in cancer cells that rely on a high translational output. In Aim 2, I will (1) elucidate the mechanism of GCN2 activation by ribosomal collisions and its interplay with mTOR signaling during translational stress, and (2) determine how aggressive malignancies like MYC-driven lung cancers and acute lymphoblastic leukemia exploit the GCN2-ISR axis to bypass ZAK-mediated apoptosis and tolerate translational dysregulation. Together, these experiments will: (1) elucidate the fundamental principles of how status of ribosomes on and off the mRNA can function as molecular cues for translational stress sensors, (2) characterize ribosome-mediated signaling pathways that regulate protein synthesis defects
