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

> **NIH NIH K99** · JOHNS HOPKINS UNIVERSITY · 2024 · $116,100

## Abstract

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.

## Key facts

- **NIH application ID:** 10900612
- **Project number:** 5K99GM146031-02
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Niladri K Sinha
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $116,100
- **Award type:** 5
- **Project period:** 2023-08-10 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10900612

## Citation

> US National Institutes of Health, RePORTER application 10900612, Molecular mechanisms of ZAK- and GCN2-mediated signaling in response to ribotoxic stress (5K99GM146031-02). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10900612. Licensed CC0.

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