Molecular mechanisms of ribosome-mediated kinase signaling

The National Institute of General Medical Sciences (NIGMS) awarded The Johns Hopkins University a $2.14 million Project Grant (CFDA 93.859: Biomedical Research and Research Training) effective September 1, 2025 through August 31, 2029 to conduct fundamental research on the molecular mechanisms of ribosome-mediated kinase signaling. The research project will investigate how ribosomes directly sense cellular stress and initiate downstream signaling pathways, specifically examining the interactions between kinases GCN2 (general control nonderepressible 2) and ZAK with colliding ribosomes. The award supports biochemical research utilizing both in vivo and in vitro approaches, including mass spectrometry-based proteomics to identify binding partners and characterize kinase activation mechanisms throughout their signaling lifecycle.

This research addresses fundamental gaps in understanding how cells detect and respond to translational distress caused by damaged messenger ribonucleic acids (mRNAs) and protein synthesis disruptions. The project will advance the scientific foundation for comprehending cellular stress responses and adaptation mechanisms, supporting NIGMS's mission to fund investigator-initiated basic research that increases understanding of biological processes and establishes the foundation for advances in disease diagnosis, treatment, and prevention. Research performance will be conducted at The Johns Hopkins University's facilities in Maryland.

Abstract

Cells must adapt to shifting environmental conditions to ensure survival. And, while we know that many of these insults target the ribosome and protein synthesis, we have recently begun to appreciate the extent to which such cellular insults are directly sensed by ribosomes themselves to initiate stress signaling pathways. Pioneering studies established that the status of translating ribosomes is monitored to identify signs of global translational distress leading to activation of the several signaling pathways including the ribotoxic stress response (RSR) and the integrated stress response (ISR). Normally, the orchestration of translation initiation, elongation, and termination rates enables maintenance of ribosome equilibrium on messenger rnas (mrnas). However, cellular insults leading to the accumulation of damaged mrnas inevitably result in prolonged stalling of ribosomes within coding sequences and eventually to ribosomal collisions which have been shown to be a key determinant for signaling translational distress. Low-level collisions prompt a ribosome-mediated quality control, while abundant transcriptome-wide collisions overwhelm the ribosomal rescue mechanisms and serve as a platform for initiating general stress response pathways. Our previous studies showed that ribosome collisions recruit and activate at least two cellular kinases, GCN2 and ZAK, triggering two distinct downstream signaling cascades. In this proposal, we propose to define molecular mechanisms for how these two kinases, GCN2 and ZAK, interact with ribosomes to activate downstream signaling. We will use primarily biochemical approaches both in vivo and in vitro to identify binding partners of these proteins and specific interactions critical to their output along the lifetime of their activity. Our in vivo focused studies will primarily rely on mass spectrometry approaches at low and high resolution. Our in vitro focused studies will evolve around a reconstitution approach using purified proteins and diverse ribosome populations. The centrality of these pathways in determining cell fate makes them critical to an understanding of cellular homeostasis in health and disease.