In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2's nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B's β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.
All data generated or anaysed during this study are included in the manuscript and source data files. The final structural model has been deposited in PDB under the accession code 7TRJ. Amplicon sequencing data for the CRISPR clones has been deposited in NCBI's Sequence Read Archive (SRA) under accession number PRJNA821864.
A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulationNCBI's Sequence Read Archive, PRJNA821864.
- Peter Walter
- Peter Walter
- Morgane Boone
- Lan Wang
- Rosalie Lawrence
- Adam Frost
- Adam Frost
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Nahum Sonenberg, McGill University, Canada
- Received: December 8, 2021
- Accepted: April 11, 2022
- Accepted Manuscript published: April 13, 2022 (version 1)
© 2022, Boone et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
The dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 has emerged as a critical regulator of cellular processes. We took a chemical biology approach to gain further insights into its function. We developed C17, a potent small-molecule DYRK2 inhibitor, through multiple rounds of structure-based optimization guided by several co-crystallized structures. C17 displayed an effect on DYRK2 at a single-digit nanomolar IC50 and showed outstanding selectivity for the human kinome containing 467 other human kinases. Using C17 as a chemical probe, we further performed quantitative phosphoproteomic assays and identified several novel DYRK2 targets, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and stromal interaction molecule 1 (STIM1). DYRK2 phosphorylated 4E-BP1 at multiple sites, and the combined treatment of C17 with AKT and MEK inhibitors showed synergistic 4E-BP1 phosphorylation suppression. The phosphorylation of STIM1 by DYRK2 substantially increased the interaction of STIM1 with the ORAI1 channel, and C17 impeded the store-operated calcium entry process. These studies collectively further expand our understanding of DYRK2 and provide a valuable tool to pinpoint its biological function.
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