Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry
- Peking University, China
- Southern University of Science and Technology, China
- Beijing Normal University, China
- Zhejiang University, China
Abstract
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 cocrystallized 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.
Data availability
The structural coordinates of DYRK2 in complex with compounds 5, 6, 7, 8, 10, 13, 14, 17, 18, 19, and 20 have been deposited in the Protein Data Bank with accession codes 7DH3, 7DG4, 7DH9, 7DHV, 7DHC, 7DHK, 7DHO, 7DJO, 7DL6, 7DHH, and 7DHN, respectively.All the raw mass spectrometry data as well as the identified and significantly regulated phosphosites tables have been deposited in the public proteomics repository MassIVE and are accessible at ftp://massive.ucsd.edu/MSV000087106/.
Article and author information
Author details
Funding
National Key Research and Development Plan (2017YFA0505200)
- Tiantian Wei
- Jue Wang
- Ruqi Liang
- Wendong Chen
- Yilan Chen
- Mingzhe Ma
- An He
- Yifei Du
- Wenjing Zhou
- Zhiying Zhang
- Xin Zeng
- Chu Wang
- Jin Lu
- Xing Guo
- Xiao-Wei Chen
- Youjun Wang
- Junyu Xiao
- Xiaoguang Lei
National Natural Science Foundation of China (91853202,21625201,21961142010,21661140001,and 21521003)
- Tiantian Wei
- Jue Wang
- Ruqi Liang
- Wendong Chen
- Yilan Chen
- Mingzhe Ma
- An He
- Yifei Du
- Wenjing Zhou
- Zhiying Zhang
- Xin Zeng
- Chu Wang
- Jin Lu
- Xing Guo
- Xiao-Wei Chen
- Youjun Wang
- Junyu Xiao
- Xiaoguang Lei
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Wei 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.
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Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry
eLife 11:e77696.
https://doi.org/10.7554/eLife.77696
Further reading
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
