Landrock Maria Friederike, Krutyhołowa Rościsław, Böhnert Pauline, Mazur Jarosław, Honc Małgorzata, Hammermeister Alexander, Bessler Larissa, Scherf David, Elms Anna, Radczuk Natalia, Skupien-Rabian Bozena, Jankowska Urszula, Herberg Friedrich W, Helm Mark, Klassen Roland, Glatt Sebastian, Schaffrath Raffael
Department of Microbiology, Institute of Biology, University of Kassel, 34132 Kassel, Germany.
Małopolska Centre of Biotechnology, Jagiellonian University, 30387 Krakow, Poland.
Nucleic Acids Res. 2025 Sep 5;53(17). doi: 10.1093/nar/gkaf881.
Casein kinase 1 (CK1) family members are crucial for ER-Golgi trafficking, calcium signalling, DNA repair, transfer RNA (tRNA) modifications, and circadian rhythmicity. Whether and how substrate interactions and kinase autophosphorylation contribute to CK1 plasticity remains largely unknown. Here, we undertake a comprehensive phylogenetic, cellular, and molecular characterization of budding yeast CK1 Hrr25 and identify human CK1 epsilon (CK1ϵ) as its ortholog. We analyse the effect of Hrr25 depletion and catalytically inactive mutants in vivo and show that perturbations in CK1 activity lead to stress-induced growth defects, morphological abnormalities, and loss of Elongator-dependent tRNA modification. We use purified Hrr25 protein to identify distinct autophosphorylation patterns and phospho-sites on several physiological substrates in vitro and find only human isozyme CK1ϵ can replace yeast Hrr25 functions essential for tRNA modification and cell proliferation in vivo. Furthermore, we demonstrate that human and yeast CK1 orthologs share conserved autophosphorylation sites within the kinase domains, which regulate their activities and mutually exclusive interactions with Elongator subunit Elp1 and Sit4, a phosphatase antagonist of Hrr25. Thus, autophosphorylation controls CK1 activity and regulates the tRNA modification pathway. Our data offer mechanistic insights into regulatory roles of CK1 that are conserved between yeast and human cells and reveal a complex phosphorylation network behind CK1 plasticity.
酪蛋白激酶1（CK1）家族成员对于内质网-高尔基体运输、钙信号传导、DNA修复、转运RNA（tRNA）修饰以及昼夜节律至关重要。底物相互作用和激酶自身磷酸化是否以及如何促成CK1的可塑性在很大程度上仍不清楚。在此，我们对芽殖酵母CK1 Hrr25进行了全面的系统发育、细胞和分子特征分析，并确定人类CK1ε（CK1ϵ）为其直系同源物。我们在体内分析了Hrr25缺失和催化失活突变体的影响，结果表明CK1活性的扰动会导致应激诱导的生长缺陷、形态异常以及依赖延伸因子的tRNA修饰丧失。我们使用纯化的Hrr25蛋白在体外鉴定了几种生理底物上不同的自身磷酸化模式和磷酸化位点，并且发现只有人类同工酶CK1ϵ能够替代酵母Hrr25在体内tRNA修饰和细胞增殖所必需的功能。此外，我们证明人类和酵母CK1直系同源物在激酶结构域内共享保守的自身磷酸化位点，这些位点调节它们的活性以及与延伸因子亚基Elp1和Sit4（Hrr25的磷酸酶拮抗剂）相互排斥的相互作用。因此，自身磷酸化控制CK1活性并调节tRNA修饰途径。我们的数据为酵母和人类细胞之间保守的CK1调节作用提供了机制性见解，并揭示了CK1可塑性背后复杂的磷酸化网络。
