Maria Friederike Landrock Maria Friederike Landrock Roscislaw Krutyholowa Roscislaw Krutyholowa Pauline Bohnert Pauline Bohnert Jaroslaw Mazur Jaroslaw Mazur Malgorzata Honc Malgorzata Honc Alexander Hammermeister Alexander Hammermeister Larissa Bessler Larissa Bessler David Scherf David Scherf Anna Elms Anna Elms Natalia Radczuk Natalia Radczuk Bozena Skupien-Rabian Bozena Skupien-Rabian Urszula Jankowska Urszula Jankowska Friedrich W. Herberg Friedrich W. Herberg Mark Helm Mark Helm Roland Klassen Roland Klassen Sebastian Glatt Sebastian Glatt Raffael Schaffrath Raffael Schaffrath Oxford University Press 2025 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. PDFDocument eng 2025-11-03T13:20:11.663728Z 2025 2656091 b application/pdf http://creativecommons.org/licenses/by/4.0/