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Updated: Jan 18, 2026
Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates
Published on: May 8, 2020
Autophosphorylation of conserved yeast and human casein kinase 1 isozymes regulates Elongator-dependent tRNA
Maria Friederike Landrock1, Rościsław Krutyhołowa2,3, Pauline Böhnert1
1Department of Microbiology, Institute of Biology, University of Kassel, 34132 Kassel, Germany.
Abstract:
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.
Insights
Casein kinase 1 (CK1) regulates transfer RNA (tRNA) modification through autophosphorylation. This study identifies conserved regulatory mechanisms in yeast and human cells, revealing a complex CK1 plasticity network.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Casein kinase 1 (CK1) family proteins are vital for numerous cellular processes, including ER-Golgi trafficking and DNA repair.
- The precise roles of substrate interactions and autophosphorylation in CK1's functional plasticity are not well understood.
Purpose of the Study:
- To characterize the budding yeast CK1 Hrr25 and its human ortholog, CK1 epsilon (CK1ϵ).
- To investigate the impact of autophosphorylation and substrate interactions on CK1 activity and regulation, particularly in tRNA modification.
Main Methods:
- Phylogenetic, cellular, and molecular characterization of yeast Hrr25 and human CK1ϵ.
- In vivo analysis of Hrr25 depletion and catalytically inactive mutants.
- In vitro studies using purified Hrr25 to identify autophosphorylation sites and substrate interactions.
- Functional complementation assays using human CK1ϵ in yeast.
Main Results:
- CK1 activity perturbations cause growth defects, morphological changes, and impaired Elongator-dependent tRNA modification in yeast.
- Conserved autophosphorylation sites in CK1 orthologs regulate activity and interactions with Elp1 and Sit4.
- Human CK1ϵ can functionally replace yeast Hrr25 in tRNA modification and cell proliferation.
Conclusions:
- Autophosphorylation is a key mechanism controlling CK1 activity and regulating the tRNA modification pathway.
- Conserved regulatory mechanisms highlight the importance of CK1 plasticity in both yeast and human cells.
- A complex phosphorylation network underlies CK1's diverse cellular functions.
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