Dataset Information

Autophosphorylation of the CK1 kinase domain regulates enzyme activity and substrate specificity

ABSTRACT: CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe a new mechanism of CK1 regulation conserved from yeast to human – autophosphorylation of a threonine in the mobile L-EF loop proximal to the active site – that inhibits kinase activity. Consequently, yeast and human CK1 enzymes with phosphoablating mutations at this site are hyperactive in vitro. We used quantitative phosphoproteomics to show that disruption of this regulatory mechanism rewires CK1 signaling in Schizosaccharomyces pombe. In accord, we found that a known CK1 pathway, a mitotic checkpoint, is downregulated in these mutants, while new pathways that confer heat shock resistance and suppress meiotic transcripts are upregulated. Molecular dynamics simulations demonstrated that phosphorylation on the L-EF loop alters the conformation of the substrate docking site, and we propose that this affects which CK1 substrates can be phosphorylated. Due to the functional importance of the L-EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Schizosaccharomyces Pombe 927

SUBMITTER: Jose Navarrete Perea

LAB HEAD: Kathleen L. Gould, Ph.D.

PROVIDER: PXD026766 | Pride | 2023-07-20

REPOSITORIES: Pride

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Publications

Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes.

Cullati Sierra N SN Chaikuad Apirat A Chen Jun-Song JS Gebel Jakob J Tesmer Laura L Zhubi Rezart R Navarrete-Perea Jose J Guillen Rodrigo X RX Gygi Steven P SP Hummer Gerhard G Dötsch Volker V Knapp Stefan S Gould Kathleen L KL

Molecular cell 20220329 11

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased ...[more]

PMID: 35353987

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Project description:The rates of mRNA synthesis and degradation determine cellular mRNA levels and can be monitored by comparative Dynamic Transcriptome Analysis (cDTA) that uses non-perturbing metabolic RNA labeling. Here we present cDTA data for 46 yeast strains lacking genes involved in mRNA degradation and metabolism. In these strains, changes in mRNA degradation rates are generally compensated by changes in mRNA synthesis rates, resulting in a buffering of mRNA levels. We show that buffering of mRNA levels requires the RNA exonuclease Xrn1. The buffering is rapidly established when mRNA synthesis is impaired, but is delayed when mRNA degradation is impaired, apparently due to Xrn1-dependent transcription repressor induction. Cluster analysis of the data defines the general mRNA degradation machinery, reveals different substrate preferences for the two mRNA deadenylase complexes Ccr4-Not and Pan2-Pan3, and unveils an interwoven cellular mRNA surveillance network. For this purpose, cDTA was carried out as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). S. cerevisiae cultures were grown at 30¡ãC in 50 ml aliquots of YPD medium. S. pombe cultures were grown at 32¡ãC in YES medium. For inhibitor perturbation, BY4741 cells (OD600=0.1) were inoculated from a saturated overnight culture in two 100 ml aliquots of YPD liquid medium, incubated at 30¡ãC and grown to early-log phase (OD600=0.8). Cells were labeled for 6 minutes (sample 0 min). Then cycloheximide was added and cells were labeled for 10 minutes and 60 minutes. Cells were harvested and treated as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). 1,10-Phenanthroline was added to the culture to a final concentration of 25 _g/ml as described (Dori-Bachash et al., 2012), and labeled for 6 minutes after 18 minutes treatment. Cells were treated as above. For the anchor-away analysis, S. cerevisiae cells (OD600=0.1) were cultured in two 200 ml aliquots of YPD containing 1_g/ml rapamycin and incubated at 30¡ãC untill OD600 reached 0.8. Due to the instability of rapamycin, we added 1_g/ml rapamycin every two hours. Then a 20 ml sample was taken and labeled with 4sU. The cells were then treated as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111).

Dataset Information

Autophosphorylation of the CK1 kinase domain regulates enzyme activity and substrate specificity

Publications

Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes.

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