Abstract
Cell cycle events are ordered by cyclin-dependent kinases (CDKs), which phosphorylate hundreds of substrates. Multiple phosphatases oppose these CDK substrates, yet their collective role in regulating phosphorylation timing in vivo remains unclear. Here, we show that four phosphatases (PP2A-B55, PP2A-B56, CDC14, and PP1) each target distinct subsets of CDK substrate sites in vivo in fission yeast, influencing when phosphorylation occurs during G2 and mitosis. On average, sites dephosphorylated by CDC14 and PP2A-B56 are phosphorylated earlier during G2, followed by sites dephosphorylated by PP1 and PP2A-B55. This suggests that these phosphatases set different phosphorylation thresholds at the G2/M transition. Consistent with this, depleting PP2A-B55 or CDC14 accelerates mitotic onset, likely by advancing phosphorylation of their respective CDK substrates, suggesting these phosphorylation thresholds are important for regulating mitotic onset. Our findings establish in vivo phosphatase substrate specificity as a key factor regulating the timing of CDK substrate phosphorylation throughout the cell cycle.
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Data availability
All mass spectrometry data generated for this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with dataset identifier PXD060298, and sample names are listed in Supplementary Data 5. Mass spectrometry data for Fig. 4 and Supplementary Fig. 10 can be found on PRIDE with dataset identifier PXD0035982. Source data are provided with this paper.
Code availability
Previously described custom scripts used to analyse fluorescent time-lapse data can be found at https://github.com/nkapadia27/Spatiotemporal-Orchestration-of-Mitosis66.
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Acknowledgements
We thank J.Curran, J. Greenwood, B. Whyte, S. Willich and T. Hammond for their comments on the manuscript, J. Curran for help with plasmid construction, T. Carr and A. Watson for S. pombe strains with the OsTIR1(F74A)-NLS construct, L.Du for the auxin-tagging plasmid and I. Hagan for sharing antibodies. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (CC2003), the UK Medical Research Council (CC2003), and the Wellcome Trust (CC2003). In addition, this work was supported by the Wellcome Trust Grant to P.N. (grant number 214183), The Lord Leonard and Lady Estelle Wolfson Foundation, Woosnam Foundation and The Breast Cancer Research Foundation (BCRF-23-117). T.U.Z. received funding from the Boehringer Ingelheim Fonds. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.
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T.U.Z. and P.N. initiated the study. T.U.Z. designed and performed all experiments. T.A. and T.U.Z. performed mass spectrometry experiments. E.L.R. developed the Spo15 protein and phospho-antibodies. T.U.Z. performed data analysis. T.U.Z. and P.N. wrote the manuscript with input from all authors.
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Zeisner, T.U., Auchynnikava, T., Roberts, E.L. et al. Phosphatase specificity influences phosphorylation timing of CDK substrates during the cell cycle. Nat Commun (2025). https://doi.org/10.1038/s41467-025-66547-5
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DOI: https://doi.org/10.1038/s41467-025-66547-5
