Abstract
The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase that initiates anaphase and mitotic exit. APC/C is activated by Cdc20 and inhibited by the mitotic checkpoint complex (MCC), which delays mitotic exit when the spindle assembly checkpoint (SAC) is activated. We previously identified apcin as a small molecule ligand of Cdc20 that inhibits APC/CCdc20 and prolongs mitosis. Here we find that apcin paradoxically shortens mitosis when SAC activity is high. These opposing effects of apcin arise from targeting of a common binding site in Cdc20 required for both substrate ubiquitination and MCC-dependent APC/C inhibition. Furthermore, we found that apcin cooperates with p31comet to relieve MCC-dependent inhibition of APC/C. Apcin therefore causes either net APC/C inhibition, prolonging mitosis when SAC activity is low, or net APC/C activation, shortening mitosis when SAC activity is high, demonstrating that a small molecule can produce opposing biological effects depending on regulatory context.
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Data availability
Raw data associated with Figs. 1a–e, 2b,c, 3a,b, 4b, 5a,d,f and 6b–e are available from the corresponding author upon reasonable request. All MS data generated during this study have been deposited to the ProteomeXchange Consortium via the PRIDE56 partner repository with dataset identifier PXD013786.
Code availability
MATLAB code used to calculate mitotic fraction is available upon request from the corresponding author.
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Acknowledgements
We extend our gratitude in memoriam to J. Hoyt, who first observed the phenotype of apcin-induced premature mitotic exit in the presence of nocodazole. We thank X. Zeng for contributing to early investigations on the mechanism of apcin-induced mitotic slippage and for comments on the manuscript. We thank T. Mitchison and D. Finley for helpful discussions. We thank the Nikon Imaging Facility at Harvard Medical School for assistance with time-lapse microscopy, the Image and Data Analysis Core at Harvard Medical School for assistance with immunofluorescence and time-lapse microscopy analysis, the ICCB-Screening Facility at Harvard Medical School for assistance with high-throughput imaging in fixed cell experiments and the Harvard Medical School Cell Biology Initiative for Molecular Trafficking and Neurodegeneration for help with MiSeq CRISPR knock-in cell line verification. This work was supported by NIH grant R35GM127032 to R.W.K. T.B. was supported by NSF grant DGE-1650116 and NIH grant T32GM00857. N.G.B. was supported by NIH grant R35GM128855 and UCRF. B.A.S., N.G.B. and M.Y. were supported by ALSAC/St. Jude and NIH grant 5P30CA021765 (St. Jude Cancer Center).
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K.V.R. designed and performed experiments to characterize mitotic slippage induced by apcin in mammalian cells. K.V.R. characterized the effect of apcin in high-throughput fixed cell assays, performed immunoprecipitation for immunoblot and MS analysis as well as immunofluorescence and investigated cyclin B1–GFP levels through fluorescence time-lapse microscopy. K.V.R. generated the Cdc20D177A CRISPR knock-in cell line and investigated the effect of apcin in the mutant background. T.B. and M.Y. performed in vitro ubiquitination assays with recombinant proteins in the presence of MCC, apcin, p31comet and/or Cdc20D177A with advice from B.A.S. and N.G.B. T.B. additionally investigated APC/C and MCC binding in vitro in response to MCC, apcin and p31comet with advice from N.G.B. K.L.S. performed initial experiments characterizing apcin-induced mitotic slippage via time-lapse microscopy and identified conditions for immunoprecipitation from cellular extracts. J.A.P. and S.P.G. performed MS analysis for the Cdc20 and Cdc27 immunoprecipitation samples. R.W.K. conceived the project and assisted with experimental design and data analysis. R.W.K. and K.V.R. wrote the manuscript, with assistance from all authors.
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Richeson, K.V., Bodrug, T., Sackton, K.L. et al. Paradoxical mitotic exit induced by a small molecule inhibitor of APC/CCdc20. Nat Chem Biol 16, 546–555 (2020). https://doi.org/10.1038/s41589-020-0495-z
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DOI: https://doi.org/10.1038/s41589-020-0495-z
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