Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit

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Abstract

APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-counteracting phosphatases PP1 and PP2A-B55, allowing wide-spread dephosphorylation of substrates. Meanwhile, continued APC/C activity promotes proteolysis of other mitotic regulators. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution proteomics, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid proteolysis of cyclin B, securin and geminin at the metaphase-anaphase transition, followed by slow proteolysis of other substrates. Dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent categories with unique sequence motifs. We conclude that dephosphorylation initiated by selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.

Data availability

Source data files have been provided for Figures 2, 3, 6 and 7. These include a processed form of the raw data, where some extraneous metadata has been removed.Full raw data is available at PRIDE with the following accession numbers: FIgure 2 Total Proteome PXD019791, Figure 2 Phospho-proteome PXD019788, Figure 3 PXD019795, Figure 6 PXD019787, Figure 7 PXD019786

The following data sets were generated

(2020) PP1 depletion phospho-proteome - Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit
Proteome Xchange, PXD019786.

http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD019786
(2020) APC/C inhibition phospho-proteome - Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit
Proteome Xchange, PXD019787.

http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD019787
(2020) Control phospho-proteome - Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit
Proteome Xchange, PXD019788.

http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD019788
(2020) Control total-proteome - Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit
Proteome Xchange, PXD019791.

http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD019791
(2020) Fractionated Control total-proteome - Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exithibition phospho-proteome - Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit
Proteome Xchange, PXD019795.

http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD019795

The following previously published data sets were used

(2019) CDK1-CCNB1 creates a spindle checkpoint-permissive state by enabling MPS1 kinetochore localization
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446832/.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446832/

Article and author information

Author details

James Holder

Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

For correspondence
james.holder@path.ox.ac.uk

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-7597-3104
Shabaz Mohammed

Biochemistry Department, University of Oxford, Oxford, United Kingdom

Competing interests
The authors declare that no competing interests exist.
Francis A Barr

Department of Biochemistry, University of Oxford, Oxford, United Kingdom

For correspondence
francis.barr@bioch.ox.ac.uk

Competing interests
The authors declare that no competing interests exist.

Funding

Cancer Research UK (Program Grant)

James Holder
Francis A Barr

Wellcome (PhD Award)

James Holder

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.