Abstract
Protein phosphatase 2A (PP2A) in complex with B55 regulatory subunits reverses cyclin-dependent kinase 1 (Cdk1) phosphorylations at mitotic exit1,2,3,4,5. Interestingly, threonine and serine residues phosphorylated by Cdk1 display distinct phosphorylation dynamics, but the biological significance remains unexplored. Here we demonstrate that the phosphothreonine preference of PP2A–B55 provides an essential regulatory element of mitotic exit. To allow rapid activation of the anaphase-promoting complex/cyclosome (APC/C) co-activator Cdc20, inhibitory phosphorylation sites are conserved as threonines while serine substitutions delay dephosphorylation and Cdc20 activation. Conversely, to ensure timely activation of the interphase APC/C co-activator Cdh1, inhibitory phosphorylation sites are conserved as serines, and threonine substitutions result in premature Cdh1 activation. Furthermore, rapid translocation of the chromosomal passenger complex to the central spindle is prevented by mutation of a single phosphorylated threonine to serine in inner centromere protein (INCENP), leading to failure of cytokinesis. Altogether, the findings of our work reveal that the inherent residue preference of a protein phosphatase can provide temporal regulation in biological processes.
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Acknowledgements
We thank T. Mayer and A. Heim (Universitat Konstanz, Germany), J. Pines (ICR, United Kingdom), D. Hermida Aponte and C. Lukas (Novo Nordisk Foundation Center for Protein Research, Denmark) for reagents and discussion, and the CPR protein production facility and, in particular, G. Cazzamali, M. Williamson, A. L. L. Vala and H. Koc in the eukaryotic and prokaryotic expression teams for purifying Cdc20 proteins. The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, is supported financially by the Novo Nordisk Foundation (grant agreement NNF14CC0001). In addition, this work was supported by grants from the Danish Cancer Society (R72-A4351-13-S2 and R124-A7827-15-S2), a grant from the Danish Council for Independent Research (DFF-4183-00388) and a grant from the Novo Nordisk Foundation (NNF16OC0022394) to J.N.
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All authors contributed to project planning and data analysis of their respective experiments and J.N. coordinated the work. J.B.H. performed all analysis of Cdc20 and Cdh1, except in vitro APC/C ubiquitylation assays performed by D.H.G. E.P.T.H. performed Michaelis–Menten kinetics analysis of PP2A–B55 and all in vitro kinase and phosphatase assays and phosphorylation of Arpp19. T.K. performed in vivo analysis of INCENP. J.B.H. and D.H.G. performed analysis of phosphoantibodies. All authors contributed to the writing of the paper.
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Supplementary Figure 1
(A) Michaelis-Menten kinetic parameters of purified PP2A-B55α holoenzyme were determined against 16-mer phospho-peptides originating from Cdh1 144-158 (pS151: WDVSPYSL(pS)PVSNKSQ; pT151 WDVSPYSL(pT)PVSNKSQ) using malachite green phosphatase assay. Mean and standard deviation shown in plot (n = 3 independent experiments). (B) Endogenous Cdc20 was depleted by RNAi in HeLa cells using the outlined protocol and complemented with the indicated YFP tagged forms of Cdc20 and mitotic duration was determined using live cell microscopy (n values are shown in the graph and represent single cells analyzed per condition, Median and interquartile range indicated by red lines from 2 independent experiments). Representative still images are shown below. Scale bar; 10 μm. (C) Mitotic APC/C devoid of Cdc20 was incubated with the indicated purified Strep tagged Cdc20 proteins. After washing the amount of Cdc20 remaining/bound was determined by western blot.
Supplementary Figure 2
(A) Conservation of APC1 residues in different species. (B) Purified GST or indicated GST-Arpp19 proteins were phosphorylated with purified MASTL in the presence of [γ-32P] labeled ATP and reactions separated by SDS-PAGE and analyzed by autoradiography. GST-Arpp19 proteins phosphorylated with MASTL using ATPγS was incubated with a mitotic extract from HeLa cells expressing myc-tagged PP2AC. Next, the GST-tagged proteins were purified and analyzed for bound PP2A-C. The experiment was performed once. (C) B55 isoforms were depleted as indicated and Cdc20 immunopurified following release from a nocodazole block at the indicated times. The level of Cdc20 T70 phosphorylation and total Cdc20 purified was determined (data represent 1 out 3 experiments with similar results). Quantification of Cdc20 T70 phosphorylation for the specific experiment is shown below. (D) Analysis of the experiments in Fig. 2a measuring the time from nuclear envelope breakdown (NEBD) to metaphase and the time from metaphase to anaphase. The timing of mitotic events was determined from the DIC images and only cells allowing clear determination of metaphase were analyzed. (n = 30 cells analyzed per condition from 3 independent experiments, median with interquartile range indicated by red lines). (E) Analysis of the localization of YFP tagged Cdc20 wt and Cdc20 3SP from cells progressing through an unperturbed mitosis. Representative images from a cell in prometaphase and metaphase are shown. More than 20 cells were analyzed. Scale bars; 10 μm.
Supplementary Figure 3
(A) Calibration of Cdc20 T70 phospho-specific antibody. GST-Cdc20 47–78 wt was phosphorylated in vitro with cdk1-cyclinB using [γ-32P] labeled ATP. The sample was blotted on PVDF and the Cdc20 T70 signal determined by quantitative western blot and then analyzed by autoradiography. Cdc20 T70p antibody signal was normalized to [32P] signal and the value obtained from the Cdc20 3S was multiplied by the Cdc20 wt to determine how well the T70p antibody recognizes the S70p epitope. The obtained value was used to calculate to what extent Cdc20 3SP was phosphorylated in cell extracts. Values are quantified signal intensities. The experiment was performed once. (B) The APC/C was purified from nocodazole arrested cells using a monoclonal APC4 antibody and then control treated or treated with lambda phosphatase as indicated. Different amounts of purified APC/C was analyzed by western-blot and probed with the indicated antibodies. Data show 1 out of 2 independent experiments with similar results. (C) As in B with cells depleted of either APC1 or APC3 using specific RNAi oligoes. Note that depletion of APC1 destabilizes the APC/C. Data show 1 out of 2 independent experiments with similar results. (D) HeLa cells synchronized using a double thymidine arrest and release protocol were released from the final thymidine block (t = 0) and the APC/C purified at the indicated times. The samples were analyzed by western-blot with the indicated antibodies. The level of APC1 S355p intensity was normalized to APC4 intensity on the diagram to the right. Data show 1 out of 2 independent experiments with similar results. (E) HeLa cells were transfected with YFP-Cdh1 wt or mutants thereof and then YFP-Cdh1 proteins were isolated from mitotic cells using a YFP affinity resin. YFP-Cdh1 wt sample was treated with lambda phosphatase as indicated and then different amounts of purified YFP-Cdh1 proteins were analyzed by western-blot using the indicated antibodies. The quantification is shown for 8 ul sample as this allows for more accurate quantification. Data show 1 out of 2 independent experiments with similar results.
Supplementary Figure 4
(A) Hela cells stably expressing YFP tagged Cdh1 were synchronized by double thymidine treatment, depleted of Cdc20 and mitotic duration was determined using live cell microsopy (n values are shown in the graph and represent single cells analyzed per condition from 3 independent experiments, Median and interquartile range indicated by red lines, Mann–Whitney Test P < 0.0001). (B) As in A but in the presence of 30 ng ml−1 nocodazole (n values are shown in the graph and represent single cells analyzed per condition from 3 independent experiments, Median and interquartile range indicated by red lines, Mann–Whitney Test (two-tailed) P < 0.0001). Scale bar, 10 μm.
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Hein, J., Hertz, E., Garvanska, D. et al. Distinct kinetics of serine and threonine dephosphorylation are essential for mitosis. Nat Cell Biol 19, 1433–1440 (2017). https://doi.org/10.1038/ncb3634
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DOI: https://doi.org/10.1038/ncb3634
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