When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells1. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates2,3,4. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5,6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A–B55α complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A–B55α functions downstream of Cdk1 inactivation. PP2A–B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-β1, and RNAi depletion of importin-β1 delayed mitotic exit synergistically with PP2A–B55α. This demonstrates that PP2A–B55α and importin-β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.
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The authors thank F. Uhlmann and B. Novak for critical comments on the manuscript. We thank S. Maar, the ETHZ Light Microscopy Centre (LMC), the ETHZ RNAi Screening Centre (RISC), M. Augsburg (The Max Planck Institute of Molecular Cell Biology and Genetics; MPI-CBG), M. Leuschner (MPI-CBG) and A. Ssykor (MPI-CBG) for technical assistance. We thank U. Kutay (ETHZ) for anti-importin-β1 and anti-nucleolin antibodies, J. Rohrer (University of Zurich) for providing GalT–eGFP plasmid, J. Ellenberg (EMBL, Heidelberg) for IBB–eGFP plasmid, M.C. Cardoso (Technical University, Darmstadt) for eGFP–PCNA plasmid and Sanofi Aventis and the National Cancer Institute for providing flavopiridol. This work was supported by Swiss National Science Foundation (SNF) research grant 3100A0-114120, SNF ProDoc grant PDFMP3_124904, a European Young Investigator (EURYI) award of the European Science Foundation to D.W.G., a MBL Summer Research Fellowship by the Evelyn and Melvin Spiegel Fund to D.W.G., a Roche Ph.D. fellowship to M.H.A.S. and a Mueller fellowship of the Molecular Life Sciences Ph.D. programme Zurich to M.H. M.H. and M.H.A.S. are fellows of the Zurich Ph.D. programme in Molecular Life Sciences. V.J. and J.G. were supported by grants of the 'Geconcerteerde OnderzoeksActies' of the Flemish government, the 'Interuniversitary Attraction Poles' of the Belgian Science Policy P6/28 and the 'Fonds voor Wetenschappelijk Onderzoek-Vlaanderen'. A.I.L. is a Wellcome Trust Principal Research Fellow. A.A.H. acknowledges funding by the Max Planck Society, MitoCheck (the EU-FP6 integrated project), and a BMBF (Bundesministerium für Bildung and Forschung) grant, DiGtoP (01GS0859). Work in the groups of K.M. and J.M.P. was supported by MitoCheck (the EU-FP6 integrated project), Boehringer Ingelheim, the GEN-AU programme of the Austrian Federal Ministry of Science and Research (Austrian Proteomics Platform III), MeioSys within the Seventh Framework Programme of the European Commission and by Chromosome Dynamics, which is funded by the Austrian Science Foundation (FWF).
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Dephosphorylation of the HIV-1 restriction factor SAMHD1 is mediated by PP2A-B55α holoenzymes during mitotic exit
Nature Communications (2018)