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Ubiquitylation-dependent localization of PLK1 in mitosis

Nature Cell Biology volume 15, pages 430–439 (2013)Cite this article

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Abstract

Polo-like kinase 1 (PLK1) critically regulates mitosis through its dynamic localization to kinetochores, centrosomes and the midzone. The polo-box domain (PBD) and activity of PLK1 mediate its recruitment to mitotic structures, but the mechanisms regulating PLK1 dynamics remain poorly understood. Here, we identify PLK1 as a target of the cullin 3 (CUL3)-based E3 ubiquitin ligase, containing the BTB adaptor KLHL22, which regulates chromosome alignment and PLK1 kinetochore localization but not PLK1 stability. In the absence of KLHL22, PLK1 accumulates on kinetochores, resulting in activation of the spindle assembly checkpoint (SAC). CUL3–KLHL22 ubiquitylates Lys 492, located within the PBD, leading to PLK1 dissociation from kinetochore phosphoreceptors. Expression of a non-ubiquitylatable PLK1-K492R mutant phenocopies inactivation of CUL3–KLHL22. KLHL22 associates with the mitotic spindle and its interaction with PLK1 increases on chromosome bi-orientation. Our data suggest that CUL3–KLHL22-mediated ubiquitylation signals degradation-independent removal of PLK1 from kinetochores and SAC satisfaction, which are required for faithful mitosis.

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Figure 1: The BTB adaptor protein KLHL22 regulates SAC-dependent chromosome alignment during mitosis.
Figure 2: The CUL3–KLHL22 E3 ligase interacts directly with PLK1 and regulates its kinetochore localization during mitosis.
Figure 3: KLHL22 regulates PLK1-mediated phosphorylation of BUBR1 and stable kinetochore–microtubule attachments.
Figure 4: CUL3–KLHL22-mediated ubiquitylation of PLK1 within the PBD domain regulates faithful chromosome alignment during mitosis.
Figure 5: Ubiquitylation of PLK1 by CUL3–KLHL22 regulates PBD-mediated phospho-interactions at kinetochores and stable kinetochore–microtubule attachments.
Figure 6: KLHL22 accumulates at the mitotic spindle and its association with PLK1 peaks with chromosome bi-orientation.

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  • 12 March 2013

    In the version of this Article that was originally published, the acknowledgement to S. Elowe was omitted in error.

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Acknowledgements

We thank W. Piwko, T. Courtheoux, P. Meraldi, D. Gerlich, A. Smith, O. Pourquié and M. Labouesse for helpful discussions and editing of the manuscript, E. A. Nigg, S. Elowe, J-M. Peters, P. Meraldi, U. Kutay and F. Barr for antibodies, D. Gerlich and U. Kutay for cell lines and G. Csucs, J. Kusch, O. Biehlmeier and T. Schwarz from the D-BIOL Light Microscopy Center for help with microscopy. J.B. was granted an EMBO Short Term Fellowship, and S.M. was funded by ETHZ and the Boehringer Ingelheim Fonds. I.S. was supported by the ETHZ and the Swiss National Science Foundation (SNF), and research in D.R. and M.P.’s laboratories by the Canadian Institute of Health Research (CIHR), the European Research Council (ERC), the SNF and the ETHZ, respectively. Research in I.S.’s laboratory is supported by the IGBMC, the ATIP-AVENIR program, CNRS, INSERM and Sanofi-Aventis.

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Author notes

  1. Sarah Maerki

    Present address: Present address: Tecan Schweiz AG, 8708 Maennedorf, Switzerland,

  2. Jochen Beck and Sarah Maerki: These authors contributed equally to this work

Authors and Affiliations

  1. Institute of Biochemistry, ETH Zurich, Schafmattstrasse 18, 8093 Zurich, Switzerland

    Jochen Beck, Sarah Maerki, Matthias Peter & Izabela Sumara

  2. Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK

    Markus Posch & Jason R. Swedlow

  3. Institute of Genetics and Molecular and Cellular Biology (IGBMC), 67400 Illkirch, France

    Thibaud Metzger & Izabela Sumara

  4. The Hospital for Sick Children, Toronto M5S 1A8, Canada

    Avinash Persaud & Daniela Rotin

  5. Miltenyi Biotec GmbH, 51429 Bergisch Gladbach, Germany

    Hartmut Scheel

  6. Institute for Genetics, University of Cologne, 50674 Cologne, Germany

    Kay Hofmann

  7. SCILLS, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK

    Patrick Pedrioli

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Contributions

J.B., S.M. and T.M. conceived ideas, performed experiments, and analysed and interpreted the data. H.S. and K.H. performed bioinformatic analysis of protein microarray data. A.P. and D.R. collaborated on protein microarray experiments. P.P. conducted the MS analysis of PLK1 ubiquitylation sites. M. Posch and J.R.S. collaborated on live-cell video microscopy techniques. M. Peter and I.S. conceived ideas. J.B., M. Peter and I.S. wrote the manuscript.

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Correspondence to Matthias Peter or Izabela Sumara.

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GFP–KLHL22 spindle and centrosome localization 1.

Live video microscopy of cells expressing GFP–KLHL22, showing localization of GFP–KLHL22 during mitosis. Movies were generated from maximum intensity projections through Z-stacks spanning a total depth of 15 μm at 1 μm increments. Projected images were normalized to 0.5% saturated pixels per frame to balance visual effects of photobleaching. Time resolution is 30 s per frame. (AVI 303 kb)

GFP–KLHL22 spindle and centrosome localization 2

Live video microscopy of cells expressing GFP–KLHL22, showing localization of GFP–KLHL22 during mitosis. Movies were generated from maximum intensity projections through Z-stacks spanning a total depth of 15 μm at 1 μm increments. Projected images were normalized to 0.5% saturated pixels per frame to balance visual effects of photobleaching. Time resolution is 30 s per frame. (AVI 667 kb)

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Beck, J., Maerki, S., Posch, M. et al. Ubiquitylation-dependent localization of PLK1 in mitosis. Nat Cell Biol 15, 430–439 (2013). https://doi.org/10.1038/ncb2695

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