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Degradation of Cep68 and PCNT cleavage mediate Cep215 removal from the PCM to allow centriole separation, disengagement and licensing

Nature Cell Biology volume 17, pages 3143 (2015) | Download Citation

Abstract

An intercentrosomal linker keeps a cell’s two centrosomes joined together until it is dissolved at the onset of mitosis. A second connection keeps daughter centrioles engaged to their mothers until they lose their orthogonal arrangement at the end of mitosis. Centriole disengagement is required to license centrioles for duplication. We show that the intercentrosomal linker protein Cep68 is degraded in prometaphase through the SCFβTrCP (Skp1–Cul1–F-box protein) ubiquitin ligase complex. Cep68 degradation is initiated by PLK1 phosphorylation of Cep68 on Ser 332, allowing recognition by βTrCP. We also found that Cep68 forms a complex with Cep215 (also known as Cdk5Rap2) and PCNT (also known as pericentrin), two PCM (pericentriolar material) proteins involved in centriole engagement. Cep68 and PCNT bind to different pools of Cep215. We propose that Cep68 degradation allows Cep215 removal from the peripheral PCM preventing centriole separation following disengagement, whereas PCNT cleavage mediates Cep215 removal from the core of the PCM to inhibit centriole disengagement and duplication.

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Acknowledgements

The authors thank B. D. Dynlacht, J. R. Skaar and M. F. B. Tsou for critical reading of the manuscript; K. Lee and S. Kim for helpful advice; Y. Deng at the NYU SOM Microscopy core for image processing assistance; A. North (Rockefeller University) for use of the DeltaVision OMX V4/Blaze system, supported by award S10RR031855 from the National Center for Research Resources; K. Rhee for the PCNT constructs, and I. Hoffmann for the FLAG–CPAP construct. M.P. is grateful to T. M. Thor for continuous support. This work was funded by grants from the National Institutes of Health (R01-GM057587 and R37-CA076584) and New York State Health Department (NYSTEM-N11G-255) to M.P. and fellowships from the National Health and Medical Research Council of Australia and the Lymphoma Research Foundation to J.K.P. A.S., L.F. and M.P.W. are supported by the Stowers Institute for Medical Research. P.V.J. and M.J.K.J. were supported by NIH grant R01 GM094972 and an award from the Mathers Foundation. M.P. is an Investigator with the Howard Hughes Medical Institute.

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Affiliations

  1. Department of Pathology, Laura and Isaac Perlmutter Cancer Center, New York University School of Medicine, New York 10065, USA

    • Julia K. Pagan
    • , Antonio Marzio
    •  & Michele Pagano
  2. Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue New York 10065, USA

    • Mathew J. K. Jones
    •  & Prasad V. Jallepalli
  3. The Stowers Institute of Medical Research, 1000 East 50th Street, Kansas City Missouri 64110, USA

    • Anita Saraf
    • , Laurence Florens
    •  & Michael P. Washburn
  4. Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City Kansas 66160, USA

    • Michael P. Washburn
  5. Howard Hughes Medical Institute, 522 First Avenue New York 10016, USA

    • Michele Pagano

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Contributions

J.K.P. planned and performed most experiments and co-wrote the manuscript. M.P. coordinated the study, oversaw the results, and co-wrote the manuscript. A.M. helped with many biochemical experiments. M.J.K.J. helped with several experiments and provided intellectual advice. P.V.J. provided reagents and intellectual advice. A.S., L.F. and M.P.W. performed the mass spectrometry analyses of the purifications performed by J.K.P. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Michele Pagano.

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https://doi.org/10.1038/ncb3076

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