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The regulated assembly of a PKCɛ complex controls the completion of cytokinesis

Nature Cell Biology volume 10, pages 891–901 (2008)Cite this article

Abstract

The cell cycle is exquisitely controlled by multiple sequential regulatory inputs to ensure fidelity. Here we demonstrate that the final step in division, the physical separation of daughter cells, is controlled by a member of the PKC gene superfamily. Specifically, we have identified three phosphorylation sites within PKCɛ that control its association with 14-3-3. These phosphorylations are executed by p38 MAP kinase (Ser 350), GSK3 (Ser 346) and PKC itself (Ser 368). Integration of these signals is essential during mitosis because mutations that prevent phosphorylation of PKCɛ and/or PKCɛ binding to 14-3-3 also cause defects in the completion of cytokinesis. Using chemical genetic and dominant-negative approaches it is shown that selective inhibition of PKCɛ halts cells at the final stages of separation. This arrest is associated with persistent RhoA activation at the midbody and a delay in actomyosin ring dissociation. This study therefore identifies a new regulatory mechanism that controls exit from cytokinesis, which has implications for carcinogenesis.

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Figure 1: 14-3-3-β binding is mediated by phospho-Ser 346 and -Ser 368 on PKCɛ.
Figure 2: PKC, p38–MAPK and GSK3 phosphorylate Ser 368, Ser 350 and Ser 346, respectively.
Figure 3: Ser 346 phosphorylation occurs during mitosis and subsequent 14-3-3 association regulates cytokinesis.
Figure 4: A PKCɛ–14-3-3 complex is crucial for cytokinesis in HeLa cells.
Figure 5: 14-3-3 siRNA and dominant-negative 14-3-3 expression inhibits mitosis/cytokinesis.
Figure 6: Inhibiting PKCɛ activity prevents cytokinesis.
Figure 7: PKCɛ inhibits RhoA and prevents its association at the furrow during late cytokinesis.
Figure 8: Schematic representation summarizing how GSK3, p38 and PKC converge on the PKCɛ–14-3-3 complex to regulate abscission.

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Acknowledgements

We thank Robert Messing for the initial characterization and constructs required to develop the chemical genetic approach for specific PKCɛ inhibition. We thank David Barford for providing purified GSK3β, Erik Sahai for cherry-RhoA, Andrey Shaw for DN-14-3-3, Alastair Aitken for GST–14-3-3-β and Myc–14-3-3-β, and Brenda Kostelecky for 14.3.3ζ. We would also like to acknowledge staff at the CRUK London Research Institute FACS laboratory and light microscopy facility. This work was supported by a British Heart Foundation Intermediate Research Fellowship, British Heart Foundation Project Grant and Cancer Research UK.

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Authors and Affiliations

  1. Protein Phosphorylation Laboratory, London Research Institute, Cancer Research UK, London, WC2A 3PX, UK

    Adrian T. Saurin, Joanne Durgan, Angus J. Cameron, Amir Faisal & Peter J. Parker

  2. Department of Cardiology, The Cardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, London, SE1 7EH, UK

    Michael S. Marber

  3. Division of Cancer Studies, King's College London, New Hunt's House, St Thomas Street, London, SE1 1UL, UK

    Peter J. Parker

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Contributions

A.T.S. carried out all experiments (except those depicted in Fig. 2b and Supplementary Information, Fig. S1c, which were performed by A.F.); A.T.S. jointly conceived the project (with P.J.P.) and wrote the manuscript with the help of P.J.P.; J.D. identified the Ser 368 autophosphorylation site and made the relevant antibodies and mutant constructs. A.J.C. helped in generating PKCɛ knockout MEFs expressing various PKCɛ mutants. M.S.M. provided the SR-p38 constructs and helpful advice. All authors contributed to editing of the manuscript.

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Correspondence to Peter J. Parker.

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Saurin, A., Durgan, J., Cameron, A. et al. The regulated assembly of a PKCɛ complex controls the completion of cytokinesis. Nat Cell Biol 10, 891–901 (2008). https://doi.org/10.1038/ncb1749

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