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Three Cdk1 sites in the kinesin-5 Cin8 catalytic domain coordinate motor localization and activity during anaphase

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Abstract

The bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics. We previously demonstrated that phosphorylation of at least one of the Cdk1 sites in the catalytic domain of the Saccharomyces cerevisiae kinesin-5 Cin8 (S277, T285, S493) regulates its localization to the anaphase spindle. The contribution of these three sites to phospho-regulation of Cin8, as well as the timing of such contributions, remains unknown. Here, we examined the function and spindle localization of phospho-deficient (serine/threonine to alanine) and phospho-mimic (serine/threonine to aspartic acid) Cin8 mutants. In vitro, the three Cdk1 sites undergo phosphorylation by Clb2-Cdk1. In cells, phosphorylation of Cin8 affects two aspects of its localization to the anaphase spindle, translocation from the spindle-pole bodies (SPBs) region to spindle microtubules (MTs) and the midzone, and detachment from the mitotic spindle. We found that phosphorylation of S277 is essential for the translocation of Cin8 from SPBs to spindle MTs and the subsequent detachment from the spindle. Phosphorylation of T285 mainly affects the detachment of Cin8 from spindle MTs during anaphase, while phosphorylation at S493 affects both the translocation of Cin8 from SPBs to the spindle and detachment from the spindle. Only S493 phosphorylation affected the anaphase spindle elongation rate. We conclude that each phosphorylation site plays a unique role in regulating Cin8 functions and postulate a model in which the timing and extent of phosphorylation of the three sites orchestrates the anaphase function of Cin8.

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Acknowledgements

We thank Vladimir Fridman, Yael Nissenkorn and Maria Podolskaya from the LG laboratory for providing plasmids for this study. We thank Liam Holt, NYU, for critical reading of this manuscript and fruitful discussions, and Ken Kaplan, UC Davis, for fruitful discussions. This work was supported by the Israel Science Foundation grant number 165/13, awarded to LG; the ERC Consolidator Grant 649124 and a Grant from Estonian Research Council IUT2-21 awarded to ML.

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Authors and Affiliations

  1. Department of Chemistry and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, PO Box 653, 84105, Beer-Sheva, Israel

    Alina Goldstein, Nurit Siegler, Darya Goldman, Haim Judah & Larisa Gheber

  2. Institute of Technology, University of Tartu, Tartu, Estonia

    Ervin Valk, Mardo Kõivomägi & Mart Loog

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  1. Alina Goldstein
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  2. Nurit Siegler
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  3. Darya Goldman
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  4. Haim Judah
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  5. Ervin Valk
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  6. Mardo Kõivomägi
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  7. Mart Loog
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  8. Larisa Gheber
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Corresponding author

Correspondence to Larisa Gheber.

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Goldstein, A., Siegler, N., Goldman, D. et al. Three Cdk1 sites in the kinesin-5 Cin8 catalytic domain coordinate motor localization and activity during anaphase. Cell. Mol. Life Sci. 74, 3395–3412 (2017). https://doi.org/10.1007/s00018-017-2523-z

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  • DOI: https://doi.org/10.1007/s00018-017-2523-z

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