Letter | Published:

Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

Nature volume 480, pages 128131 (01 December 2011) | Download Citation

Abstract

Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response1,2,3,4. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In Saccharomyces cerevisiae, the initiation of S phase is thought to be governed by complexes of Cdk1 and Cln cyclins that phosphorylate six or more sites on the Clb5–Cdk1 inhibitor Sic1, directing it to SCF-mediated destruction1,5,6,7,8. The resulting Sic1-free Clb5–Cdk1 complex triggers S phase9. Here, we demonstrate that Sic1 destruction depends on a more complex process in which both Cln2–Cdk1 and Clb5–Cdk1 act in processive multiphosphorylation cascades leading to the phosphorylation of a small number of specific phosphodegrons. The routes of these phosphorylation cascades are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1, the phospho-adaptor subunit of Cdk1. Our results indicate that Clb5–Cdk1-dependent phosphorylation generates positive feedback that is required for switch-like Sic1 destruction. Our evidence for a docking network within clusters of phosphorylation sites uncovers a new level of complexity in Cdk1-dependent regulation of cell cycle transitions, and has general implications for the regulation of cellular processes by multisite phosphorylation.

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Acknowledgements

We thank D. Kellogg for strains and L. Peil for advice on mass spectrometry. This work was supported by International Senior Research Fellowship No. 079014/Z/06/Z from the Wellcome Trust (M.Lo.), an installation grant from EMBO and HHMI (M.Lo.), no. 1253, grants no. 6766 from the Estonian Science Foundation (M.Lo.) and SF0180071s07 from Estonian Ministry of Education and Research (M.Lo.), EMP grant no. 08071N from the Norwegian government, and grants from the National Institute of General Medical Sciences (D.O.M.) and National Cancer Institute (S.M.R.).

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Affiliations

  1. Institute of Technology, University of Tartu, Tartu 50411, Estonia

    • Mardo Kõivomägi
    • , Ervin Valk
    • , Rainis Venta
    • , Anna Iofik
    • , Martin Lepiku
    •  & Mart Loog
  2. Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA

    • Eva Rose M. Balog
    •  & Seth M. Rubin
  3. Department of Physiology, University of California, San Francisco, California 94158, USA

    • David O. Morgan

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Contributions

M.K., E.V., R.V., A.I., M.Le. and M.Lo. designed and performed the experiments, except for the isothermal calorimetry experiments, performed by E.R.M.B. and S.M.R.; M.Lo. coordinated the project and wrote the manuscript with assistance from D.O.M. and S.M.R.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mart Loog.

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    Supplementary Information

    This file contains Supplementary Equations, a Supplementary Discussion, Supplementary References, Supplementary Tables 1-4 and Supplementary Figures 1-8 with legends.

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https://doi.org/10.1038/nature10560

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