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

Nature volume 480, pages 128–131 (2011)Cite this article

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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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Figure 1: The phospho-adaptor subunit Cks1 provides processivity for the multiphosphorylation of Sic1 by Cln2–Cdk1 and Clb5–Cdk1.
Figure 2: Phosphorylated priming sites provide docking interactions for efficient phosphorylation of suboptimal sites in phosphodegrons.
Figure 3: Differential roles of Cln2 and Clb5 in Sic1 multiphosphorylation and degradation.

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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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Authors and 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, 95064, California, USA

    Eva Rose M. Balog & Seth M. Rubin

  3. Department of Physiology, University of California, San Francisco, 94158, California, USA

    David O. Morgan

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  1. Mardo Kõivomägi
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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.

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Correspondence to Mart Loog.

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This file contains Supplementary Equations, a Supplementary Discussion, Supplementary References, Supplementary Tables 1-4 and Supplementary Figures 1-8 with legends. (PDF 2236 kb)

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Kõivomägi, M., Valk, E., Venta, R. et al. Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase. Nature 480, 128–131 (2011). https://doi.org/10.1038/nature10560

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