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Sequential primed kinases create a damage-responsive phosphodegron on Eco1

Nature Structural & Molecular Biology volume 20, pages 194–201 (2013)Cite this article

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Abstract

Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation, which triggers ubiquitination by SCFCdc4. We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7–Dbf4 and the GSK-3 homolog Mck1. These kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing establishment of cohesion after S phase. This elaborate regulatory system, involving three independent kinases and stringent substrate selection by a ubiquitin ligase, enables robust control of cohesion establishment during normal growth and after stress.

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Figure 1: Analysis of the contribution of individual Cdk1 sites to Eco1 degradation.
Figure 2: Mass spectrometry reveals non-Cdk1 phosphorylation of Eco1.
Figure 3: Cdc7–Dbf4 and Mck1 are necessary for full degradation of Eco1 and phosphorylate Eco1 peptides in vitro.
Figure 4: Cdc4 binding depends on precise spacing of phosphorylation sites in Eco1.
Figure 5: Correct phosphate spacing is essential for Eco1 turnover in the cell.
Figure 6: Cdc7–Dbf4 activity limits Eco1 degradation to late S phase.
Figure 7: Cdc7–Dbf4 inhibition allows damage-induced cohesion establishment.

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Acknowledgements

We thank S. Bell (Massachusetts Institute of Technology, Cambridge, Massachusetts, USA), D. Toczyski (University of California, San Francisco, California, USA), H. Madhani (University of California, San Francisco, California, USA) and K. Shokat (University of California, San Francisco, California, USA) for strains and reagents, M. Loog for advice with kinase assays, A. Ikui for discussion of unpublished results, J. Wohlschlegel for advice with mass spectrometry, J. Mugridge for assistance with fluorescence anisotropy and E. Edenberg and S. Foster for critical review of the manuscript. This work was supported by funding from the US National Institute of General Medical Sciences (R01-GM069901 to D.O.M. and P41-GM103533 to J.R.Y.) and the National Center for Research Resources (P41-RR011823 to J.R.Y.).

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  1. Department of Physiology, University of California, San Francisco, San Francisco, California, USA.,

    Nicholas A Lyons & David O Morgan

  2. Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.,

    Bryan R Fonslow, Jolene K Diedrich & John R Yates III

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Contributions

N.A.L. and D.O.M. conceived the experiments, N.A.L. conducted the biological and biochemical experiments, B.R.F. performed mass spectrometry, and B.R.F. and J.K.D. analyzed mass spectra under the guidance of J.R.Y. N.A.L. and D.O.M. wrote the manuscript.

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Correspondence to David O Morgan.

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Lyons, N., Fonslow, B., Diedrich, J. et al. Sequential primed kinases create a damage-responsive phosphodegron on Eco1. Nat Struct Mol Biol 20, 194–201 (2013). https://doi.org/10.1038/nsmb.2478

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