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Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation

Nature Methods volume 10, pages 676–682 (2013)Cite this article

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Abstract

Cross-talk between different types of post-translational modifications on the same protein molecule adds specificity and combinatorial logic to signal processing, but it has not been characterized on a large-scale basis. We developed two methods to identify protein isoforms that are both phosphorylated and ubiquitylated in the yeast Saccharomyces cerevisiae, identifying 466 proteins with 2,100 phosphorylation sites co-occurring with 2,189 ubiquitylation sites. We applied these methods quantitatively to identify phosphorylation sites that regulate protein degradation via the ubiquitin-proteasome system. Our results demonstrate that distinct phosphorylation sites are often used in conjunction with ubiquitylation and that these sites are more highly conserved than the entire set of phosphorylation sites. Finally, we investigated how the phosphorylation machinery can be regulated by ubiquitylation. We found evidence for novel regulatory mechanisms of kinases and 14-3-3 scaffold proteins via proteasome-independent ubiquitylation.

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Figure 1: Overview of methodology and PTMs identified.
Figure 2: Evolution and functional enrichment of modification sites.
Figure 3: The effect of proteasome inhibition on protein and PTM site abundance, and properties of regulated phosphorylation sites.
Figure 4: Validation of a new phosphodegron and ubiquitin–mediated regulation of phosphorylation machinery.

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Acknowledgements

We acknowledge J. Hsu for experimental assistance, R.A. Rodriguez-Mias for helpful discussions, and members of the Villén lab for critical reading of the manuscript. This work is supported in part by US National Institutes of Health (NIH) grants R00CA140789 (to J.V.) and P50 GM082250, P01 AI090935, P50 GM081879 and P01 AI091575 (to N.J.K.). S.F. is supported by the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    Danielle L Swaney, Lea Starita, Stanley Fields & Judit Villén

  2. European Molecular Biology Laboratory–European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, UK

    Pedro Beltrao

  3. Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA

    Lea Starita & Stanley Fields

  4. Cell Signaling Technology Inc., Danvers, Massachusetts, USA

    Ailan Guo & John Rush

  5. Department of Medicine, University of Washington, Seattle, Washington, USA

    Stanley Fields

  6. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA

    Nevan J Krogan

  7. California Institute for Quantitative Biosciences (QB3), San Francisco, California, USA

    Nevan J Krogan

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  1. Danielle L Swaney
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Contributions

D.L.S. and J.V. designed research. D.L.S. and L.S. performed research. D.L.S. and P.B. analyzed data. S.F., N.J.K. and J.V. supervised research. A.G. and J.R. provided reagents. D.L.S., P.B. and J.V. wrote the paper. All authors discussed the results and edited the manuscript.

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Correspondence to Judit Villén.

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Competing interests

A.G. and J.R. are employees of Cell Signaling Technology Inc., which makes the diGly-lysine antibody commercially available.

Supplementary information

Combined PDF

Supplementary Figures 1–5 and Supplementary Table 2 (PDF 556 kb)

Supplementary Table 1

The enrichment of gene ontology (GO) terms in protein isoforms co-modified with ubiquitin and phosphorylation (Ub-phos) vs. those modified only with phosphorylation (NonUb-phos) (XLSX 32 kb)

Supplementary Data 1

Peptide, protein, and PTM site identifications and quantifications (XLSX 10615 kb)

Supplementary Data 2

Co-regulated phosphorylation and ubiquitylation sites (XLSX 61 kb)

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Swaney, D., Beltrao, P., Starita, L. et al. Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation. Nat Methods 10, 676–682 (2013). https://doi.org/10.1038/nmeth.2519

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