Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways

  1. Spencer Hill
  2. Kurt Reichermeier
  3. Daniel C Scott
  4. Lorena Samentar
  5. Jasmin Coulombe-Huntington
  6. Luisa Izzi
  7. Xiaojing Tang
  8. Rebeca Ibarra
  9. Thierry Bertomeu
  10. Annie Moradian
  11. Michael J Sweredoski
  12. Nora Caberoy
  13. Brenda A Schulman
  14. Frank Sicheri
  15. Mike Tyers
  16. Gary Kleiger  Is a corresponding author
  1. University of Nevada, Las Vegas, United States
  2. Genentech Inc, United States
  3. St Jude Children's Research Hospital, United States
  4. University of Montreal, Canada
  5. Mount Sinai Hospital, Canada
  6. California Institute of Technology, United States

Abstract

The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1a. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners.

Data availability

High through-put sequence data can be found at the GEO repository: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE136175

The following data sets were generated

Article and author information

Author details

  1. Spencer Hill

    Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, United States
    Competing interests
    No competing interests declared.
  2. Kurt Reichermeier

    Departments of Discovery Proteomics and Discovery Oncology, Genentech Inc, South San Francisco, United States
    Competing interests
    Kurt Reichermeier, is an employee of the Genetech Biotechnology Compnay.
  3. Daniel C Scott

    Department of Structural Biology, St Jude Children's Research Hospital, Memphis, United States
    Competing interests
    No competing interests declared.
  4. Lorena Samentar

    School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, United States
    Competing interests
    No competing interests declared.
  5. Jasmin Coulombe-Huntington

    Institute for Research in Immunology and Cancer, Department of Medicine, University of Montreal, Montreal, Canada
    Competing interests
    No competing interests declared.
  6. Luisa Izzi

    Institute for Research in Immunology and Cancer, Department of Medicine, University of Montreal, Montreal, Canada
    Competing interests
    No competing interests declared.
  7. Xiaojing Tang

    Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada
    Competing interests
    No competing interests declared.
  8. Rebeca Ibarra

    Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, United States
    Competing interests
    No competing interests declared.
  9. Thierry Bertomeu

    Institute for Research in Immunology and Cancer, Department of Medicine, University of Montreal, Montreal, Canada
    Competing interests
    No competing interests declared.
  10. Annie Moradian

    Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, California Institute of Technology, Pasadena, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0407-2031
  11. Michael J Sweredoski

    Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, California Institute of Technology, Pasadena, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0878-3831
  12. Nora Caberoy

    School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, United States
    Competing interests
    No competing interests declared.
  13. Brenda A Schulman

    Department of Structural Biology, St Jude Children's Research Hospital, Memphis, United States
    Competing interests
    No competing interests declared.
  14. Frank Sicheri

    Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada
    Competing interests
    Frank Sicheri, is a founder and consultant for Repare Therapeutics.
  15. Mike Tyers

    Institute for Research in Immunology and Cancer, Department of Medicine, University of Montreal, Montreal, Canada
    Competing interests
    No competing interests declared.
  16. Gary Kleiger

    Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, United States
    For correspondence
    gary.kleiger@unlv.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3924-1680

Funding

National Institutes of Health (2 R15 GM117555-02)

  • Spencer Hill
  • Rebeca Ibarra
  • Gary Kleiger

National Institutes of Health (R37GM069530)

  • Daniel C Scott
  • Brenda A Schulman

National Institutes of Health (P30CA021765)

  • Daniel C Scott
  • Brenda A Schulman

St. Jude Children's Research Hospital (ALSAC)

  • Daniel C Scott
  • Brenda A Schulman

Max-Planck-Gesellschaft

  • Brenda A Schulman

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2019, Hill et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Spencer Hill
  2. Kurt Reichermeier
  3. Daniel C Scott
  4. Lorena Samentar
  5. Jasmin Coulombe-Huntington
  6. Luisa Izzi
  7. Xiaojing Tang
  8. Rebeca Ibarra
  9. Thierry Bertomeu
  10. Annie Moradian
  11. Michael J Sweredoski
  12. Nora Caberoy
  13. Brenda A Schulman
  14. Frank Sicheri
  15. Mike Tyers
  16. Gary Kleiger
(2019)
Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways
eLife 8:e51163.
https://doi.org/10.7554/eLife.51163

Share this article

https://doi.org/10.7554/eLife.51163

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