Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways
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
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Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathwaysNCBI Gene Expression Omnibus, GSE136175.
Article and author information
Author details
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.
Reviewing Editor
- Ivan Dikic, Goethe University Frankfurt, Germany
Version history
- Received: August 17, 2019
- Accepted: December 22, 2019
- Accepted Manuscript published: December 23, 2019 (version 1)
- Version of Record published: January 22, 2020 (version 2)
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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