The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3–dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade.
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We thank M. Brunner, R. VanderLinden and B. Schulman (St. Jude Children’s Research Hospital/HHMI/Max Planck Institute of Biochemistry) for providing reagents, E. Salmon (University of North Carolina) for providing cell lines and T. Kenakin for helpful discussions. Our work is supported by NIH T32GM008570 and NSF DGE-1650116 (T. Bodrug and M.E.G.); NIH T32CA009156 (G.D.G.); NIH P30CA016086 (UNC High-throughput Peptide Synthesis Facility and Array Facility); NIH R01GM083024, NIH R01GM102413 and the W.M. Keck Foundation (J.G.C.); Hertha Firnberg Program of the Austrian Science Fund (R.Q.); Boehringer Ingelheim, the Austrian Research Promotion Agency (Headquarter grant FFG-852936), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (GA no. 693949) and Human Frontier Science Program grant RGP0057/2018 (J.-M.P.); NIH R01AG011085 (J.W.H.); UNC University Cancer Research Fund (UCRF), NIH R01GM120309 and the American Cancer Society RSG-18-220-01-TBG (M.J.E.); and NIH R35GM128855 and UCRF (N.G.B.).
The authors declare no competing interests.
Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Current model of structural architectures of APC/CCDH1–UBE2C and APC/CCDH1–UBE2S.
Schematic depicting the interplay of substrate ubiquitination mechanisms, including substrate priming, multiubiquitination, and Ub chain elongation, by APC/CCDH1, UBE2C, and UBE2S. Upon APC/C activation by CDH1 binding, catalytic core APC2WHB–APC11RING is mobile and exposed for UBE2C recruitment (1). APC/CCDH1–UBE2C structural architecture for substrate priming and multiubiquitination (2 and 3). APC/CCDH1–UBE2S structural architecture where APC11RING is repurposed and binds acceptor ubiquitin for K11-linked Ub chain elongation (4).
UBE2S extends Ub chains on substrates, CycBNTD* and Securin*, and increases the rate of substrate modification in a dose-dependent manner. Fluorescence scan of full SDS-PAGE gels used in (1b). * Represents contaminant present in substrate stock. Uncropped images are available as source data.
a, Catalytically inactive UBE2SC95K enhances rapid turnover of CycBNTD* by the APC/CCDH1 and UBE2C. b, Quantification depicts relative fraction of remaining unmodified CycBNTD* shown in Extended Data Figure Fig. 3a. Average of n=3 independent experiments ± s.e.m. c, UBE2S UBC domain (UBE2S core) is defective for Ub chain elongation in an APC/C-dependent manner. d, UBE2SCTP enhances the rapid turnover of CycBNTD* by the APC/CCDH1 and UBE2C. Fluorescence scan of full SDS-PAGE gels used in (2b). Uncropped images for panels (a,c-d) and data for the graph in (b) are available as source data.
Extended Data Fig. 4 UBE2SCTP accelerates substrate modification in every UBE2C-dependent ubiquitination reaction condition using multiple substrates, APC/C variants, and different coactivators.
a, Fluorescence scan of full SDS-PAGE gels used in (2c) showing the effect of the UBE2SCTP on the polyubiquitination of multiple substrates, CycBNTD*, Ub-CycBNTD*, Securin*, Ub-Securin*, by APC/C and UBE2C. b, “No encounter” control assay done as in (2d) with the exception of swapping fluorescent substrate and cold substrate in the mixtures to prevent a reaction to occur. c, Ubiquitination reaction monitoring role of UBE2SCTP on UBE2C-dependent APC/C substrate priming using a single lysine substrate, CycBNTD*(1K), and methylated Ub (meUb). The unmodified substrate and monoubiquitinated product are followed over time in the absence or presence of UBE2SCTP. Quantitation shows effect of UBE2SCTP on substrate priming by APC/CCDH1-UBE2C, graphs depicts fraction of unmodified substrate and meUb~CycBNTD*(1K) over time. Average of n=3 independent experiments ± s.e.m. d, UBE2C-dependent substrate turnover is accelerated by UBE2SCTP when non-phosphorylated, APC/C-pA, or phopho-mimetic APC/C, APC/C-pE, is used. Fluorescence scan of full SDS-PAGE gels used in (2e). e, Substrate turnover by phosphomimetic APC/C, APC/C-pE, and CDC20 is enhanced by the addition of UBE2SCTP in UBE2C-dependent reactions. Fluorescence scan of full SDS-PAGE gels used in (2f). Uncropped images for panels (a-b, d-e) and data for the graph in (c) are available as source data.
a, UBE2SCTP promotes a substrate-independent APC/C activation of UBE2C, monitored by the hydrolysis of UBE2S~Ub over time into UBE2C and Ub. Detected using Coomassie-stained gels, full gel from (3d). b, Western blot of (3e) showing successful knockdown of UBE2S and CDC20 in HeLa H2B-GFP. c, Time necessary for the metaphase to anaphase transition for different U2OS cells. Data were collected in 2 independent experiments, total number of analyzed cells n = 67 (control), n = 76 (UBE2S knockdown), n = 67 (CDC20 knockdown) and n = 62 (UBE2S and CDC20 knockdown). Median, 25% and 75% percentiles are shown. ** indicates p<0.001, **** indicates p<0.0001 as calculated with Kruskal Wallis test followed by Dunn’s multiple comparison test. Right panel, western blot showing successful knockdown of UBE2S and CDC20 in U2OS H2B-mCherry. d, Western blot in (3g) with the addition of levels of endogenous or recombinant variants of UBE2S. Uncropped images for panels (a,d) and data for the graph in (c) are available as source data.
a, As previously reported, APC/CΔAPC1-WD40 is defective for UBE2C-dependent polyubiquitination, but rescued by the addition of the purified APC1-WD40 domain added in trans, monitored by SDS-PAGE and fluorescent scanning12. b, Assays as in (4b), showing the effects of the UBE2SCTP on the polyubiquitination of Securin* by APC/CCDH1 (wild-type and indicated variant), UBE2C, and UBE2S. c, Assays as in (4c), showing the effects of the UBE2SCTP on Ub chain elongation of Ub-Securin by APC/CCDH1 (wild-type and indicated variant) and UBE2S. d, Assays as in (4d-e), showing the effects of the UBE2SCTP on the polyubiquitination of multiple substrate by APC/CCDH1 (wild-type and indicated variant) and UBE2C. e, Representative fluorescent scans of full SDS-PAGE gels in (4f) for data used to determine kinetic parameters upon titrating UBE2C in assays measuring UBE2C-dependent ubiquitination for Ub-CycBNTD* with APC/CCDH1 (wild-type and indicated variant). f, Heat map of Ub-linkages from reactions in (4g) analyzed by label free mass spectrometry. Average of n=3 independent experiments. g, APC/C activation by UBE2SCTP bypasses the requirement for the APC1-WD40 domain. Representative full gel of hydrolysis of UBE2C~Ub monitored by Sypro-stained SDS-PAGE gels (4h). Uncropped image for panel (g) and data for the graph in (f) are available as source Data.
a, Representative SDS-PAGE gels in (5b) for data used to determine kinetic parameters in titrating the UBE2SCTP in assays measuring Securin* polyubiquitination by APC/CΔAPC1-WD40, CDH1, and UBE2C. b, As in assays in (5e), UBE2SCTP restores UBE2C-dependent substrate polyubiquitination in APC/C variant defective in CDH1-dependent activation. c, Mutation of the APC2–APC4 groove prevents Ub chain elongation of Ub-CycBNTD* by APC/C (wild-type and indicated variants) and UBE2S. d, Coomassie-stained SDS-PAGE gel of purified wild-type and mutant recombinant APC/Cs. Uncropped image for panel (b) is available as source data.
Unprocessed gels Fig. 1b.
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Unprocessed gels Fig. 2b–f.
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Unprocessed gels Fig. 3b,d and unprocessed western blots Fig. 3f,g.
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Unprocessed gels Fig. 4b–e,g,h.
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Unprocessed gels Fig. 5d–f.
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Unprocessed gels Extended Data Fig. 2.
Unprocessed gels Extended Data Fig. 3a,c,d.
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Unprocessed gels Extended Data Fig. 4a,b,d,e.
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Unprocessed gel Extended Data Fig. 5a and unprocessed western blot Extended Data Fig. 5d.
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Unprocessed gel Extended Data Fig. 6g.
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Unprocessed gel Extended Data Fig. 7b.
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Martinez-Chacin, R.C., Bodrug, T., Bolhuis, D.L. et al. Ubiquitin chain-elongating enzyme UBE2S activates the RING E3 ligase APC/C for substrate priming. Nat Struct Mol Biol 27, 550–560 (2020). https://doi.org/10.1038/s41594-020-0424-6
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