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Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology

An Erratum to this article was published on 01 August 2006

Abstract

Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. In the classical model, a uniform polyubiquitin chain that is linked through Lys 48 is required for recognition and degradation by the 26S proteasome. Here, we used a reconstituted system and quantitative mass spectrometry to demonstrate that cyclin B1 is modified by ubiquitin chains of complex topology, rather than by homogeneous Lys 48-linked chains. The anaphase-promoting complex was found to attach monoubiquitin to multiple lysine residues on cyclin B1, followed by poly-ubiquitin chain extensions linked through multiple lysine residues of ubiquitin (Lys 63, Lys 11 and Lys 48). These heterogeneous ubiquitin chains were sufficient for binding to ubiquitin receptors, as well as for degradation by the 26S proteasome, even when they were synthesized with mutant ubiquitin that lacked Lys 48. Together, our observations expand the context of what can be considered to be a sufficient degradation signal and provide unique insights into the mechanisms of substrate ubiquitination.

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Figure 1: Ubiquitin-AQUA allows quantitative analysis of poly-ubiquitin chains.
Figure 2: Ubiquitin-AQUA internal standard peptides and workflow.
Figure 3: In vitro ubiquitination of cyclin B1 by the anaphase-promoting complex and either Ubc4 or UbcH10.
Figure 4: Anaphase-promoting complex catalyses heterogeneous poly-ubiquitin linkages on cyclin B1.
Figure 5: Kinetic analysis of cyclin B1 ubiquitination.
Figure 6: Mono and poly-ubiquitination of cyclin B1.
Figure 7: Binding of ubiquitinated cyclin B1 by ubiquitin receptor proteins.
Figure 8: Deubiquitination and degradation of ubiquitinated cyclin B1 by purified proteasomes.

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Acknowledgements

The authors wish to thank S. Gerber for technical advice and helpful discussions, and D.O. Morgan for the gift of cyclin B1 and cdc2 baculovirus. Additionally, the authors thank members of the Gygi, Finley and King labs for helpful discussions. R.W.K. is supported by a National Institutes of Health (NIH) grant (GM66492) and the McKenzie Family Foundation, and is a Damon Runyon Scholar. Work in the lab of D.F. is funded by a NIH grant (GM065592). Work in the lab of S.P.G. is supported by NIH grants (HG3456 and GM67945).

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Correspondence to Randall W. King or Steven P. Gygi.

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Supplementary Figures S1, S2, S3, Supplementary Table S1 and Supplementary Text (PDF 398 kb)

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Kirkpatrick, D., Hathaway, N., Hanna, J. et al. Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nat Cell Biol 8, 700–710 (2006). https://doi.org/10.1038/ncb1436

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