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APC/C-mediated multiple monoubiquitylation provides an alternative degradation signal for cyclin B1

Nature Cell Biology volume 14, pages 168–176 (2012)Cite this article

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Abstract

The anaphase-promoting complex or cyclosome (APC/C) initiates mitotic exit by ubiquitylating cell-cycle regulators such as cyclin B1 and securin. Lys 48-linked ubiquitin chains represent the canonical signal targeting proteins for degradation by the proteasome, but they are not required for the degradation of cyclin B1. Lys 11-linked ubiquitin chains have been implicated in degradation of APC/C substrates, but the Lys 11-chain-forming E2 UBE2S is not essential for mitotic exit, raising questions about the nature of the ubiquitin signal that targets APC/C substrates for degradation. Here we demonstrate that multiple monoubiquitylation of cyclin B1, catalysed by UBCH10 or UBC4/5, is sufficient to target cyclin B1 for destruction by the proteasome. When the number of ubiquitylatable lysines in cyclin B1 is restricted, Lys 11-linked ubiquitin polymers elaborated by UBE2S become increasingly important. We therefore explain how a substrate that contains multiple ubiquitin acceptor sites confers flexibility in the requirement for particular E2 enzymes in modulating the rate of ubiquitin-dependent proteolysis.

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Figure 1: UbVS inhibits cyclin B1 degradation by depleting the amount of available ubiquitin.
Figure 2: Ubiquitin-chain formation is not essential for cyclin B1 degradation in UbVS-treated Xenopus extract.
Figure 3: Cyclin B1 proteolysis depends on Lys 11-linked ubiquitin-chain formation only when the number of available lysine residues is restricted.
Figure 4: UBCH10 and APC/C catalyse rapid multiple monoubiquitylation of cyclin B1 that is sufficient for binding ubiquitin receptors.
Figure 5: Multiple monoubiquitylated cyclin B1 is rapidly degraded by purified proteasomes and in Xenopus extract.
Figure 6: UBE2S is required for cyclin B1 proteolysis only when ubiquitylation is constrained to a single lysine.

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Acknowledgements

We thank D. Morgan (UCSF, USA) for baculoviruses encoding human cyclin B1 and CDK1. Human ubiquitin cloned in pET3a was a gift from C. M. Pickart (Johns Hopkins University, USA). Antibody for UBCH10 immunodepletion was a gift from H. Yu (UT Southwestern, USA). We thank M. Aguiar (Gygi laboratory, Harvard Medical School, USA) for carrying out mass spectrometry to confirm the presence of UBE2S and USP14 in Xenopus egg extract, and H. Besche and S. Elsasser (Harvard Medical School, USA), as well as K. Sackton and F. Sigoillot and the remaining members of the King laboratory, for helpful discussions. This work was financially supported by NIH GM66492 to R.W.K. and GM095526 to D.F.

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  1. Nathaniel A. Hathaway & Donald S. Kirkpatrick

    Present address: Present addresses: Howard Hughes Medical Institute, Department of Pathology, Stanford University, Stanford, California 94305, USA (N.A.H.); Department of Protein Chemistry, Genetech Inc., 1 DNA Way, South San Francisco, California 94080, USA (D.S.K.),

Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA

    Nevena V. Dimova, Nathaniel A. Hathaway, Byung-Hoon Lee, Donald S. Kirkpatrick, Marie Lea Berkowitz, Steven P. Gygi, Daniel Finley & Randall W. King

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Contributions

N.V.D. and R.W.K. designed and interpreted the experiments. N.V.D. carried out and analysed all experiments except those outlined below. N.A.H. carried out cyclin B1 ubiquitylation for ubiquitin-AQUA analysis and degradation assays with these species in APC/C-depleted extract. D.S.K. carried out the ubiquitin-AQUA analysis on cyclin B1 ubiquitylated in vitro with the E2 UBC4 and different ubiquitin types in the laboratory of S.P.G. B-H.L. provided purified human proteasomes with oversight from D.F. M.L.B. helped with cloning of different cyclin B1 mutants. The manuscript was written by N.V.D. and R.W.K. with input from all authors.

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Correspondence to Nathaniel A. Hathaway, Donald S. Kirkpatrick or Randall W. King.

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Dimova, N., Hathaway, N., Lee, BH. et al. APC/C-mediated multiple monoubiquitylation provides an alternative degradation signal for cyclin B1. Nat Cell Biol 14, 168–176 (2012). https://doi.org/10.1038/ncb2425

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