Article abstract
Nature Cell Biology 8, 700 - 710 (2006)
Published online: 25 June 2006 | doi:10.1038/ncb1436
There is an Erratum (August 2006) associated with this Article.
There is a Corrigendum (March 2007) associated with this Article.
Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology
Donald S. Kirkpatrick1,4, Nathaniel A. Hathaway1,4, John Hanna1, Suzanne Elsasser1, John Rush2, Daniel Finley1, Randall W. King1 & Steven P. Gygi1,3
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.
- Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
- Cell Signaling Technology, Inc., Beverly, MA 01915, USA.
- Taplin Biological Mass Spectrometry Facility, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
- These authors contributed equally to this work.
Correspondence to: Randall W. King1 e-mail: randy_king@hms.harvard.edu
Correspondence to: Steven P. Gygi1,3 e-mail: steven_gygi@hms.harvard.edu
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