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Kirkpatrick, D.S. et al. Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nat. Cell Biol. 8, 700-710

Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Nature Cell Biology (Impact Factor: 20.06). 08/2006; 8(7):700-10. DOI: 10.1038/ncb1436
Source: PubMed

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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    • "Low concentrations of Ube2C formed heterotypic chains predominantly containing K11/48 linkages, but at high concentrations Ube2C formed complex chains with six different Ub linkages. This ability of the APC/ C to form multiple Ub linkages also has been observed in Xenopus extracts, where APC/C in combination with high concentrations of the E2 UBC4 (up to 4 mM) formed polyUb chains on cyclin B1 containing K11, K48, and K63 linkages (Kirkpatrick et al., 2006). Whether this degree of complex Ub-chain formation is physiologically relevant, or simply due to the high concentrations of E2s used in the in vitro assays, remains to be determined. "
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    • "A variety of substrate modifications are possible in the conjugation cascade, including the addition of a single ubiquitin molecule (monoubiquitination), the attachment of multiple ubiquitin molecules to different lysines on the same target protein (multiubiquitination), or the addition of different types of polyubiquitin chains (polyubiquitination). There are seven lysine residues (K6, K11, K27, K29, K31, K48, and K63) in ubiquitin, any of which are available for ubiquitin attachment to produce polyubiquitin chains (Kim et al., 2007; Kirkpatrick et al., 2006). The structure of the attached polyubiquitin chains seems to affect the fate of the target protein. "
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    • "Digestion of a ubiquitinated protein with trypsin leads to a signature mass addition of 114 Da on the modified Lys, corresponding to the C-terminal GlyGly sequence of Ub. Spiking of tryptic digest mixtures with isotope-labeled GlyGlymodified standard peptides derived from each linkage site allows quantification of all chain types (Kirkpatrick et al, 2006). To account for the presence of phosphorylation at Ub Ser65, AQUA peptides incorporating pSer65 as well as (GG)Lys63/pSer65 were also included (see Supplementary Materials and Methods and Supplementary Table S1). "
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