Kirkpatrick, D.S. et al. Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nat. Cell Biol. 8, 700-710
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. "
ABSTRACT: Proteasome-mediated degradation occurs with proteins principally modified with lysine-48 polyubiquitin chains. Whether the proteasome also can bind atypical ubiquitin chains, including those linked by lysine-11, has not been well established. This is critically important, as lysine-11 polyubiquitination has been implicated in both proteasome-mediated degradation and non-degradative outcomes. Here we demonstrate that pure homotypic lysine-11-linked chains do not bind strongly to the mammalian proteasome. By contrast, heterotypic polyubiquitin chains, containing lysine-11 and lysine-48 linkages, not only bind to the proteasome but also stimulate the proteasomal degradation of the cell-cycle regulator cyclin B1. Thus, while heterotypic lysine-11-linked chains facilitate proteasomal degradation, homotypic lysine-11 linkages adopt conformations that prevent association with the proteasome. Our data demonstrate the capacity of the proteasome to bind ubiquitin chains of distinct topology, with implications for the recognition and diverse biological functions of mixed ubiquitin chains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.Cell Reports 07/2015; DOI:10.1016/j.celrep.2015.06.061 · 8.36 Impact Factor
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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. "
ABSTRACT: Ubiquitination is a major modifier of signaling in all eukaryotes that results in the conjugation of ubiquitin to the lysine residues of acceptor proteins. The targeted protein is then subjected to degradation by the 26S proteasome, the major protein degradation system in eukaryotes. The ubiquitin–proteasome system (UPS) greatly influences plant growth and development by modulating the activity, localization, and stability of proteins. Plants are frequently exposed to various abiotic stresses during their life cycles; they rely on proteomic plasticity achieved by the UPS to adapt to unfavorable environmental conditions. In stress signal pathways, a large number of components are modified by specific ubiquitination machinery. In this review, we highlight recent advances in understanding the roles of ubiquitination in plant responses to abiotic stresses, including salt and drought, temperature, ultraviolet (UV), and nutrient availability. The review focuses primarily on the roles of the UPS. In salt and/or drought stress signaling, a number of E3 ligases mediate the stress response in both abscisic acid (ABA)-dependent and ABA-independant pathways. The UPS-mediated regulation of several key ABA-regulated transcriptional factors, e.g. ABI3 and ABI5, has been well documented. In cold signaling, the transcription factor ICE1 is targeted by E3 ligase HOSI for proteosomal degradation. Under UV stress, CUL4-DDB1A-DDB2 E3 ligase participates in DNA excision repair, and COP1 interacts with the UVR8 mediated UV response. The UPS is also involved in the uptake, transport, and homeostasis of nutrients such as iron, phosphorus, and nitrogen. SIZ1-mediated sumoylation, a ubiquitin-like modification, is necessary for a number of processes involved in plant responses to abiotic stresses. A challenge moving forward for researchers is to define more UPS components and to characterize their functions in plant responses to stress conditions; there is particular interest in identifying the ubiquitination targets that function in specific stress signaling pathways.Environmental and Experimental Botany 06/2015; 114. DOI:10.1016/j.envexpbot.2014.07.005 · 3.00 Impact Factor
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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). "
ABSTRACT: The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which β5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.The EMBO Journal 12/2014; 34(3). DOI:10.15252/embj.201489847 · 10.75 Impact Factor