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

Kirkpatrick, Donald S.; Hathaway, Nathaniel A.; Hanna, John; Elsasser, Suzanne; Rush, A. John; Finley, Daniel; King, Randall W.; Gygi, Steven P.

doi:10.1038/ncb1436

Cited by 391 publications

(441 citation statements)

References 37 publications

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“…In cases where ubiquitination was examined, the gly-gly tag on lysine residues (ϩ114.1 Da) was included as a variable modification as described [10]. The interpretation and presentation of MS/MS data were performed according to published guidelines [25].…”

Section: Data Processing and Analysismentioning

confidence: 99%

“…Analysis by tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID) can often detect this modification and hence be used to map ubiquitination sites that are identified by an additional mass tag of 114.1 Da on lysine residues [7,8]. In addition, stable isotope derivatives of such orthogonal peptides have been used for the quantitation of protein ubiquitination on specific sites [9,10].…”

mentioning

confidence: 99%

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Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination

Sobott

Watt

Smith

et al. 2009

J. Am. Soc. Mass Spectrom.

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Ubiquitination has emerged as one of the major post-translational modifications that decide on protein fate, targeting, and regulation of protein function. Whereas the ubiquitination of proteins can be monitored with classic biochemical methods, the mapping of modified side chains proves to be challenging. More recently, mass spectrometry has been applied to identify ubiquitinated proteins and also their sites of modification. Typically, liquid chromatography tandem mass spectrometry (LC-MS/MS) based approaches, including collision-induced fragmentation (CID), have been successfully used in the past. However, a potential difficulty arises from the unstable nature of this modification, and also that the isopeptide bond linkage between C-terminal glycine and the N( ) lysyl side chain is susceptible to fragmentation under these conditions. Here we investigate the utility of electron-transfer dissociation (ETD)-based fragmentation to detect ubiquitination sites in proteins. Our results indicate that ETD can provide alternative fragmentation patterns that allow detection of gly-gly-modified lysyl side chains, in particular zϩ1 fragment ions derived from triply charged precursor ions. We subsequently applied ETD fragmentation-based analysis and detected novel ubiquitination sites on DNA polymerase B1 that were not easily observed using CID. We conclude that ETD can provide significant alternative fragmentation information that complements CID-derived data to improve the coverage when mapping ubiquitination sites in proteins. (J Am Soc Mass

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Section: Data Processing and Analysismentioning

confidence: 99%

mentioning

confidence: 99%

Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination

Sobott

Watt

Smith

et al. 2009

J. Am. Soc. Mass Spectrom.

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“…The human APC/C triggers the proteasomal degradation of its substrates by modifying them with K11-linked ubiquitin chains (10,11). Its specific E2, UbcH10, initiates chain formation by recognizing TEK-boxes in substrates and ubiquitin (11).…”

Section: Ube2smentioning

confidence: 99%

“…Loss of APC/C activity arrests cells at metaphase and results in severe aberrations in the structure of the mitotic spindle (6)(7)(8). Human APC/C regulates progression through mitosis by modifying a large family of substrates with K11-linked ubiquitin chains, which triggers their degradation by the proteasome (10)(11)(12). Its specific E2, UBE2C/UbcH10, initiates chain formation by recognizing a substrate motif, the TEK-box, which is homologous to residues around K11 in ubiquitin (11).…”

mentioning

confidence: 99%

Identification of a physiological E2 module for the human anaphase-promoting complex

Williamson

Wickliffe

Mellone

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

263

373

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Ubiquitination by the anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The human APC/C promotes the degradation of mitotic regulators by assembling K11-linked ubiquitin chains, the formation of which is initiated by its E2 UbcH10. Here, we identify the conserved Ube2S as a K11-specific chain elongating E2 for human and Drosophila APC/C. Ube2S depends on the cell cycledependent association with the APC/C activators Cdc20 and Cdh1 for its activity. While depletion of Ube2S already inhibits APC/C in cells, the loss of the complete UbcH10/Ube2S-module leads to dramatic stabilization of APC/C substrates, severe spindle defects, and a strong mitotic delay. Ube2S and UbcH10 are tightly co-regulated in the cell cycle by APC/C-dependent degradation. We conclude that UbcH10 and Ube2S constitute a physiological E2-module for APC/C, the activity of which is required for spindle assembly and cell division.K11-linked chain ͉ proteasome ͉ ubiquitin

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“…Gel slices containing the "ubiquitinated" receptor and the corresponding region in the vector control lane representing the Coomassie-stained gel were used for analysis of polyubiquitin linkages according to previously established methods (23,47). Briefly, the proteins in the gel slices were digested by trypsin with the addition of eight stable isotope-labeled peptides, including all seven GG peptides and one ubiquitin peptide (Thr55-Lys63), as internal standards.…”

Section: Antibodiesmentioning

confidence: 99%

Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway

Varghese

Barrière

Carbone

et al. 2008

Molecular and Cellular Biology

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The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48-and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling.Endocytosis of signaling receptors is a major mechanism used by cells to restrict the magnitude and duration of signal transduction induced by extracellular ligands. Ligand-induced endocytosis of cell surface receptors may occur through clathrin-dependent or -independent pathways. The clathrin-dependent pathway links receptors with clathrin-coated vesicles, which are specialized invaginations of the plasma membrane that concentrate receptors that become internalized. This pathway relies on the interaction of the assembly polypeptide 2 (AP2) clathrin adaptor complexes with specific endocytic signals located within the cytoplasmic domain of the receptors (5).AP2 complexes, which are involved in the assembly of clathrin triskelions at the plasma membrane, are composed of four components, including two adaptin subunits (␣ and ␤2) and two smaller subunits ( 2 and 2); among these subunits, each has different biological functions (34). There are specific endocytic motifs that are essential for receptor clustering on the membrane and clathrin-dependent internalization of receptors. For example, both tyrosine-and leucine-based motifs can be recognized by the AP2 complex through the interaction with its 2 subunit and with the ␤2 or ␣/ 2 hemicomplexes, respectively (5,8,13).The process of receptor endocytosis is not exclusively regulated by the linear motifs located on the cytoplasmic tail of receptors. Posttranslational modification by ubiquitination has also emerged as an important factor in the endocytosis and sorting of surface receptors. Ubiquitin is a 76-amino-acid protein that forms an isopeptide...

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

Cited by 391 publications

References 37 publications

Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination

Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination

Identification of a physiological E2 module for the human anaphase-promoting complex

Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway

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