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      BubR1 Promotes Bub3-Dependent APC/C Inhibition during Spindle Assembly Checkpoint Signaling

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          Summary

          The spindle assembly checkpoint (SAC) prevents premature sister chromatid separation during mitosis. Phosphorylation of unattached kinetochores by the Mps1 kinase promotes recruitment of SAC machinery that catalyzes assembly of the SAC effector mitotic checkpoint complex (MCC). The SAC protein Bub3 is a phospho-amino acid adaptor that forms structurally related stable complexes with functionally distinct paralogs named Bub1 and BubR1. A short motif (“loop”) of Bub1, but not the equivalent loop of BubR1, enhances binding of Bub3 to kinetochore phospho-targets. Here, we asked whether the BubR1 loop directs Bub3 to different phospho-targets. The BubR1 loop is essential for SAC function and cannot be removed or replaced with the Bub1 loop. BubR1 loop mutants bind Bub3 and are normally incorporated in MCC in vitro but have reduced ability to inhibit the MCC target anaphase-promoting complex (APC/C), suggesting that BubR1:Bub3 recognition and inhibition of APC/C requires phosphorylation. Thus, small sequence differences in Bub1 and BubR1 direct Bub3 to different phosphorylated targets in the SAC signaling cascade.

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          Highlights

          • The molecular basis of kinetochore recruitment of Bub1 and BubR1 is dissected

          • Bub1 and BubR1 modulate the ability of Bub3 to recognize phosphorylated targets

          • A newly identified BubR1 motif targets Bub3 to the anaphase-promoting complex

          • The newly identified motif of BubR1 is required for checkpoint signaling

          Abstract

          In spindle assembly checkpoint (SAC) signaling, the phospho-amino acid adaptor Bub3 forms complexes with Bub1 and BubR1 paralogs. Whether Bub3-Bub1 and Bub3-BubR1 bind distinct targets has been unclear. Overlack et al. demonstrate that this is the case and identify a motif in BubR1 that directs Bub3 to the SAC target, the anaphase-promoting complex.

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          Most cited references 89

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          Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

          Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.
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            Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2

            The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. We have examined the mechanism by which the checkpoint inhibits the APC/C by purifying an APC/C inhibitory factor from HeLa cells. We call this factor the mitotic checkpoint complex (MCC) as it consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, as it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells was sensitive to inhibition by MCC. We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. We propose that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.
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              The Molecular Biology of Spindle Assembly Checkpoint Signaling Dynamics.

              The spindle assembly checkpoint is a safeguard mechanism that coordinates cell-cycle progression during mitosis with the state of chromosome attachment to the mitotic spindle. The checkpoint prevents mitotic cells from exiting mitosis in the presence of unattached or improperly attached chromosomes, thus avoiding whole-chromosome gains or losses and their detrimental effects on cell physiology. Here, I review a considerable body of recent progress in the elucidation of the molecular mechanisms underlying checkpoint signaling, and identify a number of unresolved questions.
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                Author and article information

                Contributors
                Journal
                Curr Biol
                Curr. Biol
                Current Biology
                Cell Press
                0960-9822
                1879-0445
                09 October 2017
                09 October 2017
                : 27
                : 19
                : 2915-2927.e7
                Affiliations
                [1 ]Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
                [2 ]Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstrasse, 45141 Essen, Germany
                [3 ]Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Campus-Vienna-Biocenter 1, 1030 Vienna, Austria
                Author notes
                [4]

                Present address: Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstrasse, 45141 Essen, Germany

                [5]

                Lead Contact

                Article
                S0960-9822(17)31071-0
                10.1016/j.cub.2017.08.033
                5640511
                28943088
                © 2017 The Author(s)

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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