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      UBE2S elongates ubiquitin chains on APC/C substrates to promote mitotic exit

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          Abstract

          The anaphase-promoting complex (APC/C) ubiquitin ligase is the target of the spindle-assembly checkpoint (SAC), ubiquitylating protein substrates whose degradation regulates progress through mitosis 1- 3. The identity of the ubiquitin-conjugating (E2) enzymes that work with the APC/C is unclear. In an RNA interference screen for factors that modify release from drug-induced SAC activation, we identify here the E2 enzyme, UBE2S, as an auxillary factor for the APC/C that promotes mitotic exit. UBE2S is dispensable in a normal mitosis, but its depletion prolongs drug-induced mitotic arrest and suppresses mitotic slippage. In vitro, UBE2S elongates ubiquitin chains initiated by the E2 enzymes UBCH10 and UBCH5, enhancing the degradation of APC/C substrates by the proteasome. Indeed, following release from SAC arrest, UBE2S-depleted cells neither degrade crucial APC/C substrates, nor silence this checkpoint, whereas SAC bypass via BUBR1 depletion or Aurora-B inhibition negates the requirement for UBE2S. Thus, UBE2S acts with the APC/C in a two-step mechanism controlling substrate ubiquitylation that is essential for mitotic exit after prolonged SAC activation, providing a new model for APC/C function in human cells.

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          Most cited references28

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          The spindle-assembly checkpoint in space and time.

          In eukaryotes, the spindle-assembly checkpoint (SAC) is a ubiquitous safety device that ensures the fidelity of chromosome segregation in mitosis. The SAC prevents chromosome mis-segregation and aneuploidy, and its dysfunction is implicated in tumorigenesis. Recent molecular analyses have begun to shed light on the complex interaction of the checkpoint proteins with kinetochores--structures that mediate the binding of spindle microtubules to chromosomes in mitosis. These studies are finally starting to reveal the mechanisms of checkpoint activation and silencing during mitotic progression.
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            Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores

            The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.
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              Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen.

              Small molecules that perturb specific protein functions are valuable tools for dissecting complex processes in mammalian cells. A combination of two phenotype-based screens, one based on a specific posttranslational modification, the other visualizing microtubules and chromatin, was used to identify compounds that affect mitosis. One compound, here named monastrol, arrested mammalian cells in mitosis with monopolar spindles. In vitro, monastrol specifically inhibited the motility of the mitotic kinesin Eg5, a motor protein required for spindle bipolarity. All previously known small molecules that specifically affect the mitotic machinery target tubulin. Monastrol will therefore be a particularly useful tool for studying mitotic mechanisms.
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                Author and article information

                Journal
                100890575
                21417
                Nat Cell Biol
                Nat. Cell Biol.
                Nature cell biology
                1465-7392
                1476-4679
                1 October 2009
                11 October 2009
                November 2009
                24 May 2010
                : 11
                : 11
                : 1363-1369
                Affiliations
                [1 ] University of Cambridge, Department of Oncology & The Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 OXZ, UK.
                [2 ] Wellcome/CR UK Gurdon Institute and Department of Zoology, Tennis Court Road, Cambridge, CB2 1QN, UK
                Author notes
                Correspondence should be addressed to M.J.G and A.R.V., arv22@ 123456cam.ac.uk
                [3]

                These authors contributed equally to this work

                AUTHOR CONTRIBUTIONS M.J.G, A.R.V. and P.R. designed the siRNA screen; M.J.G. and C.G. performed the screen; M.J.G, C.G., P.R. and J.W. analyzed the screen data. M.J.G performed the analysis of UBE2S cellular function. J.M. performed the APC/C in vitro activity assays, and T.M., the Cyclin-B1 degradation assays and microinjection studies. M.J.G and T.M performed the time-lapse studies, which J.W. helped quantify. All authors analyzed and interpreted the data. The manuscript was written by M.J.G and A.R.V.

                Article
                UKMS27884
                10.1038/ncb1983
                2875106
                19820702
                ee82d804-0dce-47ad-880b-7b0b2175e8b8

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                History
                Funding
                Funded by: Medical Research Council :
                Award ID: U.1053(U.1053) || MRC_
                Funded by: Medical Research Council :
                Award ID: G0600332(77629) || MRC_
                Categories
                Article

                Cell biology
                Cell biology

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