BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner

The spindle assembly checkpoint (SAC) is a signalling network that delays anaphase onset until all the chromosomes are attached to the mitotic spindle through their kinetochores. The downstream target of the spindle checkpoint is the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that targets several anaphase inhibitors for proteolysis, including securin and cyclin B1. In the presence of unattached kinetochores, the APC/C is inhibited by the mitotic checkpoint complex (MCC), a tetrameric complex composed of three SAC components, namely BubR1, Bub3 and Mad2, and the APC/C co-activator Cdc20. The molecular mechanisms underlying exactly how unattached kinetochores catalyse MCC formation and how the MCC then inhibits the APC/C remain obscure. Here, using RNAi complementation and in vitro ubiquitylation assays, we investigate the domains in BubR1 required for APC/C inhibition. We observe that kinetochore localisation of BubR1 is required for efficient MCC assembly and SAC response. Furthermore, in contrast to previous studies, we show that the N-terminal domain of BubR1 is the only domain involved in binding to Cdc20–Mad2 and the APC/C. Within this region, an N-terminal KEN box (KEN1) is essential for these interactions. By contrast, mutation of the second KEN box (KEN2) of BubR1 does not interfere with MCC assembly or APC/C binding. However, both in cells and in vitro, the KEN2 box is required for inhibition of APC/C when activated by Cdc20 (APC/CCdc20). Indeed, we show that this second KEN box promotes SAC function by blocking the recruitment of substrates to the APC/C. Thus, we propose a model in which the BubR1 KEN boxes play two very different roles, the first to promote MCC assembly and the second to block substrate recruitment to APC/CCdc20.

[1]  J. Peters,et al.  Substrate binding on the APC/C occurs between the co-activator CDH1 and the processivity factor DOC1 , 2010, Nature Structural &Molecular Biology.

[2]  G. Chan,et al.  Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2 , 2001, The Journal of cell biology.

[3]  Stephen S. Taylor,et al.  Protein Phosphatase 2A and Separase Form a Complex Regulated by Separase Autocleavage* , 2007, Journal of Biological Chemistry.

[4]  Karl Mechtler,et al.  Mitotic regulation of the human anaphase‐promoting complex by phosphorylation , 2003, The EMBO journal.

[5]  J. Peters,et al.  Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1 , 2002, The Journal of cell biology.

[6]  Xianshu Wang,et al.  The Mitotic Checkpoint Protein hBUB3 and the mRNA Export Factor hRAE1 Interact with GLE2p-binding Sequence (GLEBS)-containing Proteins* , 2001, The Journal of Biological Chemistry.

[7]  R. Goorha,et al.  Spindle checkpoint function requires Mad2-dependent Cdc20 binding to the Mad3 homology domain of BubR1. , 2006, Experimental cell research.

[8]  Edward P. Morris,et al.  Structures of APC/CCdh1 with substrates identify Cdh1 and Apc10 as the D-box co-receptor , 2010, Nature.

[9]  J. Peters,et al.  Large-scale purification of the vertebrate anaphase-promoting complex/cyclosome. , 2005, Methods in enzymology.

[10]  E. Nigg,et al.  Uncoupling of the spindle-checkpoint and chromosome-congression functions of BubR1 , 2010, Journal of Cell Science.

[11]  Stephen S. Taylor,et al.  Bub1 and aurora B cooperate to maintain BubR1-mediated inhibition of APC/CCdc20 , 2005, Journal of Cell Science.

[12]  K. Hardwick,et al.  Mad3 KEN Boxes Mediate both Cdc20 and Mad3 Turnover, and Are Critical for the Spindle Checkpoint , 2007, PloS one.

[13]  E. Salmon,et al.  The spindle-assembly checkpoint in space and time , 2007, Nature Reviews Molecular Cell Biology.

[14]  G. Neale,et al.  The mitotic checkpoint gene BubR1 has two distinct functions in mitosis. , 2005, Experimental cell research.

[15]  Stephen S. Taylor,et al.  Truncating APC mutations have dominant effects on proliferation, spindle checkpoint control, survival and chromosome stability , 2004, Journal of Cell Science.

[16]  Bert Vogelstein,et al.  Mutations of mitotic checkpoint genes in human cancers , 1998, Nature.

[17]  N. Rahman,et al.  Constitutional aneuploidy and cancer predisposition caused by biallelic mutations in BUB1B , 2004, Nature Genetics.

[18]  Stephen S. Taylor,et al.  Cdc20 is required for the post-anaphase, KEN-dependent degradation of centromere protein F , 2010, Journal of Cell Science.

[19]  T. Mitchison,et al.  Evidence that mitotic exit is a better cancer therapeutic target than spindle assembly. , 2009, Cancer cell.

[20]  Hongtao Yu,et al.  Mad2-Independent inhibition of APCCdc20 by the mitotic checkpoint protein BubR1. , 2001, Developmental cell.

[21]  Jimi Kim,et al.  BubR1 acetylation at prometaphase is required for modulating APC/C activity and timing of mitosis , 2009, The EMBO journal.

[22]  Stephen S. Taylor,et al.  Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells. , 2001, Journal of cell science.

[23]  T. Blundell,et al.  Defining the Molecular Basis of BubR1 Kinetochore Interactions and APC/C-CDC20 Inhibition* , 2010, The Journal of Biological Chemistry.

[24]  Andrew W. Murray,et al.  MAD3 Encodes a Novel Component of the Spindle Checkpoint Which Interacts with Bub3p, Cdc20p, and Mad2p , 2000, The Journal of cell biology.

[25]  Mitsuhiro Yanagida,et al.  Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1. , 2007, Developmental cell.

[26]  Jeremy Minshull,et al.  The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction , 2008, Nature Cell Biology.

[27]  J. Peters The anaphase promoting complex/cyclosome: a machine designed to destroy , 2006, Nature Reviews Molecular Cell Biology.

[28]  R. Medema,et al.  Elevating the frequency of chromosome mis-segregation as a strategy to kill tumor cells , 2009, Proceedings of the National Academy of Sciences.

[29]  Andrea Musacchio,et al.  The Mad1/Mad2 Complex as a Template for Mad2 Activation in the Spindle Assembly Checkpoint , 2005, Current Biology.

[30]  M. Kirschner,et al.  The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. , 2000, Genes & development.

[31]  Stephen S. Taylor,et al.  The Human Homologue of Bub3 Is Required for Kinetochore Localization of Bub1 and a Mad3/Bub1-related Protein Kinase , 1998, The Journal of cell biology.

[32]  J. Peters,et al.  Methods to measure ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex. , 2006, Methods.

[33]  T. Blundell,et al.  The conserved N-terminal region of the mitotic checkpoint protein BUBR1: a putative TPR motif of high surface activity. , 2005, Biophysical journal.

[34]  Karl Mechtler,et al.  Structure of the Anaphase-Promoting Complex/Cyclosome Interacting with a Mitotic Checkpoint Complex , 2009, Science.

[35]  Stephen S. Taylor,et al.  Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores , 2008, The Journal of cell biology.

[36]  M. Solomon,et al.  Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint. , 2007, Genes & development.

[37]  David O. Morgan,et al.  Analysis of activator-binding sites on the APC/C supports a cooperative substrate-binding mechanism. , 2009, Molecular cell.

[38]  G. Fang,et al.  Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex. , 2002, Molecular biology of the cell.

[39]  Avram Hershko,et al.  Roles of the anaphase-promoting complex/cyclosome and of its activator Cdc20 in functional substrate binding , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[40]  T. Kapoor,et al.  The human mitotic checkpoint protein BubR1 regulates chromosome–spindle attachments , 2005, Nature Cell Biology.

[41]  J. Pines,et al.  How APC/C–Cdc20 changes its substrate specificity in mitosis , 2011, Nature Cell Biology.

[42]  Stephen S. Taylor,et al.  Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores , 2003, The Journal of cell biology.

[43]  J. V. van Deursen,et al.  BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/C(Cdc20) in interphase. , 2009, Developmental cell.

[44]  Stephen S. Taylor,et al.  Cyclin-B1-mediated inhibition of excess separase is required for timely chromosome disjunction , 2006, Journal of Cell Science.

[45]  Paul Russell,et al.  UBE2S elongates ubiquitin chains on APC/C substrates to promote mitotic exit , 2009, Nature Cell Biology.

[46]  Geert J P L Kops,et al.  Lethality to human cancer cells through massive chromosome loss by inhibition of the mitotic checkpoint. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[47]  Stephen S. Taylor,et al.  p31comet-mediated extraction of Mad2 from the MCC promotes efficient mitotic exit , 2011, Journal of Cell Science.

[48]  Rey-Huei Chen,et al.  BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1 , 2002, The Journal of cell biology.

[49]  R. Karess,et al.  Separating the spindle, checkpoint, and timer functions of BubR1 , 2009, The Journal of cell biology.

[50]  D. Cleveland,et al.  Unattached kinetochores catalyze production of an anaphase inhibitor that requires a Mad2 template to prime Cdc20 for BubR1 binding. , 2009, Developmental cell.

[51]  Anna Feoktistova,et al.  The Spindle Checkpoint Functions of Mad3 and Mad2 Depend on a Mad3 KEN Box-mediated Interaction with Cdc20-Anaphase-promoting Complex (APC/C)*S⃞♦ , 2008, Journal of Biological Chemistry.

[52]  Hongtao Yu,et al.  Functional analysis of the spindle-checkpoint proteins using an in vitro ubiquitination assay. , 2004, Methods in molecular biology.

[53]  Stephen S. Taylor,et al.  Kinetochore Localization of Murine Bub1 Is Required for Normal Mitotic Timing and Checkpoint Response to Spindle Damage , 1997, Cell.