title: BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner.
abstract: 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/C(Cdc20)). 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/C(Cdc20).
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT,  UK.
authors.forename: Pablo
authors.initials: P
authors.lastname: Lara-Gonzalez
authors.forename: Maria I F
authors.initials: MI
authors.lastname: Scott
authors.forename: Maria
authors.initials: M
authors.lastname: Diez
authors.forename: Onur
authors.initials: O
authors.lastname: Sen
authors.forename: Stephen S
authors.initials: SS
authors.lastname: Taylor
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D059566:M Phase Cell Cycle Checkpoints; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D018797:Cell Cycle Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases