4332 Research Article BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner

Pablo Lara-Gonzalez, Maria I. F. Scott, Maria Diez, Onur Sen and Stephen S. Taylor* Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK *Author for correspondence ([email protected])

Accepted 10 October 2011 Journal of Cell Science 124, 4332–4345 ß 2011. Published by The Company of Biologists Ltd doi: 10.1242/jcs.094763

Summary 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.

Key words: Mitosis, Kinetochore, Spindle assembly checkpoint, Anaphase-promoting complex, Cdc20, Mad2

Journal of Cell Science Introduction BubR1 has a C-terminal kinase domain, but this does not The spindle assembly checkpoint (SAC) is a surveillance appear to be required for SAC control; rather, it promotes the mechanism that delays anaphase until all the chromosomes second function of BubR1 in mitosis, namely regulating have stably attached to spindle microtubules through their kinetochore–microtubule interactions (Ditchfield et al., 2003; kinetochores (Musacchio and Salmon, 2007). The goal of the Harris et al., 2005; Lampson and Kapoor, 2005; Rahmani et al., SAC is to inhibit the anaphase-promoting complex/cyclosome 2009). Note that in yeast, Mad3 does not possess a kinase (APC/C), an E3 ubiquitin ligase (Peters, 2006). Specifically, the domain. In the N-terminus, BubR1 and Mad3 share a region of SAC interferes with the ability of Cdc20 to activate the APC/C. homology with Bub1 (Cahill et al., 1998; Taylor et al., 1998), and Once the SAC is satisfied, the APC/C becomes active, promoting this TPR-like domain binds the kinetochore component Blinkin/ the polyubiquitylation and subsequent degradation of anaphase KNL-1 (Bolanos-Garcia et al., 2005; Kiyomitsu et al., 2007; inhibitors such as securin and cyclin B1, thereby triggering D’Arcy et al., 2010). In the central region, BubR1/Mad3 shares anaphase onset and mitotic exit (Musacchio and Salmon, 2007). another short region of homology with Bub1, and this domain Unattached kinetochores recruit numerous SAC components mediates binding to Bub3 (Taylor et al., 1998; Hardwick et al., including Bub1, BubR1 (Mad3 in yeast), Bub3, Mad1, Mad2 and 2000). The Bub3 binding site is also required for kinetochore Mps1. These proteins in turn catalyse the release of a diffusible localisation of BubR1 (Taylor et al., 1998). However, the inhibitor that prevents APC/C activation (Musacchio and requirement for Bub3 binding and/or kinetochore localisation in Salmon, 2007). It is now generally accepted that this inhibitor terms of SAC signalling is unclear (Malureanu et al., 2009; corresponds to the ‘mitotic checkpoint complex’, or MCC Elowe et al., 2010). (Sudakin et al., 2001), which is comprised of Mad2, BubR1, The Mad3 and BubR1 proteins also contain two KEN boxes; the Bub3 and Cdc20. Although Mad2 is essential for MCC first (KEN1) is in the extreme N-terminus, the second (KEN2) lies formation, it can be present at sub-stoichiometric amounts with between the TPR domain and the Bub3 binding site. Because KEN respect to the other proteins (Nilsson et al., 2008). Thus, it has boxes can act as APC/C degrons (Pfleger and Kirschner, 2000), been proposed that the main function of Mad2 is to promote the these two sequences have attracted considerable attention. Indeed, Cdc20–BubR1 interaction, after which Mad2 is released to create these motifs are required for the APC/C-dependent degradation the final APC/C inhibitor (Nilsson et al., 2008; Kulukian et al., of both Mad3 and BubR1, but because this appears to occur in G1 2009; Westhorpe et al., 2011). Exactly how BubR1 then inhibits or after checkpoint satisfaction, respectively, this mechanism does the APC/C remains to be determined. not seem to contribute to APC/C inhibition (King et al., 2007; Choi BubR1 blocks substrate binding to APC/CCdc20 4333

et al., 2009). By contrast, studies of yeast, flies and mammalian localised to kinetochores, as evidenced by co-staining with anti- cells demonstrate that KEN1 is essential for inhibition of centromere antibodies (ACAs) (Fig. 1C). Deletion of the N-terminal APC/CCdc20, where it promotes SAC function by mediating the domain and the kinase domain did not affect this localisation binding of BubR1 to the Mad2–Cdc20 subcomplex (Davenport pattern. By contrast, deletion or mutation of the Bub3 binding et al., 2006; Burton and Solomon, 2007; King et al., 2007; domain abolished kinetochore localisation (Fig. 1C). Thus, these Sczaniecka et al., 2008; Malureanu et al., 2009; Rahmani et al., data confirm that kinetochore localisation of BubR1 is mediated by 2009; Elowe et al., 2010). Several studies suggest that BubR1 has a the Bub3 binding site (Taylor et al., 1998). second Cdc20 binding site, but the significance of this is undefined Next, we evaluated which domains in BubR1 are important (Tang et al., 2001; Davenport et al., 2006; Malureanu et al., 2009; for the interaction with MCC components and the APC/C. Elowe et al., 2010). The role of KEN2 is also not understood; Mitotically arrested HeLa cells were harvested, lysed and the although required for SAC function, its precise function remains BubR1 mutants immunoprecipitated using anti-Myc antibodies. unclear (Burton and Solomon, 2007; King et al., 2007; Sczaniecka Under these conditions, wild-type BubR1 and the DKD mutant et al., 2008; Elowe et al., 2010). were able to immunoprecipitate Bub3, Cdc20, Mad2 and the In checkpoint-arrested cells, BubR1 – as part of the MCC – is APC/C (Fig. 1D,E). As expected, the D42 and EK mutants were bound to the APC/C; however, when the SAC becomes satisfied, unable to interact with Bub3. These mutants also bound less BubR1 dissociates (Morrow et al., 2005). Importantly, when Cdc20 and Mad2, and the interaction with the APC/C was engaged by the MCC, the APC/C recruits less substrate (Herzog similarly diminished (Fig. 1D,E), suggesting that Bub3 binding et al., 2009). Two explanations could account for this: either and/or kinetochore localisation is required for efficient MCC MCC binding induces a conformational change in the APC/C that assembly. Deletion of the N-terminal domain abolished the prevents substrate docking; or perhaps an MCC component acts interaction of BubR1 with Cdc20, Mad2 and the APC/C, as a pseudo-substrate, binding as a substrate would but without although Bub3 binding was unaffected (Fig. 1D,E). These itself becoming ubiquitylated. Indeed, it was suggested that Mad3 observations indicate that the N-terminus of BubR1 is essential does act as a pseudo-substrate in budding yeast (Burton and for MCC assembly and strongly suggests that in the context of a Solomon, 2007). This notion was based on the observation that mitotic cell, there might not be a second Cdc20 binding site Mad3 and a substrate, Hsl1, can compete for Cdc20 binding. downstream of the Bub3 binding region (Tang et al., 2001; However, it now appears that binding to Cdc20 alone is not Davenport et al., 2006; Malureanu et al., 2009). Indeed, when we sufficient for efficient ubiquitylation; rather, substrates need characterised a BubR1 fragment encompassing only the N- to bind to both Cdc20 and the APC/C (Eytan et al., 2006; terminal domain and the Bub3 binding site (N484) it bound to Matyskiela and Morgan, 2009), and at present there is no direct Cdc20 as efficiently as the wild-type protein (supplementary evidence to suggest that Mad3/BubR1 directly inhibits substrate material Fig. S1). binding to APC/CCdc20. Based on the observations described above, a prevailing model BubR1 N-terminus is essential for the SAC suggests that the MCC is assembled from two sub-complexes, Next, we evaluated the consequences of these mutations on spindle namely BubR1–Bub3 and Mad2–Cdc20, and that this process is checkpoint function. For this, we co-transfected the stable HeLa

Journal of Cell Science critically dependent on first KEN box of BubR1 (Musacchio and cell lines with plasmids encoding a BubR1 shRNA to repress the Salmon, 2007). The MCC then binds the APC/C and inhibits endogenous protein (Kops et al., 2004) and a GFP-tagged histone substrate recruitment by a yet undefined mechanism. To delineate H2B to monitor chromosome segregation. Note that the shRNA this mechanism, we established an RNAi-based complementation plasmid typically reduced BubR1 levels by ,90% (Fig. 2A), and assay to define the domains of BubR1 required for SAC function. that the Myc-tagged BubR1 transgenes were rendered resistant In parallel, we established an in vitro ubiquitylation assay to to the siRNA encoded by the shRNA sequence (Fig. 2B). After monitor inhibition APC/CCdc20. Here, using these assays, we first 48 hours, cells were exposed to nocodazole and analysed by clarify a number of conflicting observations in the literature time-lapse microscopy to determine how long they remained regarding BubR1 structure and function. More importantly, we arrested in mitosis. Whereas controls arrested for the duration then show that the second KEN box promotes SAC function by of the experiment (.400 minutes), BubR1 depletion resulted Cdc20 blocking the recruitment of substrates to APC/C . in 50% of the cells exiting mitosis within ,30 minutes (i.e. T50 ,30 minutes), indicating penetrant SAC override (Fig. 2C). This Results phenotype was largely rescued by expression of wild-type BubR1 BubR1 binds to Mad2–Cdc20 and the APC/C through its (T50 ,674 minutes). However, cells expressing the DNT mutant N-terminal region behaved similarly to BubR1-depleted cells, with a T50 of To assess which domains in BubR1 are important for SAC function, ,32 minutes, confirming that the BubR1 N-terminus is essential we generated HeLa cell lines stably transfected with different Myc- for the SAC function (Fig. 2C). Interestingly, the Bub3 binding tagged BubR1 mutants (Fig. 1A). BubR1 DNT lacks the entire mutants elicited a partial rescue, restoring the T50 to ,60 minutes, N-terminal region, including the TPR-like domain and the two KEN again suggesting that Bub3 binding and/or kinetochore localisation boxes; D42 contains a deletion in the Bub3 binding domain (Taylor is required for efficient BubR1 function. Although the DKD et al., 1998); EK harbours a point mutation in the Bub3 binding mutant also gave a partial rescue, we suspect that this reflects the domain (Harris et al., 2005; Elowe et al., 2010); and DKD lacks the reduced expression level of this mutant (Fig. 2B). entire kinase domain. Western blot analysis showed that upon We then evaluated the ability of these mutants to rescue tetracycline induction, all these mutants, with the exception of DKD, mitotic timing during an unperturbed mitosis (Fig. 2D). Control were expressed to similar levels as the endogenous protein (Fig. 1B cells spent on average ,55 minutes in mitosis, but BubR1- and see also Fig. 2B). We next analysed their localisation in depleted cells exited within ,19 minutes. Expression of wild- prometaphase by immunofluorescence. Wild-type Myc–BubR1 type BubR1 and the DKD mutant restored mitotic timing to 4334 Journal of Cell Science 124 (24)

Fig. 1. BubR1 binds Cdc20 through its N-terminus. (A) Schematic of full- length BubR1 and the various mutants. (B) Immunoblot showing expression of Myc-tagged BubR1 mutants in HeLa cells. Note that the anti-BubR1 antibody does not bind the DNT mutant. (C) Immunofluorescence images of mitotic HeLa cells showing kinetochore localisation of Myc- tagged wild-type BubR1, DNT and DKD. Insets show higher magnification view of selected kinetochores. Scale bars: 5 mm. (D) Immunoblots of Myc–BubR1 Journal of Cell Science immune complexes isolated from HeLa cells treated with nocodazole for 16 hours and probed with antibodies against Myc, Bub3, Cdc20, Mad2 and Cdc27. (E) Bar graph quantifying the experiment in D. Values represent the mean ± s.e.m. of three independent experiments.

,40 minutes. By contrast, cells expressing the DNT mutant maximal binding to MCC components and the APC/C (Fig. 1D). exited mitosis in ,20 minutes, with similar kinetics as BubR1- Indeed, BubR1 mutants that cannot bind Bub3 do rescue depleted cells, again confirming the importance of the N- checkpoint function and mitotic timing, but only partially. The terminus in supporting SAC function. Interestingly, the D42 and simplest explanation for this is that by binding Bub3, BubR1 EK mutants did restore mitotic timing, albeit not as well as is recruited to kinetochores, thereby increasing its local the wild-type protein did, confirming that Bub3 binding and/or concentration at the site where Mad2–Cdc20 complexes are kinetochore localisation is indeed important in terms of the generated, thus facilitating efficient assembly of the MCC. spindle checkpoint function of BubR1. Note that the N484 mutant could also rescue the SAC, both in the presence of BubR1 TPR domain promotes SAC function in a nocodazole and during an unperturbed mitosis (supplementary blinkin-independent manner material Fig. S1C,F,G). To determine how the N-terminus of BubR1 promotes SAC Together, these results show that the N-terminus of BubR1 is function, we turned to the TPR-like domain, which has essential for SAC function, most likely because it is required for previously been implicated in SAC function through its the interaction(s) with Mad2, Cdc20 and the APC/C (Fig. 1D). interaction with blinkin/KNL-1 (Kiyomitsu et al., 2007). To Although Bub3 might not be required for MCC assembly and test the significance of this domain, we generated four stable inhibition of Cdc20 in the APC/C in vitro (Tang et al., 2001; lines expressing two types of BubR1 TPR mutants (Fig. 3A,B). Fang, 2002; Kulukian et al., 2009), it is required in cells for The first type, represented by A159W and F175G, are predicted BubR1 blocks substrate binding to APC/CCdc20 4335

Fig. 2. The BubR1 N-terminus is essential for SAC. (A) Immunoblots of HeLa cells transfected with an shRNA plasmid targeting BubR1 estimating RNAi efficiency. Bub3 is used as a loading control. (B) Immunoblots showing expression of BubR1 mutants upon tetracycline addition in cells depleted of the endogenous protein. (C) A cumulative plot showing the amount of time nocodazole-treated cells arrest in mitosis following BubR1 complementation. At least 40

Journal of Cell Science cells were analysed in each case. (D) Box and whisker plots showing the time cells take from nuclear envelope break down (NEBD) to anaphase onset. At least 30 cells were analysed for each condition.

to disrupt the TPR structure and thus abolish blinkin binding required not only for blinkin binding, as shown previously (Kiyomitsu et al., 2007), whereas the second type, L126A and (Kiyomitsu et al., 2007), but also for the efficient binding of E161A/R165A, are predicted to disrupt blinkin binding without Mad2–Cdc20. As such, the TPR-like domain is essential for BubR1 disrupting the TPR structure (D’Arcy et al., 2010). To test blinkin function, but not for the reasons suggested previously (Kiyomitsu binding, we generated a novel anti-blinkin antibody suitable for et al., 2007; D’Arcy et al., 2010). immunoblotting (Fig. 3C) and immunofluorescence (supplementary material Fig. S2A). Importantly, all four mutants failed to bind BubR1 KEN boxes are required for the spindle checkpoint blinkin (Fig. 3E), which is consistent with previous reports We next turned our attention to the two KEN boxes within the (Kiyomitsu et al., 2007; D’Arcy et al., 2010). Note however that BubR1 N-terminus, which have previously been shown to be all four mutants bound Bub3 as efficiently as the wild-type protein important for BubR1 SAC function (Burton and Solomon, 2007; did (Fig. 3E). Moreover, all four TPR mutants localised to King et al., 2007; Sczaniecka et al., 2008; Malureanu et al., 2009; kinetochores (Fig. 3D). Interestingly, the A159W and F175G Rahmani et al., 2009; Elowe et al., 2010). Again, we generated mutations, which disrupt TPR structure, also abrogated binding to HeLa lines stably transfected with Myc-tagged, tetracycline- Cdc20, Mad2 and the APC/C (Fig. 3E). By contrast, the L126A and inducible, RNAi-resistant BubR1 mutants in which either of the E161A/R165A mutants bound Cdc20, Mad2 and the APC/C as KEN boxes was deleted, namely DK1 and DK2. These mutants efficiently as wild-type BubR1 did (Fig. 3E and supplementary were expressed at similar levels as the endogenous protein material S2B). Consistent with these binding patterns, the A159W (Fig. 4A) and localised to kinetochores normally (supplementary and F175G mutants failed to restore the mitotic timing defect material Fig. S3A). In immunoprecipitations, wild-type BubR1 induced by BubR1 RNAi (Fig. 3F,G). By contrast, the L126A and and the two KEN mutants bound similar amounts of Bub3 E161A/R165A mutants restored mitotic timing as efficiently as the (Fig. 4B and supplementary material Fig. S3B). However, wild-type protein. Taken together, these observations demonstrate deletion of the first KEN box abrogated the interactions with that blinkin binding is not required for kinetochore localisation of Cdc20, Mad2 and the APC/C. By contrast, the DK2 mutant BubR1, or for the ability of BubR1 to promote SAC function. Our bound to all these proteins as efficiently as the wild-type BubR1 data do show however that the integrity of the TPR domain is did (Fig. 4B and supplementary material Fig. S3B), which is 4336 Journal of Cell Science 124 (24) Journal of Cell Science

Fig. 3. Integrity of the BubR1 TPR domain is required for SAC function. (A) Model of the BubR1 TPR motifs (D’Arcy et al., 2010), generated using PyMOL, highlighting two residues required for maintaining the TPR structure (red) and three in the blinkin-binding groove (blue). (B) Predicted effects different mutations have on TPR structure and blinkin binding. (C) Immunoblots of HeLa cells transfected with siRNAs then probed with either anti-Blinkin or Tao1 antibodies. (D) Immunofluorescence images of mitotic HeLa cells showing kinetochore localisation of the BubR1 TPR mutants. Insets show higher magnification view of selected kinetochores. Scale bars: 5 mm. (E) Immunoblots of Myc–BubR1 immune complexes isolated from HeLa cells treated with nocodazole for 16 hours and probed with antibodies against blinkin, Bub3, Cdc20, Mad2 and Cdc27. (F) Immunoblots of HeLa cells showing the expression of Myc-tagged BubR1 TPR mutants after BubR1 RNAi treatment and tetracycline induction. (G) Box and whisker plot showing the time taken from NEBD to anaphase onset. A minimum of 40 cells were analysed in each condition.

consistent with the observations made with yeast Mad3 (Burton ,56 minutes (Fig. 4C). It also restored mitotic timing during an and Solomon, 2007; King et al., 2007; Sczaniecka et al., 2008). unperturbed mitosis to ,28 minutes, which is lower than wild- Using the BubR1 RNAi complementation assay, we next type BubR1-expressing cells, which spent ,40 minutes from evaluated the functional importance of these motifs. Consistent NEBD to anaphase onset (Fig. 4D). To probe the partial function with the fact that it is unable to interact with Cdc20, Mad2 and of DK2 in more detail, we analysed the rate of degradation of the APC/C, the BubR1DK1 mutant could not rescue the SAC, securin and cyclin B1. HeLa cells were co-transfected with either in the presence of nocodazole (Fig. 4C) or during an plasmids encoding the BubR1 shRNA and either DsRed–securin unperturbed mitosis (Fig. 4D). BubR1DK2 had a weak ability to (Holland and Taylor, 2006) or Venus–cyclin B1 (Garnett et al., restore the SAC in the presence of nocodazole, with a T50 of 2009) and then imaged by time-lapse microscopy. As expected BubR1 blocks substrate binding to APC/CCdc20 4337 Journal of Cell Science

Fig. 4. BubR1 KEN boxes are required for SAC function. (A) Immunoblots of HeLa cells showing induction of the Myc-tagged BubR1 KEN mutants following RNAi-mediated repression of the endogenous protein. (B) Immunoblots of Myc immune complexes isolated from HeLa cells arrested in mitosis with nocodazole for 16 hours, probed with antibodies against Myc, Bub3, Cdc20, Mad2 and the APC/C subunit Cdc27. (C) A cumulative plot showing the time arrested in mitosis. At least 50 cells were analysed for each curve. (D) Box and whisker plot showing the amount of time cells take from NEBD to anaphase onset. At least 40 cells were analysed in each case. (E,F) Graphs measuring securin–DsRed fluorescence with maximum fluorescence intensity was normalised to 100%. The data in E and F are the same, but normalised such that in E, T50 represents NEBD whereas in F, T50 represents metaphase. Note that when BubR1 function is not rescued, anaphase occurs without obvious metaphase, so only the control, WT and DK2 are shown in F. Data are plotted as mean ± s.e.m. of three to five cells in each case.

(Hagting et al., 2002), control cells initiated the degradation Similar results were obtained when cyclin B1 degradation was of securin immediately upon metaphase (Fig. 4E,F). In cells measured (supplementary material Fig. S3C,D). Thus, as has depleted of BubR1, degradation initiated immediately after been described previously (Burton and Solomon, 2007; King nuclear envelope break down (NEBD) (Fig. 4E,F). Whereas et al., 2007; Sczaniecka et al., 2008; Malureanu et al., 2009; expression of wild-type BubR1 restored the initiation of securin Rahmani et al., 2009; Elowe et al., 2010), both KEN boxes degradation to metaphase, the BubR1 DK1 mutant had no effect, of BubR1 are required for SAC function; however, their consistent with it being completely deficient in SAC. The DK2 contributions are somewhat different. Whereas the first KEN mutant did delay the rate of securin degradation somewhat box is essential for MCC assembly and APC/C binding, the compared with BubR1 DK1, but nevertheless, initiation of BubR1 mutant lacking the second KEN box does assemble into degradation occurred immediately following NEBD (Fig. 4F). the MCC and can bind the APC/C. However, despite binding the 4338 Journal of Cell Science 124 (24)

APC/C as well as the wild-type protein did (Fig. 4B), BubR1 APC/CCdc20 inhibitor when used in combination with Mad2, the DK2 is largely checkpoint deficient, suggesting that at best, it can DK1 mutant was largely ineffective (Fig. 6A,F). By contrast, only weakly inhibit APC/CCdc20. BubR1 DK2 could partially inhibit APC/CCdc20 in the presence of Mad2 (Fig. 6A,F). Importantly, this is entirely consistent with the Establishment of an assay to measure APC/C activity cell-based observations described above; whereas BubR1 DK1 is in vitro entirely checkpoint deficient, the DK2 mutant can restore mitotic We reasoned that analysing the DK2 mutant in more detail would timing to some extent (Fig. 4D). provide novel insights into how the MCC actually inhibits the It was previously suggested that the BubR1 N-terminal domain APC/C. To more directly probe the role of the second KEN box of is sufficient to mediate inhibition of APC/CCdc20 (Malureanu BubR1 in APC/C inhibition, we analysed the BubR1 KEN mutants et al., 2009). To test this, we generated a fragment of BubR1 in an in vitro APC/C ubiquitylation assay. In contrast to previously containing the N-terminal 370 amino acids (BubR1 N370; described assays that used APC/C from Xenopus egg extracts and/ Fig. 6B), and assayed its ability to inhibit the APC/C. In or interphase cells (Tang and Yu, 2004; Herzog and Peters, 2005; combination with Mad2, both full-length BubR1 and N370 were Kraft et al., 2006), we wanted to establish an assay that more equally potent APC/CCdc20 inhibitors (Fig. 6C,F), confirming closely reflected the cell-based RNAi complementation assay that this domain is indeed sufficient for APC/CCdc20 inhibition. described above; note that the APC/C undergoes extensive Note that this domain lacks the Bub3 binding site, consistent with phosphorylation in mitosis, which contributes to its regulation observations demonstrating that Bub3 is not essential for (Kraft et al., 2003). Therefore, we aimed to isolate APC/C from APC/CCdc20 inhibition in vitro (Tang et al., 2001; Fang, 2002; mitotically arrested HeLa cells. However, we anticipated that a Kulukian et al., 2009). When we deleted the KEN boxes within significant fraction of this APC/C would be bound by the MCC BubR1 N370, we observed that DK1 totally failed to inhibit the (Morrow et al., 2005). To address this, we reasoned that first APC/CCdc20, whereas the DK2 mutant retained weak inhibitory immunodepleting BubR1 would allow us to then purify the activity (Fig. 6D,F). Similar results were obtained when securin remaining MCC-free APC/C, i.e. apo-APC/C (Herzog et al., 2009). was used as an APC/C substrate (Fig. 6E,F). Thus, these results To isolate the APC/C, we raised an anti-Cdc27 antibody show that in the context of either full-length BubR1 or in N370, against a C-terminal peptide (Herzog and Peters, 2005). This APC/CCdc20 inhibition in vitro is completely dependent on the antibody detected a single band of approximately 100 kDa first KEN box and partially on KEN2. (Fig. 5A), which migrated more slowly in mitosis, consistent with hyperphosphorylation (Kraft et al., 2003). Mass BubR1 KEN2 is required for blocking APC/CCdc20 spectrometry revealed that this antibody could efficiently substrate recruitment immunopurify the entire APC/C (Fig. 5B), and as anticipated, Taken together, the cell-based data and the in vitro observations when we immunopurified APC/C from mitotically arrested HeLa presented so far suggest the following: the BubR1 first KEN cells, MCC proteins were bound (Fig. 5C). However, by first box is essential for MCC assembly and APC/CCdc20 binding, immunodepleting BubR1, we could obtain mitotic APC/C with explaining why the DK1 mutant is unable to restore the SAC in very little MCC bound (Fig. 5C). BubR1-deficient cells, and why it cannot inhibit APC/CCdc20 in

Journal of Cell Science To activate the apo-APC/C in vitro, recombinant Cdc20 was vitro. By contrast, BubR1 DK2 can assemble into the MCC and generated (Fig. 5D) and assayed using the N-terminal 90 amino bind the APC/C, and consistent with its ability to partially inhibit acids of cyclin B1 (CycB N90) as a substrate (Fig. 5E). APC/CCdc20 in vitro, it can partially restore mitotic timing in Interestingly, APC/C that had not been subjected to BubR1 cells. To confirm that the cell-based binding data was indeed depletion had high basal activity and recombinant Cdc20 had being recapitulated in the in vitro assay, we incubated MCC only a minor stimulatory effect (Fig. 5F). We suspect that this components with the APC/C for 45 minutes and then asked reflects dissociation of BubR1 and Mad2 from purified MCC– whether they were bound to the APC/C (Fig. 7A). Consistent APC/C complexes, leaving behind active APC/CCdc20. Consistent with previous observations (Kulukian et al., 2009), Mad2 with this idea, if BubR1 was first depleted, the resulting apo- stimulated binding of BubR1 N370 to APC/CCdc20 (Fig. 7B, APC/C had lower basal activity and addition of recombinant compare lanes 4 and 7). More significantly however, whereas Cdc20 stimulated it markedly (Fig. 5F). Thus, prior removal of BubR1 N370 DK1 did not bind APC/CCdc20, the DK2 mutant MCC-bound APC/C clearly provides a good source of mitotically bound as well as the wild-type protein (Fig. 7B, lanes 8 and 9; phosphorylated apo-APC/C for in vitro assays. To reconstitute and supplementary material Fig. S4A), confirming that the in MCC-mediated inhibition, we then purified recombinant Mad2 vitro assay does indeed reflect the cell-based observations. Note and BubR1 (Fig. 5D). Once again, apo-APC/C had very low that even though we did not immunodeplete BubR1 for these activity that was significantly stimulated by addition of Cdc20 assays before APC/C immunopurification, binding of BubR1 (Fig. 5G). Moreover, although addition of either Mad2 or BubR1 N370 and Mad2 to the APC/C was still dependent on the addition in stoichiometric amounts with Cdc20 had little effect, in of recombinant Cdc20 (supplementary material Fig. S4B,C). combination, they almost completely reverted the ability of Finally, to understand why the DK2 mutant could not Cdc20 to activate the APC/C, consistent with previous efficiently inhibit APC/CCdc20 despite the fact that it binds as observations (Fang, 2002; Kulukian et al., 2009). well as the wild type, we analysed substrate binding. The APC/C was first incubated with MCC proteins, followed by addition of BubR1 KEN boxes are important for direct inhibition of the CycB N90 substrate. The APC/C was then re-isolated and APC/CCdc20 in vitro bound proteins analysed by western blot (Fig. 7A). Importantly, Having established the in vitro ubiquitylation assay, we sought to CycB N90 only bound the APC/C pre-incubated with Cdc20 determine the importance of the BubR1 KEN boxes in direct (Fig. 7C,D; compare lanes 1 and 2). Note that this binding APC/CCdc20 inhibition. Although wild-type BubR1 was a potent was dependent on the presence of the D-box in CycB N90 BubR1 blocks substrate binding to APC/CCdc20 4339 Journal of Cell Science

Fig. 5. Measuring APC/C ubiquitylation activity in vitro. (A) Immunoblot of HeLa cell lysates probed with the anti-Cdc27 antibody (RC27.3). (B) Colloidal Coomassie Blue gel of anti-Cdc27 immune complexes isolated from asynchronous HeLa cells indicating the APC/C subunits identified by mass spectrometry. The asterisk denotes a non-specific band. (C) Immunoblots of Cdc27 immune complexes isolated from mitotic extracts, with or without prior immunodepletion of BubR1. Membranes were blotted with antibodies against BubR1, Bub3, Mad2, Cdc20 and Cdc27. Dilutions of the control-depleted extract were included to estimate the immunodepletion efficiency. (D) Coomassie Blue gel of purified Cdc20, Mad2 and BubR1 proteins. The asterisk denotes a contaminant band. (E) Schematic showing the in vitro ubiquitylation assays. (F) Immunoblot of an APC/C ubiquitylation assay probed with anti-Myc antibodies to detect cyclin B1 N90 conjugates. Note that prior depletion of BubR1 reduces the basal activity of the APC/C. (G) Immunoblot of a ubiquitylation assay showing that only the combination of Mad2 and BubR1 can inhibit Cdc20-mediated activation of APC/C.

(supplementary material Fig. S4F). Addition of BubR1 or Mad2 APC/C binding, it is essential for blocking substrate recruitment alone did not affect substrate binding, but in combination they to APC/CCdc20. prevented CycB N90 binding to APC/CCdc20 (Fig. 7C,D; lane 6). Crucially, however, in the presence of Mad2, the BubR1 N370 Discussion DK2 mutant did not block substrate recruitment (Fig. 7C,D; lane BubR1 is an essential component of the spindle checkpoint; by 7). Similar results were obtained when we used securin as a virtue of its Bub3 binding site, it is recruited to unattached substrate (Fig. 7E,F). Thus, it appears that although the second kinetochores early during mitosis and, upon binding Cdc20– KEN box of BubR1 is not required for MCC assembly and Mad2, forms the MCC, which is a potent inhibitor of the APC/C. 4340 Journal of Cell Science 124 (24) Journal of Cell Science

Fig. 6. BubR1 KEN boxes are important for APC/C inhibition. (A) Ubiquitylation assay showing that, in the presence of Mad2, the BubR1 DK1 mutant cannot inhibit APC/CCdc20.(B) Schematic showing the N370 fragment which encompasses the TPR-like domain and both KEN boxes; plus a Coomassie Blue gel showing purified BubR1 N370 proteins (arrow). The asterisk denotes a contaminant band. (C) Ubiquitylation assay showing inhibition of APC/CCdc20 with either BubR1 FL or N370, in the presence of Mad2. (D,E) Ubiquitylation assays showing that in the presence of Mad2, BubR1 N370 DK1 does not inhibit APC/CCdc20 whereas the DK2 mutant only elicits a partial inhibition. The assays were performed using CycB N90 (D) or securin (E) as APC/C substrates. (F) Bar graph quantifying the experiments in A, D and E. In each case, values were normalised to the activity of APC/C in the presence of only Cdc20 (100%) and represent the mean ± s.e.m. of at least two independent experiments each.

Exactly how the MCC inhibits APC/C activity is unknown. Here, below we address these ambiguities and present a novel model using a cell-based RNAi complementation assay and an in vitro explaining how BubR1 inhibits APC/CCdc20. APC/C ubiquitylation assay, we report a structure–function analysis of BubR1, with particular emphasis on its two KEN How is the MCC assembled? boxes. Although BubR1 has already been subjected to several The MCC, which is by definition composed of BubR1, Bub3, structure–function studies, there are a number of ambiguities in Cdc20 and Mad2, is a potent inhibitor of the APC/C (Sudakin the literature and the mechanism by which BubR1 contributes to et al., 2001). Although Mad2 might subsequently dissociate from APC/CCdc20 inhibition is still unclear. Based on our new findings, the MCC (Nilsson et al., 2008; Westhorpe et al., 2011), in terms BubR1 blocks substrate binding to APC/CCdc20 4341 Journal of Cell Science

Fig. 7. BubR1 second KEN box is required to inhibit substrate binding to the APC/C. (A) Schematic showing the strategy used to evaluate the ability of recombinant proteins to bind the APC/C in vitro. (B) Immunoblot of an APC/C binding assay, probed with antibodies against the APC/C subunit Apc2 and His (note that all the recombinant proteins are His tagged), showing that whereas the DK1 mutant binds poorly under all conditions, Mad2 stimulates binding of BubR1 N370 and DK2 to APC/CCdc20.(C,E) Immunoblot of an APC/C substrate binding assay showing that the BubR1 N370 DK2 mutant cannot prevent substrate binding when added in combination with Mad2. The assays were performed using CycB N90 (C) or securin (E) as APC/C substrates. (D,F) Bar graph quantifying the experiments in C and E, respectively. Values represent the mean ± s.e.m. of three independent experiments.

of assembly, the MCC is probably formed by the association interaction between Mad3 and Mad2–Cdc20 (Burton and of two subcomplexes, namely BubR1–Bub3 and Mad2–Cdc20. Solomon, 2007; King et al., 2007; Sczaniecka et al., 2008). Whereas BubR1 and Bub3 appear to bind constitutively This simple picture is complicated by data from mammalian throughout the cell cycle (Taylor et al., 1998; Chen, 2002), systems, which appear to suggest that BubR1 contains a second the binding of Mad2 to Cdc20 is catalysed by unattached Cdc20 binding site in its C-terminal domain (Davenport et al., kinetochores during mitosis (De Antoni et al., 2005; Kulukian 2006; Malureanu et al., 2009; Elowe et al., 2010). However, our et al., 2009). Presumably therefore, upon generation of the data are entirely consistent with observations from the yeast Mad2–Cdc20 subcomplex, the BubR1–Bub3 subcomplex binds systems: when we deleted KEN1 from full-length BubR1, its to form the MCC (Musacchio and Salmon, 2007). Data from both binding to Mad2, Cdc20 and the APC/C was completely budding and fission yeast show that the N-terminal KEN box abolished (Fig. 4B). Note that at least some of the data in Mad3, the fungi equivalent of BubR1, is essential for the supporting a second Cdc20 binding site come from experiments 4342 Journal of Cell Science 124 (24)

where the rescue constructs were grossly overexpressed with APC/CCdc20 inhibition per se. Furthermore, recent observations respect to the endogenous BubR1 (Malureanu et al., 2009). By suggest that following MCC assembly, Mad2 can be removed contrast, in the system we describe here, our mutants were (Nilsson et al., 2008; Kulukian et al., 2009; Westhorpe et al., expressed at a level comparable with the endogenous protein 2011), suggesting that once Cdc20 has been handed over to (Fig. 4A). Whether overexpression accounts for other studies BubR1, Mad2 is also not essential for APC/C inhibition. Because where Cdc20 binding is observed independently of KEN1 Cdc20 is the APC/C activator being inhibited by the checkpoint, remains to be seen (Elowe et al., 2010). However, because the key question in terms of understanding how the SAC works Mad3 lacks the entire C-terminal region, and because the BubR1 thus boils down to defining how BubR1 inhibits APC/CCdc20.A N-terminus is sufficient to inhibit APC/CCdc20 in vitro (Fig. 6C), key observation is that when the MCC is bound to the APC/C, the relevance of a potential second Cdc20 binding site – if it less substrate is bound (Herzog et al., 2009), suggesting that exists – is questionable. Indeed, the study that originally the MCC, and by extension BubR1, somehow interferes with identified this second site concluded that the binding of Cdc20 substrate recruitment. It was suggested that Mad3 acts a to this domain was Mad2 independent and did not play a role in pseudosubstrate inhibitor for the APC/C (Burton and Solomon, the spindle checkpoint (Davenport et al., 2006). The emerging 2007). This notion was based on the observation that budding view is that the BubR1 C-terminal kinase domain is involved in yeast Mad3 could compete for binding to Cdc20 with the APC/C regulating kinetochore–microtubule interactions independently of substrate Hsl1 in a KEN1-dependent manner. However, whether its role in the checkpoint (Harris et al., 2005; Rahmani et al., this reflects a bona fide pseudosubstrate mechanism is 2009; Elowe et al., 2010). Consistent with this notion, in our questionable because these assays were performed in the hands, the BubR1 DKD and N484 mutants appreciably rescued absence of the APC/C, and it has been shown that efficient the SAC defect induced by BubR1 RNAi (Fig. 2C,D and substrate binding requires both the co-activator and the APC/C supplementary material Fig. S1F,G), despite being expressed at (Eytan et al., 2006; Matyskiela and Morgan, 2009). In other low levels. In summary, synthesising all the available data from words, whether Mad3 can compete Hsl1 from the substrate- yeast, flies and mammalian cells, the most likely scenario is that binding site of APC/CCdc20 was not demonstrated. the KEN box closest to the N-terminus of Mad3 or BubR1 is the Significantly, several reports demonstrate that KEN2 is key determinant in terms of Mad2–Cdc20 binding and as such important for SAC function, but the mechanism underlying this plays an essential role in MCC assembly. was unclear (Burton and Solomon, 2007; King et al., 2007; Another key feature of BubR1 is the Bub3 binding site. Sczaniecka et al., 2008; Malureanu et al., 2009; Elowe et al., However, a region containing the TPR-like domain and the two 2010). We confirm the importance of KEN2: in cells KEN boxes, but lacking the Bub3 binding site, is sufficient to reconstituted with BubR1 DK2, the SAC is effectively dead inhibit APC/CCdc20 in vitro (Fig. 5C). Consistently, full-length (Fig. 4E and supplementary material Fig. S3C). Strikingly BubR1 can efficiently inhibit the APC/CCdc20 in vitro in the however, this mutant assembles into the MCC and binds the absence of Bub3 (Fig. 5G) (Tang et al., 2001; Fang, 2002; APC/C as well as wild-type BubR1 does (Fig. 4B). Here, we Kulukian et al., 2009). Because the Bub3 binding site is essential define how the second KEN box mediates APC/CCdc20 inhibition: for kinetochore localisation of BubR1 (Taylor et al., 1998), these we show that BubR1 blocks substrate binding to the APC/CCdc20,

Journal of Cell Science data suggest that kinetochore localisation of BubR1 might not be and moreover this depends on the second KEN box (Fig. 7C,E). required for its checkpoint function, and indeed this argument We therefore propose a two-step mechanism to account for how was recently put forward (Malureanu et al., 2009). However, we BubR1 KEN boxes contribute to inhibition of APC/CCdc20 show here that, in a cellular context, when BubR1 cannot bind (Fig. 8). First, in a TPR-domain- and KEN1-dependent manner, Bub3, binding to Mad2–Cdc20 and the APC/C is compromised BubR1 binds Cdc20–Mad2 subcomplexes as they are generated (Fig. 1D). Furthermore, the Bub3 binding mutants only very at the kinetochores to form the MCC. Second, this complex binds poorly restore checkpoint function (Fig. 2C). So, although Bub3 the APC/C and allows the second KEN box of BubR1 to engage binding might not be essential for the physical interactions with the APC/C to block substrate recruitment. In agreement with that result in MCC assembly and APC/C inhibition, in the this, we observed that if a substrate is pre-bound to the APC/C, cellular context, Bub3 binding is very important for maximal subsequent binding of BubR1 cannot displace it (supplementary BubR1 function. Importantly, the Bub3 binding site and BubR1 material Fig. S4H), suggesting that binding and inhibition of the kinetochore localisation domain are synonymous. Thus, it seems APC/C are two separate functions of BubR1. to us that the most plausible explanation to account for the Interestingly, recent structural studies have shown that existing data is that, through its ability to bind Bub3, BubR1 substrates bind the APC/C at the interface between the co- is recruited to kinetochores, thereby increasing its local activator and the APC/C subunit Apc10 (Buschhorn et al., 2010; concentration at the site where Mad2–Cdc20 complexes are da Fonseca et al., 2010). Therefore, it is tempting to speculate being generated, thus promoting efficient assembly of the MCC. that KEN2 is positioned so that it engages with this same Note, because the Bub3 binding site is important for kinetochore- interface, thereby preventing substrate binding. This could based signalling, we suggest that it is not called the GLEBS motif, as explain why the MCC does not inhibit the degradation of this term implies Gle2/Rae1 binding (Wang et al., 2001), and prometaphase substrates, such as cyclin A and Nek2A, because although Bub3 might share extensive homology with Gle2/Rae1 Apc10 is not required for the recognition of these proteins as (Taylor et al., 1998), there is no compelling evidence to suggest a substrates (Izawa and Pines, 2011). Although this KEN2- functionally significant connection between BubR1 and Gle2/Rae1. mediated inhibition could reflect a bona fide pseudosubstrate mechanism, such a conclusion might be premature. Indeed, we How does the MCC inhibit the APC/C? cannot rule out the possibility that other mechanisms are at play. Once assembled, the MCC somehow binds and inhibits the For example, cryo-electron microscopy analysis of the APC/C APC/C. As discussed above, Bub3 is not essential for with and without the MCC bound show that the MCC proteins BubR1 blocks substrate binding to APC/CCdc20 4343

Fig. 8. Schematic explaining how the two KEN boxes in BubR1 promote MCC assembly and APC/C inhibition. The Cdc20–Mad2 subcomplex is generated at unattached kinetochores, and then binds the BubR1–Bub3 subcomplex in a KEN1-dependent manner to form the MCC. In turn, the MCC then binds the APC/C and inhibits substrate recruitment in a KEN2-dependent manner, possibly by occupying the substrate-binding pocket between Cdc20 and Apc10.

induce a relocation of Cdc20 within the APC/C (Herzog et al., (Morrow et al., 2005) and pSUPER/BubR1 (Kops et al., 2004) plasmids using Lipofectamine 2000 (Invitrogen). Transgenes were induced with 1 mg/ml 2009). Second, binding of Cdc20 to the APC/C is dependent on tetracycline then imaged by time-lapse microscopy 48 hours later (Morrow et al., Cdc27 in metaphase, whereas in prometaphase it is independent 2005). To monitor degradation kinetics, cells co-transfected with securin–DsRed of this subunit, presumably because of the presence of the MCC (Holland and Taylor, 2006) or cyclin-B1–Venus (Garnett et al., 2009) were imaged proteins (Izawa and Pines, 2011). Finally, despite the fact that as described (Gurden et al., 2010). To repress Blinkin, cells were transfected with siRNAs (Dharmacon) (supplementary material Table S2) using Interferin (PolyPlus) deletion of KEN2 does not affect substrate recognition, the DK2 and analysed 24 hours later. mutant can still partially inhibit APC/CCdc20 in vitro (Fig. 6) and in cells (Fig. 4E,F). Thus, it is possible that binding of BubR1 to Antibody techniques Cdc20 through KEN1 is responsible for some inhibition by The sheep anti-blinkin antibody was generated as described (Taylor et al., 2001) using amino acids 1319–1386 fused to GST. The anti-Cdc27 antibody was raised changing the conformation of Cdc20, whereas KEN2 acts by in a rabbit (Eurogentec) against a C-terminal peptide (Herzog and Peters, 2005) Journal of Cell Science inhibiting substrate binding. Nevertheless, further testing the and affinity purified using Sulfolink columns (Pierce Biotechnology). All other role of the second KEN box of BubR1 might provide further antibodies are described in supplementary material Table S3. Immunoblots were insights into how the checkpoint prevents APC/CCdc20 from done as described (Tighe et al., 2004) and visualised using either film (Biomax MR; Kodak) or a Biospecturm 500 imaging system (UVP). Western blots were ubiquitylating securin and cyclin B1, while targeting quantified using a VisionWorks LS software (UVP). Immunofluorescence was prometaphase substrates for proteolysis. done as described previously (Tighe et al., 2008). Before fixation, cells were treated with 3.3 mM nocodazole (Sigma) and 20 mM MG132 (Sigma) for 2 hours. Immunoprecipitations were performed as described (Holland et al., 2007). Closing remarks

The importance of BubR1 in terms of maintaining genome Protein expression stability is highlighted by the observation that it is mutated in the BL21(DE3)pLysS E. coli cells were transformed with pET28a plasmids (Merck) human cancer predisposition syndrome mosaic variegated encoding N- or C-terminal His-tag fusions, induced with 0.4 mM IPTG for aneuploidy (Hanks et al., 2004). The primary role of BubR1 in 16 hours at 20˚C, harvested then sonicated in lysis buffer [0.1% Triton X-100, 50 mM phosphate, pH 7.4, 300 mM NaCl, 10 mM imidazole, 0.4 mM PMSF and maintaining the fidelity of chromosome segregation is to inhibit protease inhibitor cocktail (Roche)]. Soluble proteins were incubated with TALON the APC/CCdc20. Although interfering with protein–protein beads (Clontech) for 1 hour at 4˚C then after washing, proteins were eluted using interactions using small molecules has traditionally been 300 mM imidazole. For baculovirus expression, Cdc20 and full-length BUBR1 Cdc20 cDNAs were cloned into pBAC-2cp (Merck) and co-transfected into Sf9 cells with difficult, modulating the BubR1-APC/C interaction might flashBAC Gold (GenWay Biotech) using Insect GeneJuice (Merck). Following open up new opportunities for drug discovery, with antagonists two rounds of amplification, 4 ml of baculovirus was used to infect 400 ml of Sf9 driving cells out of an aberrant mitosis and agonists inducing a cells (,26106 cells/ml) for 72 hours at 28˚C. Cells were then collected and lysed prolonged mitotic arrest, both of which can lead to cell death [1% Triton X-100, 50 mM phosphate, pH 7.4, 150 mM NaCl, 10 mM imidazole, 0.4 mM PMSF and protease inhibitor cocktail (Roche)] and the His-tagged (Huang et al., 2009; Janssen et al., 2009). proteins purified as described above.

Materials and Methods In vitro assays Cell lines and RNAi HeLa cells (Taylor and McKeon, 1997) were treated with 0.2 mg/ml nocodazole Stable HeLa cell lines were generated as described (Tighe et al., 2008) using a full- for 16 hours and mitotic cells lysed (0.1% Triton X-100, 100 mM NaCl, 10 mM length BubR1 cDNA (Taylor et al., 1998) cloned into a pcDNA5/FRT/TO-Myc- Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 20 mM b-glycerol phosphate, based vector (Invitrogen) and mutated to render it RNAi resistant. The various 10 mM NaF, 1 mM DTT, 0.2 mM PMSF and protease inhibitors). BubR1 was BubR1 mutants (supplementary material Table S1) were generated by site-directed depleted using anti-BubR1 antibodies coupled to Protein-G–Sepharose beads (GE mutagenesis (Quikchange, Stratagene) or PCR amplification. To repress endogenous Healthcare), for 1 hour at 4˚C. Apo-APC/C was then isolated from the supernatant BubR1, HeLa cells were co-transfected in 12-well plates with GFP-histone H2B using anti-Cdc27 antibodies bound to Protein-A beads (GE Healthcare). After 4344 Journal of Cell Science 124 (24)

washes, beads were incubated with recombinant proteins, each at a final Eytan, E., Moshe, Y., Braunstein, I. and Hershko, A. (2006). Roles of the anaphase- concentration of 1.5 mM in PBS containing 10% glycerol and 4 mg/ml BSA, for promoting complex/cyclosome and of its activator Cdc20 in functional substrate 45 minutes at room temperature. After washing, the beads were resuspended in binding. Proc. Natl. Acad. Sci. USA 103, 2081-2086. 15 ml of a reaction mixture (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 100 nM E1, Fang, G. (2002). Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit 1 mM UbcH10, 1.25 mg/ml ubiquitin, 7.5 mM creatine phosphate, 0.1 mg/ml anaphase-promoting complex. Mol. Biol. Cell 13, 755-766. Garnett, M. J., Mansfeld, J., Godwin, C., Matsusaka, T., Wu, J., Russell, P., Pines, creatine phosphokinase, 2 mM ATP and 2 mM MgCl2). The E1 enzyme, UbcH10 and ubiquitin were purchased from Boston Biochem. 0.2 mM of Myc–cyclin B1 J. and Venkitaraman, A. R. (2009). UBE2S elongates ubiquitin chains on APC/C N90 or Myc–securin were used as substrates. Reactions were incubated at 37˚Cin substrates to promote mitotic exit. Nat. Cell Biol. 11, 1363-1369. a Thermomixer (Eppendorf) for 20 minutes, stopped by addition of SDS sample Gurden, M. D., Holland, A. J., van Zon, W., Tighe, A., Vergnolle, M. A., Andres, buffer, then resolved on a 4–12% NuPAGE Bis-Tris gel (Invitrogen), transferred to D. A., Spielmann, H. P., Malumbres, M., Wolthuis, R. M., Cleveland, D. W. et al. a PVDF membrane (Millipore) and blotted with anti-Myc antibodies. To measure (2010). Cdc20 is required for the post-anaphase, KEN-dependent degradation of centromere protein F. J. Cell Sci. 123, 321-330. substrate binding, immunoprecipitated APC/C was incubated with recombinant Hagting, A., Den Elzen, N., Vodermaier, H. C., Waizenegger, I. C., Peters, J. M. and proteins, 1.5 mM each, for 45 minutes at room temperature, then washed in PBS Pines, J. (2002). Human securin proteolysis is controlled by the spindle checkpoint with 0.1% Triton X-100 and the APC/C eluted using 1 mg/ml peptide in PBS and reveals when the APC/C switches from activation by Cdc20 to Cdh1. J. Cell Biol. overnight at 4˚C. Eluted proteins were analysed by western blot. For substrate 157, 1125-1137. binding, immunoprecipitated apo-APC/C was first incubated with recombinant Hanks, S., Coleman, K., Reid, S., Plaja, A., Firth, H., Fitzpatrick, D., Kidd, A., proteins and then with 0.1 mM Myc–cyclin-B1 N90 or Myc–securin in PBS plus Mehes, K., Nash, R., Robin, N. et al. (2004). Constitutional aneuploidy and cancer 10% glycerol and 4 mg/ml BSA. After washes, bound proteins were eluted in predisposition caused by biallelic mutations in BUB1B. Nat. Genet. 36, 1159-1161. sample buffer and analysed by western blot. Hardwick, K. G., Johnston, R. C., Smith, D. L. and Murray, A. W. (2000). MAD3 encodes a novel component of the spindle checkpoint which interacts with Bub3p, Acknowledgements Cdc20p, and Mad2p. J. Cell Biol. 148, 871-882. Harris, L., Davenport, J., Neale, G. and Goorha, R. (2005). The mitotic checkpoint We thank Geert Kops for the BubR1 shRNA plasmid, Bill Earnshaw gene BubR1 has two distinct functions in mitosis. Exp. Cell Res. 308, 85-100. for the ACA antibody, Takahiro Matsusaka and Jonathon Pines Herzog, F. and Peters, J. (2005). Large-scale purification of the vertebrate anaphase- for the cyclin-B1–Venus construct, Jan-Michael Peters for APC/C promoting complex/cyclosome. Meth. Enzymol. 398, 175-195. antibodies, Eddie McKenzie for help with protein expression, and the Herzog, F., Primorac, I., Dube, P., Lenart, P., Sander, B., Mechtler, K., Stark, H. members of the Taylor lab for technical advice, helpful discussions and Peters, J. (2009). Structure of the anaphase-promoting complex/cyclosome interacting with a mitotic checkpoint complex. 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