© 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 2975-2982 doi:10.1242/jcs.169821

SHORT REPORT Dissecting the roles of human BUB1 in the spindle assembly checkpoint Mathijs Vleugel1, Tim A. Hoek1,*, Eelco Tromer1,2,*, Tale Sliedrecht1, Vincent Groenewold1, Manja Omerzu1 and Geert J. P. L. Kops1,3,‡

ABSTRACT (SAC) activity. The SAC is activated at unattached and Mitotic segregation is initiated by the results in the formation of a soluble inhibitor of the anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20 promoting complex/cyclosome (APC/C) with its co-activator CDC20 (forming APC/CCDC20). APC/CCDC20 is inhibited by the spindle CDC20 (APC/C ), a multisubunit E3 ligase of which the assembly checkpoint (SAC) when have not attached activity initiates chromosome segregation and mitotic exit by to spindle . Unattached kinetochores catalyze the targeting key mitotic regulators for proteasomal destruction (Pines, formation of a diffusible APC/CCDC20 inhibitor that comprises 2011). The soluble inhibitor of the APC/C is known as the mitotic BUBR1 (also known as BUB1B), BUB3, (also known as checkpoint complex (MCC) and comprises MAD2 (also known as MAD2L1) and a second molecule of CDC20. Recruitment of these MAD2L1), BUBR1 (also known as BUB1B), BUB3 and CDC20 to the , as well as SAC activation, rely on the (Chao et al., 2012; Izawa and Pines, 2015; Kulukian et al., 2009; mitotic kinase BUB1, but the molecular mechanism by which BUB1 Sudakin et al., 2001). accomplishes this in human cells is unknown. We show that The SAC response is initiated at the KMN network through kinetochore recruitment of BUBR1 and BUB3 by BUB1 is phosphorylation of multiple motifs in repeat sequences of KNL1 dispensable for SAC activation. Unlike its yeast and nematode (also known as CASC5), which is mediated by MPS1 (also known orthologs, human BUB1 does not associate stably with the MAD2 as TTK) (London et al., 2012; Shepperd et al., 2012; Vleugel et al., activator (also known as MAD1L1) and, although required for 2015; Yamagishi et al., 2012). When phosphorylated, these motifs – accelerating the loading of MAD1 onto kinetochores, BUB1 is recruit BUB1 BUB3 dimers that are essential for SAC signaling dispensable for the maintenance of steady-state levels of MAD1 and chromosome bi-orientation (Krenn et al., 2014; Primorac et al., there. Instead, we identify a 50-amino-acid segment that harbors the 2013; Vleugel et al., 2013, 2015; Zhang et al., 2014). Since the recently reported ABBA motif close to a KEN box as being crucial for discovery of BUB1, the molecular mechanism by which it the role of BUB1 in SAC signaling. The presence of this segment participates in SAC activation has been a matter of controversy. correlates with SAC activity and efficient binding of CDC20 but not of Studies in mice, budding yeast and fission yeast have shown that MAD1 to kinetochores. BUB1 kinase activity is required for SAC activation (Kawashima et al., 2010; Ricke et al., 2012; Yamaguchi et al., 2003), but this has KEY WORDS: BUB1, Kinetochores, , Spindle assembly been contradicted by other studies using the same model organisms checkpoint (Baker et al., 2009; Fernius and Hardwick, 2007; London and Biggins, 2014; Perera et al., 2007; Rischitor et al., 2007; Warren INTRODUCTION et al., 2002). Human cell studies have proven equally inconsistent – During mitosis, all chromosomes have to attach to microtubules of one study proposes that BUB1 directly phosphorylates and inhibits the mitotic spindle and become bi-orientated before cells are CDC20 (Kang et al., 2008), whereas another shows that a truncated allowed to proceed into anaphase. Chromosome attachment is BUB1 lacking the kinase domain significantly restored SAC mediated by kinetochores, large multi-protein structures that form activity to BUB1-depleted cells (Klebig et al., 2009). Recent the bridge between chromosomes and spindle microtubules (Foley studies show that in budding yeast and Caenorhabditis elegans, and Kapoor, 2013). binding activity at kinetochores is BUB1 directly binds to MAD1 through a region preceding the mainly provided by the KMN network, a 10-subunit (S. cerevisiae), or through the kinase domain itself assembly comprising three subcomplexes KNL1-C–MIS12-C– (C. elegans) (London and Biggins, 2014; Moyle et al., 2014). NDC80-C (Cheeseman et al., 2006). Whether this function of BUB1 is conserved in human cells is In addition to the formation of kinetochore–microtubule unknown. In addition to MAD1 and MAD2 as well as BUB3, attachments, the KMN network plays a key role in coupling BUB1 is essential for recruiting BUBR1 and CDC20 to kinetochore attachment status to spindle assembly checkpoint kinetochores (Johnson et al., 2004; Kang et al., 2008; Klebig et al., 2009; Sharp-Baker and Chen, 2001). It is at present unknown how BUB1 accomplishes this, and it is unclear if this is functionally 1Molecular Cancer Research, University Medical Center Utrecht, Utrecht 3584 CG, relevant for SAC signaling. 2 The Netherlands. Theoretical Biology and Bioinformatics, Department of Biology, Here, we set out to identify the molecular role of BUB1 in SAC Science Faculty, Utrecht University, Utrecht 3584 CH, The Netherlands. 3Cancer Genomics Netherlands, University Medical Center Utrecht, Utrecht 3584 CG, signaling in human cells. We provide evidence that promoting The Netherlands. localization of BUBR1 to kinetochores is not a crucial aspect of the *These authors contributed equally to this work function of BUB1 in the SAC and that BUB1-mediated recruitment ‡ Author for correspondence ([email protected]) of MAD1 to kinetochores is insufficient for SAC function. We instead find a strong correlation between SAC activity, binding of

Received 5 February 2015; Accepted 2 July 2015 CDC20 to kinetochores and the presence of 50 residues in BUB1 Journal of Cell Science

2975 SHORT REPORT Journal of Cell Science (2015) 128, 2975-2982 doi:10.1242/jcs.169821 that harbor two motifs that, in other proteins, are known to bind to 2014). LAP–BUB1WT that had been immunoprecipitated from CDC20. -treated cells associated with known interacting proteins – such as BUB3, BUBR1 and KNL1 – as determined by RESULTS AND DISCUSSION using mass spectrometry (Fig. 2A). However, no MAD1 peptides BUBR1 kinetochore localization is dispensable for the SAC were identified in these BUB1 purifications (Fig. 2A). Furthermore, To uncover the contribution of BUB1 to the SAC, we examined ectopic tethering of BUB1 to a LacO array (LacI–BUB1) was which region of BUB1 is required to localize BUBR1 to insufficient to recruit MAD1, whereas endogenous BUBR1 was kinetochores. We therefore removed various domains and motifs readily detected at those LacO foci (Fig. 2B). These observations that have been previously implicated in SAC function from GFP- contradict the notion of the existence of a stable BUB1–MAD1 based localization and affinity purification (LAP)-tagged wild-type complex in human cells. We next wanted to determine the extent to BUB1 (LAP–BUB1WT), creating LAP–BUB11–318,LAP–BUB11–500 which BUB1 contributes to MAD1 kinetochore localization. Cells and LAP–BUB11–778 (Fig. 1A). All of the RNA interference that had been depleted of BUB1 were treated with nocodazole for (RNAi)-resistant constructs were stably integrated into HeLa Flp 3 h, fixed and stained for MAD1 by using immunofluorescence. recombination target (HeLa-FRT) cells at a single doxycycline- Although BUB1 kinetochore levels were reduced to <5% in both inducible locus (Klebig et al., 2009). Recruitment of BUBR1 to the HeLa and RPE-1 cells, we observed no difference in MAD1 kinetochores in nocodazole-treated cells was abolished through kinetochore localization compared with that in control cells RNAi-mediated depletion of BUB1, and this was rescued by (Fig. 2C–F). SAC sensitization through partial MPS1 inhibition expression of LAP–BUB1WT (Fig. 1B,C). All BUB1 truncation (250 nM reversine) reduced the levels of MAD1 at kinetochores but, mutants localized to kinetochores to a similar degree (Fig. 1B,C), as even in this case, RNAi-mediated BUB1 depletion did not further expected (Krenn et al., 2012), and were able to restore BUBR1 reduce MAD1 levels (Fig. 2C–F). Taken together, these data argue levels at kinetochores upon BUB1-targeted RNAi (Fig. 1B,C). We against a role of human BUB1 as the predominant receptor for thus conclude that the N-terminal 318 amino acids of BUB1, MAD1 in human cells. encompassing a TPR domain and GLEBS motif, are sufficient for Although our observed lack of correlation between BUB1 and BUBR1 kinetochore recruitment. This redefines the region in BUB1 MAD1 at kinetochores agrees with some reports (Hewitt et al., that is required for BUBR1 recruitment to a conserved α-helix 2010; Vleugel et al., 2013), it contrasts with others (Kim et al., following the GLEBS motif (amino acid residues 271–318) 2012; Klebig et al., 2009). We noted that although we assessed the (Overlack et al., 2015). levels of MAD1 at kinetochores in cells that had been treated with We next asked whether the BUB1–BUB3 interaction is required nocodazole for several hours, others did so in unperturbed cells, to recruit BUBR1 to kinetochores. To circumvent the complicating which are likely to have spent less time in mitosis (Klebig et al., issue that disrupting this interaction (by mutating the GLEBS motif) 2009). To test the effects of this, we measured MAD1 kinetochore also prevents kinetochore binding of BUB1 (Taylor et al., 1998), levels at different times after kinetochores that were BUB1 was artificially tethered to kinetochores by fusing it to the devoid of MAD1 had been forced to detach and to recruit MAD1, KMN network protein MIS12 (LAP–MIS12–BUB1WT) (Fig. 1A). through the addition of nocodazole (Fig. 2G,H). In control cells, BUBR1 localization was recovered when endogenous BUB1 was BUB1 and MAD1 rapidly accumulated at kinetochores, with replaced with MIS12–BUB1, but not when the mutation E252K kinetochore levels peaking at 10 and 20 min (of BUB1 and MAD1, was introduced into the GLEBS motif (MIS12–BUB1E252K) respectively) (Fig. 2G,H). As expected, BUB1-depleted cells had (Fig. 1D,E) (Overlack et al., 2015). accumulated similar amounts of MAD1 as control cells after To determine whether BUBR1 kinetochore localization is 40 min. Strikingly, however, in the first 20 min, MAD1 levels were required for SAC signaling, cells expressing MIS12–BUB1E252K substantially lower in BUB1-depleted cells (Fig. 2G,H). BUB1 thus were analyzed for SAC activity by filming mitotic progression in the accelerates efficient loading of MAD1 onto kinetochores, presence of nocodazole and a low dose of the MPS1 inhibitor potentially through the previously identified CD1 motif (Klebig reversine (250 nM) (Santaguida et al., 2010). Cells that had been et al., 2009), but is not essential for it. These findings show that the depleted of BUB1 exited mitosis within 1 h, whereas control cells mechanism of MAD1 binding to kinetochores in human cells is, at and BUB1-depleted cells that expressed MIS12–BUB1WT were least in part, different from that in budding yeast and C. elegans,in able to maintain a functional SAC for over 5 h (Fig. 1F). which BUB1 is the primary kinetochore receptor for MAD1 Surprisingly, although MIS12–BUB1E252K did not recruit (London and Biggins, 2014; Moyle et al., 2014). Human BUB1 detectable levels of BUBR1 to kinetochores (Fig. 1D,E), it fully appears to catalyze recruitment of MAD1 to kinetochores, perhaps restored SAC function (Fig. 1F). Conversely, a BUB1 fragment that by forming transient interactions to bring MAD1 to kinetochores or was sufficient for BUBR1 kinetochore localization (LAP–MIS12– by licensing kinetochores for MAD1 binding. BUB11–318, supplementary material Fig. S1A,B) could not support SAC activation (Fig. 1F). Taken together, these observations Amino acids 501–550 in human BUB1 are essential for SAC suggest that a primary role of kinetochore BUBR1 lies not in SAC activation activation but most likely in other processes, such as chromosome To assess whether accelerating MAD1 kinetochore loading through bi-orientation through recruitment of the phosphatase PP2-B56 BUB1 affects SAC signaling, we analyzed various BUB1 truncation (Kruse et al., 2013; Suijkerbuijk et al., 2012; Xu et al., 2013). mutants (BUB11–318, BUB11–500 and BUB11–778) for their ability to rescue SAC function and MAD1 loading after BUB1 depletion. The Human BUB1 is not required for the stable association of SAC defect that was induced by BUB1 depletion was rescued by – – – MAD1 to unattached kinetochores but does accelerate its expression of BUB1WT, BUB11 778, BUB11 696 and BUB11 555, – – loading but not by BUB11 318 or BUB11 500, suggesting that that the region We next asked whether human BUB1 promotes SAC activation between amino acids 501–555 is essential for SAC function by forming a stable complex with MAD1, as it does in budding (Fig. 3A). In our analyses, therefore, the BUB1 kinase domain was yeast and C. elegans (London and Biggins, 2014; Moyle et al., not required for the SAC. Importantly, although BUB1-depleted Journal of Cell Science

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Fig. 1. BUB1-mediated recruitment of BUBR1 to kinetochores is not required for the SAC. (A) Schematic representation of BUB1 truncation mutants used in B–E, asterisk indicates E252K mutation. (B,C) Representative images (B) and quantification (C) of BUBR1 localization at kinetochores in HeLa Flp-in cells that expressed LAP–BUB1 constructs. The quantification in C shows kinetochore intensity (means+s.d.) as a ratio over the intensity of CENP-C at kinetochores; n=10 cells. Levels at the kinetochores were normalized to 1 in control cells for BUBR1 and in BUB1WT cells for LAP–BUB1. (D,E) As in A,B, with the indicated – constructs. (F) Time-lapse analysis of HeLa Flp-in cells that expressed LAP–MIS12–BUB1WT, LAP–MIS12–BUB1E252K and LAP–MIS12–BUB11 318. Cells were transfected with siRNAs against luciferase (siLUC) or BUB1 (siBUB1), synchronized with thymidine, and treated with nocodazole and 250 nM reversine. Time from nuclear envelope breakdown to mitotic exit was scored by examining the cell morphology using differential interference contrast imaging. Data show the cumulative fraction of cells that had exited mitosis. n=50 cells, representative of three experiments. cells had reduced levels of MAD1 at kinetochores 10 min after the (Fig. 3B,C). Poor SAC signaling in BUB11–500-expressing cells addition of nocodazole to metaphase cells, cells that expressed cannot therefore be explained by impaired MAD1 loading, or, as 1–500 BUB1 recruited normal amounts of MAD1 in that time span shown in Fig. 1, by impaired binding of BUBR1 to kinetochores. Journal of Cell Science

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Fig. 2. BUB1 promotes efficient kinetochore loading of MAD1. (A) Mass spectrometry analysis of LAP–BUB1 following immunoprecipitation from nocodazole- treated cells (Noco). Coverage represents amino-acid-sequence coverage. (B) Immunolabeling of MAD1 (top) and BUBR1 (bottom) in U2OS-LacO cells that had been transfected with LacI–LAP–BUB1WT. Insets show enlargement of indicated areas. (C–F) Representative images (C,E) and quantification (D,F) of BUB1 and MAD1 localization at kinetochores of HeLa Flp-in (C,D) or RPE (E,F) cells. Quantification was performed as described in Fig. 1C. n=10 cells, representative of two experiments. Protein levels at the kinetochores were normalized to 1 in siLUC cells without reversine (Rev). (G,H) Representative images (G) and quantification (H) of BUB1 and MAD1 localization to kinetochores in HeLa Flp-in cells after 1 h of treatment with MG132 followed by the addition of nocodazole for the indicated times. Quantification was performed as described in Fig. 1C. n=20 cells, representative of two experiments. Protein levels at the kinetochores were normalized to

1 at the time point showing the highest localization. siLUC and siBUB1, siRNAs against luciferase and BUB1, respectively. Data are means+s.d. Journal of Cell Science

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Fig. 3. Recruitment of CDC20 by BUB1 to kinetochores correlates with SAC activity. (A) Time-lapse analysis of HeLa Flp-in cells that expressed LAP–BUB1 variants, as in Fig. 1F. (B,C) Representative images (B) and quantification (C) of HeLa Flp-in cells that expressed LAP–BUB1 variants and were treated with MG132 for 1 h followed by 10 min of nocodazole. Quantification was performed as described in Fig. 1C. n=10 cells, representative of three experiments. – (D) Sequence logo of metazoan BUB1501 555 depicting the extent of sequence conservation in the region of residues 501–550. (E,F) Representative images (E) and quantification (F) of nocodazole-treated HeLa Flp-in cells that expressed LAP–BUB1 variants and had been stained for LAP–BUB1 (GFP), CDC20 and CENP-C. Quantification was performed as described in Fig. 1C. n=10 cells, representative of three experiments. Proteins levels at the kinetochores were normalized to 1 in control cells for CDC20 and in BUB1WT cells for LAP–BUB1. (G) Immunolabeling of CDC20 in U2OS-LacO cells that had been transfected with LacI–LAP–BUB1WT (top) or LacI–LAP–BUB1E252K (bottom). siLUC and siBUB1, siRNAs against luciferase and BUB1, respectively. Data are means+s.d. Insets show enlargement of indicated areas. Journal of Cell Science

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Fig. 4. Model of BUB1 functional regions. Check marks denote that the region is essential for the indicated process. BUB3/BUBR1, a complex of BUB3 and BUBR1; KT, kinetochore; TPR, tetratricopeptide repeat domain; CD1, conserved domain 1; H2A, histone H2A.

The SAC function of BUB1 correlates with its ability to recruit CDC20 formation before nuclear envelope breakdown (Rodriguez- CDC20 Bravo et al., 2014). Alternatively, by ensuring CDC20 kinetochore Amino acids 501–555 of BUB1 harbor KEN-box motif 1 (Kang localization, BUB1 might promote SAC signaling by allowing et al., 2008). In addition, our alignment of metazoan BUB1 kinetochore-driven modification of CDC20 or by mediating a orthologs revealed the presence of a conserved [F/Y]xx[F/Y]x[D/E] conformational change in CDC20 that allows it to bind to C-MAD2. motif (amino acids 527–532) (Fig. 3D). The yeast protein Acm1 Because BUBR1 is not required at unattached kinetochores for SAC utilizes a similar motif known as the ‘A motif’ in addition to its KEN activity, we propose a model in which C-MAD2–CDC20 dimers box to interact with and thereby inhibit the CDC20 yeast ortholog that have formed at kinetochores interact with cytoplasmic BUBR1– Cdh1 (Enquist-Newman et al., 2008; He et al., 2013). Because BUB3 dimers. recent reports have shown the existence of a similar motif in BUBR1 (dubbed the Phe box or ICDC20BD) that is important for a stable MATERIALS AND METHODS BUBR1–CDC20 interaction and for SAC activity (Diaz-Martinez Plasmids et al., 2015; Lischetti et al., 2014), we tested whether this region in pCDNA5-LAP-BUB1WT was generated by ligation of LAP–BUB1WT into BUB1 is involved in the localization of CDC20 to kinetochores. the Xho1 and Hpa1 site of pCDNA5-LAP-KNL1 (Vleugel et al., 2013) and – CDC20 localizes strongly to kinetochores in nocodazole-treated encodes full-length siRNA-resistant BUB1. LAP BUB1-truncation WT constructs were generated by introducing stop codons into LAP– cells, and this depends on BUB1 (Fig. 3E,F). Although BUB1 WT – – WT 1–555 1–500 BUB1 . LAP MIS12 BUB1 was generated by PCR-mediated and BUB1 restored CDC20 kinetochore levels, BUB1 did amplification of MIS12 to include two Xho1 sites, and then ligation into not (Fig. 3E,F), showing that, indeed, the region between residues the Xho1 site of LAP–BUB1WT. LAP–MIS12–BUB1E252K was generated 501–555 is responsible for kinetochore-targeting of CDC20. by introduction of a lysine substitution into LAP–MIS12–BUB1WT using Furthermore, LacI–BUB1 efficiently recruited CDC20 to ectopic site-directed mutagenesis. sites in U2OS-LacO cells (Fig. 3G). This depended on the GLEBS motif (Fig. 3G) and, therefore, most likely required the presence of Cells culture and transfection BUBR1–BUB3, although we cannot exclude a potential separate Tissue culture and transfection were performed as described previously function of the GLEBS-motif (Fig. 3G). Thus, the A-box- (Vleugel et al., 2013). Expression of the constructs was induced by containing segment, as well as BUBR1, was required for the treatment with 2 µg/ml doxycycline for 24 h. The siRNA against BUB1 ′ ′ ability of BUB1 to recruit CDC20. This agrees with a recent study (5 -GAAUGUAAGCGUUCACGAA-3 ) was transfected into cells twice by the Pines laboratory, published while our manuscript was under using HiPerfect (Qiagen) for HeLa cells or Lipofectamine RNAiMAX (Life Technologies) for RPE cells for 24 h at 20 nM. revision, which shows that recruitment of CDC20 to kinetochores requires the A-box-like motifs (referred to as the ABBA motif in that Immunofluorescence and antibodies study) of both BUB1 and BUBR1 (Di Fiore et al., 2015). To retain Cells were grown on 12-mm coverslips and treated as indicated in the figure consistency in nomenclature, we will hereafter refer to the A-box- legends with nocodazole (830 nM), MG132 (5 μM) and reversine like motif in BUB1 as the ABBA motif. Although direct (Di Fiore (250 nM). Cells were fixed and stained as described previously (Vleugel et al., 2015), the BUB1–CDC20 interaction might be transient et al., 2013). All images were acquired on a DeltaVision RT microscope because CDC20 peptides were not detected in BUB1 (Applied Precision) with a 100×1.40 NA U Plan S Apochromat objective immunoprecipitations by using mass spectrometry (Fig. 4). We (Olympus) using Softworx software (Applied Precision). Images are conclude that, although BUB1 promotes loading of MAD1 to maximum intensity projections of deconvolved stacks and were quantified unattached kinetochores, its ability to activate the SAC correlates as described previously (Vleugel et al., 2013). The primary antibodies used more strongly with its ability to recruit CDC20 to kinetochores. We were GFP-Booster (ChromoTek), rabbit anti-BUB1 and rabbit anti-BUBR1 thus propose that this constitutes the essential role of BUB1 in SAC (Bethyl Laboratories), mouse anti-MAD1 (a gift from Andrea Musacchio, MPI, Dortmund, Germany) rabbit anti-CDC20 (Santa Cruz), guinea pig signaling. anti-CENP-C (MBL International) and CREST (anti-centromere In order to understand SAC activation and MCC assembly, it will antibodies) (Cortex Biochem). Secondary antibodies were Alexa-Fluor- be important to study the ABBA motif and KEN box motifs in 488, -568 and -647 (Molecular Probes). BUB1, and the role of CDC20 kinetochore binding in the SAC. CDC20 recruitment to kinetochores through BUB1 might bring Live-cell imaging CDC20 in close proximity to newly formed closed (C-)MAD2 and Cells were grown on 24-well plates and synchronized using 2 mM thus facilitate the formation of C-MAD2–CDC20 dimers, as thymidine for 24 h. Cells were released into nocodazole (830 nM) and has recently been proposed for nuclear-pore-mediated C-MAD2– reversine (250 nM). Filming started 4–6 h after thymidine release and was Journal of Cell Science

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performed at 37°C under 5% CO2 with a 20×0.5 NA UPFLN objective on a Kawashima, S. A., Yamagishi, Y., Honda, T., Ishiguro, K.-I. and Watanabe, Y. microscope (model IX-81; Olympus) controlled by Cell-M software (2010). Phosphorylation of H2A by Bub1 prevents chromosomal instability Science (Olympus). Images were acquired using a CCD camera (ORCA-ER; through localizing shugoshin. 327, 172-177. Kim, S., Sun, H., Tomchick, D. R., Yu, H. and Luo, X. (2012). Structure of human Hamamatsu Photonics) and processed using ImageJ software. Mad1 C-terminal domain reveals its involvement in kinetochore targeting. Proc. Natl. Acad. Sci. USA 109, 6549-6554. Mass spectrometry Klebig, C., Korinth, D. and Meraldi, P. (2009). Bub1 regulates chromosome Cells were synchronized in mitosis by using a 24-h thymidine block, segregation in a kinetochore-independent manner. J. Cell Biol. 185, 841-858. followed by overnight treatment with nocodazole. LAP–BUB1 expression Krenn, V., Wehenkel, A., Li, X., Santaguida, S. and Musacchio, A. (2012). was induced for 24 h, and cells were harvested, followed by Structural analysis reveals features of the spindle checkpoint kinase Bub1- kinetochore subunit Knl1 interaction. J. Cell Biol. 196, 451-467. immunoprecipitation and mass spectrometry analyses, as described Krenn, V., Overlack, K., Primorac, I., van Gerwen, S. and Musacchio, A. (2014). previously (Vleugel et al., 2013). KI motifs of human Knl1 enhance assembly of comprehensive spindle checkpoint complexes around MELT repeats. Curr. Biol. 24, 29-39. Conserved motif analysis Kruse, T., Zhang, G., Larsen, M. S. Y., Lischetti, T., Streicher, W., Kragh Nielsen, Conservation of BUB1 residues was determined by a Jackhmmer run with T., Bjorn, S. P. and Nilsson, J. (2013). Direct binding between BubR1 and B56- J. Cell Sci. human BUB1 as query on the UniProt database. Only metazoan species PP2A phosphatase complexes regulate mitotic progression. 126, 1086-1092. were included in the analysis. Kulukian, A., Han, J. S. and Cleveland, D. W. (2009). Unattached kinetochores catalyze production of an anaphase inhibitor that requires a Mad2 template to Acknowledgements prime Cdc20 for BubR1 binding. Dev. Cell. 16, 105-117. We thank members of the Kops laboratory for discussions. Lischetti, T., Zhang, G., Sedgwick, G. G., Bolanos-Garcia, V. M. and Nilsson, J. (2014). The internal Cdc20 binding site in BubR1 facilitates both spindle assembly Competing interests checkpoint signalling and silencing. Nat. Commun. 5, 5563. The authors declare no competing or financial interests. London, N. and Biggins, S. (2014). Mad1 kinetochore recruitment by Mps1- mediated phosphorylation of Bub1 signals the spindle checkpoint. Dev. 28, Author contributions 140-152. London, N., Ceto, S., Ranish, J. A. and Biggins, S. (2012). Phosphoregulation of M.V., T.H., E.T., T.S., V.G. and M.O. performed experiments. M.V. and G.J.P.L. Spc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores. Curr. Biol. designed experiments and wrote the manuscript with input from the other authors. 22, 900-906. Moyle, M. W., Kim, T., Hattersley, N., Espeut, J., Cheerambathur, D. K., Oegema, Funding K. and Desai, A. (2014). A Bub1-Mad1 interaction targets the Mad1-Mad2 This work was supported by grants from the Netherlands Organization for Scientific complex to unattached kinetochores to initiate the spindle checkpoint. J. Cell Biol. Research [grant number NWO-Vici 865.12.004]; and from the European Research 204, 647-657. Council [grant number ERC-StG KINSIGN to G.J.P.L.K.]. Overlack, K., Primorac, I., Vleugel, M., Krenn, V., Maffini, S., Hoffmann, I., Kops, G. J. P. L. and Musacchio, A. (2015). A molecular basis for the differential roles of Supplementary material Bub1 and BubR1 in the spindle assembly checkpoint. 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