The mitotic checkpoint gene BubR1 has two distinct functions in mitosis

Experimental Cell Research 308 (2005) 85 – 100 www.elsevier.com/locate/yexcr

The mitotic checkpoint gene BubR1 has two distinct functions in mitosis Loleta Harrisa,1, James Davenportb, Geoffrey Nealec, Rakesh Goorhab,d,* a

Department of Virology and Molecular Biology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794, USA b Department of Pathology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794, USA c The Hartwell Center, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794, USA d Department of Pathology, University of Tennessee, Memphis, TN 38163, USA Received 23 September 2004, revised version received 10 February 2005 Available online 23 May 2005

Abstract BubR1 is one of two putative vertebrate homologs of the yeast spindle checkpoint protein Bub1. We have used deletion and point mutants to elucidate the functions of BubR1 in mitosis. The nocodazole-activated spindle checkpoint of HeLa cells was disrupted by expression of a 39 amino acid fragment (residues 382 – 420) of BubR1 containing the Bub3-binding GLEBS motif. In contrast, we observed normal checkpoint function in a truncation mutant comprising residues 1 – 477, despite the lack of the C-terminal BubR1 kinase domain. In the absence of nocodazole, expression of the 477 amino acid fragment slowed progress through prometaphase of mitosis, causing accumulation of mitotic cells. This accumulation was also seen in a kinase dead mutant. The prolongation of mitosis required both kinetochore binding and an intact, functional spindle checkpoint. The prolongation of mitosis by kinase deficient BubR1 constructs indicates a crucial role for the BubR1 C-terminal kinase domain in chromosome movement, in addition to the role of the N-terminus in the checkpoint. D 2005 Elsevier Inc. All rights reserved. Keywords: BubR1; Bub1; HeLa cells; Mitotic checkpoint; Spindle checkpoint

Introduction In eukaryotes, the mitotic checkpoint ensures faithful transmission of chromosomes to daughter cells by preventing mitotic progression until all the chromosomes are properly attached to spindle microtubules. The presence of even a single unattached kinetochore can generate an inhibitory ‘‘wait anaphase’’ signal that prevents the cell’s entry into anaphase [1]. Proper alignment of the chromosomes inactivates checkpoint signaling and allows cells to enter anaphase to complete mitosis (reviewed in [2 –4]). The mitotic checkpoint genes were first identified in the budding yeast Saccharomyces cerevisiae and encode the

* Corresponding author. Department of Pathology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN 381052794, USA. Fax: +1 901 495 2032. E-mail address: [email protected] (R. Goorha). 1 Current address: Department of Urology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA. 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.03.036

checkpoint component proteins Mad1, Mad2, Mad3, Bub1, Bub3, and Mps1 (reviewed by [5,6]). The vertebrate homologs of these proteins associate with kinetochores during mitosis, most especially with kinetochores that are not attached to spindle microtubules (reviewed by [3,7]). Several complexes between the checkpoint proteins have been described, and these complexes are thought to generate the wait anaphase signal at unattached kinetochores [3,4,8]. The mitotic checkpoint function of the Bub and Mad proteins and their importance in normal mitosis is well established. For example, yeast cells with null mutations in Bub1 or Bub3 genes are viable, but they grow slowly, show chromosome mis-segregation, and develop aneuploidy [9– 13]. Similarly, fibroblasts grown in culture from mice with reduced levels of BubR1 show weakened mitotic arrest and aneuploidy, while the mice themselves show enhanced tumor development [14,15]. Humans with reduced BubR1 function also develop tumors at an elevated rate [16]. The mammalian mitotic checkpoint proteins differ in some ways from those in yeast. For example, there is a

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second presumed mouse homologue of Bub1 termed Bub1b or BubR1 [17 –20] whose mRNA expression is similar to that of the previously identified mouse Bub1. Sequence analysis [17] indicates that Bub1 and BubR1 proteins share four conserved regions (domains A, B, C, and D). Domain D is homologous to the catalytic domain of protein kinases, and Bub1 proteins have kinase activity [11,18,21– 23]. Domain B is necessary and sufficient for kinetochore binding and interaction with Bub3 [18,24]. The amino acid sequences of vertebrate Bub1 and BubR1 are comparably similar to yeast Bub1, making it plausible that both vertebrate proteins are homologs of the yeast protein and thus that they play similar, if distinct, roles during mitosis. However, it has also been suggested that BubR1 is a functional homologue of yeast Mad3, a smaller protein that shares homology to the amino terminal halves of Bub1 and BubR1 [6,18]. Since the functional characteristics of yeast Mad3 are distinct from those of Bub1 [12], this line of reasoning suggests that the sequence similarity between BubR1 and Bub1 may be misleading. Suppression of the activity of either Bub1 or BubR1 alone weakens the checkpoint of vertebrate cells, indicating that their functions are at least partially non-overlapping [8,21,25,26]. However, we have a very limited understanding of the functions of these proteins at the molecular level, making the assignment of one or another as homologs difficult. To better understand the role of BubR1 in the mitotic checkpoint and to identify key portions of the protein for further study, we have performed a mutation analysis of BubR1 function during mitosis and determined which domains are required for (these) function(s). Enforced expression of several BubR1 mutants, particularly those containing the B domain, disrupted the mitotic checkpoint. More importantly, we found that the amino terminal half of BubR1, homologous to Mad3, supported checkpoint activation by nocodazole and thus appears to have checkpoint function. Surprisingly, we also found that despite its normal checkpoint function, expression of the same N-terminal mutant delayed the passage of cells through mitosis in the absence of nocodazole, suggesting that the C-terminal half of BubR1 is required for the proper movement of chromosomes during mitosis. Overall, our results show that BubR1 has multiple functions during mitosis and strengthen the argument that it is more closely related to yeast Mad3 than to Bub1.

genes to be repressed by addition of doxycycline to the growth medium. However, we did not use this feature in the experiments reported in this paper. Nocodazole (Sigma, St. Louis MO) was diluted from a 1 mg/ml stock solution in DMSO to a final concentration of 0.1 Ag/ml. For synchronization purposes, cells were subjected to a single overnight incubation in medium containing 2 mM thymidine (Sigma). Plasmid construction A full-length cDNA clone of mouse BubR1 [17] was used as a template for PCR to create inserts for expression experiments. The inserts were cloned into pBI-EGFP or pTRE2hyg (BD Clontech, Palo Alto, CA). Inserts were created both with and without an amino terminal FLAG epitope. GFP fusions were constructed by inserting the EGFP coding sequence between the BamHI and MluI sites of pTRE2hyg. The BubR1 fragments were then inserted into the MluI site, carboxyl-terminal to the EGFP. EGFP expressed in parallel with BubR1 in pBI-EGFP allowed identification of cells transfected with untagged or FLAGtagged protein, while pTRE2hyg was used to express the EGFP fusions that are intrinsically fluorescent. The parent vector for GST fusions was constructed by inserting sequence encoding residues 1 – 226 of GST from pGEX4T-1 (Amersham, Piscataway, NJ) between the PvuII and MluI sites of pBI-EGFP. Mouse Bub1, Bub3, and Mad2 cDNAs were generated by RT-PCR using Superscript II and Platinum Pfx (Invitrogen, San Diego, CA) as directed by the suppliers. The QuickChange protocol (Stratagene, La Jolla, CA) was used for site-directed mutagenesis. The Bub1BubR1 hybrids were generated using the class IIS restriction enzyme BsmBI [27]. For this purpose, the B domains were considered to comprise residues 392– 477 of BubR1 and residues 237– 331 of Bub1. Transfection 2  106 HeLa cells were seeded onto 10 cm plates and transfected the following day with 6 Ag of plasmid DNA using PolyFect (Qiagen, Valencia, CA) according to the suppliers’ instructions. HeLa cells were chosen because they show no contact inhibition, allowing transfection at high cell density to avoid toxicity problems and yet providing cell proliferation that continued through the course of an experiment.

Materials and methods Cell cycle analysis Cell culture HeLa Tet-Off cells were obtained from Clontech (Palo Alto, CA) and grown in DMEM (Mediatech, Herndon, VA) supplemented with 10% fetal calf serum (HyClone, Logan, UT). In combination with the vectors used (see next section) these cells allow expression of exogenous

For flow cytometric analysis of cell cycle progression, cells were usually harvested 48 h after transfection and fixed on ice for 10 min with 1% paraformaldehyde and 0.1% sodium azide in PBS [28]. This was necessary because the GFP that marks transfected cells is soluble in 70% ethanol. The cells were permeabilized by overnight incubation in

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70% ethanol in Tris-buffered saline. When indicated, cells were incubated with rabbit antibodies to phospho (ser 10)histone H3 (Upstate, Lake Placid, NY), which were detected with APC-labeled goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR). DNA was stained with propidium iodide (40 Ag/ml). Cells were analyzed on a FACSCalibur flow cytometer using Cell Quest software (Becton Dickinson, San Jose, CA). GFP fluorescence was used to select successfully transfected cells for analysis. To avoid potential toxicity problems, analysis was limited to those cells that were no more than 10-fold brighter in the GFP channel than untransfected cells. Immunofluorescence microscopy 2  105 cells were grown on Lab-Tek two-well chamber slides (Nalge Nunc, Naperville, IL) and synchronized by thymidine block and release. Ten hours after release the cells were fixed for 30 min at room temperature in 3.7% paraformaldehyde in PBS. Cells were permeabilized by a 7min incubation with 20-C acetone, blocked by incubation with 10% normal donkey serum, and incubated 1 h with a 1:200 dilution of human CREST serum (gift of Dr. B. R. Brinkley). The bound CREST antibodies were detected with Texas red-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA). DNA was stained with TOPRO3 (Molecular Probes, Eugene, OR). The slides were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and imaged by a Leica TCS SP confocal microscope using the manufacturer’s software and a PL APO 100  oil objective (NA 1.4). Cells expressing low levels of EGFP were selected for imaging to minimize nonspecific GFP fluorescence. For microscopic analysis of transfected cells in the G2/ M phase of the cell cycle (Table 1), HeLa cells were fixed, permeabilized with 70% ethanol, and stained with propidium iodide as described for cell cycle analysis. GFP-expressing cells with 4N DNA content were then collected using a Mo-Flo cell sorter (Cytomation, Fort Collins, CO). The sorted population was deposited onto a slide using a Cytospin (Shandon, Pittsburg, PA), and observed as above.

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Results General considerations To identify the function of BubR1 domains in the mitotic checkpoint, we made several site-specific mutants or mutants lacking one or more of the domains (Fig. 2A). Each mutant was generated with either the FLAG epitope or EGFP affixed to the amino terminus or without a tag. It has not been possible to establish cell lines lacking BubR1 [14,15], so we transfected the mutants into HeLa cells and looked for perturbations that arose as the transfected mutant competed with endogenous BubR1. Transfected cells were identified by GFP fluorescence arising either from marker gene EGFP or directly from EGFP in the fusions. Mutants with or without tags caused similar biological effects, but EGFP-tagged mutants showed some quantitative differences in phenotype as compared to un-tagged or FLAG-tagged mutants (discussed later). Mutants were examined for their effects on the mitotic checkpoint by two different assays. We activated the checkpoint by treating the cells with a microtubule depolymerizing drug, nocodazole. Cells with a defective mitotic checkpoint that aberrantly exit from mitosis with a 4N DNA content may enter a new S phase and acquire a DNA content greater than 4N. In the first assay, we measured the DNA content of the transfected cells by flow cytometry. In the second assay we examined the exit from mitosis by measuring chromosome decondensation using an antibody that binds to histone H3 when serine 10 is phosphorylated; this phosphorylation correlates with chromatin condensation in mitosis [28]. In our hands, enumeration of condensed chromosomes provided more reproducible and sensitive measure of mitotic exit than the measurement of DNA content greater than 4N (data not shown). Several BubR1 mutants disrupt the mitotic checkpoint We found that several deletion mutants disrupted the mitotic checkpoint. For example, in Fig. 1 HeLa cells were transfected either with FLAG-tagged BubR1 deletion mutant AB (BubR148 – 477) or with the vector alone. Thirty

Table 1 Bub1b mutant cells are delayed in prometaphase

Control 1 Control 2 Control 3 A*AB 1 A*AB 2 A*AB 3

G2 cells (% total)

Mitotic cells (% total)

Prometaphase cells (% mitotic)

Metaphase cells (% mitotic)

Anaphase cells (% mitotic)

Telophase cells (% mitotic)

597 725 643 199 171 208

111 105 104 109 106 105

82 80 86 104 100 103

23 23 10 4 6 1

5 1 5 1 0 1

1 1 3 0 0 0

(84.3) (87.3) (86.1) (64.6) (61.7) (66.5)

(15.7) (12.7) (13.9) (35.4) (38.3) (33.5)

(73.9) (76.2) (82.7) (95.4) (94.3) (98.1)

(20.7) (21.9) (9.6) (3.7) (5.7) (0.9)

(4.5) (1.0) (4.8) (0.9) (0.0) (0.9)

(0.9) (1.0) (2.9) (0.0) (0.0) (0.0)

HeLa cells were harvested 48 h after transfection with either empty vector (‘‘control’’) or vector expressing BubR11 – 477 (‘‘A*AB’’). The cells were fixed, stained, and then sorted by flow cytometry to select transfected (GFP positive) cells that contained 4N DNA. The mitotic stage of the cells was then determined by microscopy. The number of cells observed in each phase is reported. The results of three separate experiments are shown in the table. The differences between control and mutant samples were significant for percent mitotic cells (P = 0.0082), prophase cells (P = 0.0092), and metaphase cells (P = 0.0346).

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Fig. 1. FACS analysis of nocodazole treated cells transfected with Bub1 or BubR1 AB deletion mutants. HeLa cells were transfected with empty vector (panel A) or vector encoding the AB deletion mutant of either BubR1 (panel B) or Bub1 (panel C). 32 h later nocodazole was added to the medium and cells were grown for another 18 h. After harvesting, cells were permeabilized, incubated with antibody to phosphorylated histone H3, and then with APC-labeled secondary antibody. DNA was detected by staining with propidium iodide. Successfully transfected cells were selected for analysis on the basis of GFP expression. Histone staining is indicated by the solid peaks on the histograms. Panel D shows the percentage of cells with DNA content greater than 4N. Panel E shows the percentage of cells positive for phosphorylated histone H3, representing mitotic cells with condensed chromosomes.

hours after transfection nocodazole was added and the cells were then harvested 18 h later. This treatment arrested more than 70% of control cells in mitosis. Cells expressing BubR1 AB were twice as likely to have a DNA content greater than 4N than were cells transfected with empty vector (Fig. 1D). Similarly, fewer than half as many cells transfected with the mutant stained with the phosphohistone antibody (Fig. 1E), indicating that cells expressing the mutant protein decondensed their chromosomes and exited mitosis earlier than did control transfected cells. The above results suggest that enforced expression of BubR1 mutant protein disrupts the mitotic checkpoint and allows faster exit of cells from mitosis. Enforced expression of a comparable AB fragment of Bub1 (Bub11 – 331) also caused faster exit from mitosis by both measures (Fig. 1C), as expected [29]. We examined a series of deletion mutants to identify the minimal BubR1 region(s) that may disrupt the mitotic checkpoint. Fig. 2B shows that in the presence of nocodazole, expression of FLAG-tagged mutants lacking the B domain in HeLa cells has no effect on their mitotic checkpoint. For example, cells expressing either BubR1490 – 679 (deletion mutant ‘‘C’’), BubR1645 – 1052 (‘‘D’’), or BubR1490 – 1052 (‘‘CD’’) contained condensed chromosomes at the same frequency as control cells. In contrast, mutants expressing either the B domain alone (BubR1357 – 477, ‘‘B’’) or the B region fused to downstream sequences of BubR1 (e.g., BubR1357 – 679, mutant ‘‘BC’’ or BubR1357 – 1052, mutant ‘‘BCD’’) disrupted the mitotic checkpoint and caused faster exit of the transfected cells through mitosis as indicated by the

severely reduced number of cells with condensed chromosomes (Fig. 2B). In yeast, overexpression of Mad3 disrupts checkpoint function by depleting Bub3, but inhibition in our system is a consequence of the mutant displacing the endogenous protein: (1) the A*AB mutant, which binds Bub3, does not weaken the checkpoint; (2) our constructs bind to kinetochores (Fig. 3; [30]); and (3) co-transfection with excess Bub3 does not prevent checkpoint disruption (Supplemental Fig. 2). Western blotting of whole-cell lysates with antibodies to the FLAG epitope confirmed comparable expression of most deletion constructs (not shown). However, larger proteins were often less-well represented, at least in part because transfection efficiency dropped. To avoid this effect, we often used EGFP as a fusion tag instead of coexpressing it as a marker; during FACS analysis when we selected cells with similar levels of GFP fluorescence, we were directly selecting for similar levels of EGFP-BubR1 expression. Furthermore, use of EGFP fusions allowed direct sub-cellular localization of the mutant protein. The overall pattern of checkpoint disruption by BubR1 mutants fused to EGFP is similar to that of untagged (data not shown) or FLAG-tagged mutants but the disruption is generally less severe in cells expressing EGFP-fused mutants (Fig. 2C). We generated several smaller deletion mutants of the B domain to determine the minimum region required for disruption of the mitotic checkpoint. As shown in Fig. 2D, expression of a 39 amino acid region of B domain (BubR1382 – 420, mutant B1) fused with EGFP effectively

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Fig. 2. BubR1 deletion mutants containing the B domain disrupt the nocodazole-activated mitotic checkpoint. (A) Structure of BubR1 and the deletion mutants. The regions A (residues 49 – 194), B (375 – 449), C (562 – 632), and D (740 – 1020) were defined by their homology to sequences in Bub1. The A* (10 – 43) region is not found in Bub1, but homologous sequences are found at the amino terminus of yeast Mad3 and presumed BubR1 homologs in Drosophila, Xenopus, and chicken (see Supplemental Fig. 1). (B – D) Cells were transfected with the indicated BubR1 mutants with either (B) FLAG or (C and D) EGFP Nterminal tags. Cells were treated with nocodazole and analyzed as in Fig. 1 for mitotic cells, i.e., cells with phosphorylated histone H3. To maintain a consistent scale this percentage was normalized to that in control cells. In panel D, B1 refers to a portion of the B domain extending from residue 382 to 420 that contains the GLEBS motif. Deletion mutants containing the E406K mutation, which disrupts interaction with Bub3 and kinetochore binding, fail to have an effect upon the checkpoint. Data points represent the mean of 3 different experiments, with standard deviation indicated.

disrupted the mitotic checkpoint in these cells. Surprisingly, expression of the FLAG-tagged BubR1382 – 420 mutant had only weak effects upon the checkpoint. However, Western blotting revealed little or no expression of this construct, indicating that without a large fusion partner this small peptide is unstable (data not shown). Further deletion in this region abolished checkpoint disruption (data not shown). These 39 amino acids form the core of the B domain and contain the GLEBS motif that is required for Bub3 binding and the localization of Bub1 and BubR1 proteins to kinetochores [24]. To examine the role of the GLEBS motif in mitotic checkpoint function of BubR1, we generated a mutant in which a conserved glutamic acid residue in the GLEBS motif is mutated to lysine (mutant B E406K). Fig. 2D shows that while expression of EGFP-BubR1357 – 477 (‘‘B’’) resulted in severe disruption of the mitotic checkpoint, expression of mutant EGFP-BubR1357 – 477 E406K failed to disrupt the checkpoint. Fluorescence microscopy studies revealed that EGFP-BubR1357 – 477, as expected, localized to the kinetochores in prometaphase cells (Fig. 3C). In contrast, the E406K mutation suppressed BubR1 localization to the kinetochores in mitotic cells (Fig. 3B; L.D.H. and R.G, manuscript in preparation). These results correlate checkpoint disruption with kinetochore binding and therefore suggest that BubR1357-477 competes with wild-type, endogenous BubR1 for binding to kinetochores but that once bound is unable to provide wild-type checkpoint function.

A*AB portion of BubR1 performs mitotic checkpoint function Although expression of domain B (BubR1357 – 477) drastically disrupted the mitotic checkpoint, a mutant in which the B domain was extended to include the amino terminus (BubR11 – 477, ‘‘A*AB’’) dramatically reduced the disruptive effect of the B domain on the mitotic checkpoint. For example, FLAG-tagged BubR1357-477 expression reduced the number of cells with condensed chromosomes by ¨70% whereas FLAG-tagged BubR11-477 expression caused very little reduction in the number with condensed chromosomes (Fig. 2B). In other experiments (Figs. 2C, 4B, and 5B), there was also no significant difference between cells transfected with BubR11-477 or with the vector alone (control), indicating that this deletion mutant is able to provide the normal function of BubR1 in the mitotic checkpoint. Alternatively, it is possible that fusion of the A*A domains to the B domain prevents binding of the mutant protein to the kinetochores and thus allows the endogenous BubR1 to function. However, confocal microscopic examination of mitotic cells shows that BubR11 – 477 efficiently binds kinetochores (Fig. 3A). In fact, EGFP-BubR11 – 477 binds kinetochores as efficiently as full-length EGFP-BubR1 protein (not shown; L.D.H. and R.G., in preparation). These results indicate that BubR11 – 477 binds kinetochores and provides normal mitotic checkpoint function.

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Fig. 3. The BubR1 B domain governs binding to kinetochores. Cells were transfected with plasmids encoding the indicated EGFP-BubR1 fusions. The cells were fixed 10 h after release from thymidine arrest, permeabilized and incubated with CREST serum to identify kinetochores (red) and TO-PRO to stain DNA (pseudo-colored blue), as described in Materials and methods. (A) EGFP-BubR11 – 477, ‘‘A*AB’’ binding is strong. (B) EGFP-BubR11 – 477 E406K binds at or perhaps slightly above cytosolic background levels (C) EGFP-BubR1357 – 477, ‘‘B’’ binds well (D) EGFP-BubR11 – 363, ‘‘A*A’’ does not bind appreciably. Green reports EGFP fluorescence, the red shows CREST staining of kinetochores detected with a Texas-red-labeled antibody and the blue (pseudo-colored) TOPRO. The E406K mutation also disrupts kinetochore binding by EGFP-B, but the effect is more dramatic in the context of A*AB, which binds kinetochores more strongly than B.

A considerable degree of the mitotic checkpoint activity is apparently mediated by the 47 N-terminal amino acids of BubR1, the A* region that shares sequence similarity with yeast MAD3 but that is absent in Bub1 sequences. Expression of a mutant lacking this region (BubR148 – 477, mutant ‘‘AB’’) only partially (¨50%) supported the mitotic checkpoint (Fig. 4A). Progressive removal of amino acids from N-terminal end of BubR148 – 477 further reduced the mitotic checkpoint function (not shown). To further demon-

strate the role of the A*A region in mitotic checkpoint function, we generated several site-specific mutations in BubR11 – 477. Replacement with alanine of a highly conserved lysine residue at position 19 in the A* region (mutant A*AB K19A) reduced the mitotic checkpoint function by ¨60% (Fig. 4A). Similarly, replacement of the highly conserved aspartate 67 and leucine 69 by lysine and alanine, respectively (mutant A*AB D67K, L69A), or replacement by alanines of the highly conserved GIG motif at residues

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Fig. 4. (A) BubR11 – 477 (A*AB) possess checkpoint function. Cells were transfected with FLAG-tagged deletion or site-specific mutants and then treated with nocodazole as in Fig. 1. The K19A mutant replaces a lysine conserved between Mad3 and BubR1 with alanine. In point mutant ‘‘D67K’’ the motif DPL at residues 67 – 69 is replaced with KPA. Mutant ‘‘G140A’’ has the GIG motif between residues 140 and 142 replaced with alanines. The three-point mutants in the A* and A regions inhibit the A*AB fragment from functioning in the checkpoint, underscoring the important role these two regions play in checkpoint function. Data points represent the mean of 4 different experiments. (B) Point mutations in A* and A disrupt checkpoint function in full-length BubR1. Cells were transfected with FLAG-tagged mutants and treated with nocodazole as described in Fig. 1. Note that cells expressing the kinase dead mutant (K784A) possess normal checkpoint function. Data points represent the mean of 2 different experiments.

140 –142 (mutant A*AB GIG142AAA) in the A region resulted in more than 50% reduction in the mitotic checkpoint function by the expression of these mutants. To examine whether the A*A region plays a similar role in the context of the full-length BubR1, we made the same sitespecific mutations in wild-type BubR1. As shown in Fig. 4B, all three mutants cause mitotic checkpoint disruption as evidenced by a 35 –60% reduction in the number of cells with condensed chromosomes as compared to vector alone or wild-type BubR1 expressing cell. Overall, our results strongly suggest that the A*A region plays an important role in the mitotic checkpoint function of A*AB and BubR1. Kinase-dead BubR1 mutant provides normal mitotic checkpoint function The ability of BubR11 – 477 to maintain the mitotic checkpoint indicates that the protein kinase activity of BubR1 may not be required for its checkpoint function. To directly examine the role of BubR1 kinase activity in the mitotic checkpoint, we mutated the conserved lysine (residue 784) in the catalytic site to alanine (Kinase Dead or KD mutant) to abolish the kinase activity [11,22,

23,25,31]. Fig. 4B shows that chromosome condensation was not significantly altered in cells expressing the KD mutant as compared to control cells expressing vector alone or full-length wild-type BubR1. Thus, cells expressing the kinase dead mutant have a normal mitotic checkpoint, indicating that BubR1 kinase activity may not be required for its mitotic checkpoint function. The A domain of Bub1 does not confer checkpoint function on the B domain of Bub1 The B domain of Bub1 (Bub1204 – 331) disrupted the mitotic checkpoint (Fig. 5A), as expected. More importantly, this effect was not weakened by extension of the B fragment to include the amino terminus (the AB deletion fragment, Bub11 – 331) (Fig. 5A). The inability of the Nterminus of Bub1 to confer checkpoint function to the Bub1 B domain may indicate that the B domain of Bub1 is functionally distinct from that of BubR1, despite their sequence similarity (44% similarity over the region of clear homology), or it may reflect differences in the amino termini of the two proteins (e.g., the lack of an A* domain in Bub1). To examine the latter possibility we generated a hybrid

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Fig. 5. Bub1 and BubR1 are functionally distinct. (A) The N-terminus of Bub1 does not restore checkpoint function to the B domain of Bub1. Cells were transfected with plasmids encoding untagged Bub1 deletion mutants or FLAG-tagged B domain of BubR1. Nocodazole treatment and cell analysis were as in Fig. 1. Data points represent the mean of 3 different experiments. (B) The N-terminus of BubR1 does not restore checkpoint function to the B domain of Bub1. Cells were transfected with EGFPtagged controls or with hybrid constructs and then treated with nocodazole. Bub1 structure is indicated with more darkly filled rectangles and the letter B, while BubR1 structure is indicated with lightly filled rectangles and the letter R. The N-terminus of Bub1 does not restore checkpoint function to the B domain of BubR1 (hybrid BR), nor can the A*A fragment of BubR1 confer checkpoint function upon the B domain of Bub1 (construct RB). Data points represent the mean of 3 different experiments.

mutant that contained the A*A portion of BubR1 fused to the B domain of Bub1. As seen in Fig. 5B, disruption of the mitotic checkpoint in cells expressing this mutant (‘‘RB’’) was similar to those expressing the mutant B of Bub1. Thus the A*A domain of BubR1 lacks the ability to suppress checkpoint disruption by the B domain of Bub1. Similarly, the A domain of Bub1 was unable to suppress checkpoint disruption by the B fragment of BubR1 (Fig. 5B). BubR1 mutants cause slower passage of cells through mitosis Expression of FLAG-tagged BubR11 – 477 in the absence of nocodazole increased the proportion of cells in the G2/ M phases of the cell cycle (Figs. 6A and B). A similar increase was observed in cells transfected with non-tagged BubR11 – 477 (data not shown). Analysis of the mitotic cell population by measuring histone condensation (Figs. 6A and B) showed that FLAG-tagged BubR1 mutant-express-

ing cells contained eight times as many cells in mitosis as did control cells. A survey of all our deletion mutants showed that only BubR11 – 477 and BubR148 – 477 (mutants A*AB and AB) caused cells to accumulate in mitosis (Figs. 8A and B). We also observed that the EGFP-tagged mutant, as compared to the FLAG mutant, caused more cells to accumulate in mitosis (compare Figs. 8A and B). Expression of BubR11 – 477 in 293T cells caused similar effects (not shown). In contrast, the Bub1 deletion mutant Bub11 – 331 (‘‘AB’’) did not cause accumulation of cells either in G2/M or in mitosis (Fig. 6C). To determine how expression of BubR11 – 477 affects the cell cycle, we monitored cell-cycle progression in synchronised cell populations. Thirty hours after transfection, HeLa cells were synchronised by overnight treatment with thymidine. Aliquots of cells were analyzed for their cell cycle distribution at various times following thymidine removal (Fig. 6D). At the end of thymidine treatment, both control and mutant A*AB transfected cells were effectively synchronised in G1 and early S phase. Following release, both control cells as well as mutant A*AB expressing cells resumed cell-cycle activity and began to enter G2/M at 4 h (Figs. 6D and 7A). However, differences between the two cell cultures were seen from 9 h onwards. In the control population, most of the cells completed mitosis by 11 h, and virtually all had returned to G1 at 14 h. In the BubR11 – 477 population, cells continued to accumulate in G2/M at 11 h, and at 14 h only half of the cells had returned to G1, with the remainder still in G2/M (Fig. 6D). These data show that cells expressing mutant A*AB traverse the cell cycle normally, but they take longer to pass through G2/M than do control cells. Analysis of a synchronized population for mitotic cells confirmed that expression of BubR11 – 477 delayed the passage of cells through mitosis (Fig. 7B). Cells expressing either BubR11 – 477 or full-length BubR1 both entered mitosis around 7 h after thymidine removal. However, while the number of mitotic cells in the control culture peaked at 9 h, the number of mitotic cells in the BubR11 – 477 culture continued to accumulate and did not reach a peak until 10 h and then slowly declined. Our data show that BubR11 – 477 caused more cells to accumulate in mitosis than did control constructs, and that BubR11 – 477 cells exit mitosis later than control cells (Fig. 7B). Thus, the longer time spent in G2/M by cells expressing BubR11 – 477 is mainly, if not exclusively, due to their slower passage through mitosis. Expression of BubR11 – 477 delays prometaphase To determine which phase(s) of mitosis were prolonged by BubR11 – 477 expression, transfected G2/M cells were isolated by FACS and their mitotic index (MI) was evaluated by confocal microscopy. The proportion of the deletion mutantexpressing cells undergoing mitosis was 2.5-fold higher than that of the EGFP control cells (Table 1). Within mitotic cells, the proportion of mutant-expressing cells in prometaphase (96 T 2%) was significantly higher than for control cells (78 T

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Fig. 6. Expression of BubR11 – 477 (A*AB) causes accumulation of mitotic cells in the absence of nocodazole. Cells were transfected with either empty vector (panel A) or FLAG-tagged deletion mutant A*AB of BubR1 (panel B) or AB of Bub1 (panel C) and harvested 48 h later, without nocodazole treatment. Cell harvesting, staining, and analysis were as in Fig. 1. Note that the AB deletion mutant from Bub1 (panel C) does not prolong mitosis. (D) Expression of BubR1 A*AB prolongs the G2M phase but cells pass through S and normally traverse cell cycle. Cells were transfected as in panel A and split one plate into four at 24 h after transfection. The cells were arrested in G1/S by treatment with 2 mM thymine for 18 h, and then grown for the indicated period of time after thymine was removed.

5%; P = 0.009). Equally striking, the proportion of mutantexpressing cells in metaphase (4 T 3%) was approximately 5fold lower than controls (18 T 7%, P = 0.038). These data indicate that the condensed chromosomes in BubR11 – 477 cells take longer to align on the metaphase plate, thereby resulting in a higher proportion of prometaphase cells. The proportion of metaphase cells decreases because prolonged prometaphase allows correspondingly fewer cells to reach metaphase at any given time. We could not determine the effect of mutant expression on chromosome movement during anaphase or telophase as the number of cells in these phases of mitosis was very low (Table 1). Both domains A*A and B are required for the slower passage of cells through mitosis Expression of neither EGFP-BubR11 – 363 (‘‘A*A’’) nor EGFP-BubR1357 – 477 (‘‘B’’) was sufficient to cause accumulation of cells in mitosis (Fig. 8A), indicating that only mutants possessing both A*A and B domains prolong mitosis. We observed a similar result with Flag-tagged

mutants, but expression of FLAG-tagged mutants caused accumulation of fewer cells in mitosis than did EGFP fusions (Fig. 8B). To define the region(s) of the amino terminus that play a role in slowing passage of cells through mitosis, we generated a series of mutants in which progressively more amino acids were deleted from the Nterminus of BubR11 – 477. Deletion of the A* region (the 47 N-terminal amino acids) from A*AB to give BubR148 – 477 (AB) resulted in at least 50% reduction in the capacity to cause accumulation of cells in mitosis (Fig. 9A). Progressive removal of more amino acids from the N-terminal end of AB further reduced the ability to prolong mitosis in incremental steps. If we assume that transfected protein is replacing the cell’s endogenous BubR1, this indicates that most if not all of the A*A region of BubR1 is required for the proper movement of chromosomes during mitosis (Fig. 9A). Site-specific mutations in the A* and A domains reduced the accumulation of mitotic cells, further establishing their importance in the slowing effect (Fig. 9A). To determine the minimum region of the B domain required for the transfected gene to perturb the normal

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Fig. 7. Expression of BubR11 – 477 (A*AB) causes accumulation of cells in G2M and prolongs mitosis. Cells were transfected with plasmid expressing either A*AB or full-length BubR1. Cells were synchronized by a single thymine treatment as described in Fig. 5D and harvested at the indicated times after thymine removal. Both DNA content and histone H3 phosphorylation were assessed by flow cytometry. (A) Percentage of cells with 4N DNA, i.e., in G2 or mitosis. (B) Percentage of cells positive for phosphorylation of histone H3 on serine 10, i.e., mitotic cells with condensed chromosomes.

movement of chromosomes during mitosis, we progressively deleted amino acids from the C-terminal end of BubR11 – 477. Fig. 9B shows that removal of 27 amino acids (mutant A*AB1, BubR11 – 450) did not affect the ability to perturb mitosis but removal of a further 10 (mutant A*AB2, BubR11 – 440) or 30 amino acids (mutant A*AB3, residues BubR11 – 420) reduced the accumulation of mitotic cells. Finally, mutant A*AB4 (BubR11 – 402) did not cause any slowing of mitosis. These results indicate that all of the conserved sequence of the B domain of BubR1 is required to maximally prolong mitosis. To examine whether or not kinetochore binding was necessary for this effect, we generated a site-specific mutation (E406K) that eliminated binding of EGFP-A*AB to the kinetochores (Fig. 3B; L.D.H. and R.G, in preparation). As shown Fig. 9B, mitosis was completely unaffected in cells transfected with this mutant. These results suggest that the BubR11 – 477 deletion mutant protein must bind to kinetochores for it to cause slower passage of cells through mitosis. An intact mitotic checkpoint is required for BubR11 – 477 to cause slower passage of cells through mitosis We noticed that the ability of BubR1 mutants to cause slower passage of cell through mitosis is inversely proportion to their ability to disrupt the mitotic checkpoint. In general, EGFP-tagged BubR1 deletion constructs gave reduced disruption of the mitotic checkpoint (Fig. 2) and

more pronounced slowing of mitotic progress (e.g., Fig. 8) than did FLAG-tagged mutants. Furthermore, when compared to parental BubR11 – 477, BubR11 – 477 mutants K19A, D67K, L69A, and GIG142AAA display both weaker checkpoint function in the presence of nocodazole (Fig. 4) and less dramatic slowing of mitosis in the absence of nocodazole (Fig. 9A). To directly examine the relationship between the mitotic checkpoint and slower progress of cells through mitosis, we evaluated the effect of BubR11 – 477 in cells co-transfected with constructs that disrupt the checkpoint. We employed either a C-terminal truncation mutant of Mad2 [32] or the B fragment of Bub1 to disrupt the mitotic checkpoint (Fig. 10A). When cells were transfected with Flag-tagged BubR11 – 477 or control vector and simultaneously co-transfected with an empty ‘‘background’’ vector, BubR11 – 477 caused nearly a 5-fold increase in the abundance of mitotic cells, as expected. However, when BubR11 – 477 was co-transfected into cells expressing either MAD2DC or the B domain of Bub1 from the background vector, the increase in mitotic cells was completely abrogated (Fig. 10B). These results imply that an intact mitotic checkpoint is essential for BubR11 – 477 to cause increased accumulation of cells in mitosis. Since expression of Bub11 – 331 in cells did not prolong mitosis (Fig. 6C), we examined the possibility that either replacement of the A domain of Bub1 with A*A from BubR1 or replacement of the Bub1 B domain with the B domain of BubR1 could confer the ability to retard passage through mitosis. As shown in Fig. 11, replacement of the B domain of Bub1 with that from BubR1 resulted in a modest increase (¨2) in accumulation of cells in mitosis whereas replacement of the A domain of Bub1 with A*A from BubR1 did not significantly affect the proportion of cells in mitosis. These results indicate that expression of fragments containing both A*A and B domains of BubR1 in cells is required for the maximum prolongation of mitosis. Kinase dead BubR1mutant slows the passage of cells through mitosis Since BubR11 – 477 lacks the kinase domain, our results are consistent with the notion that BubR1 kinase activity is essential for normal progression of cells through mitosis; lack of kinase activity results in slower progression through mitosis. Fig. 8A shows that as compared to control cells, four times as many cells transfected with the KD full-length BubR1 construct accumulated in mitosis. These results suggest that BubR1 kinase activity is required for the normal passage of cells through mitosis.

Discussion We have tested the effects of expressing a variety of BubR1 mutants to better define the function of this protein.

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Fig. 8. Expression of the A*AB or AB deletion mutants of BubR1 prolongs mitosis. Cells were transfected with plasmids expressing the indicated BubR1 deletion mutants fused at the amino terminus with either (A) EGFP or (B) a FLAG epitope. Cells were harvested 48 h after transfection and analyzed by flow cytometry for the proportion of cells exhibiting phosphorylation of histone H3, i.e., cells in mitosis. Comparison of panels A and B shows that mutants expressing EGFP fusions experience a more profound lengthening of mitosis. Note that the kinase dead mutant results in accumulation of mitotic cells, though to a lesser extent than in cells expressing A*AB (panel A). Data points represent the mean of 4 (A) or 3 (B) different experiments.

Our results confirm earlier finding that BubR1 is a checkpoint protein [8,21,26,30,31] and provide a coherent structural analysis of its checkpoint function. We show that expression of the B or AB domains of either BubR1 or Bub1 disrupts the spindle checkpoint but that checkpoint function appears to be restored by extending BubR1 to include the A* domain. More importantly, we find that although A*AB from BubR1 (i.e., BubR11 – 477) possesses checkpoint function, its expression prolongs or arrests mitosis, apparently by preventing microtubule capture or subsequent alignment. Our results implicate the BubR1 amino terminus in checkpoint function and the carboxyl terminus in chromosome movement. These results provide a framework for molecular descriptions of checkpoint function and for predicting the effects of mutations in BubR1 found in some tumors. We tested the effect of BubR1 mutants on the checkpoint-dependent arrest induced by treating cells with nocodazole. Arrest was evaluated 18 h after nocodazole addition, at which time the mitotic index is maximal in control cells, and accordingly the time at which checkpoint disruption is most apparent. Using this assay we found that

expression of the B domain was sufficient to disrupt the checkpoint, while expression of A*A, C, or D deletion fragments had little, if any, effect. It has recently been reported that BubR11 – 354, which lacks the B domain and corresponds closely to our A*A mutant, disrupts the checkpoint in an assay that measures the appearance of cells with elevated DNA content at times on the order of 48 h [33,34]. We have occasionally seen similar effects when we evaluate cells at similarly long times. However, the generation of cells with more than 4N DNA content requires cells not only to prematurely exit mitosis, but to avoid entering senescence or activating cell death programs and to replicate their DNA. In fact, Shin and coworkers conclude that BubR1 may exert a direct influence on these other processes, independent of effects upon the mitotic checkpoint [34]. Given these complexities, we believe the measurement of mitotic cells at earlier time points is a more reliable measure of effects specific to the spindle checkpoint. We found that the B domain governs kinetochore binding of BubR1 (Fig. 3; L.D.H. and R.G., in preparation), as reported by others [18,24]. A*AB (BubR11 – 477) binds more

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Fig. 9. Maximum prolongation of mitosis by BubR11 – 477 (A*AB) requires both N and C termini of the deletion mutant. Cells were transfected with EGFPtagged BubR1 deletion mutants as indicated, and harvested 48 h later. (A) Removal of the amino terminus of A*AB progressively decreases the increase in mitotic cells caused by expression in HeLa cells. Mutant AB spans residues 48 – 477; AB1, residues 143 – 477; AB2, residues 220 – 477; AB3, residues 313 – 477. Data points represent the mean of 5 different experiments. (B) Removal any part of the conserved B domain weakens mitotic retardation by A*AB. The region with apparent homology to the B domain of Bub1 extends from residues 375 to residue 449 of BubR1. Mutant A*AB1 comprises residues 1 – 450; A*AB2, residues 1 – 440; A*AB3, residues 1 – 420 and A*AB4, residues 1 – 402. Mutation of the conserved E406K disrupts kinetochore binding (see Fig. 3) and prevents the slowing of mitosis. Data points represent the mean of 4 different experiments.

strongly than the B domain, raising the possibility that A*A (BubR11-363) can also bind kinetochore proteins. However, if present, binding by A*A in isolation was too weak to compete effectively with endogenous full-length BubR1 or to be apparent against background fluorescence (e.g., Figs.

3B and D), though such binding has been reported by Baek et al. [33]. We also found that a 39 residue fragment containing the most highly conserved region of the B domain (residues 382 – 420) is sufficient to disrupt the mitotic checkpoint. This fragment contains the GLEBS

Fig. 10. Accumulation of cells in mitosis by BubR11 – 477 (A*AB) requires an active checkpoint. (A) Expression of either Mad2DC or the B domain of Bub1 disrupts the mitotic checkpoint. Cells were transfected with FLAG-tagged deletion mutants, treated with nocodazole, and analyzed as described in Fig. 1. Data points represent the mean of 3 different experiments. (B) Mitotic slowing by A*AB is eliminated by checkpoint disruption. Cells were transfected with 4 Ag of either empty pTRE2hyg or pTRE2hyg expressing either Mad2DC or the B domain of Bub1, as indicated. The cells were co-transfected with 2 Ag of either pBIEGFP (‘‘vector’’) or pBI-EGFP expressing A*AB from BubR1 (‘‘A*AB’’). Data points represent the mean of 4 different experiments.

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Fig. 11. The B domains of Bub1 and BubR1 are not functionally equivalent. Cells were transfected with hybrid constructs in which the B domains of Bub1 and BubR1 were exchanged. Cells were harvested after 48 h and analyzed as in Fig. 6. Bub1 structure is indicated with more darkly filled rectangles and the letter B, while BubR1 structure is indicated with lightly filled rectangles and the letter R. The Bub1 AB deletion mutant reduces the mitotic cell population below that seen in controls, consistent with its ability to disrupt the spindle checkpoint. The BubR1 N-terminus attached to the B domain of Bub1 (construct RB) had little effect, but the Bub1 N-terminusBubR1 B domain hybrid (construct BR) show a modest (¨2-fold) increase. Data points represent the mean of 3 different experiments.

motif, first identified in nucleoporins and more recently shown to mediate Bub3 and kinetochore binding by Bub1 and BubR1 [24]. Replacement of a highly conserved glutamate in the GLEBS motif with lysine (E406K mutation) drastically reduced binding of the EGFP fusion proteins to kinetochores and at the same time abolished effects upon the checkpoint, just as similar mutations in yeast Mad3 and Bub1 weaken interaction with Bub3 and consequently prevent kinetochore binding [12,35]. Because kinetochore binding appears to depend upon Bub3 binding, it is possible that the checkpoint disruption we attribute to kinetochore binding is really caused by excess exogenous BubR1 mutants binding and depleting free Bub3 [12]. However, this is not the case since co-transfecting cells with excess Bub3 did not prevent checkpoint disruption (Supplemental Fig. 2). Furthermore, cells transfected with A*AB could not have the normal checkpoint they exhibit if Bub3 depletion was an issue. Thus, our results indicate that kinetochore binding by BubR1 deletion mutants is essential to their ability to disrupt the mitotic checkpoint. By extension, these results argue that normal BubR1 function requires kinetochore binding. Unexpectedly, expression of BubR1 deletion mutant A*AB (BubR11 – 477) caused little or no disruption of the mitotic checkpoint (Figs. 2, 4, and 5). This suggests that the A*AB fragment is able and sufficient to perform mitotic checkpoint function of BubR1. A* is only 34 residues long, but our results show that it plays an important role in BubR1 checkpoint function. A* is absent from Bub1 but present in yeast and plant Mad3 and presumed BubR1 homologs in invertebrates. Accordingly, BubR1 has been regarded as a Mad3-like kinase [18]. Further support for homology

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between Mad3 and BubR1 is provided by studies showing both proteins can form a complex with Cdc20 and Mad2 [22,31,35 – 37]. Our observation of checkpoint activity in the portion of BubR1 homologous to Mad3 adds to the evidence of true homology. The similarity between the Bub1 and BubR1 kinase domains in both protein sequence and intron – exon structure suggests gene duplication followed by secondary loss of the kinase domain in yeast to create Mad3. Our experiments with chimeric hybrids between Bub1 and BubR1 indicate that the functions of homologous regions in the two proteins are now quite distinct (Figs. 5 and 11). Because we only see phenotypic effects of transfection with mutants that can bind to the kinetochore, we have interpreted our data to mean that mutant proteins with intact B domains bind Bub3 and then displace endogenous BubR1 from functionally critical sites on kinetochores. It is instead possible that inhibition of checkpoint function results from assembly of BubR1 deletion and point mutants into the MCC, the complex thought to inhibit Cdc20 activity [7,36,37]. The requirement for the B domain could then indicate a role for Bub3 in assembly of BubR1 into the MCC. Extension of the B domain to include the N-terminus presumably restores checkpoint activity because the Nterminus contains the functional sequences needed for the MCC to inhibit Cdc20, as it does for direct Cdc20 inhibition by BubR1 in vitro [31]. The absence from BubR11 – 477 of potential negative regulatory elements in the C-terminus of BubR1 could then explain retarded exit from mitosis without the need to invoke a distinct role for BubR1 in kinetochore – BubR1 interactions. We do not currently favor this interpretation for several reasons. (1) Extension of the B domain to the C terminus confers Cdc20-inhibition to BubR1 in vitro [31], but this is not reflected in checkpoint activity of transfected cells (Fig. 2). (2) BubR11 – 525, similar to but slightly larger than our A*AB (BubR11 – 477), does not inhibit Cdc20 in vitro [31]. (3) Chronic checkpoint activation should arrest cells in metaphase, not prometaphase as does A*AB (Table 1). (4) Depletion of BubR1 by RNAi would not be expected to perturb chromosome alignment, yet such an effect has been reported [26]. (5) Finally, if Bub3 helps assemble BubR1 into the MCC, we might also expect Bub1 to be present in the MCC, which it is not [31]. Nonetheless, our understanding of the MCC is incomplete, so it remains possible that BubR1 deletion mutants exert some of their effects through the MCC. Our results indicate that the checkpoint function of BubR1 resides in the N-terminal half of the protein. The kinase dead mutant and BubR11 – 477 both appear to support normal checkpoint function (Figs. 2 and 4), and the checkpoint function of BubR11 – 477 responds to the same mutations that partially disrupt checkpoint function in fulllength BubR1 (Fig. 4). This is consistent with results from yeast where kinase activity of Bub1 is dispensable for checkpoint function and where Mad3 completely lacks kinase activity [12,38]. Furthermore, BubR1 kinase activity

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is not needed for in vitro BubR1 inhibition of Cdc20, the presumed product of checkpoint activity [31,36]. However, kinase dead BubR1 fails to restore checkpoint function to T98G cells depleted of endogenous BubR1 by siRNA treatment [39]. A possible explanation for conflicting results in vertebrates may be supplied by the observation that only 10 –20% of endogenous kinase-active BubR1 can cooperate with added kinase dead BubR1 to support normal checkpoint function in Xenopus oocyte extracts [8,30]. In all our experiments an excess of mutant protein must compete with the endogenous, wild-type BubR1 to interfere with its function and thus to give the observed phenotype. Since only a small amount of BubR1 protein is required to fulfill the kinase requirement, it is possible that in cells transfected with BubR11 – 477 or kinase dead mutant that endogenous BubR1 is able to provide the requisite kinase activity despite competition from mutant protein. Recently it was shown that patients with mosaic variegated aneuploidy have a single functional BubR1 allele with C-terminal missense mutations [16]. This suggests that essential function resides in the N-terminus, while also indicating an important role for the C-terminus. Characterization of the mitotic defects in cells from these patients may bear on the role of kinase activity. More definitive results for vertebrate cells may be provided by the use of recently described conditional BubR1 knockout cell lines [15]. Surprisingly, we found evidence that BubR1, in addition to its requirement for the mitotic checkpoint, plays a role in the movement of chromosomes during mitosis. Cells expressing BubR11 – 477 accumulated in prometaphase, indicating that they require more time to complete prometaphase and align their chromosomes at the metaphase plate (Fig. 7B and Table 1). These results strongly imply that BubR1 is necessary for the optimal movement of chromosomes during prometaphase. A functional checkpoint is necessary to observe the slower movement of chromosomes during mitosis; BubR11 – 477 expression failed to slow passage through mitosis in cells with a disrupted checkpoint (Fig. 10). Thus, in cells expressing BubR11 – 477 the normal function of BubR1 in chromosome movement is compromised, activating the mitotic checkpoint (which BubR11 – 477 supports) and therefore prolonging prometaphase and causing accumulation of mitotic cells. This requirement for intact BubR1 for efficient microtubule interaction with chromosomes likely underlies the observation of aberrant chromosome binding to the spindle in cells depleted of BubR1 [26]. Ditchfield et al. treated cells with siRNA and observed both checkpoint loss and perturbed chromosome – spindle interactions. However, because BubR1 siRNA treatment weakened the checkpoint, they could not observe any mitotic delay caused by the treatment (see Fig. 10). The structural resolution afforded by out transfection experiments allowed us to separate the two effects and directly observe the delay in cells expressing BubR11 – 477, which supports checkpoint function. While

checkpoint disruption might explain some of the aberrations observed in the siRNA experiments, our observation of perturbed chromosome alignment in checkpoint-competent cells indicates the BubR1 siRNA treatment did in fact interfere with chromosome movement. While this paper was in revision Lampson and Kapoor reported that loss of BubR1 destabilizes chromosome-spindle attachments, further demonstrating a role in for BubR1 in chromosome mechanics as well as in the checkpoint proper [40]. Expression of the KD mutant that has lost its kinase activity also caused slower progression of cells through mitosis (Fig. 8A), though the effect is smaller than that created by BubR11 – 477. The fact that mitotic cells accumulate in response to expression of both BubR11 – 477, which lacks the kinase domain, and to the KD mutant indicates that kinase activity is required for normal movement of chromosomes during mitosis. We do not yet understand why the KD mutant has a smaller effect than BubR11 – 477. Perhaps sequences missing from BubR11 – 477 but not involved in kinase activity play a role in chromosome movement. Alternatively, BubR11 – 477 may compete more efficiently with endogenous BubR1 for sites involved in chromosome movement than does the KD mutant. It is noteworthy that the same KD mutant efficiently substituted the wild-type BubR1 for the normal mitotic checkpoint function (Fig. 4B). Apparently, the requirement for the BubR1 kinase activity for chromosome movement during mitosis is much more stringent than for the mitotic checkpoint. It is likely that the effect of BubR1 on chromosome movement is mediated through its interaction with the kinetochore-associated kinesin-like motor protein centromere-associated protein E (CENP-E). The kinetochores of HeLa cells normally bind 20 or more microtubules (reviewed by [3]). CENP-E is required for efficient capture and stabilization of these microtubules by the kinetochores; depletion of CENP-E reduces the number of bound microtubules, slows chromosome congression, and consequently prolongs mitosis [41]. CENP-E binds to kinetochores through the kinase domain of BubR1 [20,30]. We propose that loss of BubR1 kinase activity impairs the CENP-E function resulting in failure to efficiently capture microtubules or in the slower movement of chromosomes during mitosis.

Acknowledgments We thank Josie Harris-Chambers, Rashmi Tiwari, Daniela Seminara, and Trushar Jeevan for providing excellent technical assistance. We also thank Dr. Richard Cross for assistance in flow cytometric analysis of cells, Ken Barnes and Dr. Gopal Murti for confocal microscopy, and Dr. Katsumi Kitagawa for helpful discussions. CREST serum was a generous gift of Dr. B. R. Brinkley of Baylor College of Medicine.

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This work was supported by grant CA092597 from NCI to R.G. and by the American Lebanese and Syrian Associated Charities (ALSAC).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2005. 03.036.

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