Spindle checkpoint function requires Mad2-dependent Cdc20 binding to the Mad3 homology domain of BubR1

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Research Article

Spindle checkpoint function requires Mad2-dependent Cdc20binding to the Mad3 homology domain of BubR1

James Davenporta, Loleta D. Harrisb,1, Rakesh Goorhaa,c,⁎aDepartment of Pathology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794, USAbDepartment of Virology and Molecular Biology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis,TN 38105-2794, USAcDepartment of Pathology, University of Tennessee, Memphis, TN 38163, USA

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. Department of PathoE-mail address: [email protected]

1 Current address: Department of Urology,

0014-4827/$ – see front matter © 2006 Elsevidoi:10.1016/j.yexcr.2006.02.018

A B S T R A C T

Article Chronology:Received 13 October 2005Revised version received11 February 2006Accepted 13 February 2006Available online 4 April 2006

The mitotic spindle assembly checkpoint delays anaphase until all chromosomes achievebipolar attachment to the spindle microtubules. The spindle assembly checkpoint proteinBubR1 is thought to act by forming an inhibitory complex with Cdc20. We here identify twoCdc20 binding sites on BubR1. A strong Cdc20 binding site is located between residues 490and 560, but mutations that disrupt Cdc20 binding to this region have no effect uponcheckpoint function. A second Cdc20 binding site present between residues 1 and 477 ishighly specific for Cdc20 already bound to Mad2. Mutation of a conserved lysine in thisregion weakened Cdc20 binding and correspondingly reduced checkpoint function. Ourresults indicate that there may be more than one checkpoint complex containing BubR1,Mad2, and Cdc20. They also lead us to propose that in vivo checkpoint inhibition of Cdc20 isa two-step process in which prior binding of Mad2 to Cdc20 is required to make Cdc20sensitive to inhibition by BubR1. Thus, Mad2 and BubR1 must cooperate to inhibit Cdc20activity.

© 2006 Elsevier Inc. All rights reserved.

Abbreviations:APC, Anaphase promoting complexMCC, Mitotic checkpoint complex

Keywords:BubR1Cdc20Mad2Bub1Bub3HeLa cellMitotic checkpointSpindle assembly checkpointMCCMitotic checkpoint complex

Introduction

During mitosis in eukaryotic cells, the spindle assemblycheckpoint inhibits progression into anaphase until all chro-

logy, St. Jude Children's R(R. Goorha).University of Texas M.D.

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mosomes are properly attached to microtubules of themitoticspindle [1]. Genetic screens in yeast first identified Mad1,Mad2, Mad3, Bub1, Bub3 and Mps1 as components of thecheckpoint machinery [2,3]. Several years later, it was estab-

esearch Hospital, Memphis, TN 38105, USA. Fax: +1 901 495 2032.

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http://dx.doi.org/10.1016/j.yexcr.2 006.02.018

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lished that the Anaphase Promoting Complex (APC) drivesprogression into anaphase and through the later stages ofmitosis bydirecting theproteolysis of securin and cyclin B [4,5].Shortly thereafter, the spindle checkpointwas shownto inhibitthe APC and consequently prevent progression into anaphaseby in vitro experiments withXenopus oocyte extracts [6] and bygenetic experiments in yeast [7]. More detailed studiesidentified Cdc20, the substrate recognition factor of the APC,as the immediate target of checkpoint inhibition [8,9].

Vertebrate homologs of Mad1, Mad2, Bub1, Bub3 and Mps1have been identified. These proteins assemble into severalcomplexes on vertebrate cell kinetochores during prophase[10–12]. Yeast Bub1 apparently has two vertebrate homolo-gues, Bub1 and BubR1. These three proteins share homologyover an amino terminal region (“A”), a Bub3 binding domain(“B”) and a carboxyl-terminal kinase domain (“D”). A homologyregion preceding the kinase domain is evident only in ver-tebrate Bub1 and BubR1 and has been associated with BubR1binding to Cdc20 (“C”) [13]. However, BubR1may instead be thevertebrate homolog of a different yeast checkpoint protein,Mad3 [14]. Mad3 is shorter than Bub1 and lacks the kinasedomain and C domain of Bub1 and BubR1, but possesses boththe A and B domains (Supplemental Fig. 1). The suggestion of acloser relationship between BubR1 and Mad3 is based both onthe presence of a sharedN-terminal extension of theA domain(“A*”) that is absent from Bub1 and on the lack of any othercandidate for the vertebrate Mad3 homolog [14]. This notionhas been strengthened by observations that suggest bothproteins participate directly in Cdc20 inhibition along withanother checkpoint protein, Mad2. In yeast, checkpoint acti-vation correlates well with formation of a complex betweenMad3, Cdc20 and Mad2. Bub3, which constitutively binds toMad3, is also part of the complex [15–17]. Although no similarin vivo experiments have been reported for vertebrate BubR1,in vitro experiments show that vertebrate Cdc20 can be in-hibited independently by either Mad2 [18] or BubR1 [13] andthat inhibition can be synergistic when both are present at lowconcentrations [19].

Despite the evidence suggesting vertebrate BubR1andyeastMad3are homologs, there are differences between theproteinsthat may indicate they are divergent. Cdc20 has been reportedto bind to residues close to theCdomainof BubR1, a region thatis absent from yeast Mad3 [13]. Furthermore, BubR1 has up-streamfunction in the checkpoint as exemplified by its bindingto CENP-E, a kinesin-like motor protein involved in stabilizingkinetochore attachment tomicrotubules [20–22]. This suggestsa closer relationship to Bub1, which also has a role in chro-mosome dynamics beyond its checkpoint function [23,24].

We have assessed the checkpoint activity of deletion andsite specific mutants of BubR1 by transfecting them into HeLacells. The pattern suggests that BubR11–477, the N-terminalpart of BubR1 that is homologous to Mad3, can supportcheckpoint function [25] despite its reported failure to bindCdc20 [13]. Since Cdc20 binding is thought to be essential forBubR1 checkpoint function, we have made a detailed study ofCdc20 binding to BubR1. We find that Cdc20 binds tightly toresidues adjacent to the C domain, and that point mutationsof the conserved FDE motif between residues 524 and 526abolish binding. Interestingly, however, loss of Cdc20 bindingto this region does not interfere with checkpoint function in

our transfection assay. We have also observed that in thepresence of Mad2, Cdc20 binds to the N-terminus of BubR1,resulting in formation of a Mad2-Cdc20-BubR11–477–Bub3complex. This binding depends critically upon the A* domainof BubR1, as does checkpoint function. Our results indicatethat BubR1 can form different complexes with Cdc20, and thatonly those involving Mad2 and the amino terminus of BubR1are inhibitory. They also suggest that in vivo checkpoint inhi-bition of Cdc20 is a two-step process in which prior binding ofMad2 to Cdc20 is required to make Cdc20 sensitive to inhi-bition by BubR1. Our results also build the case for functionalhomology between BubR1 andMad3, and open the question asto the function of the Cdc20 binding site in the C domains ofBubR1 and Bub1.

Materials and procedures

Cell culture and transfection

HeLa Tet-Off cells (Clontech, Palo Alto, CA) were grown inDMEM (Mediatech, Herndon, VA) supplementedwith 10% fetalcalf serum (HyClone, Logan, UT). 2 × 106 HeLa cells wereseeded onto 10 cm plates and transfected the following daywith 6 μg of plasmidDNAusing PolyFect (Qiagen, Valencia, CA)according to the supplier's instructions. When used, nocoda-zole (Sigma, St. Louis MO) was diluted from a 1 mg/ml stocksolution in DMSO to a final concentration of 0.1 μg/ml.

Plasmid construction

A full length cDNA clone of mouse BubR1 was used as atemplate for PCR to create inserts for expression experiments[26]. We cloned the inserts into pBI-EGFP (BD Clontech, PaloAlto, CA). The parent vector for GST fusions was constructedby inserting sequence encoding residues 1–226 of GST frompGEX-4T-1 (Amersham, Piscataway, NJ) between the PvuII andMluI sites of pBI-EGFP. The bi-directional promoter on pBI-EGFP drives expression of EGFP in parallel with expression ofthe cloned insert, allowing identification of transfected cells.Mouse Bub1, Cdc20, Bub3 and Mad2 cDNAs were generated byRT-PCR using Superscript II, Platinum Pfx (Invitrogen, SanDiego, CA) and RNA from C57B/6mice [27]. They were insertedinto pBI-EGFP that had been modified to contain a triple FLAGepitope tag 5′ to the cDNA. Mad3 defective yeast strains KH45and KH160 were gifts from Kevin Hardwick, as was the Mad3genomic clone pKH502. The QuickChange protocol (Strata-gene) was used for site-directed mutagenesis.

Checkpoint assay

For checkpoint assays, we added nocodazole to the cell culturemedium 30 h after transfection and cultured the cells foranother 18 h. The cells were harvested and fixed on ice for10 min with 1% paraformaldehyde and 0.1% sodium azide inPBS to prevent loss of marker GFP from transfected cells. Thecells were permeabilized by overnight incubation in 70%ethanol in Tris-buffered saline (“TBS”). 2 × 106 cells werewashed twice in TBS and then incubated at room temperaturewith 100 μl of 1:100 rabbit antibody to phospho (ser 10)-histone

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H3 (Upstate, Lake PlacidNY). After 2 h at room temperature thecells were washed with 5 ml PBS–1%BSA and incubated in100 μl of 1:30 APC-labeled goat anti-rabbit secondary antibody(Molecular Probes, Eugene OR) for 30min in the dark. DNAwasstainedwith propidium iodide (40 μg/ml) after washing in PBS–1%BSA.Weanalyzed the cells on a FACSCalibur flow cytometerusing Cell Quest software (BectonDickinson, San Jose, CA). GFPfluorescence was used to select transfected cells for analysis.

Pull down assays

We transfected 2 × 106 cells 1 day after plating with 3 μg ofplasmid encoding a GST–BubR1 fusion and 1 μg each of tripleFLAG-taggedBub3, Cdc20 andMad2. Non-specific binding to thebeads increases with the amount of captured GST, so we typi-cally used only 0.5–1.0 μg of GST control plasmid because GST ismoreefficiently captured than the fusions. The cellswere lysed48 h after transfectionwith 1ml of lysis buffer (50mMTris–HCl(7.3), 150mMNaCl, 1%NP-40, and1mMEDTAcontainingRocheprotease inhibitor cocktail) per 107 cells. The GST fusions andassociated proteins were captured by incubating 0.5 ml ofclarified lysate with 25 μl of Glutathione Sepharose 4B beads(Amersham) for 4 h at room temperaturewith steady agitation.Thebeadswere collectedby centrifugation andwashed6 timeswith lysis buffer. Proteins were eluted with SDS sample buffer.FLAG-tagged proteins co-purifying with the GST fusions weredetected by probing Western blots with M2 antibody to theFLAG epitope (1:4000; Sigma, St. Louis MO) followed by HRPconjugated α-mouse antibody (1:4000; Amersham). The GSTfusions were detected with 26H1 antibody (1:4,000; Cell Signal-ing, Beverly, MA). α-tubulin was detected with DM A1 mono-clonal antibody (1:1000; Sigma). We detected the HRP on thesecondary antibodies with ECL Plus (Amersham).

Yeast two-hybrid

pBridge-Mad2 was created by inserting the mouse Mad2 ORFbetween the NotI and BglII sites of pBridge (BD Clontech, PaloAlto, CA). BubR1 inserts were created by PCR and ligated

Fig. 1 – BubR11–477 has checkpoint function. (A) Structure of BubR1(375–449), C (562–632) and D (740–1020) were defined by their homin Bub1, but homologous sequences are found at the amino termvertebrates and Drosophila. (B) BubR11–477 supports checkpoint fuindicated BubR1 deletion mutants. The cells were treated with ndetermine themitotic index, as described inMaterials and procednumber of mitotic cells, while BubR11–477 does not.

between the BamHI and SalI sites of either pGBT9 (BDClontech) or pBridge-Mad2. Mouse Cdc20 was inserted intothe EcoRI site of pACT2 (BD Clontech). AH109 yeast cells (BDClontech) were co-transformedwith bait and prey plasmids assuggested by the supplier. Cells were selected for growth onmedium lacking both leucine and tryptophan. Colonies fromthese plates were then streaked onto medium lackinghistidine, leucine, and tryptophan, but containing 5 mM 3-amino-triazole (Sigma, St. Louis MO).

Results

We have recently reported that the amino terminal half ofBubR1 (BubR11–477; Fig. 1A) is sufficient for spindle checkpointfunction [25]. To determine which parts of BubR1 are directlyinvolved in checkpoint function we assessed the effect ofnocodazole upon HeLa cells transfected with BubR1 deletionmutants. Nocodazole disrupts the mitotic spindle, causing acheckpoint-dependent arrest in prometaphase. We evaluatedcheckpoint function by exposing cells to nocodazole for 18 hand then measuring the number of mitotic cells by FACS ana-lysis of phospho-histone H3, a marker of condensed chromo-somes which identifies mitotic cells [25,28]. As shown in Fig.1B, transfectionwith BubR1357–477 reduced the accumulation ofmitotic cells in response to nocodazole treatment, indicatingthat it weakened checkpoint function. This portion of BubR1contains the B domain that directs both Bub3 binding andkinetochore localization [14,25,29]. Presumably BubR1357–474

togetherwith Bub3 is assembled into a kinetochore checkpointcomplex in place of full length BubR1, but cannot provide pro-per function [25]. However, BubR11–477, which extends the Bdomain to the N-terminus, does not disrupt the checkpointresponse. Since BubR11–477 binds to kinetochores as well orbetter than BubR1357–477, the simplest explanation for thispattern is that transfected BubR11–477 can perform the check-point functions of full length BubR1 [25].

Because Cdc20 binding is thought to be an essential com-ponent of BubR1 checkpoint function, we expected BubR11–477

and the deletionmutants. The regions A (residues 49–194), Bology to sequences in Bub1. The A* (10–43) region is not foundinus of yeast Mad3 and presumed BubR1 homologs innction. HeLa cells were transfected with vector encoding theocodazole for 18 h before harvesting and FACS analysis toures. BubR1357–477 disrupts checkpoint arrest and reduces the


to bind Cdc20, despite a report that residues between positions525 and 700 govern Cdc20 binding [13]. We thereforemeasuredCdc20 binding to a series of C-terminal deletion mutants ofBubR1. We transfected different GST–BubR1 fusions into HeLacells along with FLAG-tagged Cdc20. Bub3, which binds cons-titutively to BubR1 through the B domain, was also included asa control. The GST fusions were captured from cell lysates onglutathione beads and the binding of Bub3 and Cdc20 wasdetected by Western blotting with anti-FLAG antibodies. Al-though binding of large proteins to glutathione beads is poor[30], we could detect Cdc20 binding to GST fused to full lengthBubR1 (not shown). Cdc20 did not bind to BubR11–477, but didbind weakly to BubR11–560 andmore strongly to BubR11–649 andto BubR11–679 (Fig. 2A). This confirms the earlier report [13] thatascribed Cdc20 binding to a region corresponding to residues

Fig. 2 – Cdc20 binds to BubR1 between residues 490 and 560. (A) Ccotransfected with plasmids encoding GST–BubR1 fusions and trco-purifying with the fusion on glutathione beads were detectedfusions were detected with antibody to GST (lower panel). Note tselectively enriched on the beads because they are smaller. Theirthe relatively low levels of the larger fusions. Note also that Cdc2normalized to the yield of Bub3 or the GST fusion. The last three laas compared to 20% of the bead fractions in the rest of the blot. Thonly a small fraction co-purifies with the beads. Proportionatelyfusions. (B) Alignment of the chicken (accession number AY24543sequences. (C) Mutation of the 519–529motif disrupts Cdc20 bindithe “F524A” mutant. The GST pull-down experiment was perfordoes not weaken checkpoint function. Cells were transfected withas in Fig. 1B.

518 through 689 of mouse BubR1. Further confirmation wasafforded by an internal deletion mutant lacking residues 525–700, which did not bind Cdc20, though it could bind Bub3 andCENP-E (not shown). We cotransfected FLAG-tagged Bub3along with the Cdc20 and GST–BubR1 fusions in these experi-ments. Bub3 binds constitutively to the B domain of BubR1(residues 375–449) [14,29] and thus serves as a positive control.The high sensitivity and low background of the antibody to theFLAGepitope also allowsus to use the Bub3 as ameasure of theamount of theGST fusion bound to beadswhen the signal fromthe GST antibody is too weak (e.g., Fig. 2A, the BubR11–649 lane).

Because the BubR1 sequence conferring Cdc20 binding in-cludes theCdomain (residues 562–632),weusedGST fusions tointernal segments of BubR1 to determinewhether this domainplayed a role in Cdc20 binding. As shown in Fig. 2A, the GST

dc20 binds to residues between 490 and 560. HeLa cells wereiple FLAG-tagged Bub3 and Cdc20. Cdc20 and Bub3by immunoblotting for the FLAG epitope (upper panel). Thehat minor proteolysis fragments of the GST fusion areintensity is exaggerated by overexposure needed to bring out0 binding to BubR11–649 appears low, but is strong oncenes on top and bottom represent 2% of the whole cell lysates,e composition of the FLAG-tagged proteins does not vary, andmore of the GST fusion is purified, especially of the smaller3), mouse (AF107296) and Xenopus BubR1 (AY095442) proteinng. Residues 524–526 (FDE)were allmutated to alanine to givemed as in part B. (D) Mutation or deletion of the F524A motifplasmids encoding the indicated BubR1mutants and treated


fusion to a large 190 residue segment that includes theC domain and at least 45 residues on each side (GST–BubR1490–679) bound Cdc20 well, as did the smaller fragmentGST–BubR1490–648 truncated to end just after the C homologyregion. Surprisingly, removal of the entire C homology re-gion weakened but did not eliminate Cdc20 binding (GST–BubR1490–560), while GST–BubR1530–679, which lacks residuesupstream of the C homology domain, did not bind Cdc20 at all(Fig. 2A).A smaller fusion,GST–BubR1500–539, bound low levelsofCdc20 in some experiments, but the binding was inconsistent.Thus residues between490 and560 are necessary and sufficientfor BubR1 binding to Cdc20. The beads selectively capturesmaller degradation products of most GST–BubR1 fusions,creating a family of lower mass bands on the blots for the GSTfusions in Figs. 2A and C. Alignment of the BubR1 sequences in

Fig. 3 – Mad2 can bind BubR1490–679 through Cdc20. (A) Mad2 co-pwere co-transfected with plasmids encoding GST or GST–BubR14

Mad2 and Cdc20.Mad2 and Cdc20 co-purifyingwith the GST fusioin Fig. 2A. (B) BubR1490–679 can bind a Cdc20 deletion that cannotwere tested for their ability to bind BubR1490–679. The Cdc20 deletexperiment was performed without Mad2, the same result obtaiMad2 binding to BubR1490–679. Cdc20154–499 was cotransfected wiCdc20 C-terminus prevents binding to BubR1. FLAG-tagged Cdc20The different mutants were expressed at comparable levels, but

this region frommouse, Xenopus, and chicken revealed a singlestretchof conservedaminoacidsbetweenpositions519 and529of the mouse sequence (Fig. 2B). Replacement of the invariantFDE motif within this region with three alanines (“F524A”mutant) abolished the strongbindingofCdc20 to both the largerfragment containing the C domain, GST–BubR1490–679, and tothe smaller GST–BubR490–560 (Fig. 2C). A single point mutanttargeting F524 had a similar effect, while a D525A mutantretained a low level of Cdc20 binding (not shown).

Yeast Cdc20 can bind both Mad3 and Mad2 simultaneously[15–17]. We therefore tested to see if Mad2 could bind to GST–BubR1 fusions via Cdc20.We used the GST–BubR1490–679 fusionbecause it binds more Cdc20 (Fig. 2C) and so gives a bettersignal. Cdc20 binds well to GST–BubR1490–679 whether or notMad2 is present (Fig. 3A, lanes 2 and 3). In contrast, Mad2 did

urifies with GST–BubR1490–679 in the presence of Cdc20. Cells90–679 and the indicated triple FLAG-tagged combination ofns on glutathione beadswere detected byWestern blotting asinteract with Mad2. Two Cdc20 N-terminal deletion mutantsion mutants were stable when expressed [31]. Although thisns in the presence of Mad2. (C) Cdc20154–499 does not supportth BubR1490–679 with and without Mad2. (D) Deletion of thedeletionmutants were cotransfected with GST–BubR1490–679.only the full length Cdc20 bound to the BubR1 fusion.


not co-purify with GST–BubR1490–679 when Cdc20 was absent(Fig. 3A, lane 4), indicating as expected that Mad2 does notbind directly to BubR1490–679. However, Mad2 is known to bindCdc20 [13,31] and so may co-purify with Cdc20 if its bindingsite does not overlap with that for BubR1. Confirming thisspeculation, when both Mad2 and Cdc20 are present together,Mad2 co-purifies with GST–BubR1490–679 (Fig. 3A, lane 3). Thus,Cdc20 can bindMad2 and BubR1 simultaneously, linkingMad2to BubR1. Mad2 binding to Cdc20 does not appreciably changeCdc20 binding to BubR1 (compare lanes 2 and 3 Fig. 3A). Therelative amount of Mad2 revealed on the blot can significantlyexceed that of Cdc20 at higher levels of transfected Mad2,suggesting that Mad2 oligomers may bind Cdc20 under suchconditions (not shown). Consistent with the idea that Cdc20bridges Mad2 and BubR1490–679, the F524A triple mutationeliminated co-purification of Mad2 with the GST-C, just as iteliminated Cdc20 binding to GST–BubR1490–679 (not shown).Since Mad2 binding is not required for Cdc20 binding toBubR1490–679, we suspected that Cdc20154–499, a deletionmutantlacking the N-terminalMad2 binding region [31], would be able

Fig. 4 – BubR11–477 binds to Cdc20–Mad2. (A) Mad2 stimulates Cdthe samemanner as in Fig. 2A. (B) Mad2 binds to GST–BubR11–477

still bind to BubR11–477, and do so without Mad2. The yield of GSyield of the Cdc20 deletion when compared to lane 5. (D) The A*GST–BubR1 fusions were co-transfected into HeLa cells along wi

to bind BubR1. In fact, Cdc20154–499 binds BubR1490–679 aswell asdoes full length Cdc20, confirming the Mad2 independence ofthis interaction (Fig. 3B). As expected, Cdc20154–499 bindingdoesnot support co-purificationofMad2withGST–BubR1490–679 (Fig.3C), providing further support for the indirect nature of theinteraction between Mad2 and BubR1.

The WD domain of Cdc20 comprises residues 173–471 [32]and thus accounts for most of Cdc20154–499. The binding ofCdc20154–499 to BubR1490–679 therefore suggested to us that thebinding site for BubR1490–679 might reside in theWD domain ofCdc20. Consistent with this, Cdc20211–499, in which the WDdomain of Cdc20 has been disrupted, fails to bind BubR1490–679

(Fig. 3B), as do Cdc20174–499 and Cdc20311–499 (not shown). Asimple BubR1490–679 binding motif on Cdc20 between residues154 and 175 can be ruled out because the C-terminal deletionsCdc201–174, Cdc201–210 and Cdc201–310 were also unable to bindBubR1 (Fig. 3D). Thus the binding of Mad2 to Cdc20 allows it toco-purify with GST–BubR1490–679, but Mad2 binds to a part ofCdc20 distinct from the BubR1 binding site and does notinfluence Cdc20 binding to BubR1490–679. In particular, our

c20 binding to BubR11–477. The experiment was performed inthrough Cdc20. (C) Cdc20 lacking theMad2 binding region canT fusion in lane 4 was low, probably accounting for the lowerdomain of BubR11–477 governs Cdc20 binding. Variousth a mixture of triple FLAG-tagged Cdc20, Bub3 and Mad2.


results suggest that the binding site for BubR1490–679 resides ina region of the WD domain of Cdc20 that is well separatedfrom the Mad2 binding motif in the amino terminal domain.Cdc20 binding to BubR1490–679 was insensitive to loss of the Cmotif (residues 74–86; Fig. 3B) or the IR motif from Cdc20(residues 494–499; Supplemental Fig. 2), both of whichcontribute to APC binding [32,33]. Thus APC and BubR1 bindingto Cdc20 may not be mutually exclusive.

Since Cdc20 binding is expected to be critical for BubR1function, we tested the nocodazole response of HeLa cellstransfected with full length BubR1 either with or without theF524A triplemutation. Themutationhadno apparent effect oncheckpoint function in our usual assay (Fig. 2D). Similarly,deletion of residues between 525 and 700 from full lengthBubR1 did not weaken checkpoint mediated arrest. In adifferent measure of checkpoint function, we observed cellsafter 36 h treatment with nocodazole, at which point the cellshave bypassed the checkpoint arrest and begun to developpolyploidy. The cell cycle profiles were uninfluenced by thepresence of the F524Amutation (not shown). Furthermore, thecell cycle profile of cells grown without nocodazole wasidentical in cells expressing BubR1 with or without the F524Atriple mutation, as was the rate of loss of transfected cellsduring 6 days of growth after transfection (not shown). Thus,Cdc20 binding to this motif is not required for normal BubR1checkpoint function in our assays.

Although we did not usually observe Cdc20 binding toBubR11–477 (e.g., Figs. 2A and B), we did occasionally detectsuch binding. We tested variations in the pull down protocol,including the effect of phosphatase inhibitors, but did notfind conditions that strengthened this inconsistent signal.However, during experiments designed to measure Mad2

Fig. 5 – A Cdc20 mutation that weakens Mad2 binding also disrubinds Mad2 poorly. GST fused to Cdc20 (“wt”) or its double mutaFLAG-taggedMad2 and co-purification of theMad2with the Cdc20background, but very weak. (B) Cdc20 K129A/P137A binds poorlybinds substantial levels of Cdc20 in the absence of exogenous Mpresumably because the concentration of endogenous Mad2 is toCo-transfection with Mad2 enhances Cdc20 binding (more obvioan even stronger effect on the mutant (lane 5), presumably by elebut significant fraction of the mutant binds Mad2.

binding to GST–BubR1490–679, we also examined Mad2 bindingto GST–BubR11–477. These experiments show consistentlystronger binding of Cdc20 to GST–BubR11–477 in the presenceof Mad2 (e.g., Figs. 4A, B, and C). In early experiments thedependence on exogenous Mad2 was nearly absolute (Fig.4A), while in more recent experiment we typically observesome binding in the absence of exogenous Mad2 (e.g., Fig. 4Blane 2 and C lane 3), perhaps reflecting current use of earlierpassage cells that may express higher levels of endogenousMad2. Cotransfection with Mad2 often reduced the yield ofGST fusion and correspondingly of Cdc20, suggesting thattransfected cells were dying during Mad2-induced mitoticarrest. We have recently found that inclusion of thymidine inthe culture medium for the last 18 h before cell harvestminimizes this apparent Mad2 toxicity (e.g., SupplementalFig. 4), and so makes the Mad2 stimulation of Cdc20 bindingconsistently apparent. The Mad2 stimulation of Cdc20binding to BubR11–477 is not secondary to cell cycle effectsof Mad2 since it persists in cells cultured in the presence ofeither nocodazole or thymidine (Supplemental Fig. 4). Theexogenous Mad2 does not bind BubR11–477 in the absence ofCdc20 (Fig. 4B, lane 4), but does copurify in the presence ofCdc20 (Fig. 4B, lanes 3 and 5), indicating that like BubR1490–679,BubR11–477 cannot bind Mad2 directly, but it can bind Mad2indirectly through Cdc20. However, in contrast to BubR1490–679,BubR11–477 binding to Cdc20 depends upon Mad2 beingbound to Cdc20 (Fig. 4A; Fig. 4B, lanes 2 and 3; Fig. 4C,lanes 2 and 3). This phenomenon is also observed when theGST tag is moved to Cdc20: GST–Cdc20 binding of FLAG-tagged BubR11–477 depends upon Mad2 (Supplemental Fig. 5).

We suspected that endogenous Mad2 supports the weakbinding of Cdc20 seen in the absence of transfected Mad2. To

pts BubR1 binding. (A) Cdc20 K129A/P137A (“KP/AA”)nt K129A/P137A (“KP/AA”) was cotransfected with triplefusion assessed.Mad2 binding to the doublemutant is aboveto GST–BubR11–477. In this experiment, GST–BubR11–477

ad2 (lane 2) but very little of the double mutant (lane 4),o low to allow appreciable binding to the mutant.usly if normalized to the amount of fusion; lane 3), but hasvating cellular Mad2 concentrations to the point that a small


test this, we mutated the Mad2 binding motif of Cdc20 byconverting both K129 and P137 to alanine [34]. The ability of aGST fusion of Cdc20 to pull down FLAG-tagged Mad2 wasmuch reduced, but not eliminated, by the mutations (Fig. 5A),indicating a reduced affinity for Mad2. We also expected thatbinding of endogenous Mad2 to the mutant Cdc20 proteinwould be weaker, and so would not support efficient bindingof the mutant to GST–BubR11–477. This was confirmed (Fig. 5B,lane 2 vs. lane 4). When cells were cotransfected with Flag-tagged Mad2 to increase cellular Mad2 and consequently theconcentration ofmutant Cdc20with boundMad2, the ability ofthe mutant to bind BubR11–477 was enhanced (Fig. 5B, lane 5).The binding of mutant Cdc20 to BubR11–477 was accompaniedby Mad2 co-purification, indicating that the BubR11–477 strong-ly selects the modest population of mutant Cdc20 that hadbound Mad2.

We interpret these results to indicate that Cdc20must bindMad2 before it can bind BubR11–477.We therefore expected thatcomplete removal of the Mad2 binding region of Cdc20 wouldeliminate binding to BubR11–477. However, Cdc20154–499, whichlacks the Mad2 binding motif, binds BubR11–477 (Fig. 4C). Thisbinding, as expected, does not require Mad2 (compare lanes 4and 5). This suggests that Mad2 binding to full length Cdc20exposes a BubR1 binding site C-terminal to residue 154 that isunavailable for binding to BubR1 in the absence of Mad2. Theability of Cdc20 to bind to BubR11–477 was abolished when theWD domain of Cdc20 was disrupted (Fig. 4C), just as shownabove for its binding to BubR1490–679.

We have also used deletion and point mutants to narrowthe Cdc20 binding region in GST–BubR11–477. Cdc20 bindingwas maintained in GST–BubR11–363, indicating that the Bub3and kinetochore binding are not needed (Fig. 4D, lane 2).However, Cdc20 binding was significantly weakened bydeletion of the A* homology region (GST–BubR148–477, lane 6

Fig. 6 – Yeast two hybrid assay confirmsMad2 is required for BubpGBT9 containing the DNA binding domain of GAL4 fused to varco-transfected with a pACT2 plasmid to cause expression of a GMad2 is indicated, the pGBT9 plasmid was replaced with the derMad2 in addition to the GAL–BubR1 fusion. Binding is assayed a

Fig. 4D) or mutation of the conserved K19 of the A* region(GST–BubR11–477 K19A, lane 3 Fig. 4D). Cdc20 binding wasvirtually eliminated by removal of residues between 219 and363 of BubR1 (not shown). Mutagenesis of conserved residuesin this region (R217A in Fig. 4D, lane 4; also F268, K297, andE338, not shown) has not yet identified any particular residuesessential for Cdc20 binding.

We believe that theweak and inconsistent binding of Cdc20to BubR1 observed in the absence of exogenously expressedMad2 is supported by endogenous Mad2. To eliminate theproblem posed by the endogenous protein in HeLa cells, weused a yeast two hybrid assay to confirm Mad2-dependentCdc20 binding site to BubR11–477. Control experiments showedthat expression of yeast Mad2 does not cause mitotic arrest inHeLa cells and that it does not bind to mouse Cdc20 in our GSTpull down experiments. Similarly, yeast Cdc20, with orwithout yeast Mad2, does not bind to BubR11–477 (not shown).Having confirmed the lack of interaction between mouse andyeast proteins, we expressed BubR1 as a fusion to the DNAbinding domain of GAL4 in either pGBT9 or in pBridge-Mad2, aderivative of pGBT9 that also expresses mouse Mad2. Little orno interaction occurred between BubR11–477 and mouse Cdc20in the absence of Mad2 (Fig. 6, panel 5), but a strong interactionwas apparent in the presence of Mad2 (panel 8). Theinteraction persisted in BubR11–363 and to a much lesserextent, BubR11–218 (panels 7 and 6). However, deletion ormutation of the A* domain greatly weakened or abolished theinteraction (panels 9 and 10). As expected, the interaction ofBubR1490–679 with Cdc20 differed from that of BubR11–477 in thatit did not require Mad2 (samples 11 and 12). As in the pull-down experiments in HeLa cells, both BubR11–477 andBubR1490–679 bound to Cdc20154–499 in the absence of Mad2(samples 13–16). Surprisingly, co-expression of Mad2 reducedthe strength of interaction between Cdc20154–499 and BubR1–477

R11–477 binding to Cdc20. Yeast was transfected with plasmidious BubR1 deletion or point mutants. The cells wereAL4 activation domain-Cdc20 fusion. Where the presence ofivative plasmid pBridge-Mad2, which drives expression ofs growth in the absence of histidine.


(samples 15 and 16). We do not have an explanation for thiseffect, but it is consistent with the results some of the GST pulldown experiments (e.g., Fig. 4C). Not surprisingly given thepoor interactions between yeast and vertebrate checkpointproteins, initial attempts to complement Mad3 deletion inyeast with either BubR1 or BubR11–477 have failed (KatsumiKitagawa and Rashid Abdulle, personal communication).

To assess the functional relevance of the binding of Cdc20to BubR11–477 we tested for an effect of the mutations thatdisrupt Cdc20 binding to BubR11–477 upon checkpoint functionin nocodazole-treated HeLa cells. As seen in Fig. 7A, transfec-tionwith the BubR11–477 K19Amutantweakens the checkpointresponse, as does the BubR148–477 mutant. As these BubR1mutants also show reduced binding of Cdc20, this resultimplies that the strength of the checkpoint depends upon theextent of Cdc20 binding to BubR1, which in turn depends uponMad2 binding to Cdc20. The K19A mutation in full lengthBubR1 also weakened checkpoint function in transfected cells(Fig. 7B), indicating that Cdc20 binding by the 490–560 sitecannot compensate for loss of amino-terminal Cdc20 binding.In contrast, there are other mutations, of which R217A is anexample, that do not influence Cdc20 binding to BubR1 (Fig.4D) but which like K19A disrupt checkpoint response tonocodazole (Fig. 7A). Although these mutations do notappreciably alter Cdc20 binding by BubR1, they may disruptformation of the complex at limiting concentrations of Cdc20or Mad2, or they may disrupt the functions of the assembledBubR1–Cdc20–Mad2 complex [35].

Discussion

We have identified two Cdc20 binding sites on BubR1. A sitebetween residues 490 and 560 can bind Cdc20 tightlyregardless of the presence of Mad2 bound to the Cdc20.Mutation of conserved residues in this region of BubR1disrupts Cdc20 binding but has no effect upon checkpointfunction. A second binding site between residues 1 and 477 isspecific for Cdc20 bound to Mad2. Mutations that weakenCdc20 binding to BubR11–477 also weaken checkpoint function,demonstrating that this amino terminal Cdc20 binding site is

Fig. 7 – Mutation of BubR1 K19 to alanine weakens checkpoint fshow a weakened checkpoint. The experiment was performed afunction in full length BubR1.

essential for checkpoint function. Our results provide astructural basis for the checkpoint activity of BubR11–477.They also show that there may be various BubR1–Cdc20–Mad2 complexes, but that only those involving the N-terminalbinding site and Mad2 are directly relevant to checkpointfunction. They further suggest that Mad2 and Cdc20 mustcooperate in a two-step mechanism of checkpoint inhibitionof Cdc20 in which first Mad2 and then BubR1 bind to Cdc20.

Binding of Cdc20 between residues 490–560 of BubR1 isindependent of Mad2 and does not play a role in the spindlecheckpoint

Previous in vitro studies established that recombinant humanBubR1 can inhibit Cdc20-dependent APC activity indepen-dently of Mad2, and indicated that there was a Cdc20 bindingsite between residues 525 and 700 of BubR1 [13]. Subsequent invitro experiments confirmed that Mad2 and BubR1 can eachinhibit Cdc20 activity independently of the other, but alsoshowed that at low concentrations inhibition by BubR1 andMad2 was synergistic [19]. The synergism was explained bythe observation that low concentrations of BubR1 enhancedMad2 binding to Cdc20 and vice versa, though the impliedternary complex in which Cdc20 binds both Mad2 and BubR1simultaneously was not detected. To better understand thecheckpoint activity of BubR11–477 we re-investigated Cdc20binding to BubR1 using in vivo expression of BubR1 mutants.We confirmed Cdc20 binding to residues between 490 and 679of mouse BubR1, a region corresponding to that identified inthe in vitro studies [13]. We have extended this result to showthat a smaller peptide corresponding to residues betweenpositions 490 and560 canbindCdc20, and that a conserved FDEmotif between residues 524 and 526 plays a critical role inbinding. Finally, we found that Cdc20 bound to this region ofBubR1 can simultaneously bind exogenous Mad2, resulting information of a complex containing BubR1, Cdc20, and Mad2.Although it could bind to BubR1 throughCdc20, the presence ofMad2 was not necessary for Cdc20 binding to BubR1490–679 anddid not influence the amount of Cdc20 bound to BubR1490–679.Point mutations and deletions that eliminate Cdc20 binding tothis region do not disrupt the checkpoint function, indicating

unction. (A) HeLa cells transfected with BubR11–477 K19As in Fig. 1B. (B) The K19A mutation also perturbs checkpoint

Fig. 8 – Checkpoint inhibition of Cdc20 is a two-step processin which Mad2 must bind before BubR1. We have depicted asimplemechanism for this in which the N-terminus of Cdc20physically blocks the BubR11–477 binding site until Mad2binding causes a conformational change. We ignore theC-terminal binding site for Cdc20 on BubR1. We illustrateassembly of Cdc20–Mad2 onto BubR1–Bub3 as occurring atthe kinetochore, but this step may not require kinetochores[16]. If assembly is independent of kinetochores, theimportance of kinetochore binding for checkpoint functionmay be explained if kinetochores mediate association of theMCC with the APC [35].


that Cdc20 binding to this region does not play a role in thecheckpoint. This conclusion is supportedby the failure ofMad2to stimulateCdc20 binding by this regionof BubR1, since suchastimulation would be expected from their cooperative effectsin vitro [19]. The conclusion is also consistent with the failureof the human BubR1351–700 to inhibit Cd20-dependent APCactivity in vitro [13]. Like theC region itself, the conservedmotifbetween residues 521 and 529 of BubR1 that is critical for Cdc20binding to this region is not present in yeast Bub1 or Mad3 andso is unlikely to play a conserved, central role in checkpointfunction. At this point the role of this Cdc20 binding site isunclear, though we note that the same region of Bub1 alsobinds Cdc20 (not shown).

Mad2-dependent Cdc20 binding to BubR11–477 is required forcheckpoint function

Although we occasionally observed low levels of Cdc20binding to GST fused to BubR1–477, exogenous expression ofMad2 was necessary to observe consistent, strong Cdc20 bind-ing.We inferred that Cdc20 binding observed in the absence ofexogenous Mad2 is supported by endogenous Mad2, since amutation that weakens the affinity of Cdc20 for Mad2 alsoweakens its ability to bind GST–BubR11–477 (Fig. 5B). Thisconjecture was confirmed by our yeast two-hybrid experi-ments which show complete dependence of Cdc20 binding toBubR11–477 uponMad2 (Fig. 6). We speculated that Mad2 boundto Cdc20 might help form the binding site for BubR11–477, andso we were surprised to discover that Cdc20154–499, whichentirely lacks the Mad2 binding region, can bind to BubR11–477

(Fig. 4C). This observation suggests that BubR11–477 binds toCdc20 on a surface that is sterically or allosterically obstructedby the amino terminal domain, and only revealed when theamino terminus is perturbed by Mad2 binding. Consistentwith this, studies of Mad2 binding to Cdc20 indicate thatCdc20 may adopt a more extended or open conformationwhen bound to Mad2 [13,31,34]. Thus, a simple interpretationis that BubR11–477 selectively binds Cdc20 in an open con-formation created by Mad2 binding. This behavior is consis-tent with the Mad2 dependence of Cdc20 association withBubR1 in Xenopus oocyte extracts [36]. It is also similar to theinteraction between Cdc20 and Mad3 in yeast in which, asdiscussed later, it appears that Mad3 binding to a Cdc20–Mad2complex is required for inhibition of APC activity [15–17]. Thedifference in the Cdc20 binding mechanism of BubR11–477 ascompared to that of BubR1490–679 is consistent with the lack ofsequence homology between BubR11–477 and the C-terminalCdc20 binding region, but also reflects the distinctive func-tional character of Cdc20 binding to this region, as discussedbelow.

We found that the N-terminal 47 amino acid residues ofBubR11–477 play an important role in Cdc20–Mad2 binding.Deletion of this region to generate BubR148–477 reduced theability to bind Cdc20–Mad2, and also reduced checkpointfunction. Furthermore, alanine mutagenesis of lysine 19, aresidue in this region that is conserved between BubR1 andyeast Mad3, disrupted both Cdc20 binding by BubR11–477 andcheckpoint function. These results argue that Cdc20 binding toBubR11–477 is critical for checkpoint function. More impor-tantly, the same mutation in full length BubR1 also weakened

the checkpoint function, showing that the downstream Cdc20binding site cannot provide checkpoint function and confirm-ing the relevance of observations of checkpoint functionmadewith the BubR11–477 deletionmutant. While our observation ofCdc20 binding to BubR11–477 provides a mechanistic basis forthe checkpoint activity we previously inferred for BubR11–477,the parallel effects of mutations in the A* domain on Cdc20binding and checkpoint activity strengthens our argumentthat checkpoint function resides in the amino terminus andlinks it to Mad2-dependent Cdc20 binding. More generally, ourresults suggest that checkpoint function responds to theamount of BubR1 bound through its amino terminus toCdc20. The dependence of this interaction upon Mad2 bindingto Cdc20 in turn implicates Mad2 in BubR1 checkpointfunction.

Checkpoint inhibition of Cdc20 is a two-step process

The checkpoint activity of BubR11–477, the dependence ofCdc20 binding to BubR11–477 upon Mad2, and the coordinatedinhibition by the K19A mutation of both checkpoint functionand Cdc20 binding together suggest that inhibition of Cdc20 byan activated spindle checkpoint is a two-step process (Fig. 8).First, Mad2 must bind Cdc20, changing the Cdc20 conforma-tion and thereby permitting subsequent binding by the N-terminus of BubR1 and inhibition. This model identifiesBubR11–477 as the functional homolog of yeast Mad3 [15–17].The functioning of this simple pathway in vertebrate cellsmaybe obscured by the formation of compositionally similar but


apparently non-functional complexes between the C-termi-nus of BubR1 and Cdc20. Our results indicate that the BubR1–Cdc20–Mad2 complex is required to establish mitotic check-point function, which it presumably does by preventingactivation of the APC. Similar complexes generically referredto as themitotic checkpoint complex (MCC) have been isolatedfrom both mitotic and interphase HeLa cells [13,35,37] andshown to inhibit the APC activity in vitro [35]. Thus our resultsare consistent with previous biochemical evidence for controlof APC activity by a BubR1–Cdc20–Mad2 complex and providefurther functional evidence for the relevance of these com-plexes to the vertebrate checkpoint in vivo. Furthermore, theobligate cooperation betweenMad2 and BubR1 implied by thistwo-step model also implies that if different arms of thecheckpoint independently activate Mad2 and BubR1 [10–12,38,39], these arms must nonetheless converge to inhibitCdc20.

The argument for the relevance of the N-terminal Cdc20binding is strengthened by our observation that mutants thatdisrupt the interaction between the N-terminus of BubR1 andCdc20 also disrupt the checkpoint, even if expressed in thecontext of the full-length protein. These checkpoint assaysdepend upon expression of exogenous genes in the context offunctional endogenous protein, since cells without BubR1function cannot be maintained. Assessment of functiondepends upon an excess of the transfected gene productdisplacing the endogenous protein. In the current case, thesensitivity of BubR11–477 function to point and deletionmutants argues strongly that it is functional (as discussed inHarris et al., 2005). However, we cannot exclude the possibilitythat the endogenous protein may provide some key functionthe transfected mutant does not, and so distort results [20].Emerging techniques that deplete endogenous BubR1 protein[40,41] may allow us to replace the endogenous gene with ourmutants and more effectively study the biochemistry of thecheckpoint in living cells. The various mutants we havecharacterized in this paper will be useful tools in this effort todetail the function BubR1 and to distinguish between poten-tially different types of MCC.

In summary, our results show that two different siteswithin BubR1 can form complexes with Cdc20 and Mad2, butthat only the amino-terminal site is involved in checkpointfunction. In addition, we show that inhibition of Cdc20 by theN-terminus of BubR1 is likely to be a two-step process inwhichMad2 must first bind Cdc20 and change its conformationbefore BubR1 can bind and inhibit Cdc20.

Acknowledgments

We thank Josie Harris-Chambers and Trushar Jeevan forproviding excellent technical assistance.We thankDr. RichardCross for assistance in flow cytometric analysis of cells. Wealso thank Dr. Katsumi Kitagawa and Rashid Abdulle fortesting for suppression by BubR1 of the mad3Δ phenotype inyeast and for helpful discussions. Mad3-deficient yeast celllines and MAD3 genomic clone were generously provided byDr. Kevin Hardwick. This work was supported by grantCA092597 from NCI to R.G. and by the American Lebaneseand Syrian Associated Charities (ALSAC).

Appendix A. Supplementary data

Supplementary data associated with this article can be foundin the online version at doi:10.1016/j.yexcr.2006.02.018.

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Spindle checkpoint function requires Mad2-dependent Cdc20 binding to the Mad3 homology domain of BubR1