The Journal of Cell Biology ��� The Rockefeller University Press, 0021-9525/2003/04/267/14 $8.00 The Journal of Cell Biology, Volume 161, Number 2, April 28, 2003 267���280 http://www.jcb.org/cgi/doi/10.1083/jcb.200208091 JCB Article 267 Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores Claire Ditchfield, 1 Victoria L. Johnson, 1 Anthony Tighe, 1 Rebecca Ellston, 2 Carolyn Haworth, 2 Trevor Johnson, 2 Andrew Mortlock, 2 Nicholas Keen, 2 and Stephen S. Taylor 1 1 School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK 2 Cancer and Infection Research Area, AstraZeneca Pharmaceuticals, Mereside, Cheshire SK10 4TG, UK he Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. How- ever, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A T or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint compo- nents BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle check- point function, but is also required for chromosome align- ment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset. Introduction Accurate chromosome segregation in mitosis requires that sister kinetochores attach to microtubules emanating from opposite spindle poles. Because kinetochore attachment is a stochastic process, it is error prone and can result in chromo- some malorientation (Nicklas, 1997 Rieder and Salmon, 1998). Although it is well established that a surveillance mechanism, the spindle checkpoint, delays anaphase until all chromosomes correctly bi-orient (for review see Musacchio and Hardwick, 2002), the mechanisms that detect and resolve orientation errors are poorly understood. The Ipl1/Aurora family of protein kinases plays multiple roles in mitosis (Bischoff and Plowman, 1999 Giet and Prigent, 1999 Adams et al., 2001a Nigg, 2001). In budding yeast, Ipl1 ensures accurate chromosome segregation by resolv- ing syntelic orientations, possibly by monitoring tension at centromeres and destabilizing inappropriately bound micro- tubules (Tanaka et al., 2002). Ipl1 phosphorylates the ki- netochore component Ndc10 in vitro, suggesting that it may regulate kinetochore���microtubule interactions directly (Biggins et al., 1999). However, budding yeast are atypical in that centromeres connect to the unduplicated spindle pole body (SPB) in G1. Because centromeres replicate before SPB duplication, budding yeast cells then enter mitosis with both kinetochores attached to the old SPB (Tanaka et al., 2002). In higher eukaryotes, syntelic orientations are rare during mitosis (Nicklas, 1997), and therefore, it is unclear whether the kinetochore���SPB resolving activity exhibited by Ipl1 is a universal feature of the Aurora kinase family. Further evidence suggesting that Ipl1 monitors tension at centromeres comes from analyzing budding yeast mutants that lack sister chromatid cohesion or enter mitosis without replicating their DNA. In such mutants, anaphase is prevented in an Ipl1-dependent manner (Biggins and Murray, 2001). Because kinetochores in these cells lack sisters, they fail to come under tension despite microtubule attachment, arguing that Ipl1 is required for spindle checkpoint activation in response to loss of tension at centromeres. However, budding yeast kineto- chores only attach a single microtubule. Therefore, if the The online version of this article includes supplemental material. Address correspondence to Stephen S. Taylor, School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Rd., Manchester M13 9PT, UK. Tel.: 44-161-275-5100. Fax: 44-161-275- 5763. E-mail: stephen.taylor@man.ac.uk Key words: mitosis spindle checkpoint chemical biology aneuploidy ZM447439

The Journal of Cell Biology 268 The Journal of Cell Biology | Volume 161, Number 2, 2003 primary role of Ipl1 is to destabilize bound microtubules, the apparent role of Ipl1 in checkpoint activation may be simply a secondary consequence of exposing microtubule binding sites (Tanaka et al., 2002). Furthermore, although the analysis of replication and cohesion mutants suggests that Ipl1 monitors tension at centromeres, these interpreta- tions are complicated by the fact that centromeric localiza- tion of the fission yeast Aurora kinase, Ark1, requires sister chromatid cohesion (Morishita et al., 2001). Therefore, there is a clear need to analyze Aurora kinase function under conditions where replication and cohesion are normal. Higher eukaryotes express two or more Aurora kinases. Aurora A and C localize to spindle poles, and Aurora A is re- quired for bipolar spindle formation in a variety of systems (Bischoff and Plowman, 1999 Giet and Prigent, 1999 Ad- ams et al., 2001a Nigg, 2001). Inhibition of Aurora B, an inner centromere protein, affects multiple mitotic events in- cluding histone H3 phosphorylation, chromosome segrega- tion, and cytokinesis (Bischoff and Plowman, 1999 Giet and Prigent, 1999 Adams et al., 2001a Nigg, 2001). The role of Aurora B kinase activity has been addressed by ectop- ically expressing mutants in mammalian cells. However, these studies have yielded conflicting results. In two cases, cells expressing Aurora B K109R completed mitosis, but failed to undergo cytokinesis, suggesting that Aurora B activ- ity is not required for chromosome segregation (Tatsuka et al., 1998 Terada et al., 1998). However, another report in- dicates that Aurora B K109R prevents chromosome align- ment due to the failure of kinetochore���microtubule interac- tions (Murata-Hori and Wang, 2002). These experiments are complicated by the fact that expression of wild-type Au- rora B can itself affect cell division (Tatsuka et al., 1998), and therefore, it is not clear whether these phenotypes are due to reduced Aurora B kinase activity or the disruption of Aurora B protein complexes. Xenopus cells injected with anti-Aurora B antibodies exit mitosis prematurely, consis- tent with a role for Aurora B in the spindle checkpoint (Kal- lio et al., 2002). However, in contrast to Ipl1 deficient strains, mitotic exit also occurred when microtubule poly- merization was inhibited, suggesting that Aurora B monitors microtubule attachment, not just tension. Thus, although many roles have been attributed to Aurora B, the emerging picture is confusing, and molecular explanations for these phenotypes are currently lacking. Aurora A and B are overexpressed in human tumors, and ectopic overexpression in cultured cells leads to transforma- tion, centrosome abnormalities, and aneuploidy (Bischoff et al., 1998 Tatsuka et al., 1998 Zhou et al., 1998 Adams et al., 2001b Meraldi et al., 2002). In addition, cells overex- pressing Aurora A, but not a kinase mutant, readily form tu- mors in nude mice (Bischoff et al., 1998). Therefore, ele- vated Aurora kinase activity may promote tumor evolution either by providing a growth advantage or by promoting ge- netic instability. To develop novel anti-cancer drugs, we have generated small molecule inhibitors of Aurora kinase activity. Here, we describe ZM447439, which selectively inhibits the kinase activity of Aurora A and B. Using ZM447439 as a research tool, we directly address the role of Aurora kinase activity in human cells. We show that inhibi- tion of Aurora kinase activity does not prevent progression through interphase, mitotic entry, bipolar spindle forma- tion, or kinetochore���microtubule interactions. Rather, Au- rora kinase activity is required for correct chromosome alignment and spindle checkpoint function. Using RNA in- terference (RNAi * Elbashir et al., 2001), we demonstrate that these phenotypes are due to inhibition of Aurora B, not Aurora A. Results ZM447439, a novel inhibitor of Aurora A and Aurora B To identify novel Aurora inhibitors, 250,000 compounds were screened for the ability to inhibit the kinase activity of recombinant human Aurora A against an artificial peptide substrate. One inhibitor identified was further modified to produce ZM447439 (4-(4-( N -benzoylamino)anilino)-6- methoxy-7-(3-(1-morpholino)propoxy)quinazoline Fig. 1 A). In in vitro kinase assays using purified recombinant pro- teins, ZM447439 inhibited Aurora A and B with IC 50 val- ues of 110 and 130 nM, respectively (Fig. 1 B). In contrast, the majority of other protein kinases assayed were not in- hibited by ZM447439. Based on data from model systems, we predicted that an Aurora inhibitor should prevent cell division and inhibit phosphorylation of histone H3 on serine 10. To determine whether ZM447439 inhibits cell division, a panel of human cell lines was treated with 2 M ZM447439 for up to 96 h (Fig. 1 C). After 18 h, the vast majority of cells in all the lines had 4N DNA contents. All the lines analyzed then endoreduplicated, accumulating cells with DNA contents greater than 4N, demonstrating that ZM447439 completely inhibits cell division. To deter- mine whether ZM447439 inhibits phosphorylation of his- tone H3 on serine 10, untreated and ZM447439-treated cells were stained with an anti-phosphohistone H3 anti- body (Hsu et al., 2000). In untreated cells, chromosomes stained positive for phosphohistone H3 (Fig. 1 D). How- ever, after brief exposures to ZM447439, phosphohistone H3 was not detectable, demonstrating that ZM447439 does inhibit mitotic phosphorylation of histone H3. Con- sistent with previous observations (Adams et al., 2001c), the lack of histone H3 phosphorylation did not appear to affect chromosome condensation. ZM447439-induced endoreduplication is enhanced in the absence of p53 function Although a significant fraction of A549 and HME cells en- doreduplicated in the presence of ZM447439, after 48 h vir- tually all the cells arrested with either 4N or 8N DNA con- tents (Fig. 1 C). In contrast, HeLa cells, which lack a functional p53 response, continued DNA synthesis and rap- idly lost viability. This raises the possibility that the 4N/8N arrest exhibited by the A549 and HME cells was not directly due to ZM447439, but was rather due to activation of the p53-dependent post-mitotic checkpoint that occurs after an aberrant mitosis and/or cytokinesis (Andreassen et al., 2001). Consistently, in the presence of ZM447439, U2OS cells expressing a dominant-negative p53 mutant endoredu- *Abbreviation used in this paper: RNAi, RNA interference.