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Cell Biology International (2011) 35, 575–578 (Printed in Great Britain)
Chromosome ‘by-Aurora-ientation’ during mitosis
Sally P Wheatley1
School of Biomedical Sciences, University of Nottingham, Queens Medical School, Nottingham NG7 2UH, U.K.

New evidence from three separate laboratories, published recently in Science, has shown that centromere positioning of the CPC (chromosomal passenger complex) during early mitosis is achieved through direct interaction between the CPP (chromosomal passenger protein) survivin and histone H3. In essence, an acidic pocket in the BIR (baculovirus inhibitor of apoptosis repeat) domain of survivin binds to the NH2 tail of histone H3 specifically when it is phosphorylated at threonine 3, a mark that is placed by the mitotic kinase, haspin. These data are significant, as they describe a fundamental mechanism, conserved throughout eukaryotes, which is essential for chromosome biorientation and the maintenance of genome stability during mitosis.

Key words: Aurora-B, centromere, haspin kinase, histone H3, survivin

Abbreviations: BIR, baculovirus inhibitor of apoptosis repeat, cdk1, cyclin-dependent kinase 1, CPC, chromosomal passenger complex, CPP, chromosomal passenger protein, IAP, inhibitor of apoptosis protein, INCENP, inner centromeric protein, MCAK, mitotic chromosome-associated kinesin, sgo2, shugoshin 2, T3, threonine 3


1. The CPC (chromosomal passenger complex)

The CPC is an essential mitotic complex that facilitates chromosome alignment during prometaphase, delineates the cleavage plane in anaphase and is required for cytokinesis. It is an assembly of four proteins, Aurora-B, borealin, INCENP (inner centromeric protein) and survivin, which are mutually dependent on each other for their localization, function and stability [reviewed by Ruchaud et al. (2007) and Vagnarelli and Earnshaw (2004)]. Their choreographed movements from the centromeres in early mitosis to the midzone and equatorial cortex during anaphase and telophase reflect their roles as co-ordinators of mitosis and cytokinesis (Figure 1). The CPC can be separated into two modules: the catalytic module comprising Aurora-B kinase and its primary activator, the C-terminally placed IN-box of INCENP and the centromere targeting module consisting of survivin and borealin bound to the NH2-terminus of INCENP (Adams et al., 2000; Vader et al., 2006a) (see Figure 2). The principal concern of the CPC is the function of its catalytic component Aurora-B kinase: INCENP activates it, while survivin and borealin act as its chaperones (Carmena et al., 2009; Vader et al., 2006b). Aurora-B reversibly phosphorylates multiple proteins during mitosis and is charged with the responsibility of correcting maloriented chromosomes during prometaphase. Its activity is spatially regulated by tension across the centromeres (Liu et al., 2009), which depends on chromosome orientation and is detected by the spindle checkpoint protein, BubR1. During chromosome congression, one important substrate of Aurora-B is the microtubule depolymerase, MCAK (mitotic chromosome-associated kinesin). Both the localization of MCAK to the centromere and its ability to disassemble microtubules are Aurora-B dependent (Andrews et al., 2004). As microtubules attached to the kinetochores of maloriented chromosomes must be destabilized to permit reorientation; regulation of MCAK by Aurora-B is pivotal for correct chromosome alignment during mitosis. Clearly, in order to accomplish its tasks during prometaphase, Aurora-B must first be targeted to the centromere, but how this is achieved has remained unclear until recently.

2. Survivin targets the CPC to the centromere

The recent findings of Wang et al. (2010), Kelly et al. (2010) and Yamagishi et al. (2010) have unlocked significant new insight of this key event. In an earlier study, Higgins and co-workers identified haspin as a conserved mitotic kinase that phosphorylates T3 (threonine 3) at the NH2 terminus of histone H3 (Dai et al., 2005), hereafter denoted as H31–21T3P. In their more recent study, they investigated the effect of depleting haspin from cultured human cells by siRNA (small interfering RNA) (Wang et al., 2010). Eliminating haspin reduced Aurora-B and MCAK accumulation at the centromeres to just 5% that of the controls, a level insufficient to promote error correction or to maintain the spindle assembly checkpoint in response to taxol. They also discovered that the CPC can bind NH2-terminal H31–21 peptides and do so preferentially when phosphorylated at T3. Importantly, using recombinant proteins, they showed that CPC–H31–21T3P interaction is mediated exclusively through survivin. As it was previously reported that the CPC failed to localize to the centromere in cells expressing a survivin BIR (baculovirus inhibitor of apoptosis repeat)-domain mutant, survivinD70A,D71A (Cao et al., 2006; Yue et al., 2008), Wang et al. (2010) then asked whether these residues were responsible for the interaction with H31–21T3P. Not only did the phenotype of cells expressing survivinD70A,D71A mimic haspin depletion as expected (Cao et al., 2006; Yue et al., 2008), but this double substitution also prevented the direct binding of survivin to the H31–21T3P peptide in vitro. From these data, they concluded that centromere targeting of Aurora-B and MCAK is achieved by phosphorylation of H3 at T3, which is mediated by haspin kinase, creating a docking site for survivin that facilitates CPC recruitment.

Through an entirely different route, Funabiki and co-workers also arrived at the same conclusion. In searching for histone-interacting proteins present in CSF (cytostatic factor)-metaphase-II arrested Xenopus egg extracts, these authors returned the CPPs (chromosomal passenger proteins) as positive hits (Kelly et al., 2010). As S10 (serine 10) of H3 is phosphorylated by Aurora-B during mitosis (Crosio et al., 2002), they initially focused their attention on the NH2 terminus of H3. Subsequent in vitro analysis, however, revealed that the CPC preferentially binds H31–21 peptides phosphorylated at T3, rather than at S10, or without modification. Moreover, they too found that survivin was the CPC receptor for H31–21, with its binding significantly enhanced by phosphorylation at T3. Using nuclear magnetic resonance profiling to compare the structure of survivin when bound and not bound to phosphorylated (T3) H31–21, they identified D71 (aspartate 71) as a key residue in this interaction. Demonstrating the functional significance of these findings, immunodepleting haspin or erasing the H3T3P mark inhibited Aurora-B-dependent events in their Xenopus extracts. Finally, by inducing mitotic exit with Ca2+, they noted that dephosphorylation of H3 at T3 occurs at the metaphase–anaphase transition, coincident with loss of haspin activity and the departure of the CPC from the centromere. Thus, these data corroborate with the findings of Wang et al. (2010), supporting the claim that this is a fundamental mechanism for targeting Aurora-B to the centromere.

In a third paper, Yamagishi et al. (2010) provide genetic data to support the link between haspin-related kinase, Hrk1, and survivin's homologue, Bir1, in the fission yeast, Schizosaccharomyces pombe. As in the human and Xenopus studies (Kelly et al., 2010; Wang et al., 2010), they found that Bir1 was recruited to sites enriched with Hrk1 phosphorylated H3 (Yamagishi et al., 2010). However, in contrast with the vertebrate studies, they found that this pathway was linked to chromosome cohesion rather than specifically targeting the CPC to the centromeres. Furthermore, they found that loss of Hrk1 was synthetic lethal both with the HP1 (heterochromatin protein 1) homologue, swi6 (switching gene 6) and sgo2 (shugoshin 2), a CPC adaptor found at mitotic centromeres. Earlier in 2010, these authors had shown that centromere-targeting of sgo2 is influenced by Bub1-mediated phosphorylation of histone 2A and that this event, which occurs at Ser121 in yeast H2A (Thr120 in human cells) is confined to the centromere (Kawashima et al., 2010). In their most recent paper, they ask how the Hrk1 and Bub1 pathways might be related. By artificially tethering Bir1 to the centromeres in S. pombe-bearing mutations in either of the key sites, H3T3A or H2AS120A, Yamagishi et al. (2010) were able to restore biorientation of chromosomes, genomic integrity and cell viability. Hence, they conclude that targeting of the CPC to the nucleosome is achieved by two independent pathways, one regulated by Bub1 via swi6 and H2A, the other by Hrk1, Bir1 and H3 and that these pathways converge at the centromere to achieve Aurora-B recruitment. Consistent with the localization data in yeast, in human cells, the H2A T121 mark is confined to the centromere from prophase through to early anaphase (Kawashima et al., 2010), while the H3T3 mark is also present along the chromosome arms during prometaphase and metaphase, with enrichment at the centromeres apparent in preparations of chromosome spreads (Dai et al., 2005). Thus, although this work underscores the importance of the haspin–survivin–H3 cascade in targeting Aurora-B to the centromere, it also highlights that it does not operate alone and that it collaborates with at least one other pathway, mediated by Bub1, to facilitate CPC recruitment to the centromere.

3. Regulating survivin–centromere association

For successful biorientation of all chromosomes during mitosis, the activity of Aurora-B at the centromeres must be regulated. As the H3T3P mark is present throughout prometaphase and only lost when haspin is inactivated at anaphase onset (Kelly et al., 2010), how is this achieved? Elegant work from the Lens and Lampson laboratories has revealed that one mechanism is mechanochemical, with tension across the centromeres spatially regulating the proximity of Aurora-B to its substrates (Liu et al., 2009). Another route is probably through the cyclical association and dissociation of survivin with H3T3P. Indeed, fluorescence recovery after photobleaching has shown that survivin mobility at the centromere is highly dynamic and sensitive to Lys63-mediated polyubiquitination (Vong et al., 2005) and phosphorylation by Aurora-B (Beardmore et al., 2004; Delacour-Larose et al., 2007; Wheatley et al., 2007). While a number of other post-translational modifications may influence the residence of survivin at the centromere, it is intriguing to note that the unique cdk1 (cyclin-dependent kinase 1) target site of survivin, Thr34, sits directly opposite Asp70 and Asp71 at the extreme tip of the BIR domain, which resembles a lobster claw (Figure 3). Although mutational analyses have shown that cdk1 phosphorylation of survivin is dispensable for cell proliferation (Yue et al., 2008; Barrett et al., 2009), the fact that Thr34 is phosphorylated during prometaphase (Nousiainen et al., 2006) and a phosphomimetic, T34E, is not recruited to the centromeres in the absence of the endogenous protein, and does not support mitosis (Nousiainen et al., 2006), suggest that cdk1 may be an important regulator of survivin and H3T3P association. Further possibilities include the influence of other factors such as the Rac GEF (guanine-nucleotide-exchange factor) and regulator of chromosome condensation TD60 (telophase disk protein of 60 kDa) [alias RCC2 (regulator of chromosome condensation 2)] (Mollinari et al., 2003) and the origin of replication protein, Orc6 (origin of replication complex protein 6) (Prasanth et al., 2002). Whatever the intricacies that govern the regulation of survivin interaction with H3T3P, the findings covered here clearly demonstrate that the BIR domain is the receptor for the association (Kelly et al., 2010; Wang et al., 2010). This, in itself, is noteworthy, as BIR domains were originally considered to be present only in IAPs (inhibitors of apoptosis proteins). Although survivin is an IAP family member, these studies contribute to a growing body of evidence supporting the notion that BIR domains mediate a variety of protein–protein interactions and are not exclusively involved in subverting apoptosis (Dubrez-Daloz et al., 2008).

4. Concluding remarks

Directing Aurora-B to the centromere is crucial in achieving biorientation of all chromosomes and their safe passage through mitosis. The recent investigations of Kelly et al. (2010), Wang et al. (2010) and Yamagishi et al. (2010), discussed herein, are the first to identify the interaction between survivin and H3T3P as the connection responsible for centromere-targeting of Aurora-B. Having described the physical bridge mediating Aurora-B localization, we now need to determine how the survivin–H3T3P link is regulated to ensure absolute fidelity in genome transmission from one cell generation to the next.


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Received 27 December 2010/8 March 2011; accepted 15 March 2011

Published as Cell Biology International Immediate Publication 15 March 2011, doi:10.1042/CBI20100911

© The Author(s) Journal compilation © 2011 Portland Press Limited

ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)