BIOLOGICAL OVERVIEW

Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression (Giet, 1999). Although it has been possible to use conventional genetic analysis to dissect the function of aurora, the founding family member in Drosophila, the lack of mutations in a second aurora-like kinase gene, IplI-aurora-like kinase (here referred to as aurora B), precluded this approach. Depleting Aurora B kinase using double-stranded RNA interference in cultured Drosophila cells results in polyploidy (Giet, 2001 and Adams, 2001a). aurora B encodes a passenger protein that associates first with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. Cells depleted of the Aurora B kinase show only partial chromosome condensation at mitosis. This is associated with a reduction in levels of the serine 10 phosphorylated form of Histone H3 and a failure to recruit the Barren condensin protein onto chromosomes. These defects are associated with abnormal segregation resulting from lagging chromatids and extensive chromatin bridging at anaphase, similar to the phenotype of barren mutants (Bhat, 1996). The majority of treated cells also fail to undertake cytokinesis and show a reduced density of microtubules in the central region of the spindle (Giet, 2001). RNAi for either Aurora B or Inner centromere protein (Incenp) dramatically inhibits the ability of cells to achieve a normal metaphase chromosome alignment. These experiments reveal that Incenp is required for Aurora B kinase function and confirm that the chromosomal passengers have essential roles in mitosis (Adams, 2001a).

The segregation of chromosomes with high fidelity requires exquisite coordination of cellular processes. The mechanisms that coordinate the cycle of chromosome condensation and decondensation with the assembly, function, and subsequent disassembly of the mitotic spindle are poorly understood. Highly conserved genes essential for chromosome condensation have been found through genetic screens in yeasts and Drosophila. For example, five members of a protein complex known as condensin, have been identified that are functionally and structurally conserved. Mutants exhibit incomplete chromosome condensation associated with failure of segregation and the stretching of chromatin upon the spindle. Biochemical approaches also identified the protein complex in Xenopus and showed that it can promote chromatin condensation by directing the supercoiling of the DNA in an ATP-dependent manner. Chromosome condensation is also accompanied by phosphorylation of histones H1 and H3. Indeed, mutation of the mitotic phosphorylation site of histone H3 of Tetrahymena leads to both chromosome condensation and segregation defects. A direct link between histone H3 phosphorylation and condensin recruitment onto chromosomes has recently been suggested by the colocalization of members of the condensin complex with phosphorylated histone H3 during the early stages of mitotic chromosome condensation. However, the generality of the requirement for the phosphorylation of histone H3 for chromosome condensation and segregation must be questioned by the finding that budding yeast cells in which serine 10 of histone H3 is replaced with alanine show no apparent defects in cell cycle progression or chromosome transmission. Nevertheless, maximal chromosome condensation in meiosis does correlate with maximal levels of phospho-histone H3 in wild-type cells. The enzyme required for histone H3 phosphorylation in Saccharomyces cerevisiae is the aurora-related protein kinase Ipl1p (Hsu, 2000). Moreover, one of its two counterparts from Caenorhabditis elegans, the air-2 protein kinase, has been shown to have the same function (Giet, 2001 and references therein).

The Aurora- and Ipl1-like protein kinases form a conserved family of enzymes, the founding members of which are encoded by the S. cerevisiae and Drosophila genes, IPL1 and aurora, respectively. While the yeast genome encodes only one such kinase required for accurate chromosome segregation, metazoan genomes have at least two subfamilies of aurora-like kinases. One is associated with centrosomes and is activated in early mitosis, and a second is associated with chromosomes and the spindle midbody and is activated later. These families are referred to as Aurora-like kinases A and B, respectively. The precise effects of loss of function of either of these enzymes varies a little between different organisms and cell types. Broadly speaking, however, the A-type enzymes are required to maintain the separation of centrosomes to give normal bipolar spindle structure. This is shown, for example, in Drosophila from the phenotype of aurora mutants (termed here aurora A); or in Xenopus, where the corresponding pEg2 kinase can be eliminated using antibodies or inactive mutants. In contrast, the B-type Aurora-like kinases appear to be required for cytokinesis, as shown, for example, by transfection of an inactive kinase mutant into cultured mammalian cells (Tatsuka, 1998; Terada, 1998). An affect on cytokinesis has also been reported in mutants of the gene for the C. elegans B-type enzyme, Air-2, or after RNA interference (Schumacher, 1998; Kaitna, 2000; Severson, 2000). The air-2 encoded kinase is required for the positioning of Zen-4, a kinesin-like protein required at the midzone of the late central spindle for cytokinesis. Abnormal chromosome segregation is also observed after reduction of air-2 function (Giet, 2001 and references therein).

The dynamics of the localization of the Aurora B class of enzymes can be partially explained by recent findings showing they exist in a complex with an inner centromere protein (Incenp) (Adams, 2000; Kaitna, 2000). Incenps are one example of so-called 'passenger proteins' that localize to the centromeric regions of chromosomes at metaphase and are then redistributed to the central spindle during cytokinesis. Defects in Incenp function lead to failure of chromosome congression and cytokinesis defects. These findings, and the fact that B-type Aurora kinase becomes incorrectly localized in human cells expressing mutant Incenps that fail to localize, has led to the idea that Incenp functions to target the B-type kinases, first to chromosomes and then to the spindle midzone (Adams, 2000). A physical interaction is also seen between the Air-2 kinase and the counterpart of Incenp in C. elegans, ICP-1. Moreover, the disruption of icp-1 function by RNAi leads to the same phenotype as air-2 RNAi (Kaitna, 2000). This direct functional interaction between the Aurora-like kinases and Incenp occurs not only in metazoan cells, but also in budding yeast where the counterpart of Incenp, Sli15p (Kim, 1999), was identified through a screen for genes that interact with Ipl1 (Giet, 2001 and references therein).

Although a B-type Aurora kinase gene has been identified in Drosophila, the lack of mutants at this locus has prevented any analysis of its potential mitotic function (Reich, 1999). Levels of the Aurora B kinase can be reduced by RNAi in cultured Drosophila S2 cells. This leads to cytokinesis failure, together with chromosome condensation and segregation defects strikingly similar to those that have been described for mutations in the condensin gene barren (Bhat, 1996). The segregation defects are accompanied by aberrant chromatin condensation, a reduction in the phosphorylated form of histone H3, and a failure to recruit the Barren protein onto condensed chromosomes (Giet, 2001 and references therein).

Using double stranded RNA interference it has been shown that the Aurora B kinase is required for mitotic chromosome condensation and segregation, and subsequently for cytokinesis. The Aurora B enzyme becomes perfectly positioned to execute these processes as mitosis proceeds. It is distributed throughout the chromatin as it condenses at prophase, then becomes concentrated around the centromeric regions of the condensed chromosomes at metaphase, and finally leaves for the company of the central spindle region during anaphase. As such it behaves as a so-called passenger protein. It appears from recent studies to be in an intimate relationship with a travelling companion Incenp (Adams, 2000; Kaitna, 2000). The interaction of Incenp, or its yeast counterpart Sli5p, with Aurora-like kinases in yeast, C. elegans, and Xenopus suggests that this interaction is universal. The dynamic association of Incenp with chromosome arms at prometaphase, the centromeric region at metaphase, and then the spindle midzone at anaphase makes it an attractive candidate for targeting the Aurora B kinase to these regions. Indeed, dominant mutants of Incenp in human cells disrupt the localization of the Aurora B-like kinase AIL2 (Adams, 2000). The finding of abnormal chromosome segregation and cytokinesis after depletion of either the C. elegans Incenp, Icp-1, or its Aurora B-like kinase, Air-2 (Schumacher, 1998; Woolard, 1999; Kaitna, 2000), suggests the two passengers perform similar functions (Giet, 2001).

One striking effect of aurB RNAi is to permit progression through mitosis with improperly condensed chromosomes. It was possible to account for these condensation defects by a diminution of the phosphorylation of serine 10 of histone H3 and a failure to localize condensin on the chromosomes. The former finding is consistent with several studies that now implicate a requirement for the phosphorylation of the NH₂-terminal region of histone H3 at this residue for chromosome condensation. Not only does the formation of mitotic chromosomes in a Xenopus cell-free extract by a nucleosome-associated kinase correlate with histone H3 phosphorylation, but when the serine 10 residue is mutated to alanine it results in abnormal segregation and chromosome loss during mitosis and meiosis in Tetrahymena. One enzyme credited with the ability to phosphorylate histone H3 at mitosis is the NIMA kinase of Aspergillus. However, the finding that levels of histone H3 phosphorylation are reduced after aurB RNAi in Drosophila cells is more in keeping with the report that the Aurora-like kinase homologs, Ipl1 of yeast and Air-2 (but not Air-1) of C. elegans, are required for histone H3 phosphorylation in these organisms (Hsu, 2000). The finding of some residual histone H3 phosphorylation either could reflect the incomplete elimination of Aurora B by RNAi, or could indicate that an alternative kinase has this capability, offering an explanation of the partial chromosome condensation seen in the RNAi-treated cells. The current data are important in emphasizing the importance of histone H3 phosphorylation for chromosome transmission and as such are in line with the findings in Tetrahymena. This differs from the effects seen in budding yeast cells that continue through division cycles in the absence of histone H3 phosphorylation without showing defects in chromosome transmission. As an explanation, it has been suggested that other histones could be phosphorylated in addition to the histone H3 in the yeast cell and that such phosphorylation events could be sufficient to ensure normal chromosome dynamics. A major role of the yeast enzyme Ipl1p is to regulate the function of the kinetochore-associated protein Ndc10p through its phosphorylation (Biggins, 1999; Sassoon, 1999). Therefore, the increase in ploidy reported in ipl1 mutant cells has been attributed more to inappropriate kinetochore function, and consequently the effects of Air-2 depletion upon chromosome condensation in C. elegans have been a little overshadowed. It seems likely that the abnormal chromosome segregation in Drosophila cells after aurB RNAi is due to incomplete condensation, since a similar phenotype is seen in mutants of the condensin subunit Barren (Bhat, 1996). Of course, this does not exclude the possibility that defects in the organization of the centromeric regions and kinetochores arise directly as a result of aurB RNAi or as either a direct or indirect consequence of condensation defects. The increase in ploidy seen after aurora B RNAi is reminiscent of the Ipl1 phenotype in budding yeast, but differs in that it arises from both chromosome segregation and cytokinesis defects (Giet, 2001).

The resemblance of the mitotic phenotype of cells after RNAi with aurB to that previously reported for Drosophila barren mutants (Bhat, 1996) can be further explained by the failure of Barren protein to be recruited to the mitotic chromosomes after aurB RNAi. Originally recognized through this mutant defect, it was later realized that Barren is the fly homolog of a member of the pentameric complex, condensin, first shown to be required for mitotic chromosome condensation in Xenopus. It is possible that Barren or other members of the condensin complex could themselves be directly phosphorylated by Aurora B during chromosome condensation. However, the process seems likely to involve a plethora of phosphorylation events: the nuclear A-kinase anchoring protein (AKAP95) appears to target the human hCAP-D2 condensin to chromosomes and phosphorylation of condensin subunits by cdk1 has been associated both with their nuclear accumulation and activation. It has been proposed that phosphorylation of the NH₂ terminus of histone H3 leads to the recruitment or the activation of the condensin complex to the chromosome, where it can modify DNA topology. The data presented here indicate that phosphorylation of histone H3 by the Aurora B kinase and the localization of Barren onto chromosomes are associated events in mitosis. They support and extend a recent observation that human condensin proteins hCAP-E, hCAP-C, and hCAP-D2 colocalize with phosphorylated histone H3 in clusters in partially condensed regions of chromosomes in early prophase. The similarity of the effects seen on chromosome condensation resulting from loss of either aurora B or barren function is striking and points to the value of studying these processes in a single model organism amenable to both genetic manipulation and RNAi. It is perhaps surprising that in both cases partial chromosome condensation is achieved and that there can be some degree of segregation of chromatin to the poles (Giet, 2001 and references therein).

The second major mitotic abnormality observed after aurB RNAi in Drosophila cells is a failure of cytokinesis. Thus, like its mammalian and nematode counterparts AIM-1 and AIR-2, the enzyme encoded by aurora B appears essential for this process. Two proteins that play a role in cytokinesis have recently been shown to associate with the Aurora B-like kinases: Incenp, as discussed above (Adams, 2000; Kaitna, 2000), and the Zen-4 kinesin-like protein of C. elegans (Kaitna, 2000; Severson, 2000). The localization of the latter is disrupted after disruption of air-2 function using RNAi or conditional mutant alleles. Zen-4 is the C. elegans homolog of the Pavarotti KLP of Drosophila, which likewise is mislocalized on the central spindle from anaphase onwards after aurB RNAi. Pav-KLP also cooperates with Polo kinase to achieve its localization and function in Drosophila, suggesting that multiple mitotic kinases may be required to coordinate central spindle formation before cytokinesis, just as several kinases appear to be required for centrosome maturation and separation and chromosome condensation (Giet, 2001).

It is striking that aurB RNAi cells are not arrested by a mitotic checkpoint, given the abnormalities that they show in chromosome alignment at metaphase and the subsequent disorganization of the later mitotic spindle. However, the treated cells do undergo multiple cell cycles, as is clearly demonstrated in this cell culture system in which one can monitor the shift in ploidy by FACS analysis and the increase in chromosome and centrosome complements by immunocytology. It is possible that these abnormalities arise too late in the mitotic cycle to trigger checkpoint arrest, although this seems unlikely for the chromosome segregation defect. Although it is possible that Aurora B is itself required for checkpoint functions, it could also be that the kinetochore regions of chromosomes are insufficiently well organized after aurB RNAi to promote the checkpoint activity of the complex of Bub/Mad proteins that associate with unaligned centromeres. It is noteworthy that the C. elegans baculovirus inhibitor of apoptosis (IAP)-related repeat protein Bir-1 appears to be required for the localization of Air-2. Bir-1 localizes to chromosomes and then the spindle midzone and Air-2 fails to localize to these same sites in the absence of Bir-1 (Speliotes, 2000). These IAP proteins, also known as survivin, are caspase inhibitors and as such counteract apoptosis. Is it possible that B-type Aurora kinases might play a role alongside survivin in an apoptotic checkpoint to promote mitosis (Giet, 2001)?

It is of considerable interest to know the multiple substrates of Aurora B kinase and to understand its mode of regulation in mitotic progression. It seems that subcellular localization of the enzyme could be one critical means of controlling access to its substrates. The enzyme localizes throughout condensing chromosomes when phosphorylation of histone H3 is required. Aurora B's subsequent concentration at centromeres could direct enzyme activity toward specific chromosomal proteins at these sites, but may be instrumental in its movement onto the central spindle at anaphase, thereby providing an effective way of removing the enzyme from the chromatin to facilitate chromosome decondensation at telophase. Understanding the intricacies of these processes will be a future challenge (Giet, 2001).

Additional conclusions were reached by Adams (2001a) in their analysis of Aurora B function. Adams concludes that Aurora B is required for some, but not all, aspects of Incenp localization in mitosis. In the absence of Aurora B, Incenp localizes normally to chromosomes during pro-phase; however, it is subsequently unable to concentrate at centromeres and transfer to the central spindle or midbody. As predicted from previous studies, Incenp is essential for Aurora B targeting: after Incenp RNAi, Aurora B does not localize to chromosomes, midzone microtubules, or midbodies. Thus, the chromosomal passenger proteins are interdependent on one another for proper targeting during mitosis (Adams, 2001a).

This interdependence, plus the fact that the two proteins are stockpiled in an 11S complex in Xenopus eggs, suggests that they could function in vivo in a protein complex. Incenp binds microtubules in vitro and may be responsible for targeting Aurora B to the central spindle, as the kinase appears to lack microtubule binding activity of its own. However, the differences in centromere targeting in Drosophila early embryos suggest that the two proteins may not function in an obligate complex, at least during prophase (Adams, 2001a).

S. cerevisiae aurora/Ipl1p and C. elegans aurora B/AIR-2 are required for H3-serine¹⁰ phosphorylation in mitosis (Hsu, 2000). Not only is Incenp essential for the proper targeting of Aurora B in mitotic cells, but this targeting is required for normal levels of histone H3 phosphorylation on serine¹⁰. This is the first evidence that Incenp is an essential cofactor required for Aurora B kinase function in vivo (Adams, 2001a).

The availability of mitotic cells containing chromosomes with a range of levels of H3 phosphorylated on serine¹⁰ has enabled an assessment of the widely held hypothesis that H3 phosphorylation is correlated with the degree of chromatin condensation. When phospho-H3 levels and the degree of chromatin compaction were compared by quantitative fluorescence microscopy, only a weak correlation between the two values was observed. Instead, interference with Incenp and Aurora B function appears to correlate much more strongly with difficulties in assembling mitotic chromosomes of normal morphology. Mitotic chromosomes deficient in phospho-H3 have a characteristic dumpy morphology, with no evidence of resolved sister chromatids. This resembles the defects seen in Drosophila mutants in the SMC4 subunit of condensin (Steffensen, 2001) and also those of a ts mutant in C. elegans aurora B/AIR-2 when it enters mitosis at nonpermissive temperature (Severson, 2000). Phosphorylation of histone H3 or another chromosomal substrate by Aurora B might be required for the binding of condensins or other chromosomal proteins that give mitotic chromosomes their characteristic morphology (Adams, 2001a).

At later times, after addition of dsRNA, a dramatic increase is seen in the percentage of mitotic cells in prometaphase coupled with a corresponding decrease in the number of metaphase cells. This is particularly dramatic in the Incenp RNAi, where no Incenp-negative cells in metaphase are seen. Surprisingly, this did not lead to an increase in the mitotic index of the cultures. Therefore, in the absence of Incenp and/or Aurora B function, Drosophila Dmel2 cells must exit mitosis from prometaphase. Elimination of Incenp and Aurora B function does not trigger a mitotic checkpoint in Dmel2 cells. However, since these cells do not arrest in mitosis in colcemid, they appear to lack a robust metaphase checkpoint in any case (Adams, 2001a).

What is the ultimate fate of these prometaphase cells? They are not removed by cell death. An alternative explanation for the lack of an increase in mitotic index would be if the cells transit directly from prometaphase into anaphase or telophase, as is the case for budding yeast cells mutant in the essential kinetochore protein Ndc10p. Consistent with this, a variety of striking abnormalities were seen in cells either undergoing anaphase, or early in the next cell cycle. Although anaphase/telophase cells with kinetochores at opposite poles of the chromatin mass could be seen, the kinetochores were often not clustered as tightly as normal This may reflect the initiation of anaphase movement without prior alignment of the chromosomes at a metaphase plate (Adams, 2001a).

Why does abrogation of Incenp and/or Aurora B function prevent cells from attaining a stable metaphase chromosome alignment? One obvious possibility is that kinetochore function is impaired. In budding yeast, the aurora kinase Ipl1p phosphorylates the essential kinetochore component Ndc10p (Biggins, 1999). It is therefore possible that, in metazoans, one or more kinetochore components must be phosphorylated by Aurora B in order for kinetochores to function in mitosis. An obvious candidate for this is CENP-A/Cid. CENP-A retains a site homologous to serine¹⁰, which is serine⁵ in Cid. It will be important to determine whether CENP-A/Cid is phosphorylated in an Aurora B kinase-dependent manner (Adams, 2001a).

Arguing against this model is the observation that kinetochores assemble correctly, at least as far as CENP-A/Cid binding is concerned, and move to the spindle poles at anaphase/telophase. This implies that the ability of kinetochores to bind microtubules and to undergo anaphase A movement are preserved after abrogation of Incenp and Aurora B function. However, other aspects of kinetochore function, namely the ability to form bipolar spindle attachments and disjoin at anaphase, appear to be defective. How RNAi of Incenp or Aurora B leads to defects in chromosome biorientation is unknown, but this is unlikely to be a result of interference with binding of the condensin subunit barren, since barren mutants successfully complete metaphase chromosome alignment. Furthermore, the possibility that some of the abnormal aspects of chromosome behavior reflect an impairment of microtubule and/or spindle function cannot be excluded. The detailed behavior of the mitotic spindle after RNAi of Incenp and Aurora B requires further analysis (Adams, 2001a).

Anaphase/telophase cells after RNAi for Incenp or Aurora B exhibit three highly unusual chromosomal phenotypes: (1) they often have one or more pairs of sister kinetochores located in the central spindle or flanking the midbody; (2) the foci of CENP-A/Cid staining at or near the spindle poles is often present as pairs, suggesting that sister kinetochores remain paired despite having undergone anaphase A-like poleward movement; (3) separated masses of chromatin are often connected by a mass of lagging chromatin. This is referred to as chromatin and not chromosomes because the material is amorphous, and little or no evidence of a condensed mitotic chromosome morphology can be observed (Adams, 2001a).

The first two phenotypes can be explained if centromeres fail to disjoin at anaphase onset. Under these circumstances, centromeres of bioriented chromosomes would tend to accumulate near the spindle equator -- later, near the midbody -- and be stretched apart by the spindle forces. Mono-oriented kinetochores would move as pairs to one or the other spindle pole. If this occurred in cells that had attained metaphase, then the bulk of kinetochores would remain as pairs in the spindle midzone. However, abrogation of Incenp and/or Aurora B function prevents cells from reaching metaphase and would therefore be expected to lead to the observed phenotype, with most centromeres at the poles and only a few remaining in the midzone. Defects in sister kinetochore disjunction could arise if Incenp and/or Aurora B were involved in regulation of the cohesin complex at centromeres; experiments are under way to determine whether cohesin components are substrates for Aurora B (Adams, 2001a).

The presence of massive amounts of lagging chromatin is highly characteristic of anaphase/telophase after loss of Incenp and/or Aurora B function. This lagging chromatin might arise from difficulties in sister chromatid disjunction, but it is more likely that it represents a failure of the chromosomes to move as integral units under the physical stress of anaphase movement. If the dumpy chromosomes observed in prometaphase cells lack an organized infrastructure then when centromeres begin to move polewards, the chromatin of the arms might simply unravel and be left behind as a smear of amorphous chromatin. This would be consistent with the observation that interference with Aurora B function in Drosophila cells interferes with the binding of the condensin subunit barren to mitotic chromosomes (Giet, 2001). Indeed, barren mutants exhibit dramatic chromatin bridges during syncytial mitosis, however such a dramatic defect was not seen in mutants affecting the condensin subunit SMC4 in Drosophila (Steffensen, 2001). It is possible that action of Incenp/aurora B on other chromosomal components, in addition to condensin subunits, contributes to a loss of chromosomal integrity during anaphase (Adams, 2001a).

GENE STRUCTURE

cDNA clone length - 1127

Bases in 5' UTR - 79

Exons - 3

Bases in 3' UTR - 58

PROTEIN STRUCTURE

Amino Acids - 329

Structural Domains

The members of the Ipl1-aurora like kinase (IARK) subfamily are conserved serine/threonine kinases that play a key role in the control of chromosome segregation, centrosome separation, and cytokinesis from yeast to mammals. A new Drosophila member of the family, designated Ipl1-aurora-like kinase (ial), has been isolated. Phylogenetic analysis of kinase domains has established that ial is more divergent from known mammalian IARKs than is aurora (Reich, 1999).

date revised: 30 May 2002

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