IplI-aurora-like kinase/aurora B
Chromosome segregation depends on kinetochores, the structures that mediate chromosome attachment to the mitotic spindle. Mutants in IPL1, which encodes a protein kinase, were isolated in a screen for budding yeast mutants that have defects in sister chromatid separation and segregation. Cytological tests show that ipl1 mutants can separate sister chromatids but are defective in chromosome segregation. Kinetochores assembled in extracts from ipl1 mutants show altered binding to microtubules. Ipl1p phosphorylates the kinetochore component Ndc10p in vitro and it is proposed that Ipl1p regulates kinetochore function via Ndc10p phosphorylation. Ipl1p localizes to the mitotic spindle and its levels are regulated during the cell cycle. This pattern of localization and regulation is similar to that of Ipl1p homologs in higher eukaryotes, such as the human aurora2 protein. Because aurora2 has been implicated in oncogenesis, defects in kinetochore function may contribute to genetic instability in human tumors (Biggins, 1999).
The conserved Ipl1 protein kinase is essential for proper chromosome segregation and thus cell viability in the budding yeast Saccharomyces cerevisiae. Sister chromatids that have separated from each other are not properly segregated to opposite poles of ipl1-2 cells. Failures in chromosome segregation are often associated with abnormal distribution of the spindle pole-associated Nuf2-GFP protein, thus suggesting a link between potential spindle pole defects and chromosome missegregation in ipl1 mutant cells. A small fraction of ipl1-2 cells also appears to be defective in nuclear migration or bipolar spindle formation. Ipl1 associates, probably directly, with the novel and essential Sli15 protein in vivo, and both proteins are localized to the mitotic spindle. Conditional sli15 mutant cells have cytological phenotypes very similar to those of ipl1 cells, and the ipl1-2 mutation exhibits synthetic lethal genetic interaction with sli15 mutations. sli15 mutant phenotype, like ipl1 mutant phenotype, is partially suppressed by perturbations that reduce protein phosphatase 1 function. These genetic and biochemical studies indicate that Sli15 associates with Ipl1 to promote its function in chromosome segregation (Kim, 1999).
Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP; see Drosophila Inner centromere protein). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1-Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome-spindle interactions or in the movement of kinetochores along microtubules (Kang, 2001).
The spindle checkpoint prevents cell cycle progression in cells that have mitotic spindle defects. Although several spindle defects activate the spindle checkpoint, the exact nature of the primary signal is unknown. The budding yeast member of the Aurora protein kinase family, Ipl1p, is required to maintain a subset of spindle checkpoint arrests. Ipl1p is required to maintain the spindle checkpoint that is induced by overexpression of the protein kinase Mps1. Inactivating Ipl1p allows cells overexpressing Mps1p to escape from mitosis and segregate their chromosomes normally. Therefore, the requirement for Ipl1p in the spindle checkpoint is not a consequence of kinetochore and/or spindle defects. The requirement for Ipl1p distinguishes two different activators of the spindle checkpoint: Ipl1p function is required for the delay triggered by chromosomes whose kinetochores are not under tension, but is not required for arrest induced by spindle depolymerization. Ipl1p localizes at or near kinetochores during mitosis, and it is proposed that Ipl1p is required to monitor tension at the kinetochore (Biggins, 2001).
Metazoans contain three aurora-related kinases. Aurora A is required for spindle formation while aurora B is required for chromosome condensation and cytokinesis. Less is known about the function of aurora C. S. pombe contains a single aurora-related kinase, Ark1. Although Ark1 protein levels remain constant as cells progress through the mitotic cell cycle, its distribution alters during mitosis and meiosis. Throughout G2 Ark1 is concentrated in one to three nuclear foci that are not associated with the spindle pole body/centromere complex. Following commitment to mitosis, Ark1 associates with chromatin and is particularly concentrated at several sites, including kinetochores/centromeres. Kinetochore/centromere association diminishes during anaphase A, after which it is distributed along the spindle. The protein becomes restricted to a small central zone that transiently enlarges as the spindle extends. As in many other systems mitotic fission yeast cells exhibit a much greater degree of phosphorylation of serine 10 of histone H3 than interphase cells. A number of studies have linked this modification with chromosome condensation. Ark1 immuno-precipitates phosphorylate serine 10 of histone H3 in vitro. This activity is highest in mitotic extracts. The following all suggest that Ark1 phosphorylates serine 10 of histone H3 in vivo: the absence of the histone H3 phospho-serine 10 epitope from mitotic cells in which the ark1(+) gene has been deleted (ark1.Delta1); the inability of these cells to resolve their chromosomes during anaphase, and the co-localization of this phospho-epitope with Ark1 early in mitosis. ark1.Delta1 cells also exhibit a reduction in kinetochore activity and a minor defect in spindle formation. Thus the enzyme activity, localization and phenotype arising from manipulations of this single fission yeast aurora kinase family member suggest that this single kinase is executing functions that are separately implemented by distinct aurora A and aurora B kinases in higher systems (Petersen, 2002).
How sister kinetochores attach to microtubules from opposite spindle poles during mitosis (bi-orientation) remains poorly understood. In yeast, the ortholog of the Aurora B-INCENP protein kinase complex (Ipl1-Sli15) may have a role in this crucial process, because it is necessary to prevent attachment of sister kinetochores to microtubules from the same spindle pole. IPL1 function was investigated in cells that cannot replicate their chromosomes but nevertheless duplicate their spindle pole bodies (SPBs). Kinetochores detach from old SPBs and reattach to old and new SPBs with equal frequency in IPL1+ cells, but remain attached to old SPBs in ipl1 mutants. This raises the possibility that Ipl1-Sli15 facilitates bi-orientation by promoting turnover of kinetochore-SPB connections until traction of sister kinetochores toward opposite spindle poles creates tension in the surrounding chromatin (Tanaka, 2002).
The spindle checkpoint inhibits anaphase until all chromosomes have established bipolar attachment. Two kinetochore states trigger this checkpoint. The absence of microtubules activates the attachment response, while the inability of attached microtubules to generate tension triggers the tension/orientation response. The processes regulated by the single aurora kinase of fission yeast, Ark1, represent a combination of the events that are regulated by aurora-A and aurora-B kinases in higher systems. The aurora kinase of budding yeast, Ipl1, is required for the tension/orientation, but not attachment, response. In contrast, the single aurora kinase of fission yeast, Ark1, is required for the attachment response. Having established that the initiator codon assigned to ark1+ was incorrect and that Ark1-associated kinase activity depends upon survivin function and phosphorylation, it was found that the loss of Ark1 from kinetochores by either depletion or use of a survivin mutant overides the checkpoint response to microtubule depolymerization. Ark1/survivin function is not required for the association of Bub1 (see Drosophila Bub1) or Mad3 with the kinetochores. However, it is required for two aspects of Mad2 function that accompany checkpoint activation: full-scale association with kinetochores and formation of a complex with Mad3. Neither the phosphorylation of histone H3 that accompanies chromosome condensation nor condensin recruitment to mitotic chromatin is seen when Ark1 function is compromised. Cytokinesis is not affected by Ark1 depletion or expression of the 'kinase dead' ark1.K118R mutant (Petersen, 2003).
At anaphase onset, the protease separase triggers chromosome segregation by cleaving the chromosomal cohesin complex. Cohesin destruction in metaphase was shown to be sufficient for segregation of much of the budding yeast genome, but not of the long arm of chromosome XII that contains the rDNA repeats. rDNA in metaphase, unlike most other sequences, remains in an undercondensed and topologically entangled state. Separase, concomitantly with cleaving cohesin, activates the phosphatase Cdc14. Cdc14 exerts two effects on rDNA, both mediated by the condensin complex. Lengthwise condensation of rDNA shortens the chromosome XII arm sufficiently for segregation. This condensation depends on the aurora B kinase complex. Independently of condensation, Cdc14 induces condensin-dependent resolution of cohesin-independent rDNA linkage. Cdc14-dependent sister chromatid resolution at the rDNA could introduce a temporal order to chromosome segregation (Sullivan, 2004).
A balance in the activities of the Ipl1 Aurora kinase and the Glc7 phosphatase is essential for normal chromosome segregation in yeast. This balance is modulated by the Set1 methyltransferase. Deletion of SET1 suppresses chromosome loss in ipl1-2 cells. Conversely, combination of SET1 and GLC7 mutations is lethal. Strikingly, these effects are independent of previously defined functions for Set1 in transcription initiation and histone H3 methylation. Set1 is required for methylation of conserved lysines in a kinetochore protein, Dam1. Biochemical and genetic experiments indicate that Dam1 methylation inhibits Ipl1-mediated phosphorylation of flanking serines. These studies demonstrate that Set1 has important, unexpected functions in mitosis. Moreover, these findings suggest that antagonism between lysine methylation and serine phosphorylation is a fundamental mechanism for controlling protein function (Zhang, 2005).
The data reveal unexpected functional connections between the Set1 methyltransferase and phosphorylation events governed by the Ipl1 kinase and the Glc7 phosphatase. Loss of Set1 suppresses chromosome segregation defects caused by the ipl1-2 allele and is synthetic lethal with the glc7-127 allele. The mitotic functions of Set1 require Bre2, Swd1, and Sdc1, indicating that Set1 functions in the context of the COMPASS complex to modulate Ipl1-Glc7 functions in chromosome segregation (Zhang, 2005).
Previous studies have revealed a role for Set1 and COMPASS (Complex Proteins Associated with Set1) in gene transcription that requires Paf1 and ubiquitylation of histone H2B at K123. However, the data demonstrate that deletion of PAF1 or mutation of H2B K123 cannot suppress ipl1-2. Therefore, the suppression of ipl1-2 upon deletion of SET1 is independent of COMPASS functions in transcription initiation and early elongation (Zhang, 2005).
Prior to these studies, histone H3 K4 was the only known substrate of Set1. However, loss of H3 K4 methylation is not likely the molecular basis for the observed genetic interactions between SET1, IPL1, and GLC7: (1) mutations in SET1, PAF1, or histone H2B K123 all globally diminish H3 K4 methylation, yet only SET1 deletion suppresses ipl1-2; (2) no correlation was found between the effects of deletion of other COMPASS components on H3 K4 methylation and suppression of ipl1-2; (3) mutation of H3 K4 to R suppresses ipl1-2 more weakly than does deletion or mutation of SET1, and the H3 K4R mutation is not synthetic lethal with glc7-127; (4) chromatin immunoprecipitation results indicate that little or no H3 K4 methylation occurs at centromeres in S. cerevisiae, consistent with the replacement of H3 with Cse4 in centromeric nucleosomes (Zhang, 2005).
Unlike centromeres in S. pombe and most other organisms, centromeres in S. cerevisiae are not flanked by heterochromatic repeat elements, and this yeast does not contain HP1-like proteins or Suv39 methyltransferases. H3 K9 is not methylated in S. cerevisiae, and mutations in H3 S10 do not affect chromosome segregation. Moreover, no evidence was found of global changes in phosphorylation of S10 in the absence of Set1. Therefore, the effects of Set1 loss on Ipl1 functions do not likely reflect indirect effects on modifications at S10 or K9 in H3. Rather, the results indicate that these effects are mediated through Set1-mediated methylation of at least one nonhistone substrate, Dam1 (Zhang, 2005).
How might Dam1 methylation at histone H2B K233 or K194 contribute to proper chromosome segregation? By analogy to the effects of histone methylation on the occurrence of other posttranslational modifications of the histones, K233 methylation might directly affect phosphorylation of neighboring serines. This model is consistent with the observation that Ipl1-mediated phosphorylation of methylated Dam1 peptides is inhibited in vitro, as well as genetic data that reveal functional connections between K233 and S232, S234, and S235. The data indicate that prevention of K233 methylation by set1Δ allows improved phosphorylation of Dam1 by the cripples ipl1-2 kinase, as reflected by suppression of the ipl1-2 phenotype, but might allow too much or too persistent phosphorylation by wild-type Ipl1. Conversely, the suppression of the lethality of the DAM1 K233A allele by flanking S to A mutations or by the ipl1-2 mutation (but not by S to D mutations) strongly suggests that negative effects associated with loss of Dam1 methylation can be countered by decreased phosphorylation at these sites. Several previous findings that indicate a balance in the phosphorylation and dephosphorylation of IPL1 and GLC7 substrates is essential for normal cell growth and chromosome segregation. These strongly suggest that the region between K194 and S235 is a critical module in Dam1 that is regulated by both phosphorylation and methylation (Zhang, 2005).
Several protein kinases collaborate to orchestrate and integrate cellular and chromosomal events at the G2/M transition in both mitotic and meiotic cells. During the G2/M transition in meiosis, this includes the completion of crossover recombination, spindle formation, and synaptonemal complex (SC) breakdown. Ipl1/Aurora B kinase was identified as the main regulator of SC disassembly. Mutants lacking Ipl1 or its kinase activity assemble SCs with normal timing, but fail to dissociate the central element component Zip1, as well as its binding partner, Smt3/SUMO, from chromosomes in a timely fashion. Moreover, lack of Ipl1 activity causes delayed SC disassembly in a cdc5 as well as a CDC5-inducible ndt80 mutant. Crossover levels in the ipl1 mutant are similar to those observed in wild type, indicating that full SC disassembly is not a prerequisite for joint molecule resolution and subsequent crossover formation. Moreover, expression of meiosis I and meiosis II-specific B-type cyclins occur normally in ipl1 mutants, despite delayed formation of anaphase I spindles. These observations suggest that Ipl1 coordinates changes to meiotic chromosome structure with resolution of crossovers and cell cycle progression at the end of meiotic prophase (Jordan, 2009).
The Shugoshin/Aurora circuitry that controls the timely release of cohesins from sister chromatids in meiosis and mitosis is widely conserved among eukaryotes, although little is known about its function in organisms whose chromosomes lack a localized centromere. This study shows that C. elegans chromosomes rely on an alternative mechanism to protect meiotic cohesin that is shugoshin-independent and instead involves the activity of a new chromosome-associated protein named LAB-1 (Long Arm of the Bivalent). LAB-1 preserves meiotic sister chromatid cohesion by restricting the localization of the C. elegans Aurora B kinase, AIR-2, to the interface between homologs via the activity of the PP1/Glc7 phosphatase GSP-2. The localization of LAB-1 to chromosomes of dividing embryos and the suppression of mitotic-specific defects in air-2 mutant embryos with reduced LAB-1 activity support a global role of LAB-1 in antagonizing AIR-2 in both meiosis and mitosis. Although the localization of a GFP fusion and the analysis of mutants and RNAi-mediated knockdowns downplay a role for the C. elegans shugoshin protein in cohesin protection, shugoshin nevertheless helps to ensure the high fidelity of chromosome segregation at metaphase I. It is proposed that, in C. elegans, a LAB-1-mediated mechanism evolved to offset the challenges of providing protection against separase activity throughout a larger chromosome area (de Carvalho, 2009).
Kinetochores of sister chromatids attach to microtubules emanating from the same pole (coorientation) during meiosis I and microtubules emanating from opposite poles (biorientation) during meiosis II. The Aurora B kinase Ipl1 regulates kinetochore-microtubule attachment during both meiotic divisions, and a complex known as the monopolin complex ensures that the protein kinase coorients sister chromatids during meiosis I. Furthermore, the defining of conditions sufficient to induce sister kinetochore coorientation during mitosis provides insight into monopolin complex function. The monopolin complex joins sister kinetochores independently of cohesins, the proteins that hold sister chromatids together. It is proposed that this function of the monopolin complex helps Aurora B coorient sister chromatids during meiosis I (Monje-Casas. 2007).
To date, four components of the monopolin complex have been identified. Mam1 is a meiosis-specific protein present at kinetochores from pachytene to metaphase I. The monopolin complex components Csm1 and Lrs4 are expressed during both mitosis and meiosis. They reside in the nucleolus until G2, when they are released by the Polo kinase Cdc5. After their release, Csm1 and Lrs4 form a complex with Mam1 and bind to kinetochores, Mam1 recruits the ubiquitously expressed casein kinase 1Δ/epsilon Hrr25, which is also required for sister kinetochore coorientation, to kinetochores during meiosis I. The meiosis-specific protein Spo13 is also necessary for kinetochore coorientation. In its absence, the monopolin complex initially associates with kinetochores but cannot be maintained there. How the monopolin complex and proteins that regulate its association with kinetochores bring about sister kinetochore coorientation is poorly understood (Monje-Casas. 2007 and references therein).
Aurora B kinases play an essential role in biorienting sister kinetochores during mitosis. It was therefore possible that factors promoting the coorientation of sister kinetochores during meiosis I would be inhibitors of Aurora B function. However, these studies indicate that this is not the case. Rather, they point toward Ipl1 performing the same function during meiosis I and II as it does during mitosis—that is, severing microtubule-kinetochore attachments that are not under tension. The monopolin complex modifies sister kinetochores so that they are only under tension when homologs are bioriented. How does the monopolin complex accomplish this? Several lines of evidence indicate that the complex functions as a link between sister kinetochores that is distinct from cohesins. When overproduced during mitosis, Cdc5 and Mam1 induce the cosegregation of sister chromatids, with the two sisters being tightly associated near centromeres but not at arm regions. The tight association of sister centromeres is not observed in other mutants that cosegregate sister chromatids to the same pole during anaphase, such as ipl1-321 mutants or cells depleted for cohesins. Importantly, high levels of Cdc5 and Mam1 are capable of linking cosegregating sister chromatids in cells lacking IPL1 or cohesin. Even in the absence of the cohesin subunit REC8, 91% of sister chromatids are associated at centromeres during prophase I (ndt80Δ block) and preferentially (85%) cosegregate to the same pole during anaphase I. During this cosegregation, centromeric sequences appear tightly paired, whereas arm sequences do not. Importantly, this association of sister chromatids in spo11Δ rec8Δ cells is in part dependent on MAM1, indicating that the protein has sister centromere-connecting abilities not only when overproduced during mitosis but also during meiosis I (Monje-Casas. 2007).
How could the joining of sister kinetochores force them to attach to microtubules emanating from the same pole? The fusion of sister kinetochores could put steric constraints on the kinetochores, hence favoring attachment of both kinetochores to microtubules emanating from the same spindle pole. Ultrastructural analyses of meiosis I spindles in the salamander Amphiuma tridactylum and several grasshopper species support this hypothesis. The idea is favored that, at least in yeast, the monopolin complex, in addition to joining sister kinetochores, prevents attachment of microtubules to one of the two sister kinetochores because this model is more consistent with ultrastructural analyses of meiosis I spindles in budding yeast. In S. cerevisiae, in which kinetochores bind to only one microtubule, the number of microtubules in the meiosis I spindle is more consistent with one microtubule attaching to one homolog. It is noted that in other organisms such as Drosophila and mouse, sister kinetochores also appear to form a single microtubule-binding surface during metaphase I. The second observation leading to the model in which the monopolin complex links sister centromeres and prevents one kinetochore from attaching to microtubules is that overexpression of a functional monopolin complex allows 35% of cells treated with the microtubule-depolymerizing drug nocodazole, which causes activation of the spindle checkpoint, to escape the checkpoint arrest (Monje-Casas. 2007).
The mechanisms whereby the monopolin complex links sister kinetochores remain to be determined. It is proposed that, after DNA replication, sister chromatids are initially topologically linked due to catenation even in the absence of cohesins. Mam1 assembles onto the kinetochores of these sisters, joining them at centromeres. Whether this link is able to withstand the pulling forces exerted by microtubules is unclear, but it is envisioned that the monopolin complex bridges the sister kinetochores in a way that ensures their concerted movement and conceals one of the two microtubule attachment sites. The monopolin complex could itself bridge sister chromatids or induce changes in kinetochore substructures to induce their interaction with each other. In this regard, it is interesting to note that a component of the monopolin complex, Hrr25, forms multimers only during meiosis I, potentially providing a bridging function. In S. pombe, coorientation factors appear to bring about sister kinetochore coorientation through cohesin complexes. These results suggest that, in S. cerevisiae, coorientation factors themselves have the ability to join sister chromatids. It is proposed that this function is important to promote sister kinetochore coorientation. Whether these linkages simply impose steric constraints or additionally control the attachment of microtubules to kinetochores will be an important question to examine in the future (Monje-Casas. 2007).
Homologue segregation during the first meiotic division requires the proper spatial regulation of sister chromatid cohesion and its dissolution along chromosome arms, but its protection at centromeric regions. This protection requires the conserved MEI-S332/Sgo1 proteins that localize to centromeric regions and also recruit the PP2A phosphatase by binding its regulatory subunit, Rts1. Centromeric Rts1/PP2A then locally prevents cohesion dissolution possibly by dephosphorylating the protein complex cohesin. This study shows that Aurora B kinase in Saccharomyces cerevisiae (Ipl1) is also essential for the protection of meiotic centromeric cohesion. Coupled with a previous study in Drosophila, this meiotic function of Aurora B kinase appears to be conserved among eukaryotes. Furthermore, Sgo1 recruits Ipl1 to centromeric regions. In the absence of Ipl1, Rts1 can initially bind to centromeric regions but disappears from these regions after anaphase I onset. It is suggested that centromeric Ipl1 ensures the continued centromeric presence of active Rts1/PP2A, which in turn locally protects cohesin and cohesion (Yu, 2007).
It is critical to elucidate the pathways that mediate spindle assembly and therefore ensure accurate chromosome segregation during cell division. Studies of a unique allele of the budding yeast Ipl1/Aurora protein kinase revealed that it is required for centrosome-mediated spindle assembly in the absence of the BimC motor protein Cin8. In addition, it was found that the Ase1 spindle midzone-associated protein is required for bipolar spindle assembly. The cin8 ipl1 and cin8 ase1 double mutant cells exhibit similar defects, and Ase1 overexpression completely restores spindle assembly in cin8 ipl1 strains. Consistent with the possibility that Ipl1 regulates Ase1, an ase1 mutant lacking the Ipl1 consensus phosphorylation sites cannot assemble spindles in the absence of Cin8. In addition, Ase1 phosphorylation and localization are altered in an ipl1 mutant. It is therefore proposed that Ipl1/Aurora and Ase1 constitute a previously unidentified spindle assembly pathway that becomes essential in the absence of Cin8 (Kotwaliwale, 2007).
An emerging family of kinases related to the Drosophila Aurora and budding yeast Ipl1 proteins has been implicated in chromosome segregation and mitotic spindle formation in a number of organisms. Unlike other Aurora/Ipl1-related kinases, the C. elegans ortholog AIR-2 is associated with meiotic and mitotic chromosomes. AIR-2 is initially localized to the chromosomes of the most mature prophase I-arrested oocyte residing next to the spermatheca. This localization is dependent on the presence of sperm in the spermatheca. After fertilization, AIR-2 remains associated with chromosomes during each meiotic division. However, during both meiotic anaphases, AIR-2 is present between the separating chromosomes. AIR-2 also remains associated with both extruded polar bodies. In the embryo, AIR-2 is found on metaphase chromosomes, moves to midbody microtubules at anaphase, and then persists at the cytokinesis remnant. Disruption of AIR-2 expression by RNA- mediated interference produces entire broods of one-cell embryos that have executed multiple cell cycles in the complete absence of cytokinesis. The embryos accumulate large amounts of DNA and microtubule asters. Polar bodies are not extruded, but remain in the embryo where they continue to replicate. The cytokinesis defect appears to be late in the cell cycle because transient cleavage furrows initiate at the proper location, but regress before the division is complete. Additionally, staining with a marker of midbody microtubules reveals that at least some of the components of the midbody are not well localized in the absence of AIR-2 activity. These results suggest that during each meiotic and mitotic division, AIR-2 may coordinate the congression of metaphase chromosomes with the subsequent events of polar body extrusion and cytokinesis (Schumacher, 1998).
A new sterile uncoordinated C. elegans mutant, stu-7, has been isolated that is defective in post-embryonic cell divisions in a regionally-specific fashion. The anterior of the worm is relatively unaffected whereas the mid-body and/or posterior are markedly thin, often resulting in worms having a central 'waist'. stu-7 encodes a member of the recently expanding aurora sub-family of serine/threonine kinases. Elimination of maternal as well as zygotic stu-7 expression reveals that stu-7 is essential for mitosis from the first embryonic cell cycle onwards and is required for chromosome segregation though not for centrosome separation or for setting up a bipolar spindle. Multicopy expression of stu-7 also causes mitotic defects, suggesting that the level of this protein must be tightly controlled in order to maintain genetic stability during development (Woollard, 1999).
In animal cells, cytokinesis begins shortly after the sister chromatids move to the spindle poles. The inner centromere protein (Incenp) has been implicated in both chromosome segregation and cytokinesis, but it is not known exactly how it mediates these two distinct processes. Two Caenorhabditis elegans proteins, ICP-1 and ICP-2, with significant homology in their carboxyl termini to the corresponding region of vertebrate Incenp, have been identified. Embryos depleted of ICP-1 by RNA-mediated interference have defects in both chromosome segregation and cytokinesis. Depletion of the Aurora-like kinase AIR-2 results in a similar phenotype. The carboxy-terminal region of Incenp is also homologous to that in Sli15p, a budding yeast protein that functions with the yeast Aurora kinase Ipl1p. ICP-1 binds C. elegans AIR-2 in vitro, and the corresponding mammalian orthologs Incenp and AIRK2 can be co-immunoprecipitated from cell extracts. A significant fraction of embryos depleted of ICP-1 and AIR-2 completed one cell division over the course of several cell cycles. ICP-1 promotes the stable localization of ZEN-4 (also known as CeMKLP1), a kinesin-like protein required for central spindle assembly. It is concluded that ICP-1 and AIR-2 are part of a complex that is essential for chromosome segregation and for efficient completion of cytokinesis. It is proposed that this complex acts by promoting dissolution of sister chromatid cohesion and the assembly of the central spindle (Kaitna, 2000).
The Aurora/Ipl1p-related kinase AIR-2 is required for mitotic chromosome segregation and cytokinesis in early C. elegans embryos. Previous studies have relied on non-conditional mutations or RNA-mediated interference (RNAi) to inactivate AIR-2. It has therefore not been possible to determine whether AIR-2 functions directly in cytokinesis or if the cleavage defect results indirectly from the failure to segregate DNA. One intriguing hypothesis is that AIR-2 acts to localize the mitotic kinesin-like protein ZEN-4 (also known as CeMKLP1), which later functions in cytokinesis. Using conditional alleles, it has been established that AIR-2 is required at metaphase or early anaphase for normal segregation of chromosomes, localization of ZEN-4, and cytokinesis. ZEN-4 is first required late in cytokinesis, and also functions to maintain cell separation through much of the subsequent interphase. DNA segregation defects alone were not sufficient to disrupt cytokinesis in other mutants, suggesting that AIR-2 acts specifically during cytokinesis through ZEN-4. AIR-2 and ZEN-4 share similar genetic interactions with the formin homology (FH) protein CYK-1, suggesting that AIR-2 and ZEN-4 function in a single pathway, in parallel to a contractile ring pathway that includes CYK-1. Using in vitro co-immunoprecipitation experiments, it has been found that AIR-2 and ZEN-4 interact directly. It is concluded that AIR-2 has two functions during mitosis: one in chromosome segregation, and a second, independent function in cytokinesis through ZEN-4. AIR-2 and ZEN-4 may act in parallel to a second pathway that includes CYK-1 (Severson, 2000).
Baculoviral IAP repeat proteins (BIRPs) may affect cell death, cell division, and tumorigenesis. The C. elegans BIRP BIR-1 localizes to chromosomes and to the spindle midzone. Embryos and fertilized oocytes lacking BIR-1 have defects in chromosome behavior, spindle midzone formation, and cytokinesis. Indistinguishable defects are observed in fertilized oocytes and embryos lacking the Aurora-like kinase AIR-2. AIR-2 is not present on chromosomes in the absence of BIR-1. Histone H3 phosphorylation and HCP-1 staining, which marks kinetochores, are reduced in the absence of either BIR-1 or AIR-2. It is proposed that BIR-1 localizes AIR-2 to chromosomes and perhaps to the spindle midzone, where AIR-2 phosphorylates proteins that affect chromosome behavior and spindle midzone organization. The human BIRP survivin, which is upregulated in tumors, can partially substitute for BIR-1 in C. elegans. Deregulation of bir-1 promotes changes in ploidy, suggesting that similar deregulation of mammalian BIRPs may contribute to tumorigenesis (Speliotes, 2000).
Mitotic chromosome segregation depends on bi-orientation and capture of sister kinetochores by microtubules emanating from opposite spindle poles and the near synchronous loss of sister chromatid cohesion. During meiosis I, in contrast, sister kinetochores orient to the same pole, and homologous kinetochores are captured by microtubules emanating from opposite spindle poles. Additionally, mechanisms exist that prevent complete loss of cohesion during meiosis I. These features ensure that homologs separate during meiosis I and sister chromatids remain together until meiosis II. The mechanisms responsible for orienting kinetochores in mitosis and for causing asynchronous loss of cohesion during meiosis are not well understood. During mitosis in C. elegans, aurora B kinase (AIR-2) is not required for sister chromatid separation, but it is required for chromosome segregation. Condensin recruitment during metaphase requires AIR-2; however, condensin functions during prometaphase is independent of AIR-2. During metaphase, AIR-2 promotes chromosome congression to the metaphase plate, perhaps by inhibiting attachment of chromatids to both spindle poles. During meiosis in AIR-2-depleted oocytes, congression of bivalents appears normal, but segregation fails. Localization of AIR-2 on meiotic bivalents suggests this kinase promotes separation of homologs by promoting the loss of cohesion distal to the single chiasma. Inactivation of the phosphatase that antagonizes AIR-2 causes premature separation of chromatids during meiosis I, in a separase-dependent reaction. It is concluded that aurora B functions to resolve chiasmata during meiosis I and to regulate kinetochore function during mitosis. Condensin mediates chromosome condensation during prophase, and condensin-independent pathways contribute to chromosome condensation during metaphase (Kaitna, 2002).
The faithful segregation of chromosomes to daughter cells during cell division is critical for propagation of species and for the health of individual organisms. In sexually reproducing species, two modes of chromosome segregation occur: mitotic divisions, in which sister chromatids segregate from one another, and meiotic divisions, in which homologous chromosomes segregate away from one another. Failure of mitotic chromosome segregation causes aneuploidies that might contribute to the generation of cancer. However, failures of meiotic chromosome segregation generally result in the formation of an inviable zygote. In rare cases, aneuploid gametes can develop into viable individuals, albeit with developmental abnormalities, as is the case with trisomy 21 in humans. Although meiosis and mitosis are of great fundamental and medical importance, the molecular mechanisms that mediate these events are not fully understood (Kaitna, 2002).
In recent years, major insights have been made into some aspects of mitotic chromosome segregation. Notably, the cohesin complex has been identified that maintains the association between sister chromatids from the time of their synthesis to the time of their separation at the metaphase to anaphase transition. Moreover, a protease, separase, has been identified that is responsible for cleaving one subunit of the cohesin complex, thereby allowing the two sister chromatids to be separated by the pulling forces imposed on the kinetochores by the microtubules of the mitotic spindle. Finally, this proteolytic event has been shown to be regulated both at the level of the protease and at the level of the substrate. Thus, the formation and destruction of the ties that bind sisters together are well characterized. Less well understood is the mechanism by which microtubules emanating from each pole of the mitotic spindle manage to attach to one and only one of the kinetochores on the two sister chromatids (Kaitna, 2002).
During meiosis, the arrangement of chromosomes on the meiotic spindle is radically different from the situation during mitosis. Several critical differences have been found. (1) Bivalents (homologous chromosomes linked as a consequence of meiotic crossover) are aligned on the spindle rather than pairs of sister chromatids. Thus, bivalents are arranged with one pair of sister chromatids facing one spindle pole and the other homologous pair facing the other spindle pole. (2) The linkage between the two kinetochores of homologous chromosomes is less direct than is the case in mitotic chromosomes; it is mediated by chiasmata that can be many megabases away. (3) During mitosis, sister chromatid cohesion is lost synchronously upon anaphase onset along the entire length of the chromosome, whereas, during meiosis, chromosome arm cohesion is lost at anaphase I, and cohesion in the vicinity of the kinetochore is maintained until anaphase II. Kinetochore proximal cohesion enables sister chromatids to separate from one another during meiosis II. The mechanism by which specific subsets of cohesin are maintained throughout the first meiotic division remains obscure. However, the cohesin subunit that is cleaved by separase may be one target for this regulation. This is suggested by the observation that, in budding yeast, the mitotic isoform of this protein, Scc1p, can partially substitute for the meiotic-specific isoform Rec8p. However, the mitotic isoform is incompetent to maintain the linkage between sister chromatids after meiosis I (Kaitna, 2002 and references therein).
Thus, mitosis and meiosis are two related but clearly distinct variants of a single process in which related DNA sequences are segregated from one another. As such, it is not surprising that some of the same components participate in both processes. Thus, cohesin and the cohesin cleaving protease separase are essential for both processes and play much the same role in both cases. Given that the two processes are different, it is also not surprising that there are some factors that are required for one process but completely dispensable for the other (Kaitna, 2002).
This study describes the role of the aurora B kinase, AIR-2, in mitotic and meiotic chromosome segregation in the nematode C. elegans. A variety of studies indicate that aurora B functions with at least two other proteins, BIR-1/survivin and Incenp, to form a complex, hereafter referred to as the ABI complex. Components of the ABI complex are required for chromosome segregation in budding and fission yeast, nematodes, and Drosophila. In a number of organisms, the ABI complex localizes to chromosomes during prometaphase, becomes restricted to centromeric regions during metaphase, and then binds to the central spindle during anaphase. Aurora B is the major mitotic kinase that phosphorylates histone H3 at serine 10. The function of aurora B in chromosome segregation has been studied in most detail in budding yeast. Results thus far seem to implicate aurora B in regulating kinetochore function. There are no data available yet concerning the function of aurora B in meiosis (Kaitna, 2002).
In both Drosophila cultured cells and in S. pombe, depletion or inactivation of ABI complex members prevents the binding of condensin, a multiprotein complex previously implicated in chromosome condensation. The condensin complex has as its core two SMC (structural maintenance of chromosomes) subunits and three additional subunits. Immunodepletion and reconstitution experiments have established that condensin is essential for chromosome condensation. The role of condensin in chromosome condensation has also been studied genetically in yeast and Drosophila. In budding yeast, loss of condensin is associated with defects in chromosome segregation; chromatin compaction, which is modest even in wild-type cells, is somewhat reduced in the absence of condensin subunits. In fission yeast, chromosome condensation is observable in mitotic cells and requires condensin. However, in Drosophila, mutations in condensin subunits cause a surprisingly mild condensation phenotype. Chromosomes shorten along the longitudinal axis, but resolution of sister chromatids is impaired, leading to extensive chromosome bridges when sister chromatids separate at anaphase. Condensin has also been implicated in chromosome segregation in nematodes, but this role has not been studied in detail; rather, a condensin-related complex has been intensively studied with respect to dosage compensation. Although these data are consistent with the possibility that the mitotic chromosome segregation phenotype seen in the absence of aurora B is caused by the failure of condensin binding to chromatin, this possibility has not yet been directly addressed (Kaitna, 2002 and references therein).
In this study, it is shown that, while AIR-2 is indeed required for both meiotic and mitotic chromosome segregation, there appear to be fundamental differences in the mechanisms by which this kinase contributes to these processes. Separation of sister chromatids is not impaired in C. elegans air-2(or207ts); in nematodes as in Drosophila and S. pombe, the Aurora B kinase is required for the recruitment of the condensin complex to mitotic chromosomes. In addition, the condensin complex functions during prophase to condense chromosomes; remarkably, this function appears to be AIR-2 independent. AIR-2 has an additional role in mitosis that is not due to the failure to recruit condensin: AIR-2 appears to be essential for bi-orientation of sister kinetochores. In striking contrast, during meiosis, AIR-2 is not required for the orderly alignment of bivalents on the metaphase plates, but it is required for the separation of homologous chromosomes. AIR-2 is discretely localized to the region of the bivalents where sister chromatid cohesion is lost during the first meiotic division. Inactivation of the phosphatase that acts antagonistically to the AIR-2 kinase, GLC-7alpha,ß, allows premature separation of chromatids during meiosis in a separase-dependent manner. Based on these results, it is speculated that AIR-2 acts during meiosis I to spatially regulate cleavage of a subset of cohesin, thereby allowing homologous pairs of chromosomes to separate (Kaitna, 2002).
Thus, during mitosis, AIR-2 promotes the association of condensin with chromosomes. Since depletion of condensin subunits does not mimic the chromosome segregation phenotype caused by inactivation of AIR-2, AIR-2 must serve additional function(s). Cytological analysis suggests that resolution/organization of kinetochores into pairs of oriented lateral elements occurs, albeit with a slight delay, in AIR-2-depleted embryos. In prometaphase, chromosomes with resolved kinetochores containing a variety of kinetochore antigens, including HCP-3, HCP-4, and MCAK, are observed. However, these chromosomes fail to congress properly, and they fail to segregate during mitosis, though they elongate on the mitotic spindle. This chromosome elongation requires interactions between microtubules and kinetochore, and, during anaphase, cohesion between sister chromatids is resolved. Given these results, it is suggested that in the absence of AIR-2 activity each chromatid becomes attached to both spindle poles, assuming a so-called merotelic configuration, and therefore chromosome segregation is inhibited (Kaitna, 2002).
In budding yeast, chromosome segregation also requires an aurora family kinase, Ipl1p. Cells lacking Ipl1p activity or Sli15p (the yeast Incenp homolog) activity exhibit massive defects in chromosome segregation. Unlike the situation in C. elegans, the hallmark of this mutant phenotype is the segregation of both sister chromatids to a single pole. Chromosomes marked at defined loci have been used to show that, in ipl1ts mutant cells, both separation of sister chromatids and dissociation of the cohesin complex occur with normal or near normal kinetics. One explanation for these findings is that Ipl1p may be required for kinetochore assembly or function, a speculation that is supported by the fact that Ipl1p seems to inhibit binding of kinetochores to microtubules in vitro and genetic and biochemical interactions have been detected between the yeast orthologs of the ABI complex members (Ipl1p, Bir1p, Sli15p) and a variety of kinetochore components. However, since some chromosomes do segregate normally in ipl1ts cells, it is not clear if defects in MT attachment to kinetochores can fully account for the phenotype observed in vivo. Recent data have provided a new explanation for the chromosome segregation defects caused by loss of Ipl1p function. Ipl1p appears to destabilize kinetochore-microtubule interactions. This activity is particularly apparent in yeast, since kinetochores are bound by microtubules during G1, and, in the absence of Ipl1p, this results in the frequent mono-orientation of sister chromatids. However, it is important to investigate if aurora B kinase has a similar function in organisms in which microtubules gain access to kinetochores only upon nuclear envelope breakdown (Kaitna, 2002).
In C. elegans, AIR-2 may perform a similar function to that described in budding yeast, albeit with a different morphological endpoint. C. elegans chromosomes are holocentric with multiple microtubule attachment sites along the length of the chromosome. This structure must be organized so that each attachment site on a sister chromatid is engaged by microtubules emanating from a single spindle pole, to prevent attachment of a single chromatid to both spindle poles (merotelic configuration). Once a chromatid is oriented toward a spindle pole, steric constraints would likely inhibit attachment of this chromatid to the other spindle pole. However, before chromatid orientation occurs, there seems to be no obvious mechanism that inhibits improper attachments. One possibility is that, as in budding yeast, AIR-2 destabilizes kinetochore-microtubule interactions. Thus, in the absence of AIR-2 activity, the holocentric chromosome can be engaged by microtubules from both spindle poles and, as a result, become stretched along the spindle axis (Kaitna, 2002).
If AIR-2 generally destabilizes kinetochore-microtubule interactions, it is not clear how correct attachments could be established. One hypothesis is that aurora kinase may selectively destabilize microtubule-kinetochore interactions that do not generate tension. This may be true in C. elegans as well, but, if so, it is suggested that AIR-2 must detect tension exerted across a pair of sister chromatids rather than simply the presence of tension at a kinetochore. Merotelic attachments could generate tension at kinetochores on a single chromatid, but, in wild-type embryos, this configuration is apparently not stable. It is speculated therefore that tension must be exerted on the structure between sister kinetochores, which is precisely where the ABI complex is localized. Importantly, merotelic attachments are a significant cause of chromosome loss in mammalian cells (Kaitna, 2002).
To investigate the possible significance of the AIR-2-dependent recruitment of condensin to chromatin, the consequences of loss of condensin were analyzed using RNAi to deplete the core SMC subunits, either alone or in combination. Since the condensin complex has not been biochemically analyzed and since the associated subunits of this complex are not well conserved in nematodes, focus was placed on the core SMC subunits, which are orthologs of SMC2 and SMC4 in other organisms. This live cell analysis demonstrates that condensin has a role in chromosome condensation in C. elegans. Moreover, condensin largely performs this function prior to nuclear envelope breakdown. Condensin-dependent compaction of chromosomes during prophase is not accompanied by a striking recruitment of MIX-1, one of the SMC family members, to chromatin. The failure to detect condensin may indicate that this compaction is mediated by limited amounts of the condensin complex, analogous to the small amounts of the cohesin complex that mediates sister chromatid cohesion during metaphase in animal cells (Kaitna, 2002).
There are multiple condensin-related complexes in early C. elegans embryos. In addition to the condensin complex, whose associated subunits have not yet been defined, embryos also contain the dosage compensation complex that also has two SMC proteins as core components. MIX-1 is present in both the condensin complex and the dosage compensation complex, whereas DPY-27 (a SMC-4-related protein) and DPY-26 (a protein with limited homology to Dm Barren) are solely involved in dosage compensation. Localization of MIX-1 to mitotic chromatin is independent of DPY-26, yet its localization to X chromosomes in hermaphrodites is DPY-26 dependent. The existence of multiple modes of MIX-1 binding to chromatin is compatible with the finding that condensin appears to have both AIR-2-independent and AIR-2-dependent interactions with chromatin (Kaitna, 2002).
Surprisingly, condensin function prior to nuclear envelope breakdown is AIR-2 independent, even though the mitotic recruitment of condensin to chromatin is AIR-2 dependent. The tools are currently unavailable to test if the mitotic accumulation of condensin is of physiological significance. Importantly, it is also observed that chromatin condensation occurs after nuclear envelope breakdown in condensin-depleted embryos. While it is possible that residual condensin remains after RNAi depletion and mediates this condensation, this seems unlikely, since depletion of either subunit alone or both together caused the same fully penetrant phenotype. It appears more likely that condensin-independent pathways contribute to chromosome condensation (Kaitna, 2002).
Several lines of evidence suggest that AIR-2 kinase activity promotes resolution of sister chromatid cohesion during meiosis I. This includes the remarkable concordance between AIR-2 localization and the sites where sister chromatid cohesion is lost. In addition, a phosphatase, GLC7, that is antagonistic to AIR-2 is required to prevent precocious separation of bivalents into chromatids. While the mechanism that targets AIR-2 to this discrete site is not yet clear, AIR-2 localization in diakinesis is strictly dependent upon the presence of sister chromatid cohesion, and its discrete localization requires recombination between homologs. It will be important to decipher why AIR-2 localizes to the region distal to the recombination event and why GLC-7 activity appears to predominate elsewhere. The meiotic function of AIR-2 contrasts sharply with its mitotic function, where it is dispensable for resolution of sister chromatid cohesion. The fact that the air-2(or207ts) allele exhibits penetrant defects in mitotic divisions yet no defects during meiosis provides additional evidence for mechanistic differences in AIR-2 function during mitosis and meiosis (Kaitna, 2002).
Although AIR-2 is required for proper congression of mitotic chromosomes to the metaphase plate, it is not required for this process during meiosis. However, there are significant differences in the organization of mitotic and meiotic kinetochores in C. elegans. Whereas mitotic chromosomes are holocentric, meiotic chromosomes are functionally monocentric in both meiotic divisions. The kinetic end of the chromosome is not predetermined -- rather, it is thought to be positioned at the end most distant from the crossover. The other end adopts the function of the centromere at meiosis II. Several organisms that have holocentric mitotic chromosomes have functionally monocentric meiotic chromosomes, including a variety of nematodes and arthropods. Recent ultrastructural studies have revealed a common appearance of the kinetic faces of chromosomes during meiosis and mitosis in C. elegans. However, earlier studies using different methods demonstrated a distinct kinetochore structure at the ultrastructural level during mitosis, yet, during meiosis, microtubules appear to insert directly into chromatin. Functional evidence also indicates differences between mitotic and meiotic kinetochores in C. elegans, since some factors that are critical for mitotic chromosome segregation are not critical for meiotic chromosome segregation. As shown in this study, the condensin complex does not appear to be required for meiosis, although it is essential for mitosis. Similarly, HCP-3 (the CENP-A homolog) and HCP-4 (the CENP-C homolog) are essential during mitotic chromosome segregation but only exhibit weak meiotic phenotypes. Although HCP-3 is present on the entire bivalent at diakinesis/metaphase I, this localization does not necessarily imply that it is functional, since, in mammalian cells, ectopic localization of CENP-A is insufficient to generate a functional kinetochore (Kaitna, 2002).
During meiosis, AIR-2 marks the region of the chromosome in which cohesion is lost during the first meiotic division. This region is defined by the position of the crossover that occurs during the pachytene stage. In C. elegans, usually one crossover event occurs per chromosome, regardless of the length of the chromosome or the length of recombinogenic region. Interestingly, several organisms that are holocentric during mitosis and functionally monocentric during meiosis share the additional property that only one crossover usually occurs per bivalent. One consequence of the single crossover event is that there is a single discrete region on each bivalent in which sister chromatid cohesion must be released to allow homologs to separate at meiosis I. This is in stark contrast to the situation in organisms with multiple crossover events in which sister chromatid cohesion must be released in several noncontiguous regions to allow homologs to separate (Kaitna, 2002).
In principle, separation of homologs in meiosis I could proceed by two mechanistically distinct pathways. The challenge is to destabilize sister chromatid cohesion in a regional manner so that homologs can segregate during meiosis I, while maintaining a connection between sister chromatids so that they can segregate from each other in meiosis II. One solution is to selectively destroy sister chromatid cohesion distal to the chiasmata. The data suggest that, in C. elegans, AIR-2 may regulate this selective destruction. Whereas this subset of cohesion is well defined in C. elegans, this is not the case in organisms with multiple chiasmata, and cohesion must be destroyed in numerous regions dispersed throughout the chromosome. The alternative solution is to selectively protect cohesion in the vicinity of the kinetochore. Indeed, there is evidence for centromeric protection of cohesion in yeast and Drosophila (Kaitna, 2002).
In conclusion, these results indicate that aurora B kinase, AIR-2, mediates both meiotic and mitotic chromosome segregation. Interestingly, AIR-2 acts differently in these two processes. In mitosis, AIR-2 prevents merotelic attachments and perhaps promotes a subset of condensin-dependent processes; this function is indicated by the failure of chromosomes to properly congress to form an ordered metaphase plate in air-2(or207ts) embryos. During mitosis, AIR-2 is not required for separation of sister chromatids. In contrast, during meiosis I, AIR-2 appears to be involved in the resolution of cohesion, whereas it is not obviously required for proper positioning of the bivalents on the meiotic plate. It is speculated that the role defined in this study for aurora B in mitotic chromosome segregation may apply to many organisms. It is further speculated that the meiotic function defined for AIR-2 may be restricted to organisms that have a single crossover event per chromosome during meiotic prophase and in which the kinetic end of the meiotic chromosome is not invariant. Finally, these data suggest that, while condensin plays an important role in chromosome organization, there are likely additional factors that also contribute to the condensation of mitotic chromosomes (Kaitna, 2002).
The Aurora kinases control multiple aspects of mitosis, among them centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora activity is regulated in part by a subset of Aurora substrates that, once phosphorylated, can enhance Aurora kinase activity. Aurora A substrate activators include TPX2 and Ajuba, whereas the only known Aurora B substrate activator is the chromosomal passenger INCENP. The C. elegans Tousled kinase TLK-1 is a second substrate activator of the Aurora B kinase AIR-2. Tousled kinase (Tlk) expression and activity have been linked to ongoing DNA replication, and Tlk can phosphorylate the chromatin assembly factor Asf (see Drosophila Asf). TLK-1 is phosphorylated by AIR-2 during prophase/prometaphase, and phosphorylation increases TLK-1 kinase activity in vitro. Phosphorylated TLK-1 increases AIR-2 kinase activity in a manner that is independent of TLK-1 kinase activity but depends on the presence of ICP-1/INCENP. In vivo, TLK-1 and AIR-2 cooperate to ensure proper mitotic chromosome segregation. It is concluded that the C. elegans Tousled kinase TLK-1 is a substrate and activator of the Aurora B kinase AIR-2. These results suggest that Tousled kinases have a previously unrecognized role in mitosis and that Aurora B associates with discrete regulatory complexes that may impart distinct substrate specificities and functions to the Aurora B kinase (Han, 2005).
The Aurora B kinase is the enzymatic core of the chromosomal passenger complex, which is a critical regulator of mitosis. To identify novel regulators of Aurora B, a genome-wide screen was performed for suppressors of a temperature-sensitive lethal allele of the C. elegans Aurora B kinase AIR-2. This screen uncovered a member of the Afg2/Spaf subfamily of Cdc48-like AAA ATPases as an essential inhibitor of AIR-2 stability and activity. Depletion of CDC-48.3 restores viability to air-2 mutant embryos and leads to abnormally high AIR-2 levels at the late telophase/G1 transition. Furthermore, CDC-48.3 binds directly to AIR-2 and inhibits its kinase activity from metaphase through telophase. While canonical p97/Cdc48 proteins have been assigned contradictory roles in the regulation of Aurora B, these results identify a member of the Afg2/Spaf AAA ATPases as a critical in vivo inhibitor of this kinase during embryonic development (Heallen, 2008).
The Aurora A and B protein kinases are key players in mitotic control and the etiology of human cancer. Despite the near identity of amino acid sequence in the catalytic domain, monomeric Aurora B is 50 fold lower in activity than monomeric Aurora A, and previous studies have shown that TPX2 binding to the catalytic domain activates Aurora A but not Aurora B. This study identifies G205 in Xenopus Aurora A as a key determinant of both intrinsic activity and regulation by TPX2. Mutation of G205 in Aurora A to N, the equivalent residue in Aurora B, has no effect on autophosphorylation of the T-loop but leads to a 10-fold loss of specific activity, whereas mutation of N158 in Aurora B to G causes a 350-fold increase in specific activity. G205 N Aurora A is still activated by TPX2, but protection of pT295 from dephosphorylation by protein phosphatase 1 is abolished. Structural analysis of these effects suggests that the G205 forms a pivot point in the enzyme that results in movement of the N-terminal domain glycine-rich loop closer to the ATP binding site of the enzyme and also moves the C-helix slightly closer to the activation loop. Changes in these positions are comparable to those reported for other protein kinases and demonstrate that phosphorylation of the activation loop alone is not sufficient for enzyme activation. The generation of an activated mutant of Aurora B will be important for studying its role in cell cycle control and tumorigenesis (Eyers, 2005).
As a component of the 'chromosomal passenger protein complex,' the aurora B kinase is associated with centromeres during prometaphase and with midzone microtubules during anaphase and is required for both mitosis and cytokinesis. Ablation of aurora B causes defects in both prometaphase chromosomal congression and the spindle checkpoint; however, an understanding of the mechanisms underlying these defects remains unclear. To address this question, chromosomal movement, spindle organization, and microtubule motor distribution has been examined in NRK cells transfected with a kinase-inactive, dominant-negative mutant of aurora B, aurora B(K-R). In cells overexpressing aurora B(K-R) fused with GFP, centromeres moved in a synchronized and predominantly unidirectional manner, as opposed to the independent, bidirectional movement in control cells expressing a similar level of wild-type aurora B-GFP. In addition, most kinetochores became physically separated from spindle microtubules, which appeared as a striking bundle between the spindle poles. These defects were associated with a microtubule-dependent depletion of motor proteins dynein and CENP-E from kinetochores. These observations suggest that aurora B regulates the association of motor proteins with kinetochores during prometaphase. Interactions of kinetochore motors with microtubules may in turn regulate the organization of microtubules, the movement of prometaphase chromosomes, and the release of the spindle checkpoint (Murata-Hori, 2002).
It is proposed that a substrate of the aurora B kinase, which may be an adaptor protein or motor proteins themselves, is required for the stable association of dynein and CENP-E at kinetochores. In the absence of the kinase activity, these motors are released prematurely from kinetochores as soon as they come into contact with microtubules, whereas in control cells they are released only following chromosomal congression. Since dynein contributes to the poleward movement of the chromosomes while CENP-E may be involved in the attachment of chromosomes to microtubules, loss of these motor proteins may account for the defects in chromosomal movements and kinetochore-microtubule interactions. The residual, synchronized chromosomal movements, without kinetochore fibers, are likely dragged by the 'polar ejection forces' on chromosome arms; these forces sweep the chromosomes into elongated clusters along the spindle axis. In addition, without the attachment to kinetochores, the microtubule bundling activity responsible for the formation of kinetochore fibers would induce the formation of a single microtubule bundle between the spindle poles. Therefore, deactivation of aurora B causes premature dissociation of these motors from kinetochores and leads to separation of chromosomes from microtubules and release of the spindle checkpoint. The lack of kinetochore association also causes microtubules to form a single bundle instead of multiple kinetochore fibers (Murata-Hori, 2002).
How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, mitotic XTC cells were microinjected with inhibitory antibody and several related effects were found. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. These findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis (Kallio, 2002).
The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. The small molecule Hesperadin has been identified as an inhibitor of chromosome alignment and segregation. The data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, these data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling (Hauf, 2003).
The spindle assembly checkpoint very faithfully ensures that anaphase is not initiated before all chromosomes have achieved bipolar attachment. In striking contrast, cells treated with Hesperadin readily enter anaphase in the presence of monooriented chromosomes. Likewise, Hesperadin is able to override the checkpoint arrest caused by taxol and monastrol, but Aurora B function is not required for several hours to maintain a checkpoint arrest induced by nocodazole. This situation is reminiscent of the role of Ipl1 in budding yeast in that Aurora B appears to be required for checkpoint signaling in the absence of tension but not in the absence of kinetochore attachments. It is possible that Aurora B is required to directly activate checkpoint proteins in the absence of tension, independent of its role in the correction of syntelic attachment. But because the correction function is thought to be activated by the lack of tension, it is also conceivable that Aurora B is indirectly required for checkpoint signaling. According to this model, Aurora B would destabilize microtubule-kinetochore interactions at kinetochores that are not under proper tension or that impinge on the kinetochore at too acute an angle, and the resulting kinetochores that are either unattached or only occupied with low numbers of microtubules would then generate the primary signal for checkpoint signaling. Hesperadin-treated cells would enter anaphase only once all kinetochores had been fully attached, which would then abolish checkpoint signaling. Because kinetochore attachment is a stochastic process, this model predicts that cells enter anaphase at different times after nuclear envelope breakdown (NEB). Indeed, a high intercell variability is observed between NEB and the onset of anaphase in Hesperadin-treated cells (Hauf, 2003).
This model also fits well with the observation that cells arrested with either taxol or monastrol always contain at least one kinetochore that appears to be unattached. Taxol- and monastrol-treated cells may therefore be arrested by the spindle checkpoint because Aurora B maintains a dynamic equilibrium between attached and unattached kinetochores. Inhibition of Aurora B would overcome this arrest because all kinetochores would eventually become fully attached. As predicted by this model, Hesperadin addition to monastrol-treated Ptk1 cells decreases the number of kinetochores staining with Mad2, suggesting that the monotelic chromosomes that existed in monastrol-arrested cells had been converted into syntelic chromosomes once Aurora B was inhibited (Hauf, 2003).
The stabilization of improper microtubule attachments is sufficient to explain the precocious exit from mitosis that Hesperadin induces in monastrol- and taxol-treated cells. However, even under conditions where none of the kinetochores are attached, Aurora B function is required to maintain checkpoint signaling over prolonged periods of time, indicating that it might also have a direct role in the spindle assembly checkpoint. Consistent with this notion, kinetochore localization of the checkpoint kinases BubR1 and Bub1 was found to be impaired in Hesperadin-treated cells. It is conceivable that Mad2, which is still present at kinetochores in cells treated with nocodazole and Hesperadin, is sufficient to sustain the transient mitotic delay that is observed. In contrast, the low levels of Mad2 at kinetochores in taxol-arrested cells might not be sufficient to delay cells in mitosis when BubR1 is depleted from kinetochores by Hesperadin (Hauf, 2003).
In summary, the data suggest that Aurora B has a dual role. It acts in the destabilization of improper microtubule attachments, which indirectly keeps checkpoint signaling active, but it also could have a more direct role in the spindle assembly checkpoint. This is consistent with data from budding yeast, where it was found that Ipl1 is required for the spindle assembly checkpoint in a kinetochore-dependent, but probably also in a kinetochore-independent, manner. Likewise, Aurora B antibodies have been shown to overcome a nocodazole-induced arrest both in Xenopus egg extracts and cultured cells, also suggesting a direct role of Aurora B in the spindle assembly checkpoint (Hauf, 2003).
BubR1 and Bub1 could be Aurora B substrates that play a role in either of these pathways, or in both. It is conceivable that BubR1 and Bub1 themselves have dual roles. Both proteins have been shown to be required for checkpoint signaling in the presence of unattached kinetochores. Interestingly, in some experimental settings, their kinase activity is not required for checkpoint function, but Bub1's kinase activity is essential for a genetically separable function that may be required for microtubule-kinetochore attachments. It will therefore be interesting to test if the kinase activity of both Bub1 and BubR1 and their Aurora B-dependent recruitment to kinetochores is required to regulate kinetochore attachments (Hauf, 2003).
Sister kinetochores must bind microtubules in a bipolar fashion to equally segregate chromosomes during mitosis. The molecular mechanisms underlying this process remain unclear. Aurora B likely promotes chromosome biorientation by regulating kinetochore-microtubule attachments. MCAK (mitotic centromere-associated kinesin) is a Kin I kinesin that can depolymerize microtubules. These two proteins both localize to mitotic centromeres and have overlapping mitotic functions, including regulation of microtubule dynamics, proper chromosome congression, and correction of improper kinetochore-microtubule attachments. Aurora B is shown to phosphorylate and regulate MCAK both in vitro and in vivo. Specifically, six Aurora B phosphorylation sites map on MCAK in both the centromere-targeting domain and the neck region. Aurora B activity was required to localize MCAK to centromeres, but not to spindle poles. Aurora B phosphorylation of serine 196 in the neck region of MCAK inhibits its microtubule depolymerization activity. This key site is phosphorylated at centromeres and anaphase spindle midzones in vivo. However, within the inner centromere there are pockets of both phosphorylated and unphosphorylated MCAK protein, suggesting that phosphate turnover is crucial in the regulation of MCAK activity. Addition of anti-p-S196 antibodies to Xenopus egg extracts or injection of anti-p-S196 antibodies into cells causes defects in chromosome positioning and/or segregation. Thus there is a direct link between the microtubule depolymerase MCAK and Aurora B kinase. These data suggest that Aurora B both positively and negatively regulates MCAK during mitosis. It is proposed that Aurora B biorients chromosomes by directing MCAK to depolymerize incorrectly oriented kinetochore microtubules (Lan, 2004).
Chromosome biorientation or bipolar kinetochore attachment is essential for equal segregation of duplicated genomes. Bipolar attachment of sister kinetochores to microtubules from opposite poles (amphitelic attachment) is a key event to allow accurate chromosome segregation during anaphase. Failure of this process is the predominant cause of aneuploidy in cultured mammalian cells. Kinetochores that are bound by microtubules from both poles (merotelic attachment) generate lagging chromosomes in anaphase that are often missegregated. Chromosomes with both kinetochores bound by microtubules from a single pole (syntelic) often have difficulty in congression, and both sisters segregate to one daughter cell. How do kinetochores resolve merotelic and syntelic attachments? A model is presented for the regulation of MCAK activity by Aurora B phosphorylation. Aurora B localizes to centromeres phosphorylates MCAK on multiple sites to facilitate centromere loading of MCAK. This ensures that MCAK is placed on the centromere in an inactive state that allows kinetochores to bind microtubules during prometaphase. Syntelic or merotelic attachment would lead to the local activation of MCAK to depolymerize the incorrectly attached microtubule. This cycle repeats until proper amphitelic attachment is achieved (Lan, 2004).
A model is favored in which there are redundant pathways in vertebrates where Aurora B regulates kinetochore-microtubule binding through MCAK and additional substrates at the kinetochore. Blocking centromeric MCAK function generates chromosome congression and segregation defects that are less severe than inhibiting Aurora B, suggesting that there are additional Aurora B targets. In budding yeast, Ipl1 phosphorylates three subunits of the Dam1 microtubule-interacting complex and Mif2 of the Mtw1 complex, but how this affects biorientation is unclear. In vertebrates, the motor proteins CENP-E and dynein depend on Aurora B for proper kinetochore localization. Both proteins are involved in chromosome movements, but neither of them has been implicated in biorientation. Identifying novel kinetochore-microtubule interacting activities that are regulated by Aurora B is crucial to decipher the mechanism of chromosome biorientation in vertebrates (Lan, 2004).
Thus vertebrate Aurora B kinase regulates the microtubule depolymerase activity of the centromeric fraction of MCAK by direct phosphorylation. These data provide a working model of how MCAK activity is regulated in mitosis and a molecular explanation of how Aurora B activity is involved in regulating the biorientation of kinetochore-microtubule attachments (Lan, 2004).
Cell division is finely controlled by various molecules including small G proteins and kinases/phosphatases. Among these, Aurora B, RhoA, and the GAP MgcRacGAP have been implicated in cytokinesis, but their underlying mechanisms of action have remained unclear. MgcRacGAP is shown to colocalize with Aurora B and RhoA, but not Rac1/Cdc42, at the midbody. Aurora B phosphorylates MgcRacGAP on serine residues and this modification induces latent GAP activity toward RhoA in vitro. Expression of a kinase-defective mutant of Aurora B disrupts cytokinesis and inhibits phosphorylation of MgcRacGAP at Ser387, but not its localization to the midbody. Overexpression of a phosphorylation-deficient MgcRacGAP-S387A mutant, but not phosphorylation-mimic MgcRacGAP-S387D mutant, arrests cytokinesis at a late stage and induces polyploidy. Together, these findings indicate that during cytokinesis, MgcRacGAP, a GAP for Rac/Cdc42, is functionally converted to a RhoGAP through phosphorylation by Aurora B (Minoshima, 2003).
Cell division is regulated by protein kinases of the Cdk, Polo, and Aurora families. Although it has long been established that temporal control is central to the coordinated action of these kinases, the importance of spatial regulation has only recently been appreciated and is still poorly understood. The kinesin-6 family motor protein MKlp1 is a key regulator of cytokinesis and an ideal substrate for studying spatially regulated protein-phosphorylation events. MKlp1 is negatively regulated by Cdk1 phosphorylation during metaphase and becomes activated in anaphase when cleavage-furrow assembly commences. Aurora B phosphorylates MKlp1 during anaphase and is required for its function in cytokinesis. Another kinesin-6 family motor, MKlp2, mediates the relocation of Aurora B from the centromeres to the central spindle at the onset of anaphase. This study demonstrates that this process is required for the phosphorylation of MKlp1 at S911, an Aurora B consensus site overlapping a bipartite nuclear localization sequence (NLS). MKlp1(S911A) targets to the central spindle but is prematurely imported into the nucleus and fails to support cytokinesis. Spatial restriction of Aurora B to the central spindle by MKlp2 therefore regulates MKlp1 during cytokinesis in human cells (Neef, 2006).
Three lines of investigation have suggested that interactions between Survivin and the chromosomal passenger proteins INCENP and Aurora-B kinase may be important for mitotic progression. (1) Interference with the function of Survivin/BIR1, INCENP, or Aurora-B kinase leads to similar defects in mitosis and cytokinesis. (2) INCENP and Aurora-B exist in a complex in Xenopus eggs and in mammalian cultured cells. (3) Interference with Survivin or INCENP function causes Aurora-B kinase to be mislocalized in mitosis in both C. elegans and vertebrates. Evidence is provided that Survivin, Aurora-B, and INCENP interact physically and functionally. Direct visualization of Survivin-GFP in mitotic cells reveals that it localizes identically to INCENP and Aurora-B. Survivin binds directly to both Aurora-B and INCENP in both yeast two-hybrid and in vitro pull-down assays. The in vitro interaction between Survivin and Aurora-B is extraordinarily stable in that it resists 3 M NaCl. Finally, Survivin and INCENP interact functionally in vivo; in cells in which INCENP localization is disrupted, Survivin adheres to the chromosomes and no longer concentrates at the centromeres or transfers to the anaphase spindle midzone. The data provide the first biochemical evidence that Survivin can interact directly with members of the chromosomal passenger complex (Wheatley, 2001).
The Aurora B kinase complex is a critical regulator of chromosome segregation and cytokinesis. In Caenorhabditis elegans, AIR-2 (Aurora B) function requires ICP-1 (Incenp) and BIR-1 (Survivin). In various systems, Aurora B binds to orthologues of these proteins. Through genetic analysis, a new subunit of the Aurora B kinase complex, CSC-1, an ortholog of Borealin/Dasra (see Droosphila Borealin-related), has been identified. C. elegans embryos depleted of CSC-1, AIR-2, ICP-1, or BIR-1 have identical phenotypes. CSC-1, BIR-1, and ICP-1 are interdependent for their localization, and all are required for AIR-2 localization. In vitro, CSC-1 binds directly to BIR-1. The CSC-1/BIR-1 complex, but not the individual subunits, associates with ICP-1. CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo. ICP-1 dramatically stimulates AIR-2 kinase activity. This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase (Romano, 2003).
The function of the Aurora B kinase at centromeres and the central spindle is crucial for chromosome segregation and cytokinesis, respectively. This study investigates regulation of human Aurora B by its complex partners, inner centromere protein (INCENP) and survivin. Overexpression of a catalytically inactive, dominant-negative mutant of Aurora B impairs the localization of the entire Aurora B/INCENP/survivin complex to centromeres and the central spindle and severely disturbs mitotic progression. Similar results were also observed after depletion, by RNA interference, of either Aurora B, INCENP, or survivin. These data suggest that Aurora B kinase activity and the formation of the Aurora B/INCENP/survivin complex both contribute to its proper localization. Using recombinant proteins, it was found that Aurora B kinase activity is stimulated by INCENP and that the C-terminal region of INCENP is sufficient for activation. Under identical assay conditions, survivin does not detectably influence kinase activity. Human INCENP is a substrate of Aurora B and mass spectrometry identified three consecutive residues (threonine 893, serine 894, and serine 895) containing at least two phosphorylation sites. A nonphosphorylatable mutant (TSS893-895AAA) is a poor activator of Aurora B, demonstrating that INCENP phosphorylation is important for kinase activation (Honda, 2003).
The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway was studied. The results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3 (see Drosophila Bub3), and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other (Vigneron, 2004).
The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Borealin, a novel member of the complex, is described in this study. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin. Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore-spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B-INCENP subcomplex (Gassmann, 2004).
Proper chromosome segregation requires the attachment of sister kinetochores to microtubules from opposite spindle poles to form bi-oriented chromosomes on the metaphase spindle. The chromosome passenger complex containing Survivin and the kinase Aurora B regulates this process from the centromeres. A de-ubiquitinating enzyme, hFAM, regulates chromosome alignment and segregation by controlling both the dynamic association of Survivin with centromeres and the proper targeting of Survivin and Aurora B to centromeres. Survivin is ubiquitinated in mitosis through both Lys(48) and Lys(63) ubiquitin linkages. Lys(63) de-ubiquitination mediated by hFAM is required for the dissociation of Survivin from centromeres, whereas Lys(63) ubiquitination mediated by the ubiquitin binding protein Ufd1 is required for the association of Survivin with centromeres. Thus, ubiquitinaton regulates dynamic protein-protein interactions and chromosome segregation independently of protein degradation (Vong, 2005).
Chromatin-induced spindle assembly depends on regulation of microtubule-depolymerizing proteins by the chromosomal passenger complex (CPC), consisting of Incenp, Survivin, Dasra (Borealin), and the kinase Aurora B, but the mechanism and significance of the spatial regulation of Aurora B activity remain unclear. This study shows that the Aurora B pathway is suppressed in the cytoplasm of Xenopus egg extract by phosphatases, but that it becomes activated by chromatin via a Ran-independent mechanism. While spindle microtubule assembly normally requires Dasra-dependent chromatin binding of the CPC, this function of Dasra can be bypassed by clustering Aurora B-Incenp by using anti-Incenp antibodies, which stimulate autoactivation among bound complexes. However, such chromatin-independent Aurora B pathway activation promotes centrosomal microtubule assembly and produces aberrant achromosomal spindle-like structures. It is proposed that chromosomal enrichment of the CPC results in local kinase autoactivation, a mechanism that contributes to the spatial regulation of spindle assembly and possibly to other mitotic processes (Kelly, 2007).
How does chromatin activate Aurora B-dependent phosphorylation? Four lines of evidence support a model in which Aurora B is activated by increasing the local concentration of CPC molecules on chromatin: (1) Chromatin can bind to multiple molecules of the CPC and induce Aurora B pathway activation; (2) Antibody alone can activate Aurora B kinase activity, and this activity is dependent on having multiple binding sites; (3) The responses of the small microtubule-destabilizing protein Op18 hyperphosphorylation induced by sperm nuclei and antibodies are similar and Ran independent; (4) Op18 hyperphosphorylation induced by antibody clustering is insensitive to the geometry of attachment (Kelly, 2007).
Full activation of Aurora B requires Aurora B-mediated phosphorylation of the C-terminal TSS motif of Incenp, and structural analysis suggests that this phosphorylation must occur in trans. Thus, the simplest model is that the Incenp TSS motif is actively dephosphorylated in the cytoplasm, but chromatin increases the local concentration of the CPC, resulting in initiation of a positive feedback loop among bound CPC holocomplexes. It is worth noting other possible mechanisms: clustering may also activate Aurora B independent of phosphorylation, as is the case for kinases such as Raf and EGFR, or chromatin or its associated molecules might directly induce a non-clustering-mediated structural change in Aurora B (Kelly, 2007).
It is also possible that chromatin exerts its effect on the Aurora B pathway by inhibiting protein phosphatase activities. However, the data indicate that chromatin directly stimulates the kinase activity of Aurora B, since Dasra proteins (which are required for loading of the CPC onto chromatin) are needed for spindle assembly. Importantly, more than 90% of Dasra A is associated with Incenp and Aurora B in the cytoplasm of Xenopus egg extracts. In addition, it has been reported that recombinant human Dasra B/Borealin does not affect the in vitro kinase activity of Aurora B. Thus, it is unlikely that Dasra proteins stimulate the enzymatic activity of Aurora B simply by virtue of their interactions (Kelly, 2007).
The spatial distribution of phosphorylated substrates around chromatin can be finely regulated by the level of phosphatase activity, and substrate diffusibility and stability, whereas the amplitude of the gradient is most sensitive to kinase activity. For example, the freely diffusible Op18-tubulin interaction is abrogated in the vicinity of chromosomes (4-8 microm) by a gradient of Op18 phosphorylation, the extent of which is mainly determined by phosphatase activity/concentration and the Op18 diffusion rate. Alternatively, if the substrate is immobilized on chromosomes, kinase activity dictates the behavior of the phospho-substrate. MCAK, a protein that is bound to centromeric chromatin, is more efficiently phosphorylated at Ser196 by Aurora B on centromeres of unaligned chromosomes than on aligned chromosomes. This raises the question of whether a change in chromatin status between sister kinetochores can effectively regulate Aurora B activity by modulating its local concentration. In summary, these results illustrating that Aurora B is activated by increased local concentration have important implications for the several roles of this complex throughout mitosis (Kelly, 2007).
Phosphorylation of histone H3 at serine 10 occurs during mitosis and meiosis in a wide range of eukaryotes and has been shown to be required for proper chromosome transmission in Tetrahymena. Ipl1/aurora kinase and its genetically interacting phosphatase, Glc7/PP1, are responsible for the balance of H3 phosphorylation during mitosis in Saccharomyces cerevisiae and Caenorhabditis elegans. In these models, both enzymes are required for H3 phosphorylation and chromosome segregation, although a causal link between the two processes has not been demonstrated. Deregulation of human aurora kinases has been implicated in oncogenesis as a consequence of chromosome missegregation. These findings reveal an enzyme system that regulates chromosome dynamics and controls histone phosphorylation that is conserved among diverse eukaryotes (Hsu, 2000).
Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. A role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homolog, has been examined. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. These data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis (Zeitlin, 2001).
Proper chromosome condensation requires the phosphorylation of histone and nonhistone chromatin proteins. An in vitro chromosome assembly system based on Xenopus egg cytoplasmic extracts has been used to study mitotic histone H3 phosphorylation. A histone H3 Ser(10) kinase activity associated with isolated mitotic chromosomes has been identified. The histone H3 kinase is not affected by inhibitors of cyclin-dependent kinases, DNA-dependent protein kinase, p90(rsk), or cAMP-dependent protein kinase. The activity can be selectively eluted from mitotic chromosomes and immunoprecipitated by specific anti-X aurora-B/AIRK2 antibodies. This activity is regulated by phosphorylation. Treatment of X aurora-B immunoprecipitates with recombinant protein phosphatase 1 (PP1) inhibits kinase activity. The presence of PP1 on chromatin suggests that PP1 might directly regulate the X aurora-B associated kinase activity. Indeed, incubation of isolated interphase chromatin with the PP1-specific inhibitor I2 and ATP generates an H3 kinase activity that is also specifically immunoprecipitated by anti-X aurora-B antibodies. Nonetheless, stimulation of histone H3 phosphorylation in interphase cytosol does not drive chromosome condensation or targeting of 13 S condensin to chromatin. In summary, the chromosome-associated mitotic histone H3 Ser(10) kinase is associated with X aurora-B and is inhibited directly in interphase chromatin by PP1 (Murnion, 2001).
Phosphorylation at a highly conserved serine residue (Ser-10) in the histone H3 tail is considered to be a crucial event for the onset of mitosis. This modification appears early in the G(2) phase within pericentromeric heterochromatin and spreads in an ordered fashion coincident with mitotic chromosome condensation. Although mitotic H3 phosphorylation has been long recognized, the transduction routes and the identity of the protein kinases involved have been elusive. The expression of mammalian Aurora-A and Aurora-B, two kinases of the Aurora/AIK family, is tightly coordinated with H3 phosphorylation during the G(2)/M transition. During the G(2) phase, the Aurora-A kinase is coexpressed while the Aurora-B kinase colocalizes with phosphorylated histone H3. At prophase and metaphase, Aurora-A is highly localized in the centrosomic region and in the spindle poles while Aurora-B is present in the centromeric region concurrent with H3 phosphorylation, to then translocate by cytokinesis to the midbody region. Both Aurora-A and Aurora-B proteins physically interact with the H3 tail and efficiently phosphorylate Ser10 both in vitro and in vivo, even if Aurora-A appears to be a better H3 kinase than Aurora-B. Since Aurora-A and Aurora-B are known to be overexpressed in a variety of human cancers, the findings provide an attractive link between cell transformation, chromatin modifications and a specific kinase system (Crosio, 2002).
Histones are subject to numerous post-translational modifications. Some of these 'epigenetic' marks recruit proteins that modulate chromatin structure. For example, heterochromatin protein 1 (HP1) binds to histone H3 when its lysine 9 residue has been tri-methylated by the methyltransferase Suv39h. During mitosis, H3 is also phosphorylated by the kinase Aurora B. Although H3 phosphorylation is a hallmark of mitosis, its function remains mysterious. It has been proposed that histone phosphorylation controls the binding of proteins to chromatin, but any such mechanisms are unknown. This study shows that antibodies against mitotic chromosomal antigens that are associated with human autoimmune diseases specifically recognize H3 molecules that are modified by both tri-methylation of lysine 9 and phosphorylation of serine 10 (H3K9me3S10ph). The generation of H3K9me3S10ph depends on Suv39h and Aurora B, and occurs at pericentric heterochromatin during mitosis in different eukaryotes. Most HP1 typically dissociates from chromosomes during mitosis, but if phosphorylation of H3 serine 10 is inhibited, HP1 remains chromosome-bound throughout mitosis. H3 phosphorylation by Aurora B is therefore part of a 'methyl/phos switch' mechanism that displaces HP1 and perhaps other proteins from mitotic heterochromatin (Hirota, 2005).
Histone deacetylase (HDAC) inhibitors perturb the cell cycle and have great potential as anti-cancer agents, but their mechanism of action is not well established. HDACs classically function as repressors of gene expression, tethered to sequence-specific transcription factors. This study reports that HDAC3 is a critical, transcription-independent regulator of mitosis. HDAC3 forms a complex with A-Kinase-Anchoring Proteins AKAP95 and HA95, which are targeted to mitotic chromosomes. Deacetylation of H3 in mitosis requires AKAP95/HA95 and HDAC3 and provides a hypoacetylated H3 tail that is the preferred substrate for Aurora B kinase. Phosphorylation of H3S10 by Aurora B leads to dissociation of HP1 proteins from methylated H3K9 residues on mitotic heterochromatin. This transcription-independent pathway, involving interdependent changes in histone modification and protein association, is required for normal progression through mitosis and is an unexpected target of HDAC inhibitors, a class of drugs currently in clinical trials for treating cancer (Li, 2006).
The classic role of HDAC3 has been that of a transcriptional repressor of gene expression, as part of a complex tethered to sequence-specific transcription factors. This study reports the unexpected finding that HDAC3 has a critical, transcription-independent function in mitosis. In interphase cells, AKAP95/HA95 binds to the nuclear matrix and is less associated with HDAC3. HP1 proteins are recruited to methylated H3K9 in heterochromatin. When cells enter into mitosis, AKAP95/HA95 may target the HDAC3 complex to deacetylate H3, in a reaction that is blocked by HDAC inhibitors, and thereby provides a hypoacetylated H3 tail as substrate for Aurora B to phosphorylate on S10. Phosphorylation of S10 by Aurora B then dissociates HP1 proteins from methylated H3K9 residues on mitotic heterochromatin, which has been referred to as the 'meth-phos switch'. These interdependent changes in histone modification and protein association are required for normal progression through mitosis, perhaps by facilitating chromosome condensation, or by serving as the indicator for the mitotic checkpoint to control proper cell division (Li, 2006).
While the transcriptional effect of HDAC inhibitors on specific genes, such as p21 and other cell cycle-regulated genes, has been reported to contribute to their anti-tumor actions, especially in G1-phase arrest, their direct effects on histone acetylation levels may be equally important for the anti-tumor activity because of the important functions of histones in different cellular processes, including mitosis. It is increasingly clear that HDAC inhibition induces G2/M arrest in many human cell lines and causes mitotic defects in different cancer cell lines. This effect of HDAC inhibition is independent of ongoing gene transcription, suggesting direct effects of histone hyperacetylation on mitosis. These results indicate that the hyperacetylation of histones induced by HDAC inhibitors directly interfere with mitotic progression (Li, 2006).
Global histone acetylation is reduced during mitosis. The current studies reveal that HDAC3 and its partner proteins AKAP95 and HA95 are required for global histone deacetylation during mitosis. Of note, the most dramatic change in acetylation that occurs during mitosis is hypoacetylation of Lys 5 of H4, which matches the substrate specificity of HDAC3. Moreover, the results clearly show that HDAC3 is required for normal mitotic progression. This is consistent with a recent study in which knockdown of HDAC3, but not HDAC1 or HDAC2, increased cells in G2/M phase in human colon cancer cells. Furthermore, knockdown of HDAC3 or AKAP95/HA95 also mimics the effects of nonselective HDAC inhibition on phosphorylation of H3S10 and retention of HP1β proteins on mitotic chromosomes. Inhibition of HDAC3 is therefore likely to be the mechanism by which HDAC inhibitors induce the G2/M block in the cell cycle. The transcription independence of this effect, while unexpected, is completely consistent with a direct mitotic function of HDAC3 in the context of the novel pathway that that is reported here (Li, 2006).
Specific patterns of histone modification at gene promoters regulate transcription via a 'histone code'. Notably, the transient phosphorylation of H3S10 has been reported in the promoter region of many mammalian immediate-early genes, which are rapidly induced in response to extracellular stimuli including UV radiation, growth factors, and cytokines. On these promoters, the phosphorylation of H3S10 precedes the H3K14 acetylation, resulting in multiple modifications of H3 that facilitate gene activation. On the contrary, this study found that the phosphorylation of H3S10 by Aurora B during mitosis requires the previous deacetylation of histones by HDAC3. Thus, in contrast to the phosphorylation of H3S10 by other kinases that prefer preacetylated histone tails, the mitotic phosphorylation of H3S10 by Aurora B kinase is linked to the deacetylation of H3, specifically by HDAC3. This characteristic of Aurora B may be specific to metazoans because IPL1, the yeast homolog of Aurora kinase, phosphorylated both monoacetylated and unacetylated H3. In addition to H3S10, Aurora B also phosphorylates H3S28 and other proteins including his- tone H3 variant centromere protein A (CENP-A). In human cell systems, Aurora B also seems to prefer hypoacetylated H3 and CENP-A H3 as substrate for phosphorylation of H3S28 and CENP-A Ser7, respectively. The global hypoacetylation of H3 tail lysines in mitotic cells and their proximity to the major sites of phosphorylation by Aurora B kinase suggest that deacetylation of histone substrates may be a general preference for Aurora B function. The relative importance of specific hypoacetylated lysines for phosphorylation of specific serine residues remains to be elucidated (Li, 2006).
The specificity of Aurora B toward hypoacetylated histone substrate suggests a mechanistic link between HDAC3-dependent histone deacetylation and a transcription-independent mechanism of mitotic arrest. H3S10 phosphorylation during mitosis is characteristic of many organisms, and is dependent on Aurora B kinase, which plays a central role throughout different stage of mitosis, including chromosome condensation, alignment, and segregation, spindle assembly, and cytokinesis. The recent finding that Aurora-dependent phosphorylation of H3S10 dissociates HP1 from mitotic heterochromatin provides molecular insight into the function of Aurora B. The current findings implicate AKAP95/HA95 and HDAC3 as upstream regulators of this "meth-phos switch", and provide a molecular mechanism to explain the anti-cancer effects of HDAC inhibitors. Aurora B kinase itself is overexpressed in a large number of cancers. The finding that Aurora B is present in HDAC3 complexes and that its kinase activity is dramatically greater when the H3 tail is hypoacetylated suggests that the interdependence of Aurora B and HDAC3 may be a novel and specific target for cancer therapies that would overcome the toxicity of nonspecific HDAC inhibitors (Li, 2006).
CPEB (Drosophila homolog: Orb) is an mRNA-binding protein that stimulates polyadenylation-induced translation of maternal mRNA once it is phosphorylated on Ser 174 or Thr 171 (species-dependent). Disruption of the CPEB gene in mice causes an arrest of oogenesis at embryonic day 16.5 (E16.5), when most oocytes are in pachytene of prophase I. CPEB undergoes Thr 171 phosphorylation at E16.5, but dephosphorylation at the E18.5, when most oocytes are entering diplotene. Although phosphorylation is mediated by the kinase aurora, the dephosphorylation is due to the phosphatase PP1. The temporal control of CPEB phosphorylation suggests a mechanism in which mRNA translation of CPE-containing messages is stimulated at pachytene and metaphase I (Tay, 2003).
The results presented here suggest a mechanism by which the translation of maternal mRNAs is differentially controlled during murine meiosis. As oogenesis progresses into pachytene, the kinase aurora is activated, perhaps by phosphorylation. The upstream kinase that phosphorylates aurora is unclear, although some evidence indicates that PKA is involved. Activated aurora then phosphorylates CPEB Thr 171, which stimulates the polyadenylation and translation of SCP1 and SCP3 mRNAs. The translation of other mRNAs might also be stimulated by CPEB at this time. SCP1 and SCP3 help form the synaptonemal complex, which is necessary for meiotic progression to diplotene. At diplotene, PP1 dephosphorylates and inactivates CPEB, an event that allows key CPE-containing mRNAs such as mos to accumulate but remain translationally dormant. As the fully grown (GV) oocytes begin to mature, aurora again becomes active and phosphorylates CPEB, which in turn induces the polyadenylation and translation of mos, and other mRNAs with encoded products that either stimulate maturation or lead to cytostatic factor (CSF)-mediated meta-phase II arrest (Tay, 2003).
Two additional points of upstream CPEB regulation should be considered. (1) Although aurora, in addition to CPEB, appears to be inactive in E18.5 diplotene oocytes (i.e., no T171 phosphorylation), it is plausible that the kinase is active at this time but its ability to phosphorylate CPEB is overcome by a very active PP1. To investigate this possibility, the phosphorylation experiments were perfomed except that E18.5 ovary extracts were supplemented with I-2, the PP1 inhibitor. Neither I-2-supplemented nor un-supplemented extracts supported T171 phosphorylation. Because I-2 inhibits dephosphorylation in the extracts, the lack of T171 phoshorylation can be attributed to inactive aurora rather than overriding PP1 activity. It is also interesting to note that PP1 has been suggested to inactivate aurora as well. Perhaps PP1 acts on a CPEB and aurora-containing complex to inactivate these proteins simultaneously at diplotene. (2) It is inferred that PP1, which is present in mouse oocytes, is also regulated; it is inactive during E16.5 (pachytene) when CPEB phosphorylation is robust but active at E18.5 (diplotene) to dephosphorylate CPEB. PP1 activity is regulated by a number of modulator proteins, some of which could function during oogenesis (Tay, 2003).
Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The 'chromosomal passenger' proteins, which include the inner centromere protein (INCENP), the Aurora-related serine-threonine protein kinase AIRK2 and the unidentified human autoantigen TD-60, have been suggested to integrate mitotic events. These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase. Disruption of INCENP function affects both chromosome segregation and completion of cytokinesis, whereas interference with AIRK2 function primarily affects cytokinesis. INCENP is stockpiled in Xenopus eggs in a complex with Xenopus AIRK2 (XAIRK2), and INCENP and AIRK2 kinase bind one another in vitro. This association was found to be evolutionarily conserved. Sli15p, the binding partner of yeast Aurora kinase Ipl1p, can be recognized as an INCENP family member because of the presence of a conserved carboxy-terminal sequence region, which is termed the IN box. This interaction between INCENP and Aurora kinase was found to be biologically relevant. INCENP and AIRK2 colocalize exactly in human cells, and INCENP is required to target AIRK2 correctly to centromeres and the central spindle (Adams, 2000).
The Aurora (Ipl1)-related kinases are universal regulators of mitosis. Aurora-A, in addition to Aurora-B, regulates kinetochore function in human cells. A two-hybrid screen identified the kinetochore component CENP-A as a protein that interacts with Aurora-A. Aurora-A phosphorylates CENP-A in vitro on Ser-7, a residue also known to be targeted by Aurora-B. Depletion of Aurora-A or Aurora-B by RNA interference reveals that CENP-A is initially phosphorylated in prophase in a manner dependent on Aurora-A, and that this reaction appears to be required for the subsequent Aurora-B-dependent phosphorylation of CENP-A as well as for the restriction of Aurora-B to the inner centromere in prometaphase. Prevention of CENP-A phosphorylation also led to chromosome misalignment during mitosis as a result of a defect in kinetochore attachment to microtubules. These observations suggest that phosphorylation of CENP-A on Ser-7 by Aurora-A in prophase is essential for kinetochore function (Kunitoku, 2003).
The phosphorylation of CENP-A is involved in efficient occupancy of kinetochores with spindle fibers. Concurrent with CENP-A phosphorylation at early prophase, various proteins assemble at the outer domain of the kinetochore. Given that CENP-A is essential for this assembly process in several species, the phosphorylation of CENP-A on Ser-7 might be required to initiate it during prophase, before the kinetochores begin to attach to microtubules. Such protein recruitment triggered by CENP-A phosphorylation might be important for the establishment of kinetochore-microtubule connections. However, this modification does not appear to be necessary for generation of the spindle assembly checkpoint signal, because Mad2, BubR1, and CENP-E localizes normally to kinetochores in prometaphase cells expressing CENP-A(S7A) and these cells show a marked delay in prometaphase (Kunitoku, 2003).
Given that Aurora-B plays an important role in correcting kinetochore-microtubule attachment in mammalian cells, the mislocalization of Aurora-B might contribute to the defect in chromosome alignment in cells expressing CENP-A(S7A) or in those deficient in Aurora-A. However, because Aurora-A-mediated phosphorylation of CENP-A on Ser-7 during prophase appears to be important for microtubule attachment, it was not possible to assess the possible contribution of the attachment-correcting function of Aurora-B. The many misaligned chromosomes that were found in cells in which CENP-A phosphorylation was prevented possessed either unattached or monotelic kinetochores, whereas those in Aurora-B-depleted cells exhibited syntelic attachment. Further molecular dissection of the regulation of kinetochore function by Aurora kinases could be facilitated by identification of the proteins that are recruited to the kinetochore in a manner dependent on CENP-A phosphorylation on Ser-7 (Kunitoku, 2003).
In vertebrate mitosis, cohesion between sister chromatids is lost in two stages. In prophase and prometaphase, cohesin release from chromosome arms occurs under the control of Polo-like kinase 1 and Aurora B, while Shugoshin is thought to prevent removal of centromeric cohesin until anaphase. The regulatory enzymes that act to sustain centromeric cohesion are incompletely described, however. Haspin/Gsg2, a positive regulator of centromeric cohesion, is a histone H3 threonine-3 kinase required for normal mitosis. Both H3 threonine-3 phosphorylation and cohesin are located at inner centromeres. Haspin depletion disrupts cohesin binding and sister chromatid association in mitosis, preventing normal chromosome alignment and activating the spindle assembly checkpoint, leading to arrest in a prometaphase-like state. Overexpression of Haspin hinders cohesin release and stabilizes arm cohesion. It is concluded that Haspin is required to maintain centromeric cohesion during mitosis. It is also suggested that Aurora B regulates cohesin removal through its effect on the localization of Shugoshin (Dai, 2007).
Mitosis is a highly coordinated process that assures the fidelity of chromosome segregation. Errors in this process result in aneuploidy which can lead to cell death or oncogenesis. This paper describes a putative mammalian protein kinase, AIM-1 (Aurora and Ipl1-like midbody-associated protein), related to Drosophila Aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. AIM-1 message and protein accumulate at G2/M phase. The protein localizes at the equator of central spindles during late anaphase and at the midbody during telophase and cytokinesis. Overexpression of kinase-inactive AIM-1 disrupts cleavage furrow formation without affecting nuclear division. Furthermore, cytokinesis frequently fails, resulting in cell polyploidy and subsequent cell death. These results strongly suggest that AIM-1 is required for proper progression of cytokinesis in mammalian cells (Terada, 1998).
Aurora- and Ipl1-like midbody-associated protein (AIM-1) is a serine/ threonine kinase that is structurally related to Drosophila aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. A kinase-negative form of AIM-1 inhibits the formation of cleavage furrow without affecting nuclear division, indicating that the gene controls entry into cytokinesis during M phase in mammalian cells. A human gene that encodes the protein AIM-1 was overexpressed in colorectal and other tumor cell lines. The regulation of AIM-1 expression is cell cycle dependent in normal and tumor cells, and the maximum accumulation is observed at G2-M. Exogenous overexpression of wild-type AIM-1 produces multinuclearity in human cells, suggesting that the excess amount of AIM-1 has a dominant-negative effect on the overexpressing cells. In long-term culture of AIM-1-overexpressing cells, multiple nuclei in a cell are occasionally fused, and then an increased ploidy and aneuploidy are induced. Thus, the overexpression of AIM-1 in colorectal tumor cell lines is thought to have a causal relationship with multinuclearity and increased ploidy. Cytokinesis error caused by AIM-1 overexpression is a major factor in the predisposition of tumor cells to the perturbation of chromosomal integrity that is commonly observed in human neoplasia. Thus, defects of pathways essential for mitotic regulation are important during human cancer development (Tatsuka, 1998).
The inner centromere protein (INCENP) is required for correct chromosome segregation and cytokinesis. The human INCENP gene has been idenified by library screening and reverse transcription-polymerase chain reaction (RT-PCR) and localized to chromosomal region 11q12. HsINCENP is a single-copy gene that consists of 17 exons and covers 25 kb of genomic DNA. The gene is expressed at highest levels in the colon, testis and prostate, consistent with its likely role in cell proliferation. HsINCENP encodes a highly basic protein of 915 amino acids that localizes to metaphase chromosomes and to the mitotic spindle and equatorial cortex at anaphase. It has been shown that INCENP is stockpiled in a complex with the Aurora-B/XAIRK2 kinase in Xenopus eggs. Consistent with such an interaction, the two proteins colocalize on human metaphase chromosomes. Levels of Aurora-B are increased in several human cancers, and HsINCENP protein levels are also significantly increased in several colorectal cancer cell lines (Adams, 2001b).
The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. ZM447439, a novel selective Aurora kinase inhibitor, is described. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, 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 or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components 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, it has been shown that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset (Ditchfield, 2003).
The spindle checkpoint ensures faithful chromosome segregation by linking the onset of anaphase to the establishment of bipolar kinetochore-microtubule attachment. The checkpoint is mediated by a signal transduction system comprised of conserved Mad, Bub and other proteins. Live-cell imaging coupled with RNA interference was used to investigate the functions of human Bub1. Bub1 is essential for checkpoint control and for correct chromosome congression. Bub1 depletion leads to the accumulation of misaligned chromatids in which both sister kinetochores are linked to microtubules in an abnormal fashion, a phenotype that is unique among Mad and Bub depletions. Bub1 is similar to the Aurora B/Ipl1p kinase in having roles in both the checkpoint and microtubule binding. However, human Bub1 and Aurora B are recruited to kinetochores independently of each other and have an additive effect when depleted simultaneously. Thus, Bub1 and Aurora B appear to function in parallel pathways that promote formation of stable bipolar kinetochore-microtubule attachments (Meraldi, 2005).
The central spindle regulates the formation and positioning of the contractile ring and is essential for completion of cytokinesis. Central spindle assembly begins in early anaphase with the bundling of overlapping, antiparallel, nonkinetochore microtubules, and these bundles become compacted and mature into the midbody. Prominent components of the central spindle include aurora B kinase and centralspindlin, a complex containing a Kinesin-6 protein (ZEN-4/MKLP1: Drosophila homolog - Pavarotti) and a Rho family GAP (CYK-4/MgcRacGAP) that is essential for central spindle assembly. Centralspindlin localization depends on aurora B kinase. Aurora B concentrates in the midbody and persists between daughter cells. In C. elegans embryos and in cultured human cells, respectively, ZEN-4 and MKLP1 are phosphorylated by aurora B in vitro and in vivo on conserved C-terminal serine residues. In C. elegans embryos, a nonphosphorylatable mutant of ZEN-4 localizes properly but does not efficiently support completion of cytokinesis. In mammalian cells, an inhibitor of aurora kinase acutely attenuates phosphorylation of MKLP1. Inhibition of aurora B in late anaphase causes cytokinesis defects without disrupting the central spindle. These data indicate a conserved role for aurora-B-mediated phosphorylation of ZEN-4/MKLP1 in the completion of cytokinesis (Guse, 2005 ).
Components of mitotic chromosomes assembled in Xenopus laevis egg extracts have been characterized and collectively referred to as Xenopus chromosome-associated polypeptides (XCAPs). They included five subunits of the condensin complex essential for chromosome condensation. In an effort to identify novel proteins involved in this process, XCAP-F has been isolated; it is the Xenopus ortholog of ISWI, a chromatin remodeling ATPase. ISWI exists in two major complexes in Xenopus egg extracts. The first complex contains ACF1 and two low-molecular-weight subunits, most likely corresponding to Xenopus CHRAC. The second complex is a novel one that contains the Xenopus ortholog of the human Williams syndrome transcription factor (WSTF). In the absence of the ISWI complexes, the deposition of histones onto DNA is apparently normal, but the spacing of nucleosomes is greatly disturbed. Despite the poor spacing of nucleosomes, ISWI depletion has little effect on DNA replication, chromosome condensation or sister chromatid cohesion in the cell-free extracts. The association of ISWI with chromatin is cell cycle regulated and is under the control of the INCENP-aurora B kinase complex that phosphorylates histone H3 during mitosis. Apparently contradictory to the generally accepted model, it has been found that neither chromosome condensation nor chromosomal targeting of condensin is compromised when H3 phosphorylation is drastically reduced by depletion of INCENP-aurora B (MacCallum, 2002).
Heterochromatin protein 1 (HP1 see Drosophila HP1) plays an important role in heterochromatin formation and undergoes large-scale, progressive dissociation from heterochromatin in prophase cells. However, the mechanisms regulating the dynamic behavior of HP1 are poorly understood. This study investigated the role of Aurora-B with respect to the dynamic behavior of HP1alpha. Mammalian Aurora-B, AIM-1, colocalizes with HP1alpha to the heterochromatin in G2. Depletion of Aurora-B/AIM-1 inhibits dissociation of HP1alpha from the chromosome arms at the G2-M transition. In addition, depletion of INCENP leads to aberrant cellular localization of Aurora-B/AIM-1, but it does not affect heterochromatin targeting of HP1alpha. It has been proposed in the binary switch hypothesis that phosphorylation of histone H3 at Ser-10 negatively regulates the binding of HP1alpha to the adjacent methylated Lys-9. However, Aurora-B/AIM-1-mediated phosphorylation of H3 induces dissociation of the HP1alpha chromodomain but not of the intact protein in vitro, indicating that the center and/or C-terminal domain of HP1alpha interferes with the effect of H3 phosphorylation on HP1alpha dissociation. Interestingly, Lys-9 methyltransferase SUV39H1 is abnormally localized together along the metaphase chromosome arms in Aurora-B/AIM-1-depleted cells. In conclusion, these results showed that Aurora-B/AIM-1 is necessary for regulated histone modifications involved in binding of HP1alpha by the N terminus of histone H3 during mitosis (Terada, 2006).
The spindle checkpoint delays anaphase onset in cells with mitotic spindle defects. Chk1, a component of the DNA damage and replication checkpoints, protects vertebrate cells against spontaneous chromosome missegregation and is required to sustain anaphase delay when spindle function is disrupted by taxol, but not when microtubules are completely depolymerized by nocodazole. Spindle checkpoint failure in Chk1-deficient cells correlates with decreased Aurora-B kinase activity and impaired phosphorylation and kinetochore localization of BubR1. Furthermore, Chk1 phosphorylates Aurora-B and enhances its catalytic activity in vitro. It is proposed that Chk1 augments spindle checkpoint signaling and is required for optimal regulation of Aurora-B and BubR1 when kinetochores produce a weakened signal. In addition, Chk1-deficient cells exhibit increased resistance to taxol. These results suggest a mechanism through which Chk1 could protect against tumorigenesis through its role in spindle checkpoint signaling (Zachos, 2007).
Fission yeast shugoshin Sgo1 is meiosis specific and cooperates with protein phosphatase 2A to protect centromeric cohesin at meiosis I. The other shugoshin-like protein Sgo2, which requires the heterochromatin protein Swi6/HP1 for full viability, plays a crucial role for proper chromosome segregation at both mitosis and meiosis; however, the underlying mechanisms are totally elusive. This study demonstrates that, unlike Sgo1, Sgo2 is dispensable for centromeric protection of cohesin. Instead, Sgo2 interacts with Bir1/Survivin and promotes Aurora kinase complex localization to the pericentromeric region, to correct erroneous attachment of kinetochores and thereby enable tension-generating attachment. Forced localization of Bir1 to centromeres partly restored the defects of sgo2Delta. This newly identified interaction of shugoshin with Survivin is conserved between mitosis and meiosis and presumably across eukaryotes. It is proposed that ensuring bipolar attachment of kinetochores is the primary role of shugoshin and the role of cohesion protection might have codeveloped to facilitate this process (Kawashima, 2007).
This study demonstrates that human shugoshin hSgo1 associates with Survivin and Aurora and requires these components for its centromeric localization. Together with the recent finding in Drosophila that the Aurora kinase complex is required for centromeric localization of Sgo/Mei-S332 (Resnick, 2006), these studies suggest that the linkage between shugoshin and Aurora kinase complex is conserved among eukaryotes. Studies in human cells present the strongest data to date indicating the existence of a complex including shugoshin and Survivin in vivo; hSgo1 could coprecipitate with Survivin better than Aurora in extracts prepared from chromatin fraction. This result fits with the immunoprecipitation using a cross-linker in fission yeast and with genetic results indicating that Sgo2 closely interacts with Bir1/Survivin for the centromeric localization. Although the linkage between shugoshin and the Aurora kinase complex is conserved across species, the precise manner of interaction has apparently diverged. The centromeric localization of Drosophila Mei-S332 reportedly requires phosphorylation by Aurora (Resnick, 2006); however, fission yeast Sgo2 does not require it, albeit Sgo2, like Mei-S332, is a good substrate of Ark1 in vitro. Whereas fission yeast shugoshin (Sgo2) is required for the localization and function of Aurora kinase complex at centromeres, Drosophila Mei-S332 as well as human Sgo1 is not required for the localization of the Aurora kinase complex (Resnick, 2006), albeit the centromeric function of the Aurora kinase complex might nevertheless be regulated by Mei-S332 (Kawashima, 2007).
The sole shugoshin protein in budding yeast seems to play dual roles in protecting centromeric cohesin at meiosis I (but not at mitosis) as well as in establishing tension-generating attachment at mitosis. Drosophila SGO/MEI-S332 mutants show nondisjunction of homologs at meiosis I and a reduced ratio of meta/anaphase (but only slight or little defect in cohesion) in mitosis. Therefore, it is suggested that Mei-S332, the sole shugoshin of Drosophila, is also required for establishing tension-generating attachment, like fission yeast Sgo2. The localization of the Aurora kinase complex does not depend on Mei-S332; however, it is tenable that the activation of centromeric Aurora kinase complex may somewhat depend on Mei-S332 since they physically interact in vitro (Resnick, 2006). Similarly, fission yeast Sgo2 might play an additional role in activating centromeric Aurora rather than merely promoting its localization. Given that hSgo1 associates with Survivin (and Aurora) in HeLa cells, a similar functional link is conceivable also in human cells (Kawashima, 2007).
Studies in fission yeast enabled definition of two distinct shugoshin functions or pathways that are carried out by two diverged shugoshins, Sgo1 and Sgo2; the former interacts with PP2A to protect cohesin, but the latter interacts with the Aurora kinase complex to facilitate centromeric Aurora function. It is speculated that the ancestral shugoshin molecule played dual roles at kinetochores like in budding yeast or Drosophila; fission yeast shugoshin might have divided the labor to Sgo1 and Sgo2. Thus, these findings of a functional link between Sgo2 and the Aurora kinase complex open a new view that shugoshin in general may play a role in facilitating Aurora function at centromeres, thereby ensuring tension-generating kinetochore microtubule attachment. At the centromere, microtubule attachment is ensured by tension across centromeres, which is generated depending on the cohesion between sister chromatids. Therefore, cohesion and tension are two sides of a 'coin' ensuring bipolar attachment of kinetochores. It is suggested that the original role of shugoshin was to guarantee bipolar attachment rather than to protect cohesin, because fission yeast and presumably budding yeast, two primitive eukaryotes, exhibit this role only during mitosis. The protection role, once acquired, might facilitate the generation of tension by counteracting the spindle force, improving the fidelity of chromosome segregation. This function might have been modified to evolve meiosis, in which the requirement for centromeric protection is more essential and therefore has been preserved in all eukaryotes. Whatever the validity of this view, the finding of how Sgo2 acts will contribute to understand the fundamental regulation of eukaryotic chromosome segregation (Kawashima, 2007).
The mitotic checkpoint ensures proper chromosome segregation by monitoring two critical events during mitosis. One is kinetochore attachment to the mitotic spindle, and the second is the alignment of chromosomes at the metaphase plate, resulting in tension across sister kinetochores. Mitotic-checkpoint proteins are known to accumulate at unaligned chromosomes that have not achieved proper kinetochore-microtubule attachments or established an adequate level of tension across sister kinetochores. hZW10 and hROD, two components of the evolutionarily conserved RZZ complex (Chan, 2000; Scaerou, 2001), accumulate at kinetochores in response to the loss of tension. By using live-cell imaging and FRAP, it was shown that the accumulation of hZW10 at tensionless kinetochores stems from a 4-fold reduction of kinetochore turnover rate. It was also found that cells lacking hZW10 escape loss-of-tension-induced mitotic-checkpoint arrest more rapidly than those arrested in response to the lack of kinetochore-microtubule attachments. Furthermore, it was shown that pharmacological inhibition of Aurora B kinase activity with ZM447439 in the absence of tension, but not in the absence of kinetochore-microtubule attachments, results in the loss of hZW10, hROD, and hBub1 from kinetochores. It is therefore concluded that Aurora B kinase activity is required for the accumulation of tension-sensitive mitotic-checkpoint components, such as hZW10 and hROD, in order to maintain mitotic-checkpoint arrest (Famulski, 2007).
It is concluded that human ZW10 and human ROD are tension-sensitive components of the mitotic checkpoint and that their accumulation at tensionless kinetochores is regulated by their turnover dynamics in an Aurora B kinase-dependent manner. It is proposed that Aurora B phosphorylation of the RZZ complex might reduce its kinetochore turnover rate, therefore leading to the accumulation of hp50 and the RZZ complex at tensionless kinetochores. Lowering the kinetochore turnover rate of the RZZ complex might involve modification of the interaction between the RZZ complex and dynein. This could prevent dynein-mediated transport of the RZZ complex, and other essential mitotic-checkpoint components, off kinetochores. Mitotic-checkpoint arrest in response to the loss of kinetochore tension would thus be maintained by the prevention of the 'shedding' of essential checkpoint proteins from kinetochores, even though bipolar attachment of microtubules has been achieved (Famulski, 2007).
Faithful cell-cycle progression is tightly controlled by the ubiquitin-proteasome system. A human Cullin 3-based E3 ligase (Cul3) has been identified that is essential for mitotic division. In a complex with the substrate-specific adaptors KLHL9 and KLHL13, Cul3 is required for correct chromosome alignment in metaphase, proper midzone and midbody formation, and completion of cytokinesis. This Cul3-based E3 ligase removes components of the chromosomal passenger complex from mitotic chromosomes and allows their accumulation on the central spindle during anaphase. Aurora B directly binds to the substrate-recognition domain of KLHL9 and KLHL13 in vitro, and coimmunoprecipitates with the Cul3 complex during mitosis. Moreover, Aurora B is ubiquitylated in a Cul3-dependent manner in vivo, and by reconstituted Cul3/KLHL9/KLHL13 ligase in vitro. It is thus proposed that the Cul3/KLHL9/KLHL13 E3 ligase controls the dynamic behavior of Aurora B on mitotic chromosomes, and thereby coordinates faithful mitotic progression and completion of cytokinesis (Sumara, 2007).
During division of metazoan cells, the nucleus disassembles to allow chromosome segregation, and then reforms in each daughter cell. Reformation of the nucleus involves chromatin decondensation and assembly of the double-membrane nuclear envelope around the chromatin; however, regulation of the process is still poorly understood. In vitro, nucleus formation requires p97, a hexameric ATPase implicated in membrane fusion and ubiquitin-dependent processes (Drosophila homolog: TER94). However, the role and relevance of p97 in nucleus formation have remained controversial. This study shows that p97 stimulates nucleus reformation by inactivating the chromatin-associated kinase Aurora B. During mitosis, Aurora B inhibits nucleus reformation by preventing chromosome decondensation and formation of the nuclear envelope membrane. During exit from mitosis, p97 binds to Aurora B after its ubiquitylation and extracts it from chromatin. This leads to inactivation of Aurora B on chromatin, thus allowing chromatin decondensation and nuclear envelope formation. These data reveal an essential pathway that regulates reformation of the nucleus after mitosis and defines ubiquitin-dependent protein extraction as a common mechanism of Cdc48/p97 activity also during nucleus formation (Ramadan, 2007).
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