S. cerevisiae Ipl1, Drosophila Aurora, and the mammalian centrosomal protein IAK-1 define a new subfamily of serine/threonine kinases that regulate chromosome segregation and mitotic spindle dynamics. Mutations in ipl1 and aurora result in the generation of severely aneuploid cells and, in the case of aurora, monopolar spindles arising from a failure in centrosome separation. A related, essential protein from C. elegans, AIR-1 (Aurora/Ipl1 related), is localized to mitotic centrosomes. Disruption of AIR-1 protein expression in C. elegans embryos results in severe aneuploidy and embryonic lethality. Unlike aurora mutants, this aneuploidy does not arise from a failure in centrosome separation. Bipolar spindles are formed in the absence of AIR-1, but they appear to be disorganized and are nucleated by abnormal-looking centrosomes. In addition to its requirement during mitosis, AIR-1 may regulate microtubule-based developmental processes as well. The data suggests AIR-1 plays a role in P-granule segregation and the association of the germline factor PIE-1 with centrosomes (Schumacher, 1998).
The Aurora kinase Ipl1p plays a crucial role in regulating kinetochore-microtubule attachments in budding yeast, but the underlying basis for this regulation is not known. To identify Ipl1p targets, 28 kinetochore proteins were purified from yeast protein extracts. These studies identified five previously uncharacterized kinetochore proteins and defined two additional kinetochore subcomplexes. Mass spectrometry was used to identify 18 phosphorylation sites in 7 of these 28 proteins. Ten of these phosphorylation sites are targeted directly by Ipl1p, allowing the identification of a consensus phosphorylation site for an Aurora kinase. This systematic mutational analysis of the Ipl1p phosphorylation sites demonstrates that the essential microtubule binding protein Dam1p is a key Ipl1p target for regulating kinetochore-microtubule attachments in vivo (Cheeseman, 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 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).
Chromosome segregation depends on kinetochore bi-orientation so that sister kinetochores attach to microtubules from opposite poles and come under tension. The budding yeast Ipl1/Aurora protein kinase allows the absence of tension to activate the spindle checkpoint. Checkpoint activation in the mtw1-1 kinetochore mutant requires Ipl1p, suggesting that Mtw1p promotes tension. mtw1-1 dosage suppressors were isolated and Dsn1, a kinetochore protein that immunoprecipitates with the Mif2/CENP-C and Cse4/CENP-A proteins, was identified, as well as the Mtw1, Nnf1, and Nsl1 kinetochore proteins. mtw1 and dsn1 mutant strains exhibit similar phenotypes, suggesting that Mtw1p and Dsn1p act together. Although mtw1 mutant cells contain unattached chromosomes, attachment is restored by impairing Ipl1p function. These results suggest that mtw1 mutant kinetochores are competent to bind microtubules but Ipl1p generates unattached chromosomes. It is therefore proposed that an Mtw1 complex is required for kinetochore bi-orientation that is monitored by the Ipl1p kinase (Pinsky, 2003).
An understanding of kinetochore function has been greatly aided by studies in the budding yeast where over 40 kinetochore proteins have been identified, many of which are conserved. The kinetochore is organized into distinct subcomplexes that have been placed into inner, central, and outer domains based on two criteria: (1) in vitro centromere or microtubule binding activities and (2) in vivo dependency relationships for centromere association. The inner kinetochore CBF3 complex binds centromeric DNA and is required for the centromere association of all other kinetochore proteins. The inner kinetochore also contains Cse4/CENP-A, the conserved centromeric histone H3 variant, as well as the Cbf1 and Mif2/CENP-C proteins. The central kinetochore includes the conserved Ndc80/HEC-1 complex, implicated in the stabilization of kinetochore-microtubule attachments, and the large Ctf19 complex, which can be divided into at least three subcomplexes, the only one known to require microtubules for its kinetochore association, as well as several microtubule motors and nonmotor microtubule-associated proteins (Pinsky, 2003 and references therein).
Although a number of kinetochore proteins have been identified, little is known about how their assembly and activity is regulated. The Ipl1/Aurora protein kinase is a key regulator of kinetochore function because it is required to generate bi-oriented kinetochore-microtubule attachments. In ipl1 mutant cells, kinetochores are competent to bind to microtubules, but 85% of the time sister chromatids are segregated to the same spindle pole. A similar increase in mono-oriented attachments is observed in cultured vertebrate cells when Aurora B function is compromised using inhibitory antibodies or small-molecule inhibitors, suggesting conservation of function. Because the biased segregation of chromosomes to the bud in ipl1 mutants was eliminated by the transient disruption of microtubules, it was proposed that Ipl1p promotes the turnover of mono-oriented kinetochore-microtubule interactions. A crucial in vivo target of Ipl1p is the Dam1/DASH/DDD complex. The phenotypes of a dam1 mutant thought to mimic constitutive Ipl1p phosphorylation are consistent with this complex acting as a microtubule release factor (Pinsky, 2003 and references therein).
It is not known whether there are kinetochore proteins in addition to Ipl1 and Dam1 that are required for bi-orientation. The essential Mtw1 (mis twelve-like) protein, which copurifies with the largely nonessential Ctf19 complex, is a particularly intriguing candidate for a mediator of kinetochore bi-orientation. In mtw1 mutants and cells depleted of Mtw1 protein, DNA segregates into two unequal masses, and sister centromeres frequently lose bi-orientation, suggesting that kinetochore-microtubule attachments are made but not properly regulated. Mutations in the fission yeast mis12 result in sister chromatids that are initially pulled toward the same pole, indicating mono-oriented attachments. Furthermore, when protein levels of the human homolog, hMis12, are reduced by siRNA in cell culture, chromosomes are misaligned in a manner consistent with -ientation defects. MTW1 (DSN3, NSL2) was also identified along with the essential genes DSN1 (dosage suppressor of nnf1-17) and NSL1 (nnf1-17 synthetic lethal) based on genetic interactions with NNF1 (necessary for nuclear function), an essential gene with a role in chromosome segregation. The genetic interactions and similar localization patterns suggested that these proteins might physically interact at the kinetochore (Pinsky, 2003 and references therein).
Mtw1p does associate with Dsn1, Nnf1, and Nsl1, as well as the Mif2 and Cse4 inner kinetochore proteins. Defects in Mtw1p require Ipl1p function to activate the spindle checkpoint, suggesting that Mtw1p is required for kinetochore tension. Mutations in MTW1 and DSN1 have similar phenotypes leading to spindle checkpoint activation with chromosomes unattached to the spindle. Strikingly, chromosome attachments to the spindle are completely restored in mtw1 mutants when Ipl1p function is absent, suggesting that Mtw1p is required to generate kinetochore bi-orientation but not microtubule attachments. Taken together, these data demonstrate that the Ipl1p kinase detects improper kinetochore-microtubule attachments and is required to generate unattached chromosomes in response to these defects (Pinsky, 2003).
It is proposed that Mtw1p is required for the physical tension that results from bi-orienting sister kinetochores. Although unattached chromosomes were detected in the mtw1-1 mutant, two lines of evidence are provided consistent with this being due to a bi-orientation defect that leads to Ipl1p-mediated release of microtubule attachments. First, the checkpoint arrest induced by the mtw1-1 mutant requires Ipl1p function. Because Ipl1p is required to activate the spindle checkpoint in response to defects in kinetochore tension but not attachment, this suggests that mtw1-1 mutant kinetochores are not under sufficient tension and that Ipl1p does directly monitor the state of kinetochores. This result is not necessarily specific to mtw1-1, and future work will be required to determine if defects in other kinetochore components also require Ipl1p to engage the spindle checkpoint. This tension deficiency is thought to be a direct consequence of defects in Mtw1p function and not a failure in kinetochore assembly, because kinetochore proteins that were tested remain centromere associated in mtw1-1 mutant cells. In addition, Mtw1p is required to maintain bi-orientation that is established at metaphase. Second, it was found that removing Ipl1p function in mtw1-1 mutants restores the attachment of sister chromatids to the spindle. Because both chromosome attachment and segregation in mtw1-1 ipl1-321 cells was identical to the ipl1-321 cells, the mtw1-1 attachment defect is completely suppressed by loss of Ipl1p function. It is therefore unlikely that the elimination of Ipl1p function simply strengthens weak kinetochore-microtubule interactions. In addition, this data strongly argues that mtw1-1 mutants have kinetochores that are competent to attach to microtubules. In support of this, the lack of a cell cycle delay when Mis12 is depleted by siRNA in human cells or mutated in fission yeast is consistent with microtubules attaching to kinetochores, thus satisfying the spindle checkpoint. Therefore the interpretation is favored that mtw1-1 mutant kinetochores bind to microtubules but are defective in bi-orientation so that Ipl1p generates unattached kinetochores (Pinsky, 2003).
There are several ways that Mtw1p could facilitate the bi-orientation of sister kinetochores. First, Mtw1p might contribute to the rigidity of the kinetochore and sterically inhibit mono-orientation. The placement of Mtw1p at the inner kinetochore due to interactions with the Mif2 and Cse4 proteins might reflect a role for centromeric chromatin structure in mediating proper kinetochore orientation. However, if Mtw1p facilitated sister kinetochore orientation, mtw1-1 ipl1-321 double mutants would likely be more defective for bi-orientation than either single mutant. Since the double mutant phenotype is the same as the ipl1-321 phenotype, it suggests that they act in the same pathway to ensure bi-orientation. Mtw1p cannot be required to signal Ipl1p that there is a tension defect, because Ipl1p can detach chromosomes in the absence of Mtw1p function. Therefore, one possibility is that Mtw1p acts downstream of Ipl1p to stabilize bi-oriented attachments. For example, Mtw1p could convert lateral microtubule attachments into end-on attachments that are more stable. Alternatively, Mtw1p might be important for generating tension on kinetochores that have already made bipolar attachments (Pinsky, 2003).
The data presented here suggest that Mtw1p is involved in bi-orientation and not in microtubule attachment. In addition, this work directly demonstrates that Ipl1p promotes the turnover of improper kinetochore-microtubule interactions. Evidence is provided that Mtw1p is part of an inner kinetochore complex, and in the future it will be important to elucidate the mechanism that this complex uses to promote bi-orientation. The molecular basis for the difference between mtw1 and mtw1 ipl1 mutant kinetochores is being actively pursued to understand how Ipl1p detects and responds to defects in Mtw1p function (Pinsky, 2003).
Cilia and flagella play key roles in development and sensory transduction, and several human disorders, including polycystic kidney disease, are associated with the failure to assemble cilia. The aurora protein kinase CALK in the biflagellated alga Chlamydomonas has a central role in two pathways for eliminating flagella. Cells rendered deficient in CALK are defective in regulated flagellar excision and regulated flagellar disassembly. Exposure of cells to altered ionic conditions, the absence of a centriole/basal body for nucleating flagellar assembly, cessation of delivery of flagellar components to their tip assembly site, and formation of zygotes all lead to activation of the regulated disassembly pathway as indicated by phosphorylation of CALK and the absence of flagella. It is proposed that cells have a sensory pathway that detects conditions that are inappropriate for possession of a flagellum, and that CALK is a key effector of flagellar disassembly in that pathway (Pan, 2004).
One model to explain these data is that the distal ends of the flagellar microtubules are monitored by a surveillance mechanism. A sensor ensures that flagellar components are successfully being added onto the tip of the growing flagellar axoneme. In a newly divided cell, the initial assembly would occur on the distal (plus) ends of the basal body triplet microtubules. If the proper assembly is not detected, because for example the machinery for transporting flagellar components to their site of assembly (IFT) is nonfunctional, because the intracellular ionic or osmotic conditions are inappropriate, or because the site of flagellar assembly itself (the basal body) is defective, then the sensing system is activated, CALK is phosphorylated, and flagella do not form or existing flagella are removed. Many cilia-related diseases, including some caused by aberrant cell proliferation, are associated with the absence of cilia. A further understanding of the molecular mechanisms that underlie regulation of the possession of cilia and flagella has the potential to offer important new insights into normal and pathological processes (Pan, 2004 and references therein).
Centrosomes mature as cells enter mitosis, accumulating gamma-tubulin and other pericentriolar material (PCM) components. This occurs concomitant with an increase in the number of centrosomally organized microtubules (MTs). RNA-mediated interference (RNAi) was used to examine the role of the aurora-A kinase, AIR-1, during centrosome maturation in Caenorhabditis elegans. In air-1(RNAi) embryos, centrosomes separate normally, an event that occurs before maturation in C. elegans. After nuclear envelope breakdown, the separated centrosomes collapse together, and spindle assembly fails. In mitotic air-1(RNAi) embryos, centrosomal alpha-tubulin fluorescence intensity accumulates to only 40% of wild-type levels, suggesting a defect in the maturation process. Consistent with this hypothesis, AIR-1 is found to be required for the increase in centrosomal gamma-tubulin and two other PCM components, ZYG-9 and CeGrip, as embryos enter mitosis. Furthermore, the AIR-1-dependent increase in centrosomal gamma-tubulin does not require MTs. These results suggest that aurora-A kinases are required to execute a MT-independent pathway for the recruitment of PCM during centrosome maturation (Hannak, 2001).
Aurora A kinase localizes to centrosomes and is required for centrosome maturation and spindle assembly. This study describes a microtubule-independent role for Aurora A and centrosomes in nuclear envelope breakdown (NEBD) during the first mitotic division of the C. elegans embryo. Aurora A depletion does not alter the onset or kinetics of chromosome condensation, but dramatically lengthens the interval between the completion of condensation and NEBD. Inhibiting centrosome assembly by other means also lengthens this interval, albeit to a lesser extent than Aurora A depletion. By contrast, centrosomally nucleated microtubules and the nuclear envelope-associated motor dynein are not required for timely NEBD. These results indicate that mitotic centrosomes generate a diffusible factor, which is proposed is activated Aurora A, that promotes NEBD. A positive feedback loop, in which an Aurora A-dependent increase in centrosome size promotes Aurora A activation, may temporally couple centrosome maturation to NEBD during mitotic entry (Portier, 2007).
In vertebrates, the microtubule binding protein TPX2 is required for meiotic and mitotic spindle assembly. TPX2 is also known to bind to and activate Aurora A kinase and target it to the spindle. However, the relationship between the TPX2-Aurora A interaction and the role of TPX2 in spindle assembly is unclear. TPXL-1, a C. elegans protein is the first characterized invertebrate ortholog of TPX2. An essential role of TPXL-1 during mitosis is to activate and target Aurora A to microtubules. These data suggest that this targeting stabilizes microtubules connecting kinetochores to the spindle poles. Thus, activation and targeting of Aurora A appears to be an ancient and conserved function of TPX2 that plays a central role in mitotic spindle assembly (Ozlu, 2005).
The data suggest that the most likely reason that spindles collapse in the absence of TPXL-1 is destabilization of kinetochore microtubules. One possibility is that the kinetochore-attached plus ends are unstable in the absence of TPXL-1. Unfortunately, the high-turnover rate of microtubules in C. elegans prophase precludes testing this model by FRAP. The fact that TPXL-1 operates together with Aurora A and Aurora A is localized to spindle poles suggests that the primary defect of tpxl-1(RNAi) could be in organization of the spindle pole. Supporting evidence for this is that the number of microtubules at centrosomes is quite reduced, whereas the nucleation rate seems to be the same as wild-type, and a substantial number of polymerizing microtubules exhibit freedom of movement (visible as retrograde plus-end movement) inconsistent with a stable attachment to centrosomes. Perhaps the minus ends of microtubules are unstable, and this allows the centrosomes to be pulled toward the chromosomes (Ozlu, 2005).
Therefore, it is proposed that in C. elegans mitosis, the homolog of TPX2 -- TPXL-1 -- targets Aurora A to the spindle, where Aurora A phosphorylates downstream targets required for kinetochore microtubule stabilization. These could be centrosome, spindle, or kinetochore components. One possible candidate is TAC-1, a protein implicated in microtubule stability whose homologs are known to be an Aurora A substrate. TPX2 itself is a microtubule binding protein, so it is possible that phosphorylation of TPXL-1 by Aurora A may also contribute to the stabilization activity. The other known substrate of Aurora A, the motor protein Eg5, is not required for C. elegans spindle assembly. Identification of other Aurora A substrates would provide important insights into the mechanisms of spindle assembly (Ozlu, 2005).
C. elegans Aurora A has a number of different roles in the first embryonic cell division: (1) cell polarity, (2) centrosome maturation, (3) spindle assembly, and (4) spindle elongation at anaphase. In contrast, TPXL-1 is required only for spindle assembly, and the rescue experiments with wild-type or mutant TPXL-1 indicate that all of the functions of TPXL-1 are contained within a subset of Aurora A functions. Thus, it seems that a key role for Aurora A in spindle assembly is to stabilize microtubules nucleated from centrosomes that are bound to kinetochores. Because RNAi is a run-down technique, it is possible that the difference between Aurora A phenotypes and TPX2 phenotypes represents a difference in sensitivity of different Aurora A functions to TPX2 levels. However, it is more likely that other Aurora A activation and targeting subunits exist that are responsible for the various functions of Aurora A. In this way, the cell can ensure that Aurora A is targeted at the correct time and to the correct place during the cell cycle to perform its various roles (Ozlu, 2005).
By differential screening of a Xenopus laevis egg cDNA library, a 2,111 bp cDNA has been isolated that corresponds to a maternal mRNA specifically deadenylated after fertilization. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family that includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, that corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 'invades' the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. pEg2 binds directly to taxol stabilized microtubules in vitro. In addition, the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. A catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts (Roghi, 1998).
XlEg5 is a Xenopus laevis kinesin-related protein from the bimC family. pEg2 is an Aurora-related serine/threonine kinase. Inhibition of either XlEg5 or pEg2 activity during mitosis in Xenopus egg extract leads to monopolar spindle formation. In Xenopus XL2 cells, pEg2 and XlEg5 are both confined to separated centrosomes in prophase, and then to the microtubule spindle poles. pEg2 co-immunoprecipitates with XlEg5 from egg extracts and XL2 cell lysates. Both proteins can directly interact in vitro, but also through the two-hybrid system. Furthermore immunoprecipitated pEg2 is found to remain active when bound to the beads and phosphorylates XlEg5 present in the precipitate. Two-dimensional mapping of XlEg5 tryptic peptides phosphorylated in vivo first confirms that XlEg5 is phosphorylated by p34(cdc2) and next reveals that in vitro pEg2 kinase phosphorylates XlEg5 on the same stalk domain serine residue that is phosphorylated in metabolically labeled XL2 cells. The kinesin-related XlEg5 is thought to be the first in vivo substrate ever reported for an Aurora-related kinase (Giet, 1999a).
The Xenopus laevis aurora/Ip11p-related kinase pEg2 is required for centrosome separation, which is a prerequisite for bipolar mitotic spindle formation. Inhibition of pEg2 by addition of either an inactive kinase or a monoclonal antibody destabilizes bipolar spindles previously assembled in Xenopus egg extracts. The bipolar spindles collapse to form structures such as microtubule asters with chromosome rosettes, monopolar spindles, and multipolar spindles. In collapsed spindles, chromosomes remain attached to the microtubules plus ends. The destabilization of the bipolar spindle is reminiscent of the destabilization observed after inhibition of cross-linking activities which maintain parallel and anti-parallel microtubules linked together. pEg2 phosphorylates the kinesin-related protein XlEg5 which is involved in centrosome separation but which was also reported to be involved in spindle stability. The collapse of the bipolar spindle observed after inhibition of pEg2 suggests that the kinase might regulate the cross-linking activity of XlEg5. pEg2 probably also regulates other microtubule-based motor proteins involved in bipolar spindle stability. This is the first evidence that aurora/Ip11p-related kinase activity actually participates not only in mitotic spindle formation by regulating centrosome separation but also in mitotic spindle stabilization (Giet, 2000).
The mitotic kinase Aurora A (Aur-A) is required for formation of a bipolar mitotic spindle and accurate chromosome segregation. In somatic cells, Aur-A protein and kinase activity levels peak during mitosis, and Aur-A is degraded during mitotic exit. This study has investigated how Aur-A protein and kinase activity levels are regulated, taking advantage of the rapid synchronous cell division cycles of Xenopus eggs and cell-free systems derived from them. Aur-A kinase activity oscillates in the early embryonic cell cycles, just as in somatic cells, but Aur-A protein levels are constant, indicating that regulated activation and inactivation, instead of periodic proteolysis, is the dominant mode of Aur-A regulation in these cell cycles. Cdh1, the APC/C activator that targets many mitotic proteins for ubiquitin-dependent proteolysis during late mitosis and G1 in somatic cells, is missing in Xenopus eggs and early embryos. Addition of Cdh1 to egg extracts undergoing M phase exit is sufficient to induce rapid degradation of Aur-A. Aur-A contains both of the two known APC/C recognition signals, (1) a C-terminal D box similar to those required for ubiquitin-dependent destruction of cyclin B and several other mitotic proteins, and (2) an N-terminal KEN box similar to that found on cdc20, which is ubiquitinated in response to APC/CCdh1. The D box is required for Cdh1-induced destruction of Aur-A but the KEN box is not. Destruction also requires a short region in the N terminus, which contains a newly identified recognition signal, the A box. The A box is conserved in vertebrate Aur-As and contains serine 53, which is phosphorylated during M phase. Mutation of serine 53 to aspartic acid, which can mimic the effect of phosphorylation, completely blocks Cdh1-dependent destruction of Aur-A. These results suggest that dephosphorylation of serine 53 during mitotic exit could control the timing of Aur-A destruction, allowing recognition of both the A box and D box by Cdh1-activated APC/C (Littlepage, 2002).
Aurora kinase indirectly regulates the translation of dormant maternal mRNAs. Full-grown Xenopus oocytes arrest at the G2/M border of meiosis I. Progesterone breaks this arrest, leading to the resumption of the meiotic cell cycles and maturation of the oocyte into a fertilizable egg. In these oocytes, progesterone interacts with an unidentified surface-associated receptor, which induces a non-transcriptional signaling pathway that stimulates the translation of dormant c-mos messenger RNA. Mos, a mitogen-activated protein (MAP) kinase kinase kinase, indirectly activates MAP kinase, which in turn leads to oocyte maturation. The translational recruitment of c-mos and several other mRNAs is regulated by cytoplasmic polyadenylation, a process that requires two 3' untranslated regions, the cytoplasmic polyadenylation element (CPE) and the polyadenylation hexanucleotide AAUAAA. Although the signaling events that trigger c-mos mRNA polyadenylation and translation are unclear, they probably involve the activation of CPEB, the CPE binding (Drosophila homolog: Orb). An early site-specific phosphorylation of CPEB is essential for the polyadenylation of c-mos mRNA and its subsequent translation, and for oocyte maturation. This selective, early phosphorylation of CPEB is catalysed by Eg2, a member of the Aurora family of serine/threonine protein kinases (Mendez, 2000a).
The release of Xenopus oocytes from prophase I arrest is largely driven by the cytoplasmic polyadenylation-induced translation of dormant maternal mRNAs. Cytoplasmic polyadenylation requires two cis-acting sequences and three core polyadenylation factors. The cis elements are the hexanucleotide AAUAAA, which is also essential for nuclear pre-mRNA cleavage and polyadenylation, and an upstream U-rich sequence called the cytoplasmic polyadenylation element (CPE). The core factors include CPEB, an RNA recognition motif (RRM), and zinc finger-containing protein that has a strong binding preference for the CPE, and a cytoplasmic form of cleavage and polyadenylation specificity factor (CPSF), which interacts with AAUAAA. In contrast to the nuclear form of CPSF, which consists of four subunits of 160 kDa, 100 kDa, 70 kDa, and 30 kDa, the cytoplasmic form contains the 160 kDa (or a functional equivalent thereof), the 100 kDa, and the 30 kDa subunits, but lacks the 70 kDa species. The third core factor is poly(A) polymerase (PAP). Based on the events associated with nuclear polyadenylation, the function of CPSF is probably to recruit PAP to the end of the mRNA. The molecular function of CPEB in cytoplasmic polyadenylation has not been elucidated. Although all the factors required for cytoplasmic polyadenylation are present in oocytes, they become active only at the onset of maturation. In response to progesterone-stimulated maturation, CPEB undergoes an early phosphorylation at a single residue that is essential for polyadenylation. This phosphorylation event is catalyzed by Eg2, a member of the Aurora family of Ser/Thr protein kinases. In this study the molecular function of this CPEB phosphorylation has been investigated (Mendez, 2000b and references therein).
The most proximal stimulus for polyadenylation is Eg2-catalyzed phosphorylation of CPEB serine 174. This phosphorylation event stimulates an interaction between CPEB and CPSF. This interaction is direct, does not require RNA tethering, and occurs through the 160 kDa subunit of CPSF. Eg2-stimulated and CPE-dependent polyadenylation is reconstituted in vitro using purified components. These results demonstrate that the molecular function of Eg2-phosphorylated CPEB is to recruit CPSF into an active cytoplasmic polyadenylation complex (Mendez, 2000b).
There are some key similarities between cytoplasmic and nuclear polyadenylation. In the nucleus, the affinity of CPSF for the AAUAAA is very low and must be stabilized by other RNA binding proteins. These other factors include CstF, which binds the GU-rich element downstream of the AAUAAA element, and U1A protein, which binds an upstream U-rich element present in at least one viral mRNA. While cytoplasmic polyadenylation has no downstream element, the upstream element is certainly equivalent to the CPE, and the function of CPEB is analogous to that shown for U1A, to stabilize the binding of CPSF to the AAUAAA. However, while there is no evidence that the CPSF-stabilizing activity of U1A or CstF can be modulated by phosphorylation, that of CPEB is regulated by Eg2-catalyzed phosphorylation (Mendez, 2000b and references therein).
In spite of these similarities, there are also some key differences that distinguish nuclear from cytoplasmic polyadenylation. (1) While both processes require CPSF, the cytoplasmic form of this multiprotein complex lacks the 70 kDa subunit. While the function of the 70 kDa protein in nuclear polyadenylation is unclear, it is possible that cytoplasmic CPSF contains a functional substitute, or that cytoplasmic polyadenylation has dispensed altogether with any need for a 70 kDa protein-like activity. (2) While nuclear polyadenylation is strongly coupled to pre-mRNA cleavage, the cytoplasmic process is not associated with any cleavage event. (3) Nuclear pre-mRNA polyadenylation occurs on virtually all mRNAs whereas cytoplasmic polyadenylation takes place on just those few species that contain a CPE. (4) cdc2-catalyzed phosphorylations of PAP inhibit nuclear pre-mRNA polyadenylation as cells enter M phase; cytoplasmic polyadenylation, in contrast, is induced as cells enter M phase (i.e., maturation) (Mendez, 2000b and references therein).
While Eg2-catalyzed phosphorylation of CPEB probably occurs on all CPE-containing mRNAs, there are additional processes that control the timing of polyadenylation. That is, the mRNA encoding the serine/threonine kinase Mos is the first mRNA known to undergo polyadenylation-induced translation following progesterone stimulation. Mos synthesis activates the M phase promoting factor (cyclinB/cdc2), which in turn induces the polyadenylation of other mRNAs later during maturation. What distinguishes early from late adenylating mRNAs? While additional mRNA-specific regulatory proteins could certainly affect the timing of polyadenylation, the number of CPEs appears to have an important influence. For example, the early adenylating c-mos mRNA has a single CPE while the late adenylating cyclin B1, histone B4, and cyclin A1 mRNAs have two CPEs. If one of the cyclin B1 CPEs is removed, the message adenylates early and is Mos (and cdc2) independent. It is possible that two CPEs, and thus two CPEBs, could actually inhibit the formation of an active polyadenylation complex by, for example, dimerizing or interacting with another factor that bridges the two CPEBs. While such a configuration of factors could preclude a CPEB-CPSF interaction early during maturation, a late cdc2-mediated partial destruction of CPEB could liberate one of the molecules to interact with CPSF and promote polyadenylation (Mendez, 2000b).
The polyadenylation complex includes other factors, which, while not directly influencing polyadenylation, mediate translational regulatory events associated with polyadenylation. The translational repression (masking) of CPE-containing mRNAs prior to progesterone stimulation appears to be mediated by maskin, a CPEB-associated protein that also binds the cap binding protein eIF4E. The maskin-eIF4E interaction prevents the association of eIF4G with eIF4E, thereby inhibiting translation. The dissociation of the maskin-eIF4E complex, which presumably allows eIF4G to bind eIF4E, is coincident with, and possibly the result of, cytoplasmic polyadenylation (Mendez, 2000b and references therein).
The synthesis and destruction of cyclin B drives mitosis in eukaryotic cells. Cell cycle progression is also regulated at the level of cyclin B translation. In cycling extracts from Xenopus embryos, progression into M phase requires the polyadenylation-induced translation of cyclin B1 mRNA. Polyadenylation is mediated by the phosphorylation of CPEB by Aurora, a kinase whose activity oscillates with the cell cycle. Exit from M phase seems to require deadenylation and subsequent translational silencing of cyclin B1 mRNA by Maskin, a CPEB and eIF4E binding factor, whose expression is cell cycle regulated. These observations suggest that regulated cyclin B1 mRNA translation is essential for the embryonic cell cycle. Mammalian cells also display a cell cycle-dependent cytoplasmic polyadenylation, suggesting that translational control by polyadenylation might be a general feature of mitosis in animal cells (Groisman, 2002).
Xenopus oocytes are arrested in meiotic prophase I and resume meiotic divisions in response to progesterone. Progesterone triggers activation of M-phase promoting factor (MPF) or Cdc2-cyclin B complex and neosynthesis of Mos kinase, responsible for MAPK activation. Both Cdc2 and MAPK activities are required for the success of meiotic maturation. However, the signaling pathway induced by progesterone and leading to MPF activation is poorly understood, and most of the targets of both Cdc2 and MAPK in the oocyte remain to be determined. Aurora-A is a Ser/Thr kinase involved in separation of centrosomes and in spindle assembly during mitosis. It has been proposed that in Xenopus oocytes Aurora-A could be an early component of the progesterone-transduction pathway, acting through the regulation of Mos synthesis upstream Cdc2 activation. This study addressed the question of Aurora-A regulation during meiotic maturation by using new in vitro and in vivo experimental approaches. Cdc2 kinase activity was found to be necessary and sufficient to trigger both Aurora-A phosphorylation and kinase activation in Xenopus oocyte. In contrast, these events are independent of the Mos/MAPK pathway. Aurora-A is phosphorylated in vivo at least on three residues that regulate differentially its kinase activity. Therefore, Aurora-A is under the control of Cdc2 in the Xenopus oocyte and could be involved in meiotic spindle establishment (Maton, 2003).
The success of cell division relies on the activation of its master regulator Cdc2-cyclin B, and many other kinases controlling cellular organization, such as Aurora-A. Most of these kinase activities are regulated by phosphorylation. Despite numerous studies showing that okadaic acid-sensitive phosphatases regulate both Cdc2 and Aurora-A activation, their identity has not yet been established in Xenopus oocytes and the importance of their regulation has not been evaluated. Using an oocyte cell-free system, it has been demonstrated that PP2A depletion is sufficient to lead to Cdc2 activation, whereas Aurora-A activation depends on Cdc2 activity. The activity level of PP1 does not affect Cdc2 kinase activation promoted by PP2A removal. PP1 inhibition is also not sufficient to lead to Aurora-A activation in the absence of active Cdc2. It is therefore conclude that in Xenopus oocytes, PP2A is the key phosphatase that negatively regulates Cdc2 activation. Once this negative regulator is removed, endogenous kinases are able to turn on the activator Cdc2 system without any additional stimulation. In contrast, Aurora-A activation is indirectly controlled by Cdc2 activity independently of either PP2A or PP1. This strongly suggests that in Xenopus oocytes, Aurora-A activation is mainly controlled by the specific stimulation of kinases under the control of Cdc2 and not by downregulation of phosphatase (Maton, 2005).
Segregation of chromosomes during mitosis requires interplay between several classes of protein on the spindle, including protein kinases, protein phosphatases, and microtubule binding motor proteins. Aurora A is an oncogenic cell cycle-regulated protein kinase that is subject to phosphorylation-dependent activation. Aurora A localization to the mitotic spindle depends on the motor binding protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2), but the protein(s) involved in Aurora A activation are unknown . An activator of Aurora A has been purified from Xenopus eggs and identified as TPX2. Remarkably, Aurora A that has been fully deactivated by Protein Phosphatase 2A (PP2A) becomes phosphorylated and reactivated by recombinant TPX2 in an ATP-dependent manner. Increased phosphorylation and activation of Aurora A requires its own kinase activity, suggesting that TPX2 stimulates autophosphorylation and autoactivation of the enzyme. Consistently, wild-type Aurora A, but not a kinase inactive mutant, becomes autophosphorylated on the regulatory T loop residue (Thr 295) after TPX2 treatment. Active Aurora A from bacteria is further activated at least 7-fold by recombinant TPX2, and TPX2 also impairs the ability of protein phosphatases to inactivate Aurora A in vitro. This concerted mechanism of stimulation of activation and inhibition of deactivation implies that TPX2 is the likely regulator of Aurora A activity at the mitotic spindle and may explain why loss of TPX2 in model systems perturbs spindle assembly. The finding that a known binding protein, and not a conventional protein kinase, is the relevant activator for Aurora A suggests a biochemical model in which the dynamic localization of TPX2 on mitotic structures directly modulates the activity of Aurora A for spindle assembly (Eyers, 2003)
The activated form of Ran (Ran-GTP; see Drosophila Ran) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. Since the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, it is hypothesized that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics (Tsai, 2003).
The oncogenic protein kinase Aurora A is a critical regulator of meiotic and mitotic cell cycles in eukaryotic cells. Aurora A autoactivation by autophosphorylation is promoted by specific non-catalytic binding proteins. One such protein is TPX2, a required spindle assembly factor in higher eukaryotes whose ability to activate Aurora A by direct binding to the kinase catalytic domain has been established by biochemical and structural analysis. This report clarifies the autoactivation mechanism of Aurora A by demonstrating that of seven amino acids which become autophosphorylated by Aurora A, only Thr-295 is required for activity. Association of Aurora A with TPX2 leads to activation of the kinase, in parallel with phosphorylation of TPX2. The sites are identified as three Ser residues in the N terminus of TPX2; however, mutation of these residues does not affect Aurora A activation by TPX2. In contrast, the mutation of a putative Aurora A-binding motif in TPX2 abolishes both phosphorylation of TPX2 and activation of Aurora A. The interaction between Xenopus p53 and Xenopus Aurora A was investigated. p53 blocks the activity of either full-length Aurora A or the isolated catalytic domain. Interestingly, inhibition is blocked by TPX2, suggesting that the ability of Aurora A to transform cells could be regulated by p53, TPX2, or other binding proteins (Eyers, 2004).
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).
GTP-loaded Ran induces the assembly of microtubules into aster-like and spindle-like structures in Xenopus egg extract. The microtubule-associated protein (MAP), TPX2, can mediate Ran's role in aster formation, but factors responsible for the transition from aster-like to spindle-like structures have not been described. This study identifies a complex that is required for the conversion of aster-like to spindle-like structures. The complex consists of two characterized MAPs (TPX2, XMAP215), a plus end-directed motor (Eg5), a mitotic kinase (Aurora A), and HURP, a protein associated with hepatocellular carcinoma. Formation and function of the complex is dependent on Aurora A activity. HURP protein was further characterized and shown to bind microtubules and affect their organization both in vitro and in vivo. In egg extract, anti-HURP antibodies disrupt the formation of both Ran-dependent and chromatin and centrosome-induced spindles. HURP is also required for the proper formation and function of mitotic spindles in HeLa cells. It is concluded that HURP is a new and essential component of the mitotic apparatus. HURP acts as part of a multicomponent complex that affects the growth or stability of spindle MTs and is required for spindle MT organization (Koffa, 2006).
A novel mammalian protein kinase is related to two newly identified yeast and fly kinases -- Ipl1 and aurora, respectively -- mutations in which cause disruption of chromosome segregation. This kinase has been designated Ipl1- and aurora-related kinase 1 (IAK1). IAK1 expression in mouse fibroblasts is tightly regulated temporally and spatially during the cell cycle. Transcripts first appear at G1/S boundary, are elevated at M-phase, and disappear rapidly after completion of mitosis. The protein levels and kinase activity of IAK1 are also cell cycle regulated with a peak at M-phase. IAK1 protein has a distinct subcellular and temporal pattern of localization. It is first identified on the centrosomes immediately after the duplicated centrosomes have separated. The protein remains on the centrosome and the centrosome-proximal part of the spindle throughout mitosis and is detected weakly on midbody microtubules at telophase and cytokinesis. In cells recovering from nocodazole treatment and in taxol-treated mitotic cells, IAK1 is associated with microtubule organizing centers. A wild-type and a mutant form of IAK1 cause mitotic spindle defects and lethality in ipl1 mutant yeast cells but not in wild-type cells, suggesting that IAK1 interferes with Ipl1p function in yeast. Taken together, these data strongly suggest that IAK1 may have an important role in centrosome and/ or spindle function during chromosome segregation in mammalian cells. It is suggested that IAK1 is a new member of an emerging subfamily of the serine/threonine kinase superfamily. The members of this subfamily may be important regulators of chromosome segregation (Gopalan, 1997).
Genetic and biochemical studies in lower eukaryotes have identified several proteins that ensure accurate segregation of chromosomes. These include the Drosophila aurora and yeast Ipl1 kinases that are required for centrosome maturation and chromosome segregation. Two human homologs of these genes, termed aurora1 and aurora2, have been identified; these encode cell-cycle-regulated serine/threonine kinases. The aurora2 gene maps to chromosome 20q13, a region amplified in a variety of human cancers, including a significant number of colorectal malignancies. It is proposed that aurora2 may be a target of this amplicon since its DNA is amplified and its RNA overexpressed, in more than 50% of primary colorectal cancers. Furthermore, overexpression of aurora2 transforms rodent fibroblasts. These observations implicate aurora2 as a potential oncogene in many colon, breast and other solid tumors, and identify centrosome-associated proteins as novel targets for cancer therapy (Bischoff, 1998).
The centrosomes are thought to maintain genomic stability through the establishment of bipolar spindles during cell division, ensuring equal segregation of replicated chromosomes to two daughter cells. Deregulated duplication and distribution of centrosomes have been implicated in chromosome segregation abnormalities, leading to aneuploidy seen in many cancer cell types. STK15 (also known as BTAK and aurora2), encoding a centrosome-associated kinase, is amplified and overexpressed in multiple human tumor cell types, and is involved in the induction of centrosome duplication-distribution abnormalities and aneuploidy in mammalian cells. STK15 amplification has been previously detected in breast tumor cell lines and in colon tumors; it is also amplified in approximately 12% of primary breast tumors, as well as in breast, ovarian, colon, prostate, neuroblastoma and cervical cancer cell lines. Additionally, high expression of STK15 mRNA was detected in tumor cell lines without evidence of gene amplification. Ectopic expression of STK15 in mouse NIH 3T3 cells leads to the appearance of abnormal centrosome number (amplification) and transformation in vitro. Finally, overexpression of STK15 in near diploid human breast epithelial cells has revealed similar centrosome abnormality, as well as induction of aneuploidy. These findings suggest that STK15 is a critical kinase-encoding gene, whose overexpression leads to centrosome amplification, chromosomal instability and transformation in mammalian cells (Zhou, 1998).
Aurora2 is a cell cycle regulated serine/threonine protein kinase that is overexpressed in many tumor cell lines. Aurora2 is regulated by phosphorylation in a cell cycle dependent manner. This phosphorylation occurs on a conserved residue, Threonine 288, within the activation loop of the catalytic domain of the kinase and results in a significant increase in the enzymatic activity. Threonine 288 resides within a consensus motif for the cAMP dependent kinase and can be phosphorylated by PKA in vitro. The protein phosphatase 1 is shown to dephosphorylate this site in vitro, and in vivo the phosphorylation of T288 is induced by okadaic acid treatment. Aurora2 kinase is regulated by proteasome dependent degradation and Aurora2 phosphorylated on T288 may be targeted for degradation during mitosis. These experiments suggest that phosphorylation of T288 is important for regulation of the Aurora2 kinase both for its activity and its stability (Walter, 2000).
Activity-dependent local translation of dendritic mRNAs is one process that underlies synaptic plasticity. Several of the factors known to control polyadenylation-induced translation in early vertebrate development [cytoplasmic polyadenylation element-binding protein (CPEB), maskin, poly(A) polymerase, cleavage and polyadenylation specificity factor (CPSF) and Aurora] also reside at synaptic sites of rat hippocampal neurons. The induction of polyadenylation at synapses is mediated by the N-methyl-D-aspartate (NMDA) receptor, which transduces a signal that results in the activation of Aurora kinase. This kinase in turn phosphorylates CPEB, an essential RNA-binding protein, on a critical residue that is necessary for polyadenylation-induced translation. These data demonstrate a remarkable conservation of the regulatory machinery that controls signal-induced mRNA translation, and elucidate an axis connecting the NMDA receptor to localized protein synthesis at synapses (Huang, 2002).
STK15 is an Aurora/Ipl-1 related serine/threonine kinase that is associated with centrosomes and induces aneuploidy when overexpressed in mammalian cells. Mechanisms by which STK15 activity is regulated have not been elucidated. STK15 contains two functional binding sites for protein phosphatase type 1 (PP1), and the binding of these proteins is cell cycle-regulated peaking at mitosis. Activated STK15 at mitosis phosphorylates PP1 and inhibits PP1 activity in vitro. In vivo, PP1 activity co-immunoprecipitated with STK15 is also reduced. These data indicate that STK15 inhibits PP1 activity during mitosis. Also, PP1 is shown to dephosphorylate active STK15 and abolish its activity in vitro. Furthermore, non-binding mutants of STK15 for PP1 are superphosphorylated, but their kinase activities are markedly reduced. Cells transfected with these non-binding mutants manifest aberrant chromosome alignment during mitosis. These results suggest that a feedback regulation through phosphorylation/dephosphorylation events between STK15 kinase and PP1 phosphatase operates through the cell cycle. Deregulation of this balance may contribute to anomalous segregation of chromosomes during mitotic progression of cancer cells (Katayama, 2001).
Human Aurora-A is related to a protein kinase originally identified by its close homology to Ipl1p from Saccharomyces cerevisiae and aurora from Drosophila melanogaster, both of which are key regulators of the structure and function of the mitotic spindle. Human Aurora-A is turned over through the anaphase promoting complex/cyclosome (APC/C)-ubiquitin-proteasome pathway. The association of two distinct WD40 repeat proteins known as Cdc20 and Cdh1, respectively, sequentially activates the APC/C. The present study shows that Aurora-A degradation is dependent on hCdh1 in vivo, not on hCdc20, and that Aurora-A is targeted for proteolysis through distinct structural features of the destruction box, the KEN box motifs and its kinase activity (Taguchi, 2002).
The GTPase-activating protein, RasGAP, functions as both a negative regulator and an effector of Ras proteins. In tumor cells, RasGAP is no longer able to deactivate oncogenic Ras proteins, and its effector function becomes predominant. Since RasGAP itself has no obvious enzymatic function that may explain this effector function, downstream RasGAP effectors were sought that could fulfill this role. The existence of RasGAP SH3 domain partners were sought, since this domain is involved in the regulation of cell proliferation and has an anti-apoptotic effect. Aurora is a RasGAP SH3 domain-binding protein. Drosophila Aurora Ser/Thr kinase has three human orthologs called Aurora/Ipl1-related kinase or HsAIRK-1, -2 and 3. Coimmunoprecipitation experiments in COS cells have confirmed that HsAIRK-1 and HsAIRK-2 both interact with RasGAP. RasGAP pull-down experiments showed its interaction with HsAIRK-1 in G2/M HeLa cells. RasGAP binds to the kinase domain of Aurora, and this interaction inhibits the kinase activity HsAIRK-1 and HsAIRK-2. RasGAP forms a ternary complex with HsAIRK and survivin. This complex may be involved in the regulation of the balance between cell division and apoptosis (Gigoux, 2002).
Human aurora A is a serine-threonine kinase that controls various mitotic events. The transcription of aurora A mRNA varies throughout the cell cycle and peaks during G(2)/M. To clarify the transcriptional mechanism, the 1.8-kb 5'-flanking region of aurora A including the first exon was cloned. Transient expression of aurora A promoter-luciferase constructs containing a series of 5'-truncated sequences or site-directed mutations identified a 7-bp sequence (CTTCCGG) from -85 to -79 as a positive regulatory element. Electromobility shift assays identified the binding of positive regulatory proteins to the CTTCCGG element. Anti-E4TF1-60 antibody generated a supershifted complex. Furthermore, coexpression of E4TF1-60 and E4TF1-53 markedly increases aurora A promoter activity. Synchronized cells transfected with the aurora A promoter-luciferase constructs revealed that the promoter activity of aurora A increase in the S phase and peaked at G(2)/M. In addition, a tandem repressor element, CDE/CHR, was identified just downstream of the CTTCCGG element; mutation within this element led to a loss of cell cycle regulation. It is concluded that the transcription of aurora A is positively regulated by E4TF1, a ubiquitously expressed ETS family protein, and that the CDE/CHR element is essential for the G(2)/M-specific transcription of aurora A (Tanaka, 2002).
The roles of the kinase Aurora A (AurA) in centrosome function and spindle assembly have been established in Drosophila, C. elegans, and Xenopus egg extracts. AurA acts downstream of the RanGTPase signaling pathway to stimulate spindle assembly in mitosis . However, it is still not clear whether AurA can stimulate the formation of microtubule organizing centers (MTOC) on its own. Moreover, whether AurA is essential for spindle assembly in the absence of centrosomes has remained unclear. This study reports the development of functional assays that show that activation of AurA by TPX2 is essential for Ran-stimulated spindle assembly in the presence or absence of centrosomes. Furthermore, AurA-coated magnetic beads function as MTOCs in the presence of RanGTP in Xenopus egg extracts and RanGTP stimulates AurA to recruit activities responsible for both MT nucleation and organization to the beads. The MTOC function of AurA-coated beads require both MT nucleators and motors. Compared to XMAP215-coated beads, AurA-coated beads increase the rate of bipolar spindle assembly in the presence of RanGTP, and the kinase activity of AurA is essential for the beads to function as MTOCs (Tsai, 2005).
Aurora-A is overexpressed in various types of cancer and considered to play critical roles in tumorigenesis. To better understand the pathological effect of Aurora-A activation, it is first necessary to elucidate the physiological functions of Aurora-A. This study has investigated the roles of Aurora-A in mitotic progression with the small interfering RNA, antibody microinjection, and time lapse microscopy using human cells. Suppression of Aurora-A by small interfering RNA caused multiple events to fail in mitosis, such as incorrect separation of centriole pairs, misalignment of chromosomes on the metaphase plate, and incomplete cytokinesis. Antibody microinjection of Aurora-A into late G2 cells induced dose-dependent failure in separation of centriole pairs at prophase, indicating that Aurora-A is essential for proper separation of centriole pairs. When anti-Aurora-A antibodies were injected into prometaphase cells that had separated their centriole pairs, chromosomes were severely misaligned on the metaphase plate, indicating that Aurora-A is required for proper movement of chromosomes on the metaphase plate. Furthermore, inhibition of Aurora-A at metaphase by microinjected antibodies prevented cells from completing cytokinesis, suggesting that Aurora-A also has important functions in late mitosis. These results strongly suggest that Aurora-A is essential for many crucial events during mitosis and that the phosphorylation of a series of substrates by Aurora-A at different stages of mitosis may promote diverse critical events in mitosis to maintain chromosome integrity in human cells (Marumoto, 2003).
Aurora-A is a serine-threonine kinase implicated in the assembly and maintenance of the mitotic spindle. Human Aurora-A binds to TPX2, a prominent component of the spindle apparatus. TPX2 was identified by mass spectrometry as a major protein coimmunoprecipitating specifically with Aurora-A from mitotic HeLa cell extracts. Conversely, Aurora-A could be detected in TPX2 immunoprecipitates. This indicates that subpopulations of these two proteins undergo complex formation in vivo. Binding studies demonstrated that the NH2 terminus of TPX2 can directly interact with the COOH-terminal catalytic domain of Aurora-A. Although kinase activity is not required for this interaction, TPX2 is readily phosphorylated by Aurora-A. Upon siRNA-mediated elimination of TPX2 from cells, the association of Aurora-A with the spindle microtubules is abolished, although its association with spindle poles is unaffected. Conversely, depletion of Aurora-A by siRNA has no detectable influence on the localization of TPX2. It is proposed that human TPX2 is required for targeting Aurora-A kinase to the spindle apparatus. In turn, Aurora-A might regulate the function of TPX2 during spindle assembly (Kufer, 2002).
Ran, a GTPase in the Ras superfamily, is proposed to be a spatial regulator of microtubule spindle assembly by maintaining key spindle assembly factors in an active state close to chromatin. RanGTP is hypothesized to maintain the spindle assembly factors in the active state by binding to importin beta, part of the nuclear transport receptor complex, thereby preventing the inhibitory binding of the nuclear transport receptors to spindle assembly factors. To directly test this hypothesis, two putative downstream targets of the Ran spindle assembly pathway, TPX2, a protein required for correct spindle assembly and Kid, a chromokinesin involved in chromosome arm orientation on the spindle, were analyzed to determine if their direct binding to nuclear transport receptors inhibited their function. In the amino-terminal domain of TPX2 nuclear targeting information, microtubule-binding and Aurora A binding activities were identified. Nuclear transport receptor binding to TPX2 inhibited Aurora A binding activity but not the microtubule-binding activity of TPX2. Inhibition of the interaction between TPX2 and Aurora A prevents Aurora A activation and recruitment to microtubules. In addition nuclear targeting information was identified in both the amino-terminal microtubule-binding domain and the carboxy-terminal DNA binding domain of Kid. However, the binding of nuclear transport receptors to Kid only inhibited the microtubule-binding activity of Kid. Therefore, by regulating a subset of TPX2 and Kid activities, Ran modulates at least two processes involved in spindle assembly (Trieselmann, 2003).
The related protein kinase Aurora B, whose substrates and subcellular location differ from those of Aurora A, may also be directly regulated by interaction with substrates. The Aurora B substrate Survivin exists in a complex with Aurora B and the chromosomal passenger protein INCENP, and this complex may regulate kinetochore attachment to spindle microtubules. Both Survivin and INCENP have been reported to further stimulate Aurora B activity in vitro, although the mechanism of enhancement was not determined. It will be interesting to ascertain whether other proteins also activate Aurora A by using the assay conditions described in this study, and whether TPX2 is the sole mediator of Aurora A activation in purified fractions. This work significantly advances understanding of the role of TPX2 in regulating spindle assembly because it demonstrates that TPX2 regulates Aurora A activity by directly stimulating autophosphorylation and autoactivation. It is therefore likely that TPX2 not only targets Aurora A to the mitotic spindle, but also directly regulates its activity. This regulation is likely to occur by direct binding of the N terminus of TPX2 to the Aurora A catalytic domain, where the key phosphorylated residue Thr 295 is located. The fact that TPX2 can activate a completely inactive Aurora A implies a mechanism involving autophosphorylation and autoactivation in cis. Once targeted and activated, Aurora A mediates selective phosphorylation of substrates such as the motor protein Eg5 and thereby ensures faithful bipolar spindle formation and ordered mitosis. The identification of these substrates and the regulation of TPX2 and Aurora A binding by phosphorylation represent important avenues for further study (Eyers, 2003).
Aurora-A is an oncogenic kinase essential for mitotic spindle assembly. It is activated by phosphorylation and by the microtubule-associated protein TPX2, which also localizes the kinase to spindle microtubules. The molecular mechanism of Aurora-A activation has been uncovered by determining crystal structures of its phosphorylated form both with and without a 43 residue long domain of TPX2 that was identified as fully functional for kinase activation and protection from dephosphorylation. In the absence of TPX2, the Aurora-A activation segment is in an inactive conformation, with the crucial phosphothreonine exposed and accessible for deactivation. Binding of TPX2 triggers no global conformational changes in the kinase but pulls on the activation segment, swinging the phosphothreonine into a buried position and locking the active conformation. The recognition between Aurora-A and TPX2 resembles that between the cAPK catalytic core and its flanking regions, suggesting this molecular mechanism may be a recurring theme in kinase regulation (Bayliss, 2003).
Aurora-A kinase is necessary for centrosome maturation, for assembly and maintenance of a bipolar spindle, and for proper chromosome segregation during cell division. Aurora-A is an oncogene that is overexpressed in multiple human cancers. Regulation of kinase activity apparently depends on phosphorylation of Thr-288 in the T-loop. In addition, interactions with targeting protein for Xenopus kinesin-like protein 2 (TPX2) allosterically activate Aurora-A. The Thr-288 phosphorylation is reversed by type-1 protein phosphatase (PP1). Mutations in the yeast Aurora, Ipl1, are suppressed by overexpression of Glc8, the yeast homolog of phosphatase inhibitor-2 (I-2). Human I-2 directly and specifically stimulates recombinant human Aurora-A activity in vitro. The I-2 increase in kinase activity is not simply due to inhibition of PP1 because it was not mimicked by other phosphatase inhibitors. Furthermore, activation of Aurora-A is unaffected by deletion of the I-2 N-terminal PP1 binding motif but is eliminated by deletion of the I-2 C-terminal domain. Aurora-A and I-2 were recovered together from mitotic HeLa cells. Kinase activation by I-2 and TPX2 is not additive and occurs without a corresponding increase in T-loop phosphorylation. These results suggest that both I-2 and TPX2 function as allosteric activators of Aurora-A. This implies that I-2 is a bifunctional signaling protein with separate domains to inhibit PP1 and directly stimulate Aurora-A kinase (Satinover, 2004).
Aurora-A and Plk1 are centrosomal kinases involved in centrosome maturation and spindle assembly. The microtubule-binding protein TPX2 interacts with, and activates, Aurora-A. RNA interference-mediated inactivation was used to investigate whether Aurora-A, Plk1 and TPX2 act independently or are part of one signaling cascade in spindle formation in mammalian cells. Both specific, and over- lapping, roles of each single regulator has been identified in centrosome maturation and spindle formation: (1) Aurora-A and TPX2 are required for centriole cohesion and spindle bipolarity; (2) TPX2, besides its known role in microtubule organization, is also involved in centrosome maturation; (3) finally, Plk1 controls the localization of Aurora-A to centrosomes, as well as TPX2 recruitment to microtubules. Based on these results therefore a hierarchical functional relation between Plk1 and the Aurora-A/TPX2 pathway emerges (De Luca, 2005).
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 (Drosophila homolog: Centromere identifer) 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).
Aurora family kinases contribute to regulation of mitosis. Using RNA interference in synchronized HeLa cells, Aurora-A has been shown to be required for mitotic entry. Initial activation of Aurora-A in late G2 phase of the cell cycle is essential for recruitment of the cyclin B1-Cdk1 complex to centrosomes, where it becomes activated and commits cells to mitosis. A two-hybrid screen identified the LIM protein Ajuba as an Aurora-A binding protein. Ajuba and Aurora-A interact in mitotic cells and become phosphorylated as they do so. In vitro analyses revealed that Ajuba induces the autophosphorylation and consequent activation of Aurora-A. Depletion of Ajuba prevented activation of Aurora-A at centrosomes in late G2 phase and inhibited mitotic entry. Overall, these data suggest that Ajuba is an essential activator of Aurora-A in mitotic commitment (Hirota, 2003).
Although the focal adhesion scaffolding protein HEF1 has a well-defined role in integrin-dependent attachment signalling at focal adhesions, it relocalizes to the spindle asters at mitosis. Overexpression of HEF1 causes an increase in centrosome numbers and multipolar spindles, resembling defects induced by manipulation of the mitotic regulatory kinase Aurora-A (AurA). HEF1 associates with and controls activation of AurA, and HEF1 depletion causes centrosomal splitting, mono-astral spindles and hyperactivation of Nek2, implying additional action earlier in the cell cycle. These results provide new insight into the role of an adhesion protein in coordination of cell attachment and division (Pugacheva, 2005).
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