Aurora A and Aurora B jointly coordinate chromosome segregation and anaphase microtubule dynamics

We established a conditional deletion of Aurora A kinase (AurA) in Cdk1 analogue-sensitive DT40 cells to analyze AurA knockout phenotypes after Cdk1 activation. In the absence of AurA, cells form bipolar spindles but fail to properly align their chromosomes and exit mitosis with segregation errors. The resulting daughter cells exhibit a variety of phenotypes and are highly aneuploid. Aurora B kinase (AurB)-inhibited cells show a similar chromosome alignment problem and cytokinesis defects, resulting in binucleate daughter cells. Conversely, cells lacking AurA and AurB activity exit mitosis without anaphase, forming polyploid daughter cells with a single nucleus. Strikingly, inhibition of both AurA and AurB results in a failure to depolymerize spindle microtubules (MTs) in anaphase after Cdk1 inactivation. These results suggest an essential combined function of AurA and AurB in chromosome segregation and anaphase MT dynamics.     

The functional diversity of Aurora kinases: a comprehensive review

Aurora kinases are serine/threonine kinases essential for the onset and progression of mitosis. Aurora members share a similar protein structure and kinase activity, but exhibit distinct cellular and subcellular localization. AurA favors the G2/M transition by promoting centrosome maturation and mitotic spindle assembly. AurB and AurC are chromosome-passenger complex proteins, crucial for chromosome binding to kinetochores and segregation of chromosomes. Cellular distribution of AurB is ubiquitous, while AurC expression is mainly restricted to meiotically-active germ cells. In human tumors, all Aurora kinase members play oncogenic roles related to their mitotic activity and promote cancer cell survival and proliferation. Furthermore, AurA plays tumor-promoting roles unrelated to mitosis, including tumor stemness, epithelial-to-mesenchymal transition and invasion. In this review, we aim to understand the functional interplay of Aurora kinases in various types of human cells, including tumor cells. The understanding of the functional diversity of Aurora kinases could help to evaluate their relevance as potential therapeutic targets in cancer.     

The catalytic role of INCENP in Aurora B activation and the kinetic mechanism of Aurora B/INCENP

Aurora kinases are a family of serine/threonine protein kinases that play essential roles in mitosis and cytokinesis. AurB (Aurora B kinase) has shown a clear link to cancer and is being pursued as an attractive cancer target. Multiple small molecules targeting AurB have entered the clinic for the treatment of cancer. A protein cofactor, INCENP (inner centromere protein), regulates the cellular localization and activation of AurB. In the present study, we examined the effect of INCENP on the activation kinetics of AurB and also elucidated the kinetic mechanism of AurB-catalysed substrate phosphorylation. We have concluded that: (i) substoichoimetric concentrations of INCENP are sufficient for AurB autophosphorylation at the activation loop residue Thr(232), and hence INCENP plays a catalytic role in AurB autophosphorylation; (ii) AurB/INCENP-catalysed phosphorylation of a peptide substrate proceeds through a rapid equilibrium random Bi Bi kinetic mechanism; and (iii) INCENP has relatively minor effects on the specific activity of AurB using a peptide substrate when compared with its role in AurB autoactivation. These results indicate that the effects of INCENP, and probably accessory proteins in general, may differ when enzymes are acting on different downstream targets.     

Dictyostelium Aurora kinase has properties of both Aurora A and Aurora B kinases

Aurora kinases are highly conserved proteins with important roles in mitosis. Metazoans contain two kinases, Aurora A and B, which contribute distinct functions at the spindle poles and the equatorial region respectively. It is not currently known whether the specialized functions of the two kinases arose after their duplication in animal cells or were already present in their ancestral kinase. We show that Dictyostelium discoideum contains a single Aurora kinase, DdAurora, that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box sequence and localizes at the spindle poles during early mitosis. Like Aurora B, DdAurora binds to its partner DdINCENP and localizes on centromeres at metaphase, the central spindle during anaphase, and the cleavage furrow at the end of cytokinesis. DdAurora also has several unusual properties. DdAurora remains associated with centromeres in anaphase, and this association does not require an interaction with DdINCENP. DdAurora then localizes at the cleavage furrow, but only at the end of cytokinesis. This localization is dependent on DdINCENP and the motor proteins Kif12 and myosin II. Thus, DdAurora may represent the ancestral kinase that gave rise to the different Aurora kinases in animals and also those in other organisms.     

Aurora A kinase and its substrate TACC3 are required for central spindle assembly

Cell division entails a marked reorganization of the microtubule network to form the spindle, a molecular machine that ensures accurate chromosome segregation to the daughter cells. Spindle organization is highly dynamic throughout mitosis and requires the activity of several kinases and complex regulatory mechanisms. Aurora A (AurA) kinase is essential for the assembly of the metaphase bipolar spindle and, thus, it has been difficult to address its function during the last phases of mitosis. Here, we examine the consequences of inhibiting AurA in cells undergoing anaphase, and show that AurA kinase activity is necessary for the assembly of a robust central spindle during anaphase. We also identify TACC3 as an AurA substrate essential in central spindle formation.     

Characterization of plant Aurora kinases during mitosis

The Aurora kinase family is a well-characterized serine/threonine protein kinase family that regulates different processes of mitotic events. Although functions of animal and yeast Aurora kinases have been analyzed, plant aurora kinases were not identified and characterized. We identified three Aurora kinase orthologs in Arabidopsis thaliana and designated these as AtAUR1, AtAUR2, and AtAUR3. These AtAURs could phosphorylate serine 10 in histone H3, in vitro. Dynamic analyses of GFP-fused AtAUR proteins revealed that AtAUR1 and AtAUR2 localized at the nuclear membrane in interphase and located in mitotic spindles during cell division. AtAUR1 also localized in the cell plates. AtAUR3 showed dot-like distribution on condensed chromosomes at prophase and then localized at the metaphase plate. At late anaphase, AtAUR3 is evenly localized on chromosomes. The localization of AtAUR3 during mitosis is very similar to that of phosphorylated histone H3. Interestingly, an overexpression of AtAUR3 induces disassembly of spindle microtubules and alteration of orientation of cell division. Our results indicate that plant Aurora kinases have different characters from that of Aurora kinases of other eukaryotes.†These authors equally contributed to this work     

Synthesis and biological evaluation of 2,4-disubstituted phthalazinones as Aurora kinase inhibitors

A series of 2,4-disubstituted phthalazinones were synthesized and their biological activities, including antiproliferation, inhibition against Aurora kinases and cell cycle effects were evaluated. Among them, N-cyclohexyl-4-((4-(1-methyl-1H-pyrazol-4-yl)-1-oxophthalazin-2(1H)-yl) methyl) benzamide (12c) exhibited the most potent antiproliferation against five carcinoma cell lines (HeLa, A549, HepG2, LoVo and HCT116 cells) with IC₅₀ values in range of 2.2–4.6?μM, while the IC₅₀ value of reference compound VX-680 was 8.5–15.3?μM. Moreover, Aurora kinase assays exhibited that compound 12c was potent inhibitor of AurA and AurB kinase with the IC₅₀ values were 118?±?8.1 and 80?±?4.2?nM, respectively. Molecular docking studies indicated that compound 12c forms better interaction with both AurA and AurB. Furthermore, compound 12c induced G2/M cell cycle arrest in HeLa cells by regulating protein levels of cyclinB1 and cdc2. These results suggested that 12c is a promising pan-Aurora kinase inhibitor for the potential treatment of cancer.     

Binding of TPX2 to Aurora A alters substrate and inhibitor interactions

The Aurora kinases are a family of serine/threonine kinases involved in mitosis. The expression of AurA is ubiquitous and cell cycle regulated. It is overexpressed in many tumor types, including breast, colon, and ovarian. TPX2 is a binding partner and activator of AurA. A fragment of TPX2 (residues 1-43) has been shown to be sufficient for binding, kinase activation, and protection from dephosphorylation. We have shown that the addition of TPX2(1-43) increases the catalytic efficiency of AurA. While TPX2 binding has no effect on the turnover number of AurA and does not change the reaction mechanism (characterized here to be a rapid equilibrium random mechanism), it increases the binding affinity of both ATP and a peptide substrate. We have also demonstrated differences in the inhibitor structure-activity relationship (SAR) in the presence or absence of TPX2(1-43). To better understand the differential SAR, we carried out computer modeling studies to gain insight into the effect of TPX2 on the binding interactions between AurA and inhibitors. Our working hypothesis is that TPX2 binding decreases the size and accessibility of a hydrophobic pocket, adjacent to the ATP site, to inhibitors.     

Aurora kinases and spindle assembly: Variations on a common theme?

The Aurora A (AurA) serine/threonine kinase controls multiple aspects of cell division and plays a key role in centrosome maturation and bipolar spindle assembly. The pleiotropic functions of AurA depend on its interaction with several cofactors, the best known of which is TPX2. TPX2 targets AurA to spindle microtubules (MTs) and activates it, both allosterically and by protecting the activation loop (T-loop) of the kinase domain from dephosphorylation. Although several factors have been implicated in the regulation of AurA at centrosomes, the underlying mechanism has remained elusive, and the existence of a distinct centrosome-specific AurA activator has been proposed. Our recent study has identified this activator as Cep192/Spd-2, one of the key factors in centrosome biogenesis. Cep192 targets AurA to centrosomes, where it promotes its activation by a novel, oligomerization-dependent mechanism characterized by extensive T-loop phosphorylation and high kinase activity. This process is key to the function of centrosomes as microtubule-organizing centers. Here, our findings are discussed in the context of other recent studies on the Aurora kinases, with an emphasis on their role in spindle assembly. The collected evidence suggests that the ‘hot spots’ of MT nucleation, centrosomes and kinetochores, rely on the oligomerization-mediated mechanism of activation of AurA and AurB, respectively.     

Novel E3 ligase component FBXL7 ubiquitinates and degrades Aurora A,causing mitotic arrest

Aurora family kinases play pivotal roles in several steps during mitosis. Specifically, Aurora A kinase is an important regulator of bipolar mitotic spindle formation and chromosome segregation. Like other members of the Aurora family, Aurora A kinase is also regulated by post-translational modifications. Here, we show that a previously undescribed E3 ligase component belonging to the SCF (Skp-Cullin1-F-box protein) E3 ligase family, SCFFBXL7, impairs cell proliferation by mediating Aurora A polyubiquitination and degradation. Both Aurora A and FBXL7 co-localize within the centrosome during spindle formation. FBXL7 ectopic expression led to G₂/M phase arrest in transformed epithelia, resulting in the appearance of tetraploidy and mitotic arrest with circular monopolar spindles and multipolar spindle formation. Interestingly, FBXL7 specifically interacts with Aurora A during mitosis but not in interphase, suggesting a regulatory role for FBXL7 in controlling Aurora A abundance during mitosis.     

Dissecting the role of Aurora A during spindle assembly

The centrosomal kinase Aurora A (AurA) is required for cell cycle progression, centrosome maturation and spindle assembly. However, the way it participates in spindle assembly is still quite unclear. Using the Xenopus egg extract system, we have dissected the role of AurA in the different microtubule (MT) assembly pathways involved in spindle formation. We developed a new tool based on the activation of AurA by TPX2 to clearly define the requirements for localization and activation of the kinase during spindle assembly. We show that localized AurA kinase activity is required to target factors involved in MT nucleation and stabilization to the centrosome, therefore promoting the formation of a MT aster. In addition, AurA strongly enhances MT nucleation mediated by the Ran pathway through cytoplasmic phosphorylation. Altogether, our data show that AurA exerts an effect as a key regulator of MT assembly during M phase and therefore of bipolar spindle formation.     

Evidence toward a Dual Phosphatase Mechanism That Restricts Aurora A (Thr-295) Phosphorylation during the Early Embryonic Cell Cycle

The mitotic kinase Aurora A (AurA) is regulated by a complex network of factors that includes co-activator binding, autophosphorylation, and dephosphorylation. Dephosphorylation of AurA by PP2A (human, Ser-51; Xenopus, Ser-53) destabilizes the protein, whereas mitotic dephosphorylation of its T-loop (human, Thr-288; Xenopus, Thr-295) by PP6 represses AurA activity. However, AurA(Thr-295) phosphorylation is restricted throughout the early embryonic cell cycle, not just during M-phase, and how Thr-295 is kept dephosphorylated during interphase and whether or not this mechanism impacts the cell cycle oscillator were unknown. Titration of okadaic acid (OA) or fostriecin into Xenopus early embryonic extract revealed that phosphatase activity other than PP1 continuously suppresses AurA(Thr-295) phosphorylation during the early embryonic cell cycle. Unexpectedly, we observed that inhibiting a phosphatase activity highly sensitive to OA caused an abnormal increase in AurA(Thr-295) phosphorylation late during interphase that corresponded with delayed cyclin-dependent kinase 1 (CDK1) activation. AurA(Thr-295) phosphorylation indeed influenced this timing, because AurA isoforms retaining an intact Thr-295 residue further delayed M-phase entry. Using mathematical modeling, we determined that one phosphatase would be insufficient to restrict AurA phosphorylation and regulate CDK1 activation, whereas a dual phosphatase topology best recapitulated our experimental observations. We propose that two phosphatases target Thr-295 of AurA to prevent premature AurA activation during interphase and that phosphorylated AurA(Thr-295) acts as a competitor substrate with a CDK1-activating phosphatase in late interphase. These results suggest a novel relationship between AurA and protein phosphatases during progression throughout the early embryonic cell cycle and shed new light on potential defects caused by AurA overexpression.     

Novel E3 ligase component FBXL7 ubiquitinates and degrades Aurora A,causing mitotic arrest

Aurora family kinases play pivotal roles in several steps during mitosis. Specifically, Aurora A kinase is an important regulator of bipolar mitotic spindle formation and chromosome segregation. Like other members of the Aurora family, Aurora A kinase is also regulated by post-translational modifications. Here, we show that a previously undescribed E3 ligase component belonging to the SCF (Skp-Cullin1-F-box protein) E3 ligase family, SCFFBXL7, impairs cell proliferation by mediating Aurora A polyubiquitination and degradation. Both Aurora A and FBXL7 co-localize within the centrosome during spindle formation. FBXL7 ectopic expression led to G₂/M phase arrest in transformed epithelia, resulting in the appearance of tetraploidy and mitotic arrest with circular monopolar spindles and multipolar spindle formation. Interestingly, FBXL7 specifically interacts with Aurora A during mitosis but not in interphase, suggesting a regulatory role for FBXL7 in controlling Aurora A abundance during mitosis.Key words: F-box protein, centrosome, mitosis, Aurora A     

Roles of Aurora kinases in mitosis and tumorigenesis

Aurora kinases, which have been implicated in several vital events in mitosis, represent a protein kinase family highly conserved during evolution. The activity of Aurora kinases is delicately regulated, mainly by phosphorylation and degradation. Deregulation of Aurora kinase activity can result in mitotic abnormality and genetic instability, leading to defects in centrosome function, spindle assembly, chromosome alignment, and cytokinesis. Both the expression level and the kinase activity of Aurora kinases are found to be up-regulated in many human cancers, indicating that these kinases might serve as useful targets for the development of anticancer drugs. This review focuses on recent progress on the roles of Aurora kinases in mitosis and tumorigenesis.     

Late mitotic functions of Aurora kinases

The coordination between late mitotic events such as poleward chromosome motion, spindle elongation, DNA decondensation, and nuclear envelope reformation (NER) is crucial for the completion of chromosome segregation at the anaphase-telophase transition. Mitotic exit is driven by a decrease of Cdk1 kinase activity and an increase of PP1/PP2A phosphatase activities. More recently, Aurora kinases have also emerged as master regulators of late mitotic events and cytokinesis. Aurora A is mainly associated with spindle poles throughout mitosis and midbody during telophase, whereas Aurora B re-localizes from centromeres in early mitosis to the spindle midzone and midbody as cells progress from anaphase to the completion of cytokinesis. Functional studies, together with the identification of a phosphorylation gradient during anaphase, established Aurora B as a major player in the organization of the spindle midzone and in the spatiotemporal coordination between chromosome segregation and NER. Aurora A has been less explored, but a cooperative role in spindle midzone stability has also been proposed, implying that both Aurora A and B contribute to accurate chromosome segregation during mitotic exit. Here, we review the roles of the Aurora kinases in the regulation of late mitotic events and discuss how they work together with other mitotic players to ensure an error-free mitosis.     

Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores

The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. 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, we show 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.     

Spatiotemporal regulation of Ipl1/Aurora activity by direct Cdk1 phosphorylation

Oscillating cyclin-dependent kinase 1 (Cdk1) activity is the major regulator of cell-cycle progression, whereas the Aurora B kinase, as part of the chromosome passenger complex (CPC), controls critical aspects of mitosis such as chromosome condensation and biorientation on the spindle. How these kinases mechanistically coordinate their important functions is only partially understood. Here, using budding yeast, we identify a regulatory mechanism by which the Cdk1 kinase Cdc28 directly controls the Aurora kinase Ipl1. We show that Cdk1 phosphorylates Ipl1 on two serine residues in the N-terminal domain, thereby suppressing its association with the microtubule plus-end tracking protein Bim1 until the onset of anaphase. Failure to phosphorylate Ipl1 leads to its premature targeting to the metaphase spindle and results in constitutive Bim1 phosphorylation, which is normally restricted to anaphase. Cells expressing an Ipl1-Sli15 complex that cannot be phosphorylated by Cdk1 display a severe growth defect. Our work shows that Ipl1/Aurora is not only the catalytic subunit of the CPC but also an important regulatory target that allows Cdk1 to coordinate chromosome biorientation with spindle morphogenesis.     

Urochordate Ascidians Possess a Single Isoform of Aurora Kinase That Localizes to the Midbody via TPX2 in Eggs and Cleavage Stage Embryos

Aurora kinases are key proteins found throughout the eukaryotes that control mitotic progression. Vertebrate Aurora-A and B kinases are thought to have evolved from a single Aurora-kinase isoform closest to that found in present day urochordates. In urochordate ascidians Aurora binds both TPX2 (a vertebrate AURKA partner) and INCENP (a vertebrate AURKB partner) and localizes to centrosomes and spindle microtubules as well as chromosomes and midbody during both meiosis and mitosis. Ascidian Aurora also displays this localization pattern during mitosis in echinoderms, strengthening the idea that non-vertebrate deuterostomes such as the urochordates and echinoderms possess a single form of Aurora kinase that has properties of vertebrate Aurora-kinase A and B. In the ascidian, TPX2 localizes to the centrosome and the spindle poles also as in vertebrates. However, we were surprised to find that TPX2 also localized strongly to the midbody in ascidian eggs and embryos. We thus examined more closely Aurora localization to the midbody by creating two separate point mutations of ascidian Aurora predicted to perturb binding to TPX2. Both forms of mutated Aurora behaved as predicted: neither localized to spindle poles where TPX2 is enriched. Interestingly, neither form of mutated Aurora localized to the midbody where TPX2 is also enriched, suggesting that ascidian Aurora midbody localization required TPX2 binding in ascidians. Functional analysis revealed that inhibition of Aurora kinase with a pharmacological inhibitor or with a dominant negative kinase dead form of Aurora caused cytokinesis failure and perturbed midbody formation during polar body extrusion. Our data support the view that vertebrate Aurora-A and B kinases evolved from a single non-vertebrate deuterostome ancestor. Moreover, since TPX2 localizes to the midbody in ascidian eggs and cleavage stage embryos it may be worthwhile re-assessing whether Aurora A kinase or TPX2 localize to the midbody in eggs and cleavage stage embryos.     

Polo-like kinase-activating kinases: Aurora A,Aurora B and what else?

The events of cell division are regulated by a complex interplay between kinases and phosphatases. Cyclin-dependent kinases (Cdks), polo-like kinases (Plks) and Aurora kinases play central roles in this process. Polo kinase (Plk1 in humans) regulates a wide range of events in mitosis and cytokinesis. To ensure the accuracy of these processes, polo activity itself is subject to complex regulation. Phosphorylation of polo in its T loop (or activation loop) increases its kinase activity several-fold. It has been shown that Aurora A kinase, with its co-factor Bora, activates Plk1 in G₂, and that this is essential for recovery from cell cycle arrest induced by DNA damage. In a recent article published in PLoS Biology, we report that Drosophila polo is activated by Aurora B kinase at centromeres, and that this is crucial for polo function in regulating chromosome dynamics in prometaphase. Our results suggest that this regulatory pathway is conserved in humans. Here, we propose a model for the collaboration between Aurora B and polo in the regulation of kinetochore attachment to microtubules in early mitosis. Moreover, we suggest that Aurora B could also function to activate Polo/Plk1 in cytokinesis. Finally, we discuss recent findings and open questions regarding the activation of polo and polo-like kinases by different kinases in mitosis, cytokinesis and other processes.     

A small C-terminal sequence of Aurora B is responsible for localization and function

Aurora B, a protein kinase required in mitosis, localizes to inner centromeres at metaphase and the spindle midzone in anaphase and is required for proper chromosome segregation and cytokinesis. Aurora A, a paralogue of Aurora B, localizes instead to centrosomes and spindle microtubules. Except for distinct N termini, Aurora B and Aurora A have highly similar sequences. We have combined small interfering RNA (siRNA) ablation of Aurora B with overexpression of truncation mutants to investigate the role of Aurora B sequence in its function. Reintroduction of Aurora B during siRNA treatment restored its localization and function. This permitted a restoration of function test to determine the sequence requirements for Aurora B targeting and function. Using this rescue protocol, neither N-terminal truncation of Aurora B unique sequence nor substitution with Aurora A N-terminal sequence affected Aurora B localization or function. Truncation of unique Aurora B C-terminal sequence from terminal residue 344 to residue 333 was without effect, but truncation to 326 abolished localization and function. Deletion of residues 326-333 completely abolished localization and blocked cells at prometaphase, establishing this sequence as critical to Aurora B function. Our findings thus establish a small sequence as essential for the distinct localization and function of Aurora B.