Aurora Kinases Family Members and their Synonyms
Aurora-A: AIK, ARK1, AURA, AURORA2, BTAK, MGC34538, STK15, STK6, STK7
Aurora-B: AIK2, AIM-1, AIM1, ARK2, AurB, IPL1, STK12, STK5
Aurora-C: AIE2, AIK3, ARK3, AurC, STK13, Aurora-C
Every new cell is generated through division of an existing cell (one cell generates two cells), and survival of all organisms depends on reliable transmission of the genetic information from the mother cell to daughter cells. Consequently, all cellular components must be duplicated before cell divides, and these duplications must be achieved with extreme precision and reliability over generations. The eukaryotic cell (cell with a nucleus) has set up complex network of regulatory mechanisms to ensure a correct sequence of events that eventually leads to cell division. This network called the cell cycle control system consists mainly in a complex assembly of oscillating phosphorylation/dephosphorylation reactions. Several protein kinases and phosphatases are involved in these signaling cascades. Aurora kinases that belong to a family of serine/threonine kinases play such a role; they are critical for the establishment of mitotic spindle, centrosome duplication, and separation as well as maturation, chromosomal alignment, spindle assembly checkpoint, and cytokinesis. In the early 1990s, Chan and Botstein (1993) identified the first Aurora kinase in the budding yeast Saccharomyces cerevisiae. The kinase was originally named Ipl1 for Increase in Ploidy 1. Indeed, conditional temperature-sensitive ipl1 ts-mutant cells reveal abnormal ploidy induction, suggesting that the Ipl1 kinase is involved in controlling chromosomes segregation. Glover et al. (1995) then discovered a Drosophila homolog that they named Aurora. The kinase was identified during a search of mutants that affect centrosome cycle in Drosophila. The first human Aurora kinase was isolated simultaneously by two groups, one was searching homology with yeast Ipl1 and Drosophila Aurora in randomly sequenced cDNAs (they identified Aik “Aurora Ipl1-related Kinase”) and the other one was mapping a chromosome region commonly amplified in breast cancer to search for potential oncogenes (they identified BTAK “breast tumor-activated kinase”). Rapidly, homology studies revealed several genes encoding Aurora kinases in the genome of multicellular organisms. They were first called AIRK for Aurora/Ipl1-related kinases. Thus, while yeast genome encodes only one Aurora kinase, a second gene was found in D. melanogaster through a genome sequencing program, and simultaneously two new genes were found in Homo sapiens: STK12 and AIE2. Because all these kinases have been isolated simultaneously in different laboratories, they carried “esoteric” names strongly requiring a new nomenclature. This was achieved by dividing the Aurora family into three submembers (Nigg 2001): Aurora-A, Aurora-B, and Aurora-C. Both Aurora-A and Aurora-B are expressed in proliferating cells, yet Aurora-A is associated predominantly with centrosomes and the spindle apparatus from prophase through telophase, whereas Aurora-B is prominent at the midzone during anaphase and in postmitotic bridges during telophase. Aurora-C seems to be restricted to germlines, is highly expressed in testis, but is also found overexpressed in tumors. Interestingly enough Aurora-C can fulfill Aurora-B function suggesting a close relationship. Chromosome localizations of Aurora kinase genes are as follows: Aurora-A (AURKA) on chromosome 20q13.2, Aurora-B (AURKB) on chromosome 17p13.1, and Aurora-C (AURKC) on chromosome 19q13.3.
Aurora Kinases Structure and Functions
Structure of Aurora Kinases
Aurora-A is involved in mitotic entry. On the one hand, Aurora-A phosphorylates and promotes the activation of the CDC25B phosphatase at centrosomes. This event is dependent on the targeting of Aurora-A to the centrosome via the LIM protein Ajuba. Phosphorylation of CDC25B is required for initial centrosomal activation of Cyclin-B/Cdk-1. On the other hand, it was recently shown that Aurora-A promotes the G2 activation of Polo-like kinase-1 (Plk-1) through phosphorylation within the T-loop of Plk-1. Active Plk1 controls Cyclin-B/Cdk1 activity through phosphorylation of the Cdc25B phosphatase and degradation of the Cdk1 inhibitory kinase Wee1. Aurora-A might also be involved in both the DNA damage checkpoint and in the spindle assembly checkpoint. Marumoto et al. (2003) showed that loss of function of Aurora-A through microinjection of antibodies delays mitotic entry, while gain of function through overexpression leads to premature entry into mitosis in the presence of DNA damage. Anand et al. (2003) showed that overexpression of Aurora-A at levels equivalent to those observed in cancers leads to an override of the spindle assembly checkpoint (SAC). The function of this checkpoint is to prevent chromosome segregation until every one of them is bioriented through kinetochore/microtubule attachment. This is achieved by controlling the activity of the anaphase-promoting complex/cyclosome (APC/C). Once the checkpoint is satisfied, the inhibition is relieved on the APC/C that promotes anaphase and mitotic exit by targeting securin and cyclin-B for degradation by the proteasome.
In one word, in the presence of spindle defects induced by taxol treatment, cells should arrest in prometaphase because of the SAC. Cells overexpressing Aurora-A are resistant to taxol treatment, they exit mitosis despite an active SAC. This might explain centrosome amplification and polyploidy frequently observed after Aurora-A overexpression (Meraldi et al. 2002; Marumoto et al. 2003). More recently, Caous et al. (2015) showed in Drosophila that disrupting the SAC in an Aurora-A mutant did not prevent tumor formation or chromosomes segregation. The monitoring of Mad2 and cyclin B dynamics in these cells revealed that SAC was satisfied without any degradation of Cyclin B. This suggests that, in the absence of the SAC, impaired cyclin B degradation could compensate for the defect in chromosome segregation when Aurora-A is defective and that Aurora-A would eventually be involved in Cyclin B degradation during mitotic exit. Aurora–A was also shown to be involved in cytokinesis. Hegarat and colleagues (Hegarat et al. 2011) using a conditional knock out of Aurora-A and a pharmacological inhibition of Aurora-B have shown that these two kinases likely cooperate to control microtubules depolymerization at the end of anaphase. Lioutas and Vernos (2013) used the Aurora A kinase inhibitor MLN8237 to show that Aurora-A participates in the stabilization of noncentrosomal microtubules through phosphorylation of TACC3 (Lioutas and Vernos 2013). We previously used a chemical genetics approach to demonstrate that Aurora-A is involved in central spindle stabilization through P150Glued phosphorylation (Reboutier et al. 2013).
To fulfill its functions, Aurora-A needs to be activated. Several Aurora-A activators have been suggested, yet data are quite controversial. For example, Bora and Ajuba have been first described as Aurora-A activators. However, Bora turns out not to be a direct and general activator of Aurora-A but rather an intermediate stimulating PLK1 phosphorylation by Aurora-A. And Ajuba was demonstrated not to be an Aurora-A activator in Drosophila. TPX2, HEF1, and, more recently, CEP192 and Arpc1b were also described as Aurora-A activators. Activation mechanism through binding to TPX2 and CEP192 is well documented. TPX2 activates Aurora-A through conformational changes triggering protection of the T288 residue from dephosphorylation by PP1 phosphatase (Bayliss et al. 2003). CEP192 recruits Aurora-A to centrosomes and favors oligomerization leading to a strong activation.
Aurora-B is a chromosome passenger protein, localized on chromosome and kinetochores in prophase, on kinetochores during prophase and metaphase, and at the midbody at the end of mitosis (Fig. 2). At the chromosome level, Aurora-B fulfills various functions. Aurora-B phosphorylates histone H3 at serine-10 during late G2/prophase thought to be required for chromosome condensation. Mutation of H3 serine-10 in Tetrahymena thermophila or Schizosaccharomyces pombe indeed caused chromosome condensation defects and subsequent segregation anomalies. Yet, the same mutation does not cause any similar defect in Saccharomyces cerevisiae, and there is actually no evidence it could work in a similar way in human. Within condensin complexes, that play a major role in chromosomes condensation, the three non-SMC subunits are phosphorylated by Aurora-B. This phosphorylation is required for loading of condensin I complex on chromosomes.
Aurora-B is also involved in sister chromatid cohesion. This cohesion is maintained by a ring structure formed by the cohesin complex that surrounds two sister chromatids. The ring is then broken at the metaphase-anaphase transition through a mechanism that involved both kinases Plk-1 and Aurora-B.
Aurora-B activity is required for the localization of the centromeric protein shugoshin-1 (Sgo1). In the absence of Aurora-B, instead of concentrating on centromeres, Sgo1 localizes diffusely along chromosome arms. The role of Sgo1 during mitosis is to protect centromeric cohesin from being degraded before the SAC has been satisfied. To protect cohesin, Sgo1 must bind to PP2A that locally counteracts PLK1 activity. Redistribution of Sgo1 from centromeres to chromosome arms in the absence of Aurora-B causes inappropriate cohesin protection along chromosome arms eventually inhibiting sister chromatids separation.
Aurora-B also participates in the SAC by regulating an enzyme involved in microtubule dynamics and that specifically controlled the biorientation of each chromosomes as well as the stability of the bipolar mitotic spindle. In a perfect spindle, each pair of every kinetochore is attached to microtubules emanating from different centrosomes building a bipolar spindle. Abnormal attachments can take various forms: (1) synthelic (in a kinetochore pair, both bind microtubules emanating from the same pole or centrosome) or (2) merothelic attachments (in a kinetochore pair, one binds to both centrosomes). Those two abnormal attachments are at the origin of a signal “lack of tension” in the spindle that will trigger a correction corresponding to an elimination of the abnormal attachment. The unattached kinetochores can then enter a new cycle of microtubule attachment until bipolar attachment is obtained. Two mechanisms involving Aurora-B in such corrections have been described. In the first one, Aurora-B phosphorylates the microtubule-depolymerizing enzyme MCAK and increases its recruitment to centromere. In the second one, Aurora-B phosphorylates kinetochore microtubule-capture factors such as Ncd80/Hec1 and Dam1. Phosphorylation reduces kinetochore affinity for microtubules whereas dephosphorylation stabilizes microtubule-kinetochore interactions. When biorientation is achieved, mitotic spindle comes under tension. This tension results in a movement of the kinetochores away from the kinase Aurora-B that is not able anymore to phosphorylate its target at the kinetochores. In summary, a single unattached kinetochore is sufficient to keep the SAC on in prometaphase. Aurora-B activity is required to arrest cell cycle progression in response to taxol that induces a lack of tension in the mitotic spindle. Attachments that do not generate tension are removed in an Aurora-B-dependent manner that triggers the recruitment of checkpoint proteins to kinetochores, resulting in inhibition of APC/C Cdc20.
After metaphase/anaphase transition, Aurora-B leaves kinetochores to concentrate at the spindle midzone and at the equatorial cortex to finally accumulate at the midbody. This protein localization from chromosome to midbody depends on microtubules and is characteristic of chromosome passenger protein essential for late mitotic events. Once established at the spindle midzone, Aurora-B produces a phosphorylation gradient and the kinase phosphorylates its substrates depending on their distance from the source. This gradient is now emerging as a major regulator of late mitotic events. Aurora-B is the catalytic activity of the chromosomal passenger complex (CPC) consisting of Aurora-B itself, inner centromere protein (INCENP), borealin, and survivin. CPC plays an essential role in cytokinesis in a wide range of organisms. At the midbody Aurora-B phosphorylates the centralspindlin complex composed of the kinesin MKLP1/ZEN4 and the Rac GTPase-activating protein 1 (MgcRacGAP). This phosphorylation is required to signal the positioning of the cleavage furrow via phosphorylation. The centralspindlin complex then regulates events leading to RhoA activation positioning the acto-myosin contracting ring. Aurora-B is also responsible for the regulation of the size of the central spindle through the phosphorylation of the microtubule depolymerizing factors Kif2A and Kif4A. Later during cytokinesis, the Aurora B phosphorylation gradient eventually allows for the spatial regulation of several substrates involved in chromosome decondensation and nuclear envelope reformation. In budding yeast, Aurora-B (Ipl1) plays an additional role during cytokinesis by controlling the NoCut pathway that prevents abscission (the final step of cytokinesis) when chromosomes have not been fully segregated and remain present at the site of abscission (Norden et al. 2006). Ipl1 controls the localization of the anillin-related proteins Boi1 and Boi2 to the ingression site; they both act as abscission inhibitors and prevent premature abscission and concomitant chromosome breakage.
The third member of the Aurora family, Aurora-C, is a close relative to Aurora-B. Ectopically expressed Aurora-C shows the same mitotic localization pattern as Aurora-B. Aurora-C can also interact with INCENP and survivin, two CPC proteins. Overexpression of a kinase-dead mutant of Aurora-C interfered with Aurora-B function by displacing its binding partners. In normal physiological conditions, Aurora-C mRNA and protein were initially described to be expressed only in testis. Aurora-C is required for spermatogenesis and male fertility in mice. A recent study identified a homozygous mutation within the Aurora-C gene in a group of infertile men. The mutated Aurora-C gene yielded a truncated kinase activity-deficient Aurora-C protein. The spermatozoa in these men were polyploid, again indicating a role for Aurora-C in maintaining a stable karyotype during male meiosis. Aurora-C mRNA was detected in several human adult tissues, yet in significantly lower levels than in testis.
Aurora Kinases and Cancer
The chromosomal region in which Aurora-A gene is located is frequently amplified in human cancers. Many studies show a significant incidence of Aurora-A amplification and overexpression in human breast, bladder, ovarian, colon, and pancreatic cancers. Ectopic overexpression of Aurora-A transforms NIH3T3 cells and Rat 1 fibroblasts in vitro, and introduction of these transformed cells into nude mice results in tumor growth. Aurora-A mRNA and protein overexpression is not systematically correlated with the gene amplification (i.e., amplification of Aurora-A was detected in 3% of hepatocellular carcinoma (HCC), whereas more than 60% of HCCs overexpressed Aurora-A mRNA and protein). Apart from gene amplification, transcriptional activation and inhibition of protein degradation can also contribute to the elevated levels of Aurora-A expression. It is still unclear how Aurora-A triggers cellular transformation and tumorigenesis, and how important its kinase activity is during this process. For instance, two different studies showed contradictory results concerning the effect of overexpression of a kinase-dead version of Aurora-A (Meraldi et al. 2002; Anand et al. 2003). The oncogenic effect of Aurora-A overexpression is likely due to chromosome instability and directly due to its functions during mitosis. Aurora-A overexpression triggers centrosome amplification likely through a cytokinesis defect. Subsequent abnormal spindles (monopolar or multipolar) formation are precursors of aneuploidy that could contribute to genomic instability and to tumorigenesis. Also, abnormal spindle usually results in the maintenance of the SAC, which leads to abortive mitosis and cell death through apoptosis. Yet, in many tumor cells, there is a tight relation between Aurora-A overexpression and the tumor suppressor p53. Aurora-A directly phosphorylates p53 and controls its stability and transcriptional activity. Additionally, p53 directly inhibits Aurora-A function, potentially via its binding to the catalytic domain of Aurora-A. The effect of Aurora-A overexpression on tetraploidization and centrosome amplification thus depends on the p53 status. When Aurora-A is overexpressed in cells lacking p53, the newly generated tetraploid cells would continue their cell cycle progression, thus giving rise to polyploid cells with four centrosomes and finally leading to genomic instability. Aurora-A was also shown to physically bind to and phosphorylate BRCA1. BRCA1, like p53, is a tumor suppressor; its phosphorylation by Aurora-A causes a loss of function, making cells resistant to DNA damage and override checkpoint response.
Two recent studies in Drosophila and mouse showed that Aurora-A could yet function as a tumor suppressor. In Drosophila, Aurora-A is required for correct spindle orientation during asymmetric neuroblast division. Asymmetric stem cell division is required for the correct balance between stem cell self-renewal and differentiation. Disruption of asymmetric stem cell division through Aurora-A inactivation can give rise to stem cell overproduction and concomitant tumor growth. In mouse, Aurora-A heterozygosity results in a significant increased tumor incidence suggesting that Aurora-A may act as a haplo-insufficient tumor suppressor. Furthermore, Aurora-A heterozygous mouse embryonic fibroblasts have higher rates of aneuploidy. These findings, together with the observation that Aurora-A levels are low in certain tumors, strongly suggest that a balanced Aurora-A level is critical for maintaining genomic stability. These data are important to keep in mind regarding the current efforts that are made to exploit Aurora-A inhibition by chemical inhibitors as an antitumor therapeutic.
The role of Aurora-B in tumorigenesis is not as clear as concerning Aurora-A. Only one study showed that overexpression of Aurora-B in CHO cells can promote aneuploidy and increase invasiveness in xenograft experiments. Yet, the chromosomal region containing Aurora-B has never been associated with amplification in tumors. Reports show that Aurora-B is overexpressed in certain tumor types, but it is not clear whether the observed overexpression of Aurora-B is due to the high proliferative index of cancerous cells or whether it is really related to tumorigenesis. Aurora-B overexpression was also shown to strongly enhance cellular transformation in cells expressing oncogenic Ras-V12. Thus, Aurora-B is probably not directly oncogenic but could participate in cell transformation in a particular cellular context.
Although the chromosomal region in which the Aurora-C gene is located is known to be deleted or translocated in certain human cancers cell lines, it is unclear whether Aurora-C deletion or overexpression plays a causative role in tumorigenesis even if a correlation was made between expression and aggressiveness of thyroid cancer.
Pharmacological Inhibitors of Aurora Kinases
The clearly established role for Aurora kinases in mitosis, accompanied by the evidence suggesting that deregulated Aurora-A and, to a lesser extent, Aurora-B expression is linked to tumorigenesis, raised the hypothesis that inhibiting these kinases might be a powerful antitumor strategy. The aim of this entry is not to give exhaustive data about Aurora kinase inhibitor (exhaustive details are given in these two excellent reviews: Karthigeyan et al. 2010; Bavetsias and Linardopoulos 2015) but to bring clear information important to know about pharmacological Aurora inhibition. Globally, and because the catalytic site is well conserved in Aurora kinases family, there does not exist any inhibitor to specifically target Aurora-A or Aurora-B. All inhibitors target the ATP-binding site to inhibit the catalytic activity of the kinase. The only way to specifically inhibit one particular member of the Aurora kinases family is to use the chemical genetics strategy that consists in genetically modifying the catalytic site of the kinase to make it sensitive to a drug that has no other target into the cell. This was in particular used in Reboutier et al. (2013) to show for first time the involvement of the Aurora-A kinase during cytokinesis. Concerning more conventional Aurora kinases inhibitors, Hesperadin and ZM447439 were the first proven small-molecule inhibitors of Aurora kinases. If Hesperadin is more effective on Aurora kinase B than on Aurora-A, ZM447439 inhibits both Aurora kinases A and B. Both compounds never entered clinical trials, probably due to the emergence of more potent and specific inhibitors of the Aurora kinases family. There are actually several new molecules that entered phase I and even phase II clinical trials. Some molecules are pan-Aurora inhibitors (i.e., VX-680, PHA-739358…), others are more selective for Aurora-B (i.e., AZD1152) or Aurora-A (i.e., MLN8054, MLN8237). Concerning pan-Aurora inhibitors, results suggest that effect is mainly directed against Aurora-B. Regarding these data, it is important to keep in mind that in vitro tests designed to assess inhibitor specificity do not take into account Aurora-interacting proteins (i.e., inhibitors or activators). Indeed, it is now known that binding of TPX2 to Aurora-A alters inhibitor interaction (Anderson et al. 2007). Specificity revealed in vitro thus does not reflect the real effect of the drug in vivo. Overall, the responses seen in the phase I studies reported to date in patients with solid tumors are rather disappointing. The majority of reports had only disease stabilization as best response, with very few exceptions. This highlights the fact that to increase the antitumor activity of the Aurora kinase inhibitors in the clinic, combination therapy with cytotoxic anticancer agents, radiotherapy, or other targeted agents might be used in the future (Boss et al. 2009). Regarding the possible role of Aurora-A as a tumor suppressor, one can also ask whether it is really judicious to specifically target the Aurora-A kinase or, in other words, whether there is a risk to trigger tumorigenesis of noncancer cells in a patient treated with an Aurora kinase inhibitor. That’s why more recent works tried to identify molecules that are able to disrupt the interaction between the Aurora kinases and one of their specific ligand. It was notably the case for the interaction between TPX2 and Aurora-A and INCENP and Aurora-B. Further studies are nevertheless needed to fully address these questions, and there is a strong need to generate pertinent biomarkers to treat patients that really need these molecules.
Aurora proteins belong to a family of kinases involved in cell cycle regulation. In mammals, there exist three different Aurora kinases named Aurora-A, Aurora-B, and Aurora-C. These proteins share a common structure but present different functions and subcellular localizations. Aurora-A is associated predominantly with centrosomes and the spindle apparatus from prophase through telophase and is mainly involved in mitotic spindle assembly. Aurora-B is prominent at the midzone during anaphase and in postmitotic bridges during telophase and participates in chromosomes segregation and cytokinesis. Aurora-C appears to possess functions similar to Aurora-B but its expression is restricted to testis. Due to their prominent roles in cell cycle regulation, Aurora kinases are frequently involved in tumorigenesis and are targets of pharmaceutical industry that aims at generating specific Aurora kinase inhibitors to treat cancer.
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