A novel mechanism for activation of the protein kinase Aurora A

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 [1-4]. Aurora A is an oncogenic cell cycle-regulated protein kinase that is subject to phosphorylation-dependent activation [5-11]. 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 [11-13]. Here, we purify an activator of Aurora A from Xenopus eggs and identify it 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 [14-16]. Our 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.     

Regulation of Xenopus Aurora A activation by TPX2

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. In this report we clarify 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. We identify the sites 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. We have also investigated the interaction between Xenopus p53 and Xenopus Aurora A. 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.     

Abrogation of AuroraA-TPX2 by novel natural inhibitors: molecular dynamics-based mechanistic analysis

Abstract

Introduction: Cancer is characterized by uncontrolled cell growth and genetic instabilities. The human Aurora-A kinase protein plays a crucial role in spindle assembly during mitosis and is activated by another candidate oncogene, targeting protein for Xklp2 (TPX2). It has been proposed that dissociation of Aurora A–TPX2 complex leads to disruption of mitotic spindle apparatus, thereby preventing cell division and further tumor growth. Materials and methods: A large natural compound library was docked against the active site of Aurora A–TPX2 complex. The protein–ligand complexes were subjected to molecular dynamics simulation to ascertain their binding stability. The drug properties of the compounds were analyzed to observe their drug-like properties. Results: The virtual screening of natural compound library yielded two high scoring compounds, the first compound CTOM [ZINC ID: 38143674] (Glide score: –9.49) was stable for 17 ns while the second TTOM (Glide score: ?9.07) was stable for 15 ns. While CTOM interacted with His280, Thr288 of Aurora A and Tyr34, Lys38 of TPX2, TTOM interacted with Arg285 and Arg286 in addition to the residues involved with CTOM. Conclusions: We report two natural compounds as potential drugs leads for the disruption of this complex. These ligands show a preferable docking score and have many drugs like properties within in the range of 95% of known drugs. The study provides evidence that CTOM and TTOM can efficiently inhibit the TPX2-mediated activation of Aurora A. Thus, it paves way for an elaborate investigation and establishes the importance of computational approaches as time- and cost-effective techniques.     

Mechanism of Aurora B activation by INCENP and inhibition by hesperadin

Aurora family serine/threonine kinases control mitotic progression, and their deregulation is implicated in tumorigenesis. Aurora A and Aurora B, the best-characterized members of mammalian Aurora kinases, are approximately 60% identical but bind to unrelated activating subunits. The structure of the complex of Aurora A with the TPX2 activator has been reported previously. Here, we report the crystal structure of Aurora B in complex with the IN-box segment of the inner centromere protein (INCENP) activator and with the small molecule inhibitor Hesperadin. The Aurora B:INCENP complex is remarkably different from the Aurora A:TPX2 complex. INCENP forms a crown around the small lobe of Aurora B and induces the active conformation of the T loop allosterically. The structure represents an intermediate state of activation of Aurora B in which the Aurora B C-terminal segment stabilizes an open conformation of the catalytic cleft, and a critical ion pair in the kinase active site is impaired. Phosphorylation of two serines in the carboxyl terminus of INCENP generates the fully active kinase.     

Ashwagandha derived withanone targets TPX2-Aurora A complex: computational and experimental evidence to its anticancer activity

Cancer is largely marked by genetic instability. Specific inhibition of individual proteins or signalling pathways that regulate genetic stability during cell division thus hold a great potential for cancer therapy. The Aurora A kinase is a Ser/Thr kinase that plays a critical role during mitosis and cytokinesis and is found upregulated in several cancer types. It is functionally regulated by its interactions with TPX2, a candidate oncogene. Aurora A inhibitors have been proposed as anticancer drugs that work by blocking its ATP binding site. This site is common to other kinases and hence these inhibitors lack specificity for Aurora A inhibition in particular, thus advocating the need of some alternative inhibition route. Previously, we identified TPX2 as a cellular target for withanone that selectively kill cancer cells. By computational approach, we found here that withanone binds to TPX2-Aurora A complex. In experiment, withanone treatment to cancer cells indeed resulted in dissociation of TPX2-Aurora A complex and disruption of mitotic spindle apparatus proposing this as a mechanism of the anticancer activity of withanone. From docking analysis, non-formation/disruption of the active TPX2-Aurora A association complex could be discerned. Our MD simulation results suggesting the thermodynamic and structural stability of TPX2-Aurora A in complex with withanone further substantiates the binding. We report a computational rationale of the ability of naturally occurring withanone to alter the kinase signalling pathway in an ATP-independent manner and experimental evidence in which withanone cause inactivation of the TPX2-Aurora A complex. The study demonstrated that TPX2-Aurora A complex is a target of withanone, a potential natural anticancer drug.     

A novel role for TPX2 as a scaffold and co-activator protein of the Chromosomal Passenger Complex

Aurora B kinase forms the enzymatic core of the Chromosomal Passenger Complex (CPC) and is a master regulator of mitosis. Understanding the regulation of Aurora B is critical to illuminate its role in mitosis. INCENP, Survivin and Borealin have all been known to promote Aurora B activation. In this study, we have identified the Aurora A activator protein TPX2 as a novel scaffold and co-activator protein of the CPC. Studies utilizing M-phase Xenopus egg extracts (XEE) revealed that the immunodepletion of endogenous TPX2 from XEE decreases Aurora B-Survivin and Aurora B-INCENP interactions, leading to a consequent reduction in Aurora B activity. Further, residues 138 to 328 of Xenopus TPX2 (TPX2 B) are sufficient to enhance Aurora B-Survivin association and Aurora B kinase activity in vitro. Importantly, experiments with pancreatic cancer cell lines suggest that this mechanism of Aurora B activation by TPX2 is likely to be conserved in human cells. Strikingly, the overexpression of human TPX2 B in HeLa cells causes defects in metaphase chromosome alignment and INCENP localization. Thus, in addition to its already established role as an Aurora A activator, our data support the role of TPX2 as a novel co-activator of Aurora kinase B.     

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.     

An essential function of the C. elegans ortholog of TPX2 is to localize activated aurora A kinase to mitotic spindles

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. Here, we identify TPXL-1, a C. elegans protein that is the first characterized invertebrate ortholog of TPX2. We demonstrate that an essential role of TPXL-1 during mitosis is to activate and target Aurora A to microtubules. Our 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.     

Phosphorylation of TPX2 by Plx1 enhances activation of Aurora A

Entry into mitosis requires the activation of mitotic kinases, including Aurora A and Polo-like kinase 1 (Plk1). Increased levels of these kinases are frequently found associated with human cancers, and therefore it is imperative to understand the processes leading to their activation. We demonstrate that TPX2, but neither Ajuba nor Inhibitor-2, can activate Aurora A directly. Moreover, Plx1 can induce Aurora A T-loop phosphorylation indirectly in vivo during oocyte maturation. We identify Ser204 in TPX2 as a Plx1 phosphorylation site. Mutating Ser204 to alanine decreases activation of Aurora A, whereas a phosphomimetic Asp mutant exhibits enhanced activating ability. Finally, we show that phosphorylation of TPX2 with Plx1 increases its ability to activate Aurora A. Taken together, our data indicate that Plx1 promotes activation of Aurora A, most likely through TPX2. In light of the current literature, we propose a model in which Plx1 and Aurora A activate each other in a positive feedback loop.     

Spindle pole regulation by a discrete Eg5-interacting domain in TPX2

Targeting protein for Xklp2 (TPX2) activates the Ser/Thr kinase Aurora A in mitosis and targets it to the mitotic spindle [1, 2]. These effects on Aurora A are mediated by the N-terminal domain of TPX2, whereas a C-terminal fragment has been reported to affect microtubule nucleation [3]. Using the Xenopus system, we identified a novel role of TPX2 during mitosis. Injection of TPX2 or its C terminus (TPX2-CT) into blastomeres of two-cell embryos led to potent cleavage arrest. Despite cleavage arrest, TPX2-injected embryos biochemically undergo multiple rounds of DNA synthesis and mitosis, and arrested blastomeres have abnormal spindles, clustered centrosomes, and an apparent failure of cytokinesis. In Xenopus S3 cells, transfection of TPX2-FL causes spindle collapse, whereas TPX2-CT blocks pole segregation, resulting in apposing spindle poles with no evident displacement of Aurora A. Analysis of TPX2-CT deletion peptides revealed that only constructs able to interact with the class 5 kinesin-like motor protein Eg5 induce the spindle phenotypes. Importantly, injection of Eg5 into TPX2-CT-arrested blastomeres causes resumption of cleavage. These results define a discrete domain within the C terminus of TPX2 that exerts a novel Eg5-dependent function in spindle pole segregation.     

Aurora A on the mitotic spindle is activated by the way it holds its partner

An exciting study in this issue reveals how binding of the microtubule associated protein TPX2 to the mitotic kinase Aurora A induces a conformational change. This moves the phosphorylated activation domain into a more compact position within the kinase core, providing a better substrate binding platform and hiding the activating phosphoryl group from attack by PP1.     

Computational Investigation of Cancer-Associated Molecular Mechanism in Aurora A (S155R) Mutation

Centrosomes are the key-regulating element of cell cycle progression. Aberrations in their functional mechanism lead to several cancer-related disorders. Aurora A protein is a centrosome-associated protein that regulates the centriole duplication and its abberations are associated with multiple cases of aneuploidy and cancer-related disorders. S155R mutation in Aurora A is reported to induce cancer like phenotype and disrupt its binding with TPX2 protein. In this study, we have demonstrated the structural consequences of Aurora A S155R mutation and the atomic changes that influenced the loss of TPX2-binding affinity. Docking and molecular dynamics simulation results suggested significant loss in atomic contacts between mutant Aurora A and TPX2 protein. Further, we observed a notable changes in conformation of mutant Aurora A–TPX2 docked complex as compared to the native. Loss of binding affinity rendered the TPX2 domain free which then induced unfolding in its coiled region and enabled the overall expansion of mutant complex as compared to the native. The significant outcomes obtained from this study will facilitate in future cancer researches and in developing the potent drug therapies.     

A Cancer-associated Aurora A Mutant Is Mislocalized and Misregulated Due to Loss of Interaction with TPX2

Mutations in protein kinases can drive cancer through alterations of the kinase activity or by uncoupling kinase activity from regulation. Changes to protein expression in Aurora A, a mitotic Ser/Thr kinase, are associated with the development of several human cancers, but the effects of somatic cancer-associated mutations have not been determined. In this study we show that Aurora A kinase activity is altered in different ways in three somatic cancer-associated mutations located within the catalytic domain; Aurora A(V174M) shows constitutively increased kinase activity, Aurora A(S155R) activity is decreased primarily due to misregulation, and Aurora A(S361*) activity is ablated due to loss of structural integrity. These alterations suggest vastly different mechanisms for the role of these three mutations in human cancer. We have further characterized the Aurora A(S155R) mutant protein, found that its reduced cellular activity and mislocalization are due to loss of interaction with TPX2, and deciphered the structural basis of the disruption at 2.5 Å resolution. Previous studies have shown that disruption of the Aurora A/TPX2 interaction results in defective spindles that generate chromosomal abnormalities. In a panel of 40 samples from microsatellite instability-positive colon cancer patients, we found one example in which the tumor contained only Aurora A(S155R), whereas the normal tissue contained only wild-type Aurora A. We propose that the S155R mutation is an example of a somatic mutation associated with this tumor type, albeit at modest frequency, that could promote aneuploidy through the loss of regulated interactions between Aurora A and its protein partners.     

Characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly in Xenopus egg extracts

TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.     

Spatial regulation of Aurora A activity during mitotic spindle assembly requires RHAMM to correctly localize TPX2

Construction of a mitotic spindle requires biochemical pathways to assemble spindle microtubules and structural proteins to organize these microtubules into a bipolar array. Through a complex with dynein, the receptor for hyaluronan-mediated motility (RHAMM) cross-links mitotic microtubules to provide structural support, maintain spindle integrity, and correctly orient the mitotic spindle. Here, we locate RHAMM to sites of microtubule assembly at centrosomes and non-centrosome sites near kinetochores and demonstrate that RHAMM is required for the activation of Aurora kinase A. Silencing of RHAMM delays the kinetics of spindle assembly, mislocalizes targeting protein for XKlp2 (TPX2), and attenuates the localized activation of Aurora kinase A with a consequent reduction in mitotic spindle length. The RHAMM–TPX2 complex requires a C-terminal basic leucine zipper in RHAMM and a domain that includes the nuclear localization signal in TPX2. Together, our findings identify RHAMM as a critical regulator for Aurora kinase A signaling and suggest that RHAMM ensures bipolar spindle assembly and mitotic progression through the integration of biochemical and structural pathways.     

Synthesis and biological evaluation of nitroxide labeled pyrimidines as Aurora kinase inhibitors

To find novel effective Aurora kinases inhibitors, a series of structurally interesting nitroxide labeled pyrimidines were synthesized and evaluated their anti-proliferative and Aurora kinases inhibitory activities. Among them, butyl 2-(3-((5-fluoro-2-((4-((1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl)carbamoyl) phenyl) amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)acetate (22) possessed the most potent anti-proliferative effects against four carcinoma cell lines with IC₅₀ values in range of 0.89–11.41?μM, and kinases inhibition against Aurora A and B with the IC₅₀ values were 9.3 and 2.8?nM, respectively. Furthermore, compound 22 blocked the phosphorylation of Aurora A (T288), Aurora B (Thr232) and HisH3, decreased the expression of proteins TPX2, Eg5 and Bora, as well as disrupted the mitotic spindle formation in HeLa cells. Molecular docking studies indicated that compound 22 well interact with both Aurora A and B. The results showed that compound 22 is a potential anticancer agent as promising pan-Aurora kinase inhibitor.     

A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly

The activated form of Ran (Ran-GTP) 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. We report here that 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. We show that 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. As the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, we hypothesize 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.     

The Aurora A and Aurora B Protein Kinases: A Single Amino Acid Difference Controls Intrinsic Activity and Activation by TPX2

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. Here we identify G205 in 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, had no effect on autophosphorylation of the T-loop but led to a 20-fold loss of specific activity, whereas mutation of N158 in Aurora B to G caused a 350-fold increase in specific activity. G205 N Aurora A was still activated by TPX2, but protection of pT295 from dephosphorylation by protein phosphatase 1 was abolished. Structural analysis of these effects suggests that the G198 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.     

Gain of native conformation of Aurora A S155R mutant by small molecules

Aurora A is a mitotic serine/threonine kinase protein that is a proposed target of the first-line anticancer drug design. It has been found to be overexpressed in many human cancer cells, including hematological, breast, and colorectal. Here, we focus on a particular somatic mutant S155R of Aurora kinase A protein, whose activity decreases because of loss of interaction with a TPX2 protein that results in ectopic expression of the Aurora kinase A protein, which contributes chromosome instability, centrosome amplification, and oncogenic transformation. The primary target of this study is to select a drug molecule whose binding results in gaining S155R mutant interaction with TPX2. The computational methodology applied in this study involves mapping of hotspots (for uncompetitive binding), virtual screening, protein–ligand docking, postdocking optimization, and protein–protein docking approach. In this study, we screen and validate ZINC968264, which acts as a potential molecule that can improve the loss of function occurred because of mutation (S155R) in Aurora A. Our approaches pave a suitable path to design a potential drug against physiological condition manifested because of S155R mutant in Aurora A.     

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.