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.     

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.     

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.     

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.     

Greatwall and Polo-like kinase 1 coordinate to promote checkpoint recovery

Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extracts exhibits no synergy with that of Plx1 in delaying checkpoint recovery, suggesting a distinct but related relationship between Gwl and Plx1. In further revealing their functional relationship, we found mutual dependence for activation of Gwl and Plx1 during checkpoint recovery, as well as their direct association. We characterized the protein association in detail and recapitulated it in vitro with purified proteins, which suggests direct interaction. Interestingly, Gwl interaction with Plx1 and its phosphorylation by Plx1 both increase at the stage of checkpoint recovery. More importantly, Plx1-mediated phosphorylation renders Gwl more efficient in promoting checkpoint recovery, suggesting a functional involvement of such regulation in the recovery process. Finally, we report an indirect regulatory mechanism involving Aurora A that may account for Gwl-dependent regulation of Plx1 during checkpoint recovery. Our results thus reveal novel mechanisms underlying the involvement of Gwl in checkpoint recovery, in particular, its functional relationship with Plx1, a well characterized regulator of checkpoint recovery. Coordinated interplays between Plx1 and Gwl are required for reactivation of these kinases from the G(2)/M DNA damage checkpoint and efficient checkpoint recovery.     

MPF Amplification inXenopusOocyte Extracts Depends on a Two-Step Activation of Cdc25 Phosphatase

The activation of Cdc2 kinase induces the entry into M-phase of all eukaryotic cells. We have developed a cell-free system prepared from prophase-arrestedXenopusoocytes to analyze the mechanism initiating the all-or-none activation of Cdc2 kinase. Inhibition of phosphatase 2A, the major okadaic acid-sensitive Ser/Thr phosphatase, in these extracts, provokes Cdc2 kinase amplification and concomitant hyperphosphorylation of Cdc25 phosphatase, with a lag of about 1 h. Polo-like kinase (Plx1 kinase) is activated slightly after Cdc2. All these events are totally inhibited by the cdk inhibitor p21^Cip1, demonstrating that Plx1 kinase activation depends on Cdc2 kinase activity. Addition of a threshold level of recombinant Cdc25 induces a linear activation of Cdc2 and Plx1 kinases and a partial phosphorylation of Cdc25. We propose that the Cdc2 positive feedback loop involves two successive phosphorylation steps of Cdc25 phosphatase: the first one is catalyzed by Cdc2 kinase and/or Plx1 kinase but it does not modify Cdc25 enzymatic activity, the second one requires a new kinase counteracted by phosphatase 2A. Furthermore we demonstrate that, under our conditions, Cdc2 amplification and MAP kinase activation are two independent events.     

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.     

Mitotic kinases regulate MT-polymerizing/MT-bundling activity of DDA3

Mitotic kinases orchestrate cell cycle processes by phosphorylation of cell cycle regulators. DDA3, a spindle-associated phosphor-protein, is a substrate of mitotic kinases that control chromosome movement and spindle microtubule (MT) dynamics. Through a mass spectrometry analysis, we identified phosphorylation sites on the endogenous mitotic DDA3, which include Ser22, Ser65, Ser70, and Ser223. Phosphorylation of these residues converts interphase form of DDA3 to mitotic form by changing its biochemical activity, as unphosphorylated DDA3 processed both the MT polymerizing and bundling activities, whereas phosphor-mimic mutants lost both activities, only retaining the MT-binding activity. We found that mitotic kinases, such as Cdk1, Aurora A, and Plk1, phosphorylate DDA3 in vitro. Whereas Cdk1 and Aurora A negatively regulate MT-polymerizing and MT-bundling activities, Plk1 does not affect these activities. Interestingly, the phosphorylation of DDA3 by Aurora A and Plk1 inhibits the phosphorylation by other kinases, indicating that sequential phosphorylation is important for the regulation of DDA3 function. We conclude that kinases control the function of DDA3 in the cell cycle by regulating its MT-polymerizing/bundling activities through sequential phosphorylation.     

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.     

Phosphorylation of the cyclin b1 cytoplasmic retention sequence by mitogen-activated protein kinase and Plx

The cyclin B1/Cdc2 complex regulates many of the dramatic cellular rearrangements observed at mitosis. Although predominantly cytoplasmic during interphase, this kinase complex translocates precipitously to the nucleus at the G(2)-M transition. The interphase cytoplasmic location of cyclin B1/Cdc2 reflects continuous, albeit slow, nuclear import and much more rapid nuclear export. In contrast, the sudden nuclear accumulation of the complex before entry into mitosis reflects a marked increase in the import rate, with a concomitant inhibition of cyclin B1 nuclear export. These dynamic changes in cyclin B1/Cdc2 localization are regulated by phosphorylation of four serines within a region of cyclin B1 known as the cytoplasmic retention sequence (CRS). Phosphorylation of all four serines is required for rapid nuclear entry, whereas phosphorylation of only the last in the series (Ser 113) is required to prevent nuclear export by CRM1. As these residues represent key loci of regulation, it is important to identify the kinases acting on these sites. Here we report that Xenopus cyclin B1 is regulated by both Erk and Plx kinases, and that Cdc2, counter to previous speculation, is not required for CRS phosphorylation. Phosphorylation of the first two of the CRS serines (Ser 94 and Ser 96) is catalyzed by Erk in the Xenopus system. Although it was previously reported that Ser 113 is a Plx substrate, we were unable to observe phosphorylation of this residue in isolation by purified Plx. Rather, in contrast to previously published data, we have found that the penultimate CRS serine (Ser 101) is a Plx substrate. Collectively, these data demonstrate a new role for Erk in mitotic regulation, identify the Ser 101-directed kinase, and provide a picture of cyclin B1/Cdc2 regulation by the combinatorial action of distinct kinases.     

Identification of phosphorylation sites in the polo-like kinases Plx1 and Plk1 by a novel strategy based on element and electrospray high resolution mass spectrometry

A novel strategy for the determination of protein phosphorylation sites is described and applied to the polo-like kinases Plx1 (Xenopus laevis) and Plk1 (Homo sapiens). The strategy comprises the sequential application of the following techniques: proteolytic digestion, capillary liquid chromatography (LC)-inductively coupled plasma mass spectrometry with phosphorus detection, capillary LC-electrospray mass spectrometry and electrospray tandem mass spectrometry. In this approach, phosphopeptides are generated, their elution time in capillary LC is determined, candidate phosphopeptides at the corresponding elution times are identified, and positive identification and sequencing of phosphopeptides is performed in the last step of the analysis. Using this technique, Ser25/26, Ser326, and Ser340 were identified as phosphorylation sites in recombinant Plx1, and Ser340 was identified as the major phosphorylation site in a kinase-dead mutant of Plx1 expressed in okadaic acid-treated Sf9 insect cells. A site corresponding to Ser326 in Plx1 was also shown to be phosphorylated in the human polo-like kinase Plk1 (Ser335). Element mass spectrometry with phosphorus detection provides a quantitative phosphorylation profile of all phosphorylation sites accessible by LC.     

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.     

Cell cycle-regulated phosphorylation of the Xenopus polo-like kinase Plx1

Polo-like kinases (Plks) control multiple important events during M phase progression, but little is known about their activation during the cell cycle. The activities of both mammalian Plk1 and Xenopus Plx1 peak during M phase, and this activation has been attributed to phosphorylation. However, no phosphorylation sites have previously been identified in any member of the Plk family. Here we have combined tryptic phosphopeptide mapping with mass spectrometry to identify four major phosphorylation sites in Xenopus Plx1. All four sites appear to be phosphorylated in a cell cycle-dependent manner. Phosphorylations at two sites (Ser-260 and Ser-326) most likely represent autophosphorylation events, whereas two other sites (Thr-201 and Ser-340) are targeted by upstream kinases. Several recombinant kinases were tested for their ability to phosphorylate Plx1 in vitro. Whereas xPlkk1 phosphorylated primarily Thr-10, Thr-201 was readily phosphorylated by protein kinase A, and Cdk1/cyclin B was identified as a likely kinase acting on Ser-340. Phosphorylation of Ser-340 was shown to be responsible for the retarded electrophoretic mobility of Plx1 during M phase, and phosphorylation of Thr-201 was identified as a major activating event.     

The C. elegans Tousled-like kinase contributes to chromosome segregation as a substrate and regulator of the Aurora B kinase

BACKGROUND: The Aurora kinases control multiple aspects of mitosis, among them centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora activity is regulated in part by a subset of Aurora substrates that, once phosphorylated, can enhance Aurora kinase activity. Aurora A substrate activators include TPX2 and Ajuba, whereas the only known Aurora B substrate activator is the chromosomal passenger INCENP. RESULTS: We report that the C. elegans Tousled kinase TLK-1 is a second substrate activator of the Aurora B kinase AIR-2. Tousled kinase (Tlk) expression and activity have been linked to ongoing DNA replication, and Tlk can phosphorylate the chromatin assembly factor Asf. Here, we show that TLK-1 is phosphorylated by AIR-2 during prophase/prometaphase and that phosphorylation increases TLK-1 kinase activity in vitro. Phosphorylated TLK-1 increases AIR-2 kinase activity in a manner that is independent of TLK-1 kinase activity but depends on the presence of ICP-1/INCENP. In vivo, TLK-1 and AIR-2 cooperate to ensure proper mitotic chromosome segregation. CONCLUSIONS: The C. elegans Tousled kinase TLK-1 is a substrate and activator of the Aurora B kinase AIR-2. These results suggest that Tousled kinases have a previously unrecognized role in mitosis and that Aurora B associates with discrete regulatory complexes that may impart distinct substrate specificities and functions to the Aurora B kinase.     

Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr⁷²) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr⁷², we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr⁷² does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr⁷² regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5.     

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.     

Loading of the 3F3/2 antigen onto kinetochores is dependent on the ordered assembly of the spindle checkpoint proteins

Accurate chromosome segregation is controlled by the spindle checkpoint, which responds to the lack of microtubule-kinetochore attachment or of tension across sister kinetochores through phosphorylation of kinetochore proteins by the Mps1, Bub1, BubR1, Aurora B, and Plk1/Plx1 kinases. The presence of the 3F3/2 phosphoepitope on kinetochores, generated by Plk1/Plx1-mediated phosphorylation of an unknown protein, correlates with the activation of the tension-sensitive checkpoint pathway. Using immunodepletion approach and a rephosphorylation assay in Xenopus extracts, we report here that not only the formation of the 3F3/2 phosphoepitope is dependent on the checkpoint activation but also the loading of the 3F3/2 substrate to kinetochores requires the prior assembly of Mps1, Bub1 and BubR1 onto kinetochores. Interestingly, generation of the 3F3/2 epitope in checkpoint extracts requires the kinase activities of Mps1 and Bub1 but not that of BubR1. Furthermore, we demonstrate that checkpoint proteins in Xenopus extracts are assembled onto kinetochores in a highly ordered pathway consisting of three steps. Mps1 and Bub1 are loaded first, and BubR1 and Plx1 second, followed by Mad1 and Mad2. The characterization of this ordered assembly pathway provides a framework for the biochemical mechanism of the checkpoint signaling and will aid in the eventual identification of the 3F3/2 substrate.     

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.     

Phosphorylation of multifunctional nucleolar protein nucleophosmin (NPM1) by aurora kinase B is critical for mitotic progression

The functional association of NPM1 with Aurora kinases is well documented. Surprisingly, although NPM1 is a well characterized phosphoprotein, it is unknown whether it is a substrate of Aurora kinases. We have found that Aurora kinases A and B can phosphorylate NPM1 at a single serine residue, Ser125, in vitro and in vivo. Phosphorylated-S125-NPM1 (pS125-NPM1) localizes to the midbody region during late cytokinesis where it colocalizes with Aurora B. The overexpression of mutant (S125A) NPM1 resulted in the deregulation of centrosome duplication and mitotic defects possibly due to cytokinesis failure. These data suggest that Aurora kinase B-mediated phosphorylation of NPM1 plays a critical role during mitosis, which could have wider implications in oncogenesis.     

Phosphorylation at serine 331 is required for Aurora B activation

Aurora B kinase activity is required for successful cell division. In this paper, we show that Aurora B is phosphorylated at serine 331 (Ser331) during mitosis and that phosphorylated Aurora B localizes to kinetochores in prometaphase cells. Chk1 kinase is essential for Ser331 phosphorylation during unperturbed prometaphase or during spindle disruption by taxol but not nocodazole. Phosphorylation at Ser331 is required for optimal phosphorylation of INCENP at TSS residues, for Survivin association with the chromosomal passenger complex, and for complete Aurora B activation, but it is dispensable for Aurora B localization to centromeres, for autophosphorylation at threonine 232, and for association with INCENP. Overexpression of Aurora B(S331A), in which Ser331 is mutated to alanine, results in spontaneous chromosome missegregation, cell multinucleation, unstable binding of BubR1 to kinetochores, and impaired mitotic delay in the presence of taxol. We propose that Chk1 phosphorylates Aurora B at Ser331 to fully induce Aurora B kinase activity. These results indicate that phosphorylation at Ser331 is an essential mechanism for Aurora B activation.