Glycogen synthase kinase 3 β activity is required for hBora/Aurora A-mediated mitotic entry

The synthesis and degradation of hBora is important for the regulation of mitotic entry and exist. In G₂ phase, hBora can complex with Aurora A to activate Plk1 and control mitotic entry. However, whether the post-translational modification of hBora is relevant to the mitotic entry still unclear. Here, we used the LC-MS/MS phosphopeptide mapping assay to identify 13 in vivo hBora phosphorylation sites and characterized that GSK3β can interact with hBora and phosphorylate hBora at Ser274 and Ser278. Pharmacological inhibitors of GSK3β reduced the retarded migrating band of hBora in cells and diminished the phosphorylation of hBora by in vitro kinase assay. Moreover, as well as in GSK3β activity-inhibited cells, specific knockdown of GSK3β by shRNA and S274A/S278 hBora mutant-expressing cells also exhibited the reduced Plk1 activation and a delay in mitotic entry. It suggests that GSK3β activity is required for hBora-mediated mitotic entry through Ser274 and Ser278 phosphorylation.     

Aurora A phosphorylation of WD40-repeat protein 62 in mitotic spindle regulation

Mitotic spindle organization is regulated by centrosomal kinases that potentiate recruitment of spindle-associated proteins required for normal mitotic progress including the microcephaly protein WD40-repeat protein 62 (WDR62). WDR62 functions underlie normal brain development as autosomal recessive mutations and wdr62 loss cause microcephaly. Here we investigate the signaling interactions between WDR62 and the mitotic kinase Aurora A (AURKA) that has been recently shown to cooperate to control brain size in mice. The spindle recruitment of WDR62 is closely correlated with increased levels of AURKA following mitotic entry. We showed that depletion of TPX2 attenuated WDR62 localization at spindle poles indicating that TPX2 co-activation of AURKA is required to recruit WDR62 to the spindle. We demonstrated that AURKA activity contributed to the mitotic phosphorylation of WDR62 residues Ser49 and Thr50 and phosphorylation of WDR62 N-terminal residues was required for spindle organization and metaphase chromosome alignment. Our analysis of several MCPH-associated WDR62 mutants (V65M, R438H and V1314RfsX18) that are mislocalized in mitosis revealed that their interactions and phosphorylation by AURKA was substantially reduced consistent with the notion that AURKA is a key determinant of WDR62 spindle recruitment. Thus, our study highlights the role of AURKA signaling in the spatiotemporal control of WDR62 at spindle poles where it maintains spindle organization.     

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.     

Aurora kinase is required for chromosome segregation in tobacco BY-2 cells

Post-translational modifications of core histone tails play crucial roles in chromatin structure and function. Although phosphorylation of Ser10 and Ser28 (H3S10ph and H3S28ph) of histone H3 is ubiquitous among eukaryotes, the phosphorylation mechanism during the cell cycle remains unclear. In the present study, H3S10ph and H3S28ph in tobacco BY-2 cells were observed in the pericentromeric regions during mitosis. Moreover, the Aurora kinase inhibitor Hesperadin inhibited the kinase activity of Arabidopsis thaliana Aurora kinase 3 (AtAUR3) in phosphorylating both Ser10 and Ser28 of histone H3 in vitro. Consistently, Hesperadin inhibited both H3S10ph and H3S28ph during mitosis in BY-2 cells. These results indicate that plant Aurora kinases phosphorylate not only Ser10, but also Ser28 of histone H3 in vivo. Hesperadin treatment increased the ratio of metaphase cells, while the ratio of anaphase/telophase cells decreased, although the mitotic index was not affected in Hesperadin-treated cells. These results suggest that Hesperadin induces delayed transition from metaphase to anaphase, and early exit from mitosis after chromosome segregation. In addition, micronuclei were observed frequently and lagging chromosomes, caused by the delay and failure of sister chromatid separation, were observed at anaphase and telophase in Hesperadin-treated BY-2 cells. The data obtained here suggest that plant Aurora kinases and H3S10ph/H3S28ph may have a role in chromosome segregation and metaphase/anaphase transition.     

VX-680 Inhibits Aurora A and Aurora B Kinase Activity in Human Cells

VX-680, also known as MK-0457, is a member of a diverse group of small molecules that inhibit the Aurora kinases, and has shown significant potential as an anti-cancer agent. In keeping with many protein kinase inhibitors, this compound is not a monospecific agent, and its cellular specificity remains largely unknown. In cells, VX-680 blocks mitotic Histone H3 phosphorylation and induces polyploidy and apoptosis, consistent with inhibition of the mitotic protein kinase Aurora B. In this study, we have investigated the effects of VX-680 in proliferating human cancer cells, and demonstrate that it blocks the phosphorylation and activation of both Aurora A and B. Additionally, VX-680 suppresses the phosphorylation of specific substrates of each enzyme, including the Aurora A target TACC3 on Ser558. Exposure to VX-680 induces a monopolar spindle phenotype, delays mitotic progression and rapidly overrides the spindle assembly checkpoint in the presence of spindle poisons. VX-680 also exhibits potent cytotoxicity when compared to the well documented Aurora B inhibitor ZM447439. Taken together, these data identify Aurora A and Aurora B as dual intracellular targets of VX-680.     

Aurora kinase A is essential for meiosis in mouse oocytes

The Aurora protein kinases are well-established regulators of spindle building and chromosome segregation in mitotic and meiotic cells. In mouse oocytes, there is significant Aurora kinase A (AURKA) compensatory abilities when the other Aurora kinase homologs are deleted. Whether the other homologs, AURKB or AURKC can compensate for loss of AURKA is not known. Using a conditional mouse oocyte knockout model, we demonstrate that this compensation is not reciprocal because female oocyte-specific knockout mice are sterile, and their oocytes fail to complete meiosis I. In determining AURKA-specific functions, we demonstrate that its first meiotic requirement is to activate Polo-like kinase 1 at acentriolar microtubule organizing centers (aMTOCs; meiotic spindle poles). This activation induces fragmentation of the aMTOCs, a step essential for building a bipolar spindle. We also show that AURKA is required for regulating localization of TACC3, another protein required for spindle building. We conclude that AURKA has multiple functions essential to completing MI that are distinct from AURKB and AURKC.     

Aurora A kinase activation: Different means to different ends

Aurora A is a serine/threonine kinase essential for mitotic entry and spindle assembly. Recent molecular studies have revealed the existence of multiple, distinct mechanisms of Aurora A activation, each occurring at specific subcellular locations, optimized for cellular context, and primed by signaling events including phosphorylation and oxidation.

IntroductionDuring mitosis, almost 30% of the proteome is modified by the transfer of phosphate to serine, threonine, or tyrosine residues. These phosphorylation events are responsible for the profound transformation of cellular architecture and physiology that occurs as cells progress through mitosis. Pivotal protein kinases responsible for the massive increase in protein phosphorylation as cells transit into mitosis include Aurora A kinase (AURKA), Aurora B kinase, Polo-like kinase 1 (Plk1), and Cyclin-dependent kinase 1 (Cdk1)/Cyclin A/B complexes. Their precise and coordinated activation critically defines the G2-M transition.Overexpression and aberrant activation of AURKA have been functionally linked to oncogenic transformation through centrosome amplification, aneuploidy, and chromosomal instability. Beyond its pivotal role in mitotic cell division, AURKA has numerous nonmitotic functions in tumorigenesis. AURKA thus represents a critical “druggable target” in cancer, controlling key oncogenic pathways associated with drug resistance and poor patient outcome.AURKA activation is unexpectedly complex, and a number of different mechanisms have been described, including autophosphorylation of its activation segment and binding to a variety of allosteric modulators, which recruit and locally activate AURKA at specific subcellular localizations (Fig. 1). Recent findings show that these allosteric modulators activate AURKA through surprisingly distinct mechanisms, each acting at different subcellular locations to trigger a unique event in response to different upstream signals. We summarize current views of these activation mechanisms and speculate on the reasons underlying this complexity.Open in a separate windowFigure 1.Model of AURKA activation during cell cycle progression. Left panel: AURKA is activated during the G2/M transition by binding to Bora phosphorylated on its M3 motif by Cyclin A-Cdk1 (CycA-Cdk1). Phospho-Bora (pBora) binds unphosphorylated inactive AURKA (in gray, N and C denote the N-lobe and the C-lobe, respectively) via its M1, M2 (dark blue), and phospho-M3 (green) motifs. Binding of phospho-Bora turns on the catalytic activity of AURKA (red) by substituting in trans the phosphoregulatory site on Thr288, leading to mitotic entry. P denotes phosphate. Middle panel: AURKA is activated at centrosomes via Cep192-dependent oligomerization and oxidation of Cys290 by ROS. NEDB, nuclear envelope breakdown. Right panel: AURKA is activated at spindle microtubules by Tpx2 binding through M1 and M2 motifs and by autophosphorylation at Thr288.Autophosphorylation of the activation segment and binding to the allosteric regulator Targeting protein for Xklp2 (Tpx2) synergize to locally activate AURKA at microtubulesIn eukaryotes, the transfer of phosphate from ATP to protein substrates is mediated by the protein kinase domain, a bilobal catalytic entity. The protein kinase domain possesses a complicated structure, with many flexible parts but a highly restricted catalytic mechanism (i.e., there is only one way to transfer phosphate). This affords great opportunity for the diversification of how each kinase turns on and off (Endicott et al., 2012). Like the majority of eukaryotic protein kinases, AURKA is regulated by phosphorylation of a conserved residue, Thr288, within a flexible element of the kinase domain termed the activation segment. This event leads to a reorganization of the active site that is required, but not sufficient, for full catalytic activation. This is in sharp contrast to many other protein kinases, where phosphorylation of the activation segment is sufficient for maximal catalytic activation. Similar to the closely related AGC family kinases, AURKA has evolved a dependency for its full activation on the binding of an allosteric modulator to its smaller N-terminal kinase lobe (Leroux et al., 2018). This event positions or stabilizes structural elements in the kinase active site that are not sufficiently aligned by activation segment phosphorylation alone. For most AGC family kinases, such as the exemplars PKA and AKT, the allosteric modulator represents a linear peptide sequence contained within the protein kinase itself, but distal to the protein kinase domain. In contrast, in the case of AURKA, the allosteric modulator is presented by an entirely separate protein.The best characterized allosteric modulator of AURKA is the microtubule-binding protein Txp2. Upon nuclear envelope breakdown, Tpx2 is released by RAN-GTP from importins, which then allows it to concurrently recruit and activate AURKA at microtubules to promote mitotic spindle assembly. Tpx2 uses its first N-terminal 43 amino acids to activate AURKA, in a manner synergistic with activation segment phosphorylation, by binding across the N-lobe of the AURKA kinase domain (Fig. 1). Notably, in the absence of Tpx2 binding and activation segment phosphorylation, AURKA retains marginal but detectable protein kinase activity. Binding of Tpx2 alone boosts AURKA catalytic function modestly (15-fold) while autophosphorylation alone boosts catalytic function substantially (157-fold). However, the action of both events translates into a 448-fold enhancement in activity relative to the fully repressed state (Dodson and Bayliss, 2012). This coordination of maximal activation with the recruitment of AURKA to microtubules may serve a double duty to minimize spurious phosphorylation of proteins elsewhere in the cell.Autophosphorylation and binding to Tpx2 trigger conformational changes in AURKA that are sufficiently large to be probed by time-resolved fluorescence energy transfer approaches (Ruff et al., 2018). Time-resolved fluorescence energy transfer revealed that the activation segment of AURKA adopts a wide range of conformations in solution. Notably, binding of Tpx2 to AURKA locks an inward conformation of a catalytic element termed the DFG motif (the Asp in the Asp-Phe-Gly motif coordinates a magnesium ion required for ATP binding) by rigidifying an inherently flexible helix αC (Ruff et al., 2018). In contrast, Thr288 phosphorylation promotes a large conformational change in the activation segment that enables the binding of peptide substrates. Both Tpx2 binding and autophosphorylation are required for AURKA function at microtubules. This transient “doubly activated” form of AURKA is not detectable at spindle microtubules in normal cells due to the action of the AURKA-directed protein phosphatase 6 (PP6), which specifically dephosphorylates Tpx2-bound AURKA on the activation segment.Phospho-Bora activates unphosphorylated AURKA in the cytoplasm to trigger mitotic entryIn a manner thematically similar to how Tpx2 binding and AURKA autophosphorylation synergize to activate AURKA at microtubules, recent work revealed that a phosphorylated form of Bora activates cytoplasmic AURKA during mitotic commitment (Tavernier et al., 2021).Commitment to mitosis is tightly coordinated with DNA replication to preserve genome integrity. Commitment is achieved by a tightly choreographed biochemical tug-of-war between mitotic kinases and phosphatases (PPases). To this end, AURKA activates Plk1 by phosphorylating its activation segment at Thr210. In turn, Plk1 promotes activation of the Cdk1/Cyclin B complex by phosphorylating both negative and positive regulators of Cdk1 to trigger mitotic entry. As AURKA lies at the top of this mitotic kinase cascade, the key question that arises is how is AURKA initially activated in G2?As noted above, AURKA can autophosphorylate its own activation segment at Thr288, but this form of the enzyme is rapidly dephosphorylated by counteracting PPases in G2. As dephosphorylation maintains AURKA in an inactive state, how then does AURKA overcome the repressive effect of PPases to activate Plk1?AURKA activation during mitotic commitment is critically dependent on the evolutionarily conserved protein Bora, following its own phosphorylation on a key regulatory site on Ser112. This event is essential for the phosphorylation of Plk1 on Thr210 by AURKA in vitro and for timely mitotic entry in vivo, both in Xenopus egg extracts (Vigneron et al., 2018) and in human cells (Tavernier et al., 2021). Remarkably, phospho-Bora binds to and potently activates AURKA lacking phosphorylation of its activation segment, suggesting the possibility that the phosphate on S112 of Bora may physically and/or functionally substitute for the phosphorylated activation segment on AURKA.Dissection of how Bora binds AURKA revealed at least two motifs in Bora, denoted M1 and M2, with weak similarity to AURKA-binding elements in Tpx2^1–43. Both motifs are required for the binding and activating function of Bora on AURKA, and notably, the essential Ser112 phospho-regulatory site (in the sequence Pro-Ser-Pro, denoted motif M3) lies immediately C-terminal to binding motif M2. By analogy to the mechanism of action of Tpx2, Bora motif M1 likely binds in an extended manner parallel to the top surface of helix αC, whereas motif M2 adopts a helical conformation and binds parallel to the bottom surface of helix αC. As Bora motif M3 is immediately adjacent to motif M2, this binding mode would orient the Ser112 phospho-moiety of motif M3 in close proximity to a constellation of positively charged residues that normally engage the phosphate moiety of the phosphorylated activation segment of AURKA (Fig. 1; Tavernier et al., 2021). This mode of action elegantly allows phospho-Bora to allosterically activate AURKA during mitotic commitment, when AURKA itself is catalytically repressed by dephosphorylation. The precise atomic details of how phospho-Bora binds and activates AURKA and whether other protein kinases use analogous mechanisms for activation remain to be determined.Since phosphorylation of Bora Ser112 is essential for its ability to activate AURKA and commit cells to mitosis, the upstream kinase responsible for this regulatory event represents a critical component of the AURKA activation puzzle. This function is performed by Cyclin A–Cdk1, which is active in S-G2 and known to promote mitotic entry. Consistent with this model, Bora phosphorylated on S112 is sufficient to promote mitotic commitment in Xenopus egg extracts depleted of Cyclin A (Vigneron et al., 2018). In human cells, Cyclin A–Cdk1 is confined to the nucleus during S phase, but at the S/G2 transition it is abruptly exported to the cytoplasm, allowing it to phosphorylate Bora (Silva Cascales et al., 2021). As such, Bora acts as a bridge linking Cyclin A–Cdk1 activity to the activation of the mitotic kinase cascade. Why phospho-Bora can persist under conditions that disfavor AURKA activation by autophosphorylation remains an open question worthy of further investigation. Possibilities include that the phospho-Ser112 residue is a suboptimal PPase substrate or that it is protected from dephosphorylation by cis-activating factors.Redox regulation of AURKA during mitosisRecent work indicates that AURKA activity is also regulated by oxidative signaling with both stimulatory and inhibitory outcomes. While autophosphorylation of AURKA on Thr288 is largely neutralized in the cytoplasm and at spindle microtubules by counteracting PPases, it is readily detected at centrosomes. Centrosomal AURKA autoactivation is stimulated as a consequence of Cep192-mediated oligomerization and AURKA autophosphorylation, but the underlying mechanism was poorly understood. New studies reveal that oxidative modification of a conserved cysteine residue, Cys290, located in the activation segment of the kinase domain promotes AURKA autophosphorylation during mitosis when AURKA is oligomerized.While biochemical studies using purified proteins in the absence of an oligomerizing agent revealed that oxidative modification of Cys290 inhibited AURKA kinase activity (Byrne et al., 2020; Tsuchiya et al., 2020), cell treatment with oxidizing agents such as H₂0₂ increased AURKA phosphorylation on Thr288 (Wang et al., 2017; Tsuchiya et al., 2020). This increase in Thr288 phosphorylation was accompanied by dimerization of AURKA in a manner sensitive to reducing agents such as DTT. This result hinted that disulfide bond formation between AURKA monomers might be involved in promoting AURKA trans-autophosphorylation. Consistent with this hypothesis, a crystal structure of an AURKA kinase domain obtained under disulfide bond–promoting conditions revealed a face-to-face dimer orientation of the kinase domain stabilized by a Cys290–Cys290 disulfide bond (Lim et al., 2020). In this configuration, the active site of the AURKA kinase domain adopts a productive conformation predicted to support substrate phosphorylation. Given the inherent flexibility of the activation segment itself, this face-to-face configuration of the kinase domain was also predicted to support AURKA trans-autophosphorylation on Thr288. Follow-up biochemical studies proved that the Cys290–Cys290 disulfide–linked configuration of the AURKA kinase domain is indeed compatible with trans-autophosphorylation on Thr288.An interesting feature of the Cys290-dependent activation mechanism of AURKA is the requirement for Cep192. Presumably, the ability of Cep192 to recruit and oligomerize AURKA favors the formation of the Cys290–Cys290 disulfide bond between kinase domains (Fig. 1). In support of this model, oxidation-induced Cys290–Cys290 disulfide cross-linking of AURKA could also be recapitulated in Xenopus extracts by the addition of bivalent antibodies directed at AURKA.At subcellular locations beyond centrosomes, where AURKA is not dimeric, oxidation of the activation segment would be expected to have an opposite effect on protein kinase activity. For instance, a crystal structure of monomeric AURKA covalently bound to Coenzyme A (CoAlation), a major regulator of cellular metabolism that contains both nucleotide and thiol moieties, revealed that AURKA CoAlation robustly inhibits kinase activity (Tsuchiya et al., 2020) through an ATP-competitive mechanism. Competitive binding is achieved by the nucleotide moiety of Coenzyme A engaging the nucleotide-binding pocket of ATP, while the reactive pantetheine thiol moiety forms a disulfide bond with Cys290. This dual anchoring of Coenzyme A to AURKA imparts not only affinity but also specificity toward kinase inhibition. Supporting the possibility that Coenzyme A can exert a potent inhibitory effect on AURKA under physiological conditions, microinjection of CoA into mouse oocytes caused abnormal spindles and chromosome misalignment, phenotypes typically observed upon AURKA inactivation.Interestingly, when bound to AURKA, Tpx2 exerts a protective effect against inhibition by CoAlation. Given that Bora is predicted to bind AURKA similar to Tpx2 (Tavernier et al., 2021), this could allow Bora to also protect AURKA from inhibition by CoAlation. Together, these results suggest that each distinct cellular pool of AURKA will respond differently to oxidation signals.Reactive oxygen species (ROS) are emerging as important signaling molecules. ROS and oxidative stress have been shown to increase during G2 and M phases in an otherwise unperturbed asynchronous cell cycle (Patterson et al., 2019), suggesting that oxidative modification of biomolecules, including AURKA, might regulate mitotic progression. Likewise, H₂O₂ locally released by mitochondria, where a pool of AURKA has been recently shown to localize (Bertolin et al., 2018), is implicated in symmetry breaking and polarity establishment in early Caenorhabditis elegans embryos (De Henau et al., 2020). It will be particularly exciting to determine whether AURKA, which also plays a role in setting up embryo polarity, is regulated by redox signaling in this specific context.Concluding remarksAURKA is activated by a growing list of mechanisms, with each acting at specific stages of the cell cycle and subcellular location. The ability to monitor which specific mechanism is at play at any one time in vivo presents a particular challenge. The activation state of AURKA is often measured by the use of a phospho-specific antibody targeting the phosphorylated Thr288 epitope. However, this has limited effectiveness to detect AURKA activated by Tpx2 at spindle microtubules because of the transient nature of the phospho-Thr288 epitope at this location. Furthermore, in the case of cytoplasmic AURKA activation by Bora, phosphorylation at Thr288 is not required for kinase activation. A live fluorescence energy transfer sensor has been reported for the phosphorylation status of AURKA on Thr288 that detects conformational changes induced by Thr288 phosphorylation rather than the phosphorylation motif itself (Bertolin et al., 2016). If the binding of Tpx2 to phosphorylated AURKA and the binding of phospho-Bora to dephosphorylated AURKA induces similar conformational changes to those induced by autophosphorylation, then this could represent a more generally applicable assay for monitoring the activation state of AURKA.Why is the activation of AURKA so complex? We speculate that the major reason for this complexity is related to kinase action at distinct times and in spatially distinct locations (Fig. 1). Bora acts in the cytoplasm before mitotic entry, and Cep192 acts at the centrosome before and likely after mitotic entry, whereas Tpx2 acts on spindle microtubules after mitotic entry. Thus, the allosteric regulators and their own upstream controllers (e.g., Cyclin A/Cdk1 for Bora) direct AURKA activity to execute distinct functions. In some ways, this complexity of activation represents a different solution than the one adopted by Plk1 or Protein Phosphatase 1 (PP1), which also acts at distinct time points and subcellular locations. Both Plk1 and PP1 employ docking motifs that are post-translationally controlled to dictate their time and sites of action, as well as their substrate specificity. As deeper insights are gained into the control of mitotic kinases and PPases, it will be intriguing to see what additional solutions have evolved to address the challenge of temporally and spatially restricted actions.We end by noting that the different mechanisms described above for AURKA activation are associated with specific pathologies. Doubly activated AURKA, with bound Tpx2 and Thr288 phosphorylation, is not detected on microtubules in normal cells due to the action of PP6. However, this form of AURKA is readily detected in melanoma cells bearing PP6 mutations, which gives rise to pathological chromosome instability and DNA damage (Hammond et al., 2013). Bora is overexpressed in multiple cancer types, including ovarian cancer, where it plays a pro-oncogenic role (Parrilla et al., 2020), and there is significant evidence for ROS signaling, which is involved in centrosomal AURKA activation by Cep192, contributing to a number of disease states. Finally, AURKA itself is overexpressed in numerous cancers associated with drug resistance and poor patient outcome. However, the clinical utility of AURKA inhibitors to date has been limited, likely because of essential roles of AURKA in multiple events in the cell cycle. We posit that the discovery that AURKA is activated through a variety of mechanisms to execute distinct events may afford opportunities to develop drugs targeting a subset of the biological functions of AURKA and hence enable more precise tuning of the therapeutic window.     

Aurora A is involved in central spindle assembly through phosphorylation of Ser 19 in P150Glued

Knowledge of Aurora A kinase functions is limited to premetaphase events, particularly centrosome maturation, G2/M transition, and mitotic spindle assembly. The involvement of Aurora A in events after metaphase has only been suggested because appropriate experiments are technically difficult. We report here the design of the first human Aurora A kinase (as-AurA) engineered by chemical genetics techniques. This kinase is fully functional biochemically and in cells, and is rapidly and specifically inhibited by the ATP analogue 1-Naphthyl-PP1 (1-Na-PP1). By treating cells exclusively expressing the as-AurA with 1-Na-PP1, we discovered that Aurora A is required for central spindle assembly in anaphase through phosphorylation of Ser 19 of P150Glued. This paper thus describes a new Aurora A function that takes place after the metaphase-to-anaphase transition and a new powerful tool to search for and study new Aurora A functions.     

A MEK-independent role for CRAF in mitosis and tumor progression

RAF kinases regulate cell proliferation and survival and can be dysregulated in tumors. The role of RAF in cell proliferation has been linked to its ability to activate mitogen-activated protein kinase kinase 1 (MEK) and mitogen-activated protein kinase 1 (ERK). Here we identify a MEK-independent role for RAF in tumor growth. Specifically, in mitotic cells, CRAF becomes phosphorylated on Ser338 and localizes to the mitotic spindle of proliferating tumor cells in vitro as well as in murine tumor models and in biopsies from individuals with cancer. Treatment of tumors with allosteric inhibitors, but not ATP-competitive RAF inhibitors, prevents CRAF phosphorylation on Ser338 and localization to the mitotic spindle and causes cell-cycle arrest at prometaphase. Furthermore, we identify phospho-Ser338 CRAF as a potential biomarker for tumor progression and a surrogate marker for allosteric RAF blockade. Mechanistically, CRAF, but not BRAF, associates with Aurora kinase A (Aurora-A) and Polo-like kinase 1 (Plk1) at the centrosomes and spindle poles during G2/M. Indeed, allosteric or genetic inhibition of phospho-Ser338 CRAF impairs Plk1 activation and accumulation at the kinetochores, causing prometaphase arrest, whereas a phospho-mimetic Ser338D CRAF mutant potentiates Plk1 activation, mitosis and tumor progression in mice. These findings show a previously undefined role for RAF in tumor progression beyond the RAF-MEK-ERK paradigm, opening new avenues for targeting RAF in cancer.     

RALA and RALBP1 regulate mitochondrial fission at mitosis

Mitochondria exist as dynamic interconnected networks that are maintained through a balance of fusion and fission. Equal distribution of mitochondria to daughter cells during mitosis requires fission. Mitotic mitochondrial fission depends on both the relocalization of the large GTPase DRP1 to the outer mitochondrial membrane and phosphorylation of Ser 616 on DRP1 by the mitotic kinase cyclin B-CDK1 (ref. 2). We now report that these processes are mediated by the small Ras-like GTPase RALA and its effector RALBP1 (also known as RLIP76, RLIP1 or RIP1; refs 3, 4). Specifically, the mitotic kinase Aurora A phosphorylates Ser 194 of RALA, relocalizing it to the mitochondria, where it concentrates RALBP1 and DRP1. Furthermore, RALBP1 is associated with cyclin B-CDK1 kinase activity that leads to phosphorylation of DRP1 on Ser 616. Disrupting either RALA or RALBP1 leads to a loss of mitochondrial fission at mitosis, improper segregation of mitochondria during cytokinesis and a decrease in ATP levels and cell number. Thus, the two mitotic kinases Aurora A and cyclin B-CDK1 converge on RALA and RALBP1 to promote mitochondrial fission, the appropriate distribution of mitochondria to daughter cells and ultimately proper mitochondrial function.     

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.     

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.     

Inhibitors of Histone Deacetylases Alter Kinetochore Assembly by Disrupting Pericentromeric Heterochromatin

The kinetochore, a multi-protein complex assembled on centromeric chromatin in mitosis, is essential for sister chromosome segregation. We show here that inhibition of histone deacetylation blocks mitotic progression at prometaphase in two human tumor cell lines by interfering with kinetochore assembly. Decreased amounts of hBUB1, CENP-F and the motor protein CENP-E were present on kinetochores of treated cells. These kinetochores failed to nucleate and inefficiently captured microtubules, resulting in activation of the mitotic checkpoint. Addition of histone deacetylase inhibitors prior to the end of S-phase resulted in decreased HP1-? on pericentromeric heterochromatin in S-phase and G2, decreased pericentromeric targeting of Aurora B kinase, resulting in decreased pre-mitotic phosphorylation of pericentromeric histone H3(S10) in G2, followed by assembly of deficient kinetochores in M-phase. HP1-?, Aurora B and the affected kinetochore proteins all were present at normal levels in treated cells; thus, effects of the inhibitors on mitotic progression do not seem to reflect changes in gene expression. In vitro kinase activity of Aurora B isolated from treated cells was unaffected. We propose that the increased presence in pericentromeric heterochromatin of histone H3 acetylated at K9 is responsible for the mitotic defects resulting from inhibition of histone deacetylation.     

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 by Aurora B kinase regulates caspase-2 activity and function

Mitotic catastrophe (MC) is an important oncosuppressive mechanism that serves to eliminate cells that become polyploid or aneuploid due to aberrant mitosis. Previous studies have demonstrated that the activation and catalytic function of caspase-2 are key steps in MC to trigger apoptosis and/or cell cycle arrest of mitotically defective cells. However, the molecular mechanisms that regulate caspase-2 activation and its function are unclear. Here, we identify six new phosphorylation sites in caspase-2 and show that a key mitotic kinase, Aurora B kinase (AURKB), phosphorylates caspase-2 at the highly conserved residue S384. We demonstrate that phosphorylation at S384 blocks caspase-2 catalytic activity and apoptosis function in response to mitotic insults, without affecting caspase-2 dimerisation. Moreover, molecular modelling suggests that phosphorylation at S384 may affect substrate binding by caspase-2. We propose that caspase-2 S384 phosphorylation by AURKB is a key mechanism that controls caspase-2 activation during mitosis.Subject terms: Proteases, Proteolysis     

Effects of stable knockdown of Aurora kinase A on proliferation, migration, chromosomal instability, and expression of focal adhesion kinase and matrix metalloproteinase-2 in HEp-2 cells

Overexpression of Aurora kinase A (AURKA) is frequently observed in various cancers, including laryngeal squamous cell carcinoma (LSCC). We investigated the effects of knockdown of AURKA on laryngeal cancer HEp-2 cells both in vitro and in vivo. A plasmid containing short hairpin (sh)RNA against AURKA was constructed and transfected into HEp-2. Measurements included the CCK-8 assay for viability and proliferation, flow cytometry for apoptosis and effects on the mitotic checkpoint, a trans-well assay for migration, immunofluorescence for assessment of genomic instability, and western blotting for protein expression. AURKA knockdown inhibited proliferation, migration, and colony formation in vitro and tumorigenicity in vivo. The knockdown induced the accumulation of cells in G2-M phase and eventual apoptosis. Knockdown of AURKA caused delayed entry into mitosis after treatment with nocodazole, reduced chromosomal instability, and decreased expression of focal adhesion kinase (FAK), phosphorylated FAK, and matrix metalloproteinase-2 (MMP-2), key regulators in cell adhesion and invasion. Knockdown of AURKA inhibits the growth and invasiveness of this LSCC cell line both in vitro and in vivo. These effects may partially result from the reduced expression of FAK and MMP-2. Knockdown of AURKA expression may represent a promising therapeutic strategy for the treatment of LSCC.     

Arsenic reverses glioblastoma resistance to mTOR-targeted therapies

Mitotic Aurora kinases are essential for accurate chromosome segregation during cell division. Forced over-expression of Aurora kinase results in centrosome amplification and multipolar spindles, causing aneuploidy, a hallmark of cancer. ZM447439 (ZM), an Aurora selective ATP-competitive inhibitor, interferes with the spindle integrity checkpoint and chromosome segregation. Here, we showed that inhibition of Aurora kinase by ZM reduced histone H3 phosphorylation at Ser10 in Hep2 carcinoma cells. Multipolar spindles were induced in these ZM-treated G2/M-arrested cells with accumulation of 4N/8N DNA, similar to cells with genetically suppressed Aur-B. Cells subsequently underwent apoptosis, as assessed by cleavage of critical apoptotic associated protein PARP. Hep2 cells formed a tumor-like cell mass in 3-dimensional matrix culture; inhibition of Aurora kinase by ZM either destructed the preformed cell mass or prevented its formation, by inducing apoptotic cell death as stained for cleaved caspase-3. Lastly, ZM inhibition of Aurora kinase was potently in association with decrease of Akt phosphorylation at Ser473 and its substrates GSK3&;alpha;/beta; phosphorylation at Ser21 and Ser9. Together, we demonstrated that Aurora kinase served as a potential molecular target of ZM for more selective therapeutic cancer treatment.     

Aurora Kinase A proximity map reveals centriolar satellites as regulators of its ciliary function

Aurora kinase A (AURKA) is a conserved kinase that plays crucial roles in numerous cellular processes. Although AURKA overexpression is frequent in human cancers, its pleiotropic functions and multifaceted regulation present challenges in its therapeutic targeting. Key to overcoming these challenges is to identify and characterize the full range of AURKA interactors, which are often weak and transient. Previous proteomic studies were limited in monitoring dynamic and non‐mitotic AURKA interactions. Here, we generate the proximity interactome of AURKA in asynchronous cells, which consists of 440 proteins involving multiple biological processes and cellular compartments. Importantly, AURKA has extensive proximate and physical interactions to centriolar satellites, key regulators of the primary cilium. Loss‐of‐function experiments identify satellites as negative regulators of AURKA activity, abundance, and localization in quiescent cells. Notably, loss of satellites activates AURKA at the basal body, decreases centrosomal IFT88 levels, and causes ciliogenesis defects. Collectively, our results provide a resource for dissecting spatiotemporal regulation of AURKA and uncover its proteostatic regulation by satellites as a new mechanism for its ciliary functions.