Aurora A kinase activity is required to maintain an active spindle assembly checkpoint during prometaphase

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.

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The mitosis-regulating and protein-protein interaction activities of Astrin are controlled by Aurora-A-induced phosphorylation

AJP Cell Physiology ◽

10.1152/ajpcell.00164.2014 ◽

2014 ◽

Vol 307 (5) ◽

pp. C466-C478 ◽

Cited By ~ 10

Author(s):

Shao-Chih Chiu ◽

Jo-Mei Maureen Chen ◽

Tong-You Wade Wei ◽

Tai-Shan Cheng ◽

Ya-Hui Candice Wang ◽

...

Keyword(s):

Regulatory Networks ◽

Spindle Assembly Checkpoint ◽

Morphological Changes ◽

Spindle Assembly ◽

Aurora A ◽

Protein Protein Interaction ◽

Daughter Cells ◽

Sister Chromatids ◽

Complicated Process ◽

Spindle Stability

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser115. The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

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A Novel Cell-Based, High-Content Assay for Phosphorylation of Lats2 by Aurora A

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111413923 ◽

2011 ◽

Vol 16 (8) ◽

pp. 925-931 ◽

Cited By ~ 7

Author(s):

Amy Emery ◽

David A. Sorrell ◽

Stacy Lawrence ◽

Emma Easthope ◽

Mark Stockdale ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Kinase Activity ◽

High Content Screening ◽

Aurora A Kinase ◽

Aurora A ◽

Assay Performance ◽

A Cell ◽

Significant Addition ◽

Specific Inhibitors

Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2. Using antibodies directed against phosphorylated forms of Aurora A (pThr288), P-TACC3 (pSer558), and P-Lats2 (pSer83), the authors investigate their suitability in parallel for development of a cell-based assay using several reference Aurora inhibitors: MLN8054, VX680, and AZD1152-HQPA. They validate a combined assay of target-specific phosphorylation of Lats2 at the centrosome and an increase in mitotic index as a measure of Aurora A activity. The assay is both sensitive and robust and has acceptable assay performance for high-throughput screening or potency estimation from concentration–response assays. It has the advantage that it can be carried out using a commercially available monoclonal antibody against phospho-Lats2 and the widely available Cellomics ArrayScan HCS reader and thus represents a significant addition to the tools available for the identification of Aurora A specific inhibitors.

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Aurora A kinase activity is required for localization of TACC3/ch-TOG/clathrin inter-microtubule bridges

Communicative & Integrative Biology ◽

10.4161/cib.15250 ◽

2011 ◽

Vol 4 (4) ◽

pp. 409-412 ◽

Cited By ~ 23

Author(s):

Liam P. Cheeseman ◽

Daniel G. Booth ◽

Fiona E. Hood ◽

Ian A. Prior ◽

Stephen J. Royle

Keyword(s):

Kinase Activity ◽

Aurora A Kinase ◽

Aurora A

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Aurora-A promotes the establishment of spindle assembly checkpoint by priming the Haspin-Aurora-B feedback loop in late G2 phase

Cell Discovery ◽

10.1038/celldisc.2016.49 ◽

2017 ◽

Vol 3 (1) ◽

Cited By ~ 11

Author(s):

Fazhi Yu ◽

Ya Jiang ◽

Lucy Lu ◽

Mimi Cao ◽

Yulong Qiao ◽

...

Keyword(s):

Feedback Loop ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Aurora B ◽

Aurora A ◽

G2 Phase

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Nucleophosmin/B23 activates Aurora A at the centrosome through phosphorylation of serine 89

The Journal of Cell Biology ◽

10.1083/jcb.201107134 ◽

2012 ◽

Vol 197 (1) ◽

pp. 19-26 ◽

Cited By ~ 38

Author(s):

David Reboutier ◽

Marie-Bérengère Troadec ◽

Jean-Yves Cremet ◽

Kenji Fukasawa ◽

Claude Prigent

Keyword(s):

Protein Kinase ◽

Ribonucleic Acid ◽

Kinase Activity ◽

Spindle Assembly ◽

Aurora A ◽

Local Loss ◽

Cellular Processes ◽

G2 Phase ◽

Strong Activator

Aurora A (AurA) is a major mitotic protein kinase involved in centrosome maturation and spindle assembly. Nucleophosmin/B23 (NPM) is a pleiotropic nucleolar protein involved in a variety of cellular processes including centrosome maturation. In the present study, we report that NPM is a strong activator of AurA kinase activity. NPM and AurA coimmunoprecipitate and colocalize to centrosomes in G2 phase, where AurA becomes active. In contrast with previously characterized AurA activators, NPM does not trigger autophosphorylation of AurA on threonine 288. NPM induces phosphorylation of AurA on serine 89, and this phosphorylation is necessary for activation of AurA. These data were confirmed in vivo, as depletion of NPM by ribonucleic acid interference eliminated phosphorylation of CDC25B on S353 at the centrosome, indicating a local loss of AurA activity. Our data demonstrate that NPM is a strong activator of AurA kinase activity at the centrosome and support a novel mechanism of activation for AurA.

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Searching for Biomarkers of Aurora-A Kinase Activity: Identification of in Vitro Substrates through a Modified KESTREL Approach

Journal of Proteome Research ◽

10.1021/pr050018e ◽

2005 ◽

Vol 4 (4) ◽

pp. 1296-1303 ◽

Cited By ~ 23

Author(s):

Sonia Troiani ◽

Mauro Uggeri ◽

Jürgen Moll ◽

Antonella Isacchi ◽

Henryk M. Kalisz ◽

...

Keyword(s):

Kinase Activity ◽

Aurora A Kinase ◽

Aurora A

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Inhibitors of the Bub1 spindle assembly checkpoint kinase: synthesis of BAY-320 and comparison with 2OH-BNPP1

Royal Society Open Science ◽

10.1098/rsos.210854 ◽

2021 ◽

Vol 8 (12) ◽

Author(s):

Ilma Amalina ◽

Ailsa Bennett ◽

Helen Whalley ◽

David Perera ◽

Joanne C. McGrail ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Kinase Activity ◽

Spindle Assembly ◽

Minor Role ◽

Serine Threonine Kinase ◽

Chromosome Alignment ◽

A Cell ◽

A Minor ◽

Target Effects

Bub1 is a serine/threonine kinase proposed to function centrally in mitotic chromosome alignment and the spindle assembly checkpoint (SAC); however, its role remains controversial. Although it is well documented that Bub1 phosphorylation of Histone 2A at T120 (H2ApT120) recruits Sgo1/2 to kinetochores, the requirement of its kinase activity for chromosome alignment and the SAC is debated. As small-molecule inhibitors are invaluable tools for investigating kinase function, we evaluated two potential Bub1 inhibitors: 2OH-BNPPI and BAY-320. After confirming that both inhibit Bub1 in vitro , we developed a cell-based assay for Bub1 inhibition. We overexpressed a fusion of Histone 2B and Bub1 kinase region, tethering it in proximity to H2A to generate a strong ectopic H2ApT120 signal along chromosome arms. Ectopic signal was effectively inhibited by BAY-320, but not 2OH-BNPP1 at concentrations tested. In addition, only BAY-320 was able to inhibit endogenous Bub1-mediated Sgo1 localization. Preliminary experiments using BAY-320 suggest a minor role for Bub1 kinase activity in chromosome alignment and the SAC; however, BAY-320 may exhibit off-target effects at the concentration required. Thus, 2OH-BNPP1 may not be an effective Bub1 inhibitor in cellulo , and while BAY-320 can inhibit Bub1 in cells, off-target effects highlight the need for improved Bub1 inhibitors.

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Inhibitors of the Bub1 spindle assembly checkpoint kinase: Synthesis of BAY-320 and comparison with 2OH-BNPP1

10.1101/2020.07.02.184010 ◽

2020 ◽

Author(s):

Ilma Amalina ◽

Ailsa Bennett ◽

Helen Whalley ◽

David Perera ◽

Joanne C. McGrail ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Kinase Activity ◽

Spindle Assembly ◽

Minor Role ◽

Chromosome Alignment ◽

High Concentrations ◽

A Minor ◽

Target Effects

SummaryBub1 is a serine/threonine kinase proposed to function centrally in both mitotic chromosome alignment and the spindle assembly checkpoint (SAC), however its role remains controversial. Although it is well documented that Bub1 phosphorylation of Histone 2A at T120 (H2ApT120) recruits Sgo1/2 to kinetochores, the requirement of its kinase activity for chromosome alignment and the SAC is debated. As small-molecule inhibitors can be invaluable tools for investigation of kinase function, we decided to evaluate the relative potential of two agents (2OH-BNPPI and BAY-320) as Bub1 inhibitors. After confirming that both agents inhibit Bub1 in vitro, we developed a cell based-assay to specifically measure Bub1 inhibition in vivo. For this assay we overexpressed a fusion of Histone 2B and the Bub1 kinase region (Bub1C) tethering it in close proximity to H2A, which generated a strong ectopic H2ApT120 signal along chromosome arms. The ectopic signal generated from Bub1C activity was effectively inhibited by BAY-320, but not 2OH-BNPP1. In addition, only BAY-320 was able to inhibit endogenous Bub1-mediated Sgo1 localisation. Preliminary experiments using BAY-320 suggested a minor role for Bub1 kinase activity in chromosome alignment and the SAC, however results suggest that BAY-320 may exhibit off-target effects at the concentration required to demonstrate these outcomes. In conclusion, 2OH-BNPP1 may not be an effective Bub1 inhibitor in vivo, and while BAY-320 is able to inhibit Bub1 in vivo, the high concentrations required and potential for off-target effects highlight the ongoing need for improved Bub1 inhibitors.

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Aurora A kinase activation: Different means to different ends

The Journal of Cell Biology ◽

10.1083/jcb.202106128 ◽

2021 ◽

Vol 220 (9) ◽

Author(s):

Nicolas Tavernier ◽

Frank Sicheri ◽

Lionel Pintard

Keyword(s):

Spindle Assembly ◽

Aurora A Kinase ◽

Aurora A ◽

Serine Threonine Kinase ◽

Kinase Activation ◽

Mitotic Entry ◽

Threonine Kinase ◽

Molecular Studies ◽

Signaling Events ◽

Cellular Context

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.

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