Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1

The ATR kinase controls cell cycle transitions and the DNA damage response. ATR activity is regulated through two ATR-activating proteins, ETAA1 and TOPBP1. To examine how each activator contributes to ATR signaling, we used quantitative mass spectrometry to identify changes in protein phosphorylation in ETAA1- or TOPBP1-deficient cells. We identified 724, 285, and 118 phosphosites to be regulated by TOPBP1, ETAA1, or both ATR activators, respectively. Gene ontology analysis of TOPBP1- and ETAA1-dependent phosphoproteins revealed TOPBP1 to be a primary ATR activator for replication stress, while ETAA1 regulates mitotic ATR signaling. Inactivation of ATR or ETAA1, but not TOPBP1, results in decreased Aurora B kinase activity during mitosis. Additionally, ATR activation by ETAA1 is required for proper chromosome alignment during metaphase and for a fully functional spindle assembly checkpoint response. Thus, we conclude that ETAA1 and TOPBP1 regulate distinct aspects of ATR signaling with ETAA1 having a dominant function in mitotic cells.

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Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e10-08-0673 ◽

2011 ◽

Vol 22 (9) ◽

pp. 1473-1485 ◽

Cited By ~ 29

Author(s):

Zuzana Storchová ◽

Justin S. Becker ◽

Nicolas Talarek ◽

Sandra Kögelsberger ◽

David Pellman

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Budding Yeast ◽

Spindle Pole ◽

Growth Defect ◽

Aurora B ◽

Mitotic Kinase ◽

Sister Chromatids ◽

Chromosome Alignment ◽

Chromosomal Passenger

The conserved mitotic kinase Bub1 performs multiple functions that are only partially characterized. Besides its role in the spindle assembly checkpoint and chromosome alignment, Bub1 is crucial for the kinetochore recruitment of multiple proteins, among them Sgo1. Both Bub1 and Sgo1 are dispensable for growth of haploid and diploid budding yeast, but they become essential in cells with higher ploidy. We find that overexpression of SGO1 partially corrects the chromosome segregation defect of bub1Δ haploid cells and restores viability to bub1Δ tetraploid cells. Using an unbiased high-copy suppressor screen, we identified two members of the chromosomal passenger complex (CPC), BIR1 (survivin) and SLI15 (INCENP, inner centromere protein), as suppressors of the growth defect of both bub1Δ and sgo1Δ tetraploids, suggesting that these mutants die due to defects in chromosome biorientation. Overexpression of BIR1 or SLI15 also complements the benomyl sensitivity of haploid bub1Δ and sgo1Δ cells. Mutants lacking SGO1 fail to biorient sister chromatids attached to the same spindle pole (syntelic attachment) after nocodazole treatment. Moreover, the sgo1Δ cells accumulate syntelic attachments in unperturbed mitoses, a defect that is partially corrected by BIR1 or SLI15 overexpression. We show that in budding yeast neither Bub1 nor Sgo1 is required for CPC localization or affects Aurora B activity. Instead we identify Sgo1 as a possible partner of Mps1, a mitotic kinase suggested to have an Aurora B–independent function in establishment of biorientation. We found that Sgo1 overexpression rescues defects caused by metaphase inactivation of Mps1 and that Mps1 is required for Sgo1 localization to the kinetochore. We propose that Bub1, Sgo1, and Mps1 facilitate chromosome biorientation independently of the Aurora B–mediated pathway at the budding yeast kinetochore and that both pathways are required for the efficient turnover of syntelic attachments.

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Combination Treatment of OSI-906 with Aurora B Inhibitor Reduces Cell Viability via Cyclin B1 Degradation-Induced Mitotic Slippage

International Journal of Molecular Sciences ◽

10.3390/ijms22115706 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5706

Author(s):

Yuki Ikeda ◽

Ryuji Yasutake ◽

Ryuzaburo Yuki ◽

Youhei Saito ◽

Yuji Nakayama

Keyword(s):

Cell Proliferation ◽

Cell Viability ◽

Combination Treatment ◽

Spindle Assembly Checkpoint ◽

Time Lapse ◽

Cyclin B1 ◽

Suppressive Effect ◽

Aurora B ◽

Mitotic Slippage ◽

Chromosome Alignment

Insulin-like growth factor 1 receptor (IGF1R), a receptor-type tyrosine kinase, transduces signals related to cell proliferation, survival, and differentiation. We recently reported that OSI-906, an IGF1R inhibitor, in combination with the Aurora B inhibitor ZM447439 suppresses cell proliferation. However, the mechanism underlying this suppressive effect is yet to be elucidated. In this study, we examined the effects of combination treatment with OSI-906 and ZM447439 on cell division, so as to understand how cell proliferation was suppressed. Morphological analysis showed that the combination treatment generated enlarged cells with aberrant nuclei, whereas neither OSI-906 nor ZM447439 treatment alone caused this morphological change. Flow cytometry analysis indicated that over-replicated cells were generated by the combination treatment, but not by the lone treatment with either inhibitors. Time-lapse imaging showed mitotic slippage following a severe delay in chromosome alignment and cytokinesis failure with furrow regression. Furthermore, in S-trityl-l-cysteine–treated cells, cyclin B1 was precociously degraded. These results suggest that the combination treatment caused severe defect in the chromosome alignment and spindle assembly checkpoint, which resulted in the generation of over-replicated cells. The generation of over-replicated cells with massive aneuploidy may be the cause of reduction of cell viability and cell death. This study provides new possibilities of cancer chemotherapy.

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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 B kinase controls the targeting of the Astrin–SKAP complex to bioriented kinetochores

The Journal of Cell Biology ◽

10.1083/jcb.201006129 ◽

2010 ◽

Vol 191 (2) ◽

pp. 269-280 ◽

Cited By ~ 80

Author(s):

Jens C. Schmidt ◽

Tomomi Kiyomitsu ◽

Tetsuya Hori ◽

Chelsea B. Backer ◽

Tatsuo Fukagawa ◽

...

Keyword(s):

Error Correction ◽

Spindle Assembly Checkpoint ◽

Multiple Roles ◽

Aurora B ◽

Dependent Manner ◽

Dynein Light Chain ◽

Chromosome Congression ◽

Chromosome Alignment ◽

Mitotic Delay ◽

Associated Proteins

During mitosis, kinetochores play multiple roles to generate interactions with microtubules, and direct chromosome congression, biorientation, error correction, and anaphase segregation. However, it is unclear what changes at the kinetochore facilitate these distinct activities. Here, we describe a complex of the spindle- and kinetochore-associated protein Astrin, the small kinetochore-associated protein (SKAP), and the dynein light chain LC8. Although most dynein-associated proteins localize to unaligned kinetochores in an Aurora B–dependent manner, Astrin, SKAP, and LC8 localization is antagonized by Aurora B such that they target exclusively to bioriented kinetochores. Astrin–SKAP-depleted cells fail to maintain proper chromosome alignment, resulting in a spindle assembly checkpoint–dependent mitotic delay. Consistent with a role in stabilizing bioriented attachments, Astrin and SKAP bind directly to microtubules and are required for CLASP localization to kinetochores. In total, our results suggest that tension-dependent Aurora B phosphorylation can act to control outer kinetochore composition to provide distinct activities to prometaphase and metaphase kinetochores.

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INCENP–aurora B interactions modulate kinase activity and chromosome passenger complex localization

The Journal of Cell Biology ◽

10.1083/jcb.200906053 ◽

2009 ◽

Vol 187 (5) ◽

pp. 637-653 ◽

Cited By ~ 51

Author(s):

Zhenjie Xu ◽

Hiromi Ogawa ◽

Paola Vagnarelli ◽

Jan H. Bergmann ◽

Damien F. Hudson ◽

...

Keyword(s):

Kinase Activity ◽

Aurora B ◽

Activity Levels ◽

Checkpoint Response ◽

C Terminus ◽

Chromosomal Passenger ◽

Spindle Midzone ◽

Low Levels ◽

Chromosome Passenger Complex

Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

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Cdc7 kinase stimulates Aurora B kinase in M-phase

Scientific Reports ◽

10.1038/s41598-019-54738-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Sayuri Ito ◽

Hidemasa Goto ◽

Kinue Kuniyasu ◽

Mayumi Shindo ◽

Masayuki Yamada ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Kinase Activity ◽

Phosphorylation Site ◽

Aurora B ◽

Aurora B Kinase ◽

Mitotic Kinase ◽

M Phase ◽

Phase Progression ◽

Autophosphorylation Site

AbstractThe conserved serine-threonine kinase, Cdc7, plays a crucial role in initiation of DNA replication by facilitating the assembly of an initiation complex. Cdc7 is expressed at a high level and exhibits significant kinase activity not only during S-phase but also during G2/M-phases. A conserved mitotic kinase, Aurora B, is activated during M-phase by association with INCENP, forming the chromosome passenger complex with Borealin and Survivin. We show that Cdc7 phosphorylates and stimulates Aurora B kinase activity in vitro. We identified threonine-236 as a critical phosphorylation site on Aurora B that could be a target of Cdc7 or could be an autophosphorylation site stimulated by Cdc7-mediated phosphorylation elsewhere. We found that threonines at both 232 (that has been identified as an autophosphorylation site) and 236 are essential for the kinase activity of Aurora B. Cdc7 down regulation or inhibition reduced Aurora B activity in vivo and led to retarded M-phase progression. SAC imposed by paclitaxel was dramatically reversed by Cdc7 inhibition, similar to the effect of Aurora B inhibition under the similar situation. Our data show that Cdc7 contributes to M-phase progression and to spindle assembly checkpoint most likely through Aurora B activation.

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Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1–C-Mad2 core complex

The Journal of Cell Biology ◽

10.1083/jcb.201002133 ◽

2010 ◽

Vol 190 (1) ◽

pp. 25-34 ◽

Cited By ~ 225

Author(s):

Laura Hewitt ◽

Anthony Tighe ◽

Stefano Santaguida ◽

Anne M. White ◽

Clifford D. Jones ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Aurora B ◽

Core Complex ◽

Obvious Effect ◽

Checkpoint Signaling ◽

Mitotic Entry ◽

The Core ◽

Chromosome Congression ◽

Chromosome Alignment

Mps1 is an essential component of the spindle assembly checkpoint. In this study, we describe a novel Mps1 inhibitor, AZ3146, and use it to probe the role of Mps1’s catalytic activity during mitosis. When Mps1 is inhibited before mitotic entry, subsequent recruitment of Mad1 and Mad2 to kinetochores is abolished. However, if Mps1 is inhibited after mitotic entry, the Mad1–C-Mad2 core complex remains kinetochore bound, but O-Mad2 is not recruited to the core. Although inhibiting Mps1 also interferes with chromosome alignment, we see no obvious effect on aurora B activity. In contrast, kinetochore recruitment of centromere protein E (CENP-E), a kinesin-related motor protein, is severely impaired. Strikingly, inhibition of Mps1 significantly increases its own abundance at kinetochores. Furthermore, we show that Mps1 can dimerize and transphosphorylate in cells. We propose a model whereby Mps1 transphosphorylation results in its release from kinetochores, thus facilitating recruitment of O-Mad2 and CENP-E and thereby simultaneously promoting checkpoint signaling and chromosome congression.

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The BUBR1 pseudokinase domain promotes efficient kinetochore PP2A-B56 recruitment to regulate spindle checkpoint silencing and chromosome alignment

10.1101/733378 ◽

2019 ◽

Author(s):

Luciano Gama Braga ◽

Angel F. Cisneros ◽

Michelle Mathieu ◽

Maxime Clerc ◽

Pauline Garcia ◽

...

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Aurora B ◽

Dependent Manner ◽

Regulatory Domain ◽

Checkpoint Protein ◽

Major Player ◽

Chromosome Alignment ◽

Spindle Assembly Checkpoint Protein

ABSTRACTThe balance of phospho-signalling at outer-kinetochores during mitosis is critical for the accurate attachments between kinetochores and the mitotic spindle and timely exit from mitosis. In humans, a major player in determining this balance is the PP2A-B56 phosphatase which is recruited to the Kinase Attachment Regulatory Domain (KARD) of the Spindle Assembly Checkpoint protein Budding Uninhibited by Benzimidazole 1-related 1 (BUBR1) in a phospho-dependent manner. This event unleashes a rapid, switch-like phosphatase relay that reverses phosphorylation at the kinetochore, extinguishing the checkpoint and promoting anaphase entry. Here, we conclusively demonstrate that the pseudokinase domain of human BUBR1 lacks phosphotransfer activity and that it was maintained in vertebrates because it allosterically promotes KARD phosphorylation. Mutation or removal of this domain results in decreased PP2A-B56 recruitment to the outer kinetochore, attenuated checkpoint silencing and errors in chromosome alignment as a result of imbalance in Aurora B activity. We demonstrate that the functions of the BUBR1 pseudokinase and the BUB1 kinase domains are intertwined, providing an explanation for retention of the pseudokinase domain in certain eukaryotes.

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Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores

The Journal of Cell Biology ◽

10.1083/jcb.200208091 ◽

2003 ◽

Vol 161 (2) ◽

pp. 267-280 ◽

Cited By ~ 810

Author(s):

Claire Ditchfield ◽

Victoria L. Johnson ◽

Anthony Tighe ◽

Rebecca Ellston ◽

Carolyn Haworth ◽

...

Keyword(s):

Kinase Activity ◽

Kinase Inhibitor ◽

Spindle Checkpoint ◽

Aurora Kinase ◽

Multiple Roles ◽

Aurora B ◽

Aurora B Kinase ◽

Checkpoint Proteins ◽

Anaphase Onset ◽

Chromosome Alignment

The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

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