scholarly journals Kinetochore phosphatases suppress autonomous kinase activity to control the spindle assembly checkpoint

2019 ◽  
Author(s):  
Marilia H Cordeiro ◽  
Richard J Smith ◽  
Adrian T Saurin
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Spindle Assembly ◽  
Phosphatase Regulation ◽  
Phosphatase Activation

AbstractLocal phosphatase regulation is critical for determining when phosphorylation signals are activated or deactivated. A typical example is the spindle assembly checkpoint (SAC) during mitosis, which regulates kinetochore PP1 and PP2A-B56 activities to switch-off signalling events at the correct time. In this case, kinetochore phosphatase activation dephosphorylates MELT motifs on KNL1 to remove SAC proteins, including the BUB complex. We show here that, surprisingly, neither PP1 or PP2A are required to dephosphorylate the MELT motifs. Instead, they remove polo-like kinase 1 (PLK1) from the BUB complex, which can otherwise maintain MELT phosphorylation in an autocatalytic manner. This is their principle role in the SAC, because both phosphatases become redundant if PLK1 is inhibited or BUB-PLK1 interaction is prevented. Therefore, phosphatase regulation is critical for the SAC, but primarily to restrain and extinguish autonomous kinase activity. We propose that these circuits have evolved to generate a semi-autonomous SAC signal that can be synchronously silenced following kinetochore-microtubule tension.

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

2018 ◽  
Vol 131 (7) ◽  
pp. jcs191353 ◽  
Author(s):  
Thibault Courtheoux ◽  
Alghassimou Diallo ◽  
Arun Prasath Damodaran ◽  
David Reboutier ◽  
Erwan Watrin ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Spindle Assembly ◽  
Aurora A Kinase ◽  
Aurora A

scholarly journals Kinetochore phosphatases suppress autonomous Polo-like kinase 1 activity to control the mitotic checkpoint

2020 ◽  
Vol 219 (12) ◽  
Author(s):  
Marilia H. Cordeiro ◽  
Richard J. Smith ◽  
Adrian T. Saurin
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Mitotic Checkpoint ◽  
Binding Motifs ◽  
Principal Role ◽  
Phosphatase Regulation

Local phosphatase regulation is needed at kinetochores to silence the mitotic checkpoint (a.k.a. spindle assembly checkpoint [SAC]). A key event in this regard is the dephosphorylation of MELT repeats on KNL1, which removes SAC proteins from the kinetochore, including the BUB complex. We show here that PP1 and PP2A-B56 phosphatases are primarily required to remove Polo-like kinase 1 (PLK1) from the BUB complex, which can otherwise maintain MELT phosphorylation in an autocatalytic manner. This appears to be their principal role in the SAC because both phosphatases become redundant if PLK1 is inhibited or BUB–PLK1 interaction is prevented. Surprisingly, MELT dephosphorylation can occur normally under these conditions even when the levels or activities of PP1 and PP2A are strongly inhibited at kinetochores. Therefore, these data imply that kinetochore phosphatase regulation is critical for the SAC, but primarily to restrain and extinguish autonomous PLK1 activity. This is likely a conserved feature of the metazoan SAC, since the relevant PLK1 and PP2A-B56 binding motifs have coevolved in the same region on MADBUB homologues.

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

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.

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

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.

scholarly journals Separating the spindle, checkpoint, and timer functions of BubR1

2009 ◽  
Vol 187 (5) ◽  
pp. 597-605 ◽  
Author(s):  
Zohra Rahmani ◽  
Mary E. Gagou ◽  
Christophe Lefebvre ◽  
Doruk Emre ◽  
Roger E. Karess
Keyword(s):  
Drosophila Melanogaster ◽  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Spindle Checkpoint ◽  
Spindle Assembly ◽  
Spindle Function ◽  
Microtubule Capture

BubR1 performs several roles during mitosis, affecting the spindle assembly checkpoint (SAC), mitotic timing, and spindle function, but the interdependence of these functions is unclear. We have analyzed in Drosophila melanogaster the mitotic phenotypes of kinase-dead (KD) BubR1 and BubR1 lacking the N-terminal KEN box. bubR1-KD individuals have a robust SAC but abnormal spindles with thin kinetochore fibers, suggesting that the kinase activity modulates microtubule capture and/or dynamics but is relatively dispensable for SAC function. In contrast, bubR1-KEN flies have normal spindles but no SAC. Nevertheless, mitotic timing is normal as long as Mad2 is present. Thus, the SAC, timer, and spindle functions of BubR1 are substantially separable. Timing is shorter in bubR1-KEN mad2 double mutants, yet in these flies, lacking both critical SAC components, chromosomes still segregate accurately, reconfirming that in Drosophila, reliable mitosis does not need the SAC.

scholarly journals Novel Functional Assay for Spindle-Assembly Checkpoint by Cyclin-Dependent Kinase Activity to Predict Taxane Chemosensitivity in Breast Tumor Patient

2013 ◽  
Vol 4 (9) ◽  
pp. 697-702 ◽  
Author(s):  
Yasuhiro Torikoshi ◽  
Keigo Gohda ◽  
Michelle L. Davis ◽  
W. Fraser Symmans ◽  
Lajos Pusztai ◽  
...  
Keyword(s):  
Breast Tumor ◽  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Spindle Assembly ◽  
Functional Assay ◽  
Cyclin Dependent Kinase ◽  
Tumor Patient

scholarly journals Requirement for PLK1 kinase activity in the maintenance of a robust spindle assembly checkpoint

Biology Open ◽  
2015 ◽  
Vol 5 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Aisling O'Connor ◽  
Stefano Maffini ◽  
Michael D. Rainey ◽  
Agnieszka Kaczmarczyk ◽  
David Gaboriau ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Spindle Assembly

scholarly journals Sgol2 provides a regulatory platform that coordinates essential cell cycle processes during meiosis I in oocytes

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Ahmed Rattani ◽  
Magda Wolna ◽  
Mickael Ploquin ◽  
Wolfgang Helmhart ◽  
Seamus Morrone ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Spindle Assembly ◽  
Fiber Formation ◽  
Direct Binding ◽  
Multiple Interactions ◽  
Meiosis I

Accurate chromosome segregation depends on coordination between cohesion resolution and kinetochore-microtubule interactions (K-fibers), a process regulated by the spindle assembly checkpoint (SAC). How these diverse processes are coordinated remains unclear. We show that in mammalian oocytes Shugoshin-like protein 2 (Sgol2) in addition to protecting cohesin, plays an important role in turning off the SAC, in promoting the congression and bi-orientation of bivalents on meiosis I spindles, in facilitating formation of K-fibers and in limiting bivalent stretching. Sgol2’s ability to protect cohesin depends on its interaction with PP2A, as is its ability to silence the SAC, with the latter being mediated by direct binding to Mad2. In contrast, its effect on bivalent stretching and K-fiber formation is independent of PP2A and mediated by recruitment of MCAK and inhibition of Aurora C kinase activity respectively. By virtue of its multiple interactions, Sgol2 links many of the processes essential for faithful chromosome segregation.

Control of duration of the first two mitoses in a mouse embryo

Zygote ◽  
1999 ◽  
Vol 7 (4) ◽  
pp. 293-300 ◽  
Author(s):  
Maria A. Ciemerych ◽  
Bernard Maro ◽  
Jacek Z. Kubiak
Keyword(s):  
Mouse Embryo ◽  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Metaphase Plate ◽  
Spindle Assembly ◽  
Cell Types ◽  
Histone H1 ◽  
Mitotic Apparatus ◽  
M Phase ◽  
Correct Alignment

The duration of M-phase is largely determined by the time necessary for the formation of a functional metaphase spindle and the correct alignment of all chromosomes on the metaphase plate. The spindle assembly checkpoint prevents the exit from M-phase before the proper alignment of all chromosomes on a metaphase plate in many cell types. In the present paper we show that the first mitotic M-phase of the mouse embryo lasts about 119 min, while the second embryonic M-phase lasts only about 70 min. Histone H1 kinase is activated rapidly during nuclear envelope breakdown in both mitoses. Its maximum, however, is followed by a plateau only during the first mitosis. In the second mitosis, the inactivation of histone H1 kinase activity follows its maximum directly. Histone H1 kinase is more stable in the cytoplasts obtained from mouse embryos during the first embryonic M-phase than during the second one. The stability of histone H1 kinase is greatly increased by the presence of the mitotic apparatus in both M-phases. The mitotic spindle assembly during the first and the second mitoses differs and the first metaphase spindle is stabilised during the period of maximum histone H1 kinase activity. These data show that an unknown developmentally regulated mechanism controls the duration of the two first mitoses in the mouse embryo.

scholarly journals Autophosphorylation is sufficient to release Mps1 kinase from native kinetochores

2019 ◽  
Vol 116 (35) ◽  
pp. 17355-17360 ◽  
Author(s):  
Lori B. Koch ◽  
Kwaku N. Opoku ◽  
Yi Deng ◽  
Adrienne Barber ◽  
Aimee J. Littleton ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Single Particle ◽  
Surveillance System ◽  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Spindle Assembly ◽  
Critical Aspect ◽  
Direct Competition ◽  
Spindle Microtubules ◽  
Checkpoint Signal

Accurate mitosis depends on a surveillance system called the spindle assembly checkpoint. This checkpoint acts at kinetochores, which attach chromosomes to the dynamic tips of spindle microtubules. When a kinetochore is unattached or improperly attached, the protein kinase Mps1 phosphorylates kinetochore components, catalyzing the generation of a diffusible “wait” signal that delays anaphase and gives the cell time to correct the error. When a kinetochore becomes properly attached, its checkpoint signal is silenced to allow progression into anaphase. Recently, microtubules were found to compete directly against recombinant human Mps1 fragments for binding to the major microtubule-binding kinetochore element Ndc80c, suggesting a direct competition model for silencing the checkpoint signal at properly attached kinetochores. Here, by developing single-particle fluorescence-based assays, we tested whether such direct competition occurs in the context of native kinetochores isolated from yeast. Mps1 levels were not reduced on kinetochore particles bound laterally to the sides of microtubules or on particles tracking processively with disassembling tips. Instead, we found that Mps1 kinase activity was sufficient to promote its release from the isolated kinetochores. Mps1 autophosphorylation, rather than phosphorylation of other kinetochore components, was responsible for this dissociation. Our findings suggest that checkpoint silencing in yeast does not arise from a direct competition between Mps1 and microtubules, and that phosphoregulation of Mps1 may be a critical aspect of the silencing mechanism.

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