scholarly journals Cdt1 modulates kinetochore-microtubule attachment stabilization via an Aurora B kinase-dependent mechanism

2017 ◽  
Author(s):  
Shivangi Agarwal ◽  
Kyle Paul Smith ◽  
Yizhuo Zhou ◽  
Aussie Suzuki ◽  
Richard J. McKenney ◽  
...  
Keyword(s):  
Aurora B ◽  
Deletion Mapping ◽  
Dependent Manner ◽  
Mitotic Progression ◽  
Aurora B Kinase ◽  
Winged Helix ◽  
Mitotic Kinase ◽  
Cellular Expression ◽  
Ndc80 Complex ◽  
Dna Replication Origin

AbstractRobust kinetochore-microtubule (kMT) attachment is critical for accurate chromosome segregation. G2/M-specific depletion of human Cdt1 that localizes to kinetochores in an Ndc80 complex-dependent manner, leads to abnormal kMT attachments and mitotic arrest. This indicates an independent mitotic role for Cdt1 in addition to its prototypic function in DNA replication origin licensing. Here, we show that Cdt1 directly binds to microtubules (MTs). Endogenous or transiently expressed Cdt1 localizes to both mitotic spindle MTs and kinetochores. Deletion mapping of Cdt1 revealed that the regions comprising the middle and C-terminal winged-helix domains but lacking the N-terminal unstructured region was required for efficient MT-binding. Mitotic kinase Aurora B interacts with and phosphorylates Cdt1. Aurora B-phosphomimetic Cdt1 exhibited attenuated MT-binding and its cellular expression induced defective kMT attachments with a concomitant delay in mitotic progression. Thus we provide mechanistic insight into how Cdt1 affects overall kMT stability in an Aurora B kinase phosphorylation-dependent manner; which is envisioned to augment the MT-binding of the Ndc80 complex.eTOC summary• Cdt1 binds to microtubules• The middle and the C-terminal winged-helix domains of Cdt1 are involved in MT-binding• Aurora B Kinase phosphorylates Cdt1 and influences its MT-binding• Aurora B-mediated Cdt1 phosphorylation is necessary for kMT stability and mitotic progression

scholarly journals Cdt1 stabilizes kinetochore–microtubule attachments via an Aurora B kinase–dependent mechanism

2018 ◽  
Vol 217 (10) ◽  
pp. 3446-3463 ◽  
Author(s):  
Shivangi Agarwal ◽  
Kyle Paul Smith ◽  
Yizhuo Zhou ◽  
Aussie Suzuki ◽  
Richard J. McKenney ◽  
...  
Keyword(s):  
Aurora B ◽  
Deletion Mapping ◽  
Dependent Manner ◽  
Aurora B Kinase ◽  
Mitotic Kinase ◽  
Cellular Expression ◽  
Origin Licensing ◽  
Ndc80 Complex ◽  
Dna Replication Origin ◽  
Kinase Phosphorylation

Robust kinetochore–microtubule (kMT) attachment is critical for accurate chromosome segregation. G2/M-specific depletion of human Cdt1 that localizes to kinetochores in an Ndc80 complex–dependent manner leads to abnormal kMT attachments and mitotic arrest. This indicates an independent mitotic role for Cdt1 in addition to its prototypic function in DNA replication origin licensing. Here, we show that Cdt1 directly binds to microtubules (MTs). Endogenous or transiently expressed Cdt1 localizes to both mitotic spindle MTs and kinetochores. Deletion mapping of Cdt1 revealed that the regions comprising the middle and C-terminal winged-helix domains but lacking the N-terminal unstructured region were required for efficient MT binding. Mitotic kinase Aurora B interacts with and phosphorylates Cdt1. Aurora B–phosphomimetic Cdt1 exhibited attenuated MT binding, and its cellular expression induced defective kMT attachments with a concomitant delay in mitotic progression. Thus we provide mechanistic insight into how Cdt1 affects overall kMT stability in an Aurora B kinase phosphorylation-dependent manner; which is envisioned to augment the MT-binding of the Ndc80 complex.

scholarly journals The Ndc80 complex bridges two Dam1 complex rings

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jae ook Kim ◽  
Alex Zelter ◽  
Neil T Umbreit ◽  
Athena Bollozos ◽  
Michael Riffle ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Microtubule Attachment ◽  
Sister Chromatids ◽  
Ndc80 Complex ◽  
Interaction Regions ◽  
Dam1 Complex

Strong kinetochore-microtubule attachments are essential for faithful segregation of sister chromatids during mitosis. The Dam1 and Ndc80 complexes are the main microtubule binding components of the Saccharomyces cerevisiae kinetochore. Cooperation between these two complexes enhances kinetochore-microtubule coupling and is regulated by Aurora B kinase. We show that the Ndc80 complex can simultaneously bind and bridge across two Dam1 complex rings through a tripartite interaction, each component of which is regulated by Aurora B kinase. Mutations in any one of the Ndc80p interaction regions abrogates the Ndc80 complex’s ability to bind two Dam1 rings in vitro, and results in kinetochore biorientation and microtubule attachment defects in vivo. We also show that an extra-long Ndc80 complex, engineered to space the two Dam1 rings further apart, does not support growth. Taken together, our work suggests that each kinetochore in vivo contains two Dam1 rings and that proper spacing between the rings is vital.

scholarly journals Exploring the Functional Interactions between Aurora B, INCENP, and Survivin in Mitosis

2003 ◽  
Vol 14 (8) ◽  
pp. 3325-3341 ◽  
Author(s):  
Reiko Honda ◽  
Roman Körner ◽  
Erich A. Nigg
Keyword(s):  
Chromosome Segregation ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Aurora B ◽  
Mitotic Progression ◽  
Aurora B Kinase ◽  
Kinase Activation ◽  
Central Spindle ◽  
Assay Conditions

The function of the Aurora B kinase at centromeres and the central spindle is crucial for chromosome segregation and cytokinesis, respectively. Herein, we have investigated the regulation of human Aurora B by its complex partners inner centromere protein (INCENP) and survivin. We found that overexpression of a catalytically inactive, dominant-negative mutant of Aurora B impaired the localization of the entire Aurora B/INCENP/survivin complex to centromeres and the central spindle and severely disturbed mitotic progression. Similar results were also observed after depletion, by RNA interference, of either Aurora B, INCENP, or survivin. These data suggest that Aurora B kinase activity and the formation of the Aurora B/INCENP/survivin complex both contribute to its proper localization. Using recombinant proteins, we found that Aurora B kinase activity was stimulated by INCENP and that the C-terminal region of INCENP was sufficient for activation. Under identical assay conditions, survivin did not detectably influence kinase activity. Human INCENP was a substrate of Aurora B and mass spectrometry identified three consecutive residues (threonine 893, serine 894, and serine 895) containing at least two phosphorylation sites. A nonphosphorylatable mutant (TSS893–895AAA) was a poor activator of Aurora B, demonstrating that INCENP phosphorylation is important for kinase activation.

scholarly journals EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B

2014 ◽  
Vol 204 (6) ◽  
pp. 947-963 ◽  
Author(s):  
Budhaditya Banerjee ◽  
Cortney A. Kestner ◽  
P. Todd Stukenberg
Keyword(s):  
Mitotic Chromosome ◽  
Histone H3 ◽  
Aurora B ◽  
Dependent Manner ◽  
Histone H2a ◽  
Aurora B Kinase ◽  
Chromosomal Passenger Complex ◽  
Checkpoint Signaling ◽  
Chromosomal Passenger ◽  
Spindle Microtubules

The Aurora B kinase coordinates kinetochore–microtubule attachments with spindle checkpoint signaling on each mitotic chromosome. We find that EB1, a microtubule plus end–tracking protein, is required to enrich Aurora B at inner centromeres in a microtubule-dependent manner. This regulates phosphorylation of both kinetochore and chromatin substrates. EB1 regulates the histone phosphorylation marks (histone H2A phospho-Thr120 and histone H3 phospho-Thr3) that localize Aurora B. The chromosomal passenger complex containing Aurora B can be found on a subset of spindle microtubules that exist near prometaphase kinetochores, known as preformed K-fibers (kinetochore fibers). Our data suggest that EB1 enables the spindle microtubules to regulate the phosphorylation of kinetochores through recruitment of the Aurora B kinase.

scholarly journals Phosphorylation by Aurora B kinase regulates caspase-2 activity and function

2020 ◽  
Author(s):  
Yoon Lim ◽  
Dylan De Bellis ◽  
Jarrod J. Sandow ◽  
Luisa Capalbo ◽  
Pier Paolo D’Avino ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Mitotic Catastrophe ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Mitotic Kinase ◽  
Catalytic Function ◽  
Cycle Arrest ◽  
Caspase 2 ◽  
And Function ◽  
Key Steps

AbstractMitotic catastrophe (MC) is an important oncosuppressive mechanism that serves to eliminate cells that become polyploid or aneuploidy 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.

scholarly journals Aurora B-INCENP localization at centromeres/inner kinetochores is essential for chromosome bi-orientation in budding yeast

2019 ◽  
Author(s):  
Luis J Garcia-Rodriguez ◽  
Taciana Kasciukovic ◽  
Viola Denninger ◽  
Tomoyuki U Tanaka
Keyword(s):  
Budding Yeast ◽  
Aurora B ◽  
Dependent Manner ◽  
Aurora B Kinase ◽  
Insight Into

To promote chromosome bi-orientation, Aurora B kinase weakens and disrupts aberrant kinetochore-MT interaction. It has long been debated how Aurora B halts this action when bi-orientation is established and tension is applied across sister kinetochores. Pertinent to this debate, it was shown that Bir1 (yeast Survivin), which recruits Ipl1-Sli15 (yeast Aurora B-INCENP) to centromeres, is dispensable for bi-orientation, raising the possibility that Aurora B localization at centromeres is not required for bi-orientation. Here, we show that the COMA inner kinetochore sub-complex physically interacts with Sli15, recruits Ipl1-Sli15 to the inner kinetochore and promotes chromosome bi-orientation, independently of Bir1, in budding yeast. Moreover, using an engineered recruitment of Ipl1-Sli15 to the inner kinetochore when both Bir1 and COMA are defective, we show that localization of Ipl1-Sli15 at centromeres/inner kinetochores is essential for bi-orientation, refuting the above possibility. Our results give important insight into how Aurora B disrupts kinetochore-MT interaction in a tension-dependent manner, to promote chromosome bi-orientation.

scholarly journals Cdc7 kinase stimulates Aurora B kinase in M-phase

2019 ◽  
Vol 9 (1) ◽  
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.

scholarly journals Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores

2015 ◽  
Vol 208 (2) ◽  
pp. 181-196 ◽  
Author(s):  
Soonjoung Kim ◽  
Hongtao Yu
Keyword(s):  
Spindle Checkpoint ◽  
Aurora B ◽  
Dependent Manner ◽  
Checkpoint Proteins ◽  
Multiple Strategies ◽  
Complete Inactivation ◽  
Microtubule Attachment ◽  
Ndc80 Complex ◽  
Spindle Microtubules ◽  
Multiple Assembly

During mitosis, the spindle checkpoint senses kinetochores not properly attached to spindle microtubules and prevents precocious sister-chromatid separation and aneuploidy. The constitutive centromere-associated network (CCAN) at inner kinetochores anchors the KMN network consisting of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C) at outer kinetochores. KMN is a critical kinetochore receptor for both microtubules and checkpoint proteins. Here, we show that nearly complete inactivation of KMN in human cells through multiple strategies produced strong checkpoint defects even when all kinetochores lacked microtubule attachment. These KMN-inactivating strategies reveal multiple KMN assembly mechanisms at human mitotic kinetochores. In one mechanism, the centromeric kinase Aurora B phosphorylates Mis12C and strengthens its binding to the CCAN subunit CENP-C. In another, CENP-T contributes to KMN attachment in a CENP-H-I-K–dependent manner. Our study provides insights into the mechanisms of mitosis-specific assembly of the checkpoint platform KMN at human kinetochores.

scholarly journals Borealin

2004 ◽  
Vol 166 (2) ◽  
pp. 179-191 ◽  
Author(s):  
Reto Gassmann ◽  
Ana Carvalho ◽  
Alexander J. Henzing ◽  
Sandrine Ruchaud ◽  
Damien F. Hudson ◽  
...  
Keyword(s):  
Rna Interference ◽  
Histone H3 ◽  
Aurora B ◽  
Mitotic Progression ◽  
Aurora B Kinase ◽  
Chromosomal Passenger Complex ◽  
Central Spindle ◽  
Chromosomal Passenger ◽  
Bipolar Spindles

The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Here, we describe Borealin, a novel member of the complex. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin; and Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore–spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B–INCENP subcomplex.

scholarly journals Phosphorylation by Aurora B kinase regulates caspase-2 activity and function

2020 ◽  
Vol 28 (1) ◽  
pp. 349-366 ◽  
Author(s):  
Yoon Lim ◽  
Dylan De Bellis ◽  
Jarrod J. Sandow ◽  
Luisa Capalbo ◽  
Pier Paolo D’Avino ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Mitotic Catastrophe ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Mitotic Kinase ◽  
Catalytic Function ◽  
Cycle Arrest ◽  
Caspase 2 ◽  
And Function ◽  
Key Steps

AbstractMitotic 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.

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