Protein Interaction Domain Mapping of Human Kinetochore Protein Blinkin Reveals a Consensus Motif for Binding of Spindle Assembly Checkpoint Proteins Bub1 and BubR1

In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring. In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton. In this article, we show that addition of Latrunculin A, which prevents spindle rotation, delays the separation of sister chromatids and anaphase promoting complex-mediated destruction of spindle-associated Securin and Cyclin B. Moreover, we find that whereas sister kinetochore pairs normally congress to the spindle midzone before anaphase onset, this congression is disrupted when astral microtubule contact with the actin cytoskeleton is disturbed. By analyzing the timing of kinetochore separation, we find that this anaphase delay requires the Bub3, Mad3, and Bub1 but not the Mad1 or Mad2 spindle assembly checkpoint proteins. In agreement with this, we find that Bub1 remains associated with kinetochores when spindles are mispositioned. These data indicate that, in fission yeast, astral microtubule contact with the medial cell cortex is monitored by a subset of spindle assembly checkpoint proteins. We propose that this checkpoint ensures spindles are properly oriented before anaphase takes place.

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The Spindle Assembly Checkpoint Regulates the Phosphorylation State of a Subset of DNA Checkpoint Proteins in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.00310-06 ◽

2006 ◽

Vol 26 (24) ◽

pp. 9149-9161 ◽

Cited By ~ 9

Author(s):

Céline Clémenson ◽

Marie-Claude Marsolier-Kergoat

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage Response ◽

Spindle Assembly Checkpoint ◽

Spindle Checkpoint ◽

Spindle Assembly ◽

Dna Lesions ◽

Checkpoint Proteins ◽

Cell Responses ◽

Spindle Assembly Checkpoints ◽

Damage Response

ABSTRACT The DNA and the spindle assembly checkpoints play key roles in maintaining genomic integrity by coordinating cell responses to DNA lesions and spindle dysfunctions, respectively. These two surveillance pathways seem to operate mostly independently of one another, and little is known about their potential physiological connections. Here, we show that in Saccharomyces cerevisiae, the activation of the spindle assembly checkpoint triggers phosphorylation changes in two components of the DNA checkpoint, Rad53 and Rad9. These modifications are independent of the other DNA checkpoint proteins and are abolished in spindle checkpoint-defective mutants, hinting at specific functions for Rad53 and Rad9 in the spindle damage response. Moreover, we found that after UV irradiation, Rad9 phosphorylation is altered and Rad53 inactivation is accelerated when the spindle checkpoint is activated, which suggests the implication of the spindle checkpoint in the regulation of the DNA damage response.

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Knl1 shows another face

The Journal of Cell Biology ◽

10.1083/jcb.1964if ◽

2012 ◽

Vol 196 (4) ◽

pp. 391-391

Author(s):

Mitch Leslie

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Kinetochore Protein

Kinetochore protein might not recruit key parts of spindle assembly checkpoint and instead might help silence the checkpoint.

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hZwint-1 bridges the inner and outer kinetochore: identification of the kinetochore localization domain and the hZw10-interaction domain

Biochemical Journal ◽

10.1042/bj20110137 ◽

2011 ◽

Vol 436 (1) ◽

pp. 157-168 ◽

Cited By ~ 6

Author(s):

Larissa J. Vos ◽

Jakub K. Famulski ◽

Gordon K. T. Chan

Keyword(s):

Glutathione Transferase ◽

Coiled Coil ◽

Mitotic Checkpoint ◽

Structural Protein ◽

Interaction Domain ◽

Interacting Protein ◽

Kinetochore Protein ◽

Essential Components ◽

Domain Mapping ◽

Protein Part

Accurate chromosome segregation in mitosis is required to maintain genetic stability. hZwint-1 [human Zw10 (Zeste white 10)-interacting protein 1] is a kinetochore protein known to interact with the kinetochore checkpoint protein hZw10. hZw10, along with its partners Rod (Roughdeal) and hZwilch, form a complex which recruits dynein–dynactin and Mad1–Mad2 complexes to the kinetochore and are essential components of the mitotic checkpoint. hZwint-1 localizes to the kinetochore in prophase, before hZw10 localization, and remains at the kinetochore until anaphase, after hZw10 has dissociated. This difference in localization timing may reflect a role for hZwint-1 as a structural kinetochore protein. In addition to hZw10, we have found that hZwint-1 interacts with components of the conserved Ndc80 and Mis12 complexes in yeast two-hybrid and GST (glutathione transferase) pull-down assays. Furthermore, hZwint-1 was found to have stable FRAP (fluorescence recovery after photobleaching) dynamics similar to hHec1, hSpc24 and hMis12. As such, we proposed that hZwint-1 is a structural protein, part of the inner kinetochore scaffold and recruits hZw10 to the kinetochore. To test this, we performed mutagenesis-based domain mapping to determine which regions of hZwint-1 are necessary for kinetochore localization and which are required for interaction with hZw10. hZwint-1 localizes to the kinetochore through the N-terminal region and interacts with hZw10 through the C-terminal coiled-coil domain. The two domains are at opposite ends of the protein as expected for a protein that bridges the inner and outer kinetochore.

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Aurora B prevents premature removal of spindle assembly checkpoint proteins from the kinetochore: A key role for Aurora B in mitosis

Oncotarget ◽

10.18632/oncotarget.10657 ◽

2016 ◽

Vol 9 (28) ◽

pp. 19525-19542 ◽

Cited By ~ 5

Author(s):

Mark D. Gurden ◽

Simon J. Anderhub ◽

Amir Faisal ◽

Spiros Linardopoulos

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Aurora B ◽

Checkpoint Proteins ◽

Premature Removal

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PIGN Spatiotemporally Regulates the Spindle Assembly Checkpoint Proteins in Myelodysplastic Syndromes

10.21203/rs.3.rs-130046/v1 ◽

2020 ◽

Author(s):

Emmanuel Teye ◽

Shasha Lu ◽

Fangyuan Chen ◽

Wenrui Yang ◽

Thomas Abraham ◽

...

Keyword(s):

Myelodysplastic Syndromes ◽

Chromosomal Instability ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Checkpoint ◽

Vital Role ◽

Checkpoint Activation ◽

Checkpoint Proteins ◽

Mitotic Kinase ◽

Related Proteins

Abstract Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) has been previously linked to the suppression of chromosomal instability. The spindle assembly checkpoint complex is responsible for proper chromosome segregation during mitosis to prevent chromosomal instability. In this study, the novel role of PIGN as a regulator of the spindle assembly checkpoint was unveiled in leukemic patient cells and cell lines. Transient downregulation or ablation of PIGN resulted in impaired mitotic checkpoint activation due to the dysregulated expression of spindle assembly checkpoint-related proteins including MAD1, MAD2, BUBR1, and MPS1. Moreover, ectopic overexpression of PIGN restored the expression of MAD2. PIGN regulated the spindle assembly checkpoint by forming a complex with the spindle assembly checkpoint proteins MAD1, MAD2, and the mitotic kinase MPS1. Thus, PIGN could play a vital role in the spindle assembly checkpoint to suppress chromosomal instability associated with the leukemic transformation of myelodysplastic syndromes.

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Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling

eLife ◽

10.7554/elife.01030 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 144

Author(s):

Ivana Primorac ◽

John R Weir ◽

Elena Chiroli ◽

Fridolin Gross ◽

Ingrid Hoffmann ◽

...

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Binding Mode ◽

Signaling Networks ◽

Checkpoint Signaling ◽

Checkpoint Proteins ◽

Downstream Signaling ◽

Signaling Components ◽

Insight Into

Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELTP) then promote recruitment of downstream signaling components. How MELTP motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed β-propeller, is the MELTP reader. It contains an exceptionally well-conserved interface that docks the MELTP sequence on the side of the β-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores.

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