Analysis of Ipl1-Mediated Phosphorylation of the Ndc80 Kinetochore Protein in <i>Saccharomyces cerevisiae</i>

Akiyoshi, Bungo; Nelson, Christian R.; Ranish, Jeffrey A.; Biggins, Sue

doi:10.1534/genetics.109.109041

Cited by 69 publications

(96 citation statements)

References 22 publications

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“…Kinetochore particles were purified from budding yeast strains carrying phosphomimetic mutations at previously identified Aurora B target sites. We focused on seven sites in the N-terminal "tail" of Ndc80 (threonines 21, 54, 71, and 74; serines 37, 95, and 100) (27), and four in the Dam1 protein of the Dam1 subcomplex (serines 20, 257, 265, and 292) (28), because prior work suggests that these sites are in or near key microtubulebinding interfaces (18,20,(29)(30)(31)(32)(33) and that their phosphorylation is likely to affect microtubule attachment in vivo (22,28,29,(34)(35)(36). We prepared phosphomimetic mutants in which the targeted residues were mutated to aspartic or glutamic acid (single letter codes, D or E) to mimic constitutive phosphorylation.…”

Section: Resultsmentioning

confidence: 99%

Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms

Sarangapani

Akiyoshi

Duggan

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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121

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During mitosis, multiprotein complexes called kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. The conserved kinase Aurora B phosphorylates the major microtubule-binding kinetochore subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora B phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 vs. Dam1 in the context of whole kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced lasertrapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Rupture forces were reduced by phosphomimetic mutations on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Taken together, our results suggest that, in vivo, Aurora B releases kinetochores via at least two mechanisms: by weakening the kinetochoremicrotubule interface and also by destabilizing the kinetochoreattached microtubule tip.

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Section: Resultsmentioning

confidence: 99%

Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms

Sarangapani

Akiyoshi

Duggan

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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121

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“…While Aurora B-mediated phosphorylation of many kinetochore proteins has been reported (Cheeseman et al 2002;Westermann et al 2003;Maskell et al 2010), the only substrates implicated in biorientation in budding yeast to date are the major microtubule binding complexes, Dam1 and Ndc80. Mutants in the Dam1 phosphorylation sites lead to biorientation defects in vivo (Cheeseman et al 2002) and Aurora B phosphorylation site mutants in Ndc80 exhibit biorientation defects when Aurora B function is further compromised in vivo (Akiyoshi et al 2009a). These data suggest that Ndc80 phosphorylation is important but redundant with additional substrates in yeast (Akiyoshi et al 2009a;Demirel et al 2012).…”

Section: Kinetochore Biorientationmentioning

confidence: 88%

“…Mutants in the Dam1 phosphorylation sites lead to biorientation defects in vivo (Cheeseman et al 2002) and Aurora B phosphorylation site mutants in Ndc80 exhibit biorientation defects when Aurora B function is further compromised in vivo (Akiyoshi et al 2009a). These data suggest that Ndc80 phosphorylation is important but redundant with additional substrates in yeast (Akiyoshi et al 2009a;Demirel et al 2012).…”

Section: Kinetochore Biorientationmentioning

confidence: 88%

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

2013

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The propagation of all organisms depends on the accurate and orderly segregation of chromosomes in mitosis and meiosis. Budding yeast has long served as an outstanding model organism to identify the components and underlying mechanisms that regulate chromosome segregation. This review focuses on the kinetochore, the macromolecular protein complex that assembles on centromeric chromatin and maintains persistent load-bearing attachments to the dynamic tips of spindle microtubules. The kinetochore also serves as a regulatory hub for the spindle checkpoint, ensuring that cell cycle progression is coupled to the achievement of proper microtubule-kinetochore attachments. Progress in understanding the composition and overall architecture of the kinetochore, as well as its properties in making and regulating microtubule attachments and the spindle checkpoint, is discussed. TABLE OF CONTENTS Abstract 817Introduction 818

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“…Within the KMN network the Ndc80 complex, composed of Ndc80, Nuf2, Spc24 and Spc25 plays an important role in binding to microtubules (DeLuca et al, 2002;Martin-Lluesma et al, 2002;Wigge and Kilmartin, 2001) via the calponin homology domains (CHDs) of Ndc80 and Nuf2 and the unstructured N-terminal tail of Ndc80 (Alushin et al, 2010;Ciferri et al, 2008;DeLuca et al, 2005;DeLuca et al, 2006;Guimaraes et al, 2008;Miller et al, 2008;Powers et al, 2009;Sundin et al, 2011;Tooley et al, 2011). The binding of the Ndc80 complex to microtubules is negatively regulated by the error correction machinery through the direct phosphorylation of residues in the N-terminal tail of the Ndc80 subunit by Aurora B (Akiyoshi et al, 2009;DeLuca et al, 2006;Welburn et al, 2010). In addition the KNL1 subunit of the KMN network has microtubule binding activity and both the Ndc80 complex and KNL1 contributes to the overall microtubule binding activity of the kinetochore (Cheeseman et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

The Ndc80 internal loop is required for recruitment of the Ska complex to establish end-on microtubule attachment to kinetochores.

Zhang

Kelstrup

et al. 2012

Journal of Cell Science

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SummaryThe Ndc80 complex establishes end-on attachment of kinetochores to microtubules, which is essential for chromosome segregation. The Ndc80 subunit is characterized by an N-terminal region that binds directly to microtubules, and a long coiled-coil region that interacts with Nuf2. A loop region in Ndc80 that generates a kink in the structure disrupts the long coiled-coil region but the exact function of this loop, has until now, not been clear. Here we show that this loop region is essential for end-on attachment of kinetochores to microtubules in human cells. Cells expressing loop mutants of Ndc80 are unable to align the chromosomes, and stable kinetochore fibers are absent. Through quantitative mass spectrometry and immunofluorescence we found that the binding of the spindle and kinetochore associated (Ska) complex depends on the loop region, explaining why end-on attachment is defective. This underscores the importance of the Ndc80 loop region in coordinating chromosome segregation through the recruitment of specific proteins to the kinetochore.

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Analysis of Ipl1-Mediated Phosphorylation of the Ndc80 Kinetochore Protein in Saccharomyces cerevisiae

Cited by 69 publications

References 22 publications

Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms

Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

The Ndc80 internal loop is required for recruitment of the Ska complex to establish end-on microtubule attachment to kinetochores.

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