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

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Genetics (Impact Factor: 5.96). 10/2009; 183(4):1591-5. DOI: 10.1534/genetics.109.109041
Source: PubMed


Phosphorylation of the Ndc80 kinetochore protein by the Ipl1/Aurora B kinase reduces its microtubule binding activity in vitro. We found that kinetochore-bound Ndc80 is phosphorylated on Ipl1 sites in vivo, but this phosphorylation is not essential. Instead, we show that additional Ipl1 targets contribute to segregation and the spindle checkpoint.

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Available from: Bungo Akiyoshi
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    • "Phosphorylation of the N-terminus of Ndc80 by AuroraB/Ipl1 is thought to reduce the negative charge of the microtubule binding domain and thus increase the microtubule/kinetochore dynamics necessary for the dissolution of improper kinetochore-microtubule interactions. Consistent with this model, the Ndc80-7A mutant, in which Aurora B phosphorylation sites in the N terminus of Ndc80 are mutated to alanine, is inviable under conditions of reduced Ipl1 activity [ipl1-321 background (Akiyoshi et al. 2009a)]. Our results provide direct biological support for the hypothesis that the phosphorylation of Ndc80 by Ipl1/ Aurora B reduces the affinity of the Ndc80 complex for kinetochore microtubules, thereby increasing the kinetochore/microtubule dynamics required to effect bipolar chromosome attachment on the spindle. "
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    ABSTRACT: Ipl1/Aurora B is the catalytic subunit of a protein kinase complex required for chromosome segregation and nuclear division. Before anaphase, Ipl1 is required to establish proper kinetochore-microtubule associations and to regulate the spindle assembly checkpoint (SAC). The phosphatase Glc7/PP1 opposes Ipl1 for these activities. To investigate Ipl1 and Glc7 regulation in more detail, we isolated and characterized mutations in the yeast Saccharomyces cerevisiae that raise the restrictive temperature of the ipl-2 mutant. These suppressors include three intragenic, second-site revertants in IPL1; 17 mutations in Glc7 phosphatase components (GLC7, SDS22, YPI1); two mutations in SHP1, encoding a regulator of the AAA ATPase Cdc48; and a mutation in TCO89, encoding a subunit of the TOR Complex 1. Two revertants contain missense mutations in microtubule binding components of the kinetochore. rev76 contains the missense mutation duo1-S115F, which alters an essential component of the DAM1/DASH complex. The mutant is cold sensitive and arrests in G2/M due to activation of the SAC. rev8 contains the missense mutation ndc80-K204E. K204 of Ndc80 corresponds to K166 of human Ndc80 and the human Ndc80 K166E variant was previously shown to be defective for microtubule binding in vitro. In a wild-type IPL1 background, ndc80-K204E cells grow slowly and the SAC is activated. The slow growth and cell cycle delay of ndc80-K204E cells are partially alleviated by the ipl1-2 mutation. These data provide biological confirmation of a biochemically based model for the effect of phosphorylation on Ndc80 function.
    Full-text · Article · Dec 2012 · G3-Genes Genomes Genetics
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    • "We speculate that Ipl1p could phosphorylate Mad1p or a binding partner in a manner that supports Mad1p cycling and interactions with the NPC necessary for altering Kap121p-mediated import. Though Mad1p phosphorylation by Ipl1p has not as yet been described, this event seems plausible, given that Mad1p binding to unattached KTs is likely to occur near Ndc80p, a known target of Ipl1p (Akiyoshi et al., 2009b; Martin-Lluesma et al., 2002). "
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    ABSTRACT: Nuclear pore complexes (NPCs) and kinetochores perform distinct tasks, yet their shared ability to bind several proteins suggests their functions are intertwined. Among these shared proteins is Mad1p, a component of the yeast spindle assembly checkpoint (SAC). Here we describe a role for Mad1p in regulating nuclear import that employs its ability to sense a disruption of kinetochore-microtubule interactions during mitosis. We show that kinetochore-microtubule detachment arrests nuclear import mediated by the transport factor Kap121p through a mechanism that requires Mad1p cycling between unattached, metaphase kinetochores and binding sites at the NPC. This signaling pathway requires the Aurora B-like kinase Ipl1p, and the resulting transport changes inhibit the nuclear import of Glc7p, a phosphatase that acts as an Ipl1p antagonist. We propose that a distinct branch of the SAC exists in which Mad1p senses unattached kinetochores and, by altering NPC transport activity, regulates the nuclear environment of the spindle.
    Preview · Article · Nov 2012 · Molecular cell
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    • "Numerous microtubule binding kinetochore proteins are phosphorylated by Aurora B. For example, the Ndc80 protein is regulated by Aurora B and the non-phosphorylatable mutant shows hyper-stable attachments and severe segregation defects in human and PtK1 cells, demonstrating the importance of phospho-regulation (DeLuca et al. 2006; DeLuca et al. 2011). In contrast, the non-phosphorylatable mutant is viable in budding yeast and chicken cells, implying that the regulation of additional targets is sufficient for faithful chromosome segregation in these organisms (Akiyoshi et al. 2009b; Welburn et al. 2010). KNL1 and Dsn1 are also phosphorylated by Aurora B, and their combinatorial phosphorylation appears important to regulate the microtubule binding activity of the KMN network (Welburn et al. 2010). "
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    ABSTRACT: The kinetochore is the proteinaceous complex that governs the movement of duplicated chromosomes by interacting with spindle microtubules during mitosis and meiosis. Faithful chromosome segregation requires that kinetochores form robust load-bearing attachments to the tips of dynamic spindle microtubules, correct microtubule attachment errors, and delay the onset of anaphase until all chromosomes have made proper attachments. To understand how this macromolecular machine operates to segregate duplicated chromosomes with exquisite accuracy, it is critical to reconstitute and study kinetochore–microtubule interactions in vitro using defined components. Here, we review the current status of reconstitution as well as recent progress in understanding the microtubule-binding functions of kinetochores in vivo.
    Full-text · Article · Jan 2012 · Chromosoma
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