Cdc14-Dependent Dephosphorylation of a Kinetochore Protein Prior to Anaphase in <i>Saccharomyces cerevisiae</i>

Akiyoshi, Bungo; Biggins, Sue

doi:10.1534/genetics.110.123653

Cited by 20 publications

(15 citation statements)

References 48 publications

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“…Dsn1 is the only member of the MIND complex known to contain CDK target sites (22,23), but blocking phosphorylation of these sites does not result in significant mitotic phenotypes (13). To ask whether association of Cdc14 adjacent to the MIND complex could affect phosphorylation of Dsn1, we tagged endogenous Dsn1 with the sequence encoding three HA tags.…”

Section: Resultsmentioning

confidence: 99%

“…However, the importance of these CDK phosphorylation/dephosphorylation events is unclear. For example, Dsn1 is a member of the MIND complex, which is dephosphorylated before the bulk release of Cdc14 (13). However, CDC14 conditional mutants only have a subtle defect in mitosis (25).…”

Section: Discussionmentioning

confidence: 99%

“…Another such modification is the phosphorylation and ubiquitylation of the MIND complex member Dsn1. Dsn1 is a target of both the cyclin-dependent kinase (CDK) and the Aurora kinase (Ipl1) (13,14). Ipl1-dependent phosphorylation stabilizes Dsn1 and prevents its degradation by the Mub1/Ubr2 ubiquitin pathway.…”

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confidence: 99%

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Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization

Ólafsson

Thorpe

2015

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

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The location of proteins within eukaryotic cells is often critical for their function and relocation of proteins forms the mainstay of regulatory pathways. To assess the importance of protein location to cellular homeostasis, we have developed a methodology to systematically create binary physical interactions between a query protein and most other members of the proteome. This method allows us to rapidly assess which of the thousands of possible protein interactions modify a phenotype. As proof of principle we studied the kinetochore, a multiprotein assembly that links centromeres to the microtubules of the spindle during cell division. In budding yeast, the kinetochores from the 16 chromosomes cluster together to a single location within the nucleus. The many proteins that make up the kinetochore are regulated through ubiquitylation and phosphorylation. By systematically associating members of the proteome to the kinetochore, we determine which fusions affect its normal function. We identify a number of candidate kinetochore regulators, including the phosphatase Cdc14. We examine where within the kinetochore Cdc14 can act and show that the effect is limited to regions that correlate with known phosphorylation sites, demonstrating the importance of serine phospho-regulation for normal kinetochore homeostasis.T he relocation of proteins between cellular compartments underlies many regulatory pathways. For example, protein relocation underpins most cell-signaling pathways (1) and the establishment of cell polarity and asymmetric cell division both require highly specialized relocation of proteins within the cell (2). Furthermore, the aberrant localization of proteins underlies the pathology of a number of diseases (3). However, our understanding of the effect of relocalizing members of the proteome is limited to specific studies typically concerning individual proteins, and only a select few studies have globally monitored protein relocation (4, 5).One highly localized structure within the cell is the kinetochore, which in budding yeast forms a single megadalton complex (6-8). The kinetochore attaches chromosomes to the spindle microtubules to drive accurate chromosome segregation. The kinetochore consists of at least 60 unique gene products present in multiple copies that stretch from the specialized H3 histone subunit (Cse4, CENP-A in mammals) to the microtubule binding proteins of the Dam1 complex (9). Kinetochores normally assemble hierarchically from the centromere-bound proteins (10). Once assembled, the structural homeostasis of the kinetochore is regulated by proteins that are recruited to kinetochores. For example, the histone subunit Cse4 is tightly regulated by the E3 ubiquitin ligase Psh1, which is localized at centromeres. Psh1 prevents excessive Cse4 centromere loading via ubiquitylation-dependent degradation of Cse4 (and at noncentromeric regions) (11). Cse4 is also phosphorylated by Ipl1, likely to destabilize aberrant microtubule interactions and ensure correct sister chromosome biorientation (12...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization

Ólafsson

Thorpe

2015

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

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“…Although Cdc14 is largely sequestered in the nucleolus until anaphase onset, Dsn1 kinetochore subunit dephosphorylation in metaphase is Cdc14-dependent, suggesting that sufficient levels of Cdc14 may escape nucleolar sequestration and could promote Scc2 dephosphorylation and subsequent cleavage during S phase (43). Further experimentation will be required, however, to determine whether Scc2 is a direct substrate of Cdc14 in vitro.…”

Section: Discussionmentioning

confidence: 99%

Cell cycle-specific cleavage of Scc2 regulates its cohesin deposition activity

Woodman

Fara

Dzieciątkowska

et al. 2014

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

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Sister chromatid cohesion (SCC), efficient DNA repair, and the regulation of some metazoan genes require the association of cohesins with chromosomes. Cohesins are deposited by a conserved heterodimeric loading complex composed of the Scc2 and Scc4 proteins in Saccharomyces cerevisiae, but how the Scc2/Scc4 deposition complex regulates the spatiotemporal association of cohesin with chromosomes is not understood. We examined Scc2 chromatin association during the cell division cycle and found that the affinity of Scc2 for chromatin increases biphasically during the cell cycle, increasing first transiently in late G 1 phase and then again later in G 2 /M. Inactivation of Scc2 following DNA replication reduces cellular viability, suggesting that this post S-phase increase in Scc2 chromatin binding affinity is biologically relevant. Interestingly, high and low Scc2 chromatin binding levels correlate strongly with the presence of full-length or amino-terminally cleaved forms of Scc2, respectively, and the appearance of the cleaved Scc2 species is promoted in vitro either by treatment with specific cell cycle-staged cellular extracts or by dephosphorylation. Importantly, Scc2 cleavage eliminates Scc2-Scc4 physical interactions, and an scc2 truncation mutant that mimics in vivo Scc2 cleavage is defective for cohesin deposition. These observations suggest a previously unidentified mechanism for the spatiotemporal regulation of cohesin association with chromosomes through cell cycle regulation of Scc2 cohesin deposition activity by Scc2 dephosphorylation and cleavage.M ultisubunit, ring-shaped cohesin complexes play key roles in chromosome morphogenesis that are required for faithful chromosome transmission to daughter cells. Newly replicated sister chromatids become tethered together by cohesins during S phase, which promotes chromosome biorientation on mitotic spindles (1). Cohesins also mediate efficient DNA double-strand break repair by homologous recombination (2, 3) and the formation or stabilization of chromatin loops that affect various nuclear processes, such as gene expression and Ig gene rearrangements (reviewed in refs. 4 and 5). Altered gene expression resulting from defective cohesinmediated chromatin looping is likely responsible for the pathogenesis of Cornelia de Lange Syndrome (CdLS), a dominantly inherited human developmental disorder (6).Sister chromatid cohesion (Scc) proteins form a heterodimeric cohesin deposition complex, but the complex's activity in deposition is not understood (7). Cohesins copurify with Scc2/Scc4, suggesting that Scc2/Scc4 plays a direct role in deposition (8-11). In the absence of either loader complex subunit, cohesin rings assemble, but fail to be deposited (7,12,13). ATP hydrolysis by cohesin's structural maintenance of chromosome (SMC) subunits is required for cohesin loading, and deposition is inhibited when SMC hinge domains, which mediate Smc1/3 interactions within cohesin, are artificially tethered (8,14,15). Thus, Scc2/ Scc4 may activate cohesin's ATPase activity or f...

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“…Dsn1 is also phosphorylated at three Cdk1 consensus sites (T12, S69, and S264), among which only S264 is conserved within the Saccharomyces lineage (Akiyoshi and Biggins 2010). While studying Dsn1 phosphorylation, we found that the dsn1-S240A, S250A, S264A triple mutant supports growth in the absence of wild-type DSN1 ( Figure 1B).…”

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confidence: 92%

The Aurora B Kinase Promotes Inner and Outer Kinetochore Interactions in Budding Yeast

2013

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Cdc14-Dependent Dephosphorylation of a Kinetochore Protein Prior to Anaphase in Saccharomyces cerevisiae

Cited by 20 publications

References 48 publications

Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization

Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization

Cell cycle-specific cleavage of Scc2 regulates its cohesin deposition activity

The Aurora B Kinase Promotes Inner and Outer Kinetochore Interactions in Budding Yeast

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