Global Analysis of Cdc14 Dephosphorylation Sites Reveals Essential Regulatory Role in Mitosis and Cytokinesis

Kao, Li Ting; Wang, Yiting; Chen, Yu‐Chen; Tseng, Shun‐Fu; Jhang, J. W.; Chen, Yu‐Ju; Teng, Shu-Chun

doi:10.1074/mcp.m113.032680

Cited by 25 publications

(29 citation statements)

References 70 publications

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“…More generally, phosphorylation plays an important role in kinetochore homeostasis and we noted that one Mtw1 SPI was with Cdc14. CDK phosphorylates a number of kinetochore proteins and Cdc14, when released from the nucleolus in anaphase, reverses a number of these phosphorylations (22,23,42,45). However, the importance of these CDK phosphorylation/dephosphorylation events is unclear.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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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“…S4A) and none in the other subunits of condensin. An additional Cdk1 phospho-site, Ser117, was uncovered in Smc4 in proteome-wide analyses of mitotic cells (Holt et al 2009;Kao et al 2014). Interestingly, all of these Cdk1 phosphosites were clustered in the N-terminal extension of Smc4, a region of the protein that is conserved among eukaryotic Smc4 family members but absent in the Smc1-3 families (Supplemental Fig.…”

Section: The Smc4 Subunit Of Condensin Is a Target For Cdk1 In Early mentioning

confidence: 97%

A high-sensitivity phospho-switch triggered by Cdk1 governs chromosome morphogenesis during cell division

et al. 2015

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The initiation of chromosome morphogenesis marks the beginning of mitosis in all eukaryotic cells. Although many effectors of chromatin compaction have been reported, the nature and design of the essential trigger for global chromosome assembly remain unknown. Here we reveal the identity of the core mechanism responsible for chromosome morphogenesis in early mitosis. We show that the unique sensitivity of the chromosome condensation machinery for the kinase activity of Cdk1 acts as a major driving force for the compaction of chromatin at mitotic entry. This sensitivity is imparted by multisite phosphorylation of a conserved chromatinbinding sensor, the Smc4 protein. The multisite phosphorylation of this sensor integrates the activation state of Cdk1 with the dynamic binding of the condensation machinery to chromatin. Abrogation of this event leads to chromosome segregation defects and lethality, while moderate reduction reveals the existence of a novel chromatin transition state specific to mitosis, the intertwist configuration. Collectively, our results identify the mechanistic basis governing chromosome morphogenesis in early mitosis and how distinct chromatin compaction states can be established via specific thresholds of Cdk1 kinase activity.

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“…Budding yeast Cdc14 is present at the bud neck at the time of cytokinesis (Bembenek et al , ; Palani et al , ), suggesting the possibility of a direct contribution. Although a number of cytokinetic proteins have been shown to be Cdc14 targets (Meitinger et al , ; Kao et al , ), which of these control essential cytokinetic events remains unknown (Breitkreutz et al , ; Bloom et al , ; Kao et al , ).…”

Section: Introductionmentioning

confidence: 99%

Identification of C dk targets that control cytokinesis

et al. 2014

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The final event of the eukaryotic cell cycle is cytokinesis, when two new daughter cells are born. How the timing and execution of cytokinesis is controlled is poorly understood. Here, we show that downregulation of cyclin-dependent kinase (Cdk) activity, together with upregulation of its counteracting phosphatase Cdc14, controls each of the sequential steps of cytokinesis, including furrow ingression, membrane resolution and cell separation in budding yeast. We use phosphoproteome analysis of mitotic exit to identify Cdk targets that are dephosphorylated at the time of cytokinesis. We then apply a new and widely applicable tool to generate conditionally phosphorylated proteins to identify those whose dephosphorylation is required for cytokinesis. This approach identifies Aip1, Ede1 and Inn1 as cytokinetic regulators. Our results suggest that cytokinesis is coordinately controlled by the master cell cycle regulator Cdk together with its counteracting phosphatase and that it is executed by concerted dephosphorylation of Cdk targets involved in several cell biological processes.

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Global Analysis of Cdc14 Dephosphorylation Sites Reveals Essential Regulatory Role in Mitosis and Cytokinesis

Cited by 25 publications

References 70 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

A high-sensitivity phospho-switch triggered by Cdk1 governs chromosome morphogenesis during cell division

Identification of C dk targets that control cytokinesis

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