GLOBAL ANALYSIS OF CDC14 PHOSPHATASE REVEALS DIVERSE ROLES IN MITOTIC PROCESSES

CDC14 磷酸酶的整体分析揭示了有丝分裂过程中的多种作用

• 批准号: 8361505
• 负责人: FREDERICK R. CROSS
• 金额: $ 0.26万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361505
• 关键词: Actins; Active Sites; Actomyosin; Affinity; Affinity Chromatography; Anaphase; Binding; Cell Cycle; Cell Nucleolus; Cell Nucleus; Cytoplasm; Event; Funding; Grant; In Vitro; Mitosis; Mitotic; Mitotic spindle; Mutation; National Center for Research Resources; Nucleolar Proteins; Paper; Phosphoric Monoester Hydrolases; Principal Investigator; Process; Protein Dephosphorylation; Proteins; Research; Research Infrastructure; Resources; Role; Saccharomycetales; Source; United States National Institutes of Health; Work; cost; in vivo; macromolecule; mutant; novel

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cdc14 phosphatase regulates multiple events during anaphase and is essential for mitotic exit in budding yeast. Cdc14 is regulated in both a spatial and temporal manner. It is sequestered in the nucleolus for most of the cell cycle by the nucleolar protein Net1 and is released into the nucleus and cytoplasm during anaphase. To identify novel binding partners of Cdc14, we used affinity purification of Cdc14 and mass spectrometric analysis of interacting proteins from strains in which Cdc14 localization or catalytic activity was altered. To alter Cdc14 localization, we used a strain deleted for NET1 which causes full release of Cdc14 from the nucleolus. To alter Cdc14 activity, we generated mutations in the active site of Cdc14 (C283S or D253A) which allows binding of substrates, but not dephosphorylation, by Cdc14. Using this strategy, we identified new interactors of Cdc14, including multiple proteins involved in mitotic events. A subset of these proteins displayed increased affinity for catalytically inactive mutants of Cdc14 compared to the wild-type version, suggesting they are likely substrates of Cdc14. We have also shown that several of the novel Cdc14-interacting proteins, including Kar9 (a protein that orients the mitotic spindle) and Bni1 and Bnr1 (formins that nucleate actin cables and may be important for actomyosin ring contraction) are specifically dephosphorylated by Cdc14 in vitro and in vivo. Our findings suggest the dephosphorylation of the formins may be important for their observed localization change during exit from mitosis and indicate that Cdc14 targets proteins involved in wide-ranging mitotic events. A paper describing this work is in press: J. Bloom, I.M. Cristea, A. Procko, V. Lubkov, B.T. Chait, M. Snyder, F. R. Cross Global analysis of CDC14 phosphatase reveals diverse roles in mitotic processes JBC In press

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 cdc 14磷酸酶在芽殖酵母有丝分裂后期调控多个事件，是有丝分裂退出所必需的。cdc 14在空间和时间上都受到调控。在细胞周期的大部分时间里，它被核仁蛋白Net 1隔离在核仁中，并在后期释放到细胞核和细胞质中。为了确定新的结合合作伙伴的Cdc 14，我们使用的Cdc 14亲和纯化和质谱分析的相互作用的蛋白质菌株中，Cdc 14的本地化或催化活性被改变。为了改变Cdc 14的定位，我们使用了一种删除NET 1的菌株，它会导致Cdc 14从核仁中完全释放。为了改变Cdc 14的活性，我们在Cdc 14的活性位点（C283 S或D253 A）中产生突变，其允许Cdc 14结合底物，但不脱磷酸化。使用这种策略，我们确定了新的相互作用的Cdc 14，包括多个蛋白质参与有丝分裂事件。这些蛋白质的一个子集表现出增加的亲和力的催化失活突变体的Cdc 14相比，野生型的版本，这表明他们可能是Cdc 14的基板。我们还表明，几种新的Cdc 14相互作用的蛋白质，包括Kar 9（一种蛋白质，定向有丝分裂纺锤体）和Bni 1和Bnr 1（形成核肌动蛋白电缆，并可能是重要的肌动球蛋白环收缩）特异性去磷酸化Cdc 14在体外和体内。我们的研究结果表明，formin的去磷酸化可能是重要的，他们观察到的本地化变化退出有丝分裂过程中，并表明Cdc 14的目标蛋白参与广泛的有丝分裂事件。 描述这项工作的一份文件正在印刷中： J.布鲁姆，I. M. Cristea，A. Procko，V. Lubkov，B.T. Chait，M. Snyder，F. R. CDC 14磷酸酶的交叉全局分析揭示了在有丝分裂中的不同作用 JBC印刷中