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

基本信息

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
项目类别：
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

项目摘要

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

该子项目是利用资源的众多研究子项目之一由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持并且子项目的主要研究者可能是由其他来源提供的，包括其他 NIH 来源。子项目可能列出的总成本代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 Cdc14 磷酸酶调节后期的多个事件，对于芽殖酵母的有丝分裂退出至关重要。 Cdc14 以空间和时间方式受到调节。在细胞周期的大部分时间里，它被核仁蛋白 Net1 隔离在核仁中，并在后期释放到细胞核和细胞质中。为了鉴定 Cdc14 的新结合伴侣，我们对 Cdc14 进行亲和纯化，并对 Cdc14 定位或催化活性发生改变的菌株中的相互作用蛋白进行质谱分析。为了改变 Cdc14 定位，我们使用了 NET1 删除的菌株，这会导致 Cdc14 从核仁中完全释放。为了改变 Cdc14 的活性，我们在 Cdc14 的活性位点（C283S 或 D253A）产生突变，允许 Cdc14 结合底物，但不能去磷酸化。利用这种策略，我们鉴定了 Cdc14 的新相互作用因子，包括参与有丝分裂事件的多种蛋白质。与野生型版本相比，这些蛋白质的一个子集对 Cdc14 催化失活突变体表现出更高的亲和力，表明它们可能是 Cdc14 的底物。我们还表明，一些新的 Cdc14 相互作用蛋白，包括 Kar9（一种定向有丝分裂纺锤体的蛋白）和 Bni1 和 Bnr1（使肌动蛋白缆成核的福尔马林，可能对肌动球蛋白环收缩很重要），在 Cdc14 中被特异性去磷酸化。体外和体内。我们的研究结果表明，福尔明的去磷酸化可能对其在有丝分裂退出过程中观察到的定位变化很重要，并表明 Cdc14 靶向参与广泛有丝分裂事件的蛋白质。描述这项工作的论文正在出版： J. Bloom、I.M. Cristea、A. Procko、V. Lubkov、B.T. Chait, M. Snyder, F. R. CDC14 磷酸酶的 Cross Global 分析揭示了有丝分裂中的不同作用流程 JBC 新闻中