GLOBAL ANALYSIS OF CDC14 PHOSPHATASE REVEALS DIVERSE ROLES IN MITOTIC PROCESSES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 CDC14磷酸酶在酵母生长后期调节多种事件，是芽殖酵母有丝分裂退出所必需的。CDC14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Net1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CDC14结合伙伴，我们采用亲和纯化和质谱分析的方法对CDC14定位或催化活性改变的菌株的相互作用蛋白进行了分析。为了改变CDC14的本地化，我们使用了一种Net1缺失的菌株，它会导致CDC14从核仁中完全释放。为了改变cdc14的活性，我们在cdc14的活性部位(C283S或D253A)产生了突变，允许cdc14结合底物，但不允许去磷酸化。利用这一策略，我们确定了新的CDC14相互作用因子，包括参与有丝分裂事件的多个蛋白质。与野生型相比，这些蛋白质的一个子集对CDC14的催化失活突变体显示出更高的亲和力，这表明它们很可能是CDC14的底物。我们还发现了几个新的与CDC14相互作用的蛋白质，包括Kar9(一种定位有丝分裂纺锤体的蛋白质)和Bni1和Bnr1(使肌动蛋白电缆成核的福尔马林，可能对肌动球蛋白环的收缩很重要)在体外和体内都被CDC14特异性地去磷酸化。我们的发现表明，福尔马林的去磷酸化可能是它们在退出有丝分裂过程中观察到的定位变化的重要原因，并表明CDC14以参与广泛有丝分裂事件的蛋白质为靶标。 一篇描述这项工作的论文正在印刷中： J.Bloom，I.M.Cristea，A.Procko，V.Lubkov，B.T.Chait，M.Snyder，F.R.Cross对CDC14磷酸酶的全局分析揭示了有丝分裂中的不同角色 在印刷机上加工JBC

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