Regulation of Mitotic Kinetochores by the Ran GTPase

Ran GTPase 对有丝分裂着丝粒的调节

• 批准号: 8553902
• 负责人: MARY C. DASSO
• 金额: $ 66.37万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553902
• 关键词: Anaphase; Aneuploidy; Binding; Biological Assay; Cell Cycle; Cell Cycle Regulation; Cell Nucleus; Cell physiology; Cells; Centromere; Chromatin; Chromosomal Instability; Chromosome Segregation; Chromosomes; Colorectal Cancer; Complex; Cytoplasm; Cytosol; Dactinomycin; Data; Defect; Dissociation; Essential Genes; Event; Exportins; Face; Failure; Family; Fiber; GTP Binding; GTPase-Activating Proteins; Genetic Transcription; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hela Cells; Histone H3; Histones; Human; Hydrolysis; Importins; Interphase; Investigation; Karyopherins; Kinetochores; Link; Lysine; Macromolecular Complexes; Mammalian Cell; Mediating; Metaphase; Metaphase Plate; Microtubule Bundle; Microtubules; Mitosis; Mitotic; Mitotic Chromosome; Mitotic spindle; Molecular Chaperones; Monitor; Mutation; Nuclear; Nuclear Export; Nuclear Import; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nucleoplasm; Nucleotides; Property; Protein Binding; Proteins; RNA Polymerase I; Recruitment Activity; Regulation; Regulatory Pathway; Reporting; Role; Running; Signal Transduction; Sister Chromatid; Site; Structure; System; Time; Vertebrates; Xenopus; alpha Karyopherins; anaphase-promoting complex; beta Karyopherins; cancer type; demethylation; egg; genetic regulatory protein; inhibitor/antagonist; interest; leptomycin B; novel; nucleocytoplasmic transport; prevent; rRNA Genes; ran GTP-Binding Protein; receptor; segregation; trafficking; ubiquitin ligase

The Ran GTPase is required for many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell cycle control. The sole nucleotide exchange factor for Ran, RCC1, binds chromatin throughout the cell cycle. The GTPase activating protein for Ran, RanGAP1, localizes to the cytosolic face of the nuclear pore complex (NPC) during interphase through association with RanBP2, a large nucleoporin. The interphase distribution of Ran regulators leads to a high concentration of Ran-GTP in nuclei, and low Ran-GTP in cytosol. The major effectors for Ran are a family of Ran-GTP binding proteins that were discovered as nuclear transport receptors. These receptors are collectively called Karyopherins; those that mediate import are called Importins, and those that mediate export are called Exportins. Karyopherins transit the NPC in a Ran- and cargo-independent fashion. Their cargo loading is governed by Ran-GTP levels: Importins bind to their cargo in the cytoplasm. Import complexes traverse the NPC and dissociate upon Ran-GTP-Importin binding. Exportins bind their cargo inside nuclei in complexes that contain Ran-GTP. After passage through the NPC, export complexes dissociate upon Ran-GTP hydrolysis. Notably, the capacity of Ran-GTP to bind some Importins and promote their dissociation from their cargos is conditional upon events other than nuclear import, such as the assembly of cargo into macromolecular complexes, suggesting that karyopherins can act as chaperones that assist in the targeting of cargos molecules beyond the NPC. To date, two karyopherins have been shown to act as Ran effectors during mitosis, Importin-beta and the exportin Crm1. Importin-beta binds and imports cargo with classical nuclear localization sequences (cNLSs) through an adaptor subunit, Importin-alpha. In mitotic metazoan cells, Importin-alpha/beta bind and inhibit spindle assembly factors. Elevation of diffusible Ran-GTP concentrations near mitotic chromatin releases inhibition by Importin-alpha/beta, allowing localized activation of such factors. Our studies have been particularly concerned with Ran functions at kinetochores. Kinetochores are proteinaceous structures that assemble at the centromere of each sister chromatid during mitosis, and that serve as sites of spindle microtubule (MT) attachment. The kinetochore fibers (k-fibers) that link mammalian kinetochores to spindle poles contain both MTs that are directly attached to the kinetochores at their plus ends (kMTs) and MTs that are not. Kinetochore attachment is monitored through the spindle assembly checkpoint (SAC), which prevents mitotic exit by blocking anaphase promoting complex/cyclosome (APC/C) activation until all chromosomes are attached and aligned onto the metaphase plate. The APC/C is a ubiquitin ligase that regulates the destruction of key mitotic regulatory proteins. Components of the SAC include: Mad1, Mad2, Mps1, Bub1, Bub3, BubR1, and CENP-E. Elevated levels of Ran-GTP abrogate SAC-mediated mitotic arrest in Xenopus egg extracts (XEEs) and disrupt the kinetochore localization of SAC components, suggesting that the SAC is directly responsive to the overall concentration of Ran-GTP in that system. The effector for Ran in the SAC remains an unresolved issue, and this problem is a major focus of our current interests. (Our findings indicate that the effector is neither Importin-beta nor Crm1.) Crm1 is the exportin for proteins with classical export signals (NESs). We have found that Crm1 localizes to kintochores, and inhibition of Crm1 by Leptomycin B (LMB) in mitosis results in abnormal kinetochore attachment, decreased microtubule (MT) stability and reduced spindle size. These defects are correlated with a failure to recruit the RanGAP1/RanBP2 complex onto kinetochores after MT attachment is established. We examined the proteins that bind Crm1, in order to determine whether Crm1 might regulate targets proteins through sequestration, as has been reported for Importin-beta, and to ascertain whether any targets bind Crm1 in a mitosis-specific fashion. Specifically, we examined the protein binding profile of Crm1 within interphase and mitotic HeLa cell extacts: Many nuclear export cargos bound transiently to Crm1, with only a very small fraction of these proteins bound to Crm1 at any given time. However, we also found a small set of sequestered cargos (SCs) that bind quantitatively to Crm1 in a Ran-GTP-dependent manner. This unusual property of SCs suggests that they may be regulated by Crm1 beyond simple control of their nuclear localization. A putative histone H3 lysine demethylase (KDM3B) was identified as a SC and studied further. We examined the localization of KDM3B, and found that it co-localizes with Crm1 in the nucleoplasm and in intranucleolar bodies (INBs). Strikingly, treatment with LMB caused rapid and complete loss of KDM3B from INBs, indicating that Crm1 is required for KDM3B localization at that site. Cells treated with Actinomycin D, an RNA polymerase I inhibitor, also lost KDM3B from INBs. Chromatin IP (ChIP) assays suggests that KDM3B binds to rRNA genes and qPCR data reveal that treatment with LMB causes decreased transcription of rRNA genes. Collectively, these data suggest that interaction with Crm1-RanGTP regulates KDM3B-mediated histone demethylation and rRNA gene transcription in a novel, transport independent manner.

Ran GTPase是许多细胞功能所必需的，包括核胞质运输、纺锤体组装、核组装和细胞周期控制。Ran的唯一核苷酸交换因子RCC1在整个细胞周期中与染色质结合。Ran的GTPase激活蛋白RanGAP1通过与RanBP2（一种大型核孔蛋白）结合，在间期定位于核孔复合物（NPC）的细胞质表面。Ran调节因子的间期分布导致细胞核内的Ran- gtp浓度高，细胞质内的Ran- gtp浓度低。Ran的主要效应物是被发现作为核转运受体的Ran- gtp结合蛋白家族。这些受体统称为核细胞蛋白；中介导入的称为Importins，中介导出的称为Exportins。核细胞蛋白以独立于Ran和货物的方式运输NPC。它们的货物装载是由rangtp水平控制的：进口蛋白在细胞质中与它们的货物结合。进口复合物穿过NPC并在Ran-GTP-Importin结合时解离。出口蛋白将其货物结合在含有Ran-GTP复合物的核内。通过NPC后，出口复合物在Ran-GTP水解下解离。值得注意的是，Ran-GTP结合一些进口蛋白并促进其与货物分离的能力取决于核进口以外的事件，例如货物组装成大分子复合物，这表明核细胞蛋白可以作为伴侣，协助靶向NPC以外的货物分子。迄今为止，有两种核细胞蛋白已被证明在有丝分裂过程中起Ran效应，它们是importin - β和出口蛋白Crm1。Importin-beta通过一个适配器亚基Importin-alpha与经典的核定位序列（cNLSs）结合和进口货物。在有丝分裂后生细胞中，importin - α / β结合并抑制纺锤体组装因子。有丝分裂染色质附近可扩散的Ran-GTP浓度升高，可通过importin - α / β释放抑制，从而允许这些因子的局部激活。