Regulation of Mitotic Kinetochores by the Ran GTPase

Ran GTPase 对有丝分裂着丝粒的调节

• 批准号: 8351167
• 负责人: MARY C. DASSO
• 金额: $ 66.4万
• 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/8351167
• 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. SCCR 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 GT3是许多细胞功能所必需的，包括核质运输、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RCC 1在整个细胞周期中与染色质结合。Ran的GTP酶激活蛋白RanGAP 1通过与RanBP 2（一种大的核孔蛋白）结合，在间期定位于核孔复合物（NPC）的胞质表面。Ran调节剂的间期分布导致细胞核中高浓度的Ran-GTP和细胞质中低浓度的Ran-GTP。Ran的主要效应物是Ran-GTP结合蛋白家族，其被发现为核转运受体。这些受体统称为Karyopherins;介导输入的受体称为Importins，介导输出的受体称为Exportins。Karyopherins以Ran和Cargo独立的方式转运NPC。它们的货物装载由Ran-GTP水平控制：输入蛋白在细胞质中与它们的货物结合。输入复合物穿过NPC并在Ran-GTP-输入蛋白结合后解离。输出蛋白将其货物结合在含有Ran-GTP的复合物中的细胞核内。通过NPC后，输出复合物在Ran-GTP水解后解离。值得注意的是，Ran-GTP结合一些Importins并促进其与其货物解离的能力取决于核输入以外的事件，例如货物组装成大分子复合物，这表明核粒转运蛋白可以充当分子伴侣，帮助靶向NPC以外的货物分子。到目前为止，两个karyopherins已被证明作为Ran效应器在有丝分裂，输入β和exportin Crm 1。Importin-beta通过衔接子亚基Importin-alpha与经典核定位序列（cNLS）结合并输入货物。在有丝分裂的后生动物细胞中，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Ran-GTP浓度的升高释放了Importin-α/β的抑制作用，允许这些因子的局部激活。 SCCR研究特别关注Ran在着丝粒中的功能。着丝粒是一种蛋白质结构，在有丝分裂过程中聚集在每个姐妹染色单体的着丝粒上，并作为纺锤体微管（MT）附着的位点。将哺乳动物动粒连接到纺锤体极的动粒纤维（k-纤维）包含在它们的正端（kMT）直接连接到动粒的MT和不直接连接到动粒的MT。通过纺锤体组装检查点（SAC）监测动粒附着，SAC通过阻断后期促进复合体/细胞核小体（APC/C）激活来防止有丝分裂退出，直到所有染色体附着并对齐到中期平板上。APC/C是一种泛素连接酶，调节关键有丝分裂调节蛋白的破坏。SAC的组成部分包括：Mad 1、Mad 2、Mps 1、Bub 1、Bub 3、BubR 1和CENP-E。Ran-GTP的水平升高废除SAC介导的有丝分裂阻滞在非洲爪蟾卵提取物（XEEs）和破坏的动粒定位SAC组件，这表明SAC是直接响应的Ran-GTP在该系统中的总浓度。SAC中的Ran效应器仍然是一个未解决的问题，这个问题是我们当前关注的一个主要焦点。(Our研究结果表明，效应器既不是Importin-beta也不是Crm 1。 Crm 1是具有经典输出信号（NES）的蛋白质的输出蛋白。我们发现Crm 1定位于着丝粒，在有丝分裂中，Leptomycin B（LMB）抑制Crm 1可导致着丝粒附着异常，微管（MT）稳定性降低和纺锤体尺寸减小。这些缺陷与MT附着建立后不能将RanGAP 1/RanBP 2复合物募集到着丝粒上相关。我们研究了结合Crm 1的蛋白质，以确定Crm 1是否可能通过螯合作用调节靶蛋白，如已报道的Importin-β，并确定是否有任何靶蛋白以有丝分裂特异性方式结合Crm 1。具体来说，我们研究了Crm 1的蛋白结合曲线在间期和有丝分裂HeLa细胞提取物：许多核出口货物绑定瞬时Crm 1，只有一个非常小的部分，这些蛋白质结合Crm 1在任何给定的时间。然而，我们也发现了一小部分隔离货物（SC），定量结合Crm 1的Ran-GTP依赖的方式。SC的这种不寻常的特性表明，它们可能受到Crm 1的调控，超出了简单的核定位控制。一个假定的组蛋白H3赖氨酸脱甲基酶（KDM 3B）被确定为SC和进一步研究。我们研究了KDM 3B的定位，发现它与Crm 1共定位于核质和核仁内体（INBs）中。引人注目的是，用LMB处理导致KDM 3B从INB中快速和完全丢失，表明Crm 1是KDM 3B在该位点定位所需的。用放线菌素D（RNA聚合酶I抑制剂）处理的细胞也从INB中丢失了KDM 3B。染色质IP（ChIP）测定表明，KDM 3B与rRNA基因结合，qPCR数据显示，LMB处理导致rRNA基因转录降低。总的来说，这些数据表明，与Crm 1-RanGTP的相互作用调节KDM 3B介导的组蛋白去甲基化和rRNA基因转录的一种新的，不依赖于运输的方式。