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，包括核质运输，主轴组件，核装配和细胞周期控制。 RAN RCC1的唯一核苷酸交换因子在整个细胞周期中结合染色质。 RANGAP1的GTPase激活蛋白质通过与大核孔RANBP2缔合，将其定位于核孔复合物（NPC）的胞质面（NPC）。 RAN调节剂的相间分布导致核中高浓度的RAN GTP，而细胞质中的RAN GTP低。 RAN的主要效应子是被发现为核转运受体的RAN GTP结合蛋白家族。这些受体被统称为核蛋白。介导的导入的人称为导入蛋白，介导导出的人称为导出。核蛋白以跑步和货物独立的方式过渡NPC。他们的货物负荷受RAN GTP水平的控制：进口素与其在细胞质中的货物结合。导入复合物穿越NPC并在RAN-GTP-rimettring结合上解离。导出蛋白将其货物在核中结合，其中包含RAN-GTP的复合物。通过NPC后，导出复合物将在RAN GTP水解后解离。值得注意的是，RAN-GTP结合某些进口蛋白并促进其与碳的解离的能力是有条件的，这是基于核进口以外的其他事件，例如将货物组装到大分子分子络合物中，这表明核蛋白可以作为伴侣的伴侣，从而有助于靶向NPC以外的Cargos Molecules的靶向。迄今为止，已经证明两种核蛋白在有丝分裂，Importin-Beta和Exportin CRM1期间起效应子。 Importin-Beta通过适配器亚基Imptimin-Alpha结合和进口货物与经典的核定位序列（CNLS）。在有丝分裂的后生细胞中，进口蛋白-Alpha/beta结合并抑制纺锤体组装因子。在有丝分裂染色质附近的可扩散RAN GTP浓度升高通过进口蛋白-Alpha/beta抑制，从而允许对此类因素进行局部激活。 我们的研究特别关注动力学的运行功能。动力学是蛋白质结构，在有丝分裂过程中在每个姐妹染色质剂的中心粒中组装，并且用作纺锤微管（MT）附着的位点。将哺乳动物动力学连接到主轴杆的动力学纤维（k纤维）都包含直接连接到其正末端（KMTS）和没有的MT的MTS。通过纺锤体组件检查点（SAC）监测动力学附件，该检查点（SAC）通过阻止后期促进复合物/循环体（APC/C）激活来防止有丝分裂出口，直到将所有染色体连接到中期板上。 APC/C是一种泛素连接酶，可调节关键有丝分裂调节蛋白的破坏。 SAC的组件包括：MAD1，MAD2，MP​​S1，BUB1，BUB3，BUBR1和CENP-E。 RAN-GTP水平升高消除了SAC介导的Xenopus卵提取物（XEES）中的有丝分裂停滞，并破坏了SAC成分的动力学定位，这表明该SAC直接响应该系统中RAN-GTP的整体浓度。 SAC中RAN的效应子仍然是一个尚未解决的问题，这个问题是我们当前利益的主要重点。 （我们的发现表明效应子既不是importin-beta也不是CRM1。） CRM1是具有经典导出信号（NESS）的蛋白质的导出蛋白。我们发现，CRM1定位于Kintochores，并在有丝分裂中对CRM1抑制CRM1导致动物学异常附着，降低微管（MT）稳定性和纺锤体尺寸的降低。这些缺陷与未在建立MT附件后未能募集Rangap1/Ranbp2复合物上的Rangap1/Ranbp2复合物相关。我们检查了结合CRM1的蛋白质，以确定CRM1是否可以通过固存调节靶标的蛋白质，就像据报道的Importin-Beta报道，并确定任何靶标是否以有丝分裂特异性方式结合CRM1。具体而言，我们检查了相间和有丝分裂HeLa细胞内CRM1的蛋白质结合曲线扩展：许多核出口肉类瞬时与CRM1结合，在任何给定时间，这些蛋白质中只有很小的一小部分与CRM1结合。然而，我们还发现了一组隔离的碳（SC），它们以RAN GTP依赖性方式定量与CRM1结合。 SCS的这种异常特性表明，它们可能受到CRM1的调节，而不是简单地控制其核定位。 假定的组蛋白H3赖氨酸脱甲基酶（KDM3B）被鉴定为SC，并进一步研究。我们检查了KDM3b的定位，发现它与核质和核内体（INB）中的CRM1共定位。令人惊讶的是，使用LMB的治疗导致INBS的KDM3B迅速而完全丢失，这表明该站点的KDM3B定位需要CRM1。用放线霉素D治疗的细胞（一种RNA聚合酶I抑制剂）也损失了INBS的KDM3B。染色质IP（芯片）测定表明KDM3B与rRNA基因结合，QPCR数据表明，用LMB的治疗导致RRNA基因转录降低。总的来说，这些数据表明，与CRM1-RANGTP的相互作用调节KDM3B介导的组蛋白去甲基化和RRNA基因转录以新颖的独立运输方式。