Regulation of Mitotic Kinetochores by the Ran GTPase

Ran GTPase 对有丝分裂着丝粒的调节

• 批准号: 8149305
• 负责人: MARY C. DASSO
• 金额: $ 55.62万
• 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/8149305
• 关键词: Guanosine Triphosphate Phosphohydrolases; Kinetochores; Mitotic; Regulation; Running

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. To date, two nuclear transport receptors 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 (SAFs). 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.) We have found that Crm1 localizes to kintochores, and that inhibition of Crm1 ternary complex formation blocks kinetochore recruitment of RanGAP1/RanBP2. Crm1 itself requires neither ternary complex assembly nor MTs for kinetochore binding. Under these circumstances, kinetochores dramatically failed to maintain discrete end-on attachments to single k-fibers and showed a resultant elevation in chromosome mis-segregation. The component(s) at kinetochores that is directly involved in Crm1 recruitment is a major focus of our ongoing studies. In addition, we hypothesize that Crm1 acts in mitosis through sequestration of its cargoes, as Importin-alpha/beta does. We have identified to cargos of Crm1 within lysates of HeLa cells arrested at different cell cycle stages. Western blot analysis revealed that the cargos could be categorized into two groups with their respect to Crm1 binding: low-affinity and high-affinity cargoes (LACs and HACs, respectively). LACs were defined as those that could bind to tagged Crm1 protein in the presence of Ran-GTP, but whose depletion from the lysate was limited to 1-2% of the total amount in HeLa extracts. All tested LACs showed accumulation in the nucleus upon Crm1 inhibition. HACs were defined by their depletion from lysates through affinity chromatography after incubation with tagged Crm1 and Ran-GTP. Notably, Crm1 should quantitatively sequester HACs during mitosis when it shares the same compartment with both Ran-GTP and HACs. These complexes would be dissociated after the nuclear envelope is re-established in telophase, allowing Crm1 to return to its role as an exportin. We are currently investigating the functions of HACs, the biochemistry of their high affinity binding and their mode of regulation through Crm1.

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。迄今为止，有两种核转运受体已被证明在有丝分裂过程中起Ran效应，即进口蛋白- 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（SAFs）。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。