Mitotic Roles Of Ran GTPase

Ran GTPase 的有丝分裂作用

• 批准号: 6993681
• 负责人: MARY C. DASSO
• 金额: --
• 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/6993681
• 关键词: Xenopus oocyte; cell cycle; cell growth regulation; centromere; cyclins; cytogenetics; guanine nucleotide exchange factors; guanosinetriphosphatase activating protein; guanosinetriphosphatases; mitotic spindle apparatus; protein localization; protein structure function

Ran is a small GTPase required for nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell cycle control. The nucleotide exchange factor for Ran, RCC1, is a chromatin-associated protein. The GTPase activating protein for Ran, RanGAP1, is cytoplasmic during interphase. During mitosis, the bulk of RanGAP1 is broadly distributed, although a significant fraction of RanGAP1 becomes associated with kinetochores in a SUMO-1 dependent fashion (see Z01 HD001902-10). Ran-GTP nucleotide hydrolysis also requires a family of Ran-GTP binding proteins, which act as RanGAP1 accessory factors. This family includes RanBP1 and RanBP2. The distribution of Ran?s regulators has been widely hypothesized to modulate local concentrations of Ran-GTP within cells, spatially directing processes in which Ran has been implicated. We have been examining the mechanisms through which key Ran regulators are localized within mitotic metazoan cells and the functional consequences to cells when such distribution patterns are disrupted. To look at the mitotic fate of RCC1, we examined its chromosomal association in cycling Xenopus egg extracts. Remarkably, the amount of chromatin-associated RCC1 increased drastically at anaphase onset. In order to determine the significance of this finding, we assayed whether the Ran pathway has a role in mitotic progression or checkpoint control in Xenopus egg extracts. Prior to each anaphase, chromosomes are aligned onto the metaphase through attachment between spindle microtubules and kinetochores, proteinaceous structures that assemble over the centromere of each chromosome. The spindle assembly checkpoint is a cell cycle regulatory pathway that monitors spindle assembly in all eukaryotic cells and prevents the onset of anaphase and the dissolution of sister chromatin cohesion in the presence of unattached on inappropriately attached kinetochores. Remarkably, a five- to seven-fold elevation of RCC1 concentration was sufficient to abrogate spindle checkpoint arrest in extracts containing nuclei plus the microtubule depolymerizing agent nocodazole. While assembly of centromeric structures occurred normally under these circumstances, we found that many checkpoint components were mis-localized away from kinetochores after RCC1 addition, indicating that increased RCC1 levels abolish checkpoint arrest by altering interactions between kinetochores and checkpoint regulators. Together with additional data, these observations suggest that the spindle checkpoint is directly responsive to Ran-GTP levels. Notably, the capacity of RCC1 to reverse spindle checkpoint arrest is specific, since increased RCC1 does not compromise other modes of M phase arrest (e.g. CSF arrest). Our results suggest a model wherein complete chromosome alignment on the metaphase plate triggers the increased binding of RCC1 to chromosomes, resulting in the local elevation of Ran-GTP levels and the ejection of the final population of kinetochore-associated checkpoint components. In parallel to our studies on RCC1, we have investigated the mitotic behavior and function of RanGAP1. In metazoans, RanGAP1 is conjugated with SUMO-1. Studies by this group and others have shown that SUMO-1 modification causes RanGAP1 to associate during interphase with Ubc9 and RanBP2, a large nuclear pore protein with multiple Ran-GTP binding domains and a SUMO E3 ligase domain. Through further investigation of the mitotic behavior and interactions of RanGAP1, we have found that RanGAP1 associates with kinetochores in a SUMO-1 dependent manner. Notably, RanBP2 co-localized with RanGAP1 on spindles and kinetochores. Recently, we have examined the structural requirements for targeting RanGAP1 and RanBP2, as well as their function in mitosis. We found that elimination of RanBP2 expression through RNA interference (RNAi) displaced RanGAP1 from kinetochores, supporting the notion that these proteins target to kinetochores as part of a single complex. Both proteins were displaced after RNAi of integral kinetochore components, suggesting that they require intact kinetochore structures to localize appropriately. By contrast, peripheral kinetochore proteins were not essential for correct targeting of either protein. Cells depleted of RanBP2 show abnormalities in both spindle formation and mitotic progression, substantiating the importance of correct targeting of the RanGAP1/RanBP2 complex during mitosis.

RAN是核细胞量运输，纺锤体组装，核装配和细胞周期控制所需的小GTPase。 RAN RCC1的核苷酸交换因子是一种染色质相关蛋白。 RAN，RangaP1的GTPase激活蛋白在相间期间是细胞质。在有丝分裂过程中，大部分RangaP1的分布广泛分布，尽管Rangap1的很大一部分以SUMO-1依赖性方式与动力学相关（请参阅Z01 HD001902-10）。 RAN-GTP核苷酸水解还需要RAN-GTP结合蛋白家族，该蛋白充当Rangap1辅助因子。这个家庭包括RANBP1和RANBP2。 RAN的调节剂的分布已被广泛假设，以调节细胞内RAN的局部浓度，这是涉及RAN的空间指导过程。我们一直在研究钥匙运行调节剂在有丝分裂的后生细胞中定位的机制，以及当这种分布模式被破坏时对细胞的功能后果。 为了看一下RCC1的有丝分裂命运，我们检查了其在循环爪蟾鸡蛋提取物中的染色体缔合。值得注意的是，在后期开始时与染色质相关的RCC1的量急剧增加。为了确定这一发现的重要性，我们测定了RAN途径在Xenopus卵提取物中的有丝分裂进程或检查点控制中的作用。在每个后期之前，染色体通过纺锤体微管与动力学，蛋白质结构之间的附着在每个染色体的丝粒上的蛋白质结构进行对齐。主轴组件检查点是一种细胞周期调节途径，该途径在所有真核细胞中监视主轴组件，并防止后期的发作和姐妹染色质凝聚力在不恰当地附着的动型的存在下存在的姐妹染色质凝聚力。值得注意的是，RCC1浓度的五到七倍足以消除含有核的提取物以及微管解聚剂Nocodazole中的纺锤体检查点停滞。虽然在这种情况下正常发生丝粒结构的组装，但我们发现，在添加RCC1后，许多检查点组件从动力学上误差了，这表明增加了RCC1水平通过改变动力学和检查点调节剂之间的相互作用来取消检查点的停滞。这些观察结果与其他数据一起表明，主轴检查点直接响应RAN GTP水平。值得注意的是，RCC1逆向纺锤体检查点停滞的能力是具体的，因为增加的RCC1不会损害其他M相倒置模式（例如CSF逮捕）。我们的结果表明了一个模型，其中中期板上的完全染色体对准会触发RCC1与染色体的结合增加，从而导致RAN-GTP水平的局部升高以及动脉底层相关检查点的最终种群的射出。 与我们对RCC1的研究并行，我们研究了Rangap1的有丝分裂行为和功能。在后生动物中，rangap1与SUMO-1结合。该组和其他人的研究表明，SUMO-1的修饰会导致Rangap1与UBC9和RANBP2相互关联，UBC9和RANBP2是一种具有多个RAN GTP结合域和SUMO E3连接酶结构域的大核孔蛋白。通过进一步研究Rangap1的有丝分裂行为和相互作用，我们发现Rangap1以SUMO-1依赖性方式与动力学相关。值得注意的是，RANBP2与纺锤体和动力学上的RangaP1共定位。最近，我们研究了靶向RANGAP1和RANBP2及其在有丝分裂中的功能的结构要求。我们发现，通过RNA干扰（RNAi）从动力学上取代RANGAP1，消除了RANBP2的表达，这支持了这些蛋白作为单个复合物的一部分靶向动力学的概念。两种蛋白质在整体运动学成分的RNAi后都取代，这表明它们需要完整的动力学结构才能适当定位。相比之下，周围的动力学蛋白对于正确靶向两种蛋白不是必不可少的。 RANBP2耗尽的细胞在纺锤体形成和有丝分裂进展中均表现出异常，证明了在有丝分裂过程中正确靶向RANGAP1/RANBP2复合物的重要性。