Mitotic Roles Of Ran GTPase

Ran GTPase 的有丝分裂作用

• 批准号: 6813963
• 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/6813963
• 关键词: Xenopus oocyte; 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 (see Z01 HD001902-09). Ran-GTP nucleotide hydrolysis also requires a family of accessory proteins. The best-characterized member of this family is mammalian RanBP1, which is distributed to the cytosol during interphase. RanBP1 accelerates the rate of RanGAP1-mediated Ran-GTP hydrolysis by about an order of magnitude in vitro. RanBP1 also promotes dissociation Ran-GTP from transport receptors, whose binding would otherwise block RanGAP-mediated GTP hydrolysis. The distribution of Ran's regulators has been widely hypothesized to modulate local concentrations of Ran-GTP within the cell, spatially directing the many processes in which Ran has been implicated. Ran's primary known effectors are a set of Ran-GTP binding proteins that were originally described as nuclear transport receptors. Ran-GTP binding regulates association between these proteins and their transport cargoes. Defects in the Ran pathway disrupt both the onset and the completion of mitosis, although Ran's function in cell cycle progression had not been clearly distinguished from its roles in nuclear transport and spindle assembly. We were therefore interested in examining Ran's role in mitotic regulation more closely. Mitosis is tightly controlled in eukaryotes by the activity of Cyclin B and Securin. Both Cyclin B and Securin are ubiquitinated at the metaphase-anaphase transition by an E3 ligase called the anaphase-promoting complex/cyclosome (APC/C), working in association with its activators Cdc20/FZY and Cdh1/FZR. In the presence of misassembled spindles, with kinetochores that are unattached or that lack tension from spindle microtubules, the onset of anaphase is delayed through activation of a spindle assembly checkpoint. This checkpoint pathway prevents APC/CFZY activation and thereby stabilizes APC/CFZY substrates. After all of the chromosomes have become attached and aligned within the mitotic spindle, the checkpoint is turned off, APC/CFZY becomes active and anaphase commences. Components of the spindle assembly checkpoint include: Mad1, Mad2, Mps1, Bub1, Bub3, BubR1 and CENP-E. We have examined the role of Ran in regulating mitotic checkpoints using Xenopus egg extracts, a well-established model system for checkpoint control. During normal cell cycles in cycling egg extracts, we find that the amount of chromatin-associated RCC1 increases dramatically at the onset of Cyclin B destruction. Moreover, moderate levels of exogenous RCC1 protein abrogate mitotic spindle checkpoint arrest and allow Cyclin B destruction in extracts containing nuclei plus the nocodazole, a microtubule depolymerizing agent. We find that the spindle assembly checkpoint in Xenopus is characterized by decreased APCFZY activity and addition of RCC1 to extracts with the activated checkpoint restores APCFZY activity to control levels. In order to determine the precise mechanism through which RCC1 abrogates checkpoint arrest, we examined the localization of mitotic regulators, including Mad2, CENP-E, Bub1 and Bub3. We find that these proteins are mislocalized away from kinetochores in nocodazole-treated extracts after the addition of high levels of RCC1 protein. Interestingly, displacement of Bub1 and Bub3 from kinetochores could be reversed by the addition of recombinant RanGAP1 protein, suggesting that their behavior responds directly by Ran-GTP levels. Taken together, our results indicate that the Ran pathway is normally regulated in a highly dynamic manner during mitosis. The transitions of the Ran pathway can be mimicked by addition of exogenous RCC1 protein, triggering the metaphase-anaphase transition prematurely in the presence of unattached kinetochores. Our observations suggest that changes in RCC1's chromosomal dynamics may be a key link in the chain of events between completion of metaphase spindle assembly and mitotic exit.

Ran是核质运输、纺锤体组装、核组装和细胞周期控制所需的小GT3。Ran的核苷酸交换因子RCC 1是一种染色质相关蛋白。Ran的GT3激活蛋白RanGAP 1在间期位于细胞质中。在有丝分裂过程中，大部分RanGAP 1广泛分布，尽管相当一部分RanGAP 1与动粒相关（参见Z 01 HD 001902 -09）。Ran-GTP核苷酸水解还需要辅助蛋白家族。该家族的最佳特征成员是哺乳动物RanBP 1，其在间期期间分布到胞质溶胶中。RanBP 1在体外将RanGAP 1介导的Ran-GTP水解速率加速约一个数量级。RanBP 1还促进Ran-GTP从转运受体的解离，否则其结合将阻断RanGAP介导的GTP水解。Ran的调节剂的分布已被广泛假设为调节细胞内Ran-GTP的局部浓度，在空间上指导Ran涉及的许多过程。Ran的主要已知效应物是一组最初被描述为核转运受体的Ran-GTP结合蛋白。Ran-GTP结合调节这些蛋白质和它们的运输货物之间的关联。 Ran通路的缺陷破坏了有丝分裂的开始和完成，尽管Ran在细胞周期进程中的功能与其在核运输和纺锤体组装中的作用没有明确区分。因此，我们感兴趣的是更密切地研究Ran在有丝分裂调控中的作用。在真核生物中，有丝分裂受Cyclin B和Securin活性的严格控制。细胞周期蛋白B和Securin都在中期-后期转换时被称为后期促进复合物/细胞周期体（APC/C）的E3连接酶泛素化，与其激活剂Cdc 20/FZY和Cdh 1/FZR联合作用。在错误组装的纺锤体的存在下，与着丝粒是独立的或缺乏张力的纺锤体微管，后期的开始被延迟通过激活的纺锤体组装检查点。该检查点途径防止APC/CFZY活化，从而稳定APC/CFZY底物。当所有的染色体都在有丝分裂纺锤体中附着并排列后，检查点关闭，APC/CFZY变得活跃，后期开始。纺锤体组装检查点的组件包括：Mad 1、Mad 2、Mps 1、Bub 1、Bub 3、BubR 1和CENP-E。 我们已经研究了Ran在使用非洲爪蟾卵提取物调节有丝分裂检查点中的作用，这是检查点控制的一个完善的模型系统。在正常的细胞周期中，我们发现，在周期蛋白B破坏的开始，染色质相关的RCC 1的量显着增加。此外，中等水平的外源性RCC 1蛋白消除有丝分裂纺锤体检查点阻滞，并允许细胞周期蛋白B在含有细胞核的提取物中被破坏加上诺考达唑，一种微管解聚剂。我们发现，非洲爪蟾的纺锤体组装检查点的特点是APCFZY活性降低，并添加RCC 1与激活的检查点提取物恢复APCFZY活性控制水平。为了确定RCC 1消除检查点阻滞的确切机制，我们检查了有丝分裂调节因子的定位，包括Mad 2，CENP-E，Bub 1和Bub 3。我们发现，这些蛋白质被错误定位远离着丝粒在nocodazole处理的提取物后，加入高水平的RCC 1蛋白。有趣的是，Bub 1和Bub 3从动粒的位移可以通过添加重组RanGAP 1蛋白来逆转，这表明它们的行为直接响应Ran-GTP水平。两者合计，我们的研究结果表明，Ran途径通常是在有丝分裂过程中以高度动态的方式进行调节。Ran途径的转换可以通过添加外源性RCC 1蛋白来模拟，在未连接的动粒存在下过早地触发中期-后期转换。我们的观察表明，在RCC 1的染色体动力学的变化可能是一个关键环节之间的事件链完成中期纺锤体组装和有丝分裂退出。