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，尽管Rangap1的很大一部分与动力学相关，但大部分Rangap1的分布均匀分布（请参阅Z01 HD001902-09）。 RAN GTP核苷酸水解还需要一系列辅助蛋白。该家族中特征最佳的成员是哺乳动物RANBP1，该哺乳动物ranbp1在相间期间分布在细胞质中。 RANBP1加速了RangaP1介导的RAN-GTP水解速率，大约在体外的数量级。 RANBP1还促进了从转运受体中解离RAN GTP，否则其结合将阻止rangap介导的GTP水解。已广泛认为RAN调节剂的分布以调节细胞内RAN GTP的局部浓度，从而在空间上指导了与RAN有关的许多过程。 RAN的主要已知效应子是一组RAN-GTP结合蛋白，最初被描述为核转运受体。 RAN GTP结合调节这些蛋白质与其运输货物之间的关联。 RAN途径中的缺陷破坏了有丝分裂的发作和完成，尽管RAN在细胞周期进程中的功能尚未与其在核运输和纺锤体组件中的作用明确区分开。因此，我们有兴趣更仔细地检查RAN在有丝分裂调节中的作用。有丝分裂通过细胞周期蛋白B和Secuin的活性在真核生物中严格控制。 Cyclin B和Secuin均在中期 - 解相体的转变上由称为自源促进酶的复合体/循环体（APC/C）的E3连接酶泛素化，并与其激活剂CDC20/FZY和CDH1/FZR合作。在存在错误组装的纺锤体的情况下，动力学无辅助或缺乏纺锤体微管的张力，后期的发作通过激活纺锤体组件检查点而延迟。此检查点途径可防止APC/CFZY激活，从而稳定APC/CFZY底物。在所有染色体已连接并对齐有丝分裂主轴后，关闭了检查点，APC/CFZY变得活跃，后期开始。主轴组件的组件检查点包括：MAD1，MAD2，MP​​S1，BUB1，BUB3，BUB3，BUBR1和CENP-E。 我们已经检查了RAN在使用Xenopus Egg提取物（一种完善的模型系统进行检查点控制的模型系统）中调节有丝分裂检查点的作用。在循环卵提取物中的正常细胞周期中，我们发现在细胞周期蛋白B破坏开始时，与染色质相关的RCC1的量显着增加。此外，中等水平的外源RCC1蛋白消除了有丝分裂纺锤体检查点停滞，并允许含有核的提取物加上核酸氮唑，诺科达唑（一种微管解聚剂）。我们发现，爪蟾中的主轴装配检查点的特征是APCFZY活性降低，并将RCC1添加到具有激活检查点的提取物中，将APCFZY活性恢复为控制水平。为了确定RCC1消除检查点停滞的确切机制，我们检查了包括MAD2，CENP-E，BUB1和BUB3在内的有丝分裂调节剂的定位。我们发现，在添加了高水平的RCC1蛋白后，这些蛋白质被错误地定位于牛陀唑治疗提取物中的动力学。有趣的是，通过添加重组Rangap1蛋白可以逆转BUB1和BUB3的位移，这表明它们的行为直接以RAN-GTP水平响应。综上所述，我们的结果表明，RAN途径通常在有丝分裂过程中以高度动态的方式调节。可以通过添加外源性RCC1蛋白来模仿RAN途径的过渡，从而在存在无附属动物学的情况下触发中期 - 动力学过渡。我们的观察结果表明，RCC1染色体动力学的变化可能是中期纺锤体组装和有丝分裂出口之间事件链中的关键联系。