Regulation of Mitotic Kinetochores by the Ran GTPase

Ran GTPase 对有丝分裂着丝粒的调节

• 批准号: 8941484
• 负责人: MARY C. DASSO
• 金额: $ 88.41万
• 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/8941484
• 关键词: Ablation; Adenosylhomocysteinase; Anaphase; Binding; Binding Proteins; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Nucleus; Cell physiology; Cells; Chromatin; Chromosomal Instability; Chromosomes; Complex; Cytoplasm; Cytosol; DNA Maintenance; DNA biosynthesis; Defect; Development; Dissociation; Distal; Enzymes; Equilibrium; Eukaryota; Exportins; Face; Family; Fiber; Fission Yeast; GTP Binding; GTPase-Activating Proteins; Genomics; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hela Cells; Hydrolysis; Immunoprecipitation; Importins; Interphase; Interphase Cell; Karyopherins; Kinetochores; Location; Mass Spectrum Analysis; Mediating; Metaphase; Metaphase Plate; Microtubules; Mitosis; Mitotic; Mitotic Chromosome; Mitotic spindle; Modification; Monitor; N-terminal; Nuclear; Nuclear Envelope; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nucleotides; Pathway interactions; Phosphorylation; Play; Production; Protein Binding; Proteins; Recombinants; Regulation; Regulatory Pathway; Reporting; Ribonucleotide Reductase; Role; Running; Saccharomycetales; Sister Chromatid; Site; Somatic Cell; Staining method; Stains; Structure; Time; Xenopus; Xenopus laevis; base; beta Karyopherins; egg; inhibitor/antagonist; interest; novel; nucleocytoplasmic transport; prevent; ran GTP-Binding Protein; ran-binding protein 1; receptor; segregation; trafficking; tripolyphosphate

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. 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 karyopherins have been shown to act as Ran effectors during mitosis: Importin-beta and the exportin Crm1. Proper spindle formation requires a chromatin-based gradient of Ran, with high Ran-GTP levels in the vicinity of mitotic chromosomes. This gradient is established through the activity of RCC1, which remains concentrated on mitotic chromosomes. RanBP1 is a co-activator of RanGAP1, which also forms a stable heterotrimeric complex with RCC1 and Ran (RRR complex) thereby inhibiting RCC1s nucleotide exchange activity. The function of the RRR complex has remained mysterious, however, because RanBP1 is physically separated from RCC1 during interphase. We have found that the RRR complex forms readily in M-phase Xenopus egg extracts (CSF-XEEs). RCC1 binding to chromatin and RRR complex assembly are mutually exclusive, so that promoting RRR complex formation through the addition of recombinant RanBP1 sequestered RCC1 away from chromatin. Consistent with earlier reports, RRR complex assembly inhibits RCC1s RanGEF activity in XEE. Together, these findings suggest that the RRR complex plays a key mitotic role in determining the partitioning of RCC1 between its active chromatin-bound and inactive soluble states, thereby setting both the location and magnitude of mitotic Ran-GTP production. Notably, RCC1s association to mitotic chromatin is dynamic, and there are particularly large changes during the metaphase-to-anaphase window. However, the timing of reported RCC1 modifications suggests that they do not cause such changes. We found that RanBP1 is phosphorylated during anaphase, and that this modification disrupts the RRR complex. Further analysis showed that RanBP1 phosphorylation drove increased RCC1 binding to chromatin in cycling XEE, and thereby indirectly enhanced anaphase Ran-GTP production. This modification may also contribute to Ran pathway function in early interphase, because elevated RCC1 on anaphase chromatin should provide high levels of Ran-GTP to facilitate nuclear re-assembly. Finally, separation of RCC1 and RanBP1 after RRR complex dissociation allows RCC1 sequestration to re-forming nuclei while excluding RanBP1 into the early interphase cytosol. Together, these findings document a novel role of the RanBP1 protein in controlling the localization and activity of Rans nucleotide exchange factor, RCC1. We have shown that phosphorylation of RanBP1 during anaphase drives changes in RCC1 dynamics and allows increased Ran-GTP production. These findings resolve important and long-standing questions within the Ran field regarding the function of the RanBP1/Ran/RCC1 complex and its dynamics. We have also become interested the regulation of IRBIT through its interactions with the Ran pathway. IRBIT is a conserved metazoan protein that has been implicated in a diverse set of functions. IRBIT consists of a putative enzymatic domain that has similarity to S-adenosylhomocysteine hydrolase and an essential N-terminal domain. To identify proteins that bind IRBIT, we performed immunoprecipitation from lysates of HeLa cells, followed by SDS-PAGE and protein staining. We identified prominent co-precipitating proteins and identified them by mass-spectrometry. Ribonucleotide reductase (RNR) was among the most abundant IRBIT-binding proteins, so we have investigated the relationship between these proteins. RNR supplies the balanced pools of deoxynucleotide triphosphates (dNTPs) necessary for DNA replication and maintenance of genomic integrity. RNR is subject to allosteric regulatory mechanisms in all eukaryotes, as well as to control by small protein inhibitors Sml1p and Spd1p in budding and fission yeast, respectively. We found that IRBIT forms a dATP-dependent complex with RNR, stabilizing dATP in the activity site of RNR, and thus inhibiting the enzyme. Formation of the RNR-IRBIT complex is regulated through phosphorylation of IRBIT, and ablation of IRBIT expression in HeLa cells causes imbalanced dNTP pools and altered cell cycle progression. Together, our findings provide a new mechanism for RNR regulation in higher eukaryotes that acts by enhancing allosteric RNR inhibition by dATP.

RAN GTP酶是许多细胞功能所必需的，包括核质运输、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GTP酶激活蛋白RanGAP1通过与大的核孔蛋白RanBP2结合，定位于间期核孔复合体(NPC)的胞浆面。RAN调节剂的相间分布导致核内RAN-GTP浓度较高，胞质中RAN-GTP浓度较低。RAN的主要效应物是一类RAN-GTP结合蛋白家族，它们被发现为核转运受体。这些受体统称为核粘附素；那些介导输入的受体称为Importins，而那些介导输出的受体称为Exportins。它们的货物载量受RAN-GTP水平的控制：Importins与细胞质中的货物结合。进口复合体穿过NPC并在RAN-GTP-Importin结合时解离。Exportins将它们的货物结合在含有Ran-GTP的复合体的核内。在通过NPC后，出口复合体在RAN-GTP水解时解离。到目前为止，有两种核粘附素已被证明在有丝分裂过程中作为RAN效应器：Importin-beta和Exportin CRM1。 正确的纺锤体形成需要一个基于染色质的RAN梯度，在有丝分裂染色体附近有高水平的RAN-GTP。这种梯度是通过RCC1的活性建立的，RCC1仍然集中在有丝分裂染色体上。RanBP1是RanGAP1的共激活剂，它还与RCC1和RAN形成稳定的异三聚体复合体(RRR复合体)，从而抑制RCC1的核苷酸交换活性。然而，RRR复合体的功能仍然是个谜，因为RanBP1在间期与RCC1物理上是分开的。我们发现，RRR复合体很容易在非洲爪哇卵提取液(CSF-XEEs)中形成。RCC1与染色质的结合和RRR复合体的组装是相互排斥的，因此通过加入重组RanBP1促进RRR复合体的形成将RCC1从染色质中隔离出来。与早先的报道一致，RRR复合体组装抑制了XEE中RCC1的Rangef活性。综上所述，这些发现表明，RRR复合体在决定RCC1在活性染色质结合和非活性可溶性状态之间的分配方面发挥了关键的有丝分裂作用，从而决定了有丝分裂Ran-GTP产生的位置和大小。 值得注意的是，RCC1与有丝分裂染色质的关联是动态的，在中期到后期窗口期间有特别大的变化。然而，报告的RCC1修改的时间表明它们不会导致这种变化。我们发现RanBP1在后期被磷酸化，这种修饰破坏了RRR复合体。进一步的分析表明，在XEE循环中，RanBP1的磷酸化导致RCC1与染色质结合增加，从而间接增加后期RAN-GTP的产生。这种修饰也可能有助于早期间期RAN途径的功能，因为后期染色质上RCC1的升高应该提供高水平的RAN-GTP，以促进核重组。最后，RRR复合体解离后RCC1和RanBP1的分离允许RCC1隔离重新形成核，同时将RanBP1排除到早期间期胞浆中。总之，这些发现证明了RanBP1蛋白在控制RANS核苷酸交换因子RCC1的定位和活性方面的新作用。我们已经证明，RanBP1在后期的磷酸化推动了RCC1动力学的变化，并允许增加RAN-GTP的产生。这些发现解决了RAN领域中关于RanBP1/RAN/RCC1复合体的功能及其动力学的重要和长期存在的问题。 我们也对Irbit与RAN途径相互作用的调控感兴趣。Irbit是一种保守的后生动物蛋白质，参与了一系列不同的功能。IRBIT由一个与S同型半胱氨酸腺苷水解酶相似的假定酶结构域和一个必需的N末端结构域组成。为了确定结合Irbit的蛋白质，我们从HeLa细胞的裂解物中进行了免疫沉淀，然后进行了SDS-PAGE和蛋白质染色。我们鉴定了显著的共沉淀蛋白，并用质谱仪对它们进行了鉴定。核糖核苷酸还原酶(RNR)是最丰富的IRBIT结合蛋白之一，因此我们研究了这些蛋白之间的关系。RnR提供DNA复制和维持基因组完整性所必需的平衡的脱氧核苷酸三磷酸(DNTPs)池。RnR在所有真核生物中都受到变构调节机制的控制，在芽酵母和分裂酵母中分别受到小蛋白抑制剂Sml1p和Spd1p的控制。我们发现Irbit与RNR形成dATP依赖的复合体，稳定RNR活性部位的dATP，从而抑制该酶。RNR-Irbit复合体的形成是通过Irbit的磷酸化来调节的，而在HeLa细胞中IRbit表达的缺失会导致dNTP池的不平衡和细胞周期进程的改变。综上所述，我们的发现为高等真核生物中RNR的调节提供了一种新的机制，即通过增强dATP对变构RNR的抑制来发挥作用。

项目成果

Shedding light on mysterious microtubules.

揭示神秘的微管。

• DOI: 10.1016/j.devcel.2011.02.002
• 发表时间: 2011
• 期刊: Developmental cell
• 影响因子: 11.8
• 作者: Dasso,Mary

A Mad that wears two hats: Mad1's control of nuclear trafficking.

身兼两职的疯子：Mad1 对核贩运的控制。

• DOI: 10.1016/j.devcel.2013.01.003
• 发表时间: 2013
• 期刊: Developmental cell
• 影响因子: 11.8
• 作者: Dasso,Mary

Xenopus HJURP and condensin II are required for CENP-A assembly.

• DOI: 10.1083/jcb.201005136
• 发表时间: 2011-02-21
• 期刊: The Journal of cell biology
• 作者: Bernad R;Sánchez P;Rivera T;Rodríguez-Corsino M;Boyarchuk E;Vassias I;Ray-Gallet D;Arnaoutov A;Dasso M;Almouzni G;Losada A
• 通讯作者: Losada A