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 GTPase是许多细胞功能所必需的，包括核质运输，主轴组件，核装配和细胞周期控制。 RAN RCC1的唯一核苷酸交换因子在整个细胞周期中结合染色质。 RANGAP1的GTPase激活蛋白质通过与大核孔RANBP2缔合，将其定位于核孔复合物（NPC）的胞质面（NPC）。 RAN调节剂的相间分布导致核中高浓度的RAN GTP，而细胞质中的RAN GTP低。 RAN的主要效应子是被发现为核转运受体的RAN GTP结合蛋白家族。这些受体被统称为核蛋白。介导的导入的人称为导入蛋白，介导导出的人称为导出。他们的货物负荷受RAN GTP水平的控制：进口素与其在细胞质中的货物结合。导入复合物穿越NPC并在RAN-GTP-rimettring结合上解离。导出蛋白将其货物在核中结合，其中包含RAN-GTP的复合物。通过NPC后，导出复合物将在RAN GTP水解后解离。迄今为止，已经显示两个核蛋白在有丝分裂过程中起效应子的作用：importin-beta和Exportin crm1。 正确的主轴形成需要基于染色质的RAN梯度，在有丝分裂染色体附近的RAN GTP水平高。该梯度是通过RCC1的活性建立的，RCC1仍集中在有丝分裂染色体上。 RANBP1是RANGAP1的共激活因子，它还形成具有RCC1和RAN（RRR复合物）的稳定异源三聚体配合物，从而抑制RCC1S核苷酸交换活性。但是，RRR复合物的功能仍然是神秘的，因为RANBP1在相间期间与RCC1物理分离。我们发现在M相Xenopus卵提取物（CSF-XEES）中很容易形成RRR复合物。 RCC1与染色质和RRR复合物组件的结合是相互排斥的，因此通过添加重组RANBP1的RANBP1隔离的RCC1从染色质素中启动RRR复合物的形成。与较早的报告一致，RRR复合物组装抑制XEE中的RCC1 Lajef活性。总之，这些发现表明，RRR复合物在确定RCC1在其活性染色质结合和非活性可溶性状态之间的分配中起关键作用，从而设定了有丝分裂RAN GTP的位置和幅度。 值得注意的是，RCC1与有丝分裂染色质的关联是动态的，并且在中期到时期窗口中有特别大的变化。但是，报告的RCC1修改的时机表明它们不会引起这种变化。我们发现RANBP1在后期期间被磷酸化，并且这种修饰破坏了RRR复合物。进一步的分析表明，RANBP1磷酸化促进了RCC1在循环XEE中与染色质的结合增加，从而间接增强了后期RAN GTP的产生。这种修饰也可能有助于早期相间的RAN途径功能，因为后期染色质上的RCC1升高应提供高水平的RAN GTP以促进核重新组装。最后，RRR复合物解离后的RCC1和RANBP1的分离允许RCC1固相结合核重建核，同时将RANBP1排除到早期的相互作用胞质醇中。总之，这些发现记录了RANBP1蛋白在控制RANS核苷酸交换因子RCC1的定位和活性中的新作用。我们已经表明，在后期期间，RANBP1的磷酸化驱动RCC1动力学的变化，并允许RAN GTP产生增加。这些发现解决了RANBP1/RAN/RCC1复合物及其动态的功能的重要问题。 我们还通过与RAN途径的相互作用对IRBIT的调节感兴趣。 IRBIT是一种保守的后生蛋白，与多种功能有关。 IRBIT由一个推定的酶结构域组成，该酶结构域与S-腺基质类健康水解酶和必需的N末端结构域具有相似性。为了鉴定结合IRIT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SDS-PAGE和蛋白质染色。我们确定了突出的共沉淀蛋白，并通过质谱法确定了它们。核糖核苷酸还原酶（RNR）是最丰富的IRIT结合蛋白之一，因此我们研究了这些蛋白质之间的关系。 RNR提供了DNA复制和维持基因组完整性所必需的脱氧核苷酸三磷酸（DNTP）的平衡池。 RNR在所有真核生物中都受到变构调节机制的影响，并分别在萌芽和裂变酵母中通过小蛋白抑制剂SML1P和SPD1P控制。我们发现，IRBIT与RNR形成了DATP依赖性复合物，在RNR的活性位点稳定了DatP，从而抑制了酶。 RNR-IRBIT复合物的形成是通过IRIT的磷酸化来调节的，HeLa细胞中IRBIT表达的消融会导致DNTP库不平衡并改变细胞周期进程。总之，我们的发现为高等真核生物中的RNR调节提供了一种新的机制，该机制通过增强DATP的变构RNR抑制而起作用。

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