Phosphorylation & Function of Inhibitor-2

磷酸化

• 批准号: 7859325
• 负责人: DAVID L. BRAUTIGAN
• 金额: $ 8.16万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7859325
• 关键词: Aneuploidy; Animals; Antimitotic Agents; Binding; Biological Assay; Cancer Etiology; Cell Cycle; Cell Cycle Regulation; Cell Nucleus; Cell division; Cells; Cellular Structures; Centrosome; Chromosome Segregation; Chromosomes; Cilia; Complex; Cytokinesis; Cytoskeleton; Defect; Development; Diagnosis; Dose; Drosophila genus; Drug Industry; Embryo; Enzymes; Epithelial; Equilibrium; Event; Evolution; Exhibits; Failure; Genes; Genetic; Goals; Hearing; Human; Knock-out; Life; Malignant Neoplasms; Mediating; Metaphase; Microtubules; Mitosis; Mitotic; Mitotic Chromosome; Monitor; Mus; Mutate; Nature; Oocytes; Peptidylprolyl Isomerase; Phenotype; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Plant Roots; Protein Dephosphorylation; Protein phosphatase; Proteins; Pseudogenes; RNA Interference; Regulation; Research; Role; Sensory; Signal Transduction; Site; Smell Perception; Specificity; Staging; Structure; Substrate Specificity; Surface; Testing; Tissues; Transgenic Organisms; Vision; aurora B kinase; base; clinical efficacy; developmental disease; drug development; fly; hatching; human disease; inhibitor/antagonist; inner centromere protein; insight; loss of function; mutant; neoplastic cell; novel; protein phosphatase inhibitor-1; public health relevance; reconstitution; response; tumor

DESCRIPTION (provided by applicant): Centrosomes organize the microtubule cytoskeleton and transform into the spindle poles for segregation of chromosomes during mitosis. Abnormal centrosomes produce aneuploidy in human cancers and cause other human sensory and developmental disorders. This project has a long- standing focus on protein phosphatase-1 (PP1) and inhibitor-2 (Inh2) that localize to centrosomes and regulate multiple centrosomal enzymes. Knockdown of Inh2 by RNAi in human cells causes lagging mitotic chromosomes, failure of cytokinesis and formation of multinucleated cells with supernumerary centrosomes, like seen in human tumors. The phenotype is rescued by co-expression of Inh2 that avoids the RNAi knockdown. In Drosophila there is a single Inh2 gene that is maternally expressed in oocytes and embryos during early stages of development. Flies hypomorphic for Inh2 have increased embryonic lethality, with severely reduced hatch rates. Surviving embryos exhibit loss of syncytial mitotic synchrony and have many bridged nuclei due to faulty chromosome segregation. Embryo survival is rescued by dose-dependent transgenic expression of D-Inh2. Thus, results with both human and Drosophila support a key role for Inh2 in mitotic chromosome segregation. Specific Aim 1 seeks to define mechanisms by examining the effects of Inh2 on Aurora B activation and phosphorylation of mitotic substrates that mediate proper chromosome alignment at metaphase. Inh2 is phosphorylated during mitosis at a conserved PxTP site by CDK1::cyclinB1. This phosphorylation disrupts Inh2 binding to the prolyl isomerase Pin1, which drastically alters Pin1 substrate specificity with mitotic phosphoproteins. Specific Aim 2 will determine the structure of Inh2::Pin1 by NMR and define effects of phosphorylation on the interactions and the binding of substrates. Specific Aim 3 will use rescue of RNAi knockdown cells and hypomorphic Drosophila to dissect the multifunctional nature of Inh2 as a PP1 inhibitor, mitotic kinase activator and prolyl isomerase regulator. Mitotic kinases are already designated targets for drug development by the pharmaceutical industry, but regulation by PP1 and Inh2 has heretofore been underappreciated. Effective diagnosis and treatment of human diseases and accurate monitoring of clinical efficacy of new targeted therapies will need to incorporate information from this unique project. PUBLIC HEALTH RELEVANCE Human sensory responses such as sight, hearing and smell depend on cilia on the surface of epithelial tissues. These cilia are rooted at cellular structures called centrosomes. Centrosomes fulfill multiple functions besides supporting the cilia, such as control of cell division and separation of chromosomes during division. Defects in centrosomes can result in a variety of human diseases including loss of sight and smell, and especially cancer, where too many centrosomes are seen in tumor cells that scatter chromosomes. This project studies a protein found in all animals that is critical and necessary for proper centrosome function. The goal is to understand how this protein acts to connect multiple signaling inputs into centrosome actions. Understanding cellular mechanisms is the basis for diagnosis and treatment of human diseases.

描述（由申请方提供）：中心体组织微管细胞骨架，并在有丝分裂期间转化为纺锤体极，用于染色体分离。异常的中心体在人类癌症中产生非整倍体，并导致其他人类感觉和发育障碍。本项目长期关注定位于中心体并调节多种中心体酶的蛋白磷酸酶-1（PP 1）和抑制剂-2（Inh 2）。在人类细胞中通过RNAi敲低Inh 2导致有丝分裂染色体滞后、胞质分裂失败和具有额外中心体的多核细胞的形成，如在人类肿瘤中所见。通过共表达避免RNAi敲低的Inh 2拯救表型。在果蝇中，有一个单一的Inh 2基因，在发育的早期阶段在卵母细胞和胚胎中表达。果蝇亚晶Inh 2增加了胚胎的致死率，孵化率严重降低。存活的胚胎表现出合胞体有丝分裂同步性的丧失，并且由于错误的染色体分离而具有许多桥接的核。D-Inh 2的剂量依赖性转基因表达挽救了胚胎存活。因此，人类和果蝇的结果支持Inh 2在有丝分裂染色体分离中的关键作用。具体目标1试图通过检查Inh 2对Aurora B激活和有丝分裂底物磷酸化的影响来定义机制，所述有丝分裂底物介导中期的正确染色体排列。Inh 2在有丝分裂期间在保守的PxTP位点被CDK 1：：cyclinB 1磷酸化。这种磷酸化破坏了Inh 2与脯氨酰异构酶Pin 1的结合，从而极大地改变了Pin 1与有丝分裂磷蛋白的底物特异性。具体目标2将通过NMR确定Inh 2：：Pin 1的结构，并确定磷酸化对底物相互作用和结合的影响。具体目标3将使用RNAi敲除细胞和亚形态果蝇的拯救来剖析Inh 2作为PP 1抑制剂、有丝分裂激酶激活剂和脯氨酰异构酶调节剂的多功能性质。有丝分裂激酶已经被制药工业指定为药物开发的靶点，但PP 1和Inh 2的调节迄今为止尚未得到充分重视。人类疾病的有效诊断和治疗以及新靶向治疗的临床疗效的准确监测将需要纳入这一独特项目的信息。人类的感觉反应，如视觉、听觉和嗅觉，取决于上皮组织表面的纤毛。这些纤毛扎根于称为中心体的细胞结构。除了支持纤毛外，中心体还具有多种功能，例如控制细胞分裂和分裂期间染色体的分离。中心体的缺陷会导致各种人类疾病，包括视力和嗅觉的丧失，尤其是癌症，在肿瘤细胞中看到太多的中心体，分散染色体。该项目研究在所有动物中发现的一种蛋白质，该蛋白质对中心体的正常功能至关重要。我们的目标是了解这种蛋白质如何将多个信号输入连接到中心体活动中。了解细胞机制是诊断和治疗人类疾病的基础。