Phosphorylation & Function of Inhibitor-2

描述(申请人提供)：中心体组织微管细胞骨架，并在有丝分裂过程中转变为纺锤体极，以分离染色体。异常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。该项目长期以来一直关注于定位于中心体并调节多种中心体酶的蛋白磷酸酶1(PP1)和抑制物2(InH2)。在人类细胞中，通过RNAi敲除InH2会导致有丝分裂染色体落后，胞质分裂失败，并形成多核细胞，带有多余的中心体，就像在人类肿瘤中看到的那样。通过共表达避免了RNAi敲除的InH2，拯救了表型。在果蝇中，只有一个InH2基因在发育早期的卵母细胞和胚胎中以母体形式表达。对InH2亚型的苍蝇会增加胚胎致死率，并严重降低孵化率。由于错误的染色体分离，存活的胚胎表现出合体有丝分裂同步性的丧失，并有许多桥接核。依赖剂量的D-InH2转基因表达挽救了胚胎的存活。因此，对人类和果蝇的研究结果都支持InH2在有丝分裂染色体分离中的关键作用。具体目标1试图通过检测InH2对Aurora B激活和有丝分裂底物的磷酸化的影响来确定机制，有丝分裂底物在中期调节适当的染色体对齐。InH2在有丝分裂过程中通过CDK1：：cyClinB1在保守的PxTP位点被磷酸化。这种磷酸化破坏了InH2与Prolyl异构酶Pin1的结合，从而极大地改变了Pin1底物与有丝分裂磷酸蛋白的特异性。特定目标2将通过核磁共振确定InH2：：Pin1的结构，并定义磷酸化对底物相互作用和结合的影响。具体目标3将利用拯救RNAi敲除细胞和亚型果蝇来剖析InH2作为PP1抑制因子、有丝分裂激酶激活剂和Pro异构酶调节因子的多功能性质。有丝分裂酶已经被制药行业指定为药物开发的靶点，但到目前为止，PP1和InH2的调控一直没有得到足够的重视。对人类疾病的有效诊断和治疗以及对新的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。人类的感官反应，如视觉、听觉和嗅觉，依赖于上皮组织表面的纤毛。这些纤毛植根于称为中心体的细胞结构。中心体除了支持纤毛外，还具有多种功能，如控制细胞分裂和在分裂过程中分离染色体。中心体缺陷可导致多种人类疾病，包括视力和嗅觉丧失，尤其是癌症，在分散染色体的肿瘤细胞中可见过多的中心体。这个项目研究在所有动物中发现的一种蛋白质，这种蛋白质对正常的中心体功能是关键和必要的。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。

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.