Function of the chromosomal kinase haspin in mitosis

染色体激酶 haspin 在有丝分裂中的功能

• 批准号: 7035510
• 负责人: JONATHAN M HIGGINS
• 金额: $ 29.75万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7035510
• 关键词: DNA binding protein; RNA interference; cell cycle; cell growth regulation; centromere; chromatin; cytogenetics; enzyme activity; enzyme mechanism; gene deletion mutation; gene expression; human tissue; immunofluorescence technique; mitotic spindle apparatus; molecular /cellular imaging; phosphorylation; protein binding; protein localization; protein protein interaction; protein structure function; serine threonine protein kinase

DESCRIPTION (provided by applicant): We have recently discovered a novel mitotic histone kinase, haspin, that has homologs in diverse eukaryotes. Haspin phosphorylates Thr-3 in the N-terminal tail of histone H3. In human cells, H3 Thr-3 phosphorylation is first detected on chromosome arms in late G2/early prophase, becomes focused at centromeres by prometaphase, and declines during anaphase. In vitro, haspin specifically phosphorylates histone H3 at Thr-3, and depletion of haspin by RNA interference (RNAi) reveals that it is required for H3 Thr-3 phosphorylation in mitotic cells. Haspin associates with condensed chromosomes, particularly at centromeres, and is also found at the centrosomes during mitosis. Importantly, haspin RNAi causes misalignment of metaphase chromosomes and spindle defects, and overexpression delays progression through early mitosis. Our more recent data suggest that haspin is required for the maintenance of sister chromatid cohesion and centromeric aurora B localization prior to anaphase. We have also isolated candidate haspin-binding proteins that are consistent with haspin function at the centrosome and spindle. This work reveals a new enzyme involved in composing the histone code and adds haspin to the select group of kinases that regulate chromosome dynamics and spindle activity during mitosis. We wish to determine the mechanistic basis for haspin action during mitosis. In Aim 1 we will use immunofluorescence and live cell imaging to examine the defects underlying chromosome misalignment caused by haspin depletion. We will define in detail defects in cohesion, chromosome-spindle attachment, spindle checkpoint activation, aurora B activity and spindle/centrosome function following haspin RNAi. In Aim 2 we will determine how H3 Thr-3 phosphorylation regulates centromeric chromatin. First, we will delineate the location of Thr-3 phosphorylation with respect to cohesin and other molecules, allowing refinement of current centromere structure models. Then we will use RNAi and overexpression to determine the influence of haspin on cohesin, chromosome passenger and heterochromatin protein binding at centromeres, and on patterns of histone modification. Expression of H3 molecules mutated at Thr-3 and in vitro binding studies will reveal the role of Thr-3 phosphorylation in these effects. In Aim 3 we explore functional interactions of haspin to understand its role in centrosome and spindle activity. Regulation of chromosome behavior during cell division is critical to allow accurate passage of the genome to daughter cells. Cancer cells have atypical numbers of abnormal chromosomes, suggesting that disruption of these mechanisms contributes to the generation of malignancy. A greater knowledge of the role of human haspin in this process will help us understand the defects that underlie transformation and may lead to new approaches to block the division of cancer cells.

描述（由申请人提供）：我们最近发现了一种新型的有丝分裂组蛋白激酶Haspin，该激酶具有多种真核生物的同源物。 Haspin在组蛋白H3的N末端尾部磷酸化。在人类细胞中，首先在G2晚期/早期预言的染色体臂上检测到H3 Thr-3磷酸化，通过Prometathase将聚焦于中心粒，并在后期期间下降。在体外，HASPIN在THR-3时特异性地磷酸化组蛋白H3，而RNA干扰（RNAi）耗尽了HASPIN（RNAI）表明，在有丝分裂细胞中H3 THR-3磷酸化需要它。 Haspin与冷凝的染色体相关，尤其是在丝粒中，并且在有丝分裂过程中也发现了中心体。重要的是，Haspin RNAi会导致中期染色体和纺锤体缺陷的未对准，并且过表达通过早期有丝分裂延迟了进展。我们最新的数据表明，在后期之前，维持姐妹染色单体内聚力和centromeric aurora b本地化是必需的。我们还分离了与中心体和纺锤体在haspin功能一致的HASPIN结合蛋白。这项工作揭示了一种涉及组成组蛋白代码的新酶，并将haspin添加到调节有丝分裂过程中染色体动力学和纺锤体活性的一组激酶中。我们希望在有丝分裂过程中确定Haspin作用的机械基础。在AIM 1中，我们将使用免疫荧光和活细胞成像来检查Haspin耗竭引起的染色体未对准的缺陷。我们将在Haspin RNAi后详细定义内聚，染色体主轴附着，主轴检查点激活，Aurora B活性和主轴/中心体功能。在AIM 2中，我们将确定H3 Thr-3磷酸化如何调节丝粒染色质。首先，我们将描述THR-3磷酸化在粘着蛋白和其他分子方面的位置，从而使当前的Centromere结构模型进行完善。然后，我们将使用RNAi和过表达来确定Haspin对centromeres处粘蛋白，染色体乘客和异染色质蛋白结合的影响，以及组蛋白修饰的模式。在THR-3和体外结合研究中突变的H3分子的表达将揭示THR-3磷酸化在这些作用中的作用。在AIM 3中，我们探索了Haspin的功能相互作用，以了解其在中心体和主轴活动中的作用。细胞分裂过程中染色体行为的调节对于允许基因组准确地传递给子细胞至关重要。癌细胞具有非典型数量的异常染色体，表明这些机制的破坏会导致恶性肿瘤的产生。对人haspin在这一过程中的作用的更多了解将有助于我们了解基础转化的缺陷，并可能导致阻止癌细胞分裂的新方法。