Roles of chromosomal factors in chromosome segregation

染色体因素在染色体分离中的作用

• 批准号: 8708102
• 负责人: Hironori Funabiki
• 金额: $ 34.75万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8708102
• 关键词: Address; Affect; Anaphase; Aneuploidy; Apoptosis; Apoptotic; BIR Domain; Binding; Biochemical; Biological; CDC2 Protein Kinase; Cell Proliferation; Cell division; Cells; Centromere; Chromatin; Chromosome Segregation; Chromosomes; Coiled-Coil Domain; Complex; Congenital Abnormality; Coupled; Data; Detection; Development; Embryo; Embryonic Development; Encapsulated; Ensure; Failure; Fertilization; Growth; Haspin; Histone H3; Kinetochores; Ligands; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Meiosis; Metaphase; Methods; Microtubules; Mitosis; Mitotic; Molecular; Monitor; Mutation; N-terminal; Normal Cell; Nuclear; Nuclear Envelope; Nuclear Magnetic Resonance; Outcome; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Prevention; Process; Protein Kinase; Proteins; Publishing; Regulation; Role; Signal Pathway; Signal Transduction; Sister; Spatial Distribution; Specificity; Staging; Structure; Testing; Therapeutic procedure; Threonine; Work; Xenopus; Xenopus laevis; aurora B kinase; base; cancer cell; cell type; chemical reaction; chromosome movement; egg; improved; inner centromere protein; insight; killings; mutant; neutralizing antibody; novel; pluripotency; response; signal processing; spatiotemporal; survivin; tumor

DESCRIPTION (provided by applicant): Chromosomes actively generate a variety of essential signals that promote spindle assembly and chromosome movement during mitosis and meiosis. Failure to properly complete these processes leads to aneuploidy, which is frequently associated with cancer and birth defects. In this proposal, we will focus on a critical regulator of these processes, the chromosomal passenger complex (CPC), consisting of the kinase Aurora B, and its regulatory subunits, INCENP, Dasra and Survivin. The CPC localizes to chromatin and is enriched at centromeres. Chromatin binding is directly coupled to activation of the kinase activity of Aurora B. In Xenopus laevis egg extracts, this activation mechanism is critical for spindle assembly. At centromeres, the CPC mediates spindle assembly checkpoint (SAC) signaling to delay anaphase onset in response to erroneous kinetochore attachments. To reveal the spatiotemporal mechanism that controls the Aurora B pathway, we will: 1) Address how the CPC controls the SAC. We have found that the putative coiled-coil (CC) domain of INCENP is critical for the SAC. To test if the CC domain monitors physical kinetochore changes upon microtubule attachment and converts them into Aurora B signaling, the CC domain will be systematically modulated, and changes in Aurora B-dependent phosphorylation within kinetochores will be monitored. 2) Reveal a mechanism by which a network of protein kinases controls M phase-specific activation of Aurora B by chromatin. Based on our preliminary data, we hypothesize that the histone H3 threonine 3 (H3T3) kinase Haspin is activated by the mitotic kinases Cdk1 and Plx1 to mediate the temporal control of Aurora B activation by chromatin. The precise mechanism by which these mitotic kinases activate Haspin will be analyzed by biochemical methods combined with advanced mass spectrometry. 3) Provide the structural basis for the CPC- chromosome interaction. Localization of the CPC to chromosomes is mediated by Survivin, which directly binds to histone H3 that has been phosphorylated at threonine 3 (H3T3ph). To determine how this phosphorylation regulates binding specificity, crystal structures of the Survivin-H3T3ph complex will be determined. 4) Investigate another chromatin-activated protein that was recently identified in my lab, Vespera, which appears to oppose the Aurora B pathway, inhibit microtubule assembly, and promote nuclear re- formation. Since Vespera is exclusively expressed in pluripotent cells (including eggs), it is possible that its presence is linked to the requirement for the CPC in spindle assembly in these cell types. Therefore we will investigate specific roles for Vespera during these developmental stages. The outcomes of this proposed project will provide molecular explanations for how the chromosome-associated activities of the CPC and Vespera coordinate mitosis, and will help us understand how their misregulation affects the growth of normal and cancer cells.

描述（由申请人提供）：染色体在有丝分裂和减数分裂过程中主动产生各种促进纺锤体组装和染色体运动的重要信号。未能正确完成这些过程会导致非整倍体，这通常与癌症和出生缺陷有关。在本提案中，我们将重点关注这些过程的关键调节因子，即染色体过客复合物 (CPC)，它由激酶 Aurora B 及其调节亚基 INCENP、Dasra 和 Survivin 组成。 CPC 定位于染色质并在着丝粒处富集。染色质结合直接与 Aurora B 激酶活性的激活相关。在非洲爪蟾卵提取物中，这种激活机制对于纺锤体组装至关重要。在着丝粒处，CPC 介导纺锤体组装检查点 (SAC) 信号，以响应错误的着丝粒附着来延迟后期开始。为了揭示控制 Aurora B 通路的时空机制，我们将： 1）解决 CPC 如何控制 SAC。我们发现 INCENP 的假定卷曲螺旋 (CC) 结构域对于 SAC 至关重要。为了测试 CC 结构域是否监测微管附着时的物理动粒变化并将其转化为 Aurora B 信号传导，CC 结构域将被系统地调节，并且动粒内 Aurora B 依赖性磷酸化的变化将被监测。 2) 揭示蛋白激酶网络通过染色质控制 Aurora B 的 M 期特异性激活的机制。根据我们的初步数据，我们假设组蛋白 H3 苏氨酸 3 (H3T3) 激酶 Haspin 被有丝分裂激酶 Cdk1 和 Plx1 激活，以介导染色质对 Aurora B 激活的时间控制。这些有丝分裂激酶激活 Haspin 的精确机制将通过生化方法结合先进的质谱分析进行分析。 3) 为CPC-染色体相互作用提供结构基础。 CPC 在染色体上的定位是由 Survivin 介导的，它直接与已在苏氨酸 3 (H3T3ph) 处磷酸化的组蛋白 H3 结合。为了确定这种磷酸化如何调节结合特异性，需要确定 Survivin-H3T3ph 复合物的晶体结构。 4) 研究我的实验室最近发现的另一种染色质激活蛋白 Vespera，它似乎能对抗 Aurora B 通路、抑制微管组装并促进核重组。由于 Vespera 只在多能细胞（包括卵细胞）中表达，因此它的存在可能与这些细胞类型中纺锤体组装中对 CPC 的需求有关。因此，我们将研究 Vespera 在这些发育阶段的具体作用。该项目的成果将为 CPC 和 Vespera 的染色体相关活动如何协调有丝分裂提供分子解释，并将帮助我们了解它们的失调如何影响正常细胞和癌细胞的生长。