Roles of chromosomal factors in chromosome segregation

描述(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基本信号，促进纺锤体组装和染色体运动。未能正确完成这些过程会导致非整倍体，这经常与癌症和出生缺陷有关。在这个提案中，我们将重点关注这些过程的关键调节因子-染色体乘客复合体(CPC)，它由Aurora B及其调节亚单位INCENP、Dasra和Survivin组成。CPC定位于染色质，并在着丝粒上富含。染色质结合与非洲爪哇卵提取液中极光B的激活直接相关，这种激活机制对纺锤体的组装至关重要。在着丝粒中，CPC介导纺锤体组装检查点(SAC)信号，以延迟后期开始，以响应错误的动粒附着。为了揭示控制Aurora B途径的时空机制，我们将：1)研究CPC如何控制SAC。我们发现INCENP的螺旋线圈(CC)结构域对SAC是关键的。为了测试CC结构域是否监测微管附着时物理动粒的变化并将其转换为Aurora B信号，将对CC结构域进行系统调制，并监测动点内Aurora B依赖的磷酸化的变化。2)揭示了蛋白激酶网络控制染色质对Aurora B M相特异性激活的机制。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。这些有丝分裂酶激活Haspin的确切机制将通过生化方法和先进的质谱学相结合进行分析。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，Survivin直接与组蛋白H3结合，组蛋白H3在苏氨酸3(H3T3ph)处被磷酸化。为了确定这种磷酸化如何调节结合特异性，将确定Survivin-H3T3ph复合体的晶体结构。4)研究最近在我的实验室发现的另一种染色质激活蛋白Vespera，它似乎反对Aurora B途径，抑制微管组装，并促进核重新形成。由于VesperA仅在多能细胞(包括卵子)中表达，它的存在可能与这些细胞类型的纺锤体组装中对CPC的要求有关。因此，我们将研究Vespera在这些发育阶段中的具体作用。这项拟议的项目的结果将为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错误调控如何影响正常细胞和癌细胞的生长。

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.