Roles of chromosomal factors in chromosome segregation

描述（申请人提供）：在有丝分裂和减数分裂过程中，染色体积极产生各种促进纺锤体组装和染色体运动的必要信号。不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，染色体乘客复合物（CPC），由激酶Aurora B及其调节亚基，INCENP， Dasra和Survivin组成。CPC定位于染色质并富集于着丝粒。在非洲爪蟾卵提取物中，染色质结合直接与Aurora b激酶活性的激活耦合，这种激活机制对纺锤体组装至关重要。在着丝粒中，CPC介导纺锤体组装检查点（SAC）信号传导，以延迟对着丝粒错误附着的后期开始。为了揭示控制极光B通路的时空机制，我们将：1)研究CPC如何控制SAC。我们发现假定的线圈线圈（CC）结构域对SAC至关重要。为了测试CC结构域是否在微管附着时监测着丝点的物理变化并将其转化为Aurora B信号，CC结构域将被系统地调节，并监测着丝点内Aurora B依赖性磷酸化的变化。2)揭示蛋白激酶网络通过染色质控制极光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，从而介导染色质对极光B激活的时间控制。这些有丝分裂激酶激活Haspin的确切机制将通过生物化学方法结合先进的质谱法进行分析。3)为CPC-染色体相互作用提供结构基础。CPC在染色体上的定位由Survivin介导，Survivin直接结合在苏氨酸3磷酸化的组蛋白H3 （H3T3ph）上。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活化蛋白Vespera，它似乎反对Aurora B途径，抑制微管组装，促进核重组。由于Vespera仅在多能细胞（包括卵子）中表达，因此它的存在可能与这些细胞类型的纺锤体组装中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。本项目的结果将为CPC和Vespera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节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.