Roles of chromosomal factors in chromosome segregation

描述（由申请人提供）：在有丝分裂和减数分裂期间，染色体主动产生促进纺锤体组装和染色体运动的各种必需信号。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。在这项提案中，我们将集中在这些过程的关键调节，染色体乘客复合物（CPC），由激酶极光B，及其调节亚基，INCENP，Dasra和Survivin。CPC定位于染色质并在着丝粒处富集。染色质结合与Aurora B激酶活性的激活直接相关。在非洲爪蟾卵提取物中，这种激活机制对纺锤体组装至关重要。在着丝粒，CPC介导纺锤体组装检查点（SAC）信号传导，以延迟后期发作，以响应错误的动粒附件。为了揭示控制极光B通路的时空机制，我们将：1）解决CPC如何控制SAC。我们已经发现，推定的卷曲螺旋（CC）结构域的INCENP是至关重要的SAC。为了测试CC结构域是否监测微管附着后的物理动粒变化并将其转化为极光B信号传导，将系统地调节CC结构域，并监测动粒内极光B依赖性磷酸化的变化。2)揭示了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。基于我们的初步数据，我们假设组蛋白H3苏氨酸3（H3T3）激酶Haspin被有丝分裂激酶Cdk 1和Plx 1激活，以介导染色质对极光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分析。3)为CPC与染色体相互作用提供了结构基础. 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，将确定存活素-H3T3ph复合物的晶体结构。4)研究另一种染色质激活蛋白，最近在我的实验室中发现，Vespera，它似乎反对极光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）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，我们将研究在这些发展阶段的具体作用维斯佩拉。该项目的结果将为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失调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.