The role of the NIMA kinase in mitotic regulation

描述（由申请人提供）：有丝分裂的不正确调节有助于癌症和出生缺陷的进展，关于有丝分裂调节的发现对医学研究和疾病治疗产生了积极影响。然而，新的见解和对医学的进一步影响的潜力显然存在。例如，最引人注目但最不为人所知的有丝分裂过程之一是核孔复合物（NPC）的拆卸和重新组装，这些大规模结构提供了穿过核膜的受调控通道。这个过程是如何调节和整合与其他有丝分裂事件可能涉及可逆的磷酸化，我们计划的实验将有助于测试这一假设。我们已经证明构巢曲霉的NIMA有丝分裂激酶通过促进NPC分解来启动有丝分裂。NIMA是促进NPC分解所必需的，并且足以促进NPC分解，并且显著影响脊椎动物细胞中的有丝分裂，这表明存在保守的有丝分裂底物。A.因此，nidulans为理解NPC的有丝分裂调控提供了一个强大而复杂的模型系统。本申请的目的包括使用亲和纯化和质谱法确定NPC相互作用组中的动态有丝分裂变化，以鉴定新的NPC蛋白（Nup）以及参与有丝分裂调节的Nup相关蛋白。此外，我们还将重点研究一个新的Nup相关蛋白（AN 0162），该蛋白含有一个跨膜结构域，可能编码第四个跨膜Nup。我们的数据表明，AN 0162有丝分裂功能，以维持周边mRNA输出因子Gle1的核膜周围的核仁在有丝分裂过程中，我们将测试这一假设。我们的第三个重点是Nup2及其相关蛋白NupA，importin 1和importin 2的有丝分裂功能。Nup2和NupA在有丝分裂期间也被磷酸化时从NPC易位到染色质。我们将检验NupA介导Nup2的有丝分裂染色质定位的假设，Nup2反过来控制输入蛋白1和2的功能，以调节有丝分裂进程。由于磷酸化似乎是这一过程的组成部分，最终的目标是映射Nup2，NupA和importin 2内的有丝分裂磷酸化位点，然后对特异性磷酸化位点进行体内突变分析以确定其功能。 公共卫生相关性：细胞周期是如何被调节的是基本的医学重要性，因为当这种调节出错时，疾病状态，如出生缺陷和癌症可能会导致。此外，许多目前的癌症治疗靶向细胞周期调控的不同方面，以优先杀死活跃通过细胞周期的细胞。因此，在公共卫生方面，我们越了解细胞周期是如何调节的，我们就越有能力开发针对新的化疗靶点的药物，以对抗与细胞周期缺陷相关的疾病状态。此外，所提出的实验将进一步加深我们对NIMA激酶人类直系同源物的理解，NIMA激酶人类直系同源物涉及特定的疾病状态（肾脏疾病）。最后，A. nidulans是该菌的模式生物。对A. nidulans将影响对机会曲霉属病原体的研究，以及用于产生新的生物活性化合物的成员。

DESCRIPTION (provided by applicant): Incorrect regulation of mitosis contributes to progression of cancer and birth defects and discoveries regarding the regulation of mitosis have positively impacted medical research and disease treatments. However, the potential for new insights and further impact on medicine clearly exists. For example, one of the most dramatic yet least understood mitotic processes is the disassembly and reassembly of nuclear pore complexes (NPCs), massive structures providing regulated gateways across the nuclear envelope. How this process is regulated and integrated with other mitotic events potentially involves reversible phosphorylation and our planned experiments will help test this hypothesis. We have shown the NIMA mitotic kinase of Aspergillus nidulans initiates mitosis by promoting NPC disassembly. NIMA is both required and sufficient to promote NPC disassembly and markedly affects mitosis in vertebrate cells suggesting the existence of conserved mitotic substrates. A. nidulans therefore provides a powerful and sophisticated model system in which to understand mitotic regulation of NPCs. The aims of this application include defining the dynamic mitotic changes in the NPC interactome using affinity purifications and mass spectrometry to identify new NPC proteins (Nups) as well as Nup associated proteins involved in mitotic regulation. In addition we will also focus studies on a new Nup associated protein (AN0162) which contains a transmembrane domain and potentially encodes a fourth transmembrane Nup. Our data suggest that AN0162 has a mitotic function to maintain the peripheral mRNA export factor Gle1 on the nuclear envelope surrounding the nucleolus during mitosis and we will test this hypothesis. Our third focus is on the mitotic functions of Nup2 and its associated proteins NupA, importin 1 and importin 2. Nup2 and NupA translocate from NPCs to chromatin during mitosis when they also become phosphorylated. We will test the hypothesis that NupA mediates the mitotic chromatin location of Nup2, which in turn controls the functions of importin 1 and 2 to regulate mitotic progression. As phosphorylation appears integral to this process, the final aim is to map mitotic phosphorylation sites within Nup2, NupA and importin 2 and then carry out in vivo mutational analysis of the specific phosphorylation sites to determine their function. PUBLIC HEALTH RELEVANCE: How the cell cycle is regulated is of fundamental medical importance because when this regulation goes awry disease states such as birth defects and cancer can result. In addition, many current cancer treatments target different aspects of cell cycle regulation to preferentially kill cells actively passing through the cell cycle. Therefore in terms of public health, the more we understand how the cell cycle is regulated the better positioned we will be to develop drugs against new chemotherapeutic targets to combat diseased states associated with cell cycle defects. The experiments proposed will in addition further our understanding of the NIMA kinase human orthologues of which are involved in specific disease states (kidney disease). Finally, A. nidulans is the model organism of the Aspergilli. Insights to the biology of A. nidulans will impact research on the opportunistic Aspergillus pathogens as well as members being used to generate new pharmacologically active compounds.