The regulation of PP1 in the nucleus

 描述(由申请人提供)：在细胞分裂过程中复制基因组的准确分布对基因组遗传和细胞适合性至关重要。相反，与有丝分裂(亲代细胞分裂成两个相同的子细胞的过程)相关的任何步骤的中断往往与疾病，特别是癌症有关。有丝分裂是由细胞周期蛋白和磷酸酶之间错综复杂的平衡所驱动的，特别是丝氨酸/苏氨酸磷酸酶蛋白磷酸酶1(PP1)。虽然PP1本身具有广泛的特异性，但它通过与一系列调控蛋白形成稳定的复合体(全酶)来发挥高度特异性，从而指导其活性和定位。我们的长期目标是了解在健康和疾病中控制PP1活性的结构和功能机制，我们的实验室在这一领域做出了许多基本贡献。在这里，我们描述了一个完整的研究计划，以了解PP1在细胞核中的调节。目前的研究项目使用了一种强大的综合方法，将X射线结晶学和核磁共振光谱与生化和细胞生物学实验相结合，以获得对细胞周期不同阶段调节PP1活性的分子机制的新见解。具体地说，我们重点研究了四种PP1靶向蛋白对PP1的调节：1)Repoman(在有丝分裂后期将PP1重新招募到有丝分裂染色质上)，2)PN1核靶向亚基(PP1核靶向亚基)，3)Knl1(Kinetochore空蛋白1)和4)ASPP(P53蛋白的凋亡刺激)家族蛋白。这些调控因子中的每一个的异常表达都与多种癌症的发病率和严重性的增加有关。为了全面了解这些重要的全酶，需要详细描述这些调节剂与PP1的分子相互作用，目前还没有这些分子相互作用。在我们的共同努力中，我们将：1)确定这些PP1调节剂的自由形式的结构，2)确定PP1全酶的结构，以及3)确定这些复合体如何指导和调节PP1的活性。然后，我们将利用这些蛋白质和蛋白质复合体结构在分子水平上阐明这些关键的核PP1全酶的生物学功能和作用模式。本提案中描述的研究利用了PP1研究领域主要研究人员的广泛专业知识，并利用了尽可能最好的国家和国际合作者。最后，它有初步数据表明，这项工作将为PP1的分子调控及其在细胞周期不同阶段的基本作用提供独特的、新颖的见解。

 DESCRIPTION (provided by applicant): The accurate distribution of the replicated genome during cell division is crucial for genomic inheritance and cellular fitness. Conversely, disruptios in any of the steps associated with mitosis (the process in which a parental cell divides into two identical daughter cells) are often correlated with disease, especially cancer. The rapidly emerging picture is that mitosis is driven by the intricate balance between cell cycle kinases and phosphatases, especially the ser/thr phosphatase protein phosphatase 1 (PP1). While PP1 itself has a broad specificity, it acts in a highly specific manner by forming stable complexes (holoenzymes) with a host of regulatory proteins that direct its activity and localization. Our lon-term goal is to understand the structural and functional mechanisms that control PP1 activity in health and disease, an area in which our laboratory has made many fundamental contributions. Here we describe a complete research plan to understand the regulation of PP1 in the nucleus. The presented research project uses a powerful integrated approach that combines X-ray crystallography and NMR spectroscopy with biochemical and cell biology experiments to obtain novel insights into the molecular mechanisms that regulate PP1 activity during distinct stages of the cell cycle. Specifically, we are focusing on the regulation of PP1 by four PP1-targeting proteins: 1) Repoman (recruits PP1 onto mitotic chromatin at anaphase), 2) PNUTS (PP1 nuclear targeting subunit), 3) Knl1 (Kinetochore null protein 1) and 4) the ASPP (Apoptosis-stimulating of p53 protein) family of proteins. Abnormal expression of each of these regulators is associated with the increased incidence and severity of multiple cancers. Detailed descriptions of the molecular interactions of these regulators with PP1, which are currently missing, are needed for a comprehensive functional understanding of these important holoenzymes. In our combined efforts, we will: 1) determine the structures of the free form of these PP1 regulators, 2) determine the structures of the PP1 holoenzymes and 3) determine how these complexes direct and regulate PP1 activity. We will then leverage these protein and protein complex structures to elucidate, at a molecular level, the biological functions and modes of action of these key nuclear PP1 holoenzymes. The research described in this proposal leverages the extensive expertise of leading investigators in the PP1 research field, as well as takes advantage of the best possible national and international collaborators. Finally, it has the preliminary data that demonstrates that this work will provide unique, novel insights into the molecular regulation of PP1 and its fundamental roles during distinct stages of the cell cycle.