The Regulation of PP1 in the Nucleus

描述（由申请人提供）：1/3的去磷酸化反应是由丝氨酸/苏氨酸蛋白磷酸酶1 （PP1）控制的，它存在于所有真核细胞中。PP1是一种单域金属结合蛋白，缺乏任何内在特异性。相反，它与bbb200确认的靶向蛋白的相互作用密切调节PP1，这些蛋白将PP1定位到细胞的不同区域并调节其底物特异性。虽然成千上万的细胞生物学和生物化学报告描述了PP1的关键生物学作用，但迄今为止，只有很少的结构研究取得了成功。在这里，我们描述了一个完整的研究计划，以了解PP1在细胞核中的调控。该研究项目采用强大的综合方法，将核磁共振光谱，x射线晶体学和SAXS与生物化学和体内实验相结合，以获得对调节PP1的分子机制的新见解。具体来说，我们专注于两个PP1靶向蛋白：1)PP1的核抑制剂（NIPP1）和2)PP1核靶向亚基（PNUTS）。超过1/3的PP1核库与NIPP1形成全酶。缺乏NIPP1的小鼠具有胚胎致死性，NIPP1和PP1的几种底物是致癌基因。NIPP1:PP1全酶通过染色质重塑和pre-mRNA剪接调节细胞周期进程、表观遗传沉默以及其他重要的生物学功能。因此，NIPP1:PP1全酶的解除管制导致疾病也就不足为奇了。PNUTS与染色质相关，促进染色体脱凝。它还通过p53和MDM2的翻译后修饰来控制细胞应激时的细胞死亡，并在细胞应激时调节视网膜母细胞瘤蛋白发挥重要作用，这是调节癌症的关键过程。在我们的共同努力下，我们将：1)确定这些生物关键PP1调节剂的自由形式的结构，2)确定PP1全酶的结构，3)确定这些复合物如何指导和调节PP1活性。此外，我们将利用这些蛋白质和蛋白质复合物结构来阐明，在分子水平上，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研究领域研究人员的广泛专业知识，并利用了最好的国内和国际合作者。此外，它有初步的数据表明，这项工作将为PP1的分子调控提供独特的，新颖的见解。

DESCRIPTION (provided by applicant): 1/3 of all dephosphorylation reactions are controlled by ser/thr protein phosphatase 1 (PP1), which is present in all eukaryotic cells. PP1 is a single domain metal-binding protein, which lacks any intrinsic specificity. Rather, it is closely regulated by its interaction with >200 confirmed targeting proteins, which localize PP1 to distinct regions of the cell and modulate its substrate specificity. While 1000's of cell biology and biochemical reports describe key biological roles for PP1, only very few structural efforts have so far been successful. 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 NMR spectroscopy, X-ray crystallography and SAXS with biochemical and in vivo experiments to obtain novel insights into the molecular mechanisms that regulate PP1. Specifically we are focusing on two PP1-targeting proteins: 1) the nuclear inhibitor of PP1 (NIPP1) & 2) the PP1 nuclear targeting subunit (PNUTS). More than 1/3 of the nuclear pool of PP1 forms a holoenzyme with NIPP1. Mice lacking NIPP1 are embryonic lethal and several substrate of NIPP1:PP1 are oncogenes. The NIPP1:PP1 holoenzyme regulates cell cycle progression, epigenetic silencing through chromatin remodeling and pre-mRNA splicing, among other essential biological functions. Thus it is of no surprise that deregulation of the NIPP1:PP1 holoenzyme leads to disease. PNUTS associates with chromatin and promotes chromosome de-condensation. It also controls cell death in response to cellular stresses through the post-translational modification of p53 and MDM2 and it plays essential role in the regulation of the retinoblastoma protein in response to cellular stress, critical processes for the regulation of cancer. In our combined efforts, we will: 1) determine the structures of the free form of these biologically critical PP1 regulators, 2) determine the structures of the PP1 holoenzymes and 3) determine how these complexes direct and regulate PP1 activity. Furthermore, we will 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 the investigators in the PP1 research field, as well as takes advantage of the best possible national and international collaborators. Furthermore, it has the preliminary data that demonstrates that this work will provide unique, novel insights into the molecular regulation of PP1.