The Regulation of PP1 in the Nucleus

描述(申请人提供)：1/3的去磷酸化反应由丝氨酸/苏氨酸蛋白磷酸酶1(PP1)控制，它存在于所有真核细胞中。PP1是一种单结构域金属结合蛋白，缺乏任何固有的特异性。相反，它受到其与&gt；200确认的靶向蛋白的相互作用的密切调控，后者将PP1定位到细胞的不同区域，并调节其底物特异性。虽然1000‘S的细胞生物学和生化报告描述了PP1的关键生物学作用，但到目前为止，只有极少数的结构努力取得了成功。在这里，我们描述了一个完整的研究计划，以了解PP1在细胞核中的调节。目前的研究项目使用了一种强大的综合方法，将核磁共振光谱、X射线结晶学和SAXS与生化和体内实验相结合，以获得对调节PP1的分子机制的新见解。具体地说，我们重点研究了两种PP1靶向蛋白：1)PP1核抑制因子(NIPP1)&2)PP1核靶向亚单位(PNUTS)。超过1/3的PP1核池与NIPP1形成全酶。缺乏NIPP1的小鼠是胚胎致死的，NIPP1：PP1的几个底物是癌基因。NIPP1：PP1全酶调节细胞周期进程，通过染色质重塑和前mRNA剪接等其他基本生物学功能调节表观遗传沉默。因此，NIPP1：PP1全酶的解除调控导致疾病也就不足为奇了。PNUTS与染色质结合，促进染色体去凝集。它还通过P53和MDM2的翻译后修饰来控制细胞死亡，并在调节视网膜母细胞瘤蛋白对细胞应激的反应中发挥重要作用，这是调节癌症的关键过程。在我们的共同努力中，我们将：1)确定这些生物关键的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。此外，我们将利用这些蛋白质和蛋白质的复杂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。本提案中所述的研究利用了PP1研究领域调查人员的广泛专业知识，并利用了最好的国家和国际合作者。此外，它有初步数据表明，这项工作将为PP1的分子调控提供独特的、新颖的见解。 公共卫生相关性：PP1是一种普遍存在的丝氨酸/苏氨酸蛋白磷酸酶，调节高等真核生物中所有去磷酸化反应的1/3。尽管S做了1000‘年的细胞生物学努力，但关于PP1’S调控的详细分子图像仍然缺乏。因此，我们对PP1在细胞核中的调控的分子洞察对于将PP1建立成一个强大的药物靶点是至关重要的。

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. PUBLIC HEALTH RELEVANCE: PP1 is a ubiquitous ser/thr protein phosphatase which regulates ~1/3 of all dephosphorylation reactions in higher eukaryotes. Despite 1000's of cell biological efforts, a detailed molecular picture of PP1's regulation is still missing. Therefore, our molecular insights into the regulation of PP1 in the nucleus are essential to establish PP1 into a powerful drug target.