The Regulation of PP1 in the Nucleus

描述（由申请人提供）：1/3的所有去磷酸化反应由丝氨酸/苏氨酸蛋白磷酸酶1（PP 1）控制，其存在于所有真核细胞中。PP 1是一种单结构域金属结合蛋白，缺乏任何内在特异性。相反，它通过与超过200种已确认的靶向蛋白的相互作用而受到密切调控，这些靶向蛋白将PP 1定位于细胞的不同区域并调节其底物特异性。虽然1000的细胞生物学和生物化学报告描述了PP 1的关键生物学作用，但到目前为止，只有很少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PP 1在细胞核中的调节。 该研究项目使用了一种强大的综合方法，将NMR光谱学，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PP 1的分子机制的新见解。具体而言，我们专注于两种PP 1靶向蛋白：1）PP 1的核抑制剂（NIPP 1）和2）PP 1核靶向亚基（PNUTS）。 超过1/3的PP 1核库与NIPP 1形成全酶。缺乏NIPP 1的小鼠是胚胎致死的，并且NIPP 1：PP 1的几种底物是癌基因。NIPP 1：PP 1全酶调节细胞周期进程，通过染色质重塑和前体mRNA剪接进行表观遗传沉默，以及其他基本生物学功能。因此，NIPP 1：PP 1全酶的失调导致疾病也就不足为奇了。PNUTS与染色质结合并促进染色体去凝聚。它还通过p53和MDM 2的翻译后修饰控制响应于细胞应激的细胞死亡，并且它在响应于细胞应激的视网膜母细胞瘤蛋白的调节中起重要作用，这是调节癌症的关键过程。 在我们的共同努力中，我们将：1）确定这些生物学关键PP 1调节剂的游离形式的结构，2）确定PP 1全酶的结构和3）确定这些复合物如何指导和调节PP 1活性。此外，我们将利用这些蛋白质和蛋白质复合物的结构来阐明，在分子水平上，这些关键的核PP 1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PP 1研究领域研究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，它有初步的数据表明，这项工作将提供独特的，新颖的见解PP 1的分子调控。

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.