Regulation of Protein Dephosphorylation

描述(申请人提供)：蛋白磷酸酶2A(PP2A)是蛋白质丝氨酸/苏氨酸磷酸酶基因家族中普遍存在的成员，在包括新陈代谢和细胞周期在内的基本细胞过程中发挥作用。PP2A由含有催化亚单位(C)和支架亚单位(A)的核心复合体组成，它与多种调节亚基和相互作用的蛋白质相互作用，这些蛋白质将AC核心二聚体靶向特定的底物和细胞内位置。许多不同类型的异三聚体全酶的组装解释了PP2A调节广泛的生物过程的能力。PP2A的重要靶点包括神经元微管细胞骨架、参与DNA复制的CDC6蛋白以及Wnt信号通路的组成部分。这个项目的一个总体目标是确定PP2A在这些过程中作用的分子基础。另一个主要目标是通过确定PP2A酶的新的磷酸蛋白靶标来确定PP2A的新功能。该项目有四个具体目标。1.目的1完成对转基因小鼠脑内PP2A靶向被SV4O小肿瘤抗原表达干扰的分析。这些实验将检验这样一种假设，即针对微管的PP2A形式在调节神经元特异性微管相关蛋白tau的磷酸化方面发挥着重要作用。这些实验还将测试tau的磷酸化增加是否会导致神经元退化。2.目的2将确定PR48亚基在靶向PP2A到CDC6蛋白中的作用。这些实验将检验这一假设，即在细胞周期的GI阶段，PP2A与CDC6的相互作用是维持CDC6处于去磷酸化状态所必需的。这一目的的实验还将确定钙在调节CDC6去磷酸化中的作用。3.AIM 3旨在利用蛋白质组学方法鉴定新的PP2A底物和功能。单个PP2A亚基将被使用RNAi敲除。这些亚基缺失对细胞信号的影响将通过二维电泳法、质谱学鉴定蛋白质和DNA微阵列来检验。4.Aim 4将测试PP2A的ABeta亚单位是否为肿瘤抑制蛋白。在肺癌和结肠癌中发现的野生型Abeta蛋白和含有突变的蛋白形式将被测试其下调Wnt信号通路和逆转肺癌细胞系致瘤潜力的能力

DESCRIPTION (provided by applicant): Protein phosphatase 2A (PP2A) is a ubiquitous member of the protein serine/threonine phosphatase gene family functions in fundamental cellular processes including metabolism and the cell cycle. PP2A is composed of core complex containing the catalytic subunit (C) and the scaffold subunit (A), which interacts with a wide variety of regulatory subunits and interacting proteins that target the AC core dimer to specific substrates and intracellular locations. The assembly of many different types of heterotrimeric holoenzymes accounts for the ability of PP2A to regulate a wide range of biological processes. Important targets for PP2A include the neuronal microtubule cytoskeleton, the Cdc6 protein involved in DNA replication, and components of the Wnt signaling pathway. One overall goal of this project is to determine the molecular basis for the actions of PP2A in these processes. Another major goal is to identify novel functions of PP2A by identifying new phosphoprotein targets for the enzyme. The project has four specific aims. 1. Aim 1 will complete the analysis of transgenic mice in which targeting of PP2A in the brain is disrupted by expression of SV4O small tumor antigen. The experiments will test the hypothesis that forms of PP2A targeted to microtubules play an important role in regulating the phosphorylation of the neuron-specific microtubule-associated protein tau. The experiments will also test whether increased phosphorylation of tau can lead to neuronal degeneration. 2. Aim 2 will determine the role of the PR48 subunit in targeting PP2A to the Cdc6 protein. The experiments will test the hypothesis that the interaction of PP2A with Cdc6 is required to maintain Cdc6 in a dephosphorylated state during the GI phase of the cell cycle. Experiment in this aim will also determine the role of calcium in regulating Cdc6 dephosphorylation. 3. Aim 3 is designed to identify novel PP2A substrates and functions using a proteomic approach. Individual PP2A subunits will be knocked out using RNAi. The consequences of the loss of these subunits on cellular signaling will be examined by two-dimensional electrophoresis, identification of proteins by mass spectrometry, and DNA microarrays. 4. Aim 4 will test whether the ABeta subunit of PP2A is a tumor suppressor protein. Wild type Abeta protein and forms of the protein containing mutations identified in lung and colon tumors will be tested for their abilities to downregulate the Wnt signaling pathway and to reverse the tumorigenic potential of lung cancer cell lines