Regulation of Protein Dephosphorylation

描述（由申请人提供）：蛋白磷酸酶2A （PP2A）是蛋白丝氨酸/苏氨酸磷酸酶基因家族中普遍存在的成员，在包括代谢和细胞周期在内的基本细胞过程中起作用。PP2A由包含催化亚基(C)和支架亚基(A)的核心复合物组成，其与多种调控亚基和相互作用蛋白相互作用，将AC核心二聚体靶向特定底物和细胞内位置。许多不同类型的异源三聚体全酶的组装解释了PP2A调节广泛生物过程的能力。PP2A的重要靶点包括神经元微管细胞骨架、参与DNA复制的Cdc6蛋白和Wnt信号通路的组分。该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鉴定酶的新磷酸化蛋白靶点来鉴定PP2A的新功能。该项目有四个具体目标。1. Aim 1将完成对脑内PP2A靶向被sv40o小肿瘤抗原表达中断的转基因小鼠的分析。该实验将验证靶向微管的PP2A形式在调节神经元特异性微管相关蛋白tau的磷酸化中发挥重要作用的假设。实验还将测试tau蛋白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PP2A靶向Cdc6蛋白中的作用。实验将验证PP2A与Cdc6的相互作用是维持Cdc6在细胞周期的GI期处于去磷酸化状态所必需的假设。本实验目的还将确定钙在调节Cdc6去磷酸化中的作用。3. Aim 3旨在使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RNAi敲除。这些亚基在细胞信号传导中丢失的后果将通过双向电泳、质谱鉴定蛋白质和DNA微阵列进行检查。4. 目的4将检测PP2A的β亚基是否为肿瘤抑制蛋白。在肺和结肠肿瘤中发现的野生型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能的能力

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