Regulation of PTEN Tumor Suppressor

PTEN肿瘤抑制因子的调控

• 批准号: 6785823
• 负责人: ANDREW M CHAN
• 金额: $ 27.37万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6785823
• 关键词: athymic mouse; cadherins; cell adhesion; cell growth regulation; cell motility; contact inhibition; enzyme activity; gene expression; gene induction /repression; gene targeting; immunofluorescence technique; intercellular connection; membrane proteins; metastasis; neoplasm /cancer genetics; neoplastic cell; nucleic acid sequence; phosphomonoesterases; phosphoproteins; phosphorylation; protein isoforms; protein localization; protein protein interaction; protein structure function; site directed mutagenesis; tumor suppressor genes; tumor suppressor proteins

PTEN encodes a phosphatidylinositol-3-phosphate phosphatase and is inactivated in over 30% of human cancer. The major goal of this project is to investigate the mechanism of action of PTEN at the cell junction. Loss of cell junctional integrity is a primary feature of metastatic tumors. Understanding the signaling events controlled by PTEN will lead to the identification of molecular targets for therapeutic interventions. This project is built on the important findings that PTEN is a phosphoprotein and certain phosphorylated forms of PTEN show a greater binding capacity to a scaffolding protein, MAGI-2, at the cell junction. In Specific Aim 1, the biological and biochemical properties of PTEN phosphoisoforms will be examined. A panel of PTEN phosphorylation site mutants will first be generated by site-directed mutagenesis. Their relative phosphatase activity will be assayed using water-soluble fluorescent phosphoinositides as substrates. Also, their subcellular localization will be determined by indirect immunofluorescence analysis. In Specific Aim 2, the tumor suppressing activity of MAGI-2 will be examined. The ability of MAGI-2 to alter survival, cell-cell adhesion, growth, cell motility, and tumorigenicity will be investigated using human tumor cells which lack the expression of MAGI-2. Whether the interaction between PTEN and MAGI-2 is necessary for tumor suppression will be tested using small interfering (si) RNA to down-regulate PTEN expression. In Specific Aim 3, the signaling molecules responsible for the biological functions of MAGI-2 will be delineated. The role of 13-catenin in mediating MAGI-2 actions will be tested by measuring its subcellular distribution through indirect immunofluorescence analysis. Also, 13-catenindependent transcriptional responses will be assessed by luciferase-based reporter assays using a TCF/Lef promoter element. In summary, this application focuses on an exciting and under-explored area of research into the function of PTEN at the cell junction. The proposed studies will provide mechanistic models that can be used to explain such critical physiological phenomenon as contact inhibition. In addition, novel signaling pathways will be delineated and will provide targets for tumor diagnosis and therapeutic interventions.

PTEN编码磷脂酰肌醇-3-磷酸磷酸酶，并且在超过30%的细胞中失活。 人类癌症本课题的主要目的是研究PTEN在细胞连接处的作用机制。细胞连接完整性的丧失是转移性肿瘤的主要特征。 了解由PTEN控制的信号事件将导致识别分子 治疗干预的目标。这个项目是建立在重要的发现，PTEN是一个 磷蛋白和某些磷酸化形式的PTEN显示出更大的结合能力， 支架蛋白MAGI-2在细胞连接处。 在具体目标1中，将研究PTEN磷酸化同种型的生物学和生物化学性质。 考察一组PTEN磷酸化位点突变体将首先通过定点突变产生。 诱变将使用水溶性荧光磷酸肌醇作为底物测定其相对磷酸酶活性。此外，它们的亚细胞定位将通过间接免疫荧光分析来确定。 在特定目标2中，将检查MAGI-2的肿瘤抑制活性。的能力 MAGI-2改变存活率、细胞-细胞粘附、生长、细胞运动性和致瘤性将是可能的。 使用缺乏MAGI-2表达的人肿瘤细胞进行研究。是否 PTEN和MAGI-2之间的相互作用对于肿瘤抑制是必需的，将使用 小干扰（si）RNA下调PTEN表达。 在具体目标3中，负责MAGI-2生物学功能的信号分子 将被划定。13-连环蛋白在介导MAGI-2作用中的作用将通过测量 通过间接免疫荧光分析其亚细胞分布。此外，13-连环非依赖性转录反应将通过基于TCF/Lef启动子元件的报告基因测定进行评估。 总之，该应用程序侧重于一个令人兴奋的和未开发的研究领域， PTEN在细胞连接处的功能。拟议的研究将提供机制模型 可以用来解释接触抑制等重要的生理现象。在 此外，新的信号通路将被描绘出来，并将为肿瘤诊断提供靶点 和治疗干预。