Regulation of PTEN Tumor Suppressor

PTEN编码一种磷脂酰肌醇-3-磷酸磷酸酶，在超过30%的情况下失活 人类癌症的风险。本项目的主要目的是研究PTEN在细胞连接中的作用机制。细胞连接完整性的丧失是转移性肿瘤的主要特征。 了解PTEN调控的信号转导事件将有助于分子鉴定 治疗干预的目标。该项目建立在PTEN是一项重要发现的基础上 磷酸蛋白和某些磷酸化形式的PTEN显示出更大的结合能力 细胞连接处的支架蛋白MAGI-2。 在特定目标1中，PTEN磷酸异构体的生物学和生化特性将是 检查过了。一组PTEN磷酸化位点突变体将首先通过定点产生 诱变。它们的相对磷酸酶活性将以水溶性荧光磷脂酰肌醇为底物进行测定。此外，它们的亚细胞定位将通过间接免疫荧光分析来确定。 在特定目的2中，将检测MAGI-2的抑瘤活性。的能力 改变生存、细胞间黏附、生长、细胞运动和致瘤性的MAGI-2将 使用缺乏MAGI-2表达的人肿瘤细胞进行研究。无论是 PTEN和MAGI-2之间的相互作用是肿瘤抑制所必需的，将使用 小干扰(Si)RNA下调PTEN的表达。 在特定目标3中，负责MAGI-2生物学功能的信号分子 都会被描绘出来。13-连环蛋白在介导MAGI-2作用中的作用将通过测量来检验 间接免疫荧光分析其亚细胞分布。此外，13链非依赖转录反应将通过使用TCF/LEF启动子元件的基于荧光素酶的报告分析来评估。 总而言之，这个应用程序关注的是一个令人兴奋且未被充分探索的研究领域 PTEN在细胞连接中的作用。拟议的研究将提供机械模型。 这可以用来解释接触抑制等关键的生理现象。在……里面 此外，还将描绘新的信号通路，并将为肿瘤诊断提供靶点 和治疗性干预。

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.