VIRAL ONCOGENE/GROWTH FACTOR INDUCED SIGNAL TRANSDUCTION FROM PDGF RECEPTOR

病毒癌基因/生长因子诱导 PDGF 受体的信号转导

• 批准号: 6347545
• 负责人: DEENA M KEGLER-EBO
• 金额: $ 6.95万
• 依托单位: CLARK ATLANTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6347545
• 关键词: 3T3 cells; DNA binding protein; JAK kinase; animal tissue; biological signal transduction; cell membrane; confocal scanning microscopy; fluorescence microscopy; gel mobility shift assay; gene induction /repression; growth factor receptors; human tissue; immunoprecipitation; oncogenes; oncoproteins; phosphorylation; platelet derived growth factor; protein protein interaction; receptor binding; receptor expression; transcription factor; virus protein; western blottings

Signal transduction from activated platelet derived growth factor beta receptor (PDGF receptor) is implicated in chronic myelomonocytic leukemia and in morphological transformation of rodent cells in culture. Platelet derived growth factor (PDGF-BB) and two viral oncoproteins, BPV E5 and vSIS, bind the PDGF-B receptor to activate the JAK-STAT signal transduction pathway. Inhibitory or stimulatory growth signals in this pathway depend on the receptor stimulated and cytoplasmic phosphorylation of a specific combination of the JAK kinases (JAKs) and the signal transducers and activator of transcription (STATs). Nuclear STAT protein dimers can induce transcription of a growth regulatory gene by binding to a promoter consensus element. The IRF protein family is associated with some of these STAT promoter complexes to facilitate binding or regulate signaling. Our lab made a novel finding, that a differential activation of STAT DNA binding occurs when stably expressed BPV E5 or exogenous PDGF-BB bind to the PDGF-B receptors in C127 murine fibroblast. Therefore, the hypothesis is that the protein that binds and activates PDGF-B receptors can specify differential signaling. To test this hypothesis, we will observe JAK-STAT signaling events after activation of the PDGF-B receptor signaling cascade by different receptor binding proteins, BPV E5 (intracellular receptors), vSIS (intracellular and plasma membrane) and exogenous PDGF-BB (plasma membrane receptors) in murine and human cell lines. We will examine STAT and IRF protein family members, for their protein-protein interactions, DNA binding activities, phosphorylation states and protein levels in cell lines with wild type PDGF-B receptors. We will conduct similar experiments using mutants of BP5 E5 and the PDGF receptor to determine the contribution of each molecule's structural features to STAT and IRF protein signaling or to transformation. We will use retroviruses to develop stable cell lines, then analyze nuclear and cytosolic STAT and IRF protein expression, complexes and phosphorylation by immunoprecipitation and western blotting. We will use electrophoretic mobility shift assays to examine protein binding to DNA consensus elements, and use antibody and oligonucleotide competitions to characterize the induced protein complexes. We will use immunofluorescence and confocal microscopy to localize STAT and IRF proteins in the established cell lines. The objective is to compare and contrast activated PDGF-B receptor signaling, to identify BPV E5, PDGF-BB treated or vSIS specific signaling proteins and complexes whose modulation in the JAK-STAT signal transduction pathway may be important in cell transformation and cancer. The information gained from these studies will be important for targeting responsive genes, developing therapeutic tools and regulation of PDGF receptors signaling in cancer.

活化的血小板衍生生长因子β受体（PDGF受体）的信号转导与慢性粒单核细胞白血病和培养的啮齿动物细胞的形态转化有关。血小板衍生生长因子（PDGF-BB）和两种病毒癌蛋白BPV E5和vSIS结合PDGF-B受体以激活JAK-STAT信号转导途径。该途径中的抑制性或刺激性生长信号取决于JAK激酶（JAK）和信号转导子和转录激活子（STAT）的特定组合的受体刺激和细胞质磷酸化。核STAT蛋白二聚体可以通过结合启动子共有元件来诱导生长调节基因的转录。IRF蛋白家族与这些STAT启动子复合物中的一些相关以促进结合或调节信号传导。我们实验室发现，当稳定表达的BPV E5或外源性PDGF-BB与C127小鼠成纤维细胞中的PDGF-B受体结合时，STAT DNA结合发生差异激活。因此，假设是结合和激活PDGF-B受体的蛋白质可以指定差异信号。为了验证这一假设，我们将在鼠和人细胞系中观察不同受体结合蛋白BPV E5（细胞内受体）、vSIS（细胞内和质膜）和外源性PDGF-BB（质膜受体）激活PDGF-B受体信号级联后的JAK-STAT信号传导事件。我们将研究STAT和IRF蛋白家族成员，其蛋白质-蛋白质相互作用，DNA结合活性，磷酸化状态和野生型PDGF-B受体细胞系中的蛋白质水平。我们将使用BP 5 E5和PDGF受体的突变体进行类似的实验，以确定每个分子的结构特征对STAT和IRF蛋白信号传导或转化的贡献。我们将使用逆转录病毒开发稳定的细胞系，然后通过免疫沉淀和蛋白质印迹分析核和胞浆STAT和IRF蛋白表达、复合物和磷酸化。我们将使用电泳迁移率变动分析，以检查蛋白质结合的DNA共识元素，并使用抗体和寡核苷酸竞争，以表征诱导的蛋白质复合物。我们将使用免疫荧光和共聚焦显微镜定位STAT和IRF蛋白在建立的细胞系。目的是比较和对比活化的PDGF-B受体信号传导，以鉴定BPV E5、PDGF-BB处理的或vSIS特异性信号传导蛋白和复合物，其在JAK-STAT信号转导途径中的调节可能在细胞转化和癌症中是重要的。从这些研究中获得的信息对于靶向应答基因、开发治疗工具和调节癌症中的PDGF受体信号传导将是重要的。