VIRAL ONCOGENE/GROWTH FACTOR INDUCED SIGNAL TRANSDUCTION FROM PDGF RECEPTOR

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BPVE5和vSIS与PDGF-B受体结合，激活JAK-STAT信号转导通路。该途径中的抑制性或刺激性生长信号依赖于JAK激酶(JAK)与信号转导和转录激活因子(STATs)结合的受体刺激和胞浆磷酸化。核转录调控蛋白二聚体可以通过与启动子一致元件结合来诱导生长调控基因的转录。IRF蛋白家族与其中一些STAT启动子复合体相关，以促进结合或调节信号。我们的实验室有一个新的发现，当稳定表达的BPVE5或外源PDGF-BB与C127小鼠成纤维细胞中的PDGF-B受体结合时，STAT DNA结合的差异激活发生。因此，假设结合并激活PDGF-B受体的蛋白质可以指定差异信号。为了验证这一假设，我们将观察不同受体结合蛋白BPV E5(细胞内受体)、vSIS(细胞内和质膜)以及外源PDGF-BB(质膜受体)激活PDGF-B受体信号级联后的JAK-STAT信号事件。我们将研究STAT和IRF蛋白家族成员，以了解它们在带有野生型PDGF-B受体的细胞系中的蛋白质-蛋白质相互作用、DNA结合活性、磷酸化状态和蛋白质水平。我们将使用BP5、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蛋白信号或转化的贡献。我们将利用逆转录病毒建立稳定的细胞系，然后通过免疫沉淀和Western blotting分析核和胞质STAT和IRF蛋白的表达、复合体和磷酸化。我们将使用电泳迁移率变化分析来检测蛋白质与DNA共识元件的结合，并使用抗体和寡核苷酸竞争来表征诱导的蛋白质复合体。我们将使用免疫荧光和共聚焦显微镜来定位已建立的细胞系中的STAT和IRF蛋白。目的是比较和对比激活的PDGF-B受体信号，鉴定BPV E5、PDGF-BB处理的或vSIS特异的信号蛋白和复合体，它们在JAK-STAT信号转导通路中的调节可能在细胞转化和癌症中起重要作用。从这些研究中获得的信息将对靶向反应基因、开发治疗工具和调节癌症中PDGF受体信号转导具有重要意义。

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.