VIRAL ONCOGENE/GROWTH FACTOR INDUCED SIGNAL TRANSDUCTION FROM PDGF RECEPTOR

点击翻译按钮获取中文摘要

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.