Signal transduction from type I and type II cytokine receptors plays important roles in development and function of cells regulating immune system. Although accumulating evidence suggests importance of Jak-Stat pathways in cytokine signal transduction, our earlier studies and other reports demonstrated that lymphocytes can differentiate and function even in the absence of the activation of Stat proteins, leading to a hypothesis that Stat-independent signaling pathways may play important roles in lymphocyte differentiation and function. However, the molecular nature of Stat-independent pathways is still elusive. The objective of the present research is to elucidate these elusive signaling pathways by using a novel expression cloning system, inducible translocation trap (ITT). ITT is based on translocation of a fusion protein consisting of LexA DNA-binding domain, transactivation domain of a strong transcriptional activator and a test molecule. LexA-mediated reporter gene expression is detected by flow cytometry. This system will be employed to analyze Stat-independent pathways in interferon gamma, as well as interleukin (IL)-2 and IL-3 signaling. The initial screening of cDNA library revealed that an isoform of pyruvate kinase (M2-PK), a key enzyme in glycolysis, translocates into the nucleus following IL-3 stimulation. In addition, it was found that the nuclear M2-PK plays an important role in cell proliferation. Molecular mechanisms of IL-3-induced nuclear translocation of M2-PK and enhancement of cell proliferation by the nuclear M2-PK will be analyzed by using molecular and cell biological techniques. Detailed information gained from the proposed experiments will be valuable for elucidation of molecular mechanisms of oncogenesis and autoimmune diseases caused by dysregulation of cytokine signaling.
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