Cytokine signal transduction in immune system

描述（由申请人提供）：来自I型和II型细胞因子受体的信号转导在调节免疫系统的细胞的发育和功能中起重要作用。虽然越来越多的证据表明Jak-Stat途径在细胞因子信号转导中的重要性，但我们早期的研究和其他报告表明，即使在没有Stat蛋白活化的情况下，淋巴细胞也可以分化和发挥功能，从而导致一种假设，即Stat-independent信号通路可能在淋巴细胞分化和功能中发挥重要作用。然而，Stat-independent途径的分子本质仍然是难以捉摸的。本研究的目的是利用一种新的表达克隆系统-诱导型易位陷阱（ITT）来阐明这些难以捉摸的信号通路。ITT是基于由莱克萨DNA结合结构域、强转录激活因子的反式激活结构域和测试分子组成的融合蛋白的易位。通过流式细胞术检测LexA介导的报告基因表达。该系统将用于分析干扰素γ中的Stat-independent通路，以及白细胞介素（IL）-2和IL-3信号传导。对cDNA文库的初步筛选显示，糖酵解的关键酶丙酮酸激酶（M2-PK）的一种亚型在IL-3刺激后易位到细胞核中。此外，还发现核M2-PK在细胞增殖中起重要作用。本研究将利用分子和细胞生物学技术分析IL-3诱导M2-PK核转位及核M2-PK促进细胞增殖的分子机制。从所提出的实验中获得的详细信息将是有价值的阐明肿瘤发生和自身免疫性疾病的细胞因子信号失调引起的分子机制。

DESCRIPTION (provided by applicant): 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.