Köster M, Hauser H
Department of Gene Regulation and Differentiation, GBF-National Research Institute for Biotechnology, Braunschweig, Germany.
Eur J Biochem. 1999 Feb;260(1):137-44. doi: 10.1046/j.1432-1327.1999.00149.x.
STAT proteins (signal transducers and activators of transcription) are a family of transcription factors which are used by many cytokines and cell growth factors for initiating gene expression. They are activated by tyrosine phosphorylation through the cytoplasmic domain of stimulated receptors. Upon phosphorylation STAT proteins dimerize, translocate to the nucleus and activate transcription by binding to specific recognition sites. Different cytokines activate different subsets of STATs and other signaling proteins. We have made use of green fluoresencent protein (GFP) fusion proteins to visualize the subcellular localization and trafficking of STAT1, STAT2 and p48 during interferon (IFN) stimulation and have analysed in detail STAT1-GFP trafficking in living cells. Analysis of GFP fusion proteins allowed the determination of time kinetics of subcellular trafficking in individual living cells. STAT1-GFP is indistinguishable from its wild-type protein displaying strong activity as transcriptional activator as well as the same time kinetics of transport to the nucleus and retreat to the cytoplasm. After prolonged exposure to IFN, STAT1-GFP is no longer retained in the nucleus and relocation to the cytoplasm is observed. Restimulation with the same type of IFN does not lead to repeated nuclear translocation of STAT1-GFP. STAT1 is not subject of inhibition, as restimulation with another type of IFN allows immediate reuse of previously activated STAT1-GFP. However, restimulation with the same type of IFN can be achieved when the primary stimulus is removed after a short induction period. This method of visualizing signal transduction reveals a considerable inhomogeneity with respect to the extent of STAT1-GFP shuttling within a clonal cell population, indicating that competence for full-blasted IFN response is restricted to a cellular subpopulation whereas other cells respond incompletely, retarded or not at all.
信号转导与转录激活因子（STAT）蛋白是一类转录因子，许多细胞因子和细胞生长因子利用它们来启动基因表达。它们通过受刺激受体的胞质结构域的酪氨酸磷酸化而被激活。磷酸化后，STAT蛋白二聚化，转位至细胞核，并通过结合特定识别位点来激活转录。不同的细胞因子激活不同的STAT亚群和其他信号蛋白。我们利用绿色荧光蛋白（GFP）融合蛋白来观察干扰素（IFN）刺激过程中STAT1、STAT2和p48的亚细胞定位和运输，并详细分析了活细胞中STAT1-GFP的运输情况。对GFP融合蛋白的分析使得能够确定单个活细胞中亚细胞运输的时间动力学。STAT1-GFP与其野生型蛋白没有区别，作为转录激活因子显示出强大的活性，并且向细胞核运输和返回细胞质的时间动力学相同。长时间暴露于IFN后，STAT1-GFP不再保留在细胞核中，而是观察到其重新定位到细胞质中。用同一种IFN再次刺激不会导致STAT1-GFP重复核转位。STAT1不受抑制，因为用另一种IFN再次刺激可使先前激活的STAT1-GFP立即重新利用。然而，当在短诱导期后去除初次刺激时，可以实现用同一种IFN再次刺激。这种可视化信号转导的方法揭示了在克隆细胞群体中STAT1-GFP穿梭程度方面存在相当大的不均匀性，表明完全的IFN反应能力仅限于细胞亚群，而其他细胞反应不完全、延迟或根本不反应。
