Kretzschmar Antje K, Dinger Michaela C, Henze Christian, Brocke-Heidrich Katja, Horn Friedemann
Institute of Clinical Immunology and Transfusion Medicine, University Hospital, Leipzig Johannisallee 30, D-04103 Leipzig, Germany.
Biochem J. 2004 Jan 15;377(Pt 2):289-97. doi: 10.1042/BJ20030708.
Signal transducer and activator of transcription 3 (Stat3) dimerization is commonly thought to be triggered by its tyrosine phosphorylation in response to interleukin-6 (IL-6) or other cytokines. Accumulating evidence from in vitro studies, however, suggests that cytoplasmic Stat3 may be associated with high-molecular-mass protein complexes and/or dimerize prior to its activation. To directly study Stat3 dimerization and subcellular localization upon cytokine stimulation, we used live-cell fluorescence spectroscopy and imaging microscopy combined with fluorescence resonance energy transfer (FRET). Stat3 fusion proteins with spectral variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were constructed and expressed in human hepatoma cells (HepG2) and human embryonic kidney cells (HEK-293). Like wild-type Stat3, the fusion proteins redistributed from a preferentially cytoplasmic to nuclear localization upon IL-6 stimulation and supported IL-6-dependent target gene expression. FRET studies in cells co-expressing Stat3-CFP and Stat3-YFP demonstrated that Stat3 dimers exist in the absence of tyrosine phosphorylation. IL-6 induced a 2-fold increase of this basal FRET signal, indicating that tyrosine phosphorylation either increases the dimer/monomer ratio of Stat3 or induces a conformational change of the dimer yielding a higher FRET efficiency. Studies using a mutated Stat3 with a non-functional src-homology 2 (SH2) domain showed that the SH2 domain is essential for dimer formation of phosphorylated as well as non-phosphorylated Stat3. Furthermore, our data show that visualization of normalized FRET signals allow insights into the spatiotemporal dynamics of Stat3 signal transduction.
信号转导与转录激活因子3（Stat3）的二聚化通常被认为是由其酪氨酸磷酸化引发的，以响应白细胞介素-6（IL-6）或其他细胞因子。然而，来自体外研究的越来越多的证据表明，细胞质中的Stat3可能与高分子质量蛋白复合物相关联和/或在其激活之前就已二聚化。为了直接研究细胞因子刺激后Stat3的二聚化和亚细胞定位，我们使用了活细胞荧光光谱法和成像显微镜，并结合荧光共振能量转移（FRET）。构建了带有绿色荧光蛋白（GFP）、青色荧光蛋白（CFP）和黄色荧光蛋白（YFP）光谱变体的Stat3融合蛋白，并在人肝癌细胞（HepG2）和人胚肾细胞（HEK-293）中表达。与野生型Stat3一样，融合蛋白在IL-6刺激后从优先存在于细胞质重新分布到细胞核，并支持IL-6依赖的靶基因表达。在共表达Stat3-CFP和Stat3-YFP的细胞中进行的FRET研究表明，Stat3二聚体在酪氨酸磷酸化缺失的情况下也存在。IL-6使这种基础FRET信号增加了2倍，表明酪氨酸磷酸化要么增加了Stat3的二聚体/单体比例，要么诱导了二聚体的构象变化，从而产生更高的FRET效率。使用具有无功能src同源2（SH2）结构域的突变Stat3进行的研究表明，SH2结构域对于磷酸化以及未磷酸化的Stat3的二聚体形成至关重要。此外，我们的数据表明，标准化FRET信号的可视化能够深入了解Stat3信号转导的时空动态。
