Centre for Drug Discovery, Graduate School of Pharmaceutical Science, University of Shizuoka, Suruga-ku, Shizuoka, Shizuoka, Japan.
Discovery Technology Laboratories, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda-shi, Saitama, Japan.
Sci Rep. 2018 Feb 5;8(1):2385. doi: 10.1038/s41598-018-20234-2.
Dimerization in signal transduction is a dynamically regulated process and a key regulatory mechanism. Signal transducer and activator of transcription 3 (STAT3) dimerizes after tyrosine phosphorylation upon cytokine stimulation. Because only the STAT3 dimer possesses the trans-activation activity, dimerization is an indispensable process for cytokine signaling. Here we report the detection of dynamic STAT3 dimerization in living cells using the homoFluoppi system. This method allowed us to validate the presence of an intact Src homology 2 domain and STAT3 Tyr705 phosphorylation, which facilitate puncta formation and homodimerization. Puncta formation was reversible, as determined by a decreased punctate signal after washout of oncostatin M. We analyzed STAT3 mutants, which have been reported in patients with hyper IgE syndrome and inflammatory hepatocellular adenoma (IHCA). Analysis of the IHCA mutants using homoFluoppi revealed constitutive activity independent of cytokine stimulation and novel insight into kinetics of dimer dissociation process. Next, we used homoFluoppi to screen for inhibitors of STAT3 dimerization, and identified 3,4-methylenedioxy-β-nitrostyrene as a novel inhibitor. The results of this study show that homoFluoppi is a useful research tool for the analysis of proteins like STAT3 that dynamically dimerize, and is applicable for the screening of dimerization modulators.
信号转导中的二聚化是一个动态调节的过程，也是一个关键的调节机制。信号转导子和转录激活子 3（STAT3）在细胞因子刺激下发生酪氨酸磷酸化后发生二聚化。由于只有 STAT3 二聚体具有转录激活活性，因此二聚化是细胞因子信号转导所必需的过程。在这里，我们使用 homoFluoppi 系统报告了活细胞中动态 STAT3 二聚化的检测。该方法使我们能够验证完整的Src 同源结构域 2 和 STAT3 Tyr705 磷酸化的存在，这有利于斑点的形成和同源二聚化。斑点的形成是可逆的，因为在用 Oncostatin M 冲洗后，点状信号减少。我们分析了在高 IgE 综合征和炎症性肝细胞腺瘤（IHCA）患者中报道的 STAT3 突变体。使用 homoFluoppi 对 IHCA 突变体进行分析，发现其具有独立于细胞因子刺激的组成性活性，并对二聚体解离过程的动力学有了新的认识。接下来，我们使用 homoFluoppi 筛选 STAT3 二聚化的抑制剂，并确定 3,4-亚甲二氧基-β-硝基苯乙烯是一种新型抑制剂。该研究结果表明，homoFluoppi 是一种用于分析像 STAT3 这样动态二聚化的蛋白质的有用研究工具，并且适用于二聚化调节剂的筛选。
