Division of Hematology/Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
Mol Cancer Res. 2009 Nov;7(11):1854-60. doi: 10.1158/1541-7786.MCR-09-0313. Epub 2009 Nov 10.
The epidermal growth factor (EGF) receptor activation of downstream signal transducers and activators of transcription 3 (STAT3) plays a crucial role in the pathogenesis of lung cancer. STAT3 transcriptional activity can be negatively regulated by protein inhibitor of activated STAT3 (PIAS3). We investigated the time-dependent PIAS3 shuffling and binding to STAT3 in an EGF-dependent model in lung cancer by using confocal microscopy, immunoprecipitation, luciferase reporter assay, and protein analysis of segregated cellular components. We also explored the role of phosphorylation at Tyr705 of STAT3 in the formation and intracellular shuffling of the PIAS3-STAT3 complex. In a growth factor-free state, PIAS3 was localized to the cytoplasm and unbound to STAT3 in both H520 and A549 cells. On exposure to EGF, we observed STAT3 phosphorylation and rapid formation of the PIAS3-STAT3 complex. Within 5 minutes, there was a progressive translocation of the complex to the nucleus, and by 10 minutes, PIAS3 was uniquely localized to the nuclear compartment. After 30 minutes, PIAS3 returned to the cytoplasm. Using site-directed mutagenesis, we substituted Tyr705 of STAT3 with a phenylalanine. Despite EGF stimulation, we observed a significant decrease in PIAS3-STAT3 binding and a significant reduction in nuclear translocation of PIAS3. Furthermore, there was a significant reduction in the capacity of PIAS3 to reduce STAT3-mediated gene transcription. In wild-type STAT3 cells, increasing concentrations of PIAS3 resulted in a proportional decrease in STAT3 phosphorylation. These data suggest an important role for the negative regulatory effect of PIAS3 on STAT3 in EGF-driven tumors.
表皮生长因子（EGF）受体激活下游信号转导子和转录激活子 3（STAT3）在肺癌的发病机制中起着至关重要的作用。STAT3 的转录活性可被激活 STAT3 的蛋白抑制剂 3（PIAS3）负调节。我们通过共聚焦显微镜、免疫沉淀、荧光素酶报告基因检测和分离细胞成分的蛋白分析，研究了 EGF 依赖性模型中 PIAS3 与 STAT3 的时间依赖性 shuffling 和结合。我们还探讨了 STAT3 上 Tyr705 磷酸化在 PIAS3-STAT3 复合物形成和细胞内 shuffling 中的作用。在无生长因子的状态下，PIAS3 定位于细胞质中，与 H520 和 A549 细胞中的 STAT3 不结合。暴露于 EGF 后，我们观察到 STAT3 磷酸化和 PIAS3-STAT3 复合物的快速形成。在 5 分钟内，复合物逐渐向核内转位，到 10 分钟时，PIAS3 仅定位于核区。30 分钟后，PIAS3 回到细胞质。通过定点突变，我们将 STAT3 的 Tyr705 突变为苯丙氨酸。尽管受到 EGF 的刺激，我们观察到 PIAS3-STAT3 结合显著减少，PIAS3 核内转位显著减少。此外，PIAS3 降低 STAT3 介导的基因转录的能力显著降低。在野生型 STAT3 细胞中，随着 PIAS3 浓度的增加，STAT3 磷酸化呈比例下降。这些数据表明，PIAS3 对 EGF 驱动的肿瘤中 STAT3 的负调控作用具有重要意义。
