The association and nuclear translocation of the PIAS3-STAT3 complex is ligand and time dependent.
The association and nuclear translocation of the PIAS3-STAT3 complex is ligand and time dependent.
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DOI：
10.1158/1541-7786.mcr-09-0313
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发表时间：
2009-11
期刊：
影响因子：
--
通讯作者：
Dowlati A
中科院分区：
文献类型：
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作者：
Dabir S;Kluge A;Dowlati A
表皮生长因子受体（EGFR）激活下游信号转导和转录激活因子3（STAT 3）在肺癌的发病机制中起着至关重要的作用。STAT 3的转录活性可被活化的STAT 3蛋白抑制剂（PIAS 3）负调控。我们通过共聚焦显微镜、免疫沉淀、荧光素酶报告基因分析和分离细胞组分的蛋白质分析，研究了肺癌中EGF依赖性模型中PIAS 3的时间依赖性改组和与STAT 3的结合。我们还探讨了STAT 3的Tyr 705磷酸化在PIAS 3/STAT 3复合物形成和细胞内改组中的作用。在无生长因子的状态下，PIAS 3定位于细胞质，并且在H520和A549细胞中与STAT 3未结合。暴露于EGF后，我们观察到STAT 3磷酸化和PIAS 3/STAT 3复合物的快速形成。在5分钟内，复合物进行性易位到细胞核，到10分钟PIAS 3唯一定位到细胞核区室。30分钟后，PIAS 3返回细胞质。使用定点诱变，我们用苯丙氨酸取代STAT 3的Tyr 705。尽管EGF刺激，我们观察到PIAS 3和STAT 3结合的显著减少和PIAS 3核转位的显著减少。此外，PIAS 3减少STAT 3介导的基因转录的能力显著降低。在野生型STAT 3细胞中，PIAS 3浓度增加导致STAT 3磷酸化成比例降低。这些数据表明PIAS 3对STAT 3在表皮生长因子驱动的肿瘤中的负调节作用的重要作用。
The Epidermal Growth Factor Receptor (EGFR) 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 PIAS3 time-dependent 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 of PIAS3/STAT3 complex and intracellular shuffling. In a growth factor-free state, PIAS3 was localized to the cytoplasm and unbound to STAT3 in both H520 and A549 cells. Upon 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. Thirty minutes after, 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 and STAT3 binding and a significant reduction in nuclear translocation of PIAS3. Furthermore, there was a significant reduction in PIAS3 capacity 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 epidermal growth factor driven tumors.