Selective inhibition of the function of tyrosine-phosphorylated STAT3 with a phosphorylation site-specific intrabody
Selective inhibition of the function of tyrosine-phosphorylated STAT3 with a phosphorylation site-specific intrabody
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DOI：
10.1073/pnas.1316815111
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发表时间：
2014-04-29
影响因子：
11.1
通讯作者：
Rhee, Sue Goo
中科院分区：
文献类型：
--
作者：
Koo, Mi Young;Park, Jiyoung;Rhee, Sue Goo
信号转导和转录激活因子3(STAT3)是一种多功能蛋白，参与多种生长因子和细胞因子启动的信号转导通路。它以多种形式存在，包括那些在Tyr(705)(PYSTAT3)或Ser(727)(PSSTAT3)上磷酸化的蛋白以及未磷酸化的蛋白(USTAT3)。除了pYSTAT3的典型转录调控作用外，USTAT3和pSSTAT3都通过与不同的启动子结合而发挥转录调节作用，并在胞浆或线粒体中发挥信号作用。然而，每个STAT3物种在不同生物过程中的作用还不容易调查。我们现在已经准备了一种体内，它与pYSTAT3的酪氨酸磷酸化位点特异性地结合，并具有高亲和力。腺病毒介导的体内表达在HepG2细胞和小鼠肝脏中既阻断了pYSTAT3在细胞核中的积聚，也阻断了由白细胞介素6诱导的急性时相反应蛋白的产生。PSSTAT3的体内表达不影响白介素6或佛波酯(PMA)诱导的pSSTAT3的整体积聚，也不影响PMA诱导的c-Fos基因的表达，也不影响PMA诱导的pSSTAT3在线粒体中的特异性积聚。此外，它对白介素6诱导的干扰素调节因子1基因的表达也没有影响，干扰素调节因子1是STAT1的下游靶标。我们的结果表明，工程化的体内能够特异性地阻断pYSTAT3的下游效应，而不影响pSSTAT3的下游效应，证明了体内作为工具来剖析以多种形式存在的特定修饰形式的蛋白质的细胞功能的潜力。
Signal transducer and activator of transcription 3 (STAT3) is a multifunctional protein that participates in signaling pathways initiated by various growth factors and cytokines. It exists in multiple forms including those phosphorylated on Tyr(705) (pYSTAT3) or Ser(727) (pSSTAT3) as well as the unphosphorylated protein (USTAT3). In addition to the canonical transcriptional regulatory role of pYSTAT3, both USTAT3 and pSSTAT3 function as transcriptional regulators by binding to distinct promoter sites and play signaling roles in the cytosol or mitochondria. The roles of each STAT3 species in different biological processes have not been readily amenable to investigation, however. We have now prepared an intrabody that binds specifically and with high affinity to the tyrosine-phosphorylated site of pYSTAT3. Adenovirus-mediated expression of the intrabody in HepG2 cells as well as mouse liver blocked both the accumulation of pYSTAT3 in the nucleus and the production of acute phase response proteins induced by interleukin-6. Intrabody expression did not affect the overall accumulation of pSSTAT3 induced by interleukin-6 or phorbol 12-myristate 13-acetate (PMA), the PMA-induced expression of the c-Fos gene, or the PMA-induced accumulation of pSSTAT3 specifically in mitochondria. In addition, it had no effect on interleukin-6-induced expression of the gene for IFN regulatory factor 1, a downstream target of STAT1. Our results suggest that the engineered intrabody is able to block specifically the downstream effects of pYSTAT3 without influencing those of pSSTAT3, demonstrating the potential of intrabodies as tools to dissect the cellular functions of specific modified forms of proteins that exist as multiple species.