Kim Jinsu, Won Je-Seong, Singh Avtar K, Sharma Anand K, Singh Inderjit
1 Department of Pediatrics, Medical University of South Carolina , Charleston, South Carolina.
Antioxid Redox Signal. 2014 Jun 1;20(16):2514-27. doi: 10.1089/ars.2013.5223. Epub 2014 Feb 14.
S-nitrosylation and S-glutathionylation, redox-based modifications of protein thiols, are recently emerging as important signaling mechanisms. In this study, we assessed S-nitrosylation-based regulation of Janus-activated kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway that plays critical roles in immune/inflammatory responses and tumorigenesis.
Our studies show that STAT3 in stimulated microglia underwent two distinct redox-dependent modifications, S-nitrosylation and S-glutathionylation. STAT3 S-nitrosylation was associated with inducible nitric oxide synthase (iNOS)-produced nitric oxide (NO) and S-nitrosoglutathione (GSNO), whereas S-glutathionylation of STAT3 was associated with cellular oxidative stress. NO produced by iNOS or treatment of microglia with exogenous GSNO inhibited STAT3 activation via inhibiting STAT3 phosphorylation (Tyr(705)). Consequently, the interleukin-6 (IL-6)-induced microglial proliferation and associated gene expressions were also reduced. In cell-free kinase assay using purified JAK2 and STAT3, STAT3 phosphorylation was inhibited by its selective preincubation with GSNO, but not by preincubation of JAK2 with GSNO, indicating that GSNO-mediated mechanisms inhibit STAT3 phosphorylation through S-nitrosylation of STAT3 rather than JAK2. In this study, we identified that Cys(259) was the target Cys residue of GSNO-mediated S-nitrosylation of STAT3. The replacement of Cys(259) residue with Ala abolished the inhibitory role of GSNO in IL-6-induced STAT3 phosphorylation and transactivation, suggesting the role of Cys(259) S-nitrosylation in STAT3 phosphorylation.
Microglial proliferation is regulated by NO via S-nitrosylation of STAT3 (Cys(259)) and inhibition of STAT3 (Tyr(705)) phosphorylation.
Our results indicate the regulation of STAT3 by NO-based post-translational modification (S-nitrosylation). These findings have important implications for the development of new therapeutics targeting STAT3 for treating diseases associated with inflammatory/immune responses and abnormal cell proliferation, including cancer.
蛋白质硫醇基于氧化还原的修饰——S-亚硝基化和S-谷胱甘肽化，近来正作为重要的信号传导机制出现。在本研究中，我们评估了基于S-亚硝基化对Janus激活激酶2/信号转导及转录激活因子3（JAK2/STAT3）通路的调控，该通路在免疫/炎症反应和肿瘤发生中起关键作用。
我们的研究表明，在受刺激的小胶质细胞中，STAT3经历了两种不同的氧化还原依赖性修饰，即S-亚硝基化和S-谷胱甘肽化。STAT3的S-亚硝基化与诱导型一氧化氮合酶（iNOS）产生的一氧化氮（NO）和S-亚硝基谷胱甘肽（GSNO）有关，而STAT3的S-谷胱甘肽化与细胞氧化应激有关。iNOS产生的NO或用外源性GSNO处理小胶质细胞可通过抑制STAT3磷酸化（Tyr(705)）来抑制STAT3激活。因此，白细胞介素-6（IL-6）诱导的小胶质细胞增殖及相关基因表达也降低。在使用纯化的JAK2和STAT3进行的无细胞激酶测定中，STAT3的磷酸化通过其与GSNO的选择性预孵育而受到抑制，但JAK2与GSNO的预孵育则无此作用，这表明GSNO介导的机制通过STAT3的S-亚硝基化而非JAK2来抑制STAT3磷酸化。在本研究中，我们确定Cys(259)是GSNO介导的STAT3 S-亚硝基化的靶标半胱氨酸残基。用丙氨酸取代Cys(259)残基消除了GSNO对IL-6诱导的STAT3磷酸化和反式激活的抑制作用，表明Cys(259)的S-亚硝基化在STAT3磷酸化中的作用。
小胶质细胞增殖受NO通过STAT3（Cys(259)）的S-亚硝基化和STAT3（Tyr(705)）磷酸化的抑制来调控。
我们的结果表明基于NO的翻译后修饰（S-亚硝基化）对STAT3的调控。这些发现对于开发靶向STAT3的新疗法以治疗与炎症/免疫反应及异常细胞增殖相关的疾病（包括癌症）具有重要意义。
