Cellular LanthaScreen and β-lactamase reporter assays for high-throughput screening of JAK2 inhibitors
Cellular LanthaScreen and β-lactamase reporter assays for high-throughput screening of JAK2 inhibitors
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DOI：
10.1089/adt.2008.144
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发表时间：
2008-08-01
影响因子：
1.8
通讯作者：
Bi, Kun
中科院分区：
文献类型：
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作者：
Robers, Matthew B.;Machleidt, Thomas;Bi, Kun
Janus Kinase(JAK)2/信号转导和转录激活因子(STAT)5通路负责调节细胞对多种细胞因子和生长因子的反应。在造血细胞中，粒细胞巨噬细胞集落刺激因子、白介素3和促红细胞生成素等生长因子诱导JAK2激活，从而导致STAT5蛋白的磷酸化、二聚化和反式激活。通过激活JAK2V617F等突变而导致JAK2的失调会导致STAT5的结构性磷酸化，并与许多骨髓增生性疾病有关，如真性红细胞增多症。建立了一种检测野生型JAK2活性的细胞LanthaScreen(TM)(Invitgen Corp.，CarlsbadCA)时间分辨Forester共振能量转移分析方法。本实验利用生长因子依赖型人红白血病TF1细胞株，通过基因工程表达绿色荧光蛋白-STAT5融合蛋白。此外，还建立了一种互补的β-内酰胺酶报告基因分析方法来分析JAK2下游的STAT5在TF1细胞中的转录活性。同样的技术也被应用于细胞检测的开发，用于询问与疾病相关的JAK2V617F激活突变体。一种小分子抑制剂和隐形(商标)(Invitgen Corp.)用RNA干扰寡核苷酸证实JAK2的参与。我们的结果表明，这些细胞分析和验证工具为在生理细胞环境中分析生理和疾病相关的JAK/STAT通路提供了强大的集成方法。
The Janus kinase (JAK) 2/signal tranducer and activator of transcription (STAT) 5 pathway is responsible for regulation of cellular responses to a number of cytokines and growth factors. In hematopoietic cells, growth factors such as granulocyte macrophage-colony stimulating factor, interleukin-3, and erythropoietin induce the activation of JAK2, which leads to the phosphorylation, dimerization, and transactivation of STAT5 proteins. Dysregulation of JAK2 by activating mutations such as JAK2V617F results in constitutive phosphorylation of STAT5 and has been linked to numerous myeloproliferative disorders such as polycythemia vera. A cellular LanthaScreen (TM) (Invitrogen Corp., Carlsbad, CA) time-resolved Forester resonance energy transfer assay for wild-type JAK2 activity was developed. This assay utilized the growth factor-dependent human erythroleukemia TF1 cell line engineered to express a green fluorescent protein-STAT5 fusion protein. Furthermore, a complementary beta-lactamase reporter gene assay was developed to analyze the transcriptional activity of STAT5 downstream of JAK2 in TF1 cells. The same technologies were applied to the development of cellular assays for the interrogation of the disease-relevant JAK2V617F activating mutant. A small molecule inhibitor and Stealth (TM) (Invitrogen Corp.) RNA interference oligonucleotides were used to confirm the involvement of JAK2. Our results suggest that these cellular assays and validation tools represent powerful integrated methods for the analysis of physiological and disease-relevant JAK/STAT pathways within the physiological cellular context.