Adach Alicja, Ellert-Miklaszewska Aleksandra, Kaminska Bozena
Laboratory of Transcription Regulation, Nencki Institute, Warsaw, Poland.
Methods Mol Biol. 2009;512:265-78. doi: 10.1007/978-1-60327-530-9_14.
The Janus kinases (JAK) and signal transducer and activator of transcription (STAT) signaling are strongly activated in many tumors. STAT proteins are activated by phosphorylation at the tyrosine residue, then dimerize, translocate to the nucleus and bind DNA, initiating the transcription of target genes. Activation of JAK-STAT pathway is implicated in the regulation of cell growth, differentiation, survival and cross-talk between cancer and immune cells. The activation of STATs depends on phosphorylation on a single tyrosine residue (e.g., Tyr705 in STAT3 and Tyr694 in STAT5) in the C-terminal domain. Commercially available antibodies discriminate between total and specifically phosphorylated (active) forms of different STATs, which allows to measure directly STATs activation in crude cell extracts. Nuclear translocation and transcriptional activity of STATs can be measured in transfected cells using STAT dependent promoter driving reporter luciferase gene. STAT signaling pathway and STAT-dependent gene expression in cells can be specifically modulated using oligodeoxynucleotide (ODN) STAT decoy which is a double-stranded fragment of DNA containing an overlapping ISRE/GAS binding site.
Janus激酶（JAK）和信号转导及转录激活因子（STAT）信号通路在许多肿瘤中被强烈激活。STAT蛋白通过酪氨酸残基的磷酸化而被激活，然后二聚化，转位至细胞核并结合DNA，启动靶基因的转录。JAK-STAT通路的激活与细胞生长、分化、存活以及癌症与免疫细胞之间的相互作用的调节有关。STAT的激活取决于其C末端结构域中单个酪氨酸残基（例如，STAT3中的Tyr705和STAT5中的Tyr694）的磷酸化。市售抗体可区分不同STAT的总形式和特异性磷酸化（活性）形式，这使得能够直接在粗细胞提取物中测量STAT的激活。使用驱动报告荧光素酶基因的STAT依赖性启动子，可以在转染细胞中测量STAT的核转位和转录活性。使用寡脱氧核苷酸（ODN）STAT诱饵可以特异性调节细胞中的STAT信号通路和STAT依赖性基因表达，该诱饵是一种双链DNA片段，含有重叠的ISRE/GAS结合位点。
