Lackmann M, Harpur A G, Oates A C, Mann R J, Gabriel A, Meutermans W, Alewood P F, Kerr I M, Stark G R, Wilks A F
Growth Regulation Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch), Australia.
Growth Factors. 1998;16(1):39-51. doi: 10.3109/08977199809017490.
The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA. The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferon gamma (IFN gamma) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immuno-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFN gamma-receptor alpha-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) non-activated STAT1 is present in a higher molecular weight complex(es) and, at least for IFN gamma-primed cells, is available for recruitment to the activated IFN gamma-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.
JAK/STAT信号通路的基本框架已有充分记载。潜在的细胞质STAT转录因子被招募到细胞因子受体上酪氨酸磷酸化的对接位点，以及它们由JAK介导的磷酸化，促使它们转运至细胞核并具备结合DNA的能力。该信号通路招募和激活背后的生化过程通常是在体外重组系统中，使用预先定义的重组信号成分进行研究的。我们通过实时BIAcore分析、尺寸排阻（SE）色谱法和免疫检测，剖析了野生型和STAT1阴性突变细胞系粗提物中的干扰素γ（IFNγ）信号转导通路。数据表明，在不含去污剂的细胞提取物中：（1）IFNγ受体α链含磷酸酪氨酸（Y440P）的肽基序直接与STAT1或STAT1复合物相互作用，不与其他蛋白质相互作用；（2）未激活的STAT1存在于更高分子量的复合物中，至少对于IFNγ预处理的细胞而言，仅能从这类复合物的一个子集中被招募到激活的IFNγ受体；（3）激活的STAT1以单体形式从受体释放。
