Tsuboi A, Sugimoto K, Yodoi J, Miyatake S, Arai K, Arai N
Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304-1104.
Int Immunol. 1991 Aug;3(8):807-17. doi: 10.1093/intimm/3.8.807.
Activation of T cells by antigen, lectin, or a combination of phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) leads to the induction of genes for a set of lymphokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF). We demonstrated in earlier studies that the upstream region of the mouse GM-CSF promoter at positions between -95 and -73 is essential for transcriptional activation in response to PMA/A23187. This region contains two DNA-binding motifs, GM2 and GC-box. The GM2 sequence (GGTAGTTCCC) is recognized by an inducible factor NF-GM2; the other (CCGCCC) by constitutive factors A1, A2, and B. To elucidate the mechanism of GM-CSF gene activation, we have purified the inducible factor NF-GM2 from the nuclear extract of stimulated Jurkat cells on the basis of specific DNA-binding activity. The purified NF-GM2 consists of 50 (p50) and 65 kDa (p65) polypeptides and has a binding activity specific for both the GM-CSF and immunoglobulin kappa (GGAAAGTCCC) enhancers. Electrophoretically purified p50 alone can form a protein-DNA complex, but in the mixture, p50 associates preferentially with p65 to form the NF-GM2 complex. In addition, p65 gave per se, with low affinity, a protein-DNA complex that migrated more slowly than native NF-GM2 complex. Furthermore, an antiserum against KBF1 (identical to 50 kDa NF-kappa B protein) reacted with the p50 of NF-GM2, indicating that the NF-GM2 polypeptide cannot be immunologically differentiated from the 50 kDa subunit of NF-kappa B. The purified NF-GM2 activated in vitro transcription from the kappa B enhancer, while it failed to stimulate transcription from the GM-CSF promoter harboring the GM2 sequence. This suggests that the activation mechanism of the GM-CSF gene through the GM2/GC-box sequence is different from that of genes carrying the kappa B enhancer alone.
抗原、凝集素，或佛波醇-12-肉豆蔻酸酯乙酸盐（PMA）与钙离子载体（A23187）的组合激活T细胞，会诱导一组淋巴因子的基因表达，其中包括粒细胞-巨噬细胞集落刺激因子（GM-CSF）。我们在早期研究中证明，小鼠GM-CSF启动子上游-95至-73位之间的区域对于响应PMA/A23187的转录激活至关重要。该区域包含两个DNA结合基序，即GM2和GC框。GM2序列（GGTAGTTCCC）可被诱导因子NF-GM2识别；另一个（CCGCCC）则被组成型因子A1、A2和B识别。为阐明GM-CSF基因激活的机制，我们基于特异性DNA结合活性，从受刺激的Jurkat细胞核提取物中纯化了诱导因子NF-GM2。纯化后的NF-GM2由50 kDa（p50）和65 kDa（p65）的多肽组成，对GM-CSF和免疫球蛋白κ（GGAAAGTCCC）增强子均具有特异性结合活性。单独经电泳纯化的p50可形成蛋白质-DNA复合物，但在混合物中，p50优先与p65结合形成NF-GM2复合物。此外，p65本身与蛋白质-DNA复合物的结合亲和力较低，其迁移速度比天然NF-GM2复合物慢。此外，针对KBF1（与50 kDa NF-κB蛋白相同）的抗血清与NF-GM2的p50发生反应，这表明NF-GM2多肽在免疫上无法与NF-κB的50 kDa亚基区分开来。纯化后的NF-GM2可激活κB增强子的体外转录，但无法刺激含有GM2序列的GM-CSF启动子的转录。这表明通过GM2/GC框序列激活GM-CSF基因的机制与仅携带κB增强子的基因不同。
