Herrera R, Agarwal S, Walton K, Satterberg B, Distel R J, Goodman R, Spiegelman B M, Roberts T M
Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Cell Growth Differ. 1990 Oct;1(10):483-90.
Transcription factor activator protein 1 (AP-1) is a protein fraction that contains c-fos, c-jun, and several other related proteins. Although this protein fraction can stimulate transcription in vitro, the relative contributions of c-fos and c-jun to the transcriptional effect of AP-1 are not clear. In order to approach this question, we have overexpressed both proteins using a baculovirus-mediated expression system and defined their DNA-binding and transcriptional enhancement activities in vitro. Gel mobility-shift and DNase 1 footprinting assays showed that c-jun protein specifically binds to DNA through an AP-1 binding site. Under the same conditions, no detectable binding of c-fos protein was observed. However, when the DNA binding assays were performed in the presence of both c-jun and c-fos, a marked increase in the affinity of c-jun for the AP-1 site was observed. An AP-1-dependent transcription assay was used to test the capability of both proteins to stimulate correctly initiated RNA synthesis in vitro. Under our conditions, c-jun protein was capable of stimulating specific RNA transcription in an AP-1 site-dependent manner. In contrast, c-fos protein showed no detectable transcriptional activation by itself. However, a transcription assay carried out in the presence of both c-fos and c-jun proteins showed that the c-fos/c-jun complex was more active as a transcriptional regulator than c-jun protein alone. These experimental results indicate that c-fos and c-jun proteins are required to reconstitute full AP-1-dependent transcriptional activation and directly demonstrate that c-fos is a regulator of gene expression.
转录因子激活蛋白1（AP-1）是一种包含c-fos、c-jun和其他几种相关蛋白的蛋白质组分。尽管该蛋白质组分在体外能够刺激转录，但c-fos和c-jun对AP-1转录效应的相对贡献尚不清楚。为了解决这个问题，我们使用杆状病毒介导的表达系统使这两种蛋白均过表达，并在体外确定了它们的DNA结合和转录增强活性。凝胶迁移率变动分析和DNase 1足迹分析表明，c-jun蛋白通过AP-1结合位点特异性结合DNA。在相同条件下，未观察到c-fos蛋白有可检测到的结合。然而，当在c-jun和c-fos同时存在的情况下进行DNA结合分析时，观察到c-jun对AP-1位点的亲和力显著增加。使用AP-1依赖性转录分析来测试这两种蛋白在体外刺激正确起始的RNA合成的能力。在我们的条件下，c-jun蛋白能够以AP-1位点依赖性方式刺激特异性RNA转录。相比之下，c-fos蛋白本身未显示出可检测到的转录激活。然而，在c-fos和c-jun蛋白同时存在的情况下进行的转录分析表明，c-fos/c-jun复合物作为转录调节因子比单独的c-jun蛋白更具活性。这些实验结果表明，c-fos和c-jun蛋白是重建完全AP-1依赖性转录激活所必需的，并直接证明c-fos是基因表达的调节因子。
