Nishizawa Makoto, Fu Shu-Ling, Kataoka Kohsuke, Vogt Peter K
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
Oncogene. 2003 Sep 11;22(39):7931-41. doi: 10.1038/sj.onc.1206527.
We have constructed artificial AP-1 proteins containing elements derived from yeast GCN4 and from the herpes simplex virus activator VP16. These proteins can only homodimerize but do not heterodimerize, and lacking significant homology to Jun outside the DNA-binding domain, they are largely unaffected by proteins that modulate Jun. Constructs in which the transactivation domain of GCN4 is replaced by that of VP16 induce oncogenic transformation in cultures of chicken embryo fibroblasts. The availability of transforming VP16-GCN4 fusion proteins permits an evaluation of downstream target genes, based on the hypothesis that transformation-relevant targets should be common between Jun and the artificial AP-1 proteins. In a pilot study, we examined the expression of several Jun target genes in cells transformed by the VP16-GCN4 fusions and found that some of the Jun targets are not upregulated by the GCN4-derived transforming construct, suggesting that their upregulation in Jun-transformed cells is not essential for cell transformation. We have further constructed a regulatable GCN4-VP16 protein that will permit a kinetic characterization of target gene responses and will facilitate discrimination between direct and indirect targets.
我们构建了人工AP-1蛋白，其包含源自酵母GCN4和单纯疱疹病毒激活因子VP16的元件。这些蛋白只能形成同二聚体，不能形成异二聚体，并且在DNA结合结构域之外与Jun缺乏显著同源性，它们在很大程度上不受调节Jun的蛋白质的影响。用VP16的反式激活结构域替换GCN4的反式激活结构域的构建体在鸡胚成纤维细胞培养物中诱导致癌转化。可获得的转化性VP16-GCN4融合蛋白允许基于与转化相关的靶标在Jun和人工AP-1蛋白之间应该是共同的这一假设来评估下游靶基因。在一项初步研究中，我们检测了由VP16-GCN4融合体转化的细胞中几个Jun靶基因的表达，发现一些Jun靶基因并未被源自GCN4的转化构建体上调，这表明它们在Jun转化的细胞中的上调对于细胞转化并非必不可少。我们进一步构建了一种可调节的GCN4-VP16蛋白，它将允许对靶基因反应进行动力学表征，并将有助于区分直接靶标和间接靶标。
