Hadman M, Loo M, Bos T J
Department of Microbiology and Immunology, Eastern Virginia Medical School, Norfolk 23501.
Oncogene. 1993 Jul;8(7):1895-903.
A direct comparison of the relative DNA-binding capabilities of in vivo Jun-containing complexes derived from overexpression of the highly transforming viral Jun (VJ-1 CEF), the weakly transforming chicken cellular Jun (CJ-3 CEF) or background endogenous Jun (RCAS CEF) was assessed by gel mobility-shift assays using a synthetic oligonucleotide containing the consensus sequence TGACTCA (consensus AP-1). Chicken embryo fibroblasts (CEFs) expressing background c-Jun levels (RCAS CEF) contain almost undetectable levels of c-Jun but retain significant DNA-binding activity with two distinct complexes capable of binding specifically to the consensus AP-1 site. CEFs overexpressing either v-Jun or c-Jun contain these same two complexes and, while showing marked increases in Jun protein levels, do not exhibit any increase in DNA binding or transcriptional activation activity, suggesting that much of the overexpressed protein is inactive. Gel-shift assays performed in the presence of a Jun-specific antibody revealed a reduction in binding by both complexes, suggesting that each contains Jun or a Jun cross-reactive protein. Antibodies specific for Jun B, c-Fos, Fos B and CREB failed to interact with either complex. However, antibody specific for Fra-2 caused a slight supershift, suggesting that one or both complexes may contain Fra-2. Gel-shift competition assays with 16 'AP-1- and CREB-like' target sequences revealed that, within each cell type, the two protein complexes varied in their ability to recognize the mutant target sequences. These results clearly indicate differences in potential target recognition by each specific in vivo complex, and suggest that each may preferentially bind its own subset of target DNAs. In addition, a comparison of binding by individual complexes derived from CEFs overexpressing v-Jun and c-Jun also revealed differences in target recognition. Thus, in vivo complexes formed by overexpression of v-Jun and c-Jun vary in their ability to recognize and bind to a number of 'AP-1- and CREB-like' target sequences. This has important implications with regard to the mechanisms involved in cell transformation by v-Jun.
通过凝胶迁移率变动分析，使用包含共有序列TGACTCA（共有AP-1）的合成寡核苷酸，评估了源自高转化性病毒Jun（VJ-1 CEF）、弱转化性鸡细胞Jun（CJ-3 CEF）或背景内源性Jun（RCAS CEF）过表达的体内含Jun复合物的相对DNA结合能力。表达背景c-Jun水平的鸡胚成纤维细胞（CEF，RCAS CEF）含有几乎检测不到的c-Jun水平，但保留了与两种能够特异性结合共有AP-1位点的不同复合物的显著DNA结合活性。过表达v-Jun或c-Jun的CEF含有这两种相同的复合物，并且在Jun蛋白水平显著增加的同时，DNA结合或转录激活活性没有任何增加，这表明大部分过表达的蛋白是无活性的。在Jun特异性抗体存在下进行的凝胶迁移分析显示，两种复合物的结合都减少，表明每种复合物都含有Jun或Jun交叉反应蛋白。针对Jun B、c-Fos、Fos B和CREB的特异性抗体未能与任何一种复合物相互作用。然而，针对Fra-2的特异性抗体引起了轻微的超迁移，表明一种或两种复合物可能含有Fra-2。用16个“AP-1和CREB样”靶序列进行的凝胶迁移竞争分析表明，在每种细胞类型中，这两种蛋白质复合物识别突变靶序列的能力各不相同。这些结果清楚地表明了每种特定体内复合物在潜在靶标识别上的差异，并表明每种复合物可能优先结合其自身的靶DNA子集。此外，对过表达v-Jun和c-Jun的CEF衍生的单个复合物的结合比较也揭示了靶标识别上的差异。因此，由v-Jun和c-Jun过表达形成的体内复合物在识别和结合多种“AP-1和CREB样”靶序列的能力上有所不同。这对于v-Jun参与细胞转化的机制具有重要意义。
