Cohen D R, Curran T
Department of Molecular Oncology and Virology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110.
Oncogene. 1990 Jun;5(6):929-39.
The products of two cellular proto-oncogenes c-fos and c-jun form a heterodimeric complex that contribute to the DNA-binding activity referred to as AP-1 (activator protein-1). Two domains have been proposed to be required for heterodimer formation and protein-DNA complex formation. The leucine zipper domain mediated the interaction between the two proteins and a highly basic region immediately N-terminal to the leucine zipper forms a DNA binding domain. To assess the role of these two domains in dimerization and DNA binding and to determine what contribution, if any, is made by residues outside of these regions, we carried out an extensive domain swap analysis. Restriction sites created in the fos and jun cDNAs flanking the basic region and leucine zipper allowed these domains to be swapped between the two proteins either singly or in various combinations with adjacent domains. The chimeric proteins were assayed for their ability to dimerize with each other and to interact with the AP-1 consensus sequence. It was found that two Jun leucine zipper regions could mediate protein dimerization, whereas two Fos leucine zipper regions could not. The dimers formed between two Jun leucine repeats were less stable than those formed between a Fos and a Jun leucine zipper. A conserved His residue seven amino acids C-terminal of the last leucine of the zipper region contributed to the stability of protein-protein interactions. The basic region of both Fos and Jun was found to interact with DNA without the presence of the other, i.e. the combination of two Fos- or two Jun-DNA binding domains could bind to the AP-1 site. However, replacement of the Jun N-terminus with that of Fos resulted in a decrease in DNA binding, indicating that residues outside of the Jun basic region contribute to DNA binding. The results also suggest that the dimerization and DNA binding functions of each protein are not completely independent properties, but that each exerts an influence on the other.
两个细胞原癌基因c-fos和c-jun的产物形成一种异二聚体复合物，它对被称为AP-1（激活蛋白-1）的DNA结合活性有贡献。已提出异二聚体形成和蛋白质-DNA复合物形成需要两个结构域。亮氨酸拉链结构域介导两种蛋白质之间的相互作用，并且在亮氨酸拉链紧邻的N端有一个高度碱性区域形成DNA结合结构域。为了评估这两个结构域在二聚化和DNA结合中的作用，并确定这些区域之外的残基有何贡献（如果有的话），我们进行了广泛的结构域交换分析。在fos和jun cDNA中位于碱性区域和亮氨酸拉链两侧创建的限制性位点使这些结构域能够在两种蛋白质之间单独或与相邻结构域以各种组合进行交换。检测嵌合蛋白彼此二聚化以及与AP-1共有序列相互作用的能力。发现两个Jun亮氨酸拉链区域可以介导蛋白质二聚化，而两个Fos亮氨酸拉链区域则不能。两个Jun亮氨酸重复序列之间形成的二聚体比Fos和Jun亮氨酸拉链之间形成的二聚体稳定性更低。拉链区域最后一个亮氨酸C端七个氨基酸处的一个保守His残基有助于蛋白质-蛋白质相互作用的稳定性。发现Fos和Jun的碱性区域在没有另一个存在的情况下都能与DNA相互作用，即两个Fos-或两个Jun-DNA结合结构域的组合可以结合到AP-1位点。然而，用Fos的N端取代Jun的N端导致DNA结合减少，表明Jun碱性区域之外的残基对DNA结合有贡献。结果还表明，每种蛋白质的二聚化和DNA结合功能不是完全独立的特性，而是彼此相互影响。
