Analysis of dimerization and DNA binding functions in Fos and Jun by domain-swapping: involvement of residues outside the leucine zipper/basic region

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.     

Encounters with Fos and Jun on the road to AP-1

The nuclear proto-oncogenes, c-fos and c-jun, are induced in response to a diverse array of extracellular stimuli. Their protein products, Fos and Jun, form a heterodimeric complex that interacts with the DNA regulatory element known as the AP-1 binding site. Protein dimerization occurs via a parallel interaction of leucine zipper domains and is required for DNA binding. In addition to the leucine zipper, DNA binding requires two clusters of basic amino acids adjacent to the leucine zipper domains of both Fos and Jun. The leucine zipper and DNA-binding regions are highly conserved among the c-fos and c-jun families of related inducible genes. Thus, multiple protein complexes can be formed that may interact with AP-1 binding sites in numerous genes to affect gene expression in response to environmental signals.     

Mutagenesis of the DNA contact site in Fos protein: compatibility with the scissors grip model and requirement for transformation

To elucidate the mechanisms involved in the transformation by fos we have initiated a study pertaining to the identification of molecular functions of Fos protein that are crucial for transformation. We have previously reported that the presence of an intact leucine zipper in Fos is an absolute requirement for the induction of transformation, but that the autorepression function of Fos is dispensable. We now show that Fos protein also needs an intact DNA (TRE)-binding site to be able to transform. Amino acid substitutions in this domain of Fos which impair DNA binding also destroy the transforming potential of Fos, suggesting that the interaction of Fos-Jun complexes with TREs may be a crucial part of Fos-induced transformation. This hypothesis is further strengthened by our observation that Fos and Jun can cooperate in the induction of transformation. We show that a Fos protein which contains a Jun leucine zipper and is thus capable of dimerization is still dependent on the presence of exogenous Jun to induce transformation. The critical positions in the Fos DNA-binding site include those which the 'scissors grip' model predicts to be crucial, although the DNA-binding site in Fos seems to extend beyond the basic region into an adjacent cluster of acidic amino acids.