Activator protein 1 (AP-1) transcription factors play important roles in specifying and executing essential genetic programs. Proteins that form AP-1 belong to the basic leucine zipper (bZIP) family and bind DNA as obligate homo- or heterodimers (for review, see reference 33
). The bZIP motif consists of a DNA-binding domain rich in basic amino acids and an adjacent protein-protein dimerization domain known as the leucine zipper structure, which is characterized by leucines at every seventh residue (21
). c-Jun and c-Fos are the prototype AP-1 components. The related proteins, JunB, JunD, Fra1, Fra2, and FosB, show expression patterns and transactivation potentials distinct from those of c-Jun or c-Fos. While Jun proteins can dimerize with each other and with the Fos proteins, Fos proteins cannot form homodimers (26
). The AP-1 complexes are not limited to Jun and Fos dimers, since certain Jun and Fos proteins have been shown to dimerize with other bZIP proteins, such as ATF2, MAF, and their related proteins (for review, see reference 5
). Various AP-1 dimers differ in DNA binding affinity and specificity. For example, Jun-Fos heterodimers preferentially bind to the heptamer consensus, 5′-TGA(C/G)TCA-3′, known as the TPA response element (TRE), while Jun-ATF dimers prefer the octamer consensus 5′-TGACGTCA-3′ known as the cyclic AMP response element (CRE). These binding sites have been found in the regulatory regions of a wide range of genes, including transcription factors, matrix-degrading enzymes, cell adhesion molecules, cyclins, and cytokines. Jun-Fos dimers that have similar DNA binding specificities can differ in transcriptional activity due to nonconserved domains located outside the bZIP region that can be regulated by phosphorylation. It is therefore plausible that AP-1 dimers of different composition execute specific cellular programs.
AP-1 complexes in exponentially growing mouse NIH 3T3 fibroblasts are predominantly dimers containing c-Jun, JunD, and Fra2 (19
). In serum-starved fibroblasts, JunD accumulates while the level of the other members decreases. Mitogenic stimulation induces the synthesis of c-Jun, JunB, c-Fos, Fra1, Fra2, and FosB in a defined order (19
). In addition, we have recently shown that the abundance and phosphorylation of Jun proteins vary during the cell cycle (2
The combinatorial character of the AP-1 transcriptional complex makes the interpretation of overexpression experiments difficult. Introduction of a new bZIP species into the cell can produce primary effects from homodimerization of itself or from heterodimerization with usually unidentified endogenous bZIP proteins, which cause potentially widespread effects by titrating out certain monomers and shifting the equilibrium within the AP-1 pool. Furthermore, the expression level and phosphorylation status of individual bZIP proteins in the recipient cell can be dramatically different depending on cell type and cell context. To overcome these difficulties, we developed a tethering strategy. Inspired by successful examples using other dimeric molecules, we joined two AP-1-forming monomers by a flexible polypeptide so that they formed an intramolecular dimer. In this study, we characterized the biochemical properties and biological activities of tethered Jun~Fos proteins (the tilde indicates a tethered dimer). These single-chain AP-1 molecules revealed differences in the transcriptional regulation of specific promoters and showed that c-Jun in combination with Fra2 inhibits cell cycle arrest under both confluency and low-serum conditions for cultured NIH 3T3 mouse fibroblasts.