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The c-Jun and c-Myc oncogenic transcription factors are highly unstable proteins due to polyubiquitination. Similar to c-Myc, we report here that phosphorylation of c-Jun by GSK3 creates a high-affinity binding site for the E3 ligase Fbw7, which targets c-Jun for polyubiquitination and proteasomal degradation. In keeping with this, we found that c-Jun levels were inversely related to GSK3 activity in mammalian cells that had entered the cell cycle. Importantly, phosphorylation of c-Jun by GSK3 requires a priming phosphorylation event at Ser-243. Ser-243 is mutated to phenylalanine in v-Jun and allows it to escape recognition by Fbw7. These findings explain the enhanced stability and oncogenicity of v-Jun relative to its cellular counterpart and reveal that GSK3 and Fbw7 coordinately regulate c-Jun and c-Myc.

The v-myb oncogene of avian myeloblastosis virus transforms myelomonocytic cells in vitro. The line of v-myb-transformed chicken monoblasts BM2 can be induced to terminal differentiation using phorbol esters. The fact that Jun proteins are up-regulated in the phorbol ester-treated BM2 cells prompted us to investigate the role of the Jun proteins in regulation of myeloid differentiation. We ectopically expressed v-jun and c-jun in BM2 cells and evaluated their effects on differentiation and proliferation. c-Jun up-regulated the transactivation activity of v-Myb and induced a proliferation block and differentiation of BM2 cells. In contrast, v-Jun down-regulated v-Myb transactivation causing no dramatic effects on BM2 cells. This confirms that there is no strong correlation between transcriptional activation and strength of oncogenic transformation by v-Myb. Both c-Jun and v-Jun proteins affected sensitivity of BM2 cells to retinoic acid and phorbol ester. Sensitivity of BM2 cells to retinoic acid was enhanced by both Jun proteins, while sensitivity to phorbol 12-myristate 13-acetate was reduced by v-Jun. These data suggest thate Jun plays a major role in macrophage differentiation.

Jun and Fos, b-ZIP transcription factors, form a heterodimer and bind to DNA enhancer elements, thereby regulating the expression of target genes. The present study was undertaken to investigate the molecular mechanism underlying nuclear translocation of the Jun/Fos complex. For this purpose, normal rat kidney cells were microinjected with a DNA expression vector containing wild-type or mutant c- or v-jun together with c- or v-fos, followed by detection of the subcellular localization of Jun or Fos by immunofluorescence staining. The nuclear accumulation of Fos was markedly enhanced by the presence of wildtype Jun, but not by Jun mutants lacking nuclear targeting or zipper dimerization functions, implying that Jun and Fos mutually interact via their leucine zippers and translocate from the cytoplasm to the nucleus using the markedly stronger nuclear localization signal of Jun.

The BKJ gene was originally identified based on its specific transcriptional activation in jun-transformed avian fibroblasts. We now show that BKJ is a direct transcriptional target of the AP-1 transcription factor components Jun and Fos. The complete structural organization of the quail BKJ gene was determined by nucleotide sequence analysis and transcriptional mapping. The gene contains three exons with the coding region confined to the third exon. A major mRNA species of 0.8 kb and a minor one of 1.3 kb are produced by variable usage of two transcriptional initiation sites. The BKJ promoter region contains two authentic AP-1 binding sites. By transactivation of reporter gene constructs and direct binding of Jun recombinant protein, the proximal AP-1 element was shown to be essential for BKJ promoter activation. Using polyclonal antiserum directed against recombinant BKJ protein, the activation of BKJ in jun-transformed avian fibroblasts was also demonstrated at the protein level. BKJ is a novel gene related to the avian beta-keratin gene family whose members display highly specific expression patterns during embryogenesis and epidermal development. Activation of BKJ in fibroblasts by retroviral or deregulated cellular jun or fos alleles may contribute to cell transformation.

c-jun proto-oncogene expression is extinguished in cells transformed by v-Jun; however, the mechanistic basis of this phenomenon has not been elucidated. c-jun mRNA levels are greatly reduced in v-Jun-transformed cells, and we show that this reduction is associated with a similar decrease in the rate of c-jun transcription. Transcriptional down-regulation was also evident in functional assays in which the c-jun gene promoter was approximately 10-fold less active in v-Jun-transformed cells than it was in normal cells. This reduction was largely attributable to a conserved 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE)-like motif at position -72 (the proximal junTRE) that was essential for efficient basal expression in normal cells but that conferred little, if any, detectable transcriptional activity in v-Jun-transformed cells. DNA-binding analysis showed that this element was recognized by a mixture of c-Jun/Fra and cyclic AMP-responsive element-binding protein/activating transcription factor-like complexes in normal cells but that v-Jun/Fra heterodimers predominated in v-Jun-transformed cells. Furthermore, ectopic expression of v-Jun repressed c-jun promoter activity in normal cells through the proximal junTRE. Thus, the deficit in transcription mediated by the junTRE correlates with and is most likely attributable to binding of v-Jun to this element in vivo. We also find that the c-jun promoter is refractory to induction via the stress-activated protein kinase/c-jun NH2-terminal kinase pathway in v-Jun-transformed cells, suggesting that v-Jun interferes with signal-regulated gene expression. Therefore, c-jun is an example of a cellular gene, the transcription of which is regulated negatively by v-Jun in vivo.

The c-Fos and c-Jun transcription factors are rapidly turned over in vivo. One of the multiple pathways responsible for their breakdown is probably initiated by calpains, which are cytoplasmic calcium-dependent cysteine proteases. The c-fos gene has been transduced by two murine oncogenic retroviruses called Finkel-Biskis-Jenkins murine sarcoma virus (FBJ-MSV) and Finkel-Biskis-Reilly murine sarcoma virus (FBR-MSV); c-jun has been transduced by the chicken avian sarcoma virus 17 (ASV17) retrovirus. Using an in vitro degradation assay, we show that the mutated v-FosFBR, but not v-FosFBJ or v-JunASV17, is resistant to calpains. This property raises the interesting possibility that decreased sensitivity to calpains might contribute to the tumorigenic potential of FBR-MSV by allowing greater accumulation of the protein that it encodes in infected cells. It has also been demonstrated that resistance to cleavage by calpains does not result from mutations that have accumulated in the Fos moiety of the viral protein but rather from the addition of atypical peptide motifs at its both ends. This observation raises the interesting possibility that homologous regions in viral and cellular Fos either display slightly different conformations or are differentially accessible to interacting proteins.

We compared the ability of cellular and viral Jun (c-Jun and v-Jun) to transactivate target genes. c-Jun and v-Jun bind specifically to 12-O-tetradecanoylphorbol-13-acetate responsive elements [TREs, also called activator protein 1 (AP-1) motifs]. However, whereas c-Jun activates TRE-controlled promoters, v-Jun represses them. Cotransfection of the two Jun proteins reduces c-Jun-dependent transactivation. The expression of the endogenous c-jun gene, regulated through a promoter-proximal AP-1-binding site, is repressed in v-Jun-transformed chicken embryo fibroblasts. It is suggested that an M(r) 18,000 v-Jun peptide prominent in v-Jun-transformed cells acts as a transdominant-negative regulator of AP-1 activity and of c-jun expression. In contrast to the results with TRE sites, both v-Jun and c-Jun activate transcription through the human T-cell leukemia virus type I 21-bp repeat which contains a sequence homologous to the cyclic AMP responsive element. However, full-length Jun proteins bind to this site only with low affinity, and binding of the truncated v-Jun was barely detectable. These observations show that the oncogenic viral form of Jun differs from the cellular version in promoter preference and on certain promoters acts as an antagonist to c-Jun.

We have investigated the expression of Jun family proteins and composition of AP-1 in chicken embryo fibroblasts before and after transformation by the v-Jun oncoprotein of ASV17. We show that p39 c-Jun is the predominant Jun family protein expressed in normal fibroblasts, and that heterodimers of c-Jun and Fos-related partners (Fra's) account for the majority of the AP-1 DNA binding activity. Unexpectedly, because ASV17-transformed fibroblasts do not express p39 c-Jun, v-Jun replaces c-Jun as the predominant AP-1 constituent in association with similar or identical Fra's. This substitution has little effect on the overall level of TRE-specific DNA binding activity, however it results in a profound reduction in TRE-dependent transcriptional activity and a striking defect in signal-regulated phosphorylation of the Jun component of AP-1; whilst agonists of SAPK/JNK kinases trigger transient N-terminal phosphorylation of c-Jun in normal fibroblasts, no corresponding modification of v-Jun occurs in ASV17-transformed cells. Because SAPK/JNK-mediated phosphorylation is thought to regulate c-Jun transcriptional activity and thereby cellular gene expression in response to extracellular signals, we propose that subversion of this signal transduction process by v-Jun is likely to contribute to oncogenesis by ASV17.

The role of the ubiquitin-dependent proteolysis system in c-Jun breakdown was investigated. Using in vitro experiments and a novel in vivo assay that utilizes molecularly-tagged ubiquitin and c-Jun proteins, it was shown that c-Jun, but not its transforming counterpart, retroviral v-Jun, can be efficiently multiubiquitinated. Consistently, v-Jun has a longer half-life than c-Jun. Mutagenesis experiments indicate that the reason for the escape of v-Jun from multiubiquitination and its resulting stabilization is the deletion of the delta domain, a stretch of 27 amino acids that is present in c-Jun but not in v-Jun. c-Jun sequences containing the delta domain, when transferred to the bacterial beta-galactosidase protein, function as a cis-acting ubiquitination and degradation signal. The correlation between transforming ability and the escape from ubiquitin-dependent degradation described here suggests a novel route to oncogenesis.

The v-maf oncogene, identified from AS42 avian retrovirus, encodes a nuclear bZip protein. To elucidate the molecular mechanism of cell transformation induced by this oncogene, we determined the specific binding sequences of its product. Maf protein recognized two types of relatively long palindromic consensus sequences, TGCTGACTCAGCA and TGCTGACGTCAGCA, at roughly equal efficiency. The middle parts of these Maf-binding sequences completely match with two binding sequences for AP-1 transcription factor, i.e., phorbol 12-O-tetradecanoate-13-acetate (TPA)-responsive element (TRE) and cyclic AMP responsive element, suggesting partial overlapping of the target genes for Maf and AP-1. Furthermore, Maf efficiently formed heterodimers with the components of AP-1, Fos and Jun, through their leucine zipper structures, and these heterodimers show binding specificities distinct from those for Maf-Maf and Jun-Jun homodimers. Thus, a multiple combination of the dimers should generate a greatly expanded repertoire of transcriptional regulatory potential. DNA data base search for the Maf-binding consensus sequences suggested that some of the TRE-like cis elements reported previously may actually be the targets for Maf family proteins or their heterodimers with other bZip proteins.

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.

The ability of the c-Jun protein, the main component of the transcription factor AP1, to interact directly or indirectly with the RNA polymerase II-initiation complex to activate transcription was investigated by in vivo transcription interference ("squelching") experiments. Coexpression of a Jun mutant lacking its DNA binding domain strongly represses the activity of wild-type c-Jun. Repression depends on the presence of the transactivation domains (TADs), suggesting that a limiting factor interacting with the TADs is essential to link Jun and the components of the transcriptional machinery. The activity of this intermediary factor(s) is restricted to TADs characterized by an abundance of negatively charged amino acids, as demonstrated by the abilities of the TADs of JunB, GAL4, and VP16 to repress c-Jun activity. Depending on the presence of the TADs of Jun, we found physical interaction between Jun and a cluster of three proteins with molecular masses of 52, 53, and 54 kDa (p52/54). Association between Jun and p52/54 is strongly reduced in the presence of VP16, suggesting that the two proteins compete for binding to p52/54. Transcription factors containing a different type of TAD (e.g., GHF1, estrogen receptor, or serum response factor) fail to inhibit Jun activity, suggesting that these proteins act through a different mechanism. We consider the requirement of Jun to interact with p52/54 utilized by other transcription factors a new mechanism in the regulation of transcription of Jun-dependent target genes.

c-Jun and its oncogenic counterpart v-Jun are completely conserved within the region from Ser-63 to Ser-73; these serines are sites for phorbol ester-inducible c-Jun phosphorylation. Using a U937 human leukemic cell line stably expressing v-Jun, we have demonstrated that phorbol esters stimulate the in vivo phosphorylation of c-Jun but not v-Jun. We developed an in vitro protein kinase assay to characterize the c-Jun protein kinase and to examine the determinants underlying this differential phosphorylation. Fusion proteins between glutathione S-transferase and the N terminus of c-Jun, v-Jun, or several c-Jun mutants were used as substrates. A c-Jun kinase activity was affinity-purified 5000-fold by using glutathione S-transferase-c-Jun-glutathione-Sepharose beads and was found to phosphorylate the N terminus of c-Jun but not v-Jun or c-Jun containing a 27-amino acid N-terminal deletion found in v-Jun. These effects were also observed in vivo as phorbol 12-myristate 13-acetate did not induce the phosphorylation of v-Jun or the c-Jun deletion mutant in U937 cell lines stably expressing these proteins. These findings indicate that the delta domain of c-Jun (amino acids 34-60), which is deleted in v-Jun, plays a critical role in regulating N-terminal c-Jun phosphorylation.

The Jun protein is a transcription factor of the AP-1 complex, and it is concentrated in the cell nucleus. While the cellular Jun protein is transported into the nucleus in a cell-cycle-independent fashion, the oncogenic viral version of the protein translocates into the nucleus most rapidly during the G2 phase of the cell cycle and only slowly during G1 and S phases. This cell cycle dependence of nuclear transport has been mapped to the cysteine to serine mutation in the carboxyl-terminal portion of viral Jun. We have identified a complex nuclear translocation signal located in the basic region of viral Jun. This signal has the sequence ASKSRKRKL. A peptide of this sequence synthesized in vitro and conjugated to IgG can mediate cell-cycle-dependent translocation of the microinjected conjugate from the cytoplasm into the nucleus. The nuclear translocation signal has two functional domains. The pentapeptide RKRKL is sufficient as a cell-cycle-independent nuclear address. The entire signal is needed for cell-cycle-dependent nuclear translocation. The amino-terminal tetrapeptide contains the cysteine to serine substitution responsible for cell cycle dependence. Deletion analysis of the Jun protein suggests that the nuclear translocation signal identified in the basic region is required for nuclear translocation of Jun and may be the only such signal in the Jun molecule.