We have studied the effect of the ras
oncogene on the AP-1 component Fra-1 in rat thyroid cells and found that the high constitutive activity of Fra-1 results from the conjunction of multiple mechanisms, including transcriptional induction, posttranslational stabilization, and recruitment of transcriptionally active partners (summarized in Fig. ). We have shown that such control mechanisms largely depend on the activity of the MEK/ERK phosphorylation cascade, in agreement with the essential role played by the Raf-dependent pathway in neoplastic transformation (56
), and the requirement of ERK-mediated signaling for the induction of Fra-1 synthesis (18
). However, in the rat thyroid epithelial cell system the cooperation with other pathways, in addition to the MEK- and Rac-dependent signaling, appears necessary for optimal signaling from the constitutive Ras oncoprotein to the fra-1
promoter. In addition, we have found that ERK-dependent phosphorylation plays an essential role in the stabilization of the protein in ras
-transformed cells while it is not essential for Fra-1 DNA binding activity.
FIG. 8. Model of multistep positive autoregulation of Fra-1 in ras-transformed cells. The thickness of the arrows originating from the constitutively GTP-bound oncogene (Ras*) indicates the relative contribution of different Ras-dependent pathways in (more ...)
It has been previously shown that, during the mitogenic response of mouse fibroblasts, the delayed kinetics of the serum-dependent induction of fra-1
requires the presence of intact c-fos
gene products (48
) acting through regulatory sequences within the fra-1
first intron. In this respect, fra-1
was depicted as a unique member of the fos
gene family under direct control of AP-1 activity, although a weaker AP-1-dependent induction also has been described for fra-2
). Here we show that the previously characterized intronic regulatory element is essential for the ras
-dependent induction of the fra-1
promoter, that Fra-1 is the predominant component of the AP-1 complex interacting in vitro and in vivo with the fra-1
intronic site, and that the activation of the surrounding chromatin region is reflected by the increased histone H3 acetylation. Therefore, in response to the activated ras
oncogene, Fra-1 becomes implicated in its serum-independent positive autoregulation, which is likely to play a major role in maintaining a constitutively high level of AP-1 activity in transformed cells.
The identification of Fra-1 as part of an in vivo transcriptionally active complex raised the question of its role as a bona fide transcriptional activator. The original report showing that Fra-1 lacks an autonomous transactivation domain (10
) is in apparent contradiction with a recent study that supports the evidence of its phosphorylation-dependent transactivating activity (64
). Our results, showing that the activity of the GAL4 fusion protein is strongly inducible by Ras, apparently support the hypothesis of a phosphorylation-dependent transactivation domain within the Fra-1 molecule. However, unlike c-Fos, the requirement for Fra-1 of an intact leucine zipper suggests that the Ras-dependent transactivation may depend on the recruitment of a heterodimeric partner. We propose that the major role of Fra-1 is to allow the DNA binding and transcriptional activation mediated by its Jun partners and that the ERK-dependent phosphorylation is not needed for transactivation, although it cannot be ruled out that an inducible transactivation domain, modulated by phosphorylation of the essential Thr-231 residue (64
), might act in cooperation with functional domains of the heterodimeric partner. Interestingly, a recent study showing the discrepancy between the transactivation potential and the ability to induce cellular morphological alterations by Fos family members led to the suggestion that Fra-1 biological activity might reflect its specific role within the molecular context of natural enhancers driving the expression of genes selectively upregulated by Fra-1 rather than c-Fos and Fra-2, such as the uPA (urokinase plasminogen activator) gene (3
Our analysis shows multiple roles for the ERK-dependent regulation of Fra-1, including the MAP kinase-dependent stabilization of the protein in ras
-transformed cells. The role of MAP kinase in controlling the half-life of Fra-1 in response to serum mitogens has been described previously in a detailed study on the regulation of Fra-1 and Fra-2 phosphorylation during the cell cycle of murine fibroblasts (24
). We have found that ras
transformation results in the serum-independent Fra-1 stabilization, which likely supports the constitutively high levels of the protein in transformed cells. It remains to be investigated whether the expression of the protein is totally deregulated or is still subjected to cell cycle-dependent variations in the transformed cell lines.
The relationship between phosphorylation and protein stabilization has been analyzed in detail for the two prototype AP-1 components, c-Fos and c-Jun. The first evidence on the role of MAP kinases in c-Fos stabilization came from the study of the v-mos
-dependent c-Fos stabilization (38
), which is likely unrelated to neoplastic transformation, since mos
is not involved in human tumorigenesis. Recently it has been shown that the duration of ERK signaling allows c-Fos to discriminate between sustained (mitogenic) and transient (nonmitogenic) stimuli, by a complex mechanism involving multiple sequential phosphorylation steps, required for full protein stabilization. Interestingly, it has been proposed that an identical putative DEF (ERK docking) domain, identified both in Fra-1 and Fra-2 (but not in FosB), might be implicated in similar signaling mechanisms (36
). Accordingly, the cluster of serine and threonine residues previously identified as MAP kinase phosphorylation sites in the Fra-2 carboxy-terminal region is adjacent to the putative DEF domain (34
) and includes the conserved Thr-231 residue of Fra-1 which, although not yet biochemically identified as a phosphoacceptor site, was found to be essential for ERK-dependent transactivation in response to tetradecanoyl phorbol acetate (64
). An additional level of complexity, implying a possible stimulus-specific regulation of Fra-1 phosphorylation, has been suggested from the recent analysis of Fra-1 modification in response to insulin, which showed that the activation of ERK1/ERK2 resulted in serine (but not threonine) phosphorylation of the carboxy-terminal region of Fra-1 (26
The essential role of c-Jun amino-terminal phosphorylation in ras
-mediated transformation (53
) and in protection from ubiquitin-dependent degradation (37
) has been well established. In our experimental system we have found that the increased half-life is a major determinant of ras
-dependent c-Jun accumulation, and we analyzed the mechanism of the stabilization in the transformed thyroid cell lines (unpublished data). In summary, at least for c-Jun and Fra-1, the Ras- and phosphorylation-dependent stabilization appears to be an important regulatory mechanism which might favor the accumulation of c-Jun/Fra-1 heterodimers during tumorigenesis.
Since the induction of Fra-1 and Fra-2 cannot take place in the absence of sustained MEK/ERK activation (55
), both proteins would be synthesized and immediately converted into the phosphorylated stable isoforms. Therefore, it can be speculated that another important regulatory step, potentially altered during oncogenesis, would be the dephosphorylation of Fra-1 and/or Fra-2, following their ERK-dependent synthesis and modification, to allow their turnover and interchange with other Fos family members within the AP-1 complex during the cell cycle.
It has been proposed that, both for Fra-1 and Fra-2 (but not c-Fos and FosB), MAP kinase phosphorylation might also regulate DNA binding activity (24
). In our cell system we have shown that phosphorylation is not essential for the DNA binding of Fra-1. To explain the apparent discrepancy, it can be observed that the previous study relied on the in vitro phosphorylation of recombinant proteins, while we have analyzed the in vivo phosphorylated Fra-1. Alternatively, the effect on DNA binding might be specific for c-Jun/Fra-1 (analyzed in the previous study) but not for the JunB/Fra-1 and JunD/Fra-1 heterodimers, which represent a significant component of the AP-1 complex in ras
-transformed thyroid cells.
Besides the common ERK-dependent control on their synthesis and stability, important differences exist in the ras
-dependent regulation of Fra-1 and Fra-2. In the thyroid cell system we have found a dramatic induction of the Fra-1 transcript compared to small variations of the Fra-2 mRNA (57
). Importantly, in rat fibroblasts a genome-wide survey of ras
transformation targets allowed the identification of Fra-1 as the most upregulated transcription factor (over 100-fold), pinpointing Fra-1 as the major AP-1 component in ras
-transformed fibroblasts, while Fra-2 was not detected.
The study of the role of Fra-1 in the cell cycle will take great advantage of the utilization of fra-1
knockout cell lines. The availability of primary fibroblasts derived from mouse embryos lacking individual AP-1 components allows the establishment of important relationships between AP-1 and cell cycle regulators, showing the dual effects of c-Jun, both as a positive regulator of cyclin D1 (6
) and a negative regulator of the p21 cdk cyclin inhibitor (47
), and JunB, as a negative regulator of cyclin D1 (6
) and a positive regulator of the p16 cell cycle inhibitor (41
). The analysis of c-fos
−/− and fosB
−/− mouse embryo fibroblasts revealed redundancy among these fos
family members, showing that while the lack of individual components had no effect on cell cycle control, the double knockout exhibited a proliferation defect, at least partially consequent to insufficient induction of cyclin D1 promoter (11
). Since the in vitro analysis did not show any cell-autonomous proliferation defect in fra-1
null fibroblasts (49
) and because of a mitogen-dependent regulation very similar to that of fra-2
), it will be important to explore both the potential functional overlap between the fra-1
gene products and the possibility that the two Fos family members might play nonredundant roles in cell types different from fibroblasts.
A major function of Fra-1 might be unrelated to normal cell cycle control. In light of the evidence suggesting a role for Fra-1 in tumor progression in a variety of naturally occurring neoplasms and experimental models, including breast (29
), lung (44
), epidermis (64
), and thyroid (15
), the accumulation of Fra-1 and the activation of target genes in tumor cells would require multiple genetic lesions. In this respect it can be noticed that the above-mentioned survey of ras
transformation targets was based on differential screening between a preneoplastic cell line and the malignant ras
-transformed derivative (65
). Likewise, the analysis of the Fra-1 ability to induce morphological transformation and activation of multiple transcriptional targets was based on the ectopic overexpression of the protein in a mouse mammary adenocarcinoma revertant cell line, selected for the complete loss of metastatic potential, compared to that of the parental malignant cell line (3
). Interestingly, the Fra-1-upregulated gene products (mts1, uPA, uPAR, and PAI-1) have been strongly implicated in metastasis, cell migration, and angiogenesis (1
), thus indicating the crucial role played by Fra-1 in tumor progression.