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Previous Article | Next Article 
Molecular and Cellular Biology, June 2007, p. 3936-3950, Vol. 27, No. 11
0270-7306/07/$08.00+0 doi:10.1128/MCB.01776-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Institut de Génétique Moléculaire de Montpellier, CNRS, 1919 Route de Mende, Montpellier F-34293, France,1 INSERM U540, Montpellier F-34090, France,2 University Montpellier I, Montpellier F-34000, France3
Received 20 September 2006/ Returned for modification 12 November 2006/ Accepted 10 March 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Within the variety of transcription factors that form AP-1, the best known ones are the members of the Fos family, namely, c-Fos, FosB, Fra-1, and Fra-2, and those of the Jun family, namely, c-Jun, JunB, and JunD (16, 43). They all share two adjacent, highly conserved domains: the basic DNA-binding domain and the leucine zipper (LZ), which mediates dimerization. Together, these domains are known as the bZip region. Fos proteins must heterodimerize with other AP-1 components to acquire transcriptional competence. In contrast, Jun proteins can also function as homodimers, even though heterodimerization with partners like Fos proteins is favored. Importantly, AP-1 can act as either a positive or negative regulator of transcription depending on its composition, the target gene, the cell context, and the signals received from the environment (16).
c-Fos, the prototype of the family, and Fra-1 are the most studied Fos proteins. They are expressed constitutively in a limited number of tissues (33). However, the c-fos and fra-1 genes are best characterized as immediate early genes, as they are rapidly induced, i.e., within 15 min and 2 h, respectively, in many cell types by a variety of extracellular stimuli. They also show differences in protein expression. For example, upon mitogen stimulation of quiescent cells, c-Fos accumulates transiently, disappearing within a few hours, whereas Fra-1 appears later and persists well beyond the G1 phase of the cell cycle. Importantly, variations in abundances are not limited to c-Fos and Fra-1 in cells reentering the cell cycle but also concern other AP-1 family proteins, which are responsible for continuous and dynamic changes in AP-1 dimer composition (35, 36, 38). It is noteworthy that c-Fos participates in the transcriptional activation of fra-1 (8, 68) together with other components that have yet to be fully elucidated.
The expression of c-Fos and Fra-1 is altered in many tumors (44, 68). c-Fos is oncogenic in several in vitro and in vivo settings (20, 33), while Fra-1 is not transforming on its own (20, 33). However, it is associated with tumor progression, where it can contribute to cell survival (66), proliferation (7), and invasiveness (7). To avoid the deleterious effects of improper expression, both the c-fos and fra-1 genes are regulated at multiple transcriptional and posttranscriptional levels. In particular, their transcription is activated by various mitogen-activated protein kinase (MAPK) pathways, and their protein products are targeted by the Erk1/2 (48, 68) and Erk5 (62) cascades. Physiological regulation of the c-Fos protein by the Erk1/2 pathway has been more extensively studied than that of Fra-1. Both Erk1/2 and their effector kinases Rsk1 and Rsk2 phosphorylate c-Fos (14, 15, 26, 45, 46, 50, 51, 60), which alters both its degradation rate (15, 23, 50, 51) and its transcriptional activity (45, 46, 50). Similarly, phosphorylation catalyzed by the Erk5 cascade stabilizes c-Fos and enhances its ability to activate reporter genes (62). Importantly also, the pattern of AP-1 proteins, including Fra-1, is perturbed in Ras-, Raf-, and Mek1-transformed cells, where Erk1/2 plays a key role in the expression and the phosphorylation of several of them (18, 42, 64).
As for many key cell regulators, the tight control of Fos protein degradation rates is essential to ensure the correct timing and level of expression. Thus far, only the mechanisms of c-Fos breakdown have been studied in detail. Most of the studies have been performed in two different experimental contexts where the protein is massively nuclear. One is constitutive expression during asynchronous growth, and the other is transient induction during the G0-to-G1-phase transition following mitogen stimulation of quiescent cells. In both situations, c-Fos is unstable, with a half-life in the hour range, and the bulk of the protein is degraded by the proteasome (1, 9, 23, 55) independently of prior ubiquitylation (9), although a fraction of c-Fos can undergo ubiquitylation in vivo in certain circumstances (9). This is unusual, as most substrates require polyubiquitylation to be addressed to and/or processed by the proteasome (22). In line with this observation and the fact that c-Fos is not detectably ubiquitylated in serum-stimulated cells (9), Sasaki et al. recently reported the lack of ubiquitylation of unstable, nuclear c-Fos induced by tetradecanoyl phorbol acetate (56). However, they also elegantly showed that c-Fos, when retained in the cytoplasm upon activation of the STAT3 pathway in the presence of the inactive Erk5 kinase pathway, is subjected to ubiquitylation-dependent and proteasome-dependent degradation (56). These data raise the possibility that alternative pathways may contribute to c-Fos degradation in different subcellular compartments. They may also explain why c-Fos was originally found to be more unstable in the cytoplasm than in the nucleus (53).
Another peculiarity of c-Fos is that its breakdown is controlled by differentially regulated autonomous destabilizers located at its two extremities (9, 23). A C-terminal element is functional in c-Fos during both asynchronous growth and the G0-to-G1-phase transition, whereas an N-terminal destabilizer is active only in G0/G1 cells (9, 23). Moreover, the cytoplasmic degradation of c-Fos described previously by Sasaki et al. depends on a single destabilizer colocalizing with the N-terminal one active in G0/G1 cells (56). Further work will establish whether the c-Fos N-terminal region contains a single or two distinct destabilizing elements. Importantly, the activity of the C-terminal destabilizer is reduced upon the phosphorylation of two C-terminal serines by Erk1/2 and Rsk1/2 (9, 23). As a consequence, this domain is less active in G0/G1 cells, where the Erk1/2 pathway is strongly activated, than in asynchronously growing cells, where Erk1/2 is at best weakly active (48). c-Fos turnover is, however, maintained in G0/G1 phase by the functional activation of the N-terminal destabilizer via an undefined mechanism (9, 23). Finally, the expression of Ras, Mos, and Raf oncogenes, which activate the Erk1/2 pathway (48), also inhibits c-Fos C-terminal destabilizer activity in proliferating transformed cells.
There are a number of reports suggesting that Fra-1 is also an unstable protein that is stabilized upon Erk1/2 MAPK pathway activation. Thus, in the case of high Erk1/2 pathway activity resulting from either physiological stimulation by mitogens (26, 49) or oncogenic activation of upstream signaling effectors in thyroid (13), colon (66), and breast (7) cancer cells, Fra-1 accumulates to high levels and shows a characteristic diffuse and retarded electrophoretic mobility due to phosphorylation at multiple, unmapped sites. By contrast, Fra-1 shows reduced phosphorylation and destabilization when Erk1/2 activity is reduced, as is the case in control nontransformed thyroid cells (13), in cells treated with a pharmacological inhibitor (66), or upon mitogen withdrawal (26, 49). Finally, pharmacological inhibition of the proteasome in colon tumor cells with a high content of stabilized Fra-1 led to a further overaccumulation of the latter protein. This suggested that the turnover of at least a fraction of Fra-1 is controlled by proteasomal degradation (66).
Several lines of evidence suggest that c-Fos and Fra-1 may be degraded by, at least partially, similar mechanisms. In addition to the bZip domain, the two proteins show a second region of high homology at the C terminus, where one of the two destabilizers of c-Fos is located (9, 23). Several serines and threonines phosphorylated by Erk1/2 and Rsk1/2 in c-Fos are conserved in Fra-1 (see Fig. 1A and below) (45, 46, 49, 50, 69), where only Thr231 of rat Fra-1 has been studied in depth (69). Finally, the activation of the Erk1/2 and Erk5 pathways stabilizes both c-Fos and Fra-1 (62). Nevertheless, the two proteins show differences in their turnovers. For example, in Swiss 3T3 mouse embryo fibroblasts (MEFs) undergoing a G0-to-G1-phase transition, Fra-1 has a half-life of more than 5 h, while that of c-Fos is 45 min (26). To elucidate the molecular basis of Fra-1 degradation and that of its physiological and pathological Erk1/2 pathway-dependent stabilization, we have undertaken here genetic and signaling studies of MEFs and several cancer cell lines.
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Immunoblotting analysis and antibodies. Immunoblotting analyses were carried out as previously described after the electrotransfer of sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis-fractionated proteins onto polyvinylidene difluoride membranes and using the Western Lightening chemiluminescence kit from Perkin-Elmer Life Sciences (10). Luminograms of appropriate exposures on Kodak X-OmatAR films are presented in the figures. When necessary, bioluminescent signals were quantified using the camera-based GeneGnome system from Syngene BioImaging and the GeneTool analysis program provided by the supplier. Home-prepared 9E10 monoclonal antibody was used to detect Myc-tagged proteins. All other antibodies were rabbit antisera. The anti-enhanced green fluorescent protein (EGFP) antiserum was obtained from Roche, and that against GAPDH (glyceraldehyde-3-phoshpate dehydrogenase) was homemade. Erk1/2 was detected using the anti-p42/44 MAPK antibody and phospho-Erk1/2 with the anti-phospho-p42/44 MAPK (Thr202/Tyr204) antibody from Cell Signaling. Anti-Fra-1 antibodies (sc183) were from Santa Cruz Biotechnology. The anti-phosphoserine 252 and 265 antibodies were obtained after immunization of rabbits with the CSAHRKSpSSSSGD and SSDPLGpSPTLLAL peptides coupled to keyhole limpet hemocyanin, respectively. They were produced and affinity chromatography purified by Eurogentec (Belgium). The purification procedure involved two successive steps using phosphorylated and nonphosphorylated forms of the above-described peptides. Specific blocking by phosphorylated peptides, but not by the nonphosphorylated ones, was verified both in enzyme-linked immunosorbent assays using the phosphorylated peptides as targets and by immunoblotting using extracts from HeLa cells cotransfected with plasmids for wild-type Fra-1 and Mos. The horseradish peroxidase-conjugated anti-mouse and anti-rabbit immunoglobulin secondary antibodies were obtained from Sigma.
Pulse-chase experiments. Twenty-four hours after transient transfection of pIRES2-EGFP-based vectors for Fra-1, Fra-1-2S/A, and Fra-1-2S/D, 107 cells were radiolabeled in the presence of 150 µCi of a [35S]methionine-cysteine mixture (NEG-772; Perkin-Elmer Life Sciences) per ml of culture medium for 1 h. Chases and cell lysis were conducted as described previously (9, 23). Immunoprecipitations were carried out with an anti-Fra-1 antibody (sc183; Santa Cruz Biotechnology) using 20 µl of magnetic protein G Dynabeads (Dynal). The immunocomplexes were directly resuspended in the electrophoresis loading buffer and fractionated through sodium dodecyl sulfate-containing 12% polyacrylamide gels. The quantification of signals on dried gels was performed using the 445SI PhosphorImager 5 device from Molecular Dynamics and the ImageQuant program.
Sequence alignments. Sequence alignments were performed using the Clustal W (1.83) program available from EMBL-EBI (www.ebi.ac.uk/).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Erk1/2 pathway-driven phosphorylations of serines 252 and 265, but not those of threonines 217, 223, 227, 230, and 240, stabilize Fra-1 in asynchronous cells. We first addressed the phosphorylation and stabilization of Fra-1 by the Erk1/2 pathway in exponentially growing cells to determine if the two events were linked directly or indirectly. A series of nonphosphorylatable and phosphomimetic mutants were generated in human Fra-1 at positions corresponding to known Erk1/2 and Rsk1/2 target sites in c-Fos (Fig. 1B and 2A and Table 1). The stability of these mutants was evaluated in HeLa cells that display low, albeit somewhat variable, Erk1/2 activity. We developed an assay system that is simpler than pulse-chase analysis to measure the relative stabilities of the numerous wild-type, mutant, and chimeric proteins in our study (Fig. 1C). This assay involves parallel transfections of asynchronous cells with expression vectors derived from pIRES2-EGFP (Fig. 1C) that give rise to both the proteins under study and EGFP from the same bicistronic mRNA. The steady-state levels of the proteins were analyzed using immunoblotting. EGFP, which is highly stable in our system (23), serves as an internal standard for normalization and allows the estimation relative protein stabilities, if needed (see the legend of Fig. 1C). This approach was validated (not shown) by verifying that differences in the accumulation of c-Fos mutants relative to that of the wild-type protein correlated with differences in half-lives measured by pulse-chase in a previous work (23).
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Since phosphorylation occurs preferentially at sites that are N terminal to DEF motifs after the docking of Erk1/2 (21, 50), we analyzed mutants in threonines 217, 223, 227, and 230 alone or in combination with mutants in S252 and S265 (Fig. 2C). Mutation of T217 and T223 (Fra-1-2T/D), which are conserved between Fra-1 and c-Fos, or of all four threonines (Fra-1-4T/D) into alanines curiously led to a slight stabilization. This increase (on average less than 50%) was modest relative to that found with Fra-1-2S/D, either alone or together with the four T-to-A mutants (Fra-1-4T/A-2S/D). Similarly, no increase in stability was seen upon the mutation of the four threonines into aspartic acids (Fra-1-4T/D). Taken together, these data indicate that the phosphorylation of these threonines plays no crucial role in the stabilization of Fra-1 turnover. Instead, it may slightly attenuate the stabilizing effect of S252 and S265 phosphorylation. Given this minor effect, these threonines were not investigated further.
We also studied the potential role of T240, located downstream of the DEF motif (Fig. 2D). Neither its mutation in D nor its mutation in A affected Fra-1 accumulation, either alone (Fra-1-T240D and Fra-1-T240A, respectively) or with S252 and S265 mutated to D (Fra-1-T240D-2S/D and Fra-1-T240A-2S/D) or A (Fra-1-T240D-2S/A and Fra-1-T240A-2S/A). Importantly, all stabilized constructs, like wild-type Fra-1, localized predominantly within the nucleus in indirect immunofluorescence assays (not shown), indicating that stabilization was not a direct consequence of gross intracellular protein redistribution. Moreover, expression of wild-type Fra-1, Fra-1-2S/A, and Fra-1-2S/D in three other cell lines with low Erk1/2 pathway activity, namely, BALB/c 3T3 mouse embryo fibroblasts, LS174T human colon carcinoma cells, and MCF7 human breast cancer cells, gave results similar to those described above (Fig. 2E to G). This strongly suggests that the regulation of Fra-1 stability by modification of serines 252 and 265 is not cell type specific.
Pulse-chase experiments (Fig. 2H) were then used to estimate the half-lives of wild-type Fra-1, Fra-1-2S/A, and Fra-1-2S/D in asynchronous HeLa cells. Those of Fra-1 and Fra-1-2SA were 80 ± 15 and 45 ± 15 min, respectively. This matched the twofold difference usually seen in steady-state-level assays such as the one presented in Fig. 2B and further validated that approach. Consistently, with the 10- to 20-fold higher accumulation of Fra-1-2S/D compared with Fra-1 in parallel transfection experiments, we were unable to determine the half-life of Fra-1-2S/D, as only an approximately 30% decay could be monitored in an 8-h chase.
Next, we demonstrated that the activation of the Erk1/2 pathway leads to the stabilization of Fra-1 via the phosphorylation of S252 and S265. The Erk1/2 and the Erk5 cascades are blocked by U0126, a pharmacological inhibitor of the Erk1/2-activating kinases Mek1 and Mek2 as well as of the Erk5 activator Mek5 (see references 34 and 48) (Fig. 3A). In contrast, the Erk1/2 cascade is activated selectively upon ectopic expression of the Mek1/2 kinase Mos or of an activated form of the Erk1/2-activating kinase Mek1 (MEK1DD). HeLa cells were transfected with expression vectors for single or double alanine mutants of S252 and S265 either alone or together with one for Mos or a mutant Mos lacking kinase activity (MosKD). The activation of Erk1/2 by Mos, but not by MosKD, was shown by immunoblotting using antibodies specific to residues phosphorylated by Mek1/2 (34, 48) (Fig. 3B). Mos led to increased levels of EGFP, presumably by stimulating the promoter activity of the pIRES2-EGFP-derived expression vectors. More importantly, Mos induced much stronger expression of wild-type Fra-1 than Fra-1-2S/A, indicating stabilization of the former protein but not of the latter. Interestingly, the single S mutants showed intermediate degrees of stabilization (Fig. 3B), with Fra-1-S252A being higher than Fra-1-S265A. This is consistent with the increased accumulation of Fra-1-S265D relative to that of Fra-1-S252D in Fig. 2B. Notably, all proteins, including Fra-1-2S/A, showed retarded electrophoretic mobility in the presence of Mos. This is most likely due to the phosphorylation of the DEF domain-proximal threonines T217, T223, T227, and T230, as suggested previously by other researchers (see above), since Fra-1-4T/D (Fig. 2C) and another Fra-1-4T/D-2S/A (not shown) were also subjected to electrophoretic retardation. These phosphorylations were not further investigated because of the lack of an effect on Fra-1 stability in our setting. Identical results were obtained in BALB/c MEFs and upon cotransfection of wild-type and mutant Fra-1 with expression vectors for MEK1DD and Erk1 in HeLa cells (not shown).
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Taken together, these data identify S252 and S265 as being targets of the Erk1/2 pathway and implicate their phosphorylation, and not that of other putative Erk1/2 phosphorylation sites, as the principal regulator of Fra-1 stability.
Phosphorylation of serines 252 and 265 in high-Erk1/2-activity-displaying HCT116 cancer cells is responsible for stabilization of Fra-1. HCT116 colon cancer cells display 10-fold-higher Erk1/2 activity than LS174T, as estimated from immunoblotting quantification of phospho-Erk1/2 in the two cell types (Fig. 5A). This correlates with a high level of Fra-1 showing reduced electrophoretic mobility, reflecting hyperphosphorylation (66). We used several strategies to address whether this high Fra-1 accumulation is contributed by S252 and S265 phosphorylation-dependent protein stabilization. In a first step, we used immunoblotting to show that endogenous Fra-1 is phosphorylated on S252 and S265 in HCT116 cells (Fig. 5B). Next, the addition of UO126 to HCT116 cells led to the dephosphorylation of S252 and S265, which correlated with an 8- to 10-fold reduction in protein levels in a 16-h chase (Fig. 5B). Next, before resorting to the analysis of Fra-1 mutants, we tested whether exogenous Fra-1 behaves similarly to the endogenous protein. This was the case: when overexpressed by the transfection of a bicistronic expression vector, the level of exogenous wild-type Fra-1 decreased by approximately 10-fold upon U0126 treatment, which correlated with faster electrophoretic mobility and the absence of phosphorylation of S252 and S265 (Fig. 5C). Finally, transfected Fra-1 accumulated to a level comparable to that of Fra-1-2S/D in parallel transfection experiments, in contrast to the Fra-1-2S/A level, which was much lower (Fig. 5D). Notably, the latter data contrasted with those obtained using LS174T cells, where Erk1/2 activity was low and where Fra-1 and Fra-1-2S/A showed only a modest difference in their level of accumulation, whereas Fra-1-2S/D accumulated to a high level (Fig. 2F).
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Stability of Fra-1 during the G0/G1-to-S-phase transition is due to Erk pathway-dependent phosphorylation of serines 252 and 265. Fra-1 levels are low in most nondividing, nontransformed cells. They increase within 2 h upon mitogenic stimulation and return to an intermediate level by 16 to 24 h poststimulation, depending on the cell type. During the G0/G1-to-S-phase transition, Fra-1 is phosphorylated and relatively stable but becomes destabilized if mitogens are removed (see above). Moreover, mitogenic stimulation leads to the strong activation of the Erk1/2 cascade within minutes, which then decreases to a moderate level that persists for hours. Therefore, we investigated whether the Erk1/2 pathway-dependent phosphorylation of S252 and S265 regulates the stability of endogenous Fra-1 in the G0/G1-to-S-phase transition.
First, we assessed S252 and S265 phosphorylation during a G0/G1-to-S-phase transition in quiescent BALB/c MEFs stimulated for growth by serum. The typical kinetics of endogenous Fra-1 accumulation are presented in Fig. 6A. The phosphorylation of S252 and S265 was detectable 2 h after serum addition. Thereafter, the decrease in phospho-S265 levels paralleled those of total Fra-1 and of active Erk1/2, while phospho-S252 decayed more rapidly. The addition of UO126 2 h after stimulation by serum led to (i) the rapid dephosphorylation of Fra-1, as visualized by its faster electrophoretic migration at the 4- and 8-h points, (ii) the loss of phospho-S252 and phospho-S265 signals as early as 4 h poststimulation, and (iii) the faster decay of Fra-1 starting at 8 h (Fig. 6B, left).
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Delineation of the Fra-1 destabilizer. We next delineated the regions in Fra-1 responsible for its instability. The stability of Fra-1 C-terminal truncation mutants was compared to that of the wild-type protein in asynchronous HeLa cells. The removal of 10 amino acids led to approximately a 12- to 15-fold stabilization, as deduced from densitomer scanning analysis of the luminogram shown in Fig. 7A. Further deletions did not enhance this effect. We then tested whether the fusion of Fra-1 C-terminal fragments to the C terminus of EGFP would destabilize this normally very stable protein. The addition of an N-terminal Myc6 tag (78 amino acids) allowed us to differentiate between the EGFP-Fra-1 chimeras and EGFP that served as the control for expression. A 2.2-fold destabilizing effect was already detectable with the last 30 amino acids of Fra-1, as deduced from densitomer scanning of the luminogram shown Fig. 7B, and the fusion of the last 40 or 50 amino acids further destabilized EGFP with similar efficiencies (12-fold) (Fig. 7B). Taken together, the above-described data indicate that a unique structural determinant contained within the last 30 to 40 amino acids is sufficient to destabilize Fra-1 in asynchronous cells.
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Proteasomal degradation of Fra-1. We then asked to what extent Fra-1 is degraded by the proteasome. Asynchronous HeLa cells were first transfected to express wild-type Fra-1, Fra-1-2S/A, and Fra-1-2S/D, respectively, and then treated with the proteasome inhibitors MG132 or epoxomycin for 8 h before immunoblotting analysis (Fig. 8A). Fra-1 and Fra-1-2S/A levels increased more than 20- to 30-fold, which is consistent with their half-lives in the 1- to 1.5-h range and indicates that most, if not all, Fra-1 undergoes proteasomal degradation. In contrast, there was no detectable change in Fra-1-2S/D abundance, in keeping with our observation that the half-life of phosphorylated Fra-1 exceeds 8 h (Fig. 2G). This also ruled out a significant contribution by another major proteolytic system to Fra-1 turnover under these experimental conditions. Consistently, Fra-1 decay induced by UO126 treatment in HCT116 cells (Fig. 5B) was blocked by MG132 (Fig. 8B). We also verified in two steps that the stabilization of phosphorylated Fra-1 in the G0/G1-to-S-phase transition was due to the antagonism of proteasomal degradation and not of another proteolytic pathway. First, serum-stimulated BALB/c 3T3 fibroblasts were treated with both UO126 and MG132 2 h after the addition of serum. Endogenous Fra-1 did not decay during the experiments, even though its faster electrophoretic migration indicated that it was hypophosphorylated (Fig. 8C), which contrasted with its disappearance in the absence of MG132 (compare with the 16-h time point in Fig. 6B, left). Second, quiescent BALB/c 3T3 cells stably transfected with the serum-inducible nonphosphorylatable Fra-1-2S/A mutant were serum stimulated and treated with either MG132 or epoxomycin 1 h later. Under these conditions, Fra-1-2S/A was stable for 16 h (Fig. 8D), which contrasted with its disappearance in the experiments presented in Fig. 6E. Finally, we verified that the instability of the nonphosphorylatable Fra-1 mutants was reversed in the presence of MG132. Indeed, all these mutants accumulated to a level comparable to that of the most stable phosphomimetic mutants processed similarly in parallel experiments (Fig. 8E). This confirmed that the bulk of Fra-1 undergoes proteasomal degradation under the experimental conditions used.
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-amino group of lysines and, in rare cases, the N terminus (22). We previously showed that a nonubitiquitylable c-Fos mutant with all lysines turned into arginines and with the N terminus blocked by a Myc tag undergoes properly regulated degradation by the proteasome, which thereby demonstrated that prior ubiquitylation was not necessary for c-Fos destruction (9). We therefore tested whether a similar mutant of Fra-1 (Myc2K/R-Fra-1K/R) would undergo proteasomal proteolysis and show regulated degradation like the wild-type protein. In parallel transient transfection assays in HeLa cells, Fra-1 and Myc2K/R-Fra-1K/R accumulated to the same level (Fig. 9A). Moreover, the degradation of both proteins was dependent on the proteasome, as shown by the stabilization by MG132 (Fig. 9A), and on the presence of a C-terminal destabilizer, since mutants lacking the C-terminal 10 amino acids were stabilized (Fra-1/1-261 and Myc2K/R-Fra-1K/R/1-261 in Fig. 7A and 9B, respectively). Finally, the degradation of Fra-1 and Myc2K/R-Fra-1K/R was similarly antagonized by Mos-driven activation of the Erk1/2 pathway (Fig. 9C). Mutation of S252 and S265 to A led to a slight destabilization, while mutating these two S's to D's strongly stabilized both proteins (Fig. 2B and 9D). Taken together, these data indicate that the ubiquitylation of Fra-1 is not required for regulated proteasomal degradation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fra-1 stabilization upon phosphorylation by kinases of the Erk1/2 pathway. We have combined several approaches to demonstrate that S252 and S265 are actual targets of the Erk1/2 pathway. The approaches include the analysis of site-specific mutants of Fra-1, cell signaling studies, and the use of both a pharmacological inhibitor and phosphoserine-specific antisera. The phosphorylation of one serine is not strictly dependent on that of the other, but whether one event can modulate the other cannot be excluded at this stage. The decreased electrophoretic mobility of Fra-1-2S/A upon the activation of the Erk1/2 pathway is consistent with previous suggestions by others that multiple sites are phosphorylated in Fra-1 (49, 69). This also indicates that the latter phosphorylations do not necessarily depend on prior S252 and/or S265 modifications. However, we cannot rule out that they might have an effect, as is the case with c-Fos (49, 50), under more moderate activation levels of Erk1/2. Whatever the case, these additional phosphorylations do not affect Fra-1 turnover control, a situation similar to that of c-Fos, where a mutation of T325 and T331 neither accelerates nor slows down the rate of degradation (45, 46, 50).
An important question is which kinases are responsible for S252 and S265 phosphorylation. By analogy with c-Fos and given our data, it is reasonable to assume that S265, which resides in an SP motif, is a target for Erk1 and/or Erk2. Our data do not rule out its phosphorylation on endogenous Fra-1 by Erk5 (62) in serum-induced BALB/c 3T3 cells, since this pathway is also inhibited by UO126. In contrast, S252 is not followed by a proline but instead is preceded by an arginine at position –3, a motif recognized by several kinases. The most likely candidates are members of the Rsk and Msk families, both of which are activated by Erk1/2 and phosphorylate numerous transcriptional regulatory proteins (19, 24). Accordingly, c-Fos is phosphorylated by Rsk in vitro and in vivo, a modification that regulates its activity and turnover (14, 15, 50). Phosphorylation of neither Fra-1 by Rsk's nor Fos family proteins by Msks has been described. Future studies will determine the contributions of these two kinase families in vivo.
The addition of UO126 to HeLa cells expressing phosphorylated Fra-1 due to the presence of Mos entailed a dephosphorylation of S265 that was slower than that of S252 (Fig. 4B), suggesting that the two phosphoserines are targeted by different still-to-be-identified phosphatases. Although we do not exclude the possibility that the phosphatase(s) specific for S265 may have been rate limiting under our conditions of Fra-1 overexpression, an interesting scenario is that basal Erk1/2 pathway activity may be required for the dephosphorylation of S265. The possibility that a kinase responsible for the stabilization of a protein is also necessary for the destabilization of the same protein may appear contradictory. However, it is well documented that both the nature and the intensity of the processes under the control of Erk1/2 depend on not only the timing but also the level of their activation (41, 48). Characterizing the relative contributions of phosphorylation and dephosphorylation events will help elucidate how Fra-1 turnover is regulated.
Several lines of evidence show that the principal mechanism responsible for Fra-1 stabilization in HCT116 cells is Erk1/2 pathway-dependent phosphorylation of S252 and S265 (Fig. 5). As high Fra-1 levels are observed in a variety of tumors also displaying high MAPK activity (see above), it will be interesting to determine whether the phosphorylation of S252 and S265 is also a major stabilization factor in these situations. Should this be the case, it is important to underline that other mechanisms might also contribute to Fra-1 overaccumulation, especially because the Erk1/2 pathway is already known to positively regulate fra-1 gene transcription (68), including in tumor cells (13, 66).
Proteasome-dependent, ubiquitylation-independent degradation of Fra-1. Vial and Marshall previously reported that the proteasome is responsible for the residual degradation of Fra-1 in HCT116 cells (66). Our work, even though it does not exclude possible minor contributions by other proteolytic systems, establishes that Fra-1, when unstable, is essentially degraded by the proteasome (Fig. 8C). The vast majority of substrates subjected to proteasomal hydrolysis are thought to depend on prior ubiquitylation for being addressed and/or recognized by the proteasome (22). We show that the degradation, as well as the regulation of degradation, of the nonubiquitylatable Myc2K/R-Fra-1K/R mutant is undistinguishable from that of wild-type Fra-1. Thus, Fra-1 belongs to the small group of proteins that can undergo ubiquitylation-independent proteasomal destruction. The hamster E36-ts20 cell line is thermosensitive for ubiquitylation activity due to a mutation in the first enzyme (E1) of the ubiquitin cycle (37). It is notable that Fra-1 is not stabilized upon a shift of these cells cultured asynchronously at the nonpermissive temperature (not shown). Even though caution is required for the interpretation of data from this experiment due to the usually leaky phenotype of E1-thermosensitive cell lines (54), these data are consistent with our turnover analysis of Myc2K/R-Fra-1K/R. Another point of consistency is our previous observation that the activity of the C-terminal c-Fos destabilizer, closely related to that of Fra-1, is independent of an active ubiquitin cycle (9). It remains possible that a small fraction of Fra-1 underwent ubiquitylation-dependent degradation in our experiments, as approaches such as those that we have used for studying c-Fos and Fra-1 measure the behavior of the bulk of a protein only. It is also possible that Fra-1 may undergo ubiquitin-dependent degradation under other conditions, as it is now clear that some proteins can be addressed to the proteasome via several mechanisms. This is well illustrated for the p53 oncosuppressor (4) and c-Fos, whose cytoplasmic degradation can involve the UBR1 E3 ubiquitin ligase (56).
Although the ubiquitylation of Fra-1 itself was not necessary for proteasomal degradation in our experiments, one possibility to consider is the polyubiquitin chains present in trans on a protein partner. Such an interactor cannot be an LZ dimerization partner since (i) an LZ-deficient mutant of Fra-1 was as unstable as the wild-type protein (not shown) and (ii) EGFP is destabilized by a 40-amino-acid C-terminal fragment of Fra-1 that does not contain the LZ.
Very interestingly, Hoffman et al. showed previously that transfected Fra-1 is also ubiquitylatable in an in vivo ubiquitylation assay, like that initially described by Treier et al. (63), and that Fra-1 ubiquitylation is stimulated by the activation of the Erk1/2 pathway, i.e., when the protein is stabilized (31). This poses the question regarding the actual role of this modification, as ubiquitylation is also involved in the control of a variety of protein functions independent of proteasomal proteolysis (27, 57). The regulation of Fra-1 transcriptional activity is certainly to be considered a priority, as it is stimulatable by the Erk1/2 pathway (69). Ubiquitylation is, however, not an absolute prerequisite, as the lysineless Myc2K/R-Fra-1K/R mutant was transcriptionally active in a transient luciferase transcription assay performed using HeLa cells (not shown).
Fra-1 destabilizer and degradation of other Fos proteins. We show here that Fra-1 breakdown depends on a unique C-terminal destabilizer. This helps us to understand why c-Fos expression is transient in mitogen-stimulated cells, whereas that of Fra-1 is much longer lasting. The molecular explanations are multiple. An obvious first explanation is the differential transcriptional regulations of the two genes. Thus, c-fos mRNA induction in G0/G1 phase is rapid and transient, whereas that of Fra-1 occurs later and is longer before returning to a basal level (2, 17, 68). A second mechanism is protein stability control. Using pulsed ectopic mRNA expression for wild-type and mutant Fra-1 proteins in serum-stimulated fibroblasts (Fig. 6), we show that the Erk1/2 pathway kinases are sufficiently active for at least 16 h to phosphorylate S252 and S265 and thereby prevent Fra-1 degradation during this period of time. By contrast, c-Fos protein instability is preserved by the activation of an N-terminal destabilizer even though the activity of its C-terminal destabilizer is also reduced (23).
The Fra-1 destabilizer is contained within the last 30 to 40 amino acids at the C terminus. This region contains striking similarities with the equivalent domains in the other Fos proteins (Fig. 11). It is therefore plausible that its counterparts in Fra-2 and FosB also show destabilizing activities, as these two proteins are also unstable. Work is currently under way to investigate this point and to determine whether (i) the destabilizers of the four Fos proteins are equally efficient or not and (ii) those of Fra-2 and FosB are also regulated by the Erk1/2 pathway-driven phosphorylation of the serine counterparts of S252 and S265 of human Fra-1. Because the N-terminal regions of c-Fos, Fra-2, and FosB share several homologous segments that are not found in Fra-1, it will also be important to address whether Fra-2 and FosB carry another N-terminally-located destabilizer as c-Fos. In turn, the dissimilarity between the N-terminal regions of c-Fos and Fra-1 raises the question of whether UBR1-dependent, ubiquitin-dependent degradation of c-Fos (56) may also occur on Fra-1. Preliminary studies using RNA interference to knock down UBR1 showed no increase in Fra-1 levels in asynchronous HeLa cells (not shown). However, to truly determine if Fra-1 behaves like c-Fos with respect to UBR1-mediated degradation, we must establish conditions where Fra-1 is quantitatively retained within the cytoplasm.
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/ß and PA28
) activator complexes, which can attach alone, in duplicate, or in combination at the extremities of the 20S particle (52). The 19S complex recognizes ubiquitylated as well as nonubiquitylated substrates (52). In contrast, PA28 activators, which influence the proteolytic activities of the 20S proteasome, are not capable of recognizing ubiquitylated proteins, although they may show some ubiquitylation-independent substrate selectivity (40). These observations thus leave open the possibility that the 19S and/or the PA28 regulators contribute to Fra-1 degradation. Biochemical studies involving various proteasomal complexes are under way to identify which proteasomal complex(es) is responsible for Fra-1 hydrolysis as well as whether recognition occurs directly via its C terminus or indirectly through a peptidic adaptor.
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ACKNOWLEDGMENTS |
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We are grateful to G. Bossis for critical reading of the manuscript.
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FOOTNOTES |
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Published ahead of print on 19 March 2007.
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REFERENCES |
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2. Adiseshaiah, P., S. Peddakama, Q. Zhang, D. V. Kalvakolanu, and S. P. Reddy. 2005. Mitogen regulated induction of FRA-1 proto-oncogene is controlled by the transcription factors binding to both serum and TPA response elements. Oncogene 24:4193-4205.[CrossRef][Medline]
3. Asher, G., Z. Bercovich, P. Tsvetkov, Y. Shaul, and C. Kahana. 2005. 20S proteasomal degradation of ornithine decarboxylase is regulated by NQO1. Mol. Cell 17:645-655.[CrossRef][Medline]
4. Asher, G., and Y. Shaul. 2005. p53 proteasomal degradation: poly-ubiquitination is not the whole story. Cell Cycle 4:1015-1018.[Medline]
