Title: JNK phosphorylation relieves HDAC3-dependent suppression of the transcriptional activity of c-Jun
Abstract: Article15 July 2003free access JNK phosphorylation relieves HDAC3-dependent suppression of the transcriptional activity of c-Jun Carsten Weiss Corresponding Author Carsten Weiss Department of Biomedical Genetics, University of Rochester, Rochester, NY, 14642 USA Present address: Institute of Toxicology, University of Mainz, D-55131 Mainz, Germany Search for more papers by this author Sandra Schneider Sandra Schneider Department of Biomedical Genetics, University of Rochester, Rochester, NY, 14642 USA Search for more papers by this author Erwin F. Wagner Erwin F. Wagner Research Institute of Molecular Pathology, Vienna, A-1030 Austria Search for more papers by this author Xiaohong Zhang Xiaohong Zhang Department of Molecular Oncology, H.Lee Moffitt Cancer Center, Tampa, FL, 33612 USA Search for more papers by this author Edward Seto Edward Seto Department of Molecular Oncology, H.Lee Moffitt Cancer Center, Tampa, FL, 33612 USA Search for more papers by this author Dirk Bohmann Corresponding Author Dirk Bohmann Department of Biomedical Genetics, University of Rochester, Rochester, NY, 14642 USA Search for more papers by this author Carsten Weiss Corresponding Author Carsten Weiss Department of Biomedical Genetics, University of Rochester, Rochester, NY, 14642 USA Present address: Institute of Toxicology, University of Mainz, D-55131 Mainz, Germany Search for more papers by this author Sandra Schneider Sandra Schneider Department of Biomedical Genetics, University of Rochester, Rochester, NY, 14642 USA Search for more papers by this author Erwin F. Wagner Erwin F. Wagner Research Institute of Molecular Pathology, Vienna, A-1030 Austria Search for more papers by this author Xiaohong Zhang Xiaohong Zhang Department of Molecular Oncology, H.Lee Moffitt Cancer Center, Tampa, FL, 33612 USA Search for more papers by this author Edward Seto Edward Seto Department of Molecular Oncology, H.Lee Moffitt Cancer Center, Tampa, FL, 33612 USA Search for more papers by this author Dirk Bohmann Corresponding Author Dirk Bohmann Department of Biomedical Genetics, University of Rochester, Rochester, NY, 14642 USA Search for more papers by this author Author Information Carsten Weiss 1,2, Sandra Schneider1, Erwin F. Wagner3, Xiaohong Zhang4, Edward Seto4 and Dirk Bohmann 1 1Department of Biomedical Genetics, University of Rochester, Rochester, NY, 14642 USA 2Present address: Institute of Toxicology, University of Mainz, D-55131 Mainz, Germany 3Research Institute of Molecular Pathology, Vienna, A-1030 Austria 4Department of Molecular Oncology, H.Lee Moffitt Cancer Center, Tampa, FL, 33612 USA *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2003)22:3686-3695https://doi.org/10.1093/emboj/cdg364 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The AP-1 transcription factor c-Jun is a prototypical nuclear effector of the JNK signal transduction pathway. The integrity of JNK phosphorylation sites at serines 63/73 and at threonines 91/93 in c-Jun is essential for signal-dependent target gene activation. We show that c-Jun phosphorylation mediates dissociation of an inhibitory complex, which is associated with histone deacetylase 3 (HDAC3). The subsequent events that ultimately cause increased mRNA synthesis are independent of c-Jun phosphorylation and its interaction with JNK. These findings provide an ‘activation by de-repression’ model as an explanation for the stimulatory function of JNK on c-Jun. Introduction Whereas it is well established that the interaction of MAP kinases with responsive transcription factors and their subsequent phosphorylation often coincides with changed transcriptional output, the mechanistic basis for such processes is not well understood (Treisman, 1996; Chang and Karin, 2001). We have chosen the Jun-N-terminal-kinase (JNK)/c-Jun module to study mechanisms of transcription factor activation in eukaryotic cells. JNK is a MAPK that phosphorylates and activates c-Jun in response to a range of extracellular stimuli (Leppä and Bohmann, 1999; Davis, 2000). JNK-to-c-Jun signaling has been implicated in cellular proliferation, differentiation and apoptosis. Loss of either JNK or c-Jun activity is incompatible with embryonic development and impairs proliferation of mouse embryonic fibroblasts in tissue culture (Jochum et al., 2001). Jun proteins can also contribute to oncogenesis in different settings. c-Jun cooperates with Ras and is required for Ras to transform fibroblasts. Moreover, c-Jun is essential for the development of chemically induced tumors in mice (Eferl et al., 2003). v-jun is a retrovirally transduced allele of c-jun that can cause fibrosarcoma in chickens as well as wound-induced tumors in v-jun-expressing transgenic mice (reviewed in Vogt, 2001). While the molecular biology underlying the role of Jun in cell transformation and tumorigenesis is not completely understood at present, it is believed to involve the signaling and transcriptional properties of the protein. JNK docks to c-Jun on a sequence referred to as the δ-domain (Dai et al., 1995; Kallunki et al., 1996). The relatively stable interaction between JNK and c-Jun facilitates phosphorylation of the latter when JNK is activated by its upstream kinase, JNKK. The relevant MAPK–substrate sites on c-Jun are serines 63 and 73 (Binetruy et al., 1991; Smeal et al., 1991) and either threonine 91 or 93 or both (Hibi et al., 1993; Derijard et al., 1994, Papavassiliou et al., 1995). Current models assume that phosphorylation facilitates the interaction of signal responsive transcription factors with the basal transcriptional machinery or with transcriptional co-activators, including histone acetyl transferases (HATs; Treisman, 1996; Mayr and Montminy, 2001). Examples for such scenarios include the transcription factors CREB and Smad3 where increased binding to the HAT CBP following phosphorylation seems to mediate signal-dependent gene activation (Janknecht et al., 1998; Mayr and Montminy, 2001). While CBP also appears to be required for transcriptional activation by AP-1 (Mayr and Montminy, 2001) and can bind to the N-terminal region of c-Jun in vitro, there is no evidence that this interaction is regulated by or dependent on phosphorylation (Bannister et al., 1995). More generally, no co-activators or components of the basal transcriptional machinery have yet been identified that preferentially bind to phosphorylated c-Jun. As an alternative to the phosphorylation-induced binding of a co-activator, release of transcriptional inhibitors upon phosphorylation might also explain the signal-dependent activation of transcription factors. Examples for such inhibitory activities include prominently histone deacetylases (HDACs; Khochbin et al., 2001). HDACs antagonize the stimulatory effect of HATs on chromatin remodeling. Indeed, several transcription factors, notably nuclear receptors, recruit HDACs to promoters and thereby repress transcription in the absence of appropriate signals (Glass and Rosenfeld, 2000). Once such a transcription factor is activated, HDAC-associated protein complexes are exchanged for HAT-containing complexes, resulting in increased target gene expression. Thus, the activity of a transcription factor in a given cellular context may be regulated at the level of co-activator, or repressor interaction, or both. Here we show that in the absence of JNK signaling a repressor activity that is associated with HDAC3 inhibits c-Jun. Phosphorylation of c-Jun by JNK relieves this repression, thereby enhancing the transcriptional activity of c-Jun. Our data indicate that JNK phosphorylation causes dissociation of the HDAC3-containing repressor complex, releasing c-Jun from a repressed state. The subsequent steps which lead to enhanced transactivation by c-Jun appear to be independent of JNK, as unphosphorylated c-Jun can stimulate transcription efficiently if the repressor complex is dissociated. Experiments with JNK-deficient cells show that inactive JNK cannot be an essential component of the repressor complex and that c-Jun can activate transcription in the absence of its kinase, provided that the repressor function is abrogated. Based on these findings, we propose an explanation for the increased transcriptional and transforming potential of v-Jun, which acts as an activated version of c-Jun even though it cannot be phosphorylated by JNK. Results c-Jun contains an inhibitory domain which mediates JNK responsiveness To gain insight into the mechanism by which JNK stimulates c-Jun target gene transcription, we employed different types of reporter assays in cultured cells. We intended to restrict the analysis to the transcriptional function of c-Jun itself independent of heterodimerization with other AP-1 family members. To this end, we constructed fusion proteins that replaced the basic region/leucine zipper (bZIP) domain of c-Jun with the heterologous DNA-binding domain of the yeast transcription factor GAL4 (GAL-DBD, Figure 1A). In GAL–Jun1–256 all of c-Jun, except the bZIP domain, is fused to the GAL-DBD. As a reporter we stably integrated a Gal4-responsive luciferase gene into the genome of 293 cells. We chose stably integrated reporters because, presumably due to their packaging into chromatin, they acted more reliably and displayed less variable baseline activity than comparable reporters that were transiently transfected. Figure 1.Features of c-Jun that confer JNK responsiveness. (A) Schematic structure of c-Jun and GAL–Jun: the DNA-binding and dimerization domains of c-Jun (bZIP, black box) and GAL–Jun (GAL-DBD, cross-hatched box), the δ-domain (white box) and the JNK phosphorylation sites (serines 63, 73 and threonines 91, 93) are shown. Numbers indicate amino acid positions. (B) The transcriptional activity of GAL–Jun1–256 MAPK phosphorylation site mutants (AA: serines 63, 73 and threonines 91, 93 were replaced by alanines; AT: serines 63, 73 were replaced by alanines; SA: threonines 91, 93 were replaced by alanines) or a δ- domain deletion mutant were measured in reporter assays with or without activation of the JNK pathway by ΔMEKK. The graph displays the fold increase of normalized reporter activity after transfection with plasmids coding for the indicated fusion proteins relative to GAL– Jun1–256 which was set to 1 (means ± SD of 3–5 independent experiments). (C) Activities of GAL–Jun1–256 and different deletion mutants were determined as in (B). Note that Gal–Jun1–101 is constitutively active. (D) 293 cells were transiently transfected with the indicated expression constructs together with an empty expression vector or an expression plasmid encoding ΔMEKK. Lysates of transfected cells were analyzed by immunoblotting (IB) using either antibodies specific for the GAL-DBD or phospho-c-Jun-specific antibodies against the phosphorylated serine 63 or serine 73 (diamonds indicate fusion proteins, cross-reactivity with endogenous c-Jun is indicated by an arrow). Download figure Download PowerPoint At moderate levels of expression, comparable to endogenous c-Jun, GAL–Jun1–256 did not stimulate the reporter when compared with GAL-DBD alone. However, when JNK activity in the transfected cells was stimulated by expression of a constitutively active form of the JNK kinase kinase MEKK1 (ΔMEKK), reporter activity increased 6-fold (Figure 1B). Thus, the assay system recapitulates the activation of c-Jun by JNK signaling. Replacement of all potential JNK phosphorylation sites, serines 63 and 73 and threonines 91 and 93, by alanines (Figure 1B, AA) abrogated responsiveness to JNK. Furthermore, substitution mutants in which only subsets of the JNK phosphorylation sites have been mutated (Figure 1B, AT and SA) show that both the serines and the threonines are required for signal-dependent regulation of c-Jun. Similar to the mutation of the phosphorylation sites, deletion of the δ-domain, which serves as the JNK docking site, impaired JNK-dependent transcriptional activation (Figure 1B). Next, we examined which features of c-Jun in addition to the phosphorylation sites and the δ-domain are important for JNK responsiveness. A series of truncations of the GAL–Jun1–256 protein was generated and tested in reporter assays (Figure 1C). Similar to GAL–Jun1–256, a Gal4 fusion protein containing amino acids 1–145 of c-Jun (GAL–Jun1–145) was sufficient to recapitulate JNK responsiveness (Figure 1C). In contrast, a shorter form including only residues 1–101 (GAL–Jun1–101) was already transcriptionally active in the absence of a JNK signal and activation of JNK did not increase this constitutive activity further (Figure 1C). This increased signal-independent activity is not caused by a higher expression level or constitutive phosphorylation of GAL–Jun1–101, as confirmed by western blot experiments (Figure 1D). Interestingly, phosphorylation of GAL–Jun1–101 was still signal-dependent and increased upon JNK activation by MEKK co-expression, evidently without enhancing transcriptional activity. Thus, phosphorylation of c-Jun and its transcriptional activity do not absolutely correlate and can be uncoupled from each other. The findings described above suggest that GAL– Jun1–145, but not GAL–Jun1–101, contains a negatively acting domain that suppresses transactivation of the reporter gene. JNK signaling appears to neutralize this repressing function, permitting target gene activation. A conceivable mechanistic explanation for these observations is that JNK phosphorylation displaces a repressor activity that precludes transcription activation by c-Jun. Consistent with these results, a titratable repressor of c-Jun has been postulated by Baichwal and colleagues more than a decade ago (Baichwal and Tjian, 1990; Baichwal et al., 1992). The described activity acted via the so-called ϵ-domain (amino acids 101–128 of c-Jun), which resides in the region that we now also find to be important for JNK signaling to c-Jun. Analogous to the experiments by Baichwal et al., we performed transactivation assays using different GAL–Jun fusion proteins and co-expressed them with an excess of full-length c-Jun, which acts as a competitor for the repressor as it cannot bind, and therefore not activate, the GAL-reporter (Figure 2A). Overexpression of full-length c-Jun increases the transactivation potential of GAL– Jun1–256 in our experimental system (Figure 2B). Furthermore, we find that a c-Jun-derived competitor lacking the bZIP region (ΔbZIP), which cannot bind to DNA, was sufficient to enhance GAL–Jun1–256 activity (Figure 2B). This rules out that the stimulatory effect of full-length c-Jun is caused indirectly by c-Jun target gene expression. The GAL–Jun1–101 truncation mutant, as well as a mutated version of GAL–Jun1–256 that lacks the ϵ-domain could not be further activated by co-expression of full-length c-Jun (Figure 2B). Taken together, these data are most readily explained by a repressor, which requires amino acids 101–145 of c-Jun for a stable interaction with the transcription factor. This repressor, or an essential subunit of it can be dissociated from c-Jun by titration with an excess of competitor. Interestingly, co-expressed full-length c-Jun reduces the activity of a GAL–Jun mutant devoid of the ϵ-domain. This squelching effect supports the notion that in addition to the above-described inhibitor, c-Jun also binds to titratable co-activators. However their effect only becomes apparent when the influence of the inhibitor is eliminated, in the case of our experiment by the removal of the ϵ-domain. Figure 2.A titratable repressor inhibits c-Jun transcriptional activity via the same region that mediates JNK responsiveness. (A) Schematic model to illustrate repressor function on c-Jun transcriptional activity. See Figure 1 for a legend. In its inactive state c-Jun is bound by a repressor (R). This interaction is stabilized by the ϵ-domain (shaded box) and the δ-domain (white box). (B) The transcriptional activity of GAL–Jun1–256, ‘the activator’, can be enhanced by co-expression of full-length c-Jun, ‘the competitor’, due to competition for the repressor. 293 cells with a stably integrated 5× UAS luciferase reporter were transiently transfected with expression constructs encoding various derivatives of GAL–Jun as activators. The different activators were GAL–Jun1–256, GAL–Jun1–101 or an ϵ-domain deletion mutant. In addition, an empty expression vector (white bars) or an expression plasmid encoding either full-length c-Jun (black bars) or a c-Jun deletion mutant lacking the bZIP region (gray bar) was co-transfected as ‘competitor’. The fold activation compared to GAL–Jun is represented (means ± SD of 3–5 independent experiments). The activity of GAL–Jun1–256 alone was set to 1. (C) The activation of the human collagenase I promoter by c-Jun is inhibited by a titratable repressor. 293 cells were transiently transfected with a luciferase reporter gene under the control of the human collagenase I promoter (region −517 to +63) together with an empty expression vector or an expression plasmid encoding full-length c-Jun (gray bars). For competition assays, a full-length c-Jun expression plasmid, as activator, was co-transfected with a GAL–Jun1–256 expression plasmid as the competitor. Activities were determined as in (B). The fold activation compared with the basal activity of the collagenase promoter, which was set to 1, is shown (average ± average deviation of two independent experiments). Download figure Download PowerPoint Next, we examined whether the repressor also regulates transactivation of a natural target gene by c-Jun via its own DBD. The collagenase 1 promoter can be moderately activated by expression of full-length c-Jun (Figure 2C). Parallel over-expression of GAL–Jun1–256 increased c-Jun-dependent transcription from the collagenase 1 promoter significantly. This indicates that the competition for the repressor activity between c-Jun and Gal–Jun is reciprocal. Phosphorylation of c-Jun by JNK destabilizes the interaction with repressor Based on the data described above, we proposed the hypothesis that JNK might activate c-Jun by decreasing its affinity for the ϵ-domain-specific repressor or one of its components (Figure 3A). To test this idea, we measured transactivation by GAL–Jun1–256 (‘the activator’ in this experimental setting) when expressed either together with wild-type c-Jun (‘the competitor’) or with a phosphorylation point mutant, in which all JNK substrate sites have been replaced with alanines (JunAla). Compared with wild-type c-Jun, c-JunAla more potently stimulated GAL– Jun1–256 activity when co-expressed as a competitor (Figure 3B, upper part). Upon increased JNK signaling (+ΔMEKK) the difference between the phosphorylatable and non-phosphorylatable competitor to stimulate the activator was even more pronounced. This indicates that the distribution of repressor factors between the activator and the competitor is influenced by their respective phosphorylation states (Figure 3B, right panel). If wild-type c-Jun is used as a competitor, it reduces the amount of repressor bound to the activator, resulting in enhanced transcription of the reporter. In the presence of JNK activation, the affinities of both, the activator and the competitor, are decreased by phosphorylation. The net effect is no further enhancement of activator-mediated transcription by JNK under these conditions. However, competition by non-phosphorylatable c-Jun is more effective, due to its higher affinity to the repressor compared with wild-type c-Jun phosphorylated at basal levels. If the activator's phosphorylation is increased by JNK signaling, non-phosphorylatable competitor leads to a substantial shift of the distribution manifested in a super-enhancement of transcription. Figure 3.c-Jun phosphorylation and repressor binding. (A) Hypothesis: phosphorylation of c-Jun by JNK appears to reduce the affinity to the repressor thereby enhancing c-Jun's transcriptional activity. (B) Phosphorylated c-Jun competes less efficiently for repressor than non-phosphorylated c-Jun. Left panel: 293 cells with a stably integrated 5× UAS luciferase reporter were transiently transfected with expression constructs encoding GAL–Jun1–256 or GAL–Jun1–256Ala as activators and the indicated c-Jun derivatives as competitors. To stimulate phosphorylation of c-Jun, ΔMEKK was co-transfected (indicated with +). The normalized activity of GAL–Jun1–256 alone was set to 1 (means ± SD of 3–5 independent experiments). Note broken scale for values >50 (//). Right panel: model to explain the results shown in the graph on the left (for further details see text). (C) In the absence of the repression domain GAL–Jun1–101 activates transcription independently of its MAPK phosphorylation sites. 293 cells with a stably integrated 5× UAS luciferase reporter were transiently transfected with expression constructs encoding GAL–Jun or deletion mutants with, or without, MAPK phosphorylation sites (GAL–Jun1–101 and GAL–Jun1–101Ala, respectively). Activities were determined as in (A). Fold activation (average ± average deviation of two independent experiments) relative to GAL–Jun1–256, which was set to 1, is shown. (D) Phosphorylation of GAL–Jun1–256 is not increased by co-expression of full-length c-Jun. Lysates of 293 cells transfected as indicated were analyzed by immunoblotting (IB) using either antibodies specific for the HA epitope tag or antibodies against the phosphorylated serine 63 or serine 73 of c-Jun. Note that both Gal–Jun1–256 and c-Jun are HA-tagged. Arrows demarcate hypo- and hyperphosphorylated forms of GAL–Jun1–256; diamonds indicate endogenous hypo- (open diamonds) and hyperphosphorylated c-Jun (black diamonds). (E) The δ-domain of c-Jun stabilizes the interaction with the repressor. 293 cells with a stably integrated 5× UAS luciferase reporter were transiently transfected with expression constructs encoding GAL–Jun or a mutant, GAL–Junδ−, which lacks the δ- domain, as activator, together with an expression plasmid encoding either full-length c-Jun or a δ-domain deletion mutant, c-Junδ− as competitor. Activities were determined as in (A). Percent activation compared with the activity of GAL–Jun + c-Jun which was set to 100% is shown (means ± SD of 3–5 independent experiments). Download figure Download PowerPoint To further corroborate the model that phosphorylation weakens the repressor/c-Jun interaction, we used a non-phoshorylatable activator (GAL–Jun1–256Ala) and co- expressed it, in the absence or presence of JNK signaling, with wild-type c-Jun as a competitor (Figure 3B, lower part). Without JNK signaling the wild-type-Jun competitor causes increased transactivation by GAL–Jun1–256Ala. However, increased phosphorylation of the competitor (+ΔMEKK) abolished this effect, presumably because it no longer efficiently titrates the repressor. The conclusion that the negative effect of JNK on repressor binding and titration is due to the phosphorylation of the competitor is shown when c-JunAla is used as a competitor. In this experiment the inhibition of the activator can also be relieved under conditions of active JNK signaling. These experiments show that after removal of the repressor function even non-phosphorylatable c-Jun can efficiently enhance transcription. Accordingly, in a c-Jun mutant that cannot bind the repressor or essential components of it (Figure 3C, GAL–Jun1–101) all phosphorylation sites can be replaced by alanines without affecting its activity (Figure 3C, GAL–Jun1–101Ala, AA). Once c-Jun is de-repressed, either by deleting the repressor-binding domain or by sequestering the repressor activity, it does not have to be phosphorylated to transactivate and, presumably, to interact with co-activators. Consistently, although GAL–Jun efficiently enhances transcription when the inhibitor is dissociated by co-expressed c-Jun, its phosphorylation does not increase under these conditions (Figure 3D). The above experiments show that phosphorylation of c-Jun regulates repressor binding and not vice versa. Interestingly, whereas low amounts of transfected GAL–Jun1–256 or GAL–Jun1–256Ala expression plasmid do not activate the reporter in comparison to GAL-DBD alone, higher amounts do this even in the absence of JNK signaling (data not shown). Presumably under these conditions the concentration of activator exceeds that of the repressor, precluding efficient inhibition. In conclusion, we have identified a plausible mechanism for signal-dependent enhancement of c-Jun transactivation, which relies on phosphorylation-dependent de-repression of c-Jun. JNK1 and JNK2 are not essential components of the repressor and are dispensable for c-Jun to activate transcription It has previously been reported that the ϵ-domain is essential for binding a repressor activity, and that the δ-domain stabilizes this interaction (Baichwal and Tjian, 1990; Baichwal et al., 1992). The δ-domain serves as a docking site for a stable interaction between c-Jun and JNK, which can be detected even in the absence of signaling (Dai et al., 1995; C.Weiss, unpublished data). For these reasons, it has been suggested that JNK in its inactive form might constitute the repressor or contribute to repressor function (Dai et al., 1995; Vogt, 2001). This hypothesis gained credibility with the finding that the yeast MAPK Kss1 in the inactive state can bind to and thereby inhibit its target transcription factor STE12 (Madhani et al., 1997). In agreement with earlier data, we find that a mutant of c-Jun that lacks the δ-domain can compete less efficiently for the repressor activity than wild-type c-Jun. Conversely, a δ-domain deletion mutant when it is acting as a transactivator, is more easily relieved of repression (Figure 3E). To test the role of JNK in c-Jun repression directly, we performed experiments in JNK-deficient 3T3 cell lines, which were derived from mice in which the genes encoding both JNK1 and JNK2 have been disrupted (Ouwens et al., 2002). In such jnk1−/−, jnk2−/− cells c-Jun phosphorylation in response to inducers of JNK signaling is completely absent (Tournier et al., 2000 and data not shown). The presence of the repressor activity in wild-type 3T3 cells was evident because increasing amounts of a wild-type c-Jun competitor enhanced the activity of the GAL–Jun activator as shown above for 293 cells. Strikingly, a similar result was obtained in jnk1−/−, jnk2−/− double knock-out cells, indicating that these JNK-deficient cells have repressor activity and demonstrating that JNK is not required for this function (Figure 4A). Figure 4.JNK is not the repressor and is dispensable for transcription activation by c-Jun. (A) wt-3T3 or jnk1−/−, jnk2−/− 3T3 cells were transiently transfected with a 5× UAS luciferase reporter and expression vectors for activator and competitor proteins as indicated. Fold activation compared with GAL–Jun1–256 (activity set to 1, means ± SD of three independent experiments) is shown. (B) Phosphorylation of c-Jun by JNK weakens the interaction with the inhibitor. Left panel: wt-3T3 or jnk1−/−, jnk2−/− 3T3 cells were transiently transfected with a 5× UAS luciferase reporter and the GAL–Jun1–256Ala expression construct as activator, together with full-length c-Jun expression plasmid as competitor. Where indicated, ΔMEKK, was co-transfected. Activities were determined as in (A). The relative reporter gene activities (means ± SD of three independent experiments) are shown. The activity of GAL–Jun1–256Ala in the presence of c-Jun competitor was set to 100%. Right panel: model to explain the results shown in the graph on the left (for further details see text). (C) The δ-domain of c-Jun stabilizes the interaction with the repressor in the absence of JNK. wt-3T3 or jnk1−/−, jnk2−/−3T3 cells were transiently transfected with a 5× UAS luciferase reporter and expression constructs as indicated. Activities were determined as in (A). The % activation compared with that mediated by GAL–Jun + c-Jun, which was set to 100%, is shown (means ± SD of three independent experiments). Download figure Download PowerPoint Once c-Jun is de-repressed by sequestration of the repressor or limiting components of a larger repressor complex, GAL–Jun1–256 activated transcription in wild-type and JNK-deficient cells, further supporting the interpretation that JNK binding and/or phosphorylation is not essentially required for the productive interaction of positively acting co-factors with c-Jun. Thus, the main function of the kinase appears to be the signal-dependent removal of repressor activity. To confirm this model, we investigated whether or not the interaction between c-Jun and repressor can still be destabilized by JNK signaling in the knock-out cells. As shown above (Figure 3B, lower part), the ability of over-expressed c-Jun to sequester the repressor can be suppressed by stimulation of the JNK pathway in wild-type 3T3 cells. This inhibition of repressor titration was indeed mediated via JNK, since it is almost completely abolished in jnk1−/−, jnk2−/− cells (Figure 4B). Although JNK is not an essential component of the repressor, it might mediate the stabilizing effect of the δ-domain on the interaction of repressor with c-Jun. To test this idea, we performed competition experiments in either wild-type or jnk1−/−, jnk2−/− cells. As observed in 293 cells (Figure 3C), the δ-domain stabilized the interaction with the repressor in 3T3 wild-type and also in jnk1−/−, jnk2−/− cells (Figure 4C). This finding suggests that the δ-domain facilitates repressor binding independently of JNK. HDAC3