Fig. 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–Jun_1–256 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– Jun_1–256 which was set to 1 (means ± SD of 3–5 independent experiments). (C) Activities of GAL–Jun_1–256 and different deletion mutants were determined as in (B). Note that Gal–Jun_1–101 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).

Fig. 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–Jun_1–256 or GAL–Jun_1–256^Ala 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–Jun_1–256 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–Jun_1–101 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–Jun_1–101 and GAL–Jun_1–101^Ala, respectively). Activities were determined as in (A). Fold activation (average ± average deviation of two independent experiments) relative to GAL–Jun_1–256, which was set to 1, is shown. (D) Phosphorylation of GAL–Jun_1–256 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–Jun_1–256 and c-Jun are HA-tagged. Arrows demarcate hypo- and hyperphosphorylated forms of GAL–Jun_1–256; 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).

Fig. 5. HDAC3 binds to c-Jun and represses transcription activation. (A) HDAC3 but not HDAC1 suppresses c-Jun activity dependent on the ε- domain. As described in Figure 2B, cells were transfected with the indicated expression constructs. UAS reporter activation mediated by GAL–Jun + c-Jun was measured in the absence and presence of co-expressed HDAC1 or 3. The activation compared with that mediated by GAL–Jun + c-Jun, which was set to 100%, is shown (means ± SD of 3–4 independent experiments). Lysates of transfected cells were analyzed by immunoblotting using either antibodies specific for the Flag or HA epitope (lower panel). (B) HDAC3 binds to the N-terminal region (aa 1–101) of c-Jun. 293 cells were transfected with expression constructs either coding for full-length c-Jun, HDAC3 or empty expression vector (left panel) or coding for HDAC3 and various c-Jun deletions (right panel). Lysates were subjected to immunoprecipitation (IP) and subsequent immunoblot analysis (IB) using the indicated antibodies. (C) HDAC3 suppresses c-Jun dependent on its ε-domain. UAS-reporter activation mediated by GAL–Junε^– + c-Jun (left panel) was quantitated in the presence or absence of co-expressed HDAC3 as described in (A). (D) Loss-of-HDAC3-function releases c-Jun from repression. Expression constructs either coding for GAL–Jun or GAL–Junε^– were co-transfected with empty or HDAC3 siRNA expression vectors. Reporter activation was measured three days after transfection. The fold activation compared with that mediated by GAL–Jun which was set to 1, is shown (average ± average deviation of two independent experiments). (E) Expression of HDAC3 siRNA efficiently depletes endogenous HDAC3 protein levels in 293 cells. Lysates of transfected cells were analysed three days after transfection by immunoblotting using an anti-HDAC3 antibody.

Fig. 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–Jun_1–256, ‘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–Jun_1–256, GAL–Jun_1–101 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–Jun_1–256 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–Jun_1–256 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).

Fig. 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–Jun_1–256 (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–Jun_1–256^Ala 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–Jun_1–256^Ala 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).