Article Figures & Data
Figures
- FIG. 1.
JNK and ERK pathways mediate AP-1 activation by PDGF and serum. NIH 3T3 cells were transfected with pAP-1-Luc and pRL-null, cultured under serum-free conditions, and pretreated for 30 min with inhibitors for each MAPK pathway (U0126, SP600125, and SB203580; 10 μM each) before PDGF (20 ng/ml) or FBS (10%) stimulation. Dual luciferase activities were determined 4 h after treatment, as described in Materials and Methods. The data represent the firefly luciferase activity normalized by Renilla luciferase activity present in each sample, expressed as absolute counts. Values are the average ± standard deviation of triplicate samples from a typical experiment. Nearly identical results were obtained in three additional experiments.
- FIG. 2.
ERK signaling regulates c-Fos expression and posttranslational modifications. (A) PDGF induction of c-Fos expression in NIH 3T3 cells. Cells were grown overnight in the absence of serum and then stimulated with PDGF (20 ng/ml) for the indicated times. Nuclear fractions were collected, and c-Fos was detected by Western blotting. (B) ERK dependency of c-Fos expression. Cells were serum starved overnight, incubated for 30 min in the absence (c) or presence of U0126 (U), SP600125 (SP), or SB203580 (SB) (10 μM each), and stimulated with PDGF (20 ng/ml; 2 h), and c-Fos expression in nuclear fractions was determined as described for panel A. (C) ERK dependency of c-fos mRNA expression. Cells were serum starved overnight, incubated for 30 min in the absence or presence of U0126, and stimulated with PDGF (20 ng/ml) for the indicated times. c-fos transcript levels were detected by Northern blotting as described in Materials and Methods. (D) ERK dependency of c-Fos posttranslational modifications. Cells were transiently transfected with pCEFL-AU5-c-Fos (1 μg/well) and grown in serum-free medium overnight after transfection. Cells were stimulated with PDGF (20 ng/ml; 30 min), and total lysates were processed for Western blotting using anti-c-Fos antibodies (top panel). Pretreatment with MAPK inhibitors was performed as described above. Endogenous active (middle panel) and total (bottom panel) ERK expression from the same samples served as controls for PDGF and U0126 effects on ERK phosphorylation. (E) c-Fos phosphorylation induced by PDGF. NIH 3T3 cells were transfected with pCEFL-AU5-c-Fos (1 μg/well), cultured in serum-free medium overnight, and stimulated with PDGF for 10 or 30 min. Cell lysates were subjected to PP2A digestion followed by immunoblotting using anti-c-Fos antibodies (see Materials and Methods). (F) SP600125 (SP) and SB203580 (SB) inhibitory action on JNK and p38 activities. HEK-293T cells were transfected with HA-JNK1 or HA-p38α alone or in combination with their upstream activating kinases (MEKK and MEK3EE, respectively). In vitro kinase assays were performed as described in Materials and Methods in the absence or presence of the indicated final concentrations of the inhibitors. 32P-labeled substrate (P-ATF2) is indicated. NT, nontransfected cells.
- FIG. 3.
PDGF activation of c-Fos is ERK dependent. (A) ERK dependency of c-Fos activation of AP-1 by PDGF. NIH 3T3 cells were transfected with pAP-1Luc and pRL-null along with 0.2 μg of pCEFL-AU5-c-Fos or pcDNAIII-β-gal. Where indicated (+), cells were pretreated with U0126 (10 μM) for 30 min and left untreated or stimulated with PDGF (20 ng/ml) for 4 h before reading dual luciferase activities. (B) c-Fos transcriptional response to PDGF and serum. Cells were cotransfected with pGal4-c-FosFL or pGal4-c-FosTAD (2 ng/well each) along with pGal4-Luc and pRL-null. At 24 h after transfection, cells were stimulated with PDGF (20 ng/ml) or FBS (10%), as indicated, and dual luciferase activities were determined 4 h later. (C) ERK dependency of c-Fos TAD activation and posttranslational modifications by PDGF. Results shown in the bar graph are cells that were transiently transfected with pGal4-c-FosTAD (2 ng/well), pGal4-Luc, and pRL-null; luciferase assays were performed as described above. Where indicated, cells were pretreated with U0126, SP600125, or SB203580 (10 μM each) for 30 min before PDGF stimulation. PDGF-induced modified c-Fos species are denoted by the bracket. The Western blot shows results with cells that were transfected with pGal4-c-FosTAD (1 μg/well), grown in serum-free medium overnight, and stimulated with PDGF for 10 min. Total lysates were assayed by Western blotting using anti-c-Fos antibodies (top panel). Controls for phosphorylated and total ERKs are shown in parallel (middle and bottom blots). Reporter assay data represent the firefly luciferase activity normalized by Renilla luciferase activity present in each sample, expressed as absolute counts. All values are the average ± standard deviation of triplicate samples from a typical experiment. In each case, similar results were obtained in three additional experiments.
- FIG. 4.
ERK2 activates c-Fos. (A) NIH 3T3 cells were transfected with p-Gal4c-FosFL or p-Gal4c-FosTAD (each 2 ng/well) along with pGal4-Luc, pRL-null, and MEKEE+ERK2 (0.5 μg each) or pcDNAIII-β-gal (controls). Cells were incubated in serum-deprived medium, lysed, and assayed for firefly and Renilla luciferase activities as for Fig. 3. (B) Effect of ERK2 on AP-1 activation by c-Fos. Cells were transfected with pAP-1Luc and pRL-null, alone or together with pCEFL-AU5-c-Fos (1 μg/well) pCEFL-c-Jun (1 μg/well), and/or MEKEE+ERK2 (0.5 μg each), and processed as above for AP-1-driven luciferase expression. (C) ERK2 induction of c-Fos posttranslational modifications. pGal4-c-FosTAD (1 μg/well) was expressed alone or in combination with ERK2+MEKEE (0.5 μg each) in HEK-293T cells. At 24 h posttransfection, c-Fos expression was detected by Western blotting using anti-c-Fos antibodies. The high-molecular-mass protein bands correspond to endogenous c-Fos proteins expressed in HEK-293T cells.
- FIG.5.
Candidate MAPK phosphorylation sites confer PDGF responsiveness to the c-Fos TAD. (A) Schematic representation of c-Fos primary structure with emphasis on the major functional domains and the relative location of the four conserved sites that follow the consensus motif for MAPK phosphorylation in the C-terminal TAD (above). bZIP, region that comprises the leucine zipper and DBD; N- and C-TAD, N-terminal and C-terminal TADs, respectively. Details are shown for known c-Fos sequences from diverse species, showing the conservation of all four potential MAPK phosphorylation sites (below). Note that Thr-325, Thr-331, and Ser-374 lie within sequence motifs that are also highly conserved. (B) Effect of mutations in potential MAPK phosphorylation sites on the basal activity and inducibility by PDGF of the c-Fos TAD. Where indicated, cells were transfected with pGal4-FosTAD or pGal4-c-FosTAD-m (2 ng/well each) along with pGal4-luc, pRL-null, and MEKEE+ERK2 (0.5 μg each) (right panel). Cells were serum starved and stimulated with PDGF (20 ng/ml) or FBS (10%) (left panel) for 4 h before reading firefly and Renilla luciferase activities. Total lysates from Gal4c-FosTAD- or Gal4-c-FosTAD-m-overexpressing cells were analyzed by Western blotting using anti-Gal4 DBD antibodies (inset). (C) Effect of mutations in MAPK sites on the c-Fos activation of AP-1 transcription. NIH 3T3 fibroblasts were transfected with pAP-1-Luc, pRL-null, and 1 μg (right panel) or increasing concentrations (left panel) of pCEFL-AU5-c-Fos or pCEFL-AU5-c-Fos-m. Cells were incubated overnight in the absence of serum and then stimulated with PDGF for 4 h before measuring dual luciferase activities. Total lysates of c-Fos- or c-Fos-m-overexpressing cells were analyzed by Western blotting using anti-AU5 antibodies (left panel, inset). MEKEE and ERK2 were cotransfected at 0.5 μg each (right panel). Reporter assay data represent the firefly luciferase activity normalized by Renilla luciferase activity present in each sample, expressed as absolute counts. All values are the average ± standard deviation of triplicate samples from a typical experiment. Similar results were obtained in three additional experiments.
- FIG. 6.
(A) MAPK sites are required for c-Fos phosphorylation induced by PDGF. As indicated, NIH 3T3 cells were transfected with pCEFL-AU5-c-Fos or pCEFL--AU5-c-Fos-m (2 μg/well), pretreated with U0126 (10 μM) or vehicle for 30 min, and stimulated with PDGF for an additional 30 min. Cells were lysed, resolved by SDS-PAGE, and blotted using anti-c-Fos antibodies (top panel). Phospho-ERK and total ERK immunoreactivities from the same samples are shown below (middle and bottom panels). (B) MAPK sites are required for c-Fos phosphorylation by ERK2. Cell lysates from c-Fos- or c-Fos-m-overexpressing cells in the absence or presence of MEKEE+ERK2, as indicated, were subjected to PP2A digestion as described in Materials and Methods. c-Fos expression was detected by Western blotting using anti-c-Fos antibodies.
- FIG. 7.
ERK2 phosphorylates c-Fos in vitro and in vivo. (A and B) Bacterially expressed c-Fos TADs, six-His c-FosTAD-wt, and six-His c-FosTAD-m were purified and assayed as substrates for an immunoprecipitated (A) or recombinant purified (b) preparation of ERK2. In vitro kinase assays were performed as described in Materials and Methods. The purified six-His substrates (A and B, lower panels) and the HA-tagged ERK2 (A, middle panel) used in the kinase reactions were detected by Western blotting with the indicated antibodies. (C) In vitro cold kinase assay using purified six-His c-Fos TAD as a substrate for recombinant ERK2. Reaction products were analyzed by Western blotting using anti-c-Fos antibodies or anti-phospho-T/P antibodies. (D) HEK-293T cells were transfected with pCEFLc-Fos (1 μg/well) alone or in combination with ERK2+MEKEE (0.5 μg each). Total lysates were analyzed by Western blotting using anti pCEFL-AU5-c-Fos antibodies (left panel) or immunoprecipitated with anti-c-Fos and immunoblotted using anti-phospho-T/P antibodies (right panel).
- FIG. 8.
ERK2 phosphorylation sites in the c-Fos TAD. (A) Mapping of c-Fos TAD phosphorylated sites by ERK2. The indicated purified six-His c-Fos TAD wild-type (wt) or mutant proteins bearing none (m) or a single potential ERK phosphorylation site were used as substrates for recombinant ERK2 in in vitro kinase assays. Equal amounts of purified proteins in each reaction mixture were confirmed by Western blotting with anti-six-His antibody. Phosphorylated products (P-c-FosTAD) were resolved by SDS-PAGE. (B) Evaluation of the role of ERK phospho-acceptor sites in the c-Fos TAD in the transcriptional response of the c-Fos TAD to PDGF and ERK2 activation. Cells were transiently transfected with pGal4-c-FosTAD wild-type (wt) or mutant vectors bearing none (m) or single potential ERK phosphorylation sites (2 ng/well), together with pGal4-Luc and pRL-null, and luciferase assays were performed. The top graph shows results with cells that were left untreated (control) or treated with PDGF for 5 h (20 ng/ml). The bottom graph shows results with cells that were transfected with pCDNAIII- β-gal (control) or MEKEE+ERK2 (0.5 μg each). All Gal4 constructs were expressed at comparable levels (data not shown). Reporter assay data represent the firefly luciferase activity normalized by the Renilla luciferase activity present in each sample. All values are the average ± standard deviation of triplicate samples from a typical experiment. In each case, similar results were obtained in at least three additional experiments.
- FIG. 9.
c-fos potentiates the focus-forming activity of c-sis: requirement of ERK phosphorylation sites in c-Fos. (A) NIH 3T3 fibroblasts were transfected with 1 μg of c-fos expression vector (pCEFL-AU5-c-Fos) or pCDNAIII-β-galactosidase (β-gal) alone or in combination with 0.5 μg of c-sis (pcDNAIII-c-sis), as indicated. Cells were maintained in 5% calf serum and fixed and stained 25 days after transfection. Plates from a representative experiment are shown. (B) Cells were transfected with pcDNAIII-c-sis (0.5 μg/plate) alone or together with pCEFL-AU5-c-Fos and/or MEKAA (1 μg each). Transforming efficiency was estimated by counting the number of foci per dish after 25 days of transfection and expressed as the percentage with respect to that observed in c-sis and c-fos transfections, which was taken as 100%. Results from a representative experiment out of three independent ones are shown. (C) Cells were transfected with pcDNAIII-c-sis (0.5 μg) with or without pCEFL-AU5-c-Fos or pCEFL-AU5-c-Fos-m (1 μg each). Foci were counted 25 days after transfection and are represented as described for panel B.
- FIG. 10.
Proposed model for PDGF receptor and ERK signaling to c-Fos and AP-1-regulated transcription. PDGF (c-sis) triggers a variety of signaling pathways, including those leading to the activation of the ERK cascade (Raf, MEK, and ERK). This results in the phosphorylation of the ternary complex factor (TCF) by ERK and the stimulation of the activity of the c-fos SRE, thus promoting c-fos expression. Subsequently, ERK phosphorylates c-Fos and that stimulates the transactivating activity of c-Fos, thereby promoting the expression of AP-1-regulated genes.
