p38 activation promotes MyoD/E47–DNA binding activity and E-box-mediated myogenic transcription. (A) Inhibition of p38 MAPK activity abrogates myoblast differentiation and fusion. C2C12 myoblasts were cultured in growth medium (GM) and then shifted to differentiation medium (DM) for 3 days in the presence or absence of SB203580 (10 μM). Left: Effect of SB203580 on myoblast fusion in DM. Low-power views of representative fields are shown. Magnifications are × 100. Middle: p38 activity from cell extracts corresponding to the above treatments was analyzed by in vitro kinase assay using GST-ATF₂ as a substrate. Right: The effect of inhibition of p38 by SB203580 treatment on myogenic differentiation was analyzed by Western blotting using antibodies against myogenin and MHC. (B) p38 activity and MyoD/E47–E-box complex formation are concomitantly induced in DM, compared to GM. Left: p38 phosphorylation is induced during myoblast differentiation. C2C12 myoblasts were cultured in GM and then shifted to DM for 1 and 3 days. Activation of p38 was determined by Western blotting of whole-cell extracts (WCE) using a specific anti-phospho-p38 antibody (P-p38). C2C12 myogenic differentiation was assessed by Western blotting using antibodies against myogenin and MHC. MyoD and E47 levels were also analyzed in GM and DM. Right: MyoD/E47–E-box binding activity is induced during myoblast differentiation. Nuclear extracts (NE) of C2C12 myoblasts cultured in GM and DM for 1 and 3 days were prepared and analyzed by EMSA using the E-box double-stranded oligonucleotide (corresponding to the right E box of the MCK promoter) as a probe. The arrowhead indicates specific MyoD/E47–E-box complex formation during C2C12 differentiation. Right: The specificity of the MyoD/E47 heterodimer (corresponding to C2C12 cells after 24 h in DM) was analyzed by EMSA, after incubating NE with specific antibodies against MyoD or E47, or with an irrelevant antibody. (C) MCK transcriptional activity during myoblast differentiation depends on p38 MAPK activity. C2C12 cells were transfected with pMCK-Luc and after 24 h cells were either maintained in GM or shifted to DM for an additional 48 h. Cells in DM were treated or not with the p38 MAPK inhibitor SB203580 (SB) or with the JNK MAPK inhibitor SP600125 (SP) (20 μM), and luciferase activity was measured subsequently. (D) p38 MAPK activity induces MyoD/E47-dependent transcription from the MCK or 4RE-tk promoters. NIH3T3 fibroblasts were transfected with MCK-Luc (left) or 4RE-tk-luc (right) promoter-reporter plasmids with MyoD and/or E47 expression vectors. When indicated, MKK6 expression plasmid (+) or empty vector (−) was included in the transfected DNA. Cells were maintained for 48 h in GM, in the presence or absence of SB203580, and luciferase activity was measured subsequently. All transfection experiments were normalized to β-galactosidase activity. (E) SB203580 treatment inhibits the reporter activity of p4RE-tk-luc in C2C12 differentiating cells. C2C12 cells were transfected with p4RE-tk-luc and analyzed as in panel C. (F) MyoD/E47–DNA binding activity is induced by ectopic activation of p38 MAPK. Left: NIH3T3 cells were transiently transfected with E47 (lanes 2–7) and/or MyoD (lanes 5–10), or empty vector (lane 1), and MKK6(E) (lanes 3, 4, 6, 7, 9 and 10) expression plasmids, in the absence or presence of SB203580. Protein–DNA binding was analyzed by EMSA after incubation of NE with the labeled E box from the MCK promoter (as in (B)). Right: The composition of the MyoD/E47–E-box binding activity (corresponding to lane 6) was analyzed by EMSA, after incubating NE with specific antibodies against MyoD or E47, or with an irrelevant antibody. Western blotting against ectopic E47 and MyoD proteins was performed to show equal expression of transfected plasmids.

E47, but not MyoD, phosphorylation by p38 is required for the formation of the MyoD/E47 heterodimer. (A) E47 is a better substrate than MyoD for p38 phosphorylation in vitro. Two different amounts of purified GST-E47, GST-MyoD, GST-ATF₂ and GST alone (200 and 1000 ng) were incubated with 200 ng of MKK6-activated p38α. All incubations were performed in the presence of [γ-³²P]ATP, and labeled proteins separated by SDS–PAGE and detected by autoradiography. GST-ATF₂ and GST alone were used as positive and negative p38 MAPK phosphorylation control substrates, respectively. Coomassie staining confirmed equal loading of the purified recombinant proteins. (B) Phosphorylation of E47 by p38 is necessary for the formation of the E47/MyoD heterodimer–E-box complex. Left: GST-E47 (200 ng) was in vitro phosphorylated by activated p38 in the presence or absence of ATP. Phosphorylated (E47^*) and unphosphorylated (E47) forms of E47 were incubated with 200 ng of GST-MyoD (or 200 ng of GST, as control) and proteins were allowed to form homo- or heterodimers prior to the addition of the ³²P-labeled E-box probe, and analyzed by EMSA. Right: A similar assay was performed by incubating p38-phosphorylated (MyoD^*) and unphosphorylated (MyoD) forms of MyoD with GST-E47, followed by EMSA. Arrowheads indicate the formation of the MyoD/E47 heterodimer (black) and E47/E47 homodimers (white) binding to the E box. No MyoD/MyoD homodimers were found bound to the E box, irrespective of the phosphorylation status of MyoD.

Ser140 of E47 is a regulatory p38 site in muscle differentiation in vitro and in vivo. (A) Mutation of Ser140 of E47 abrogates MyoD/E47 heterodimer formation in vitro. In all, 200 ng of GST-E47 or GST-E47-S140A was in vitro phosphorylated by activated p38 (GST-E47^* and GST-E47-S140A^*, respectively) and incubated with 200 ng of GST-MyoD, followed by addition of the ³²P-labeled E-box probe, and analyzed by EMSA, as in Figure 3B. The arrowhead indicates the formation of the MyoD/E47 heterodimer. Coomassie staining confirmed equal amounts of the purified recombinant proteins. (B) Top: Inability of activated p38 to induce MyoD/E47-S140A heterodimer binding activity in vivo. NIH3T3 cells were transiently transfected with expression plasmids for MyoD and for WT or serine 140-mutated E47, with or without MKK6(E), or with vector alone (lane 1), in the absence or presence of SB203580, as indicated. Protein–DNA binding was analyzed by EMSA as in Figure 1F. Bottom: Western blotting was performed to show equal amounts of MyoD- and E47-transfected plasmids. (C) Influence of E47 Ser140 phosphorylation by p38 on 4RE-tk and MCK promoter activities. NIH3T3 cells were cotransfected with 4RE-tk-luc (left) or MCK-Luc (right) promoter-reporter plasmids, and WT or mutated E47 and MyoD expression plasmids. When indicated, MKK6 expression plasmid (+) or empty vector (−) was included in the transfection experiment with the presence or absence of SB203580. Cells were assayed for luciferase expression 48 h following transfection under GM conditions. All transfection experiments were normalized to β-galactosidase activity. (D) Inability of E47-S140 to induce muscle-specific expression from MyoD-expressing NIH3T3 fibroblasts in differentiation-promoting conditions. Left: NIH3T3 fibroblasts were transfected with expression vectors for MyoD and for Myc-E47 or Myc-E47-S140A, or with an empty vector (−), and switched to DM for 1 and 3 days. MCK and GAPDH mRNA expression was analyzed by RT–PCR using specific primers. Ectopic expression of MyoD and E47 (WT and mutated) proteins was analyzed by Western blotting using anti-MyoD and anti-Myc antibodies, respectively. Right: Inhibition of p38 activity abrogates MCK mRNA expression from MyoD/E47-transfected fibroblasts. NIH3T3 fibroblasts were transfected with expression vectors for MyoD and for Myc-E47, and cultured in DM for 1 day, in the absence or presence of SB203580. MCK and GAPDH mRNA expression was analyzed by RT–PCR (as in (D), left).

p38 induces MyoD/E47 heterodimerization rather than its binding to DNA. (A) Effect of MKK6-activated p38 on MyoD and E47 heterodimerization. NIH3T3 cells were transiently transfected with expression plasmids for E47 and MyoD (lanes 2–4), or MyoD∼E47 (lanes 5–7), or empty vector (lane 1), and MKK6(E) (lanes 3, 4, 6 and 7), in the absence or presence of SB203580. Protein–DNA binding activity was analyzed by EMSA, as in Figure 1F. (B) Specificity of the E-box–MyoD∼E47 binding activity. The composition of the MyoD∼E47 tethered dimer binding to the E box (from lane 6) was analyzed by EMSA, after incubating NE with specific antibodies against MyoD or E47, or with an irrelevant antibody. Western blotting against ectopic E47 and MyoD protein was performed to show equal expression of transfected plasmids. (C) The transcriptional activity of the MyoD∼E47 tethered dimer is not potentiated by activated p38. MyoD∼E47 expression plasmid was cotransfected with the 4RE-tk-luc reporter plasmid in NIH3T3 cells. When indicated, MKK6 expression plasmid (+) or empty vector (−) was included in the transfection experiment in the presence or absence of SB203580. Cells were assayed for luciferase expression 48 h following transfection under GM conditions. All transfection experiments were normalized to β-galactosidase activity. In the case of mixtures of MyoD+E47 monomers compared with tethered dimers, molar normalization was based on the monomer protein-coding sequences (i.e. 1 U of MyoD plus 1 U of E47 compared with 1 U of MyoD∼E47). (D) p38 activity is necessary for MyoD and E47 dimerization during C2C12 cell differentiation. C2C12 cells were cultured in GM or differentiated for 1 day in DM, in the absence or presence of SB203580, and NE were immunoprecipitated with an anti-E47 antibody. Immunoblotting was performed with an anti-MyoD antibody. NE were subjected to immunoblotting with antibodies against MyoD and E47, as control of endogenous protein levels. (E) MyoD heterodimerizes with E47 in the absence of DNA. C2C12 cells were differentiated for 1 day in DM, and MyoD was immunoprecipitated from WCE, and the immunoprecipitate was divided in two fractions: one was left untreated and the other was subjected to 30 min of DNaseI treatment. Both fractions were analyzed for the presence of E47 by Western blotting.

p38 phosphorylates E47 at Ser140 in vitro and during myoblast differentiation in vivo. (A) Phosphorylation of E47 deletion mutants by p38 in vitro. Equal amounts of purified full-length E47 (E47) or deletion mutants (GST-0^*, GST-2^*, GST-6^*, GST-8^*, GST-13^*) (see left panel) were incubated with 200 ng of activated p38. All incubations were performed in the presence of [γ-³²P]ATP, and labeled proteins separated by SDS–PAGE and detected by autoradiography (right). GST numbering indicates the number of potential p38 phosphorylation sites in the E47 deletion fragments. The C-terminal fragment of E47, GST-0^* (containing no p38 consensus sites), was used as a negative control for the phosphorylation assays. Amino-acid sequence including Ser135 and Ser140 is shown. Coomassie staining showed that equal amounts of substrate proteins were used in the in vitro phosphorylation assays. (B) E47 is phosphorylated at Ser140 in vitro. Purified full-length E47 or E47 mutated at serines 135 or 140 were incubated with 200 ng of activated p38, and analyzed for its p38 phosphorylation in vitro, as in (A). Coomassie staining confirmed equal loading of the different purified proteins. (C) p38 MAPK interacts with full-length and mutated E47 in vitro. 293T cell lysates were incubated with the indicated GST fusion proteins bound to glutathione–Sepharose beads. Total cellular proteins (lysate) and bound cellular proteins were analyzed using an anti-p38 antibody. The lysate lane contains 1/10 of the input used in incubations. Ponceau staining showed that comparable amounts of GST proteins were used in the pull-down assays. (D) E47 is phosphorylated by p38 in vivo. Left: 293T cells were transfected with a myc-tagged E47 expression vector, pCS2-E47, together with either an empty vector (−) or MKK6 expression plasmid, as indicated, and metabolically labeled with [³²P]orthophosphate. Myc-tagged proteins were immunoprecipitated from cell extracts, separated using SDS–PAGE and blotted. Phosphorylated proteins were visualized by autoradiography. The lower panel shows immunoblotting of the membrane with an anti-Myc (9E10) antibody to confirm equal expression of E47-transfected plasmids in all lanes. Right: NE from 293T cells (transfected as above) were prepared and the differential mobility of the hypo- and hyperphosphorylated E47 proteins was resolved by SDS–PAGE under special separation conditions, as explained in Materials and methods. Myc-E47 protein was visualized by immunoblotting of the membrane with anti-Myc (9E10) antibody. (E) Mutation of Ser140 to alanine impairs phosphorylation of E47 by p38 in vivo. 293T cells were transfected with a myc-tagged expression vector for E47 WT, pCS2-E47, or Ser140-mutated E47, pCS2-E47-S140A, together with either an empty vector (−) or the MKK6 expression plasmid, as indicated. E47 proteins were resolved as in panel D. (F) Phosphorylation of E47 protein by differentiation-induced p38 in C2C12 cells in DM. C2C12 cells were transfected with the myc-tagged expression vectors pCS2-E47 and pCS2-E47-S140A. At 12 h after transfection, cells were shifted to DM for 1 day, in the absence or presence of SB203580, and NE obtained and separated, as in panel D.

p38 activity is required for MyoD/E47 heterodimer binding to muscle promoters in myocytes in vivo. (A) Inhibition of p38 activity downregulates MyoD/E47 heterodimer formation and binding to DNA. C2C12 cells differentiated for 24 h in DM were further treated (or not) with 10 μM SB203580 for 2 h. NE and WCE were prepared for EMSA and Western blotting analyses, respectively. Left: The influence of SB203580 treatment on MyoD, E47 and p38 expression levels in C2C12 differentiating cells was analyzed by immunoblotting using specific antibodies. Right: MyoD/E47–DNA binding activity in C2C12 is inhibited by SB203580 treatment. NE from C2C12 cells subjected to the indicated cell treatments were analyzed by EMSA using the MCK-E box as a probe. The arrowhead indicates specific MyoD/E47–E-box complexes. The specificity of the MyoD/E47 heterodimer (corresponding to C2C12 cells after 24 h in DM) was analyzed by EMSA, after incubating NE with specific antibodies against MyoD or E47, or with an irrelevant antibody. (B) Influence of p38 inhibition on ChIP of two muscle regulatory regions with antibodies against MyoD and E47. Chromatin derived from C2C12 cells treated as in panel A was immunoprecipitated with control IgG, MyoD, E47 or histone 3 (H3) antibodies, and subjected to PCR with primers corresponding to the MCK and MHC promoter regions containing MyoD binding sites (E boxes) (primer sequences are reported in Materials and methods). NAB, no antibody. This is a representative experiment of three, with similar results. Bottom: Graph represents experimental values of the shown experiment relative to H3 immunoprecipitation.