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. 1999 Jan;19(1):21-30.
doi: 10.1128/MCB.19.1.21.

Regulation of the MEF2 family of transcription factors by p38

M Zhao  1 , L New, V V Kravchenko, Y Kato, H Gram, F di Padova, E N Olson, R J Ulevitch, J Han
Affiliations

Regulation of the MEF2 family of transcription factors by p38

M Zhao et al. Mol Cell Biol. 1999 Jan.

Abstract

Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.

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Figures

FIG. 1
FIG. 1
Phosphorylation of bacterially expressed MEF2 proteins by p38 in vitro. (A) His-tagged MEF2A, MEF2B, MEF2C, and MEF2D proteins were phosphorylated in vitro by purified p38 (p38α) as described in Materials and Methods. Autophosphorylated p38 is indicated by an arrow. (B) Coomassie blue staining of MEF2 proteins used in the kinase assay.
FIG. 2
FIG. 2
Activation of the p38 pathway by MKK6b(E) up-regulates MEF2A and MEF2C activity. 293 cells were cultured in six-well plates and cotransfected with β-galactosidase expression vector pCMV-β-gal (0.2 μg), the reporter plasmid pG5ElbLuc (0.2 μg), GAL4-MEF2A(wt), and expression vectors for GAL4(DBD) fused to various MEF2 isoforms (0.3 μg), the MKK6b(E) expression plasmid (0.3 μg), or the control empty vector pcDNA3. Empty vector pcDNA3 was used to normalize the total DNA to 1 μg per transfection. Cell extracts were prepared 48 h following transfection. The ratio of luciferase activity to β-galactosidase activity is presented as the mean ± standard deviation (n = 3) (A). Three experiments were performed with comparable results. The results of one experiment are shown. The expression of GAL4 fusion proteins in extracts of transfected cells was determined by Western blotting using anti-GAL4(DBD) monoclonal antibody RK5C1 (B).
FIG. 3
FIG. 3
Schematic representations of MEF2A and MEF2C. The number of amino acids in each protein is indicated at the right. MEF2A and MEF2C without the last alternatively spliced exon are shown. The amino acid sequence in the regions containing p38 phosphorylation sites of MEF2C and the corresponding sequence in MEF2A are shown. Thr-293, Thr-300, and Ser-387 in MEF2C and Thr-312, Thr-319, and Ser-453 in MEF2A are underlined.
FIG. 4
FIG. 4
Tryptic phosphopeptide mapping and phosphoamino acid analysis of wild-type MEF2A [MEF2A(wt)] phosphorylated by p38. (A) Two-dimensional tryptic peptide map obtained from 32P-labeled MEF2A; (B) phosphoamino acid analysis of each phosphorylated peptide from panel A. There are three phosphorylated peptides. Peptide 1 contains phosphoserine and phosphothreonine, while peptides 2 and 3 contain only phosphoserine. The positions of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards are indicated; + designates the origin.
FIG. 5
FIG. 5
In vitro phosphorylation sites of MEF2A by p38. (A) Phosphopeptide mapping of MEF2A(T312, 319A) phosphorylated by p38 in vitro; (B) phosphopeptide mapping of MEF2A(T312, 319, S355A) phosphorylated by p38 in vitro; (C) phosphopeptide mapping of MEF2A(S479A) phosphorylated by p38 in vitro; (D) phosphopeptide mapping of MEF2A(S453A) phosphorylated by p38 in vitro. The sequences of predicted phosphopeptides are shown above panels B to D, with phosphorylated residues underlined.
FIG. 6
FIG. 6
In vivo phosphorylation of MEF2A. (A) MEF2A was immunoprecipitated from sorbitol-stimulated or nonstimulated 293 cells that were metabolically labeled with 32P. Only the region of the gel containing MEF2A is shown. (B) Phosphopeptide mapping of MEF2A isolated by SDS-PAGE shown in panel A. The only 32P-labeled peptide obtained had Rf and mr values very similar to those of phosphopeptide 1 of p38-phosphorylated MEF2A in vitro (Fig. 4). (C) Phosphoamino acid analysis of the peptide shown in panel B.
FIG. 7
FIG. 7
Enhancement of the MEF2A transactivation activity by p38 pathway is dependent on phosphorylation of Thr-312 and Thr-319. 293 cells were cotransfected with pCMV-β-gal, pG5ElbLuc, MKK6b(E), GAL-MEF2A(wt), and plasmids expressing GAL4(DBD) fused to various MEF2A isoforms, and reporter gene expression was assayed as described for Fig. 2. The expression of GAL4–MEF2A and its mutants was determined by Western blotting using anti-GAL4(DBD) monoclonal antibody RK5C1 (top). Two experiments were performed, and the results of one experiment are shown.
FIG. 8
FIG. 8
MEF2A activation is specifically mediated by the p38, but not ERK, JNK, or ERK5/BMK, pathway. 293 cells were cotransfected with pCMV-β-gal, pG5ElbLuc, and GAL4-MEF2A(wt). The p38 pathway, ERK pathway, JNK pathway, or ERK5/BMK pathway was activated by expression of MKK6b(E), MEK1(E), MKK7(D), or MEK5(D), respectively. (A) Reporter gene expression determined as described for Fig. 2; (B) positive control of ERK, JNK, or ERK5/BMK activation. Comparable results were obtained in two experiments.
FIG. 9
FIG. 9
Activation of MEF2A by MKK6b(E) is mediated by p38 in 293 cells. (A) In vitro phosphorylation of MEF2A by different p38 isoforms. Glutathione S-transferase–ATF2(1–109) protein, which is efficiently phosphorylated by all p38 isoforms, was included as a positive control. (B) 293 cells pretreated for 30 min with different doses of SB203580 were cotransfected with pCMV-β-gal, pG5ElbLuc, MKK6b(E), GAL-MEF2A(wt), MKK6b(E), or empty vector as described for Fig. 2. MKK6b(E)-induced reporter gene expression was inhibited dose dependently by SB203580. Comparable results were obtained in two experiments. (C) 293 cells were cotransfected with pCMV-β-gal (0.1 μg), pG5ElbLuc (0.1 μg), MKK6b(E) (0.1 μg), GAL-MEF2A(wt) (0.1 μg), and p38, p38β, p38γ, or p38δ in the amounts of DNA indicated. Empty vector pcDNA3 was used to normalize the total DNA to 1.6 μg per transfection. (D) 293 cells were cotransfected with pCMV-β-gal, pG5ElbLuc, MKK6b(E), GAL-MEF2A(wt), and kinase-inactive mutants of four p38 isoforms as indicated.
FIG. 10
FIG. 10
Compositions of MEF2 dimers in 293 cells. Nuclear protein extracts from 293 cells were used in EMSA. Incubation of extracts and probe with specific MEF2A, MEF2B, MEF2C, and MEF2D immune sera or an anti-p50 antiserum as a nonspecific antibody (Ab) was carried out to test for the presence of MEF2 isoforms in DNA-protein complex. Complex 2 appears to represent MEF2A-MEF2D heterodimers; complex 1 contains MEF2C-MEF2D and MEF2D-MEF2D dimers.
FIG. 11
FIG. 11
Effect of overexpression of MEF2 homo- or heterodimers on MEF2-dependent reporter gene expression in 293 cells. 293 cells were cotransfected with pCMV-β-gal (0.1 μg), pJluc (0.1 μg), MKK6b(E) (0.1 μg), and MEF2A, MEF2B, MEF2C, or MEF2D. Empty vector pcDNA3 was used to normalize the total DNA to 1.5 μg per transfection (A). 293 cells were cotransfected with pCMV-β-gal (0.1 μg), pJluc (0.1 μg), MKK6b(E) (0.1 μg), and different combinations of MEF2 isoforms with different DNA ratio as indicated (B) and with MEF2A or its mutants together with MEF2D at a 1:1 ratio (C). Reporter gene pJluc expression is shown.
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