Phosphorylation and activation of MEF2A or MEF2C by different p38 MAP kinases. (A) Diagram illustrating the domain structure of full-length MEF2C and truncated MEF2A and MEF2C proteins fused to either GST or the GAL4 DNA binding domain (open boxes). The location of the DNA binding domain (DBD), minimal TAD, and p38α (T293 and T300) and ERK5 (S387) phosphorylation sites in MEF2C are indicated. The black box represents the putative kinase docking domain (D-domain) of MEF2A/C, and the numbers of the N- and C-terminal amino acids in the MEF2A or MEF2C moiety are indicated. The sequences of MEF2A and MEF2C around the major phosphoacceptor motifs for p38α MAP kinase are shown. (B) The phosphorylation of GST-MEF2A, GST-MEF2C, and GST–Elk-310 by the p38 MAP kinase subtypes p38α (lane 1), p38β₂ (lane 2), p38γ (lane 3), and p38δ (lane 4) was examined by a protein kinase assay. The activity of each protein kinase was standardized towards the substrate GST–Elk-310 (bottom panel). Kinase assays were performed for 15 min at 30°C with 5 pmol of GST-MEF2A and GST-MEF2C as substrates. (C) COS-7 cells were cotransfected with either GAL4-MEF2A or GAL4-MEF2C expression vectors, a constitutively activated form of MKK6 (MKK6[E]), the indicated p38 MAP kinases, and a GAL4-driven luciferase reporter plasmid. (D) COS-7 cells were cotransfected with expression vectors encoding GAL4-MEF2A, an overexpressed (MKK7β) or constitutively activated form of MKK (MEK and MKK6), a MAP kinase (ERK2, JNK2, and p38β₂), and a GAL4-driven luciferase reporter plasmid. (E) HeLa cells were cotransfected with vectors encoding GAL4-MEF2A and a GAL4-driven luciferase reporter plasmid. Cells either were left unstimulated or were stimulated with IL-1 for 18 h in the absence or presence of the indicated JNK pathway (cotransfected dominant negative form of MKK4 [DN-MKK4]) or p38 pathway (SB202190) inhibitors. (F) COS-7 (for EGF stimulation of ERKs), CHO (for IL-1 stimulation of JNKs), and HeLa (for IL-1 stimulation of p38s) cells were cotransfected with vectors encoding GAL4-MEF2A or GAL4–Elk-1 and a GAL4-driven luciferase reporter plasmid. The cells either were left unstimulated (white bars) or were stimulated (black bars for Elk-1 and grey bars for MEF2A) with EGF or IL-1 as in panel E. Transfection efficiencies were monitored by using the β-galactosidase expression vector pCH110. The normalized luciferase activities (means ± standard errors; n, 2 or 3) are presented.

Identification of a domain required for efficient p38-mediated activation of MEF2A and MEF2C. (A and B) Requirement of the D-domain for phosphorylation of MEF2A and MEF2C in vitro. Diagrammatic illustrations of truncated MEF2A or MEF2C and MEF2AΔD or MEF2CΔD proteins fused to either GST or the GAL4 DNA binding domain (open boxes) are shown above each gel. The black box represents the putative kinase docking domain (D-domain) of MEF2A, and the numbers of the N- and C-terminal amino acids in MEF2A moiety are shown. (A) Kinetic analysis of GST-MEF2A phosphorylation by p38α, p38β₂, and p38γ in vitro. GST-MEF2A (lanes 1 to 4) and GST-MEF2AΔD (lanes 5 to 8) were phosphorylated by p38α, p38β₂, and p38γ MAP kinases for the times indicated above each lane. Due to the lower levels of phosphorylation by p38γ, the bottom panels were exposed for longer times than the other panels. The results are presented graphically below these panels with data obtained with GST-MEF2A (squares) and GST-MEF2AΔD (circles) as p38 substrates. Data are presented relative to the phosphorylation of GST-MEF2A after 120 min (taken as 100). (B) As described for panel A, except that the GST-MEF2C derivatives were used as substrates. (C and D) The D-domain is essential for efficient p38β₂-inducible transcriptional activation by MEF2A and MEF2C in vivo. (C) COS-7 cells were cotransfected with expression vectors encoding GAL4-MEF2A derivatives, a constitutively activated form of MKK6 (MKK6[E]), p38β₂ MAP kinase, and a GAL4-driven luciferase reporter plasmid. The expression levels of the GAL4 fusion proteins in the unstimulated cells were examined by Western blot analysis with an anti-GAL4 antibody (bottom panel). (D) Assays were carried out as described for panel C, except that GAL4-MEF2C was examined. Transfection efficiencies were monitored by using the β-galactosidase expression vector pCH110. The normalized luciferase activities (means ± standard errors; n = 3) are presented.

Mapping the residues in the D-domain of MEF2A required for targeting by p38α and p38β₂. (A) Amino acid sequences of the WT and D-domain mutants R269A/K270A (M1), L273A/V275A (M2), and I277A/P278A (M3) are shown. Numbers above the sequences represent the N- and C-terminal residues in the D-domain. (B) Kinase assays of WT and mutant GST-MEF2A fusion proteins by p38 MAP kinases were carried out as described in the legend to Fig. 1. The activity of each kinase was standardized relative to the phosphorylation of GST-MEF2A(WT). (C) p38α-inducible transcriptional activation by WT and mutant GAL4-MEF2A fusion proteins. COS-7 cells were cotransfected with expression vectors encoding GAL4 fusions to either WT or D-domain mutant (M1 to M3) MEF2A derivatives, vectors encoding MKK6(E) and p38α, and a GAL4-driven luciferase reporter plasmid. The luciferase activities relative to GAL4-MEF2A-mediated reporter activation in the absence of cotransfected kinases are presented (means ± standard errors; n = 3). (D) Assays were carried out as in panel C, except that vectors encoding p38β₂ were cotransfected where indicated. (E) Expression levels of the GAL4 fusion proteins, in the absence (lanes 1, 3, 5, and 7) and presence (lanes 2, 4, 6, and 8) of cotransfected MKK6(E) and p38α, were examined by Western blotting with an anti-GAL4 antibody.

The D-domain is required for the efficient phosphorylation of full-length MEF2A by p38β₂. (A) Diagrammatic representation of the domain structure of full-length MEF2A. The locations of the MADS-MEF DNA-binding domain (DBD), the MAP kinase docking domain (black box), and the key phosphoacceptor motifs (T304 and T311) in the TAD are shown. The locations of the mutations in MEF2A(M3) are indicated in italics. (B and C) Phosphorylation of MEF2A(WT) and MEF2A(M3) was carried out in vitro with p38β₂. Equal amounts of in vitro-translated WT and mutant MEF2A proteins were phosphorylated by p38β₂ for the indicated times in the absence (B) or presence (C) of ³²P-labelled ATP. Phosphorylated MEF2A was detected by the reduced mobility of the proteins (arrows in panel B) or by the incorporation of ³²P-labelled ATP (C).

The MEF2A D-domain acts as a binding site for the p38α and p38β₂ MAP kinases. (A) The sequences of the competitor peptides corresponding to the MAP kinase docking domains from Elk-1, SAP-1, and MEF2A. Sequences are aligned to give maximal similarities, with identical and highly conserved residues highlighted. Residues altered in the mutant MEF2A peptide (MEFD[M2]) are shown in bold. (B) Phosphorylation of GST-MEF2A (5 pmol) by p38 MAP kinases in the presence of indicated competitor peptides. The peptide competition assay was based on the kinase assays described in the legend to Fig. 1, except that the p38 MAP kinases were preincubated in the absence (lanes 1 and 8) or presence of competitor peptides (a 10- to 1,000-fold excess over MEF2A substrate) at 50 pmol (lanes 2 and 5), 500 pmol (lanes 3 and 6), and 5 nmol (lanes 4, 7, 9, and 10), respectively. Increases in the concentration of added competitor peptides are indicated schematically above each set of lanes. The activity of each kinase was standardized relative to the phosphorylation of GST-MEF2A(WT). (C) Specificity of action of inhibitory peptides and phosphorylation of transcription factor substrates (5 pmol) by MAP kinases (combinations indicated above each panel) in the presence of the MEFD(WT) peptide. Assays were carried out as in panel B in the presence of 0 pmol (lanes 1, 5, and 9), 5 pmol (lanes 2, 6, and 10), 50 pmol (lanes 3, 7, and 11), and 500 pmol (lanes 4, 8, and 12) of the competitor peptide. The quantification of the data is shown graphically below each panel, relative to the phosphorylation of each substrate in the absence of added competitor peptide.

The p38-binding domain of MEF2A (D-domain) functions in a heterologous context. The phosphorylation and activation of GST and GAL4 fusions to MEF2A–Elk-1 (A to D) and MEF2A-cJun (E to H) chimeras by MAP kinases were analyzed. (A and E) Diagrams illustrating fusions of GST to Elk-1ΔD, MEF2A–Elk-1, cJun, and MEF2A-cJun. The numbers of the N- and C-terminal Elk-1/cJun (roman type) and MEF2A (italics) amino acids included in these constructs are indicated above and below each construct, respectively. The MEF2A D-domain and cJun δ-domain are indicated by solid and open boxes, respectively. The Elk-1 and cJun transcriptional activation domains are shown by open and black boxes, respectively. (B and F) Kinase assays of GST fusion proteins (5 pmol of each substrate) for the indicated MAP kinases were carried out for 15 min as described in the legend to Fig. 1. (C and G) Phosphorylation of the GST–MEF2A–Elk-1 and GST-MEF2A-cJun chimeras by MAP kinases was analyzed in the presence of competitor peptides as described in the legend to Fig. 4. Five picomoles of each chimeric protein was used in the reactions. The indicated p38 MAP kinases were preincubated in the absence (lane 1) or presence of 500 pmol of the following peptides: MEF2A WT D-domain (lane 2; MEFD[WT]), MEF2A mutant D-domain peptide (lane 3; MEFD[M2]), SAP-1 D-domain peptide (lane 4; SAPD), and Elk-1 D-domain peptide (lane 5; ElkD). (D and H) COS-7 cells were cotransfected with vectors encoding GAL4–Elk-1ΔD, GAL4–MEF2A–Elk-1, GAL4-cJun, and GAL4-MEF2A-cJun fusions, a GAL4-driven luciferase reporter plasmid, and vectors encoding MAP kinases (ERK2, JNK2, and p38β₂) and an overexpressed (MKK7β) or constitutively activated form (MEKΔN and MKK6[E]) of the upstream MAP kinase kinases. The expression levels of the GAL4 fusion proteins in unstimulated cells were examined by Western blotting with an anti-GAL4 antibody (bottom panel).

The p38-binding domain of MEF2A directs the phosphorylation of the key phosphoacceptor motifs in MEF2A–Elk-1 chimeras. (A) In vitro kinase assays of GST fusion proteins (5 pmol of each substrate) by p38β₂ MAP kinase were carried out for 15 min. Phosphorylation of Ser383 was detected by Western blotting with an anti-phospho–Elk-1(Ser383) antibody. (B) In vivo phosphorylation of MEF2A–Elk-1 chimeras. 293 cells were transfected with vectors encoding GAL–Elk-1 or GAL–MEF2A–Elk-1 and a GAL4-driven luciferase reporter plasmid and, where indicated, p38β₂ and MKK6(E). The phosphorylation of the Elk-1 moiety at Ser383 in cell extracts was detected by Western blotting as in panel A (top panel), and the total levels of each GAL4 fusion protein were detected with an anti-GAL4 antibody (bottom panel). The locations of the bands corresponding to nonphosphorylated and phosphorylated GAL4 fusion proteins are indicated.

The identity of the kinase docking domain determines the specificity of MAP kinases towards MEF2A. (A) Diagrammatic illustration of GST fusions to MEF2B, the MEF2B-MEF2A chimera, and MEF2A. The numbers of the N- and C-terminal MEF2A (italics) and MEF2B (roman type) amino acids are indicated above and below each construct. The p38-binding motifs in MEF2A and MEF2B are indicated by black and grey boxes, respectively, the amino acid sequences of these regions in MEF2A, MEF2B, MEF2C and MEF2D are shown, and identical residues are highlighted. (B) Kinase assays with the indicated GST fusion proteins and p38 MAP kinases were carried out as described in the legend to Fig. 1. (C) Diagram illustrating fusions of GST to MEF2A and MEF2AΔD and the chimeric proteins cJunδ-MEF2A, SAPD-MEF2A, and ElkD-MEF2A. These constructs contain the transcriptional activation domain of MEF2A and the kinase docking domain of cJun (δ), SAP-1 (D), Elk-1 (white box), MEF2A (black box), or no docking site, respectively. The numbers of the N- and C-terminal c-Jun/SAP-1/Elk-1 (italics) and MEF2A (roman type) amino acids are indicated above and below each construct, respectively. (D) Kinase assays with the indicated chimeric GST fusion proteins as substrates for MAP kinases (as indicated on the right) were carried out with 5 pmol of each protein for 15-min reactions as described in the legend to Fig. 1.