Phosphorylation and activation of MEF2 transcription factors by p38 MAP kinases.
The transcription factor MEF2C is phosphorylated and activated by the p38α MAP kinase in response to inflammation (8
), thereby providing a link between the p38 pathway and a member of the MEF2 group of transcription factors. However, several distinct members of the p38 MAP kinases have been identified, including p38α, -β, -γ, and -δ (reviewed in references 1
). The abilities of these different p38 MAP kinases to phosphorylate MEF2 proteins in vitro and stimulate their transcriptional activation properties in vivo were tested (Fig. ). Firstly, the phosphorylation of a GST fusion protein containing the transcriptional activation domain (TAD) of MEF2A and MEF2C by the different p38 MAP kinases was tested (Fig. B). These fusion proteins contain the minimal TAD, which in the case of MEF2C, has been shown to be sufficient for maximal phosphorylation-inducible transcriptional activation (8
). This region contains the major p38α phosphorylation sites T293
in MEF2C (8
) and the corresponding amino acid residues T304
in MEF2A (Fig. A). An independent study has confirmed that these two residues in MEF2A are the major p38α phosphorylation sites (39
in the MEF2A isoform were used in that study). GST-MEF2A and GST-MEF2C are both phosphorylated by p38α and p38β2
(Fig. B, lanes 1 and 2). However, in comparison, neither p38γ nor p38δ can efficiently phosphorylate GST-MEF2A and GST-MEF2C (Fig. B, lanes 3 and 4). Moreover, GST-MEF2A and GST-MEF2C appear to be phosphorylated selectively by a subgroup of p38 MAP kinases but not by other classes of MAP kinases, as ERK and JNK family members only poorly phosphorylate these substrates in vitro (data not shown).
FIG. 1 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 (more ...)
In order to determine whether this differential phosphorylation by p38 MAP kinases reflects a difference in their response to these pathways in vivo, GAL4 fusion proteins containing the TADs of MEF2A and MEF2C were constructed (Fig. A) and were tested for their ability to activate a GAL4-driven luciferase reporter gene. The activation of distinct MAP kinase cascades was achieved by cotransfecting a constitutively activated form of MKK6, together with either p38α, p38β2
, p38γ, or p38δ. In the cell line used in this study (COS-7), the expression of MKK6 alone had little effect on the activity of the GAL4 fusion proteins (Fig. C), although the activation of MEF2A can be detected by using MKK6 alone in other cell lines (e.g., 293 cells [39
]). MEF2A and MEF2C were efficiently activated by p38α and p38β2
but not by p38γ and p38δ (Fig. C). To test the ability of the ERK2 and JNK2 pathways to activate MEF2A, similar experiments were performed with the cotransfection of their respective upstream kinases (MAP kinase [or Erk] kinase [MEK] and MKK7β, respectively). However, in comparison to p38β2
(~50-fold induction), both ERK2 and JNK2 had little effect on the activity of MEF2A (Fig. D). Treatment with MEK/ERK2 and MKK7β/JNK2 is sufficient to activate other nuclear substrates (see Fig. ) (37
FIG. 6 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 (more ...)
The response of MEF2A to the activation of endogenous MAP kinase pathways following stimulation by mitogens and cytokines in the absence of overexpressed pathway components was subsequently investigated. Initially, HeLa cells were stimulated with IL-1. This treatment results in the stimulation of GAL4-MEF2A via the p38 pathway, as the JNK pathway inhibitor (DN-MKK4) has little effect, while the p38 pathway inhibitor (SB202190) almost completely blocks this stimulation (Fig. E). To directly compare the effect of stimulating individual MAP kinase pathways on MEF2A activation, COS-7, CHO, and HeLa cells were transfected with GAL4-MEF2A and treated with either EGF (to activate the ERK pathway in COS-7 cells [37
]) or IL-1 (to activate the JNK pathway in CHO cells [34
] or the p38 pathway in HeLa cells [Fig. E]). While the treatment of COS-7 cells with EGF and that of CHO cells with IL-1 lead to the activation of GAL4-Elk-1 via the ERK and JNK pathways, neither of these treatments activates GAL4-MEF2A (Fig. F). However, in comparison, the stimulation of the endogenous p38 pathway by IL-1 treatment of HeLa cells results in a comparable activation of GAL4–Elk-1 and GAL4-MEF2A (Fig. F).
Taken together, these results demonstrate that like MEF2C, MEF2A is phosphorylated and activated by p38α. This is in agreement with the findings of two independent studies (18a
). Moreover, while both MEF2A and MEF2C are also targeted by p38β2
, neither appears to be a target of p38γ and p38δ. Thus, these MEF2 family proteins appear to be substrates of a subset of p38 MAP kinases in vitro and in vivo.
Requirement of the MEF2 D-domain for phosphorylation by p38α and p38β2 MAP kinases in vitro and in vivo.
We have previously identified within the transcription factor Elk-1 a kinase docking domain, the D-domain, that contains specificity determinants and therefore enhances the phosphorylation of Elk-1 by ERK and JNK MAP kinases. However, this domain does not appear to affect the phosphorylation of Elk-1 by the p38 MAP kinases (37
). Inspection of the sequence of MEF2A and MEF2C indicated the presence of a motif which exhibits limited similarity with the Elk-1 D-domain (see Fig. A). To investigate whether the efficiency of MEF2A and MEF2C phosphorylation by p38α and p38β2
is enhanced by the presence of a kinase docking domain, the GST fusion proteins GST-MEF2AΔD and GST-MEF2CΔD, which contain the TAD and associated phosphoacceptor motifs but lack the region which resembles the Elk-1 D-domain (Fig. A and B), were created. These GST fusion proteins were tested as in vitro MAP kinase substrates, in comparison to analogous proteins which contain the putative MAP kinase docking site (Fig. A and B). The activity of the kinases towards GST–Elk-1 was initially standardized, and equivalent activities were used in the kinase assays. The kinetics of phosphorylation of GST-MEF2A (Fig. A, lanes 1 to 4) and GST-MEF2C (Fig. B, lanes 1 to 4) by p38α, p38β2
, and p38γ were virtually indistinguishable. In contrast, the phosphorylation of GST-MEF2AΔD (Fig. A, lanes 5 to 8) and GST-MEF2CΔD (Fig. B, lanes 5 to 8) by p38α and p38β2
was greatly reduced over the same time period. However, the phosphorylation of these two substrates by p38γ was virtually indistinguishable from that when the putative docking site was present (compare the graphs and lanes 1 to 4 and 5 to 8 in the bottom panels of Fig. A and B).
FIG. 5 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 (more ...)
FIG. 2 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 (more ...)
To examine the requirement of the D-domain for the activation of MEF2 transcription factors by MAP kinases in vivo, GAL4 fusion proteins to MEF2A and MEF2C which either contain or lack this domain were constructed and tested for their ability to activate a GAL4-driven luciferase reporter gene in response to MAP kinase activation (Fig. C and D). In the absence of cotransfected MKK6(E) and p38β2 (Fig. C and D), all the fusion proteins activated the reporter gene to low levels. However, upon cotransfection of MKK6(E) together with p38β2 and either GAL4-MEF2A (Fig. C) or GAL4-MEF2C (Fig. D), greatly enhanced transcriptional activation was observed. In contrast, the deletion of the D-domain in both MEF2A and MEF2C caused a large reduction in the GAL4-MEF2A- and GAL4-MEF2C-mediated transcriptional activation (Fig. C and D). Western blotting indicates that the deletion of the D-domain had little effect on the levels of the GAL4 fusion proteins (Fig. C and D, bottom panels). Virtually identical results were observed for the activation of these chimeric proteins by p38α (data not shown).
These data therefore indicate that both MEF2A and MEF2C contain a domain, the D-domain, which is distinct from their phosphoacceptor motifs and is required for efficient phosphorylation in vitro and stimulation of their transcriptional activation potential in vivo by the p38α and p38β2 MAP kinases.
Identification of important residues in the D-domain required for efficient phosphorylation by p38 MAP kinases.
The D-domain plays an important role in enhancing phosphorylation and stimulation of MEF2A- and MEF2C-mediated transcriptional activation by the p38α and p38β2 MAP kinases. In order to investigate the contribution of residues within this domain of MEF2A towards this function, pairs of amino acids conserved between MEF2A and MEF2C (see Fig. A) were mutated to alanine residues (Fig. A). Such mutations should preserve any structural motifs which are present but remove side chains which are available for intermolecular interactions. The mutant proteins were examined as substrates for p38 MAP kinases (Fig. B). All three of the mutants tested (M1, M2, and M3) exhibited a reduction in the efficiency of their phosphorylation by p38α and p38β2 (Fig. B). In contrast, none of the mutations within the D-domain resulted in a decrease in the efficiency of MEF2A phosphorylation by p38γ (Fig. B). GAL4 fusion proteins were also constructed with each of the mutant MEF2A derivatives to investigate their activation by p38α and p38β2 in vivo. In comparison to the wild-type (WT) protein, the M1, M2, and M3 mutant GAL4-MEF2A fusion proteins exhibit reduced activation of transcription in response to MKK6(E) and p38α (Fig. C) or MKK6(E) and p38β2 (Fig. D) in vivo. Western blotting indicates that all the mutant proteins were expressed to equivalent levels in the presence and absence of activated upstream cascades (Fig. E). Collectively, these data demonstrate that the conserved residues within the MEF2A D-domain play key roles in determining its efficient phosphorylation and transcriptional activation by p38α and p38β2. Furthermore, the critical residues for activation by p38α and p38β2 appear to be indistinguishable.
FIG. 8 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 (more ...)
FIG. 3 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 (more ...)
In order to demonstrate the importance of the D-domain in targeting p38 MAP kinases to MEF2A in the context of a full-length protein rather than truncated fusion proteins, we created a mutant version of MEF2A with two point mutations in the D-domain (MEF2A[M3]; Fig. A), which reduce the phosphorylation and activation of truncated MEF2A derivatives by p38α and p38β2 (Fig. ). Full-length MEF2A was initially tested as a substrate for the different p38 isoforms (data not shown) with results virtually identical to those obtained with truncated GST fusion proteins (Fig. B). We then compared the ability of p38β2 to phosphorylate MEF2A(WT) and the mutant MEF2A(M3) in vitro. Phosphorylation of MEF2A(WT) was observed by the presence of a slower migrating band (Fig. B, lanes 4 and 5) and the incorporation of 32P-labelled ATP (Fig. C, lanes 2 to 4). In contrast, over the same time period, much-reduced MEF2A(M3) phosphorylation was detected by these assays (Fig. B, lanes 6 to 10, and C, lanes 5 to 8). Thus, the D-domain plays an important role in directing the efficiency of MEF2A phosphorylation by p38β2 in the context of the full-length protein.
FIG. 4 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 (more ...) The MEF2A D-domain acts as a binding motif for the p38 MAP kinases.
The ability of ERK2 to phosphorylate Elk-1 and that of JNK to phosphorylate c-Jun correlate with their ability to bind to these substrates via a docking domain (13
). However, while stable interactions are readily detectable in these cases, such stable interactions are not observed between MAP kinases and other substrates (e.g., JNK and Elk-1; 38
). Similarly, we were unable to demonstrate a physical interaction between MEF2A and p38 MAP kinases under a variety of experimental conditions (data not shown), although others have previously demonstrated such an interaction (8
). We therefore adopted a peptide competition assay to investigate the binding of the kinases to MEF2A. This approach has previously been used to allow the comparison of ERK and JNK binding to Elk-1 under identical experimental conditions and relies on the fact that peptides which bind to the kinase will act as competitors for the binding of the kinase to docking sites in the transcription factor targets (38
). Peptides were synthesized which correspond to MEF2A amino acids 266 to 283 (encompassing the D-domain) (MEFD[WT]) and the same region with two amino acid substitutions (MEFD[M2]) (Fig. A). Increasing amounts of these peptides were included in kinase assays to compete for binding of the p38 MAP kinases to MEF2A via the D-domain (Fig. B). The MEFD(WT) peptide acted as an inhibitor of p38α- and p38β2
-mediated phosphorylation of MEF2A in a concentration-dependent manner (Fig. B, lanes 1 to 4). However, little effect was seen on the efficiency of phosphorylation by p38γ except at the highest concentrations of the wild-type and mutant peptides used (Fig. B, lanes 1 to 4, bottom panel). In contrast, the mutant MEFD(M2) peptide did not act as a competitor (Fig. B, lanes 5 to 7). This is consistent with the observation that the M2 mutant GST- and GAL-MEF2A fusion proteins are poor targets for p38α and p38β2
(Fig. ). Peptides corresponding to the kinase docking domains from Elk-1 (ElkD) and SAP-1 (SAPD) (Fig. A) were also tested (Fig. B). The SAPD peptide also acted as an efficient inhibitor (Fig. B, lane 9), whereas the ElkD peptide did not act as an inhibitor of the phosphorylation of MEF2A by p38α and p38β2
(Fig. B, lane 10). These results are consistent with the observation that the SAP-1 D-domain acts as a docking site for both the p38α and p38β2
MAP kinases (5a
), whereas the Elk-1 D-domain does not act as a p38 MAP kinase docking site (38
In order to investigate the specificity of action of the MEF2A D-domain peptide as a p38α and p38β2 MAP kinase inhibitor, the ability of the D-domain peptide to inhibit MEF2A phosphorylation by p38β2 was compared to its inhibitory properties on other MAP kinase-substrate combinations (Fig. C). Of the three kinases tested, the D-domain peptide acted as a more efficient competitor of p38β2 than ERK and JNK. This is most apparent at the highest concentrations of peptide used (Fig. C, lanes 4, 8, and 12), although the inhibition of all three MAP kinases can be observed to some degree. This might reflect some conservation of the peptide binding site on the MAP kinases, as these proteins are all related and the relative order of peptide inhibition efficiency is consistent with the observation that the highest similarity is between the p38 and ERK MAP kinases.
These data therefore demonstrate that the D-domain of MEF2A preferentially acts as a binding site for p38α and p38β2 MAP kinases in vitro.
The MAP kinase targeting domain of MEF2A is sufficient to confer p38 responsiveness to Elk-1 and c-Jun.
In order to investigate whether the MEF2A D-domain can act in a heterologous context to allow p38 targeting and hence transduce signals via the p38 pathway to different substrates, chimeric proteins were created. In these proteins, the p38 targeting domain of MEF2A was fused to the minimal TAD of Elk-1 (which responds to both the ERK and JNK pathways) and c-Jun (which responds to the JNK pathway) and either GST or the GAL4-DNA-binding domain (Fig. A and E). These minimal TADs lack their natural kinase binding domains. Firstly, the efficiencies of phosphorylation of the GST fusion proteins by different MAP kinases were compared (Fig. B and F). GST–Elk-1ΔD, which lacks its own kinase targeting domain, represents a relatively poor MAP kinase substrate (Fig. B, lane 1). However in comparison, GST–MEF2A–Elk-1 was efficiently phosphorylated by p38α and p38β2 (Fig. B, lane 2). In contrast, little enhancement of Elk-1 phosphorylation by ERK2, JNK2, and p38γ was observed by the inclusion of the MAP kinase docking site from MEF2A (Fig. B, lane 2). Moreover, the fusion of the MEF2A D-domain to c-Jun in the GST-MEF2A-cJun chimera converts c-Jun into a better substrate for p38α and p38β2 but not for p38γ (Fig. F, lane 2). c-Jun can usually be efficiently phosphorylated only by JNK and not p38 MAP kinases (Fig. F, lane 1), and the propensity of c-Jun as a JNK substrate is severely reduced in the presence of the MEF2A MAP kinase docking site (Fig. F, lane 2, bottom panel).
In order to demonstrate that these changes in substrate specificity towards MAP kinases, which are dictated by the MEF2A D-domain, reflect a difference in the ability of these domains to bind to the MAP kinases, peptide competition experiments were performed with the chimeric proteins MEF2A–Elk-1 (Fig. C) and MEF2A-cJun (Fig. G). The MEFD(WT) and, to a lesser extent, SAPD peptides acted as competitors of phosphorylation of these chimeras by p38α and p38β2 (Fig. C and G, lanes 2 and 4), whereas the MEFD(M2) and ElkD peptides were ineffectual competitors (Fig. C and G, lanes 3 and 5). These results are essentially the same as those observed with wild-type GST-MEF2A (Fig. B), thereby demonstrating that the MEF2A D-domain acts in a similar manner to permit p38α and p38β2 binding in a heterologous context.
The analogous GAL4 fusion proteins were subsequently analyzed for their activation by p38α (data not shown) and p38β2
(Fig. D and H) in vivo. All these GAL4 derivatives are expressed to similar levels (Fig. D and H, bottom panels). GAL4–Elk-1ΔD, which lacks the kinase docking domain, is moderately activated by the ERK and p38 pathways (Fig. D) (37
). In comparison, GAL4–MEF2A–Elk-1 exhibits greatly enhanced transcriptional activation in response to MKK6/p38β2
-mediated stimulation in vivo (Fig. D), whereas the response to MEK/ERK-mediated stimulation barely changes (Fig. D). Similarly, c-Jun is usually activated by the JNK pathway but only poorly by the p38β2
pathway in vivo. However, reciprocal effects are seen with the MEF2A-cJun chimera, which is efficiently activated by the p38β2
pathway but in comparison is poorly activated by the JNK pathway (Fig. H).
Taken together, the results clearly demonstrate that the D-domain of MEF2A is sufficient to confer responsiveness to the p38α and p38β2 signalling pathways in heterologous contexts both in vitro and in vivo.
The p38-binding domain of MEF2A directs phosphorylation of key phosphoacceptor motifs.
The D-domain of MEF2A is sufficient to permit the targeting of p38α and p38β2
MAP kinases to heterologous substrates. In order to demonstrate that physiologically relevant residues are targeted for phosphorylation by the D-domain, we investigated the phosphorylation of Ser383 in MEF2A–Elk-1 chimeras, which has been shown to be one of the key residues which must be phosphorylated to trigger the DNA binding and transcriptional activation properties of Elk-1 (reviewed in reference 33
). The phosphorylation of Ser383 was assessed by Western blotting with a phospho–Elk-1(Ser383) antibody.
Firstly, GST–MEF2A–Elk-1 was phosphorylated by p38β2 in vitro. In comparison to GST–Elk-1ΔD, which lacks a p38 docking site, GST–MEF2A–Elk-1 was efficiently phosphorylated on Ser383 (Fig. A). In order to analyze whether the same effect could be observed in vivo, the phosphorylation of GAL4–MEF2A–Elk-1 was compared to the phosphorylation of GAL4–Elk-1 in the presence of cotransfected MKK6/p38β2 (Fig. B). The overall phosphorylation of Elk-1 was greater in the presence of the D-domain from MEF2A (Fig. B; compare lanes 2 and 4, bottom panel). Moreover, the phosphorylation of Ser383 was greatly enhanced in the GAL4–MEF2A–Elk-1 chimera (Fig. B; compare lanes 2 and 4, top panel).
FIG. 7 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 (more ...)
Furthermore, in the MEF2A-cJun chimeras analyzed in Fig. , there are only two potential phosphoacceptor motifs in the c-Jun moiety which correspond to the physiologically relevant sites. Together these results therefore demonstrate that in addition to enhancing the overall efficiency of substrate phosphorylation by p38α and p38β2, the MEF2A D-domain also directs the phosphorylation of physiologically relevant sites in vitro and in vivo.
The kinase docking domain determines the specificity of MAP kinases towards MEF2A.
The kinase docking domain of MEF2A is sufficient to change the specificity of heterologous proteins as substrates for phosphorylation and activation by different MAP kinases. Reciprocal constructs were made in which either known or putative kinase docking domains from other transcription factors were fused to the transcription activation domain of MEF2A (Fig. A and C). Firstly, the homologous region of MEF2B was substituted for the kinase docking domain of MEF2A (Fig. A), and the resulting chimeric protein was tested as a substrate for p38 MAP kinases. In contrast to MEF2A, MEF2B appears not to be phosphorylated by p38 MAP kinases (Fig. B; compare lanes 1 and 3). Moreover, the putative kinase docking domain in MEF2B is unable to permit the efficient phosphorylation of MEF2A in the GST-MEF2B-MEF2A chimera (Fig. B, lane 2). Thus, the amino acid sequence differences in this region of MEF2B inactivate its ability to act as a kinase binding motif.
The MAP kinase docking domains from c-Jun (for JNK MAP kinases) (4
), SAP-1 (for ERK, p38α, and p38β2
MAP kinases) (5a
), and Elk-1 (for JNK and ERK MAP kinases) (37
) were fused with the transcriptional activation domain of MEF2A (Fig. C) and tested as substrates for different MAP kinases (Fig. D). MEF2A is phosphorylated efficiently by p38α and p38β2
but is a poor substrate for the ERK and JNK MAP kinases (Fig. D, lane 4, and Fig. ). However, in the absence of the D-domain, MEF2A is a poor substrate for all of these MAP kinases (Fig. D, lane 5, and Fig. ). Significantly, the introduction of the c-Jun δ-domain converts MEF2A into a good JNK substrate (Fig. D, lane 1). Similarly, the ElkD-MEF2A chimera is a good JNK substrate (Fig. D, lane 3). In contrast, the fusion of the SAP-1 D-domain to MEF2A restores its ability to act as a substrate for p38α and p38β2
(Fig. D, lane 2). None of the chimeric proteins are good ERK2 substrates (Fig. D, top panel), indicating that the docking site alone is insufficient to permit the targeting of ERK MAP kinases (see Discussion).
Together these results demonstrate that the docking domains from several different transcription factors can direct the targeting of specific subsets of stress-activated MAP kinases to MEF2A. Thus, MAP kinase docking sites appear to be common, conserved elements in transcription factors which can act as independent domains and contribute significantly to the specificity of action of the diverse MAP kinase cascades.