MEF2 was originally described as a muscle-specific DNA binding protein that recognizes an A/T-rich element within the promoter regions of many muscle-specific genes (46
). Recently, the MEF2 element has been shown to play a role in growth factor- and stress stimulus-induced early gene responses (15
). Thus, the regulation of MEF2-mediated gene expression may be controlled at multiple levels: by tissue specificity and by signal transduction pathways activated by growth or stress stimuli. Tissue-specific expression of certain MEF2 family members and tissue-specific splicing have been shown to play an important role in the control of MEF2-regulated genes (28
). Phosphorylation of MEF2C has also been recently reported to play an important role in regulation of MEF2-dependent gene expression (10
). We now extend our previous observations by showing that MEF2A is regulated by the p38 pathway. We found that in 293 cells, MEF2A forms a heterodimer with MEF2D. Phosphorylation of MEF2A in the MEF2A-MEF2D dimer by p38 enhanced MEF2-dependent gene expression. These data extend the possible regulatory pathways that depend on p38-mediated phosphorylation of members of the MEF2 family of transcription factors.
Our in vitro studies demonstrated that MEF2A can be phosphorylated by p38 at two threonines and two serines. Importantly, however, we found that only the two threonines, which are phosphorylated in stress-activated cells, play a regulatory role in p38-mediated MEF2A activation in 293 cells; differences between in vitro and in vivo phosphorylation patterns were also found in our studies with MEF2C. Although three phosphorylation sites in MEF2C have been identified, cell studies using either CHO or 293 cells revealed that two threonines comprised the essential regulatory sites (10
). Nonetheless, the regulatory phosphorylation sites appear to be cell type dependent because MEF2C was phosphorylated at all three sites in macrophages in response to lipopolysaccharide stimulation (10
). By analogy with MEF2C, it is possible that the other in vitro phosphorylation sites in MEF2A are of importance in cell types other than those examined in this study. How cell-type-specific phosphorylation of MEF2 isoforms occurs remains to be determined. There may be cell-type-specific cofactors involved in the regulation of MEF2 activity. Other explanations may include differences in composition of dimers in different cell types or the possibility that tissue-specific alternatively spliced isoforms are differentially phosphorylated.
Our data support the contention that phosphorylation of one component of the MEF2A-MEF2D heterodimer is sufficient to augment transactivation activity. Since multiple MEF2 family members can be phosphorylated by one or more kinases, it would be interesting to know if there is phosphorylation of both components in vivo and the consequences of such phosphorylation. Moreover, phosphorylation of a component in a dimer can be carried out by different kinases, which can phosphorylate MEF2 family members through different phosphorylation sites. For example, p38 and ERK5 have been shown to regulate MEF2C via different phosphorylation sites (19
). Whether the transactivation activity of MEF2A is regulated by different kinases awaits further investigation. In this regard, Kato et al. (19a
) also examined phosphorylation of four MEF2 isoforms by ERK5 and found that MEF2D is the preferred substrate for ERK5. Therefore, studies to determine if the p38 and ERK5 pathways are integrated in controlling MEF2A-MEF2D activity are needed. Although MEF2 family members can be targeted by MAP kinases of two different groups, within the p38 group, only a single isoform appears to influence MEF2A activity. Thus, while there is complexity at the level of kinases, there also appears to be a high degree of selectivity at the enzyme-substrate level.
Targeted disruption of mef2c
in mice and mef2
provides evidence for a crucial role of MEF2 family transcriptional factors in both skeletal and cardiac muscle development (25
). It has been shown recently that sole activation of the p38 pathway can lead to hypertrophy of cultured cardiomyocytes (43
). Because of the pivotal role of MEF2C protein in cardiac development (26
), it will be of interest to address whether phosphorylation of MEF2 family members by p38 has a role in cardiomyocyte hypertrophy. MEF2 proteins do not function by themselves but rather cooperate with other transcription factors, such as basic helix-loop-helix (bHLH) transcription factors (1
). Although the interaction between bHLH transcriptional factors and MEF2 proteins is mediated by the DNA binding and dimerization domains (1
), the effect of phosphorylation within the MEF2 transactivation domain on the synergistic effect of these transcriptional factors could be important. Since cooperation of myogenic bHLH proteins with members of MEF2 group protein plays an essential role in the establishment of skeletal muscle lineages (31
), it is reasonable to conclude that regulation MEF2 protein activity by p38 may play a role in the process of myocyte differentiation. Indeed, Puri et al. (35a
) observed that activation of the p38 pathway by transient expression of dominant active MKK6 drove C2C12 cell differentiation to myotubes. The investigation of the potential role of the p38 pathway in the initiation of myogenesis is now a priority.
In summary, we have examined the regulatory effect of p38 pathway on four different MEF2 family members. We found that MEF2A and MEF2C can be phosphorylated and regulated by p38 and that regulation occurs when MEF2 is present as a dimer. In the future, it will be of interest to determine tissue- and/or cell-type-specific regulation of these transcription factors as well as to investigate how several MAP kinase pathways are coordinated in regulating MEF2 family member activities. Since MEF2 factors play pivotal roles in differentiation of skeletal and cardiac muscle cells, it will be especially interesting to determine whether MAP kinase signaling affects muscle gene expression through modulation of MEF2 activity.