MEKK1 signaling through p38 leads to transcriptional inactivation of E47 and repression of skeletal myogenesis.

Activation of the Raf kinase signal transduction pathway in skeletal myoblasts causes a complete cessation of myofiber formation and muscle gene expression. The negative impacts of the signaling pathway are realized through downstream activation of mitogen and extracellular kinase (MEK) phosphorylation-dependent events and MEK-independent signal transmission. MEKK1, a kinase that can physically associate with Raf, may contribute to the MEK-independent signaling in response to elevated Raf activity. Myogenic cells overexpressing activated Raf and kinase-defective MEKK1 remain differentiation-defective, suggesting that MEKK1 does not contribute to the inhibitory actions of Raf. However, constitutive activation of MEKK1 dramatically inhibits biochemical and morphological measures of muscle formation. MEKK1 inhibits MyoD-directed transcriptional activity without altering the ability of the protein to form heterodimers with E2A proteins or bind DNA. By contrast, the transcriptional activity of E47, the preferred dimer partner of the myogenic regulatory factors, is severely compromised by MEKK1-initiated signaling. Inhibition of MEK1/2 and JNK1/2 function did not reinstate E47-directed transcription, indicating that these two downstream kinases likely are not involved in the MEKK1-controlled transcriptional block. Inhibition of p38 signaling overcame the negative effects exerted by MEKK1 on the amino terminus of E47. Closer examination indicates that E47 is phosphorylated in vitro by p38, and deletion analysis predicts that the critical amino acid(s) phosphorylated by p38 lie outside of the minimal transcriptional activation domains. Thus, modification of E47 by p38 likely disrupts higher order protein complex formation that is necessary for muscle gene transcription.

tiation in myogenic cells (1)(2)(3)(4). The block to myocyte formation is inclusive of both inhibition of muscle-specific gene transcription and attainment of fusion competency. Activation of MAPK through overexpression of constitutively active mutants of Raf leads to a robust induction of the Raf/MEK/MAPK signaling module that results in suppression of skeletal myogenesis. The negative impact of Raf can often be abrogated by supplementation of culture media with chemical inhibitors to MEK1 (1). Interestingly, myocytes overexpressing Raf CAAX, a membrane-localized Raf protein, remain refractile to treatment with MEK inhibitors as measured by their inability to fuse or activate muscle gene expression (1,5,6). Thus, it is likely that secondary pathways initiated by Raf kinase contribute to the block to myogenesis.
The molecular basis for the Raf-imposed inhibition of myogenesis involves disruption of MEF2 function (4). MEF2, a MADS (MCM1, agamous, deficiens, serum response factor) box transcription factor, acts in concert with the myogenic regulatory factors (MRFs) to promote muscle gene transcription (7,8). Overexpression of activating mutants of Raf leads to sequestration of MEF2 in the cytoplasm of myoblasts (4). Relocalization of MEF2 to the nucleus through transfection of MEF2-encoding plasmids or through prevention of MEK activation leads to myoblast fusion and muscle gene expression in mouse cells. Interestingly, this phenomenon may be unique to various types of skeletal muscle cells. Coexpression of MEF2 in avian myoblasts transduced with activated Raf did not reinstitute the myogenic gene program (9). Moreover, a direct effect of MEK1 on the MRFs may contribute to inhibition of myogenesis. Nuclear localization of MEK1 allows the kinase to directly interact with MyoD and prevent the MRF from directing muscle gene expression (2). Thus, it is probable that multiple nuclear factors are perturbed in their functions by chronic Raf activities that cooperatively act to prevent skeletal myogenesis.
MEK kinase 1 (MEKK1) is a serine/threonine kinase that induces downstream activation of amino-terminal Jun kinase (JNK), and to a lesser extent, ERK1/2, via activation of MEK1 (for a review, see Ref. 10). MEKK1 falls into the same classification as Raf as a mitogen-activated kinase kinase kinase. Sequential phosphorylation and activation of MEKK1/MKK4/ JNK can lead to a plethora of events including apoptosis, cell survival, or proliferation (11)(12)(13)(14)(15)(16)(17)(18)(19). Initiation of JNK signaling in skeletal myocytes appears to represent an impediment to complete differentiation. L6 and quail myoblasts overexpessing activated forms of Rac1, an upstream inducer of MEKK1, synthesize reduced levels of skeletal muscle proteins and form myocytes that are substantially smaller in size and exhibit disorganized sarcomeric arrangements (20,21). In a similar manner, forced expression of an activated form of MKK7, a JNK-activating kinase, results in elevated levels of JNK that are accompanied by a loss of myocyte formation and muscle gene expression (22). Although high JNK activity can be asso-ciated with enhanced programmed cell death, no increase in the numbers of apoptotic muscle cells was noted.
In addition to its ability regulate JNK activity, MEKK1 physically associates with components of the Raf, MEK, and ERK1/2 signaling module (22). By serving as a scaffolding kinase, MEKK1 may collaborate with Raf to prevent muscle formation. Furthermore, an essential role for MEKK1 has been documented in Raf-mediated fibroblast transformation through its ability to modulate NFB activity, a known inhibitor of myogenesis (23,24). Inhibition of MEK/ERK activity in Raf-expressing cells does not prevent NFB activation, but forced expression of kinase-defective MEKK1 does eliminate NFB activation by Raf. Because of the mutual effects of the MEKK1 and Raf signaling modules, we assessed the capacity of MEKK1 to contribute to the Raf-imposed block to skeletal myogenesis. Our results indicate that both MEKK1 and Raf inhibit muscle differentiation but that neither kinase directly shares in the repressive actions of the other. Interestingly, myoblasts expressing MEKK1 do not direct substantial levels of downstream JNK, indicating that MEKK1 inhibition is directed through an alternate pathway. The means by which MEKK1 inhibits myogenesis include increased p38 activity that reduces the functional capabilities of E47, the obligate heterodimer partner of the MRFs. E47 retains its ability to bind to a consensus DNA E-box element but fails to transactivate a multimerized immunoglobulin E-box reporter gene. Deletion analysis reveals that p38 phosphorylates E47 within the amino terminus on a serine residue that lies outside of the transcriptional activation domain. In summary, MEKK1 inhibits skeletal myogenesis independent of the actions of Raf and through a mechanism that implicates increased p38 kinase activity.

Activated Raf Does Not Require MEKK1 for MEK-independent
Signaling-Our previous work demonstrated that Raf CAAX induces substantial levels of ERK1/2 activity that is acquired through a non-MEK mechanism (1). MEKK1 can assemble with components of the Raf/MEK/MAPK signaling module, and low levels of ERK1/2 activity are found upon MEKK1 induction (22). To measure the contribution of MEKK1 signaling to the Raf-imposed block to myogenesis, C3H10T1/2 fibroblasts were transfected with pEM-MyoD, troponin I luciferase (TnI-Luc), and expression plasmids coding for a kinase-defective MEKK1 (pcDNA-MEKK1⌬DN) and activated Raf (CMV-RafCAAX). The Renilla luciferase expression plasmid, pRL-tk, was included as a monitor of transfection efficiency. After 48 h, the cells were lysed, and luciferase activities were measured. The amount of corrected activity directed by MyoD was set to 100%. As expected, MyoD readily activated the complex muscle reporter, TnI-Luc, and this level of activity was inhibited in the presence of Raf CAAX (Fig. 1A). The kinase-inactive MEKK1 protein did not alter MyoD-directed reporter gene activation, nor did the protein reverse the negative effects of activated Raf in this system. In a similar manner, MEKK1⌬DN failed to restore MyoD-directed myofiber formation in the presence of activated Raf (Fig. 1B). Therefore, MEKK1 does not contribute significantly to inhibition of muscle formation in myoblasts overexpressing activated Raf.
MEKK1 Inhibits MyoD-directed Skeletal Myogenesis-Previous work demonstrated the ability of MEKK1 to inhibit C2C12 myofiber formation in vitro (26). Inhibition of JNK signaling in vivo partially restored muscle function in a model of muscular dystrophy (27). To clarify the mechanism by which MEKK1 alters myogenesis, C3H10T1/2 fibroblasts were transiently transfected with expression plasmids coding for MyoD, the constitutively active kinase domain of MEKK1 (MEKK1⌬), and the muscle reporter genes, TnI-Luc or 4Rtk-Luc. Following treatment for 48 h in differentiation-permissive media, the cells were lysed, and reporter gene activities were measured. Parallel plates transfected in a similar manner were fixed for immunocytochemistry or lysed for Western analysis. Overexpression of MEKK1⌬ inhibited activation of the complex muscle promoter reporter by 80% ( Fig. 2A). MEKK1⌬ failed to significantly alter transcription from the multimerized E-box reporter gene, suggesting that signaling through the kinase does not directly affect MyoD function. The complete myogenic program was disrupted; myoblasts overexpressing activated MEKK1⌬ did not fuse into multinucleated myofibers that synthesize myosin heavy chain (MyHC) (Fig. 2B). The inability to undergo myogenic conversion was not a consequence of low MyoD protein levels. Western blot analysis using an antibody specific for MyoD demonstrated the presence of abundant amounts of the transcription factor irrespective of MEKK1⌬ expression (Fig. 2C).
MEKK1 Inhibits Myogenesis Independent of JNK and ERK1/2 Activation-In numerous cell types, MEKK1 initiates downstream signaling events that involve activation of ERK1/2 and JNK (10). Both ERK1/2 and JNK activation are capable of impeding muscle formation (1,2,4,20,27). To define the mechanism through which MEKK1 inhibits myoblast differentiation, C310T1/2 fibroblasts were transiently transfected with expression plasmids encoding MyoD and MEKK1⌬. Subsequent to fixation, cells were treated for 48 h in differentiation medium containing chemical inhibitors to MEK1/2 (PD98059) and JNK (SP600125). Immunostaining for MyHC demonstrates the robust ability of MyoD to initiate myogenic conver-sion (Fig. 3A). Treatment of the cells with either PD98059 or SP600125 did not inhibit MyoD-directed myofiber formation, suggesting that MEK1/2 and JNK are not necessary for muscle formation. Fibroblasts expressing MyoD and MEKK1⌬ were devoid of MyHC immunopositive fibers. Interestingly, treatment of these cells with either 20 M PD98059 or 50 M SP600125 did not restore myofiber formation.
To verify the effective dosages of the inhibitors, skeletal myoblasts were treated with PD98059 and SP600125, and the levels of phospoERK1/2 and phosphoJNK were measured. 23A2RafER myoblasts express an inducible Raf allele that initiates downstream activation of ERK1/2 upon the addition of 4-hydroxytamoxifen to the culture medium (6). Treatment of these myoblasts with 20 M PD98059 efficiently prevented the phosphorylation of ERK1/2 in the presence or absence of Raf signaling (Fig. 3B). C2C12 myoblasts were treated with 2 mM hydrogen peroxide to induce downstream JNK activity, as described previously (29). Western analysis using an antibody specific for phosphoJNK demonstrated the activation of JNK by H 2 O 2 (Fig. 3C). Supplementation of the culture medium with 50 M SP600125 completely abolished JNK activation without altering endogenous levels of the kinase. Thus, the concentrations of PD98059 and SP600125 are sufficient to prevent activation of ERK1/2 and JNK.
E47 Transcriptional Activity Is Inhibited by MEKK1 Signaling-Optimal muscle gene expression initiated by the MRFs is achieved through heterodimer formation with E2A proteins (30). The E2A proteins, E47 and E12, are ubiquitously expressed bHLH transcription factors (31). The ability of MEKK1 to affect E47 function was examined in fibroblasts. In brief, C3H10T1/2 cells were transiently transfected with expression plasmids coding for E47 and MEKK1⌬ and a multimerized immunoglobulin E-box reporter gene (E5-Luc). pRL-tk was included as a monitor of transfection efficiency. As shown in Fig. 4A, E47 readily stimulated transcription from the luciferase reporter gene. Co-expression of MEKK1⌬ with E47 severely reduced the levels of E5-Luc activity. Loss of E47directed transcription was not a product of altered protein levels. Western analysis of cell lysates indicated substantial E47 protein synthesis in the absence and presence of MEKK1 (Fig. 4B).

MEKK1 Inhibits the Transcriptional Activity of E47 Containing Homo-and Heterodimer Complexes Independent of Their
Ability to Bind DNA-To test the hypothesis that MEKK1 inhibits myogenesis by disruption of functional MRF-E47 transcriptional complexes, pECE-MyoDϳE47, an expression plasmid coding for a forced heterodimer of the respective proteins, was employed (32). C3H10T1/2 fibroblasts were transiently transfected with expression plasmids encoding the tethered protein and MEKK1⌬ and the reporter gene, TnI-Luc or 4Rtk-Luc. After 48 h in differentiation media, the cells were lysed, and luciferase activities were measured. Levels of corrected MyoDϳE47 activity were set to 100%. As expected, the forced heterodimer readily stimulated TnI-Luc activity, which was inhibited by co-expression of MEKK1⌬ (Fig. 5A). Interestingly, MEKK1⌬ prevented efficient transactivation of 4Rtk-Luc by MyoDϳE47. Expression of the tethered protein and appropriate localization were confirmed by Western blot and immunocytochemistry. Fibroblast cultures transfected as described above were lysed in 4ϫ SDS-PAGE buffer, and protein from equal cell numbers was electrophoretically separated and transferred to nitrocellulose. Western blot analysis using an antibody-directed E47 demonstrated the presence of a single protein corresponding to MyoDϳE47 irrespective of MEKK1D expression (Fig. 5B).
The ability of E47 homo-and heterodimers to bind DNA was verified by an electrophorectic mobility shift assay. In brief, C2C12 myoblasts were transiently transfected with mammalian expression plasmids coding for E47, MyoDϳE47, and MEKK1⌬. Myofiber nuclear proteins were purified following 48 h in differentiation media. Nuclear proteins were incubated with 32 P-labeled TnI E-box probe, and protein-DNA complexes were separated through nondenaturing polyacrylamide gels. Autoradiography revealed a single MyoDϳE47 complex capable of binding the muscle E-box DNA probe (Fig. 6A). Coincubation of the nuclear extract with anti-hemagglutinin or anti-E47 caused a reduction in the mobility of the DNA binding complex, confirming the identity of the tethered protein. In a similar manner, nuclear extracts isolated from C2C12 myofibers expressing E47 demonstrated the presence of a single complex capable of binding the E5 E-box DNA probe (Fig. 6B).

FIG. 4. MEKK1⌬ inhibits E47-directed transcriptional activity.
A, C3H10T1/2 fibroblasts were transiently transfected with plasmids encoding E47 and MEKK1⌬ and E5-Luc. pRL-tk was included as a control for transfection efficiency. The amount of corrected E5-Luc activity directed by E47 was set to 100. Cells were maintained in DM for 48 h prior to lysis and measurement of luciferase activity. RLU, relative light units. B, plates transfected as described above were lysed, and the amounts of E47 proteins were detected by Western blot using an antibody against the Myc epitope (␣-Myc). Extracts from myoblasts co-expressing E47 and MEKK1⌬ revealed the existence of a single E47 homodimer with efficient DNA binding capabilities.
MEKK1-stimulated p38 Activity Targets the Amino Terminus of E47 to Inhibit Transcription-E47 protein is synthesized, localized to the nucleus, and retains its ability to contact DNA in the presence of sustained MEKK1 signal transduction. However, the protein fails to induce gene transcription. To examine the effects of MEKK1 on inherent E47 transcriptional activation, C3H10T1/2 fibroblasts were transiently transfected with expression plasmids coding for MEKK1⌬ and a fusion protein consisting of the yeast Gal4 DNA binding domain (Gal4DB) and the amino terminus of E47 (amino acids 1-355) and a multimerized Gal4 DNA binding element reporter gene (pG5T-Luc). After 48 h in culture, the cells were lysed, and luciferase activities were measured. The 355 amino-terminal residues contain the two regions necessary for transcriptional activation (33). As predicted, Gal4DB-E47-(1-355) stimulated activity of the luciferase reporter gene (Fig. 7A). Co-expression of the E47-TAD fusion protein with MEKK1⌬ resulted in an approximate 70% reduction in reporter gene expression. Fibroblasts expressing Gal4DB-MyoD-(1-66) efficiently activated the Gal4 DNA binding site reporter gene irrespective of MEKK1⌬ expression, arguing that the effects of MEKK1⌬ on the E47 TAD are not a general phenomenon. Consistent with our prior results, treatment of the transfected cells with a chemical MEK inhibitor (PD98059) or chemical inhibitor of JNK (SP600125) did not restore transcriptional activity to the E47 fusion protein in the presence of MEKK1⌬ (Fig. 7B). Unexpectedly, treatment of cells with SB202190, a p38 kinase inhibitor, completely removed the negative impact of MEKK1⌬ on Gal4DB-E47-(1-355) function. The inhibitory actions of MEKK1 on the E47 fusion protein are specific to p38. Cotransfection of C3H10T1/2 fibroblasts with expression plasmids coding for Gal4DB-E47- (1-355), MEKK1⌬, and a kinase-deficient form of MKK6, an upstream activator of p38, prevented repression of E47 transcriptional activity (Fig. 7C).

DISCUSSION
MEKK1 has been implicated as a negative regulator of skeletal myogenesis through a mechanism that remains to be identified (26). The serine/threonine kinase participates in the MAPK signaling architecture as the initial kinase in the threetiered system (10). Raf kinase shares a similar role in the prototypical MAPK activation scheme (36). Moreover, Raf and MEKK1 physically associate within several cell types, suggesting that these two kinases may modify the actions of one another (22). Previously, our group reported the ability of Raf kinase to inhibit myocyte formation through a MEK-independent mechanism (1,6). We proposed that Raf stimulation of MEKK1 may be responsible for the observed MEK-independent effects. Overexpression of dominant inhibitory forms of MEKK1 in Raf-expressing myoblasts did not relieve the repressive effects imposed by Raf. In a similar manner, kinase-defective Raf failed to reinstate the myogenic program to MEKK1expressing myoblasts. Thus, we conclude that both Raf and MEKK1 inhibit muscle development through distinct, separable mechanisms.
MEKK1 propagates downstream signal transduction through activation of MKK4, MKK6, and MEK1 in several cell types (10). The most common pathways activated by the multifunctional kinase are MKK4 and MEK1, leading to increased JNK and ERK1/2 activity, respectively. Phosphorylation events controlled by ERK1/2 and JNK are known to inhibit myoblast fusion and muscle gene expression (3,4,20,21,27,37). Unexpectedly, inhibition of either ERK1/2 or JNK in MEKK1 myoblasts did not restore myogenic capabilities. The myoblasts remained differentiation-defective as measured by both biochemical and morphological means. The inability to reverse the effects of MEKK1 is not due to the inappropriate use of inhibitors as these chemicals readily prevented the appearance of phosphoJNK and phosphoERK1/2 in myogenic cells. Moreover, overexpression of kinase-defective MEK1 and MKK4 failed to reinstate the differentiation program to MEKK1-expressing muscle cells (data not shown). These results argue that neither MEK/ERK nor MKK4/JNK signals are propagated in response to MEKK1 activity in myogenic cells.
E47, the obligate dimer partner of the MRFs, represents a regulatory target of the MAPK pathways (38). Phosphorylation of E47 by ERK1/2 and JNK in T-cells causes ubiquitination of the protein and degradation through the 26 S proteasome (39,40). In skeletal myoblasts, elevated casein kinase II activity leads to a severe reduction in E47-directed transcriptional activity (41). Thus, E47 modification in response to MEKK1 signaling may contribute to the defective myogenic program. Our data indicate that MEKK1 triggers p38 signaling that directly alters E47 function. The effects are attributed to p38 phosphorylation as E47 is a direct target in vitro and inhibition of the kinase reverses the transcriptional block imposed by MEKK1 in vivo. Moreover, the negative effects of MEKK1 on E47 are imparted on a region of the protein that does not contain either the TAD or the bHLH motif. In vitro kinase assays demonstrate that E47-(271-582), the region containing both the TAD and bHLH motifs, is phosphorylated weakly by p38␤. The low level phosphate incorporation may reflect poor accessibility of the enzyme to the phosphoacceptor site. It is likely that the GST fusion protein exists in vitro as a homodimer through HLH interactions. This conformation may mask the optimal p38 phosphorylation sites. In turn, the residual incorporation found may be due to the phosphorylation of denatured GST- E47 protein molecules. However, it is apparent from the transient transfection experiments that p38 phosphorylation of E47-(271-582) does not disrupt the functional capabilities of the DNA binding motif or the TAD. These data support the existence of a novel mechanism for regulating E47 function in myoblasts that may include inhibiting additional protein:protein interactions that are necessary for optimal myogenesis. MEKK1 signaling through p38 represents a dichotomy of contrasting effects on myogenesis. Several groups have demonstrated that treatment of myogenic cultures with a p38 inhibitor prevents myoblast fusion and muscle gene activation, clearly arguing that the kinase is a requisite for differentiation (42)(43)(44)(45). Recently, Suelves et al. (46) reported that p38 phosphorylation of the MRF4 TAD is inhibitory to muscle gene expression. The authors present evidence for a regulatory pathway whereby phosphorylation and inactivation of MRF4 by p38 lead to differential muscle gene expression. In this manner, subsets of muscle genes are expressed at varying times during development. We propose a similar model involving the phosphorylation of E47 and subsequent differential inactivation of heterodimeric complexes. In our model, the early MRF, MyoD, acts predominantly as a homodimer in muscle cells to regulate the transcription of specific sets of contractile genes. MyoD homodimers are the predominant multiplex in C2C12 myoblasts (28,47). Moreover, the presence of MyoD-MyoD transcriptional units is further supported by the inability of MEKK1⌬ to disrupt MyoD-directed transcription from a minimal muscle E-box reporter (Fig. 2). In the immature myocyte, phosphorylation of E47 by p38 leads to the inactivation of MyoD-E47 heterodimers, thereby preventing precocious differentiation. As myogenesis proceeds, myofibers are formed in response to the p38-induced up-regulation of myogenin gene expression (45). The validity of this model remains to be tested.
In summary, MEKK1 signaling is inhibitory to skeletal myogenesis through a mechanism that is independent of downstream induction of JNK or MEK activities. Overexpression of MEKK1 in myoblasts leads to increased p38 activity that disrupts E47 and MyoDϳE47 function. E47 is an in vitro target of p38 phosphorylation, and modification of the protein likely occurs outside of the bHLH or TAD motifs. Future efforts will focus on the identification of proteins that physically associate with the amino terminus of E47 and impair transcriptional activation.