Eyal Bengal

A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein.

The ubiquitin proteolytic pathway is a major system for selective protein degradation in eukaryotic cells. One of the first steps in the degradation of a protein via this pathway involves selective modification of ϵ‐NH2 groups of internal lysine residues by ubiquitination. To date, this amino group has been the only known target for ubiquitination. Here we report that the N‐terminal residue of MyoD is sufficient and necessary for promotion of conjugation and subsequent degradation of the protein. Substitution of all lysine residues in the protein did not affect significantly its conjugation and degradation either in vivoor in vitro. In cells, degradation of the lysine‐less protein is inhibited by the proteasome inhibitors MG132 and lactacystin. Inhibition is accompanied by accumulation of high molecular mass ubiquitinated forms of the modified MyoD. In striking contrast, wild‐type MyoD, in which all the internal Lys residues have been retained but the N‐terminus has been extended by fusion of a short peptide, is stable both in vivo and in vitro. In a cell‐free system, ATP and multiple ubiquitination are essential for degradation of the lysine‐less protein. Specific chemical modifications have yielded similar results. Selective blocking of the α‐NH2 group of wild‐type protein renders it stable, while modification of the internal Lys residues with preservation of the free N‐terminal group left the protein susceptible to degradation. Our data suggest that conjugation of MyoD occurs via a novel modification involving attachment of ubiquitin to the N‐terminal residue. The polyubiquitin chain is then synthesized on an internal Lys residue of the linearly attached first ubiquitin moiety.

The p38 MAPK signaling pathway: a major regulator of skeletal muscle development.

Skeletal muscle development is regulated by extracellular growth factors that transmit largely unknown signals into the cell affecting the muscle-transcription program. One intracellular signaling pathway activated during the differentiation of myogenic cell lines is p38 mitogen-activated protein kinase (MAPK). As a result of modifying the activity of p38 in myoblasts, the pathway proved essential for the expression of muscle-specific genes. P38 affects the activities of transcription factors from the MyoD and MEF2 families and participates in the remodeling of chromatin at specific muscle-regulatory regions. P38 cooperates with the myogenic transcription factors in the activation of a subset of late-transcribed genes, hence contributing to the temporal expression of genes during differentiation. Recent developmental studies with mouse and Xenopus embryos, substantiated and further extended the essential role of p38 in myogenesis. Evidence exists supporting the crucial role for p38 signaling in activating MEF2 transcription factors during somite development in mice. In Xenopus, p38 signaling was shown to be needed for the early expression of Myf5 and for the expression of several muscle structural genes. The emerging data indicate that p38 participates in several stages of the myogenic program.

Mitogen-activated Protein Kinase Pathway Is Involved in the Differentiation of Muscle Cells

The differentiation of muscle cells is controlled by the MyoD family of transcription factors. This family is regulated by extracellular growth factors that transmit largely unknown signals into the cells. Here we show that the activity of extracellular signal-regulated protein kinase (ERK), a kinase that is part of the mitogen-activated protein kinase (MAPK) cascade, is low in myoblasts and is induced with the onset of terminal differentiation of C2 cells. ERK activity is also induced in fibroblasts that were modified to express MyoD, but not in the parental fibroblast cells. Thus, ERK induction is an intrinsic property of muscle cells. A specific MAPK kinase inhibitor (PD098059) that was added to C2 cells partially inhibited the fusion of myoblasts to multinucleated myotubes without affecting the expression of muscle-specific markers. This inhibitor blocked the induction of MyoD expression that normally takes place during terminal differentiation. Two lines of evidence suggest that the MAPK cascade induces the activity of MyoD: 1) the expression of constitutively activated forms of MEK1 or Raf1 enhanced the transcriptional activity of MyoD in 10T1/2 fibroblasts; and 2) the addition of PD098059 to fibroblast cells expressing a conditional MyoD-estrogen fusion protein significantly inhibited the expression of MyoD-responsive genes. Our results indicate that the MAPK pathway is activated in differentiating muscle cells and that it positively regulates the expression and activity of MyoD protein.

CREB-binding Protein/p300 Activates MyoD by Acetylation

The myogenic protein MyoD requires two nuclear histone acetyltransferases, CREB-binding protein (CBP)/p300 and PCAF, to transactivate muscle promoters. MyoD is acetylated by PCAFin vitro, which seems to increase its affinity for DNA. We here show that MyoD is constitutively acetylated in muscle cells.In vitro, MyoD is acetylated both by CBP/p300 and by PCAF on two lysines located at the boundary of the DNA binding domain. MyoD acetylation by CBP/p300 (as well as by PCAF) increases its activity on a muscle-specific promoter, as assessed by microinjection experiments. MyoD mutants that cannot be acetylated in vitro are not activated in the functional assay. Our results provide direct evidence that MyoD acetylation functionally activates the protein and show that both PCAF and CBP/p300 are candidate enzymes for MyoD acetylationin vivo.

Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

ABSTRACT MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

Phosphoinositide 3-Kinase Induces the Transcriptional Activity of MEF2 Proteins during Muscle Differentiation

The activity of phosphoinositide 3-kinase (PI3-K) is essential for the differentiation of skeletal muscle cells by largely unknown mechanisms. Here we show that inhibition of PI3-K activity by the pharmacological agent LY294002 affects early processes of myoblast differentiation including the transcriptional activation of myogenin. Previous studies indicated that transcription of myogenin was dependent on MyoD and MEF2 proteins. We find that expression of a dominant negative form of PI3-K or growth in the presence of LY294002 inhibits cellular activity of MEF2 but not of MyoD. Evidence reveals that whereas MEF2 transcriptional activity is inhibited, its DNA binding activity remains unaffected. Recent studies demonstrated that phosphorylation by p38 mitogen-activated protein kinase (MAPK) induced transcriptional activity of MEF2 proteins. We show that the phosphorylation of MEF2 occurring during muscle differentiation is prevented if the activity of PI3-K is inhibited. However, our results also indicate that p38 MAPK is not affected by PI3-K in muscle cells. Nevertheless, p38 MAPK can substitute for PI3-K in the induction of MEF2 and muscle transcription. Together, these findings indicate that PI3-K affects skeletal muscle differentiation by inducing phosphorylation and transcriptional activity of MEF2 proteins in a parallel but distinct route from p38 MAPK.

Interaction between Acetylated MyoD and the Bromodomain of CBP and/or p300

ABSTRACT Acetylation is emerging as a posttranslational modification of nuclear proteins that is essential to the regulation of transcription and that modifies transcription factor affinity for binding sites on DNA, stability, and/or nuclear localization. Here, we present both in vitro and in vivo evidence that acetylation increases the affinity of myogenic factor MyoD for acetyltransferases CBP and p300. In myogenic cells, the fraction of endogenous MyoD that is acetylated was found associated with CBP or p300. In vitro, the interaction between MyoD and CBP was more resistant to high salt concentrations and was detected with lower doses of MyoD when MyoD was acetylated. Interestingly, an analysis of CBP mutants revealed that the interaction with acetylated MyoD involves the bromodomain of CBP. In live cells, MyoD mutants that cannot be acetylated did not associate with CBP or p300 and were strongly impaired in their ability to cooperate with CBP for transcriptional activation of a muscle creatine kinase-luciferase construct. Taken together, our data suggest a new mechanism for activation of protein function by acetylation and demonstrate for the first time an acetylation-dependent interaction between the bromodomain of CBP and a nonhistone protein.

Inhibition of Myoblast Differentiation by Tumor Necrosis Factor α Is Mediated by c-Jun N-terminal Kinase 1 and Leukemia Inhibitory Factor

The proinflammatory cytokine, TNFα plays a major role in muscle wasting occurring in chronic diseases and muscular dystrophies. Among its other functions, TNFα perturbs muscle regeneration by preventing satellite cell differentiation. In the present study, the role of c-Jun N-terminal kinase (JNK), a mediator of TNFα, was investigated in differentiating myoblast cell lines. Addition of TNFα to C2 myoblasts induced immediate and delayed phases of JNK activity. The delayed phase is associated with myoblast proliferation. Inhibition of JNK activity prevented proliferation and restored differentiation to TNFα-treated myoblasts. Studies with cell lines expressing MyoD:ER chimera and lacking JNK1 or JNK2 genes indicate that JNK1 activity mediates the effects of TNFα on myoblast proliferation and differentiation. TNFα does not induce proliferation or inhibit differentiation of JNK1-null myoblasts. However, differentiation of JNK1-null myoblasts is inhibited when they are grown in conditioned medium derived from cell lines affected by TNFα. We investigated the induced synthesis of several candidate growth factors and cytokines following treatment with TNFα. Expression of IL-6 and leukemia inhibitory factor (LIF) was induced by TNFα in wild-type and JNK2-null myoblasts. However, LIF expression was not induced by TNFα in JNK1-null myoblasts. Addition of LIF to the growth medium of JNK1-null myoblasts prevented their differentiation. Moreover, LIF-neutralizing antibodies added to the medium of C2 myoblasts prevented inhibition of differentiation mediated by TNFα. Hence, TNFα promotes myoblast proliferation through JNK1 and prevents myoblast differentiation through JNK1-mediated secretion of LIF.

Induction of Terminal Differentiation by the c-Jun Dimerization Protein JDP2 in C2 Myoblasts and Rhabdomyosarcoma Cells

Muscle cell differentiation is a result of a complex interplay between transcription factors and cell signaling proteins. Proliferating myoblasts must exit from the cell cycle prior to their differentiation. The muscle regulatory factor and myocyte enhancer factor-2 protein families play a major role in promoting muscle cell differentiation. Conversely, members of the AP-1 family of transcription factors that promote cell proliferation antagonize muscle cell differentiation. Here we tested the role of the c-Jun dimerization protein JDP2 in muscle cell differentiation. Endogenous expression of JDP2 was induced in both C2C12 myoblast and rhabdomyosarcoma (RD) cells programmed to differentiate. Ectopic expression of JDP2 in C2C12 myoblast cells inhibited cell cycle progression and induced spontaneous muscle cell differentiation. Likewise, constitutive expression of JDP2 in RD cells reduced their tumorigenic characteristics and restored their ability to differentiate into myotubes. JDP2 potentiated and synergized with 12-O-tetradecanoylphorbol-13-acetate to induce muscle cell differentiation of RD cells. In addition, JDP2 induced p38 activity in both C2 and RD cells programmed to differentiate. This is the first demonstration of a single transcription factor that rescues the myogenic program in an otherwise non-differentiating cancer cell line. Our results indicate that the JDP2 protein plays a major role in promoting skeletal muscle differentiation via its involvement in cell cycle arrest and activation of the myogenic program.

p53 protein is activated during muscle differentiation and participates with MyoD in the transcription of muscle creatine kinase gene

The p53 protein is a transcription factor involved in processes of cell growth and differentiation. The muscle creatine kinase (MCK) gene whose transcription is induced during muscle differentiation contains p53-binding sites. In this study we tested the involvement of p53 in the activation of MCK transcription during muscle differentiation of C2 cells. We have shown that the p53 protein is stabilized and its DNA binding and transcriptional activities are induced during muscle differentiation. At the stage of muscle-differentiation, p53 protein can induce the accurate transcription of a minimal p53-dependent MCK reporter gene. Moreover, p53 cooperates with MyoD in the induction of MCK transcription. The expression of a dominant negative p53 protein in muscle cells reduced the expression of endogenous MCK gene. The dominant negative p53 protein abolished the cooperativity of wild type p53 with MyoD. Amino and carboxy terminal residues of MyoD required for the cooperation with p53 in transcription were identified. The cooperativity between the two proteins occurs also at the stage of DNA binding. We suggest that p53 protein is activated during myoblast differentiation and participates with MyoD in the induction of MCK transcription.