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Akt-mediated phosphorylation controls the activity of the Y-box protein MSY3 in skeletal muscle.

De Angelis L, Balasubramanian S, Berghella L - Skelet Muscle (2015)

Bottom Line: This correlated well with the reduction of phosphorylated active Akt.Knocking down Akt expression increased the amount of dephosphorylated MSY3 and reduced myogenin expression and muscle differentiation.These results support the hypothesis that MSY3 phosphorylation by Akt interferes with MSY3 repression of myogenin circuit activity during muscle development.

View Article: PubMed Central - PubMed

Affiliation: DAHFMO, Unit of Histology and Medical Embryology, University La Sapienza, Via Scarpa 16, Rome, 00161 Italy.

ABSTRACT

Background: The Y-box protein MSY3/Csda represses myogenin transcription in skeletal muscle by binding a highly conserved cis-acting DNA element located just upstream of the myogenin minimal promoter (myogHCE). It is not known how this MSY3 activity is controlled in skeletal muscle. In this study, we provide multiple lines of evidence showing that the post-translational phosphorylation of MSY3 by Akt kinase modulates the MSY3 repression of myogenin.

Methods: Skeletal muscle and myogenic C2C12 cells were used to study the effects of MSY3 phosphorylation in vivo and in vitro on its sub-cellular localization and activity, by blocking the IGF1/PI3K/Akt pathway, by Akt depletion and over-expression, and by mutating potential MSY3 phosphorylation sites.

Results: We observed that, as skeletal muscle progressed from perinatal to postnatal and adult developmental stages, MSY3 protein became gradually dephosphorylated and accumulated in the nucleus. This correlated well with the reduction of phosphorylated active Akt. In C2C12 myogenic cells, blocking the IGF1/PI3K/Akt pathway using LY294002 inhibitor reduced MSY3 phosphorylation levels resulting in its accumulation in the nuclei. Knocking down Akt expression increased the amount of dephosphorylated MSY3 and reduced myogenin expression and muscle differentiation. MSY3 phosphorylation by Akt in vitro impaired its binding at the MyogHCE element, while blocking Akt increased MSY3 binding activity. While Akt over-expression rescued myogenin expression in MSY3 overexpressing myogenic cells, ablation of the Akt substrate, (Ser126 located in the MSY3 cold shock domain) promoted MSY3 accumulation in the nucleus and abolished this rescue. Furthermore, forced expression of Akt in adult skeletal muscle induced MSY3 phosphorylation and myogenin derepression.

Conclusions: These results support the hypothesis that MSY3 phosphorylation by Akt interferes with MSY3 repression of myogenin circuit activity during muscle development. This study highlights a previously undescribed Akt-mediated signaling pathway involved in the repression of myogenin expression in myogenic cells and in mature muscle. Given the significance of myogenin regulation in adult muscle, the Akt/MSY3/myogenin regulatory circuit is a potential therapeutic target to counteract muscle degenerative disease.

No MeSH data available.


Related in: MedlinePlus

Akt phosphorylates MSY3 and reduces its binding to myogHCE in vitro. a Phosphorylation of MSY3 by Akt. GST-MSY3 (*) was phosphorylated by Akt, (lanes 7 and 8) but not by GSK3β (11 12). Auto-phosphorylated Akt protein (°) and GSK3β (§) are present. The Comassie gel is shown (lanes 1–4). For each sample, there are two experimental replicates. b Schematic of the myogenic locus. This diagram describes the myogenin promoter (the 130 bp upstream region from the TSS) and the genomic alignments of six vertebrates. The schematic also shows the relative locations of myogHCE element—the region to which MSY3 binds and other well-known motifs (myogHCE-brown, MEF3box-light blue, MEF2box-green, TATA box-purple, and E-box-yellow. MyogHCE is MSY3 binding site [16]; MEF3box is Dach2 binding site [13]; MEF2box is MEF2/HDAC9 binding site [12]. c MSY3 phosphorylation by Akt impairs its binding at the myogenin promoter. Sequences of MyogHCE (MYOwt) and mutant myogHCE (MYOmutL) oligonucleotides used in EMSA are shown. Mutant MYOmutL disrupts the myogHCE sequence, which incudes the MSY3 site. Mobility shift assay (EMSA) of GST-MSY3 binding to EMSA assay of GST and GSTMSY3 fusion protein with myogHCE (MYOwt) and mutant myogHCE (MYOmutL) 32P labeled oligonucleotide in double-strand form without (first three lanes) and with prior treatment by Akt (lanes 4 and 5) and by GSK3β (lanes 6 and 7)
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Fig4: Akt phosphorylates MSY3 and reduces its binding to myogHCE in vitro. a Phosphorylation of MSY3 by Akt. GST-MSY3 (*) was phosphorylated by Akt, (lanes 7 and 8) but not by GSK3β (11 12). Auto-phosphorylated Akt protein (°) and GSK3β (§) are present. The Comassie gel is shown (lanes 1–4). For each sample, there are two experimental replicates. b Schematic of the myogenic locus. This diagram describes the myogenin promoter (the 130 bp upstream region from the TSS) and the genomic alignments of six vertebrates. The schematic also shows the relative locations of myogHCE element—the region to which MSY3 binds and other well-known motifs (myogHCE-brown, MEF3box-light blue, MEF2box-green, TATA box-purple, and E-box-yellow. MyogHCE is MSY3 binding site [16]; MEF3box is Dach2 binding site [13]; MEF2box is MEF2/HDAC9 binding site [12]. c MSY3 phosphorylation by Akt impairs its binding at the myogenin promoter. Sequences of MyogHCE (MYOwt) and mutant myogHCE (MYOmutL) oligonucleotides used in EMSA are shown. Mutant MYOmutL disrupts the myogHCE sequence, which incudes the MSY3 site. Mobility shift assay (EMSA) of GST-MSY3 binding to EMSA assay of GST and GSTMSY3 fusion protein with myogHCE (MYOwt) and mutant myogHCE (MYOmutL) 32P labeled oligonucleotide in double-strand form without (first three lanes) and with prior treatment by Akt (lanes 4 and 5) and by GSK3β (lanes 6 and 7)

Mentions: To determine if MSY3 is a downstream phospho-substrate for Akt in vitro, we constructed a GST-MSY3 fusion construct with GST at the N-terminal end and tested the ability of Akt and GSK3β, another kinase regulated by the PI3K pathway [47], to phosphorylate MSY3, in an in vitro kinase assay (Fig. 4a). We observed that a phospho protein with the same molecular weight as GST-MSY3 (Comassie staining lanes 3–4) is present only when the GST-MSY3 protein (but not GST alone) is incubated with Akt (Fig. 4a lanes 7–8) but not when it is incubated with GSK3β (Fig. 4a, lanes 11–12). This evidence confirms that MSY3 is specifically phosphorylated by Akt in myogenic cells. MSY3 represses myogenin by binding the highly conserved DNA cis-acting element located upstream of the myogenin promoter (myogHCE) (Fig. 4b) [16]. To analyze the functional impact of MSY3 phosphorylation, we tested if the binding of MSY3 to the myogHCE regulative element in vitro is modulated by the Akt phosphorylation of MSY3. The GST-MSY3 protein binds efficiently to the WT myogHCE oligo, (MYOwt) but to a lesser extent to the mutated oligo (MYOmutL) as expected [16] in a gel shift assay (Fig. 4c, lanes 2–3). We observed that when MSY3 is phosphorylated in vitro by Akt, its binding at the myogHCE (MYOwt) element is reduced more than 50 %, while phosphorylation by GSK3β showed no effects on binding (Fig. 4c, lanes 4–7). This result indicates that MSY3 phosphorylation by Akt shows a moderate but specific effect on the DNA binding property of the MSY3 recombinant protein.Fig. 4


Akt-mediated phosphorylation controls the activity of the Y-box protein MSY3 in skeletal muscle.

De Angelis L, Balasubramanian S, Berghella L - Skelet Muscle (2015)

Akt phosphorylates MSY3 and reduces its binding to myogHCE in vitro. a Phosphorylation of MSY3 by Akt. GST-MSY3 (*) was phosphorylated by Akt, (lanes 7 and 8) but not by GSK3β (11 12). Auto-phosphorylated Akt protein (°) and GSK3β (§) are present. The Comassie gel is shown (lanes 1–4). For each sample, there are two experimental replicates. b Schematic of the myogenic locus. This diagram describes the myogenin promoter (the 130 bp upstream region from the TSS) and the genomic alignments of six vertebrates. The schematic also shows the relative locations of myogHCE element—the region to which MSY3 binds and other well-known motifs (myogHCE-brown, MEF3box-light blue, MEF2box-green, TATA box-purple, and E-box-yellow. MyogHCE is MSY3 binding site [16]; MEF3box is Dach2 binding site [13]; MEF2box is MEF2/HDAC9 binding site [12]. c MSY3 phosphorylation by Akt impairs its binding at the myogenin promoter. Sequences of MyogHCE (MYOwt) and mutant myogHCE (MYOmutL) oligonucleotides used in EMSA are shown. Mutant MYOmutL disrupts the myogHCE sequence, which incudes the MSY3 site. Mobility shift assay (EMSA) of GST-MSY3 binding to EMSA assay of GST and GSTMSY3 fusion protein with myogHCE (MYOwt) and mutant myogHCE (MYOmutL) 32P labeled oligonucleotide in double-strand form without (first three lanes) and with prior treatment by Akt (lanes 4 and 5) and by GSK3β (lanes 6 and 7)
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Fig4: Akt phosphorylates MSY3 and reduces its binding to myogHCE in vitro. a Phosphorylation of MSY3 by Akt. GST-MSY3 (*) was phosphorylated by Akt, (lanes 7 and 8) but not by GSK3β (11 12). Auto-phosphorylated Akt protein (°) and GSK3β (§) are present. The Comassie gel is shown (lanes 1–4). For each sample, there are two experimental replicates. b Schematic of the myogenic locus. This diagram describes the myogenin promoter (the 130 bp upstream region from the TSS) and the genomic alignments of six vertebrates. The schematic also shows the relative locations of myogHCE element—the region to which MSY3 binds and other well-known motifs (myogHCE-brown, MEF3box-light blue, MEF2box-green, TATA box-purple, and E-box-yellow. MyogHCE is MSY3 binding site [16]; MEF3box is Dach2 binding site [13]; MEF2box is MEF2/HDAC9 binding site [12]. c MSY3 phosphorylation by Akt impairs its binding at the myogenin promoter. Sequences of MyogHCE (MYOwt) and mutant myogHCE (MYOmutL) oligonucleotides used in EMSA are shown. Mutant MYOmutL disrupts the myogHCE sequence, which incudes the MSY3 site. Mobility shift assay (EMSA) of GST-MSY3 binding to EMSA assay of GST and GSTMSY3 fusion protein with myogHCE (MYOwt) and mutant myogHCE (MYOmutL) 32P labeled oligonucleotide in double-strand form without (first three lanes) and with prior treatment by Akt (lanes 4 and 5) and by GSK3β (lanes 6 and 7)
Mentions: To determine if MSY3 is a downstream phospho-substrate for Akt in vitro, we constructed a GST-MSY3 fusion construct with GST at the N-terminal end and tested the ability of Akt and GSK3β, another kinase regulated by the PI3K pathway [47], to phosphorylate MSY3, in an in vitro kinase assay (Fig. 4a). We observed that a phospho protein with the same molecular weight as GST-MSY3 (Comassie staining lanes 3–4) is present only when the GST-MSY3 protein (but not GST alone) is incubated with Akt (Fig. 4a lanes 7–8) but not when it is incubated with GSK3β (Fig. 4a, lanes 11–12). This evidence confirms that MSY3 is specifically phosphorylated by Akt in myogenic cells. MSY3 represses myogenin by binding the highly conserved DNA cis-acting element located upstream of the myogenin promoter (myogHCE) (Fig. 4b) [16]. To analyze the functional impact of MSY3 phosphorylation, we tested if the binding of MSY3 to the myogHCE regulative element in vitro is modulated by the Akt phosphorylation of MSY3. The GST-MSY3 protein binds efficiently to the WT myogHCE oligo, (MYOwt) but to a lesser extent to the mutated oligo (MYOmutL) as expected [16] in a gel shift assay (Fig. 4c, lanes 2–3). We observed that when MSY3 is phosphorylated in vitro by Akt, its binding at the myogHCE (MYOwt) element is reduced more than 50 %, while phosphorylation by GSK3β showed no effects on binding (Fig. 4c, lanes 4–7). This result indicates that MSY3 phosphorylation by Akt shows a moderate but specific effect on the DNA binding property of the MSY3 recombinant protein.Fig. 4

Bottom Line: This correlated well with the reduction of phosphorylated active Akt.Knocking down Akt expression increased the amount of dephosphorylated MSY3 and reduced myogenin expression and muscle differentiation.These results support the hypothesis that MSY3 phosphorylation by Akt interferes with MSY3 repression of myogenin circuit activity during muscle development.

View Article: PubMed Central - PubMed

Affiliation: DAHFMO, Unit of Histology and Medical Embryology, University La Sapienza, Via Scarpa 16, Rome, 00161 Italy.

ABSTRACT

Background: The Y-box protein MSY3/Csda represses myogenin transcription in skeletal muscle by binding a highly conserved cis-acting DNA element located just upstream of the myogenin minimal promoter (myogHCE). It is not known how this MSY3 activity is controlled in skeletal muscle. In this study, we provide multiple lines of evidence showing that the post-translational phosphorylation of MSY3 by Akt kinase modulates the MSY3 repression of myogenin.

Methods: Skeletal muscle and myogenic C2C12 cells were used to study the effects of MSY3 phosphorylation in vivo and in vitro on its sub-cellular localization and activity, by blocking the IGF1/PI3K/Akt pathway, by Akt depletion and over-expression, and by mutating potential MSY3 phosphorylation sites.

Results: We observed that, as skeletal muscle progressed from perinatal to postnatal and adult developmental stages, MSY3 protein became gradually dephosphorylated and accumulated in the nucleus. This correlated well with the reduction of phosphorylated active Akt. In C2C12 myogenic cells, blocking the IGF1/PI3K/Akt pathway using LY294002 inhibitor reduced MSY3 phosphorylation levels resulting in its accumulation in the nuclei. Knocking down Akt expression increased the amount of dephosphorylated MSY3 and reduced myogenin expression and muscle differentiation. MSY3 phosphorylation by Akt in vitro impaired its binding at the MyogHCE element, while blocking Akt increased MSY3 binding activity. While Akt over-expression rescued myogenin expression in MSY3 overexpressing myogenic cells, ablation of the Akt substrate, (Ser126 located in the MSY3 cold shock domain) promoted MSY3 accumulation in the nucleus and abolished this rescue. Furthermore, forced expression of Akt in adult skeletal muscle induced MSY3 phosphorylation and myogenin derepression.

Conclusions: These results support the hypothesis that MSY3 phosphorylation by Akt interferes with MSY3 repression of myogenin circuit activity during muscle development. This study highlights a previously undescribed Akt-mediated signaling pathway involved in the repression of myogenin expression in myogenic cells and in mature muscle. Given the significance of myogenin regulation in adult muscle, the Akt/MSY3/myogenin regulatory circuit is a potential therapeutic target to counteract muscle degenerative disease.

No MeSH data available.


Related in: MedlinePlus