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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

Forced phosphorylation of MSY3 in skeletal muscle tissue abolishes its binding activity at the myogenin promoter. a Western blot with anti-phospho and anti-total Akt of protein extracts of limb and TA muscle isolated at different developmental stages, fetal (dpc), and post-natal days (pn) and mature 1 month (1 M). b Western blot with anti-MSY3 Ab (ZONAB), anti-phospho-Akt, and anti-myogenin of protein extracts of TA muscle, electroporated with a mock expression plasmid pcDNA3 (mock) and with myristoylated Akt (Akt). Three replicates from three independent electroporations are shown in lanes 1, 2, 3 and lanes 5, 6, 7. Normalizer is α-tubulin. c Chromatin immunoprecipitation (ChIP) analysis for control IgG and MSY3 antibodies used individually to enrich fixed chromatin from TA electroporated with a mock expression plasmid (M) and with myristoylated Akt (Akt). Replicates from three independent electroporations are shown for mock plasmid electroporation (M1, M2, M3) and for myristoylated Akt (Akt1, Akt2, Akt3). qRT-PCR is used to quantify sequences from the myogenin promoter
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Fig8: Forced phosphorylation of MSY3 in skeletal muscle tissue abolishes its binding activity at the myogenin promoter. a Western blot with anti-phospho and anti-total Akt of protein extracts of limb and TA muscle isolated at different developmental stages, fetal (dpc), and post-natal days (pn) and mature 1 month (1 M). b Western blot with anti-MSY3 Ab (ZONAB), anti-phospho-Akt, and anti-myogenin of protein extracts of TA muscle, electroporated with a mock expression plasmid pcDNA3 (mock) and with myristoylated Akt (Akt). Three replicates from three independent electroporations are shown in lanes 1, 2, 3 and lanes 5, 6, 7. Normalizer is α-tubulin. c Chromatin immunoprecipitation (ChIP) analysis for control IgG and MSY3 antibodies used individually to enrich fixed chromatin from TA electroporated with a mock expression plasmid (M) and with myristoylated Akt (Akt). Replicates from three independent electroporations are shown for mock plasmid electroporation (M1, M2, M3) and for myristoylated Akt (Akt1, Akt2, Akt3). qRT-PCR is used to quantify sequences from the myogenin promoter

Mentions: Our next step was to determine if Akt was able to regulate MSY3 phosphorylation and activity in mature skeletal muscle, as observed in myogenic cells. To answer this question, we forced Akt expression in muscle fibers by electroporating a TA muscle of an adult mouse (3 months old, 3MM), with a myristoylated Akt construct (myrsAkt) or a control plasmid (pcDNA3, mock). Phosphorylated Akt is present at very low levels in adult muscle, since it is dephosphorylated as muscle matures (Fig. 8a). When myristoylated, Akt is over-expressed in muscle and phosphorylates MSY3, as demonstrated by the shift in the MSY3 protein migration pattern (Fig. 8b). We also detected a slight increase in myogenin expression under this condition (Fig. 8b).Fig. 8


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)

Forced phosphorylation of MSY3 in skeletal muscle tissue abolishes its binding activity at the myogenin promoter. a Western blot with anti-phospho and anti-total Akt of protein extracts of limb and TA muscle isolated at different developmental stages, fetal (dpc), and post-natal days (pn) and mature 1 month (1 M). b Western blot with anti-MSY3 Ab (ZONAB), anti-phospho-Akt, and anti-myogenin of protein extracts of TA muscle, electroporated with a mock expression plasmid pcDNA3 (mock) and with myristoylated Akt (Akt). Three replicates from three independent electroporations are shown in lanes 1, 2, 3 and lanes 5, 6, 7. Normalizer is α-tubulin. c Chromatin immunoprecipitation (ChIP) analysis for control IgG and MSY3 antibodies used individually to enrich fixed chromatin from TA electroporated with a mock expression plasmid (M) and with myristoylated Akt (Akt). Replicates from three independent electroporations are shown for mock plasmid electroporation (M1, M2, M3) and for myristoylated Akt (Akt1, Akt2, Akt3). qRT-PCR is used to quantify sequences from the myogenin promoter
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4491233&req=5

Fig8: Forced phosphorylation of MSY3 in skeletal muscle tissue abolishes its binding activity at the myogenin promoter. a Western blot with anti-phospho and anti-total Akt of protein extracts of limb and TA muscle isolated at different developmental stages, fetal (dpc), and post-natal days (pn) and mature 1 month (1 M). b Western blot with anti-MSY3 Ab (ZONAB), anti-phospho-Akt, and anti-myogenin of protein extracts of TA muscle, electroporated with a mock expression plasmid pcDNA3 (mock) and with myristoylated Akt (Akt). Three replicates from three independent electroporations are shown in lanes 1, 2, 3 and lanes 5, 6, 7. Normalizer is α-tubulin. c Chromatin immunoprecipitation (ChIP) analysis for control IgG and MSY3 antibodies used individually to enrich fixed chromatin from TA electroporated with a mock expression plasmid (M) and with myristoylated Akt (Akt). Replicates from three independent electroporations are shown for mock plasmid electroporation (M1, M2, M3) and for myristoylated Akt (Akt1, Akt2, Akt3). qRT-PCR is used to quantify sequences from the myogenin promoter
Mentions: Our next step was to determine if Akt was able to regulate MSY3 phosphorylation and activity in mature skeletal muscle, as observed in myogenic cells. To answer this question, we forced Akt expression in muscle fibers by electroporating a TA muscle of an adult mouse (3 months old, 3MM), with a myristoylated Akt construct (myrsAkt) or a control plasmid (pcDNA3, mock). Phosphorylated Akt is present at very low levels in adult muscle, since it is dephosphorylated as muscle matures (Fig. 8a). When myristoylated, Akt is over-expressed in muscle and phosphorylates MSY3, as demonstrated by the shift in the MSY3 protein migration pattern (Fig. 8b). We also detected a slight increase in myogenin expression under this condition (Fig. 8b).Fig. 8

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