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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

Disruption of Akt substrate Ser126 alters MSY3 nuclear/cytoplasmatic trafficking and function in myogenic cells. a Top: FLAG expression in C2C12 myoblasts transiently transfected with FLAG-tagged MSY3 proteins mutated at Ser126 (FLAGSer126Ala) and at Ser 328 (FLAGSer328Ala). Scale bar = 150 μm. Bottom: Western blot with anti-FLAG Ab of protein extracts of C2C12 transiently transfected with FLAG-tagged MSY3 protein (FLAGMSY3), FLAGSer126Ala and FLAGSer328Ala. b Measurements of myogenin expression by qRT-PCR in myrsAkt transfected C2C12 multiclones expressing FLAG MSY3, FLAGΔCSD, and FLAGSer126Ala transfected with myristoylated Akt (Akt) in proliferation medium (GM) or upon different times cultured in differentiation medium (24–72 h DM). GAPDH was used to normalize expression levels
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Fig7: Disruption of Akt substrate Ser126 alters MSY3 nuclear/cytoplasmatic trafficking and function in myogenic cells. a Top: FLAG expression in C2C12 myoblasts transiently transfected with FLAG-tagged MSY3 proteins mutated at Ser126 (FLAGSer126Ala) and at Ser 328 (FLAGSer328Ala). Scale bar = 150 μm. Bottom: Western blot with anti-FLAG Ab of protein extracts of C2C12 transiently transfected with FLAG-tagged MSY3 protein (FLAGMSY3), FLAGSer126Ala and FLAGSer328Ala. b Measurements of myogenin expression by qRT-PCR in myrsAkt transfected C2C12 multiclones expressing FLAG MSY3, FLAGΔCSD, and FLAGSer126Ala transfected with myristoylated Akt (Akt) in proliferation medium (GM) or upon different times cultured in differentiation medium (24–72 h DM). GAPDH was used to normalize expression levels

Mentions: We mutated the Serine 126 and 328 residues to Alanine in order to remove the side chain substrates and thus prevent phosphorylation by Akt. Two mutated MSY-3 constructs (MSY3 Ser126Ala) and (MSY3 Ser328Ala) were constructed and cloned in FLAG tagged vectors and were transiently expressed in C2C12 myoblasts. Nuclear accumulation of the anti-FLAG signal was observed only in myoblasts transfected with the MSY3-S126A but not myoblasts transfected with the MSY3-S328A construct. This result demonstrates that Ser126 and not Ser328 is the phosphorylation substrate responsible for the nuclear trafficking of MSY3 (Fig. 7a). Also, only Ser126A migrated as a single band, similar to the CSD mutation, providing further evidence that the substrate responsible for MSY3 phosphorylation in C2C12 is Ser126.Fig. 7


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)

Disruption of Akt substrate Ser126 alters MSY3 nuclear/cytoplasmatic trafficking and function in myogenic cells. a Top: FLAG expression in C2C12 myoblasts transiently transfected with FLAG-tagged MSY3 proteins mutated at Ser126 (FLAGSer126Ala) and at Ser 328 (FLAGSer328Ala). Scale bar = 150 μm. Bottom: Western blot with anti-FLAG Ab of protein extracts of C2C12 transiently transfected with FLAG-tagged MSY3 protein (FLAGMSY3), FLAGSer126Ala and FLAGSer328Ala. b Measurements of myogenin expression by qRT-PCR in myrsAkt transfected C2C12 multiclones expressing FLAG MSY3, FLAGΔCSD, and FLAGSer126Ala transfected with myristoylated Akt (Akt) in proliferation medium (GM) or upon different times cultured in differentiation medium (24–72 h DM). GAPDH was used to normalize expression levels
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4491233&req=5

Fig7: Disruption of Akt substrate Ser126 alters MSY3 nuclear/cytoplasmatic trafficking and function in myogenic cells. a Top: FLAG expression in C2C12 myoblasts transiently transfected with FLAG-tagged MSY3 proteins mutated at Ser126 (FLAGSer126Ala) and at Ser 328 (FLAGSer328Ala). Scale bar = 150 μm. Bottom: Western blot with anti-FLAG Ab of protein extracts of C2C12 transiently transfected with FLAG-tagged MSY3 protein (FLAGMSY3), FLAGSer126Ala and FLAGSer328Ala. b Measurements of myogenin expression by qRT-PCR in myrsAkt transfected C2C12 multiclones expressing FLAG MSY3, FLAGΔCSD, and FLAGSer126Ala transfected with myristoylated Akt (Akt) in proliferation medium (GM) or upon different times cultured in differentiation medium (24–72 h DM). GAPDH was used to normalize expression levels
Mentions: We mutated the Serine 126 and 328 residues to Alanine in order to remove the side chain substrates and thus prevent phosphorylation by Akt. Two mutated MSY-3 constructs (MSY3 Ser126Ala) and (MSY3 Ser328Ala) were constructed and cloned in FLAG tagged vectors and were transiently expressed in C2C12 myoblasts. Nuclear accumulation of the anti-FLAG signal was observed only in myoblasts transfected with the MSY3-S126A but not myoblasts transfected with the MSY3-S328A construct. This result demonstrates that Ser126 and not Ser328 is the phosphorylation substrate responsible for the nuclear trafficking of MSY3 (Fig. 7a). Also, only Ser126A migrated as a single band, similar to the CSD mutation, providing further evidence that the substrate responsible for MSY3 phosphorylation in C2C12 is Ser126.Fig. 7

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