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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

MSY3 is phosphorylated by Akt in C2C12. a Left: multi-band migration pattern is shown in the Western blot with protein extracts of C2C12 myoblasts (GM) and myotubes (DM) treated with 1 unit of Antarctic Phosphatase (AP), probed with anti-MSY3 Ab (ZONAB). α-tubulin was used as normalizer. Right: densitometry calculations for faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. b Left: Western blot probed with anti-MSY3 Ab (ZONAB) and anti-myogenin Ab with protein extracts of C2C12 myoblasts (GM) treated with two different (20 μM and 30 μM) concentrations of LY294002 (LY). α-tubulin was used as normalizer. Right: densitometry calculations for faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. c Left: immunoblot with an anti-Akt total, an anti-MSY3 and anti-myogenin Abs in protein extracts of C2C12 grown in DM for 36 h, transfected with scrambled RNAi oligos (Ki) and RNA interfering oligos against of Akt 1 and Akt 2 (Akti), demonstrates that Akt activity is responsible for MSY3 phosphorylation. Right: densitometry calculations for Akt, myogenin, and MSY3 faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. We estimated a reduction of Akt of 75 %. Figure displays results representative from three independent RNAi experiments. Akt P <0.01; MSY3 dephosph P <0.05; MSY3 phosph P <0.01; myogenin P <0.05 by Student’s t test
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Fig2: MSY3 is phosphorylated by Akt in C2C12. a Left: multi-band migration pattern is shown in the Western blot with protein extracts of C2C12 myoblasts (GM) and myotubes (DM) treated with 1 unit of Antarctic Phosphatase (AP), probed with anti-MSY3 Ab (ZONAB). α-tubulin was used as normalizer. Right: densitometry calculations for faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. b Left: Western blot probed with anti-MSY3 Ab (ZONAB) and anti-myogenin Ab with protein extracts of C2C12 myoblasts (GM) treated with two different (20 μM and 30 μM) concentrations of LY294002 (LY). α-tubulin was used as normalizer. Right: densitometry calculations for faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. c Left: immunoblot with an anti-Akt total, an anti-MSY3 and anti-myogenin Abs in protein extracts of C2C12 grown in DM for 36 h, transfected with scrambled RNAi oligos (Ki) and RNA interfering oligos against of Akt 1 and Akt 2 (Akti), demonstrates that Akt activity is responsible for MSY3 phosphorylation. Right: densitometry calculations for Akt, myogenin, and MSY3 faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. We estimated a reduction of Akt of 75 %. Figure displays results representative from three independent RNAi experiments. Akt P <0.01; MSY3 dephosph P <0.05; MSY3 phosph P <0.01; myogenin P <0.05 by Student’s t test

Mentions: In order to determine the mechanism responsible for MSY3 phosphorylation in skeletal muscle, we analyzed MSY3 protein expression and migration patterns in C2C12 myogenic cells by Western blot analysis. MSY3 protein (long isoform), detected with the polyclonal antibody ZONAB, is expressed at the same levels in proliferating (GM) and differentiated for 48 h (DM) C2C12 cells (Fig. 2a). We observed that MSY3 migrates as multiple forms in both conditions, GM and DM. Also, densitometry measurements of untreated extracts show that the dephosphorylated form of MSY3 is more prominent in myoblasts (GM) than in myotubes (DM) (Fig. 2a). After in vitro treatment of C2C12 extracts with AP, MSY3 protein shifted to a single faster migrating form. This indicates that both phosphorylated and the dephosphorylated forms of MSY3 are present in C2C12 myogenic cells (Fig. 2a). We then tested the effects of LY294002 (LY), an inhibitor of the PI3K/Akt pathway that also inhibits myogenin expression and myogenic differentiation [52, 55], on MSY3 phosphorylation in vivo (Additional file 1: Figure S4A, B). Treatment with LY reduced MSY3 basal phosphorylation in C2C12 myoblasts (Fig. 2b). However, we observed that the highest concentration of LY tested (30 μM) resulted in a pronounced reduction of MSY3 expression. We observed no changes in the MSY3 protein phosphorylated/dephosphorylated ratio when C2C12 cells were treated with other kinase inhibitors, involved in muscle differentiation or hypertrophy such as p38 mitogen-activated protein kinase (p38MAPK) inhibitor (SB203580), mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059), or calcium/calmodulin-dependent protein kinase (CAMKII) inhibitors (KN-62; KN-93) (Additional file 1: Figure S4A, B, D, E). Inhibition of the IGF1/PI3K/Akt pathway upstream of Akt by treatment with the PI3K pathway inhibitor, wortmannin, showed a reduction in MSY3 phosphorylation (Additional file 1: Figure S4C). In addition, treatment with rapamycin, the mTOR inhibitor that blocks the PI3K pathways downstream of Akt, did not alter the ratio of phosphorylated/dephosphorylated MSY3 protein (Additional file 1: Figure S4C), although both wortmannin and rapamycin inhibit myogenic differentiation (Additional file 1: Figure S4A). The observation that only the PI3K inhibitor altered the phosphorylated/dephosphorylated MSY3 levels argues for a role for Akt in phosphorylating the Y-box protein MSY3 in myogenic cells. This conclusion is in agreement with the previously documented role of Akt in phosphorylating and regulating functions of Y-box proteins in other cell types [29, 35, 38].Fig. 2


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)

MSY3 is phosphorylated by Akt in C2C12. a Left: multi-band migration pattern is shown in the Western blot with protein extracts of C2C12 myoblasts (GM) and myotubes (DM) treated with 1 unit of Antarctic Phosphatase (AP), probed with anti-MSY3 Ab (ZONAB). α-tubulin was used as normalizer. Right: densitometry calculations for faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. b Left: Western blot probed with anti-MSY3 Ab (ZONAB) and anti-myogenin Ab with protein extracts of C2C12 myoblasts (GM) treated with two different (20 μM and 30 μM) concentrations of LY294002 (LY). α-tubulin was used as normalizer. Right: densitometry calculations for faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. c Left: immunoblot with an anti-Akt total, an anti-MSY3 and anti-myogenin Abs in protein extracts of C2C12 grown in DM for 36 h, transfected with scrambled RNAi oligos (Ki) and RNA interfering oligos against of Akt 1 and Akt 2 (Akti), demonstrates that Akt activity is responsible for MSY3 phosphorylation. Right: densitometry calculations for Akt, myogenin, and MSY3 faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. We estimated a reduction of Akt of 75 %. Figure displays results representative from three independent RNAi experiments. Akt P <0.01; MSY3 dephosph P <0.05; MSY3 phosph P <0.01; myogenin P <0.05 by Student’s t test
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: MSY3 is phosphorylated by Akt in C2C12. a Left: multi-band migration pattern is shown in the Western blot with protein extracts of C2C12 myoblasts (GM) and myotubes (DM) treated with 1 unit of Antarctic Phosphatase (AP), probed with anti-MSY3 Ab (ZONAB). α-tubulin was used as normalizer. Right: densitometry calculations for faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. b Left: Western blot probed with anti-MSY3 Ab (ZONAB) and anti-myogenin Ab with protein extracts of C2C12 myoblasts (GM) treated with two different (20 μM and 30 μM) concentrations of LY294002 (LY). α-tubulin was used as normalizer. Right: densitometry calculations for faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. c Left: immunoblot with an anti-Akt total, an anti-MSY3 and anti-myogenin Abs in protein extracts of C2C12 grown in DM for 36 h, transfected with scrambled RNAi oligos (Ki) and RNA interfering oligos against of Akt 1 and Akt 2 (Akti), demonstrates that Akt activity is responsible for MSY3 phosphorylation. Right: densitometry calculations for Akt, myogenin, and MSY3 faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. We estimated a reduction of Akt of 75 %. Figure displays results representative from three independent RNAi experiments. Akt P <0.01; MSY3 dephosph P <0.05; MSY3 phosph P <0.01; myogenin P <0.05 by Student’s t test
Mentions: In order to determine the mechanism responsible for MSY3 phosphorylation in skeletal muscle, we analyzed MSY3 protein expression and migration patterns in C2C12 myogenic cells by Western blot analysis. MSY3 protein (long isoform), detected with the polyclonal antibody ZONAB, is expressed at the same levels in proliferating (GM) and differentiated for 48 h (DM) C2C12 cells (Fig. 2a). We observed that MSY3 migrates as multiple forms in both conditions, GM and DM. Also, densitometry measurements of untreated extracts show that the dephosphorylated form of MSY3 is more prominent in myoblasts (GM) than in myotubes (DM) (Fig. 2a). After in vitro treatment of C2C12 extracts with AP, MSY3 protein shifted to a single faster migrating form. This indicates that both phosphorylated and the dephosphorylated forms of MSY3 are present in C2C12 myogenic cells (Fig. 2a). We then tested the effects of LY294002 (LY), an inhibitor of the PI3K/Akt pathway that also inhibits myogenin expression and myogenic differentiation [52, 55], on MSY3 phosphorylation in vivo (Additional file 1: Figure S4A, B). Treatment with LY reduced MSY3 basal phosphorylation in C2C12 myoblasts (Fig. 2b). However, we observed that the highest concentration of LY tested (30 μM) resulted in a pronounced reduction of MSY3 expression. We observed no changes in the MSY3 protein phosphorylated/dephosphorylated ratio when C2C12 cells were treated with other kinase inhibitors, involved in muscle differentiation or hypertrophy such as p38 mitogen-activated protein kinase (p38MAPK) inhibitor (SB203580), mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059), or calcium/calmodulin-dependent protein kinase (CAMKII) inhibitors (KN-62; KN-93) (Additional file 1: Figure S4A, B, D, E). Inhibition of the IGF1/PI3K/Akt pathway upstream of Akt by treatment with the PI3K pathway inhibitor, wortmannin, showed a reduction in MSY3 phosphorylation (Additional file 1: Figure S4C). In addition, treatment with rapamycin, the mTOR inhibitor that blocks the PI3K pathways downstream of Akt, did not alter the ratio of phosphorylated/dephosphorylated MSY3 protein (Additional file 1: Figure S4C), although both wortmannin and rapamycin inhibit myogenic differentiation (Additional file 1: Figure S4A). The observation that only the PI3K inhibitor altered the phosphorylated/dephosphorylated MSY3 levels argues for a role for Akt in phosphorylating the Y-box protein MSY3 in myogenic cells. This conclusion is in agreement with the previously documented role of Akt in phosphorylating and regulating functions of Y-box proteins in other cell types [29, 35, 38].Fig. 2

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