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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

Deletion of CSD alters MSY3 nuclear/cytoplasmatic trafficking in myogenic cells. a Graphic illustration of the deleted MSY3 protein domains tested in the mutagenesis assay and distribution of the Akt phosphorylation consensus sites in the MSY3 protein. b Western blot with anti-FLAG Ab of protein extract of C2C12 cells transiently transfected with a FLAG-tagged MSY3 protein (FLAGMSY3) and FLAG-tagged MSY3 protein deleted of the cold shock (FLAGΔCSD), splicing alternative (FLAGΔSHORT), and the carboxy (FLAGΔRP-CD) domains. c FLAG expression tested by IF with an anti-FLAG Ab in C2C12 myoblasts transiently transfected with FLAGMSY3, FLAGΔCSD, FLAGΔSHORT, and FLAGΔRP-CD proteins. The white arrow indicates a nuclear FLAG signal of the FLAGΔRP-CD protein. Scale bar = 100 μm. d N/C ratios of FLAG signal in C2C12 transfected with FLAGMSY3, FLAGΔCSD, FLAGΔSHORT, FLAGΔRP-CD, FLAGSer126Ala, and FLAGSer328Ala proteins, evaluated as a mean ± DS for 60–80 cells from a total of three independent experiments. Quantification was performed as described in Methods. e Akt phosphorylation consensus sites in the MSY3 protein
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Fig6: Deletion of CSD alters MSY3 nuclear/cytoplasmatic trafficking in myogenic cells. a Graphic illustration of the deleted MSY3 protein domains tested in the mutagenesis assay and distribution of the Akt phosphorylation consensus sites in the MSY3 protein. b Western blot with anti-FLAG Ab of protein extract of C2C12 cells transiently transfected with a FLAG-tagged MSY3 protein (FLAGMSY3) and FLAG-tagged MSY3 protein deleted of the cold shock (FLAGΔCSD), splicing alternative (FLAGΔSHORT), and the carboxy (FLAGΔRP-CD) domains. c FLAG expression tested by IF with an anti-FLAG Ab in C2C12 myoblasts transiently transfected with FLAGMSY3, FLAGΔCSD, FLAGΔSHORT, and FLAGΔRP-CD proteins. The white arrow indicates a nuclear FLAG signal of the FLAGΔRP-CD protein. Scale bar = 100 μm. d N/C ratios of FLAG signal in C2C12 transfected with FLAGMSY3, FLAGΔCSD, FLAGΔSHORT, FLAGΔRP-CD, FLAGSer126Ala, and FLAGSer328Ala proteins, evaluated as a mean ± DS for 60–80 cells from a total of three independent experiments. Quantification was performed as described in Methods. e Akt phosphorylation consensus sites in the MSY3 protein

Mentions: In order to map the putative Akt phosphorylation sites, we generated FLAG-tagged WT and mutant MSY3 constructs, carrying deletions of the domains showed in Fig. 6a: CSD, alternative splicing domain (SHORT) and the carboxy (RP-CD) domain, which includes the arginine and proline-rich conserved domain and the C-terminal proline-rich domain. We introduced these constructs by transient transfection in C2C12 myoblasts and analyzed the migration of their protein products. FLAGMSY3 WT protein migrated as double bands, as expected. Treatment with AP reduced phosphorylation levels of the FLAGMSY3 WT protein similar to the endogenous MSY3 protein (Additional file 1: Figure S6). Among the mutations assayed, only the FLAG∆CSD showed a strong reduction of the upper band, indicating that the phosphorylation site responsible for MSY3 phosphorylation in C2C12 cells is located in the CSD domain (Fig. 6b). It is known that the Akt phosphorylation site in YB-1 and MSY3 in non-muscle cells was identified in the CSD. This region is highly conserved among the cold-shock protein family and is responsible for the nucleus-cytoplasmic shuttling [29, 36, 62]. In our model, we also identified the CSD as the domain responsible for the nucleus-cytoplasm trafficking. While the FLAG∆CSD protein localized exclusively in the nucleus in myoblasts, the FLAGMSY3 and the other mutated forms localized predominantly in the cytoplasm (Fig. 6c). An accurate analysis of IF (white arrow in Fig. 6c) and distribution of phosphorylated and dephosphorylated MSY3 in the nuclear (N) and (C) cytoplasmic compartments (Fig. 6d), also revealed that the deletion of the C-terminal-domain (RP-CD) induced a partial accumulation of the FLAGΔRP-CD in the nuclei. This modest nuclear migration of FLAGΔRP-CD protein suggests that the MSY3 RP-CD domain is at least partially responsible for MSY3 nucleus-cytoplasmic trafficking. For instance, it could contain another phosphorylation target, as indicated by the residual presence of the slower (phosphorylated) band in the CSD mutant (Fig. 6b).Fig. 6


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)

Deletion of CSD alters MSY3 nuclear/cytoplasmatic trafficking in myogenic cells. a Graphic illustration of the deleted MSY3 protein domains tested in the mutagenesis assay and distribution of the Akt phosphorylation consensus sites in the MSY3 protein. b Western blot with anti-FLAG Ab of protein extract of C2C12 cells transiently transfected with a FLAG-tagged MSY3 protein (FLAGMSY3) and FLAG-tagged MSY3 protein deleted of the cold shock (FLAGΔCSD), splicing alternative (FLAGΔSHORT), and the carboxy (FLAGΔRP-CD) domains. c FLAG expression tested by IF with an anti-FLAG Ab in C2C12 myoblasts transiently transfected with FLAGMSY3, FLAGΔCSD, FLAGΔSHORT, and FLAGΔRP-CD proteins. The white arrow indicates a nuclear FLAG signal of the FLAGΔRP-CD protein. Scale bar = 100 μm. d N/C ratios of FLAG signal in C2C12 transfected with FLAGMSY3, FLAGΔCSD, FLAGΔSHORT, FLAGΔRP-CD, FLAGSer126Ala, and FLAGSer328Ala proteins, evaluated as a mean ± DS for 60–80 cells from a total of three independent experiments. Quantification was performed as described in Methods. e Akt phosphorylation consensus sites in the MSY3 protein
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491233&req=5

Fig6: Deletion of CSD alters MSY3 nuclear/cytoplasmatic trafficking in myogenic cells. a Graphic illustration of the deleted MSY3 protein domains tested in the mutagenesis assay and distribution of the Akt phosphorylation consensus sites in the MSY3 protein. b Western blot with anti-FLAG Ab of protein extract of C2C12 cells transiently transfected with a FLAG-tagged MSY3 protein (FLAGMSY3) and FLAG-tagged MSY3 protein deleted of the cold shock (FLAGΔCSD), splicing alternative (FLAGΔSHORT), and the carboxy (FLAGΔRP-CD) domains. c FLAG expression tested by IF with an anti-FLAG Ab in C2C12 myoblasts transiently transfected with FLAGMSY3, FLAGΔCSD, FLAGΔSHORT, and FLAGΔRP-CD proteins. The white arrow indicates a nuclear FLAG signal of the FLAGΔRP-CD protein. Scale bar = 100 μm. d N/C ratios of FLAG signal in C2C12 transfected with FLAGMSY3, FLAGΔCSD, FLAGΔSHORT, FLAGΔRP-CD, FLAGSer126Ala, and FLAGSer328Ala proteins, evaluated as a mean ± DS for 60–80 cells from a total of three independent experiments. Quantification was performed as described in Methods. e Akt phosphorylation consensus sites in the MSY3 protein
Mentions: In order to map the putative Akt phosphorylation sites, we generated FLAG-tagged WT and mutant MSY3 constructs, carrying deletions of the domains showed in Fig. 6a: CSD, alternative splicing domain (SHORT) and the carboxy (RP-CD) domain, which includes the arginine and proline-rich conserved domain and the C-terminal proline-rich domain. We introduced these constructs by transient transfection in C2C12 myoblasts and analyzed the migration of their protein products. FLAGMSY3 WT protein migrated as double bands, as expected. Treatment with AP reduced phosphorylation levels of the FLAGMSY3 WT protein similar to the endogenous MSY3 protein (Additional file 1: Figure S6). Among the mutations assayed, only the FLAG∆CSD showed a strong reduction of the upper band, indicating that the phosphorylation site responsible for MSY3 phosphorylation in C2C12 cells is located in the CSD domain (Fig. 6b). It is known that the Akt phosphorylation site in YB-1 and MSY3 in non-muscle cells was identified in the CSD. This region is highly conserved among the cold-shock protein family and is responsible for the nucleus-cytoplasmic shuttling [29, 36, 62]. In our model, we also identified the CSD as the domain responsible for the nucleus-cytoplasm trafficking. While the FLAG∆CSD protein localized exclusively in the nucleus in myoblasts, the FLAGMSY3 and the other mutated forms localized predominantly in the cytoplasm (Fig. 6c). An accurate analysis of IF (white arrow in Fig. 6c) and distribution of phosphorylated and dephosphorylated MSY3 in the nuclear (N) and (C) cytoplasmic compartments (Fig. 6d), also revealed that the deletion of the C-terminal-domain (RP-CD) induced a partial accumulation of the FLAGΔRP-CD in the nuclei. This modest nuclear migration of FLAGΔRP-CD protein suggests that the MSY3 RP-CD domain is at least partially responsible for MSY3 nucleus-cytoplasmic trafficking. For instance, it could contain another phosphorylation target, as indicated by the residual presence of the slower (phosphorylated) band in the CSD mutant (Fig. 6b).Fig. 6

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