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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 progressively dephosphorylated and accumulates in the nuclei in skeletal muscle after birth. a Left: Western blot with anti-MSY3 (ZONAB) protein extracts of limb and TA muscle at different postnatal days (pn). α-tubulin was used as normalizer. Right: densitometry calculations for MSY3 faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. Figure displays results representative from three independent experiments. *P <0.01 by Student’s t test. b Western blot with extracts from 15 dpc embryo limbs treated with Antarctic Phosphatase (AP), 1 unit and 5 units, and probed with anti-MSY3 Ab (ZONAB). c Western blots of nuclear and cytosolic fractions of protein extracts of limb muscle at fetal (15 dpc), postnatal (pn), and mature, 1 month (1 M), stages. Histone H3 was used as normalizer of the nucleic fraction and GAPDH as normalizer of the cytosolic fraction. d Left: expression of MSY3 evaluated by IF with anti-MSY3 Ab, ZONAB, in cross sections of limbs of 2-day-aged pups (2 pn) and TA of 1-month-aged mice (1 M). The images merge MSY3 expression (red), caveolin 3 expression (green), and Hoechst nuclei staining (blue). MSY3 localization in nuclei is labeled in purple. Scale bars = 100 μm. On the right, high magnification images (corresponding to dotted white squares) show staining of few muscle fibers. Scale bar = 40 μm. White arrows indicate sporadic MSY3 nuclear accumulation in 2 pn limb cross sections. Right Graph quantifies MSY3 positive nuclei over the total nuclei in cross sections of limbs of 2-day-aged pups (2 pn) and TA of 1-month-aged mice (1 M)
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Fig1: MSY3 is progressively dephosphorylated and accumulates in the nuclei in skeletal muscle after birth. a Left: Western blot with anti-MSY3 (ZONAB) protein extracts of limb and TA muscle at different postnatal days (pn). α-tubulin was used as normalizer. Right: densitometry calculations for MSY3 faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. Figure displays results representative from three independent experiments. *P <0.01 by Student’s t test. b Western blot with extracts from 15 dpc embryo limbs treated with Antarctic Phosphatase (AP), 1 unit and 5 units, and probed with anti-MSY3 Ab (ZONAB). c Western blots of nuclear and cytosolic fractions of protein extracts of limb muscle at fetal (15 dpc), postnatal (pn), and mature, 1 month (1 M), stages. Histone H3 was used as normalizer of the nucleic fraction and GAPDH as normalizer of the cytosolic fraction. d Left: expression of MSY3 evaluated by IF with anti-MSY3 Ab, ZONAB, in cross sections of limbs of 2-day-aged pups (2 pn) and TA of 1-month-aged mice (1 M). The images merge MSY3 expression (red), caveolin 3 expression (green), and Hoechst nuclei staining (blue). MSY3 localization in nuclei is labeled in purple. Scale bars = 100 μm. On the right, high magnification images (corresponding to dotted white squares) show staining of few muscle fibers. Scale bar = 40 μm. White arrows indicate sporadic MSY3 nuclear accumulation in 2 pn limb cross sections. Right Graph quantifies MSY3 positive nuclei over the total nuclei in cross sections of limbs of 2-day-aged pups (2 pn) and TA of 1-month-aged mice (1 M)

Mentions: We previously demonstrated that MSY3 protein expression progressively increased as muscle matures, from late stages of fetal development (15 dpc) to the mature stages of muscle growth in postnatal life (2 months old, 2MM). A Western blot analysis showed that the migration pattern of MSY3 protein (long isoform) shifts from a multi-band to a single lower molecular weight band in the nuclear extract of leg muscle, between very early stages of postnatal development (8 pn) and later stages (15 pn) [16]. We interpreted this modification as a result of the protein phosphorylation state change. We analyzed the MSY3 protein-banding pattern from 6–15 days postnatal extracts and inferred the time of shift from a multi-band to a single band migration pattern around the ninth day of postnatal life (Fig. 1a). During progression from fetal to adult stages, myogenin expression is severely reduced along the muscle fiber, as described previously [30, 57], and shown in Additional file 1: Figure S1. To investigate whether this change in MSY3 migration pattern is caused by the loss of a phosphate group, we incubated protein extracts from limbs of 16 dpc embryos with Antarctic Phosphatase (AP). Upon treatment with AP, MSY3 protein migrated as a single band compared to the multiple bands observed in the untreated sample (Fig. 1b), suggesting that MSY3 is phosphorylated during muscle development. Analysis of MSY3 immunoprecipitated protein from skeletal muscle extracts of 8 pn and 15 pn mice showed the same migration observed in skeletal muscle (Additional file 1: Figure S2A). Treatment of the immunoprecipitated MSY3 protein with AP affected its migration pattern similar to the input control (non-immunoprecipitated) MSY3 protein (Additional file 1: Figure S2B).Fig. 1


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 progressively dephosphorylated and accumulates in the nuclei in skeletal muscle after birth. a Left: Western blot with anti-MSY3 (ZONAB) protein extracts of limb and TA muscle at different postnatal days (pn). α-tubulin was used as normalizer. Right: densitometry calculations for MSY3 faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. Figure displays results representative from three independent experiments. *P <0.01 by Student’s t test. b Western blot with extracts from 15 dpc embryo limbs treated with Antarctic Phosphatase (AP), 1 unit and 5 units, and probed with anti-MSY3 Ab (ZONAB). c Western blots of nuclear and cytosolic fractions of protein extracts of limb muscle at fetal (15 dpc), postnatal (pn), and mature, 1 month (1 M), stages. Histone H3 was used as normalizer of the nucleic fraction and GAPDH as normalizer of the cytosolic fraction. d Left: expression of MSY3 evaluated by IF with anti-MSY3 Ab, ZONAB, in cross sections of limbs of 2-day-aged pups (2 pn) and TA of 1-month-aged mice (1 M). The images merge MSY3 expression (red), caveolin 3 expression (green), and Hoechst nuclei staining (blue). MSY3 localization in nuclei is labeled in purple. Scale bars = 100 μm. On the right, high magnification images (corresponding to dotted white squares) show staining of few muscle fibers. Scale bar = 40 μm. White arrows indicate sporadic MSY3 nuclear accumulation in 2 pn limb cross sections. Right Graph quantifies MSY3 positive nuclei over the total nuclei in cross sections of limbs of 2-day-aged pups (2 pn) and TA of 1-month-aged mice (1 M)
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: MSY3 is progressively dephosphorylated and accumulates in the nuclei in skeletal muscle after birth. a Left: Western blot with anti-MSY3 (ZONAB) protein extracts of limb and TA muscle at different postnatal days (pn). α-tubulin was used as normalizer. Right: densitometry calculations for MSY3 faster (dephosph) and slower (phosph) migration bands of the Western blot on the left, normalized by α-tubulin. Figure displays results representative from three independent experiments. *P <0.01 by Student’s t test. b Western blot with extracts from 15 dpc embryo limbs treated with Antarctic Phosphatase (AP), 1 unit and 5 units, and probed with anti-MSY3 Ab (ZONAB). c Western blots of nuclear and cytosolic fractions of protein extracts of limb muscle at fetal (15 dpc), postnatal (pn), and mature, 1 month (1 M), stages. Histone H3 was used as normalizer of the nucleic fraction and GAPDH as normalizer of the cytosolic fraction. d Left: expression of MSY3 evaluated by IF with anti-MSY3 Ab, ZONAB, in cross sections of limbs of 2-day-aged pups (2 pn) and TA of 1-month-aged mice (1 M). The images merge MSY3 expression (red), caveolin 3 expression (green), and Hoechst nuclei staining (blue). MSY3 localization in nuclei is labeled in purple. Scale bars = 100 μm. On the right, high magnification images (corresponding to dotted white squares) show staining of few muscle fibers. Scale bar = 40 μm. White arrows indicate sporadic MSY3 nuclear accumulation in 2 pn limb cross sections. Right Graph quantifies MSY3 positive nuclei over the total nuclei in cross sections of limbs of 2-day-aged pups (2 pn) and TA of 1-month-aged mice (1 M)
Mentions: We previously demonstrated that MSY3 protein expression progressively increased as muscle matures, from late stages of fetal development (15 dpc) to the mature stages of muscle growth in postnatal life (2 months old, 2MM). A Western blot analysis showed that the migration pattern of MSY3 protein (long isoform) shifts from a multi-band to a single lower molecular weight band in the nuclear extract of leg muscle, between very early stages of postnatal development (8 pn) and later stages (15 pn) [16]. We interpreted this modification as a result of the protein phosphorylation state change. We analyzed the MSY3 protein-banding pattern from 6–15 days postnatal extracts and inferred the time of shift from a multi-band to a single band migration pattern around the ninth day of postnatal life (Fig. 1a). During progression from fetal to adult stages, myogenin expression is severely reduced along the muscle fiber, as described previously [30, 57], and shown in Additional file 1: Figure S1. To investigate whether this change in MSY3 migration pattern is caused by the loss of a phosphate group, we incubated protein extracts from limbs of 16 dpc embryos with Antarctic Phosphatase (AP). Upon treatment with AP, MSY3 protein migrated as a single band compared to the multiple bands observed in the untreated sample (Fig. 1b), suggesting that MSY3 is phosphorylated during muscle development. Analysis of MSY3 immunoprecipitated protein from skeletal muscle extracts of 8 pn and 15 pn mice showed the same migration observed in skeletal muscle (Additional file 1: Figure S2A). Treatment of the immunoprecipitated MSY3 protein with AP affected its migration pattern similar to the input control (non-immunoprecipitated) MSY3 protein (Additional file 1: Figure S2B).Fig. 1

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