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Syntaxin 4 regulates the surface localization of a promyogenic receptor Cdo thereby promoting myogenic differentiation.

Yoo M, Kim BG, Lee SJ, Jeong HJ, Park JW, Seo DW, Kim YK, Lee HY, Han JW, Kang JS, Bae GU - Skelet Muscle (2015)

Bottom Line: Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels.Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface.Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women's University, Seoul, 140-742 Republic of Korea.

ABSTRACT

Background: Syntaxins are a family of membrane proteins involved in vesicle trafficking, such as synaptic vesicle exocytosis. Syntaxin 4 (Stx4) is expressed highly in skeletal muscle and plays a critical role in insulin-stimulated glucose uptake by promoting translocation of glucose transporter 4 (GLUT4) to the cell surface. A cell surface receptor cell adhesion molecule-related, down-regulated by oncogenes (Cdo) is a component of cell adhesion complexes and promotes myoblast differentiation via activation of key signalings, including p38MAPK and AKT. In this study, we investigate the function of Stx4 in myoblast differentiation and the crosstalk between Stx4 and Cdo in myoblast differentiation.

Methods: The effects of overexpression or shRNA-based depletion of Stx4 and Cdo genes on C2C12 myoblast differentiation are assessed by Western blotting and immunofluorescence approaches. The interaction between Cdo and Stx4 and the responsible domain mapping are assessed by coimmunoprecipitation or pulldown assays. The effect of Stx4 depletion on cell surface localization of Cdo and GLUT4 in C2C12 myoblasts is assessed by surface biotinylation and Western blotting.

Results: Overexpression or knockdown of Stx4 enhances or inhibits myogenic differentiation, respectively. Stx4 binds to the cytoplasmic tail of Cdo, and this interaction seems to be critical for induction of p38MAPK activation and myotube formation. Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels. Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface. Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it.

Conclusions: Stx4 promotes myoblast differentiation through interaction with Cdo and stimulation of its surface translocation. Both Cdo and Stx4 are required for GLUT4 translocation to cell surface and glucose uptake in myoblast differentiation.

No MeSH data available.


Related in: MedlinePlus

Stx4 and Cdo interact physically in differentiating myoblasts, and the t-SNARE domain of Stx4 mediates the interaction with Cdo. a Lysates of 293T cells transfected with Stx4-myc, Cdo, or control vector were subjected to immunoprecipitation with myc and immunoblotting with Cdo or myc antibodies. b Lysates of C2C12 cells from various differentiation time courses were immunoprecipitated with control IgG or anti-Cdo antibody and immunoblotted with antibodies to Stx4, Cdo, and pan-Cadherin as a loading control. c The schematic representation of the domain structure of Cdo. Cdo consists of five immunoglobulin, three fibronectin type III, a single transmembrane domain, and a 270-amino-acid-long intracellular region. d 293T cells were transiently cotransfected with control or Stx4-myc along with either the full length or three deletion mutants of the Cdo’s cytoplasmic region. Forty-eight hours later, cell lysates were subjected to immunoprecipitation with myc antibody followed by immunoblotting with Cdo antibody. Total lysates served as the expression controls. e The schematic representation depicts the domain structure of Stx4 and the deletion of the specific domain; Δ33-153 (the Syntaxin domain deletion), Δ195-262 (the t-SNARE region deletion), and Δ154-194 (the linker region deletion). f 293T cells were transiently cotransfected with control or Cdo along with the full length or deletion mutants of Stx4, and the lysates were pulled down with S-agarose beads followed by immunoblotting with Cdo or GFP antibodies
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Fig3: Stx4 and Cdo interact physically in differentiating myoblasts, and the t-SNARE domain of Stx4 mediates the interaction with Cdo. a Lysates of 293T cells transfected with Stx4-myc, Cdo, or control vector were subjected to immunoprecipitation with myc and immunoblotting with Cdo or myc antibodies. b Lysates of C2C12 cells from various differentiation time courses were immunoprecipitated with control IgG or anti-Cdo antibody and immunoblotted with antibodies to Stx4, Cdo, and pan-Cadherin as a loading control. c The schematic representation of the domain structure of Cdo. Cdo consists of five immunoglobulin, three fibronectin type III, a single transmembrane domain, and a 270-amino-acid-long intracellular region. d 293T cells were transiently cotransfected with control or Stx4-myc along with either the full length or three deletion mutants of the Cdo’s cytoplasmic region. Forty-eight hours later, cell lysates were subjected to immunoprecipitation with myc antibody followed by immunoblotting with Cdo antibody. Total lysates served as the expression controls. e The schematic representation depicts the domain structure of Stx4 and the deletion of the specific domain; Δ33-153 (the Syntaxin domain deletion), Δ195-262 (the t-SNARE region deletion), and Δ154-194 (the linker region deletion). f 293T cells were transiently cotransfected with control or Cdo along with the full length or deletion mutants of Stx4, and the lysates were pulled down with S-agarose beads followed by immunoblotting with Cdo or GFP antibodies

Mentions: Next, we examined whether Stx4 and Cdo physically interacts in mammalian cells. To do so, 293T cells were transiently transfected with myc-tagged Stx4 and Cdo and then lysates were immunoprecipitated with the myc-tag antibody followed by immunoblotting with Cdo and myc antibodies. Consistent with the result obtained from a previous yeast two-hybrid screening [13], Stx4 and Cdo interacted in 293T cells when coexpressed (Fig. 3a). To assess whether Stx4 and Cdo interact endogenously in myoblasts, cell lysates of differentiating C2C12 myoblasts from a total of 3 days of differentiation time course were immunoprecipitated with control IgG or an anti-Cdo antibody and analyzed by Western blotting. Stx4 was precipitated with Cdo throughout differentiation time course, and the coprecipitation was highest at D2 (Fig. 3b) when myoblasts were differentiating (Fig. 1b). These results suggest that Stx4 and Cdo can physically interact in myoblasts during differentiation.Fig. 3


Syntaxin 4 regulates the surface localization of a promyogenic receptor Cdo thereby promoting myogenic differentiation.

Yoo M, Kim BG, Lee SJ, Jeong HJ, Park JW, Seo DW, Kim YK, Lee HY, Han JW, Kang JS, Bae GU - Skelet Muscle (2015)

Stx4 and Cdo interact physically in differentiating myoblasts, and the t-SNARE domain of Stx4 mediates the interaction with Cdo. a Lysates of 293T cells transfected with Stx4-myc, Cdo, or control vector were subjected to immunoprecipitation with myc and immunoblotting with Cdo or myc antibodies. b Lysates of C2C12 cells from various differentiation time courses were immunoprecipitated with control IgG or anti-Cdo antibody and immunoblotted with antibodies to Stx4, Cdo, and pan-Cadherin as a loading control. c The schematic representation of the domain structure of Cdo. Cdo consists of five immunoglobulin, three fibronectin type III, a single transmembrane domain, and a 270-amino-acid-long intracellular region. d 293T cells were transiently cotransfected with control or Stx4-myc along with either the full length or three deletion mutants of the Cdo’s cytoplasmic region. Forty-eight hours later, cell lysates were subjected to immunoprecipitation with myc antibody followed by immunoblotting with Cdo antibody. Total lysates served as the expression controls. e The schematic representation depicts the domain structure of Stx4 and the deletion of the specific domain; Δ33-153 (the Syntaxin domain deletion), Δ195-262 (the t-SNARE region deletion), and Δ154-194 (the linker region deletion). f 293T cells were transiently cotransfected with control or Cdo along with the full length or deletion mutants of Stx4, and the lysates were pulled down with S-agarose beads followed by immunoblotting with Cdo or GFP antibodies
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Fig3: Stx4 and Cdo interact physically in differentiating myoblasts, and the t-SNARE domain of Stx4 mediates the interaction with Cdo. a Lysates of 293T cells transfected with Stx4-myc, Cdo, or control vector were subjected to immunoprecipitation with myc and immunoblotting with Cdo or myc antibodies. b Lysates of C2C12 cells from various differentiation time courses were immunoprecipitated with control IgG or anti-Cdo antibody and immunoblotted with antibodies to Stx4, Cdo, and pan-Cadherin as a loading control. c The schematic representation of the domain structure of Cdo. Cdo consists of five immunoglobulin, three fibronectin type III, a single transmembrane domain, and a 270-amino-acid-long intracellular region. d 293T cells were transiently cotransfected with control or Stx4-myc along with either the full length or three deletion mutants of the Cdo’s cytoplasmic region. Forty-eight hours later, cell lysates were subjected to immunoprecipitation with myc antibody followed by immunoblotting with Cdo antibody. Total lysates served as the expression controls. e The schematic representation depicts the domain structure of Stx4 and the deletion of the specific domain; Δ33-153 (the Syntaxin domain deletion), Δ195-262 (the t-SNARE region deletion), and Δ154-194 (the linker region deletion). f 293T cells were transiently cotransfected with control or Cdo along with the full length or deletion mutants of Stx4, and the lysates were pulled down with S-agarose beads followed by immunoblotting with Cdo or GFP antibodies
Mentions: Next, we examined whether Stx4 and Cdo physically interacts in mammalian cells. To do so, 293T cells were transiently transfected with myc-tagged Stx4 and Cdo and then lysates were immunoprecipitated with the myc-tag antibody followed by immunoblotting with Cdo and myc antibodies. Consistent with the result obtained from a previous yeast two-hybrid screening [13], Stx4 and Cdo interacted in 293T cells when coexpressed (Fig. 3a). To assess whether Stx4 and Cdo interact endogenously in myoblasts, cell lysates of differentiating C2C12 myoblasts from a total of 3 days of differentiation time course were immunoprecipitated with control IgG or an anti-Cdo antibody and analyzed by Western blotting. Stx4 was precipitated with Cdo throughout differentiation time course, and the coprecipitation was highest at D2 (Fig. 3b) when myoblasts were differentiating (Fig. 1b). These results suggest that Stx4 and Cdo can physically interact in myoblasts during differentiation.Fig. 3

Bottom Line: Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels.Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface.Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women's University, Seoul, 140-742 Republic of Korea.

ABSTRACT

Background: Syntaxins are a family of membrane proteins involved in vesicle trafficking, such as synaptic vesicle exocytosis. Syntaxin 4 (Stx4) is expressed highly in skeletal muscle and plays a critical role in insulin-stimulated glucose uptake by promoting translocation of glucose transporter 4 (GLUT4) to the cell surface. A cell surface receptor cell adhesion molecule-related, down-regulated by oncogenes (Cdo) is a component of cell adhesion complexes and promotes myoblast differentiation via activation of key signalings, including p38MAPK and AKT. In this study, we investigate the function of Stx4 in myoblast differentiation and the crosstalk between Stx4 and Cdo in myoblast differentiation.

Methods: The effects of overexpression or shRNA-based depletion of Stx4 and Cdo genes on C2C12 myoblast differentiation are assessed by Western blotting and immunofluorescence approaches. The interaction between Cdo and Stx4 and the responsible domain mapping are assessed by coimmunoprecipitation or pulldown assays. The effect of Stx4 depletion on cell surface localization of Cdo and GLUT4 in C2C12 myoblasts is assessed by surface biotinylation and Western blotting.

Results: Overexpression or knockdown of Stx4 enhances or inhibits myogenic differentiation, respectively. Stx4 binds to the cytoplasmic tail of Cdo, and this interaction seems to be critical for induction of p38MAPK activation and myotube formation. Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels. Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface. Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it.

Conclusions: Stx4 promotes myoblast differentiation through interaction with Cdo and stimulation of its surface translocation. Both Cdo and Stx4 are required for GLUT4 translocation to cell surface and glucose uptake in myoblast differentiation.

No MeSH data available.


Related in: MedlinePlus