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BAG3 regulates formation of the SNARE complex and insulin secretion.

Iorio V, Festa M, Rosati A, Hahne M, Tiberti C, Capunzo M, De Laurenzi V, Turco MC - Cell Death Dis (2015)

Bottom Line: This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the β cell, hence facilitating insulin secretion.In this manuscript, we show for the first time that BAG3 plays an important role in this process.Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Salerno, via Giovanni Paolo II, 132, Fisciano, SA, Italy.

ABSTRACT
Insulin release in response to glucose stimulation requires exocytosis of insulin-containing granules. Glucose stimulation of beta cells leads to focal adhesion kinase (FAK) phosphorylation, which acts on the Rho family proteins (Rho, Rac and Cdc42) that direct F-actin remodeling. This process requires docking and fusion of secretory vesicles to the release sites at the plasma membrane and is a complex mechanism that is mediated by SNAREs. This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the β cell, hence facilitating insulin secretion. In this manuscript, we show for the first time that BAG3 plays an important role in this process. We show that BAG3 downregulation results in increased insulin secretion in response to glucose stimulation and in disruption of the F-actin network. Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1. Upon glucose stimulation BAG3 is phosphorylated by FAK and dissociates from SNAP-25 allowing the formation of the SNARE complex, destabilization of the F-actin network and insulin release.

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Related in: MedlinePlus

BAG3 is expressed in pancreatic beta cells. (a) A representative section of human pancreas showing high expression of BAG3 in the pancreatic islets compared with the exocrine tissue. (b) Western blot of β-TC-6 lysate and BAG3 recombinant incubated with a polyclonal antibody against BAG3. (c) β-TC-6 cells were fixed and stained with a monoclonal anti-BAG3 antibody (red) followed by incubation with fluorescein-conjugated secondary antibody. DAPI was used to stain cell nuclei (scale bars, 10 μm). (d) β-TC-6 cells were fixed and stained with a monoclonal anti-BAG3 antibody (red) and with an anti-insulin antibody (green) followed by incubation with fluorescein-conjugated secondary antibodies. Yellow regions indicate co-localization (scale bars, 10 μm; zoom 2). All images are fully representative of three independent experiments. (e) β-TC-6 cells were fractionated into cytosolic, microsomal and insulin granule fraction; 15 μg of each sample was loaded for immunoblotting with anti-BAG3 antibody. Anti-GAPDH and anti-insulin antibody were used to confirm the purity of isolated subcellular fractions. These results are representative of two independent experiments
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fig1: BAG3 is expressed in pancreatic beta cells. (a) A representative section of human pancreas showing high expression of BAG3 in the pancreatic islets compared with the exocrine tissue. (b) Western blot of β-TC-6 lysate and BAG3 recombinant incubated with a polyclonal antibody against BAG3. (c) β-TC-6 cells were fixed and stained with a monoclonal anti-BAG3 antibody (red) followed by incubation with fluorescein-conjugated secondary antibody. DAPI was used to stain cell nuclei (scale bars, 10 μm). (d) β-TC-6 cells were fixed and stained with a monoclonal anti-BAG3 antibody (red) and with an anti-insulin antibody (green) followed by incubation with fluorescein-conjugated secondary antibodies. Yellow regions indicate co-localization (scale bars, 10 μm; zoom 2). All images are fully representative of three independent experiments. (e) β-TC-6 cells were fractionated into cytosolic, microsomal and insulin granule fraction; 15 μg of each sample was loaded for immunoblotting with anti-BAG3 antibody. Anti-GAPDH and anti-insulin antibody were used to confirm the purity of isolated subcellular fractions. These results are representative of two independent experiments

Mentions: While studying expression of BAG3 in PDACs we noticed that pancreatic islets in normal pancreatic tissue surrounding the tumor strongly stained for BAG3, we therefore decided to further investigate the role of this protein in endocrine pancreatic cells. Indeed we confirmed this finding in additional pancreas sections and as shown in Figure 1a, BAG3 shows high expression in pancreatic islets, while the exocrine portion of the pancreas is as expected negative. We then chose to use an established mouse insulinoma cell line, β-TC-6, to further investigate the role played by BAG3 in endocrine pancreatic cells. As shown in the western blot in Figure 1b this cell line does express BAG3 that is localized in the cytoplasm of these cells (Figure 1c). Interestingly, BAG3 appears to co-localize with insulin-containing granules as shown by co-immunostaining (Figure 1d) and by subcellular fractionation (Figure 1e) that shows the presence of BAG3 in the insulin granule fraction.


BAG3 regulates formation of the SNARE complex and insulin secretion.

Iorio V, Festa M, Rosati A, Hahne M, Tiberti C, Capunzo M, De Laurenzi V, Turco MC - Cell Death Dis (2015)

BAG3 is expressed in pancreatic beta cells. (a) A representative section of human pancreas showing high expression of BAG3 in the pancreatic islets compared with the exocrine tissue. (b) Western blot of β-TC-6 lysate and BAG3 recombinant incubated with a polyclonal antibody against BAG3. (c) β-TC-6 cells were fixed and stained with a monoclonal anti-BAG3 antibody (red) followed by incubation with fluorescein-conjugated secondary antibody. DAPI was used to stain cell nuclei (scale bars, 10 μm). (d) β-TC-6 cells were fixed and stained with a monoclonal anti-BAG3 antibody (red) and with an anti-insulin antibody (green) followed by incubation with fluorescein-conjugated secondary antibodies. Yellow regions indicate co-localization (scale bars, 10 μm; zoom 2). All images are fully representative of three independent experiments. (e) β-TC-6 cells were fractionated into cytosolic, microsomal and insulin granule fraction; 15 μg of each sample was loaded for immunoblotting with anti-BAG3 antibody. Anti-GAPDH and anti-insulin antibody were used to confirm the purity of isolated subcellular fractions. These results are representative of two independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385931&req=5

fig1: BAG3 is expressed in pancreatic beta cells. (a) A representative section of human pancreas showing high expression of BAG3 in the pancreatic islets compared with the exocrine tissue. (b) Western blot of β-TC-6 lysate and BAG3 recombinant incubated with a polyclonal antibody against BAG3. (c) β-TC-6 cells were fixed and stained with a monoclonal anti-BAG3 antibody (red) followed by incubation with fluorescein-conjugated secondary antibody. DAPI was used to stain cell nuclei (scale bars, 10 μm). (d) β-TC-6 cells were fixed and stained with a monoclonal anti-BAG3 antibody (red) and with an anti-insulin antibody (green) followed by incubation with fluorescein-conjugated secondary antibodies. Yellow regions indicate co-localization (scale bars, 10 μm; zoom 2). All images are fully representative of three independent experiments. (e) β-TC-6 cells were fractionated into cytosolic, microsomal and insulin granule fraction; 15 μg of each sample was loaded for immunoblotting with anti-BAG3 antibody. Anti-GAPDH and anti-insulin antibody were used to confirm the purity of isolated subcellular fractions. These results are representative of two independent experiments
Mentions: While studying expression of BAG3 in PDACs we noticed that pancreatic islets in normal pancreatic tissue surrounding the tumor strongly stained for BAG3, we therefore decided to further investigate the role of this protein in endocrine pancreatic cells. Indeed we confirmed this finding in additional pancreas sections and as shown in Figure 1a, BAG3 shows high expression in pancreatic islets, while the exocrine portion of the pancreas is as expected negative. We then chose to use an established mouse insulinoma cell line, β-TC-6, to further investigate the role played by BAG3 in endocrine pancreatic cells. As shown in the western blot in Figure 1b this cell line does express BAG3 that is localized in the cytoplasm of these cells (Figure 1c). Interestingly, BAG3 appears to co-localize with insulin-containing granules as shown by co-immunostaining (Figure 1d) and by subcellular fractionation (Figure 1e) that shows the presence of BAG3 in the insulin granule fraction.

Bottom Line: This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the β cell, hence facilitating insulin secretion.In this manuscript, we show for the first time that BAG3 plays an important role in this process.Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Salerno, via Giovanni Paolo II, 132, Fisciano, SA, Italy.

ABSTRACT
Insulin release in response to glucose stimulation requires exocytosis of insulin-containing granules. Glucose stimulation of beta cells leads to focal adhesion kinase (FAK) phosphorylation, which acts on the Rho family proteins (Rho, Rac and Cdc42) that direct F-actin remodeling. This process requires docking and fusion of secretory vesicles to the release sites at the plasma membrane and is a complex mechanism that is mediated by SNAREs. This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the β cell, hence facilitating insulin secretion. In this manuscript, we show for the first time that BAG3 plays an important role in this process. We show that BAG3 downregulation results in increased insulin secretion in response to glucose stimulation and in disruption of the F-actin network. Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1. Upon glucose stimulation BAG3 is phosphorylated by FAK and dissociates from SNAP-25 allowing the formation of the SNARE complex, destabilization of the F-actin network and insulin release.

Show MeSH
Related in: MedlinePlus