Limits...
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.

Show MeSH

Related in: MedlinePlus

BAG3 associates with SNAP-25 and syntaxin-1 and regulates the formation of the SNARE complex. β-TC-6 cells were stimulated with 25-mM glucose for the indicated time. Clarified whole-cell detergent extracts were prepared and immunoprecipitated with (a) anti-SNAP-25 or (b) anti-syntaxin-1 antibodies. Immune precipitates were analyzed by western blot with anti-SNAP-25, syntaxin-1, BAG3, Hsc-70 and GAPDH antibodies. Control immunoprecipitations were performed in parallel using mouse IgG. These results are representative of three sets of lysates prepared from independent experiments. (c) β-TC- 6 cells at 80% confluence were transfected with a bag3-specific small interfering (si) RNA or a non-target (si) RNA (NT siRNA); 48 h after transfection, cells were stimulated with 25-mM glucose for 15 min. Clarified whole-cell detergent extracts were prepared and immunoprecipitated with anti-syntaxin-1. Immune precipitates were subjected to western blot with, anti-SNAP-25 and GAPDH antibodies. Control immunoprecipitations were performed in a similar manner, using mouse IgG. These results are representative of two independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385931&req=5

fig3: BAG3 associates with SNAP-25 and syntaxin-1 and regulates the formation of the SNARE complex. β-TC-6 cells were stimulated with 25-mM glucose for the indicated time. Clarified whole-cell detergent extracts were prepared and immunoprecipitated with (a) anti-SNAP-25 or (b) anti-syntaxin-1 antibodies. Immune precipitates were analyzed by western blot with anti-SNAP-25, syntaxin-1, BAG3, Hsc-70 and GAPDH antibodies. Control immunoprecipitations were performed in parallel using mouse IgG. These results are representative of three sets of lysates prepared from independent experiments. (c) β-TC- 6 cells at 80% confluence were transfected with a bag3-specific small interfering (si) RNA or a non-target (si) RNA (NT siRNA); 48 h after transfection, cells were stimulated with 25-mM glucose for 15 min. Clarified whole-cell detergent extracts were prepared and immunoprecipitated with anti-syntaxin-1. Immune precipitates were subjected to western blot with, anti-SNAP-25 and GAPDH antibodies. Control immunoprecipitations were performed in a similar manner, using mouse IgG. These results are representative of two independent experiments

Mentions: Cortical F-actin remodeling is known to couple granule mobilization to the SNARE exocytosis machinery.38, 45, 46 The formation of the t-SNARE complex by the interaction of SNAP-25 with syntaxin-1 at the plasma membrane is essential for the pairing with the vesicle v-SNARE complex.31, 47, 48 It is therefore possible that BAG3 affects F-actin remodeling by interacting with components of the SNARE complex. To test this hypothesis, we performed co-immunoprecipitation experiments in basal conditions and after 5, 15 and 30 min of glucose stimulation. β-TC-6 cell extracts were immunoprecipitated using anti-SNAP-25 (Figure 3a) and anti-syntaxin-1 (Figure 3b) antibodies and revealed using a polyclonal anti-BAG3 antibody. Interestingly, while BAG3 is pulled down with the antibody against SNAP-25 at basal levels and even more after 5 min of glucose stimulation, this interaction is lost at longer time points (Figure 3a). On the contrary BAG3 appears to interact with syntaxin-1 with the same intensity throughout the stimulation (Figure 3b).


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 associates with SNAP-25 and syntaxin-1 and regulates the formation of the SNARE complex. β-TC-6 cells were stimulated with 25-mM glucose for the indicated time. Clarified whole-cell detergent extracts were prepared and immunoprecipitated with (a) anti-SNAP-25 or (b) anti-syntaxin-1 antibodies. Immune precipitates were analyzed by western blot with anti-SNAP-25, syntaxin-1, BAG3, Hsc-70 and GAPDH antibodies. Control immunoprecipitations were performed in parallel using mouse IgG. These results are representative of three sets of lysates prepared from independent experiments. (c) β-TC- 6 cells at 80% confluence were transfected with a bag3-specific small interfering (si) RNA or a non-target (si) RNA (NT siRNA); 48 h after transfection, cells were stimulated with 25-mM glucose for 15 min. Clarified whole-cell detergent extracts were prepared and immunoprecipitated with anti-syntaxin-1. Immune precipitates were subjected to western blot with, anti-SNAP-25 and GAPDH antibodies. Control immunoprecipitations were performed in a similar manner, using mouse IgG. 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

fig3: BAG3 associates with SNAP-25 and syntaxin-1 and regulates the formation of the SNARE complex. β-TC-6 cells were stimulated with 25-mM glucose for the indicated time. Clarified whole-cell detergent extracts were prepared and immunoprecipitated with (a) anti-SNAP-25 or (b) anti-syntaxin-1 antibodies. Immune precipitates were analyzed by western blot with anti-SNAP-25, syntaxin-1, BAG3, Hsc-70 and GAPDH antibodies. Control immunoprecipitations were performed in parallel using mouse IgG. These results are representative of three sets of lysates prepared from independent experiments. (c) β-TC- 6 cells at 80% confluence were transfected with a bag3-specific small interfering (si) RNA or a non-target (si) RNA (NT siRNA); 48 h after transfection, cells were stimulated with 25-mM glucose for 15 min. Clarified whole-cell detergent extracts were prepared and immunoprecipitated with anti-syntaxin-1. Immune precipitates were subjected to western blot with, anti-SNAP-25 and GAPDH antibodies. Control immunoprecipitations were performed in a similar manner, using mouse IgG. These results are representative of two independent experiments
Mentions: Cortical F-actin remodeling is known to couple granule mobilization to the SNARE exocytosis machinery.38, 45, 46 The formation of the t-SNARE complex by the interaction of SNAP-25 with syntaxin-1 at the plasma membrane is essential for the pairing with the vesicle v-SNARE complex.31, 47, 48 It is therefore possible that BAG3 affects F-actin remodeling by interacting with components of the SNARE complex. To test this hypothesis, we performed co-immunoprecipitation experiments in basal conditions and after 5, 15 and 30 min of glucose stimulation. β-TC-6 cell extracts were immunoprecipitated using anti-SNAP-25 (Figure 3a) and anti-syntaxin-1 (Figure 3b) antibodies and revealed using a polyclonal anti-BAG3 antibody. Interestingly, while BAG3 is pulled down with the antibody against SNAP-25 at basal levels and even more after 5 min of glucose stimulation, this interaction is lost at longer time points (Figure 3a). On the contrary BAG3 appears to interact with syntaxin-1 with the same intensity throughout the stimulation (Figure 3b).

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