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Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in {beta}-cells.

Trajkovski M, Mziaut H, Altkrüger A, Ouwendijk J, Knoch KP, Müller S, Solimena M - J. Cell Biol. (2004)

Bottom Line: Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic beta-cells.This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression.Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in beta-cells, whose impaired insulin production and secretion causes diabetes.

View Article: PubMed Central - PubMed

Affiliation: Experimental Diabetology, Carl Gustav Carus Medical School, Dresden University of Technology, Dresden, Germany.

ABSTRACT
Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic beta-cells. Here, we show that exocytosis of SGs and insertion of ICA512 in the plasma membrane promotes the Ca(2+)-dependent cleavage of ICA512 cytoplasmic domain by mu-calpain. This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression. Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in beta-cells, whose impaired insulin production and secretion causes diabetes.

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ICA512 is cleaved by μ-calpain at the plasma membrane. (A) Western blottings with anti-ICA512 (top) or GFP (bottom) antibodies on 40 μg of protein from INS-1 cells cotransfected with active (Ttx) or inactive (Ttx-mut.) tetanus toxin light chain and GFP. Cells were incubated in resting or stimulating buffer for 105 min after 1 h at rest. (B) Quantitation of ICA512-TMF from three independent experiments as shown in A. (C) Stimulation index of insulin secretion from INS-1 cells that were either nontransfected or transfected with Ttx or Ttx-mut. (D) Western blotting with anti-ICA512 (top) and anti-GFP (bottom) antibodies on 30 μg of protein from INS-1 cells, which were either nontransfected (control) or transiently transfected with dynamin 1-GFP or dynamin 1 (K44A)-GFP and kept in resting buffer for 1 h. White lines indicate that intervening lanes have been spliced out. (E and H) Western blotting with anti-ICA512 (top) and anti-GFP (bottom) antibodies on 20 μg of protein from INS-1 cells, which were either nontransfected (control) or transiently transfected dynamin 1-GFP or dynamin 1 (K44A)-GFP (E) and with dynamin 2-GFP or dynamin 1 (K44A)-GFP (H). Cells were stimulated for 105 min in the absence or presence of calpeptin at the indicated concentration. As the expression of dynamin 2 constructs differed, loading of comparable protein amount was verified by immunoblot for γ-tubulin. (F and I) Quantitation of ICA512-TMF from four independent experiments as shown in E and H, respectively. Error bars in B, C, F, and I show mean + SD. (G) Fluorescence microscopy on INS-1 cells transiently transfected with dynamin 1 (K44A)-GFP (top) or dynamin 1-GFP (bottom) and incubated with Alexa568-transferrin (Tfn-Alexa568). Bars, 10 μm.
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fig3: ICA512 is cleaved by μ-calpain at the plasma membrane. (A) Western blottings with anti-ICA512 (top) or GFP (bottom) antibodies on 40 μg of protein from INS-1 cells cotransfected with active (Ttx) or inactive (Ttx-mut.) tetanus toxin light chain and GFP. Cells were incubated in resting or stimulating buffer for 105 min after 1 h at rest. (B) Quantitation of ICA512-TMF from three independent experiments as shown in A. (C) Stimulation index of insulin secretion from INS-1 cells that were either nontransfected or transfected with Ttx or Ttx-mut. (D) Western blotting with anti-ICA512 (top) and anti-GFP (bottom) antibodies on 30 μg of protein from INS-1 cells, which were either nontransfected (control) or transiently transfected with dynamin 1-GFP or dynamin 1 (K44A)-GFP and kept in resting buffer for 1 h. White lines indicate that intervening lanes have been spliced out. (E and H) Western blotting with anti-ICA512 (top) and anti-GFP (bottom) antibodies on 20 μg of protein from INS-1 cells, which were either nontransfected (control) or transiently transfected dynamin 1-GFP or dynamin 1 (K44A)-GFP (E) and with dynamin 2-GFP or dynamin 1 (K44A)-GFP (H). Cells were stimulated for 105 min in the absence or presence of calpeptin at the indicated concentration. As the expression of dynamin 2 constructs differed, loading of comparable protein amount was verified by immunoblot for γ-tubulin. (F and I) Quantitation of ICA512-TMF from four independent experiments as shown in E and H, respectively. Error bars in B, C, F, and I show mean + SD. (G) Fluorescence microscopy on INS-1 cells transiently transfected with dynamin 1 (K44A)-GFP (top) or dynamin 1-GFP (bottom) and incubated with Alexa568-transferrin (Tfn-Alexa568). Bars, 10 μm.

Mentions: To directly address whether ICA512-TMF cleavage precedes or follows SG exocytosis, INS-1 cells were transiently transfected with the light chain of clostridium tetanus toxin, which blocks the last step in SG fusion with the plasma membrane by cleaving synaptobrevin 2/VAMP2 (Schiavo et al., 1992; Niemann et al., 1994; Regazzi et al., 1995). The active, but not the inactive, tetanus toxin light chain, similarly inhibits ICA512-TMF cleavage (Fig. 3 A) and insulin secretion (Fig. 3 C), thereby indicating that SG exocytosis and ICA512-TMF insertion in the plasma membrane are necessary in order for the latter to become susceptible to cleavage.


Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in {beta}-cells.

Trajkovski M, Mziaut H, Altkrüger A, Ouwendijk J, Knoch KP, Müller S, Solimena M - J. Cell Biol. (2004)

ICA512 is cleaved by μ-calpain at the plasma membrane. (A) Western blottings with anti-ICA512 (top) or GFP (bottom) antibodies on 40 μg of protein from INS-1 cells cotransfected with active (Ttx) or inactive (Ttx-mut.) tetanus toxin light chain and GFP. Cells were incubated in resting or stimulating buffer for 105 min after 1 h at rest. (B) Quantitation of ICA512-TMF from three independent experiments as shown in A. (C) Stimulation index of insulin secretion from INS-1 cells that were either nontransfected or transfected with Ttx or Ttx-mut. (D) Western blotting with anti-ICA512 (top) and anti-GFP (bottom) antibodies on 30 μg of protein from INS-1 cells, which were either nontransfected (control) or transiently transfected with dynamin 1-GFP or dynamin 1 (K44A)-GFP and kept in resting buffer for 1 h. White lines indicate that intervening lanes have been spliced out. (E and H) Western blotting with anti-ICA512 (top) and anti-GFP (bottom) antibodies on 20 μg of protein from INS-1 cells, which were either nontransfected (control) or transiently transfected dynamin 1-GFP or dynamin 1 (K44A)-GFP (E) and with dynamin 2-GFP or dynamin 1 (K44A)-GFP (H). Cells were stimulated for 105 min in the absence or presence of calpeptin at the indicated concentration. As the expression of dynamin 2 constructs differed, loading of comparable protein amount was verified by immunoblot for γ-tubulin. (F and I) Quantitation of ICA512-TMF from four independent experiments as shown in E and H, respectively. Error bars in B, C, F, and I show mean + SD. (G) Fluorescence microscopy on INS-1 cells transiently transfected with dynamin 1 (K44A)-GFP (top) or dynamin 1-GFP (bottom) and incubated with Alexa568-transferrin (Tfn-Alexa568). Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172607&req=5

fig3: ICA512 is cleaved by μ-calpain at the plasma membrane. (A) Western blottings with anti-ICA512 (top) or GFP (bottom) antibodies on 40 μg of protein from INS-1 cells cotransfected with active (Ttx) or inactive (Ttx-mut.) tetanus toxin light chain and GFP. Cells were incubated in resting or stimulating buffer for 105 min after 1 h at rest. (B) Quantitation of ICA512-TMF from three independent experiments as shown in A. (C) Stimulation index of insulin secretion from INS-1 cells that were either nontransfected or transfected with Ttx or Ttx-mut. (D) Western blotting with anti-ICA512 (top) and anti-GFP (bottom) antibodies on 30 μg of protein from INS-1 cells, which were either nontransfected (control) or transiently transfected with dynamin 1-GFP or dynamin 1 (K44A)-GFP and kept in resting buffer for 1 h. White lines indicate that intervening lanes have been spliced out. (E and H) Western blotting with anti-ICA512 (top) and anti-GFP (bottom) antibodies on 20 μg of protein from INS-1 cells, which were either nontransfected (control) or transiently transfected dynamin 1-GFP or dynamin 1 (K44A)-GFP (E) and with dynamin 2-GFP or dynamin 1 (K44A)-GFP (H). Cells were stimulated for 105 min in the absence or presence of calpeptin at the indicated concentration. As the expression of dynamin 2 constructs differed, loading of comparable protein amount was verified by immunoblot for γ-tubulin. (F and I) Quantitation of ICA512-TMF from four independent experiments as shown in E and H, respectively. Error bars in B, C, F, and I show mean + SD. (G) Fluorescence microscopy on INS-1 cells transiently transfected with dynamin 1 (K44A)-GFP (top) or dynamin 1-GFP (bottom) and incubated with Alexa568-transferrin (Tfn-Alexa568). Bars, 10 μm.
Mentions: To directly address whether ICA512-TMF cleavage precedes or follows SG exocytosis, INS-1 cells were transiently transfected with the light chain of clostridium tetanus toxin, which blocks the last step in SG fusion with the plasma membrane by cleaving synaptobrevin 2/VAMP2 (Schiavo et al., 1992; Niemann et al., 1994; Regazzi et al., 1995). The active, but not the inactive, tetanus toxin light chain, similarly inhibits ICA512-TMF cleavage (Fig. 3 A) and insulin secretion (Fig. 3 C), thereby indicating that SG exocytosis and ICA512-TMF insertion in the plasma membrane are necessary in order for the latter to become susceptible to cleavage.

Bottom Line: Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic beta-cells.This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression.Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in beta-cells, whose impaired insulin production and secretion causes diabetes.

View Article: PubMed Central - PubMed

Affiliation: Experimental Diabetology, Carl Gustav Carus Medical School, Dresden University of Technology, Dresden, Germany.

ABSTRACT
Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic beta-cells. Here, we show that exocytosis of SGs and insertion of ICA512 in the plasma membrane promotes the Ca(2+)-dependent cleavage of ICA512 cytoplasmic domain by mu-calpain. This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression. Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in beta-cells, whose impaired insulin production and secretion causes diabetes.

Show MeSH
Related in: MedlinePlus