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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-CCF is translocated to the nucleus upon stimulation. (A) Western blots with anti-GFP (top), anti-ICA512 (middle), and γ-tubulin (bottom) on 50 μg of cytoplasmic and 25 μg of nuclear protein from INS-1 ICA512-GFP cells. Cells were incubated with resting or stimulating buffer for 105 min after 1 h at rest and with or without 60 μM calpeptin. (B) 0.5-μm optical Z-sections of INS-1 ICA512-GFP cells kept at rest (R; top) or stimulated (S; bottom) as in A and immunolabeled with anti-insulin antibody (pseudored). ICA512-GFP is shown as pseudogreen, whereas nuclei were counterstained with DAPI (pseudoblue). Insets in the right panels show high magnifications of the marked areas. (C) Western blots with anti-GFP on 40 μg of cytoplasmic and 20 μg of nuclear protein from INS-1 ICA512-GFP cells incubated as in A for the indicated times. (D) Quantitation of ICA512-TMF-GFP and ICA512-CCF-GFP in cytoplasmic (black line) and nuclear (red line) extracts at the indicated time points 1–6. Values are from four independent experiments as shown in C. For graphic representation, the amounts of ICA512-TMF-GFP and ICA512-CCF-GFP after 105 min in resting buffer (time point 2) were equaled to 100% and 50%, respectively. Error bars show mean ± SD. (E) 0.5-μm optical Z-sections of INS-1 cells transiently transfected with ICA512-HA, kept at rest (R; top) or stimulated (S; bottom) as in A, and immunolabeled with rabbit anti-HA (pseudogreen) and mouse anti-insulin (pseudored) antibodies. Nuclei were counterstained with DAPI (pseudoblue). Insets in the right panels show high magnifications of the marked areas. Bars, 10 μm.
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fig5: ICA512-CCF is translocated to the nucleus upon stimulation. (A) Western blots with anti-GFP (top), anti-ICA512 (middle), and γ-tubulin (bottom) on 50 μg of cytoplasmic and 25 μg of nuclear protein from INS-1 ICA512-GFP cells. Cells were incubated with resting or stimulating buffer for 105 min after 1 h at rest and with or without 60 μM calpeptin. (B) 0.5-μm optical Z-sections of INS-1 ICA512-GFP cells kept at rest (R; top) or stimulated (S; bottom) as in A and immunolabeled with anti-insulin antibody (pseudored). ICA512-GFP is shown as pseudogreen, whereas nuclei were counterstained with DAPI (pseudoblue). Insets in the right panels show high magnifications of the marked areas. (C) Western blots with anti-GFP on 40 μg of cytoplasmic and 20 μg of nuclear protein from INS-1 ICA512-GFP cells incubated as in A for the indicated times. (D) Quantitation of ICA512-TMF-GFP and ICA512-CCF-GFP in cytoplasmic (black line) and nuclear (red line) extracts at the indicated time points 1–6. Values are from four independent experiments as shown in C. For graphic representation, the amounts of ICA512-TMF-GFP and ICA512-CCF-GFP after 105 min in resting buffer (time point 2) were equaled to 100% and 50%, respectively. Error bars show mean ± SD. (E) 0.5-μm optical Z-sections of INS-1 cells transiently transfected with ICA512-HA, kept at rest (R; top) or stimulated (S; bottom) as in A, and immunolabeled with rabbit anti-HA (pseudogreen) and mouse anti-insulin (pseudored) antibodies. Nuclei were counterstained with DAPI (pseudoblue). Insets in the right panels show high magnifications of the marked areas. Bars, 10 μm.

Mentions: The stimulation-dependent proteolysis of ICA512-TMF-GFP is calpeptin-sensitive (Fig. 5 A, top panel). Strikingly, a GFP-reacting protein, whose size of 55 kD matches the one expected for ICA512-CCF-GFP, is enriched in nuclear, but not in cytosolic, fractions of stimulated ICA512-GFP INS-1 cells, also in a calpeptin-sensitive fashion (Fig. 5 A, top panel, lanes 4–6). Predictably, this 55-kD protein is not recognized by the anti-ICA512 monoclonal antibody (Fig. 5 A, middle panel). ICA512-CCF-GFP is already present in the nuclei of cells at rest for 165 min. Conceivably, this time is insufficient to clear the nuclei from ICA512-CCF-GFP that is constantly generated in cells cultured with 11 mM glucose, which stimulates sub-maximal exocytosis.


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-CCF is translocated to the nucleus upon stimulation. (A) Western blots with anti-GFP (top), anti-ICA512 (middle), and γ-tubulin (bottom) on 50 μg of cytoplasmic and 25 μg of nuclear protein from INS-1 ICA512-GFP cells. Cells were incubated with resting or stimulating buffer for 105 min after 1 h at rest and with or without 60 μM calpeptin. (B) 0.5-μm optical Z-sections of INS-1 ICA512-GFP cells kept at rest (R; top) or stimulated (S; bottom) as in A and immunolabeled with anti-insulin antibody (pseudored). ICA512-GFP is shown as pseudogreen, whereas nuclei were counterstained with DAPI (pseudoblue). Insets in the right panels show high magnifications of the marked areas. (C) Western blots with anti-GFP on 40 μg of cytoplasmic and 20 μg of nuclear protein from INS-1 ICA512-GFP cells incubated as in A for the indicated times. (D) Quantitation of ICA512-TMF-GFP and ICA512-CCF-GFP in cytoplasmic (black line) and nuclear (red line) extracts at the indicated time points 1–6. Values are from four independent experiments as shown in C. For graphic representation, the amounts of ICA512-TMF-GFP and ICA512-CCF-GFP after 105 min in resting buffer (time point 2) were equaled to 100% and 50%, respectively. Error bars show mean ± SD. (E) 0.5-μm optical Z-sections of INS-1 cells transiently transfected with ICA512-HA, kept at rest (R; top) or stimulated (S; bottom) as in A, and immunolabeled with rabbit anti-HA (pseudogreen) and mouse anti-insulin (pseudored) antibodies. Nuclei were counterstained with DAPI (pseudoblue). Insets in the right panels show high magnifications of the marked areas. Bars, 10 μm.
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Related In: Results  -  Collection

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fig5: ICA512-CCF is translocated to the nucleus upon stimulation. (A) Western blots with anti-GFP (top), anti-ICA512 (middle), and γ-tubulin (bottom) on 50 μg of cytoplasmic and 25 μg of nuclear protein from INS-1 ICA512-GFP cells. Cells were incubated with resting or stimulating buffer for 105 min after 1 h at rest and with or without 60 μM calpeptin. (B) 0.5-μm optical Z-sections of INS-1 ICA512-GFP cells kept at rest (R; top) or stimulated (S; bottom) as in A and immunolabeled with anti-insulin antibody (pseudored). ICA512-GFP is shown as pseudogreen, whereas nuclei were counterstained with DAPI (pseudoblue). Insets in the right panels show high magnifications of the marked areas. (C) Western blots with anti-GFP on 40 μg of cytoplasmic and 20 μg of nuclear protein from INS-1 ICA512-GFP cells incubated as in A for the indicated times. (D) Quantitation of ICA512-TMF-GFP and ICA512-CCF-GFP in cytoplasmic (black line) and nuclear (red line) extracts at the indicated time points 1–6. Values are from four independent experiments as shown in C. For graphic representation, the amounts of ICA512-TMF-GFP and ICA512-CCF-GFP after 105 min in resting buffer (time point 2) were equaled to 100% and 50%, respectively. Error bars show mean ± SD. (E) 0.5-μm optical Z-sections of INS-1 cells transiently transfected with ICA512-HA, kept at rest (R; top) or stimulated (S; bottom) as in A, and immunolabeled with rabbit anti-HA (pseudogreen) and mouse anti-insulin (pseudored) antibodies. Nuclei were counterstained with DAPI (pseudoblue). Insets in the right panels show high magnifications of the marked areas. Bars, 10 μm.
Mentions: The stimulation-dependent proteolysis of ICA512-TMF-GFP is calpeptin-sensitive (Fig. 5 A, top panel). Strikingly, a GFP-reacting protein, whose size of 55 kD matches the one expected for ICA512-CCF-GFP, is enriched in nuclear, but not in cytosolic, fractions of stimulated ICA512-GFP INS-1 cells, also in a calpeptin-sensitive fashion (Fig. 5 A, top panel, lanes 4–6). Predictably, this 55-kD protein is not recognized by the anti-ICA512 monoclonal antibody (Fig. 5 A, middle panel). ICA512-CCF-GFP is already present in the nuclei of cells at rest for 165 min. Conceivably, this time is insufficient to clear the nuclei from ICA512-CCF-GFP that is constantly generated in cells cultured with 11 mM glucose, which stimulates sub-maximal exocytosis.

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