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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-GFP is correctly processed and targeted to SGs. (A and B) Western blottings with anti-GFP (A) or anti-ICA512 (B) antibodies on 40 μg of protein from INS-1 cells nontransfected (control) or stably transfected with ICA512-GFP. (C) Confocal microscopy on INS-1 ICA512-GFP cells immunostained for insulin (pseudored). Bar, 10 μm. (D) Immunoelectron microscopy on resting INS-1 ICA512-GFP cells double labeled with anti-insulin (6 nm of gold) and anti-GFP (12 nm of gold; arrowheads) antibodies. PM, plasma membrane. Bar, 200 nm. (E) Western blottings with anti-GFP, anti-ICA512, anti-CPE, and anti-Glut2 antibodies on fractions of INS-1 ICA512-GFP cells separated on a continuous sucrose density gradient. The sucrose molarity of each fraction is indicated on the top. (F) Western blots for μ-calpain (top), ICA512 (middle), and γ-tubulin (bottom) on 40 μg of protein from INS-1 cells transfected with scrambled or anti–μ-calpain siRNA oligos 1 or 3.
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fig4: ICA512-GFP is correctly processed and targeted to SGs. (A and B) Western blottings with anti-GFP (A) or anti-ICA512 (B) antibodies on 40 μg of protein from INS-1 cells nontransfected (control) or stably transfected with ICA512-GFP. (C) Confocal microscopy on INS-1 ICA512-GFP cells immunostained for insulin (pseudored). Bar, 10 μm. (D) Immunoelectron microscopy on resting INS-1 ICA512-GFP cells double labeled with anti-insulin (6 nm of gold) and anti-GFP (12 nm of gold; arrowheads) antibodies. PM, plasma membrane. Bar, 200 nm. (E) Western blottings with anti-GFP, anti-ICA512, anti-CPE, and anti-Glut2 antibodies on fractions of INS-1 ICA512-GFP cells separated on a continuous sucrose density gradient. The sucrose molarity of each fraction is indicated on the top. (F) Western blots for μ-calpain (top), ICA512 (middle), and γ-tubulin (bottom) on 40 μg of protein from INS-1 cells transfected with scrambled or anti–μ-calpain siRNA oligos 1 or 3.

Mentions: Recognition by the anti-ICA512 monoclonal antibody of a juxtamembrane epitope just upstream of the calpain cleavage site (Ort et al., 2001) precluded its use for investigating the fate of the ICA512-cleaved cytosolic fragment (ICA512-CCF). We overcame this limit by fusing GFP at the COOH terminus of ICA512. Immunoblots with anti-GFP or anti-ICA512 antibodies on extracts from stable ICA512-GFP INS-1 cells show pro-ICA512-GFP and ICA512-TMF-GFP having the expected sizes of 135 and 90 kD, respectively (Fig. 4, A and B). By confocal microscopy it appears that most ICA512-GFP colocalizes with insulin (Fig. 4 C). Specific labeling of granules with GFP antibody is also found by immunogold electron microscopy on cryosections (Fig. 4 D). In ICA512-GFP INS-1 cells, 19/87 granules were labeled, compared with 0/102 in nontransfected INS-1 cells (P < 0.001). Enrichment of ICA512-GFP on SGs has also been confirmed by subcellular fractionation on sucrose density gradients (Fig. 4 E). Moreover, limited knockdown of μ-calpain is sufficient to decrease ICA512-TMF-GFP cleavage (Fig. 4 F and Fig. S1 D). Thus, the GFP tag does neither prevent the conversion of pro-ICA512 into ICA512-TMF nor the targeting of the latter to SGs and its cleavage by μ-calpain.


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-GFP is correctly processed and targeted to SGs. (A and B) Western blottings with anti-GFP (A) or anti-ICA512 (B) antibodies on 40 μg of protein from INS-1 cells nontransfected (control) or stably transfected with ICA512-GFP. (C) Confocal microscopy on INS-1 ICA512-GFP cells immunostained for insulin (pseudored). Bar, 10 μm. (D) Immunoelectron microscopy on resting INS-1 ICA512-GFP cells double labeled with anti-insulin (6 nm of gold) and anti-GFP (12 nm of gold; arrowheads) antibodies. PM, plasma membrane. Bar, 200 nm. (E) Western blottings with anti-GFP, anti-ICA512, anti-CPE, and anti-Glut2 antibodies on fractions of INS-1 ICA512-GFP cells separated on a continuous sucrose density gradient. The sucrose molarity of each fraction is indicated on the top. (F) Western blots for μ-calpain (top), ICA512 (middle), and γ-tubulin (bottom) on 40 μg of protein from INS-1 cells transfected with scrambled or anti–μ-calpain siRNA oligos 1 or 3.
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fig4: ICA512-GFP is correctly processed and targeted to SGs. (A and B) Western blottings with anti-GFP (A) or anti-ICA512 (B) antibodies on 40 μg of protein from INS-1 cells nontransfected (control) or stably transfected with ICA512-GFP. (C) Confocal microscopy on INS-1 ICA512-GFP cells immunostained for insulin (pseudored). Bar, 10 μm. (D) Immunoelectron microscopy on resting INS-1 ICA512-GFP cells double labeled with anti-insulin (6 nm of gold) and anti-GFP (12 nm of gold; arrowheads) antibodies. PM, plasma membrane. Bar, 200 nm. (E) Western blottings with anti-GFP, anti-ICA512, anti-CPE, and anti-Glut2 antibodies on fractions of INS-1 ICA512-GFP cells separated on a continuous sucrose density gradient. The sucrose molarity of each fraction is indicated on the top. (F) Western blots for μ-calpain (top), ICA512 (middle), and γ-tubulin (bottom) on 40 μg of protein from INS-1 cells transfected with scrambled or anti–μ-calpain siRNA oligos 1 or 3.
Mentions: Recognition by the anti-ICA512 monoclonal antibody of a juxtamembrane epitope just upstream of the calpain cleavage site (Ort et al., 2001) precluded its use for investigating the fate of the ICA512-cleaved cytosolic fragment (ICA512-CCF). We overcame this limit by fusing GFP at the COOH terminus of ICA512. Immunoblots with anti-GFP or anti-ICA512 antibodies on extracts from stable ICA512-GFP INS-1 cells show pro-ICA512-GFP and ICA512-TMF-GFP having the expected sizes of 135 and 90 kD, respectively (Fig. 4, A and B). By confocal microscopy it appears that most ICA512-GFP colocalizes with insulin (Fig. 4 C). Specific labeling of granules with GFP antibody is also found by immunogold electron microscopy on cryosections (Fig. 4 D). In ICA512-GFP INS-1 cells, 19/87 granules were labeled, compared with 0/102 in nontransfected INS-1 cells (P < 0.001). Enrichment of ICA512-GFP on SGs has also been confirmed by subcellular fractionation on sucrose density gradients (Fig. 4 E). Moreover, limited knockdown of μ-calpain is sufficient to decrease ICA512-TMF-GFP cleavage (Fig. 4 F and Fig. S1 D). Thus, the GFP tag does neither prevent the conversion of pro-ICA512 into ICA512-TMF nor the targeting of the latter to SGs and its cleavage by μ-calpain.

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