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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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Related in: MedlinePlus

ICA512 cytoplasmic domain is cleaved by μ-calpain in response to stimulation of β-cells. (A) Western blot for ICA512 (top) and γ-tubulin (bottom) on 40 μg of protein from INS-1 cells that had been incubated with resting or stimulating buffer for 105 min following 1 h at rest. Protease inhibitors L-685,458 and calpeptin were added at indicated concentrations. (B) Western blots for ICA512 (top) and γ-tubulin (bottom) on 30 μg of protein from rat islets that were incubated with 0 (resting) or 25 mM (stimulated) glucose for indicated times. (C) Quantitation of ICA512-TMF from two independent experiments as shown in B. (D) Western blots for ICA512 (top) and γ-tubulin (bottom) on 30 μg of protein from rat islets that were incubated in resting or stimulating buffers as in A, with or without 60 μM calpeptin. White lines indicate that intervening lanes have beem spliced out. (E) Autoradiographies showing the turnover of newly synthesized ICA512-TMF (top left) and synaptophysin (syn.; top right) as determined by pulse-chase and immunoprecipitation from [35S]methionine-labeled INS-1 cells kept at rest or stimulated for the indicated times. Middle and bottom panels show the total amount of ICA512-TMF (left middle), synaptophysin (right middle), and IgG heavy chain (bottom panels) in each immunoprecipitate as visualized by Western blotting. (F) Levels of μ-calpain and β-actin mRNAs by semiquantitative RT-PCR in INS-1 cells transfected with scrambled or anti–μ-calpain siRNA oligos 1. (G) Western blots for μ-calpain (top), ICA512 (middle), and γ-tubulin (bottom) on 40 μg of protein from stimulated INS-1 cells transfected with scrambled or anti–μ-calpain siRNA oligos 1. (H) Quantitation of μ-calpain and ICA512-TMF from three independent experiments as shown in G. (I) Western blots for firefly luciferase (top), μ-calpain (middle top), ICA512-TMF (middle bottom), and γ-tubulin (bottom) on 30 μg of protein in INS-1 cells transfected with firefly luciferase cDNA and untreated or treated with siRNA oligos for firefly luciferase. White lines indicate that intervening lanes have beem spliced out. (J) Firefly luciferase activity and protein levels of firefly luciferase, μ-calpain, and ICA512-TMF from three independent experiments as shown in I. (K) Western blots for ICA512 (top), μ-calpain-V5 (middle), and γ-tubulin (bottom) on 40 μg of protein from INS-1 cells transfected with the indicated amount of cDNA for μ-calpain-V5 and stimulated as in A. (L) Quantitation of ICA512-TMF from three independent experiments as shown in K. In B, D, H, J, and L, protein signals were normalized for γ-tubulin and expressed as a percentage of their respective values in cells at rest (B and D), transfected with scrambled siRNA oligos (H) or firefly luciferase cDNA (J), or electroporated (L). Error bars in C, H, J, and L show mean + SD.
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fig1: ICA512 cytoplasmic domain is cleaved by μ-calpain in response to stimulation of β-cells. (A) Western blot for ICA512 (top) and γ-tubulin (bottom) on 40 μg of protein from INS-1 cells that had been incubated with resting or stimulating buffer for 105 min following 1 h at rest. Protease inhibitors L-685,458 and calpeptin were added at indicated concentrations. (B) Western blots for ICA512 (top) and γ-tubulin (bottom) on 30 μg of protein from rat islets that were incubated with 0 (resting) or 25 mM (stimulated) glucose for indicated times. (C) Quantitation of ICA512-TMF from two independent experiments as shown in B. (D) Western blots for ICA512 (top) and γ-tubulin (bottom) on 30 μg of protein from rat islets that were incubated in resting or stimulating buffers as in A, with or without 60 μM calpeptin. White lines indicate that intervening lanes have beem spliced out. (E) Autoradiographies showing the turnover of newly synthesized ICA512-TMF (top left) and synaptophysin (syn.; top right) as determined by pulse-chase and immunoprecipitation from [35S]methionine-labeled INS-1 cells kept at rest or stimulated for the indicated times. Middle and bottom panels show the total amount of ICA512-TMF (left middle), synaptophysin (right middle), and IgG heavy chain (bottom panels) in each immunoprecipitate as visualized by Western blotting. (F) Levels of μ-calpain and β-actin mRNAs by semiquantitative RT-PCR in INS-1 cells transfected with scrambled or anti–μ-calpain siRNA oligos 1. (G) Western blots for μ-calpain (top), ICA512 (middle), and γ-tubulin (bottom) on 40 μg of protein from stimulated INS-1 cells transfected with scrambled or anti–μ-calpain siRNA oligos 1. (H) Quantitation of μ-calpain and ICA512-TMF from three independent experiments as shown in G. (I) Western blots for firefly luciferase (top), μ-calpain (middle top), ICA512-TMF (middle bottom), and γ-tubulin (bottom) on 30 μg of protein in INS-1 cells transfected with firefly luciferase cDNA and untreated or treated with siRNA oligos for firefly luciferase. White lines indicate that intervening lanes have beem spliced out. (J) Firefly luciferase activity and protein levels of firefly luciferase, μ-calpain, and ICA512-TMF from three independent experiments as shown in I. (K) Western blots for ICA512 (top), μ-calpain-V5 (middle), and γ-tubulin (bottom) on 40 μg of protein from INS-1 cells transfected with the indicated amount of cDNA for μ-calpain-V5 and stimulated as in A. (L) Quantitation of ICA512-TMF from three independent experiments as shown in K. In B, D, H, J, and L, protein signals were normalized for γ-tubulin and expressed as a percentage of their respective values in cells at rest (B and D), transfected with scrambled siRNA oligos (H) or firefly luciferase cDNA (J), or electroporated (L). Error bars in C, H, J, and L show mean + SD.

Mentions: Pro-ICA512 is a glycoprotein of 110 kD. Processing of its ectodomain by a furin-like protease during SG maturation converts pro-ICA512 into the mature ICA512-transmembrane fragment (ICA512-TMF) of 65 kD, which is found on SGs (Solimena et al., 1996). Stimulation of rat insulinoma cells-1 (INS-1) with high glucose (25 mM) and high K+ (55 mM) induces pro-ICA512 biosynthesis and reduces ICA512-TMF levels (Ort et al., 2001; Fig. 1 A, lanes 1 and 2). This dual effect on ICA512 can be dissociated by stimulating INS-1 cells either with high glucose or high K+ (Ort et al., 2001). High glucose alone prompts pro-ICA512 biosynthesis, but only a modest release of insulin and decrease of ICA512-TMF. In contrast, high K+ induces a strong insulin secretion and decrease of ICA512-TMF, but it does not enhance pro-ICA512 biosynthesis. The reduction of ICA512-TMF depends on the potency of a stimulus in inducing SG exocytosis. Stimulation with high glucose alone for 30, 60, and 120 min elicits indeed a progressive reduction of ICA512-TMF in freshly isolated rat pancreatic islets (Fig. 1, B and C), which, unlike INS-1 cells, retain a virtually intact secretory responsiveness to glucose. Pro-ICA512 induction by glucose is less apparent in islets (Fig. 1 D) than in INS-1 cells because they convert faster pro-ICA512 into ICA512-TMF. This induction, indeed, is readily detected upon stimulation of islets at 19°C, which blocks protein export from the trans-Golgi network and thereby pro-ICA512 conversion into ICA512-TMF (Knoch et al., 2004). Pulse-chase [35S]methionine labelings in INS-1 cells show that newly synthesized ICA512-TMF has a faster turnover compared with synaptophysin, an intrinsic membrane protein of synaptic vesicle/synaptic-like microvesicles, which is known to participate in multiple rounds of exo-endocytosis (Johnston et al., 1989; Fig. 1 E). Notably, newly synthesized ICA512-TMF is virtually not detectable in INS-1 cells kept at rest for 2 h after 1 h labeling in medium with low glucose (5.5 mM), whereas it is readily apparent in cells only exposed for 30 min to high glucose (25 m), which is consistent with the notion that glucose rapidly up-regulates ICA512 biosynthesis (Knoch et al., 2004).


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 cytoplasmic domain is cleaved by μ-calpain in response to stimulation of β-cells. (A) Western blot for ICA512 (top) and γ-tubulin (bottom) on 40 μg of protein from INS-1 cells that had been incubated with resting or stimulating buffer for 105 min following 1 h at rest. Protease inhibitors L-685,458 and calpeptin were added at indicated concentrations. (B) Western blots for ICA512 (top) and γ-tubulin (bottom) on 30 μg of protein from rat islets that were incubated with 0 (resting) or 25 mM (stimulated) glucose for indicated times. (C) Quantitation of ICA512-TMF from two independent experiments as shown in B. (D) Western blots for ICA512 (top) and γ-tubulin (bottom) on 30 μg of protein from rat islets that were incubated in resting or stimulating buffers as in A, with or without 60 μM calpeptin. White lines indicate that intervening lanes have beem spliced out. (E) Autoradiographies showing the turnover of newly synthesized ICA512-TMF (top left) and synaptophysin (syn.; top right) as determined by pulse-chase and immunoprecipitation from [35S]methionine-labeled INS-1 cells kept at rest or stimulated for the indicated times. Middle and bottom panels show the total amount of ICA512-TMF (left middle), synaptophysin (right middle), and IgG heavy chain (bottom panels) in each immunoprecipitate as visualized by Western blotting. (F) Levels of μ-calpain and β-actin mRNAs by semiquantitative RT-PCR in INS-1 cells transfected with scrambled or anti–μ-calpain siRNA oligos 1. (G) Western blots for μ-calpain (top), ICA512 (middle), and γ-tubulin (bottom) on 40 μg of protein from stimulated INS-1 cells transfected with scrambled or anti–μ-calpain siRNA oligos 1. (H) Quantitation of μ-calpain and ICA512-TMF from three independent experiments as shown in G. (I) Western blots for firefly luciferase (top), μ-calpain (middle top), ICA512-TMF (middle bottom), and γ-tubulin (bottom) on 30 μg of protein in INS-1 cells transfected with firefly luciferase cDNA and untreated or treated with siRNA oligos for firefly luciferase. White lines indicate that intervening lanes have beem spliced out. (J) Firefly luciferase activity and protein levels of firefly luciferase, μ-calpain, and ICA512-TMF from three independent experiments as shown in I. (K) Western blots for ICA512 (top), μ-calpain-V5 (middle), and γ-tubulin (bottom) on 40 μg of protein from INS-1 cells transfected with the indicated amount of cDNA for μ-calpain-V5 and stimulated as in A. (L) Quantitation of ICA512-TMF from three independent experiments as shown in K. In B, D, H, J, and L, protein signals were normalized for γ-tubulin and expressed as a percentage of their respective values in cells at rest (B and D), transfected with scrambled siRNA oligos (H) or firefly luciferase cDNA (J), or electroporated (L). Error bars in C, H, J, and L show mean + SD.
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fig1: ICA512 cytoplasmic domain is cleaved by μ-calpain in response to stimulation of β-cells. (A) Western blot for ICA512 (top) and γ-tubulin (bottom) on 40 μg of protein from INS-1 cells that had been incubated with resting or stimulating buffer for 105 min following 1 h at rest. Protease inhibitors L-685,458 and calpeptin were added at indicated concentrations. (B) Western blots for ICA512 (top) and γ-tubulin (bottom) on 30 μg of protein from rat islets that were incubated with 0 (resting) or 25 mM (stimulated) glucose for indicated times. (C) Quantitation of ICA512-TMF from two independent experiments as shown in B. (D) Western blots for ICA512 (top) and γ-tubulin (bottom) on 30 μg of protein from rat islets that were incubated in resting or stimulating buffers as in A, with or without 60 μM calpeptin. White lines indicate that intervening lanes have beem spliced out. (E) Autoradiographies showing the turnover of newly synthesized ICA512-TMF (top left) and synaptophysin (syn.; top right) as determined by pulse-chase and immunoprecipitation from [35S]methionine-labeled INS-1 cells kept at rest or stimulated for the indicated times. Middle and bottom panels show the total amount of ICA512-TMF (left middle), synaptophysin (right middle), and IgG heavy chain (bottom panels) in each immunoprecipitate as visualized by Western blotting. (F) Levels of μ-calpain and β-actin mRNAs by semiquantitative RT-PCR in INS-1 cells transfected with scrambled or anti–μ-calpain siRNA oligos 1. (G) Western blots for μ-calpain (top), ICA512 (middle), and γ-tubulin (bottom) on 40 μg of protein from stimulated INS-1 cells transfected with scrambled or anti–μ-calpain siRNA oligos 1. (H) Quantitation of μ-calpain and ICA512-TMF from three independent experiments as shown in G. (I) Western blots for firefly luciferase (top), μ-calpain (middle top), ICA512-TMF (middle bottom), and γ-tubulin (bottom) on 30 μg of protein in INS-1 cells transfected with firefly luciferase cDNA and untreated or treated with siRNA oligos for firefly luciferase. White lines indicate that intervening lanes have beem spliced out. (J) Firefly luciferase activity and protein levels of firefly luciferase, μ-calpain, and ICA512-TMF from three independent experiments as shown in I. (K) Western blots for ICA512 (top), μ-calpain-V5 (middle), and γ-tubulin (bottom) on 40 μg of protein from INS-1 cells transfected with the indicated amount of cDNA for μ-calpain-V5 and stimulated as in A. (L) Quantitation of ICA512-TMF from three independent experiments as shown in K. In B, D, H, J, and L, protein signals were normalized for γ-tubulin and expressed as a percentage of their respective values in cells at rest (B and D), transfected with scrambled siRNA oligos (H) or firefly luciferase cDNA (J), or electroporated (L). Error bars in C, H, J, and L show mean + SD.
Mentions: Pro-ICA512 is a glycoprotein of 110 kD. Processing of its ectodomain by a furin-like protease during SG maturation converts pro-ICA512 into the mature ICA512-transmembrane fragment (ICA512-TMF) of 65 kD, which is found on SGs (Solimena et al., 1996). Stimulation of rat insulinoma cells-1 (INS-1) with high glucose (25 mM) and high K+ (55 mM) induces pro-ICA512 biosynthesis and reduces ICA512-TMF levels (Ort et al., 2001; Fig. 1 A, lanes 1 and 2). This dual effect on ICA512 can be dissociated by stimulating INS-1 cells either with high glucose or high K+ (Ort et al., 2001). High glucose alone prompts pro-ICA512 biosynthesis, but only a modest release of insulin and decrease of ICA512-TMF. In contrast, high K+ induces a strong insulin secretion and decrease of ICA512-TMF, but it does not enhance pro-ICA512 biosynthesis. The reduction of ICA512-TMF depends on the potency of a stimulus in inducing SG exocytosis. Stimulation with high glucose alone for 30, 60, and 120 min elicits indeed a progressive reduction of ICA512-TMF in freshly isolated rat pancreatic islets (Fig. 1, B and C), which, unlike INS-1 cells, retain a virtually intact secretory responsiveness to glucose. Pro-ICA512 induction by glucose is less apparent in islets (Fig. 1 D) than in INS-1 cells because they convert faster pro-ICA512 into ICA512-TMF. This induction, indeed, is readily detected upon stimulation of islets at 19°C, which blocks protein export from the trans-Golgi network and thereby pro-ICA512 conversion into ICA512-TMF (Knoch et al., 2004). Pulse-chase [35S]methionine labelings in INS-1 cells show that newly synthesized ICA512-TMF has a faster turnover compared with synaptophysin, an intrinsic membrane protein of synaptic vesicle/synaptic-like microvesicles, which is known to participate in multiple rounds of exo-endocytosis (Johnston et al., 1989; Fig. 1 E). Notably, newly synthesized ICA512-TMF is virtually not detectable in INS-1 cells kept at rest for 2 h after 1 h labeling in medium with low glucose (5.5 mM), whereas it is readily apparent in cells only exposed for 30 min to high glucose (25 m), which is consistent with the notion that glucose rapidly up-regulates ICA512 biosynthesis (Knoch et al., 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.

Show MeSH
Related in: MedlinePlus