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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 binds PIASy in the nucleus. (A) SDS-PAGE and autoradiography of in vitro transcribed and translated [35S]methionine-labeled ICA512(601–979) pulled down with glutathione sepharose beads coupled to 0.5 μg PIASy-GST, PIAS1-GST, or GST alone. Lane 1 shows the starting labeled material for the pull-down assay. White lines indicate that intervening lanes have been spliced out. (B) SDS-PAGE and autoradiography of in vitro transcribed and translated [35S]methionine-labeled PIASy pulled down with glutathione sepharose beads coupled to 0.5 μg GST-ICA512(601–979), GST-p53, or GST alone. First lane shows the starting labeled material for the pull-down assay. (C) FRET between ICA512-CFP (pseudocyan) and PIASγ-YFP (pseudogreen) in transiently cotransfected INS-1 cells incubated in resting or stimulating buffer for 105 min after 1 h at rest. The right panels show the signal for ICA512-CFP following the photobleaching of PIASy-YFP in the circled nuclei (arrows). (D) Quantitation of FRET to CFP in INS-1 cells transiently cotransfected with the indicated constructs: ICA512-CFP + PIASy-YFP; ICA512-CFP + lsm4-YFP; lsm4-CFP + PIASy-YFP. Values are from two independent experiments as shown in C. (D) Error bars show mean + SD.
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fig6: ICA512-CCF binds PIASy in the nucleus. (A) SDS-PAGE and autoradiography of in vitro transcribed and translated [35S]methionine-labeled ICA512(601–979) pulled down with glutathione sepharose beads coupled to 0.5 μg PIASy-GST, PIAS1-GST, or GST alone. Lane 1 shows the starting labeled material for the pull-down assay. White lines indicate that intervening lanes have been spliced out. (B) SDS-PAGE and autoradiography of in vitro transcribed and translated [35S]methionine-labeled PIASy pulled down with glutathione sepharose beads coupled to 0.5 μg GST-ICA512(601–979), GST-p53, or GST alone. First lane shows the starting labeled material for the pull-down assay. (C) FRET between ICA512-CFP (pseudocyan) and PIASγ-YFP (pseudogreen) in transiently cotransfected INS-1 cells incubated in resting or stimulating buffer for 105 min after 1 h at rest. The right panels show the signal for ICA512-CFP following the photobleaching of PIASy-YFP in the circled nuclei (arrows). (D) Quantitation of FRET to CFP in INS-1 cells transiently cotransfected with the indicated constructs: ICA512-CFP + PIASy-YFP; ICA512-CFP + lsm4-YFP; lsm4-CFP + PIASy-YFP. Values are from two independent experiments as shown in C. (D) Error bars show mean + SD.

Mentions: Screening by 2-hybrid assays in yeast showed that the cytoplasmic domain of ICA512 binds PIASy (unpublished data). PIASs were originally identified as PIAS (Liu et al., 1998) and have been more recently shown to have E3-sumoylating activity toward numerous transcription factors, including p53 and signal transducers and activators of transcription (STATs; Johnson and Gupta, 2001; Takahashi et al., 2001; Melchior and Hengst, 2002; Schmidt and Muller, 2002). The binding of ICA512 cytoplasmic domain to PIAS proteins, including PIASy and PIAS1, was confirmed by pull-down assays (Fig. 6 A). Specifically, 35S-labeled human ICA512 cytoplasmic domain (residues 601–979) generated by in vitro transcription and translation is recovered on glutathione-sepharose beads coupled with PIASy-GST or PIAS1-GST, but not with GST alone (Fig. 6 A). Likewise, 35S-labeled PIASy interacts specifically with GST-ICA512(601–979) and GST-p53 (Fig. 6 B). Fluorescence resonance energy transfer (FRET) analyses in INS-1 cells transiently cotransfected with ICA512-CFP and PIASy-YFP show that the two proteins interact in the nucleus (Fig. 6 C). Specifically, the ICA512-CFP nuclear signal after photobleaching (Fig. 6 C, circled areas) is increased approximately fivefold upon stimulation (Fig. 6 D). In parallel experiments, no interaction is detected between ICA512-CCF-CFP and the nuclear protein lsm4-YFP (Fig. 6 D and Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200408172/DC1) and between lsm4-CFP and PIASy-YFP (Fig. 6 D) in either resting or stimulating conditions, thereby confirming the specific interaction of ICA512-CCF with PIASy in the nucleus.


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 binds PIASy in the nucleus. (A) SDS-PAGE and autoradiography of in vitro transcribed and translated [35S]methionine-labeled ICA512(601–979) pulled down with glutathione sepharose beads coupled to 0.5 μg PIASy-GST, PIAS1-GST, or GST alone. Lane 1 shows the starting labeled material for the pull-down assay. White lines indicate that intervening lanes have been spliced out. (B) SDS-PAGE and autoradiography of in vitro transcribed and translated [35S]methionine-labeled PIASy pulled down with glutathione sepharose beads coupled to 0.5 μg GST-ICA512(601–979), GST-p53, or GST alone. First lane shows the starting labeled material for the pull-down assay. (C) FRET between ICA512-CFP (pseudocyan) and PIASγ-YFP (pseudogreen) in transiently cotransfected INS-1 cells incubated in resting or stimulating buffer for 105 min after 1 h at rest. The right panels show the signal for ICA512-CFP following the photobleaching of PIASy-YFP in the circled nuclei (arrows). (D) Quantitation of FRET to CFP in INS-1 cells transiently cotransfected with the indicated constructs: ICA512-CFP + PIASy-YFP; ICA512-CFP + lsm4-YFP; lsm4-CFP + PIASy-YFP. Values are from two independent experiments as shown in C. (D) Error bars show mean + SD.
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fig6: ICA512-CCF binds PIASy in the nucleus. (A) SDS-PAGE and autoradiography of in vitro transcribed and translated [35S]methionine-labeled ICA512(601–979) pulled down with glutathione sepharose beads coupled to 0.5 μg PIASy-GST, PIAS1-GST, or GST alone. Lane 1 shows the starting labeled material for the pull-down assay. White lines indicate that intervening lanes have been spliced out. (B) SDS-PAGE and autoradiography of in vitro transcribed and translated [35S]methionine-labeled PIASy pulled down with glutathione sepharose beads coupled to 0.5 μg GST-ICA512(601–979), GST-p53, or GST alone. First lane shows the starting labeled material for the pull-down assay. (C) FRET between ICA512-CFP (pseudocyan) and PIASγ-YFP (pseudogreen) in transiently cotransfected INS-1 cells incubated in resting or stimulating buffer for 105 min after 1 h at rest. The right panels show the signal for ICA512-CFP following the photobleaching of PIASy-YFP in the circled nuclei (arrows). (D) Quantitation of FRET to CFP in INS-1 cells transiently cotransfected with the indicated constructs: ICA512-CFP + PIASy-YFP; ICA512-CFP + lsm4-YFP; lsm4-CFP + PIASy-YFP. Values are from two independent experiments as shown in C. (D) Error bars show mean + SD.
Mentions: Screening by 2-hybrid assays in yeast showed that the cytoplasmic domain of ICA512 binds PIASy (unpublished data). PIASs were originally identified as PIAS (Liu et al., 1998) and have been more recently shown to have E3-sumoylating activity toward numerous transcription factors, including p53 and signal transducers and activators of transcription (STATs; Johnson and Gupta, 2001; Takahashi et al., 2001; Melchior and Hengst, 2002; Schmidt and Muller, 2002). The binding of ICA512 cytoplasmic domain to PIAS proteins, including PIASy and PIAS1, was confirmed by pull-down assays (Fig. 6 A). Specifically, 35S-labeled human ICA512 cytoplasmic domain (residues 601–979) generated by in vitro transcription and translation is recovered on glutathione-sepharose beads coupled with PIASy-GST or PIAS1-GST, but not with GST alone (Fig. 6 A). Likewise, 35S-labeled PIASy interacts specifically with GST-ICA512(601–979) and GST-p53 (Fig. 6 B). Fluorescence resonance energy transfer (FRET) analyses in INS-1 cells transiently cotransfected with ICA512-CFP and PIASy-YFP show that the two proteins interact in the nucleus (Fig. 6 C). Specifically, the ICA512-CFP nuclear signal after photobleaching (Fig. 6 C, circled areas) is increased approximately fivefold upon stimulation (Fig. 6 D). In parallel experiments, no interaction is detected between ICA512-CCF-CFP and the nuclear protein lsm4-YFP (Fig. 6 D and Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200408172/DC1) and between lsm4-CFP and PIASy-YFP (Fig. 6 D) in either resting or stimulating conditions, thereby confirming the specific interaction of ICA512-CCF with PIASy in the nucleus.

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