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. 2013 Dec 13;288(50):35824-39.
doi: 10.1074/jbc.M113.526632. Epub 2013 Nov 1.

Identification of the ubiquitin-like domain of midnolin as a new glucokinase interaction partner

Anke Hofmeister-Brix  1 , Katrin Kollmann, Sara Langer, Julia Schultz, Sigurd Lenzen, Simone Baltrusch
Affiliations

Identification of the ubiquitin-like domain of midnolin as a new glucokinase interaction partner

Anke Hofmeister-Brix et al. J Biol Chem. .

Abstract

Glucokinase acts as a glucose sensor in pancreatic beta cells. Its posttranslational regulation is important but not yet fully understood. Therefore, a pancreatic islet yeast two-hybrid library was produced and searched for glucokinase-binding proteins. A protein sequence containing a full-length ubiquitin-like domain was identified to interact with glucokinase. Mammalian two-hybrid and fluorescence resonance energy transfer analyses confirmed the interaction between glucokinase and the ubiquitin-like domain in insulin-secreting MIN6 cells and revealed the highest binding affinity at low glucose. Overexpression of parkin, an ubiquitin E3 ligase exhibiting an ubiquitin-like domain with high homology to the identified, diminished insulin secretion in MIN6 cells but had only some effect on glucokinase activity. Overexpression of the elucidated ubiquitin-like domain or midnolin, containing exactly this ubiquitin-like domain, significantly reduced both intrinsic glucokinase activity and glucose-induced insulin secretion. Midnolin has been to date classified as a nucleolar protein regulating mouse development. However, we could not confirm localization of midnolin in nucleoli. Fluorescence microscopy analyses revealed localization of midnolin in nucleus and cytoplasm and co-localization with glucokinase in pancreatic beta cells. In addition we could show that midnolin gene expression in pancreatic islets is up-regulated at low glucose and that the midnolin protein is highly expressed in pancreatic beta cells and also in liver, muscle, and brain of the adult mouse and cell lines of human and rat origin. Thus, the results of our study suggest that midnolin plays a role in cellular signaling of adult tissues and regulates glucokinase enzyme activity in pancreatic beta cells.

Keywords: Fluorescence Resonance Energy Transfer (FRET); Glucokinase; Insulin Secretion; Insulin Synthesis; Midnolin; Pancreatic Islets; Parkin; Two-hybrid System; Ubiquitin; Ubiquitin-like Domain.

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Figures

FIGURE 1.
FIGURE 1.
Identification of a new glucokinase interaction partner in a yeast two-hybrid library screening. A, 13 different library inserts were identified in a yeast two-hybrid library screening showing different growth on SD-selection agar plates containing Leu/Trp/His (open bars), Leu/Trp/Ade (gray bars), or Leu/Trp/His/Ade (black bars). B, growth of AH109 co-expressing pGADT7-sequence17 and pGBKT7-glucokinase (left side of agar plates) or pGBKT7-lamin (right side of agar plates) on SD-Leu/Trp/His selection agar containing 2.5 (1), 5 (2), 7.7 (3), 10 (4), 12.5 (5), or 15 (6) mmol/liter 3-amino-1,2,4-triazol. C, β-galactosidase reporter gene activity in yeast expressing pGADT7-sequence17 and pGBKT7-glucokinase (black bar) or pGBKT7-lamin (open bar). **, p < 0.05 (Student's t test). U, units. D, sequence alignment of sequence17 with midnolin (NCBI protein accession number NP_001178506.1) with homologies marked in gray. The black box frames the ULD. E, shown is a section of a MAFFT (42) alignment of sequence17 with the rat isoform (D4AE48), mouse isoform1 (Q3TPJ7-1), and isoform2 (Q3TPJ7-2), and human isoform (Q504T8) of midnolin.
FIGURE 2.
FIGURE 2.
Glucokinase interacts with the ULD of midnolin preferentially at low glucose. A, the MMTHS plasmids pGL4.EYFP, pBIND.ECFP-ULD or pBIND.ECFP-sequence17 and pACT-glucokinase or pACT were cotransfected, and MIN6 cells were cultured at 3, 10, or 25 mmol/liter glucose. Whereas ECFP is constitutively expressed, EYFP expression indicates protein-protein interaction. The EYFP/ECFP fluorescence ratio was determined every 2 h in the cell nucleus in a semi-automated microscopy approach. B, interaction of BD-sequence17 and AD-glucokinase (black bars) or AD (open bars). C, interaction between BD-ULD and AD-glucokinase (black bars) or AD (open bars). D, time course of interaction between BD-ULD and AD-glucokinase in MIN6 cells cultured at 3 mmol/liter (closed circles, solid line) or 10 mmol/liter (open circles, dashed line). Shown are normalized mean nuclear EYFP/ECFP ratios ± S.E. obtained 43–55 h after transfection of 4–8 individual experiments with a total of 624–6434 nuclei analyzed. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with control; ##, p < 0.01; ###, p < 0.001 compared with 3 mmol/liter glucose (ANOVA/Bonferroni's multiple comparison test).
FIGURE 3.
FIGURE 3.
Glucose-dependent gene expression of ULD-containing proteins. A, sequence alignment of the ULD of sequence17 with the ULD of parkin (Q9WVS6), UBL4A (P21126), and UBAC1 (Q8VDI7). Asterisks, fully conserved residues; double dots, strongly similar properties; dots, weakly similar properties. MIN6 cells (B–E) and NMRI islets (F–I) were cultured at 3, 10, or 25 mmol/liter glucose for 24 h. Gene expression levels of midnolin (Midn, B and F), Park2 (C and G), Ubl4 (D and H), and Ubac1 (E and I) were determined. Relative expression levels normalized to housekeeping gene GAPDH are shown. Data are expressed as the means ± S.E. of three individual experiments. *, p < 0.05; **, p < 0.01 compared with 3 mmol/liter glucose; #, p < 0.05; ##. p < 0.01 compared with 10 mmol/liter glucose (ANOVA/Bonferroni's multiple comparison test).
FIGURE 4.
FIGURE 4.
Homologies and differences between midnolin and parkin. A, comparison of the three-dimensional structures of ubiquitinD (Q921A3), the ULD of sequence17 (midnolin), and the ULD of parkin (Q9JK66). Images were constructed with SWISS-MODEL (47, 48, 50). B, FRETN efficiencies were calculated in COS cells transfected with ECFP-glucokinase and EYFP (white bar), EYFP-ULD (black bar) EYFP-midnolin (gray bar), or EYFP-parkin (white-striped bar). Shown are the means ± S.E. of three individual experiments. *, p < 0.05; ***, p < 0.001 compared with control (ANOVA/Bonferroni's multiple comparison test). MIN6 cells (C and D) were fixed and stained for midnolin (N-terminal part) (red, C) or parkin (red, D) and DAPI (blue, C and D). Representative images shown were obtained from z-stacks after deconvolution. Scale bars, 10 μm.
FIGURE 5.
FIGURE 5.
Effects of the ULD of midnolin, full-length midnolin, and of parkin on glucokinase activity and glucose-induced insulin secretion in MIN6 cells. A, activity of recombinant beta cell glucokinase was determined at 1, 3.125, 5, 6.25, 10, 12.5, 25, and 50 mmol/liter glucose without (closed circles, solid line) and after incubation with 100 nmol/liter recombinant ULD for 5 min (open circles, dashed line). Shown are the means ± S.E. in units/mg protein of six individual experiments. The activity of endogenous glucokinase was determined in MIN6 EYFP cells (closed circles, solid line; B–D), MIN6 EYFP-ULD cells (open circles, dashed line; B), MIN6 cells transiently transfected with EYFP-midnolin (open circles, dashed line, C), and MIN6 EYFP-parkin cells (open circles, dashed line; D). Glucokinase enzyme activity measured at 1 mmol/liter glucose was subtracted from the values obtained at 3.125, 6.25, 12.5, 25, and 50 mmol/liter glucose to exclude the cellular hexokinase activity. Shown are the means ± S.E. in milliunits/mg of cellular protein of three individual experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with control at the same glucose concentration (ANOVA/Bonferroni's multiple comparison test). MIN6 EYFP cells (E), MIN6 EYFP-ULD cells (F), MIN6 cells transiently transfected with EYFP-midnolin (G) and MIN6 EYFP-parkin cells (H) were cultured at 3 mmol/liter (gray bars) or 25 mmol/liter (black bars) glucose for 48 h. After starvation for 1 h, cells were incubated at 3 mmol/liter (open bars) or 25 mmol/liter (closed bars) glucose. Data are the means ± S.E. of 4–11 individual experiments. ***, p < 0.001 compared with 3 mmol/liter glucose; #, p < 0.05; ##, p < 0.01; ###, p < 0.001 compared with MIN6 EYFP cells (ANOVA/Bonferroni's multiple comparison test).
FIGURE 6.
FIGURE 6.
Subcellular localization of midnolin after overexpression in MIN6 cells and its effect on glucokinase expression and cell viability. A–D, MIN6 cells were transiently co-transfected with EYFP-ULD (A, left panel), EYFP-midnolin (B and D, left panels), midnolin-EYFP (C, left panel), and ECFP-glucokinase (A–C, middle panel) or ECFP-Nuc (D, middle panel). EYFP fusion proteins in red, ECFP fusion proteins in green, and the merge images (A-D, right panels) are shown. Representative images shown were obtained from z-stacks after deconvolution. Scale bars, 10 μm. E–F, MIN6 cells were transiently transfected with EYFP (white bar and white horizontal-striped bar; E and F), EYFP-ULD (black bar; E and F), EYFP-midnolin (gray bar; E and F), midnolin-EYFP (cross-striped gray bar, E), and EYFP-parkin (white striped bar; E and F) and treated with 30 μmol/liter camptothecin (white vertical-striped bar; E and F). Cell viability was determined by MTT assay (E) or quantification of cleaved caspase-3 in relation to caspase-3 and GAPDH expression (F). G, glucokinase expression is shown in relation to GAPDH expression. Data are the means ± S.E. of 3–4 individual experiments. **, p < 0.01; ***, p < 0.001 compared with EYFP transfected MIN6 cells (ANOVA/Bonferroni's multiple comparison test).
FIGURE 7.
FIGURE 7.
Co-localization of midnolin and glucokinase in insulin-secreting cells, primary beta cells, and hepatocytes. INS1E cells (A), MIN6 cells (B), primary mouse beta cells (C), mouse pancreatic islets (D), and hepatocytes (F) were fixed and stained for midnolin (N-terminal part) (red; A-C and F) and (C-terminal part) (red; D), glucokinase (green; A-D and F) and with DAPI (blue; A–D and F). G, pancreatic sections were stained for midnolin (N-terminal part) (red), glucokinase (green), and with DAPI (blue; A–D and F). Representative images shown were obtained from z-stacks after deconvolution (B, C, and F) or processed with FV10-ASW software (A, D, and E). Scale bars, 5 (C), 10 (A, B, and F), 20 (E), or 30 (D) μm.
FIGURE 8.
FIGURE 8.
Midnolin expression in different tissues. A and B, brain (open bars), liver (gray bars), and muscle (black bars) were obtained from NMRI mice. Total RNA was isolated, and midnolin (Midn; A) and Park2 (B) gene expression was determined. Shown are relative expression levels normalized to GAPDH. C–F, 40 μg of isolated protein were analyzed by SDS-PAGE and immunoblotted using antibodies against the N-terminal part of midnolin (E), GAPDH (F), and parkin. Quantifications of the 50-kDa midnolin line (C) and the 50-kDa parkin line (D) are shown in relation to the 37-kDa GAPDH line. Shown are the means ± S.E. of three individual experiments. ***, p < 0.001 compared with muscle (ANOVA/Bonferroni's multiple comparison test); #, p < 0.05, ##, p < 0.01 compared with muscle (Student's t test). G and H, 40 μg of protein isolated from MIN6, INS1E, RINm5F, MH7777A, HepG2, and HeLa cells were analyzed by SDS-PAGE and immunoblotted using antibodies against the N-terminal part (G) and C-terminal part (H) of midnolin. I–L, HeLa cells were fixed and stained for midnolin using the N-terminal (green; I and K) or C-terminal (green; J and L) midnolin antibody and for β-tubulin (red; I and J) with MitoTracker® Deep Red FM (red; K and L) and with DAPI (blue; I–L). Representative images shown were obtained from z-stacks after deconvolution. Scale bars, 10 μm.
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