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. 2009 Nov 24;24(1):171–177. doi: 10.1210/me.2009-0138

Naturally Occurring Glucokinase Mutations Are Associated with Defects in Posttranslational S-Nitrosylation

Shi-Ying Ding 1, Nicholas D Tribble 1, Catherine A Kraft 1, Michele Markwardt 1, Anna L Gloyn 1, Mark A Rizzo 1
PMCID: PMC2802892  PMID: 19934346

Abstract

Posttranslational activation of glucokinase (GCK) through S-nitrosylation has been recently observed in the insulin-secreting pancreatic β-cell; however, the function of this molecular mechanism in regulating the physiology of insulin secretion is not well understood. To more fully understand the function of posttranslational regulation of GCK, we examined two naturally occurring GCK mutations that map to residues proximal to the S-nitrosylated cysteine and cause mild fasting hyperglycemia (maturity-onset diabetes of the young; subtype glucokinase). The kinetics of recombinantly generated GCK-R369P and GCK-V367M were assessed in vitro. The GCK-R369P protein has greatly reduced catalytic activity (relative activity index 0.05 vs. 1.00 for wild type), whereas the GCK-V367M has near normal kinetics (relative activity index 1.26 vs. 1.00 for wild type). Quantitative imaging and biochemical assays were used to assess the effect of these mutants on the metabolic response to glucose, GCK activation, and S-nitrosylation of GCK in βTC3 insulinoma cells. Expression of either mutant in βTC3 cells did not affect the metabolic response to 5 mm glucose. However, expression of either mutant blocked the effects of insulin on glucose-stimulated nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate reduction, suggesting defects in posttranslational regulation of GCK. Each of these mutations blocked GCK activation, and prevented posttranslational cysteine S-nitrosylation. Our findings link defects in hormone-regulated GCK S-nitrosylation to hyperglycemia and support a role for posttranslational regulation of GCK S-nitrosylation as a vital regulatory mechanism for glucose-stimulated insulin secretion.

Point mutations that exclusively block glucokinase S-nitrosylation inhibit enhancement of glucose metabolism by insulin.

Glucose phosphorylation by glucokinase (GCK) is the rate-limiting step for insulin secretion from pancreatic β-cells and thus net cellular GCK activity is a critical determinant of β-cell glucose sensitivity (1). This is most powerfully supported by evidence from human genetics. Heterozygous inactivating mutations in the GCK gene cause decreased glucose-stimulated insulin secretion and mild fasting hyperglycemia (2), whereas heterozygous activating mutations cause congenital hyperinsulinemia (3). More than 200 disease-causing mutations in GCK have been reported, and, of these, approximately 45 have been functionally characterized (4,5). Interestingly, a number of these mutations do not have defects in their enzyme kinetics that sufficiently explain the clinical phenotypes observed in patients with these mutations (5). Consequently, there is considerable interest in exploring alternate mutational mechanisms including defects in posttranslational GCK regulation (6,7).

Evidence of posttranslational regulation of GCK in pancreatic β-cells has recently emerged. Localization of GCK to secretory granules (8,9,10) and mitochondria (11) has been observed. GCK remains tightly bound to secretory granules even during stimulation with high-glucose concentrations, suggesting that glucose itself is not a direct regulator of GCK localization (9,12). In contrast, insulin stimulates GCK translocation to the cytoplasm, and increases cellular GCK activity in βTC3 insulinoma cells (10). Regulation by insulin occurs by stimulating dissociation of a complex containing GCK and neuronal-type nitric oxide synthase (NOS) present on the surface of secretory granules (13). Activation of NOS leads to S-nitrosylation of GCK. Mutation of C371 prevents GCK S-nitrosylation, and receptor-stimulated GCK translation and activation (13), suggesting that S-nitrosylation of C371 regulates GCK activation and localization.

Although insulin is a potent regulator of GCK activation in culture, the physiology of autocrine regulation of secretion is complex, and it is unclear whether insulin plays a direct role in acute regulation of secretion (14). To test our hypothesis that GCK S-nitrosylation is a vital mechanism for regulating glucose responsiveness of pancreatic β-cells, we used naturally occurring GCK mutations that cause mild fasting hyperglycemia [maturity-onset diabetes of the young (MODY) subtype GCK] as a tool to assess defects in normal GCK S-nitrosylation. Given that the S-nitrosylation reaction is facilitated by neighboring side chains (15), we hypothesized that mutations proximal to C371 could affect GCK S-nitrosylation. Two GCK-MODY mutations, c. 1099G>A, p.V367M (16) and c. 1106G>C p.R369P (17), were examined in this study. Here we show that these mutants display different kinetics in vitro but did not affect the metabolic response to 5 mm glucose. In contrast, both mutants inhibited the stimulatory effect of insulin on the glucose metabolic response and prevented hormone-regulated GCK activation and S-nitrosylation. These findings demonstrate a deficit for the GCK-V367M mutant protein that is consistent with the clinical presentation of mild fasting hyperglycemia in patients with this mutation and suggest that hormonal control over GCK S-nitrosylation is a vital regulator of glucose homeostasis.

Results

Kinetic characterization of GCK mutant proteins

Naturally occurring GCK mutations c. 1099G>A, p.V367M (16) and c. 1106G>C p.R369P (17) are at residues proximal to the GCK S-nitrosylation site at C371. The kinetic properties of GCK-V367M have been previously reported (5,18) but not in direct comparison with GCK-R369P. Due to the inherent variability of recombinant GCK enzymatic measurements between laboratories, we expressed and purified both GCK mutant proteins to directly compare their kinetic properties (Table 1). Both mutations primarily affect the catalytic rate constant (Kcat), with GCK-V367M marginally more active than the wild type (relative activity index 1.26) and GCK-R369P 30-fold less active (relative activity index 0.05). Interestingly, we observed minimal effects on substrate binding (glucose and ATP) for either mutation (Table 1). Although the in vitro kinetics of GCK-R369P are consistent with the clinical presentation of mild fasting hyperglycemia, the near-normal kinetics of GCK-V367M do not explain the observed clinical phenotype.

Table 1.

The kinetic properties of wild-type and mutant recombinant GST-GCK proteins

Wild type R369P V367 m
Glucose affinity S0.5 (mm) 7.35 ± 0.07 7.48 ± 0.29 8.06 ± 0.10
Glucose cooperativity coefficient (Hill number) 1.69 ± 0.02 1.51 ± 0.02 1.66 ± 0.01
ATP affinity Km (mm) 0.57 ± 0.03 0.61 ± 0.03 0.48 ± 0.01
Kcata 59.70 ± 0.44 2.38 ± 0.04 79.94 ± 0.61
Kcatb 63.42 ± 0.51 2.18 ± 0.72 90.01 ± 0.72
Relative activity index 1 0.05 1.26
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Data are means ± sem The results are the kinetic analysis from two (V367 m) and three (wild-type and R369P) independent expressions of GST-GCK proteins. Kcata is calculated from glucose S0.5 assays and Kcatb from ATP assays. 

Effect of GCK-MODY mutants on the metabolic response to glucose

To characterize the metabolic response to glucose in single cells expressing GCK mutant proteins, we used quantitative fluorescence imaging of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] in living β-cells (19,20). GCK mutants were fused to the mCherry red fluorescent protein (21) because mCherry has sufficiently red fluorescence to permit quantification of NAD(P)H fluorescence. Plasmids were transiently transfected into βTC3 cells, and cells expressing GCK-mCherry mutants were identified by confocal microscopy (Fig. 1A). Two-photon fluorescence imaging was used to quantify glucose-stimulated changes in NAD(P)H fluorescence (19,20,22). We first compared the NAD(P)H responsiveness of βTC3 cells transiently transfected with GCK-mCherry to neighboring untransfected cells (Fig. 1B). Quantification of GCK-mCherry expression is shown in Fig. 1D. Control treatment with vehicle (+H2O) or insulin alone did not change NAD(P)H levels, whereas treatment with a low-glucose dose (5 mm) produced equivalent rises in transfected and untransfected cells. Both transfected and untransfected cells treated with insulin and 5 mm glucose showed an enhanced metabolic response compared with glucose alone (ANOVA; P < 0.01 for untransfected cells; P < 0.001 for transfected cells; n > 7). This is consistent with posttranslational activation of GCK by S-nitrosylation (13).

Figure 1.

Figure 1

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Effects of GCK-mCherry expression on the metabolic response of βTC3 cells to glucose. A, βTC3 cells expressing GCK-mCherry were identified by confocal imaging (red) and overlaid with time-resolved two-photon images of NAD(P)H autofluorescence (green). B, The NAD(P)H responses to various treatments was measured in cells expressing GCK-mCherry (black bars) and untransfected cells (white bars). The glucose response (5 mm, 3 min) was similar for transfected and untransfected cells (n ≥ 4 cells; *, P < 0.05; ***, P < 0.001 by ANOVA vs. initial fluorescence). Simultaneous addition of insulin (100 nm) and glucose (5 mm, 3 min) enhances the glucose response. Neither group responded to control stimulations with an equivalent amount of vehicle (+H2O, 3 min), or insulin alone (100 nm, 3 min). C, Expression of GCK-MODY mutants did not significantly affect the NAD(P)H response to glucose (5 mm, 3 min) stimulation compared with cells expressing wild-type GCK-mCherry proteins (black bars; ANOVA, P > 0.05 for all groups; n > 6 cells). However, the effects of insulin stimulation on the metabolic response to glucose was significantly inhibited in GCK-MODY-mCherry expressing cells compared with wild-type (wt) GCK-mCherry expressing cells (white bars, ANOVA; *, P < 0.05; n > 6 cells). Expression of a mutant GCK(C220S) that retains S-nitrosylation (13), but has reduced activity (23), did not negate insulin responsiveness. Changes in NAD(P)H fluorescence were normalized to prestimulation fluorescence intensities. The inhibited metabolic response is consistent with defects in GCK S-nitrosylation. Error bars indicate sem for panels B and C. D, Expression of GCK-mCherry mutants was quantified using recombinant H6mCherry as a reference standard. Bars indicate the mean, and error bars indicate sem (ANOVA; ***, P < 0.001 compared with wild type; n > 10 cells). Glc, Glucose; Ins, insulin.

To test the effect of MODY GCK mutations on insulin-enhanced metabolism of glucose, we introduced two MODY mutations into the GCK-mCherry and expressed these mutants in βTC3 cells by transient transfection (Fig. 1C). Expression of mutant GCK-R369P and GCK-V367M mCherry fusions did not affect the NAD(P)H response to 5 mm glucose compared with cells expressing wild-type GCK-mCherry (Fig. 1C; ANOVA; P > 0.05). When treated with insulin to stimulate GCK S-nitrosylation, however, cells expressing mutant GCK showed a decreased NAD(P)H response to glucose compared with wild-type GCK-expressing cells. (Fig. 1C, white bars; ANOVA, P < 0.05 compared with wild-type GCK-mCherry). Expression of GCK-V367M and GCK-R369P diminished the NAD(P)H response by a similar magnitude, despite differing glucose phosphorylation kinetics. To test whether these effects were related to defects in S-nitrosylation or decreased GCK activity, we transfected βTC3 cells with GCK(C220S)-mCherry. This mutation has diminished glucose-phosphorylating ability (23) but does not affect GCK nitrosylation or GCK activation by insulin (13). Expression of this mutant did not affect metabolism of glucose in the absence or presence of insulin compared with wild-type GCK (Fig. 1C).

Effects of GCK-MODY mutations on GCK activation

Posttranslational activation of GCK can be detected in living cells using a Förster resonance energy transfer (FRET)-based assay (10). Optimized variants of cyan and yellow fluorescent proteins (mCerulean and mVenus) were fused to opposing ends of wild-type and mutant GCK. Activation of GCK produces a conformational change in the sensor that decreases FRET between mCerulean and mVenus fluorescent proteins (24), and blockage of GCK S-nitrosylation pharmacologically or by mutation prevents the insulin-stimulated decrease in FRET (13). Insulin stimulation (100 nm, 3 min) produced a statistically significant decrease (P < 0.0001, t test) in the mVenus/mCerulean fluorescence ratio in the wild-type sensor, whereas treatment with vehicle alone (water) produced no effect (Fig. 2). No decrease in the mVenus/mCerulean fluorescence ratio was observed in cells expressing the FRET-GCK construct containing R369P or V367M. These results are consistent with decreased S-nitrosylation of GCK mutants (13).

Figure 2.

Figure 2

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GCK-MODY mutations prevent activation of the FRET-GCK sensor. Activation of GCK by S-nitrosylation produces protein conformational changes in GCK that can be measured using a FRET-based sensor (10). mCerulean and mVenus fluorescent proteins inserted on opposing ends of GCK move apart from one another after GCK activation, and decreased FRET is observed after insulin stimulation [3 min, 100 nm; black bars compare to pretreatment FRET ratio (mVenus/mCerulean fluorescence) in white]. MODY mutations were introduced into this sensor and expressed in βTC3 cells before stimulation with insulin as described in Materials and Methods. A control stimulation of an equal amount of vehicle (+H2O) without insulin is shown for the wild-type (wt) sensor. Statistical significance from prestimulation FRET ratio as indicated by * (t test; criteria set to P < 0.05; P < 0.0001for wild type; n > 14 cells). Error bars indicate the sem.

Effect of GCK-MODY mutations on GCK S-nitrosylation

To test whether defects in activation of GCK-R369P and GCK-V367M are associated with defects in S-nitrosylation, we expressed mVenus-labeled GCK in βTC3 cells. Cells were lysed 24 h after transfection, and S-nitrosylated cysteines were covalently labeled with biotin (25). mVenus-tagged GCK proteins were then isolated by immunoprecipitation with an antifluorescent protein antibody and resolved by Western blot (Fig. 3). The GCK pulldown was equally efficient for wild-type and mutant proteins. However, biotinylated protein was only detected for wild-type-GCK. GCK-V367M and GCK-R369P were equally efficient at suppressing GCK S-nitrosylation as direct mutation of the S-nitrosylation site (C371S).

Figure 3.

Figure 3

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V367M and R369P inhibit GCK S-nitrosylation. Mutant and wild-type GCK-mVenus proteins were expressed in βTC3 cells and assayed for S-nitrosylation using the biotin switch assay (25). S-nitrosylated proteins from cell lysates were labeled with biotin, and GCK-mVenus proteins were immunoprecipitated. Precipitates were analyzed by Western blot using anti-GFP antibodies (top) and streptavidin-horseradish peroxidase (bottom) to detect S-nitrosylated proteins. Strept-HRP, Streptavidin-horseradish peroxidase.

Discussion

Here we report that defects in site-specific cysteine S-nitrosylation of GCK are associated with naturally occurring mutations known to cause mild fasting hyperglycemia in humans. Both V367M and R369P GCK-MODY mutations prevent GCK S-nitrosylation and posttranslational activation and similarly affect the metabolic response to glucose in living cells even though the in vitro kinetics of these mutants are dissimilar. Notably, GCK-V367M has near normal kinetics, and the total effects of this mutation on GCK thermal stability and kinetics are insufficient to explain the clinical presentation of this mutation (4,5,18,26). Furthermore, mathematical modeling based on the kinetics and stability of GCK-V367M has calculated that β-cells expressing this mutant enzyme would have a threshold for glucose-stimulated insulin secretion of approximately 4.75 mm (5). Because the predicted threshold is very close to the wild-type threshold (∼5 mm), the kinetic effects of this mutant are insufficient to account for the mild fasting hyperglycemia seen in patients with this mutation. The inability of the in vitro kinetics of GCK-V367M to account for the clinical phenotype observed supports a critical role for GCK S-nitrosylation in regulating glucose homeostasis.

The defects in hormone-stimulated GCK S-nitrosylation observed in this study are consistent with clinical presentation of mild fasting hyperglycemia caused by these naturally occurring mutations. Our single-cell assay performed in cells expressing these GCK mutant proteins show diminished metabolic responsiveness to glucose in the presence of insulin, suggesting impaired GCK activity under these conditions. Rises in NAD(P)H fluorescence display a sigmoidal dose response to glucose that is consistent with limitation by GCK kinetics (20), and deletion of GCK from individual β-cells impairs glucose-stimulated rises in NAD(P)H fluorescence (22). Thus, glucose-stimulated rises in NAD(P)H fluorescence strongly reflect cellular GCK activity. Single-cell measures have an additional advantage because they permit quantification of the short-term effects of transiently expressed GCK mutants, thus minimizing complications that could arise from chronic metabolic disruption.

Acute regulation of GCK by S-nitrosylation suggests an obvious mechanism for postprandial regulation of insulin secretion. We find that insulin potently regulates GCK activity in culture and has been a useful agonist in elucidating the molecular mechanisms of GCK regulation at the cellular level (10,13). However, the participation of insulin in positive autocrine feedback regulation of insulin secretion is far less certain and a subject of much debate (14). Because the physiological complexities of primary culture systems are notably absent from our model system, it is unknown whether the phenotypes observed in the naturally occurring mutant GCK proteins V367M and R369P reflect defects exclusive to insulin receptor signaling in pancreatic β-cells. Alternatively, a general defect in regulating of GCK S-nitrosylation may better explain the clinical phenotype. Notably, neuronal-type NOS, which is responsible for GCK S-nitrosylation (13), is tightly regulated by rises in intracellular Ca2+ (27). Because regulated release of Ca2+ from intracellular stores is not exclusive to insulin-receptor signaling pathways, the phenomena of GCK S-nitrosylation is likely applicable to other receptor signaling systems in pancreatic β-cells. Continued exploration into the mechanisms that control GCK nitrosylation in β-cells will undoubtedly shed further light on the subject.

Our findings also point to very tight control of cellular GCK activity at the posttranslational level to an extent in the β-cell that has not been previously considered. Previous studies have focused on the role of protein-protein interactions (28) and glucose (8,12) in regulating GCK activity and localization. In contrast, our findings show critical modulation of GCK activity by cell-surface receptors that does not directly correlate with expression levels of exogenous GCK protein. Expression of GCK-mCherry did not significantly affect glucose-stimulated increases in NAD(P)H fluorescence compared with neighboring untransfected cells (Fig. 1B; t test, P > 0.05) despite overexpression of GCK. Use of the same promoter to express similar fluorescent protein-tagged GCK proteins produces approximately 10- to 14-fold (7) increase in GCK activity in MIN6 cells, and we find a similar increase in our GCK-mCherry-expressing cells. Our finding that exogenous expression of active and inactive GCK mutations do not perturb NAD(P)H production of 5 mm glucose in this cell type is also consistent with expectations based on previous findings. Overexpression of GK-green fluorescent protein (GFP) in MIN6 cells did not affect the threshold for insulin secretion (9), demonstrating that increased GCK expression alone is insufficient to alter the threshold for insulin secretion. For βTC cells, the metabolic response to 5 mm glucose is largely influenced by hexokinase activity (29), in part because of the low affinity of GCK for glucose at this dose. For the insulin-treated groups, the effect of GCK-V367M expression on the metabolic response to glucose was identical in magnitude to the effect of GCK-R369P expression, even though the in vitro activities of these molecules are very different. Yet, both mutants disrupt S-nitrosylation, and this was sufficient to blunt the effect of hormone stimulation. In contrast, expression of GCK-C220S, which does not affect GCK S-nitrosylation (13), but has reduced GCK activity (23), did not significantly affect insulin responsiveness. Taken together, a model incorporating cell surface-receptor-mediated control over posttranslational GCK activity best explains these data.

In conclusion, we report an association between GCK S-nitrosylation and mild fasting hyperglycemia. These findings point to a role for hormonal regulation in acute regulation of GCK function in pancreatic β-cells, and suggest that defects in posttranslational GCK regulation could lead to diabetes. Future work will be required to understand the molecular mechanisms that control GCK in healthy and diabetic islets, as well as the physiological context of GCK regulation by cell surface receptors.

Materials and Methods

Construct preparation

We exchanged the fluorescent proteins used in previous GCK constructs (13) for brighter monomeric variants to enhance quantification. Protein expression is regulated by the cytomegalovirus promoter that is found in the original pEGFP-C1 plasmid (CLONTECH Laboratories, Inc., Mountain View, CA). GCK-mVenus, mCerulean-GCK-mVenus, and NOS-mCerulean were used for this study. To minimize fluorescence cross talk in our NAD(P)H quantification studies, GCK-mCherry fusions were created by subcloning the GCK mutants into the pmCherry-N3 vector. V367M and R369P point mutations were introduced into GCK-mVen using QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). For V367M, we used sense primer 5′-ACTGCGATATCATGCGCCGTGC-3′ and antisense primer 5′-CACGGCGCATGATATCGCAGTC-3′. For R369P, we used sense primer 5′-ATATCGTGCGCCCTGCCTGTGAA-3′ and antisense primer 5′-TCACAGG CAGGGCGCACGATA-3′. Successful cloning was verified by DNA sequencing (University of Maryland Biopolymer Core, Baltimore, MD).

Kinetic analysis

Recombinant human islet wild type (wild type) and mutant enzymes were generated and expressed in the form of glutathionyl S-transferase (GST) fusion proteins using methods previously described, with the following modifications (30). The Escherichia coli cell line BL21 was used to improve protein yield, and the purity of wild-type and mutant GST-GCK was assessed on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Enzymatic properties of recombinant GCK proteins were analyzed spectrophotometrically as previously described (4).

Cell culture

βTC3 cells were cultured for microscopy and biochemical experiments as previously described (13). To achieve a resting baseline condition, cells were starved by washing four times with BMHH buffer (125 mm NaCl; 5.7 mm KCl; 2.5 mm CaCl2; 1.2 mm MgCl2; 10 mm HEPES, pH 7.4) containing 0.1% BSA (Sigma-Aldrich, St. Louis, MO) and incubated for 4 h before experimentation. Transfections were performed using FuGene 6 (Roche, Indianapolis, IN) according to the manufacturer’s recommended protocol. Experiments were performed approximately 24 h after transfection.

Purification of mCherry

To generate purified mCherry protein, a (His)6 variant was generated by subcloning the mCherry sequence from the pmCherry-C1 plasmid into the pQE9-N1 vector (31) using NheI and HindIII restriction sites. The H6mCherry plasmid was transformed into M15(pRep4) cells for protein production as previously described (31). Bacterial pellets were lysed as previously described (31), and protein was purified using Ni loaded Hitrap Chelating HP columns (GE Healthcare, Piscataway, NJ), as per the manufacturer’s protocol. Protein concentration was quantified using the Advanced Protein Assay (Sigma-Aldrich, St. Louis, MO) and SDS-PAGE, using BSA as a protein standard.

Quantitative imaging

Quantitative imaging was performed on a LSM510 META/NLO confocal system using a 1.3 NA, 40× oil immersion Plan-Neofluar lens (Carl Zeiss Microimaging, Thornwood, NY). Temperature was maintained at 32 C using a CO2 incubation system (Carl Zeiss Microimaging). FRET-GCK imaging of βTC3 cells expressing mCerulean-GCK-mVenus was performed using an optimized spectral imaging strategy as previously described (31). A baseline average mVenus/mCerulean fluorescence FRET ratio for individual cells was calculated from three images collected before pharmacological manipulation, and from three images collected 3 min after stimulation. These averages were used to calculate the change in ratio for individual cells. Statistical significance was determined using t tests (Prism 5; GraphPad Software, La Jolla, CA).

For quantification of NAD(P)H fluorescence, samples were illuminated using 710 nm excitation and collection with a 380–550 nm filter. Cells expressing GCK-mCherry were identified before NAD(P)H imaging using 543-nm laser excitation and 565–615 nm collection. Images were filtered using a median filter, and the average fluorescence intensities for regions of interest were extracted using Zeiss software. The stimulus- induced change in NAD(P)H fluorescence was calculated from background-subtracted values and analyzed for statistical significance using Prism 5 software. The cellular concentration of GCK-mCherry constructs was quantified by generating a standard concentration curve of purified mCherry solutions in PBS according to the method of Piston et al. (32). Standard H6mCherry solutions were prepared in Lab-Tek eight-chamber cover glass wells and imaged under equivalent conditions as GCK-mCherry fluorescence in transfected cells.

Biotin-switch assay

βTC3 cells transfected with GCK-mVenus plasmids were treated with insulin (5 min) to stimulate GCK S-nitrosylation. Nitrosylated cysteines were specifically labeled with biotin using the biotin-switch assay as previously described (25). GCK-mVenus proteins were precipitated using mouse anti-GFP antibodies (Abcam, Cambridge, MA) preconjugated to rabbit antimouse IgG-agarose (Sigma-Aldrich). GCK-mVenus and biotinylated proteins were resolved by Western blot using anti-GFP and peroxidase-conjugated streptavidin (Thermo Scientific, Rockford, IL).

Acknowledgments

We thank Drs. B. Selvakumar and S. Snyder of the Johns Hopkins University for assistance with the biotin switch assay. We also thank L. He for assistance with plasmid preparation and tissue culture and A. Nkobena for assistance with construction of the H6mCherry plasmid.

Footnotes

This work was supported by National Institutes of Health Grants R21-DK067415 and R01-DK077140 (to M.A.R.). Pilot and feasibility funds were made available from centers supported by National Institutes of Health Grants P60-DK079637 and P30-DK072488 (to M.A.R.) and an Independent New Investigator award from the University of Maryland School of Medicine. A.L.G. is a Medical Research Council New Investigator (81696). Work in Oxford was funded by the Medical Research Council, Diabetes UK, and the Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom.

Disclosure Summary: The authors have nothing to disclose.

First Published Online November 24, 2009

Abbreviations: FRET, Förster resonance energy transfer; GCK, glucokinase; GFP, green fluorescent protein; GST, glutathionyl S-transferase; NAD(P)H, reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate; NOS, nitric oxide synthase; MODY, maturity onset diabetes of the young.

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