Report |
Address correspondence to David W. Piston, Dept. of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 735 Light Hall, Nashville, TN 37232. Tel.: (615) 322-7030. Fax: (615) 322-7236. E-mail: dave.piston{at}vanderbilt.edu
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Abstract |
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Key Words: insulin; glucokinase; nitric oxide; nitric oxide synthase; fluorescence resonance energy transfer
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Introduction |
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The molecular mechanism of GK association with secretory granules and the processes that modulate this association are unknown. To address the mechanism of GK association with secretory granules, we examined the role of nitric oxide synthase in this regulation. Neuronal nitric oxide synthase (nNOS) is activated by a rise in intracellular calcium, which is a known response of ß cells to glucose or insulin stimulation (Aspinwall et al., 2000); and nNOS is also known to be localized on insulin secretory granules (Lajoix et al., 2001). Nitric oxide (NO) has also been shown to have a stimulatory affect on glucose-stimulated insulin secretion from both cultured ß cell lines and pancreatic islets (Smukler et al., 2002; Kaneko et al., 2003). However, these findings are highly controversial, and an inhibitory effect on insulin secretion has also been shown using a variety of experimental approaches. (Salehi et al., 1996; Lajoix et al., 2001; Henningsson et al., 2002). This controversy points to the need for more careful consideration of potential targets for NO in the regulation of glucose-stimulated insulin secretion in order to understand the role of NO synthase (NOS) in ß cell function. GK is one potential target for regulation by NO, since GK contains several cysteines that have been shown to be critical for maintaining catalytic activity (Tiedge et al., 2000).
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Results and discussion |
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To determine whether regulation of GK by NO is the result of direct posttranslational modification of GK, we examined whether GK was S-nitrosylated in insulin-treated cells. Nitrosylated proteins from cell lysates were chemically modified with biotin (Jaffrey et al., 2001) before isolation using neutravidin-agarose and analysis by Western blot. Nitrosylated GK was detected only in precipitates from insulin-treated cells but not from untreated cells or cells treated with L-NAME in addition to insulin (Fig. 1 F). Together with our fluorescence-based assays, these results support a model in which changes in GK localization and activity are related to S-nitrosylation of GK.
To test the role of S-nitrosylation in regulating GK, we examined whether site-directed mutagenesis of GK could block its nitrosylation and affect its regulation. Since reaction of NO with cysteines can be greatly enhanced by a consensus nitrosylation motif (Stamler et al., 1997), we examined the primary structure of GK for potential nitrosylation sites. Four such consensus sites were found in GK (C220, C364, C371, and C434), and each was subsequently mutated to serine, an amino acid that does not react with NO. The mutant GK constructs were tagged with YFP, and the S-nitrosylation of the mutated proteins was assessed (Fig. 2 A). Of the four mutations generated, only C371S eliminated GK nitrosylation, although a slight decrease in the amount of nitrosylated GK was observed for the C364S mutation. Furthermore, the C371S mutant stopped insulin-stimulated FRAP to CFP-labeled granules (Fig. 2 B) and changes in FRET (Fig. 2 C). Mutation of C364S did not have a significant effect on insulin-stimulated FRAP to CFP-labeled granules (Fig. 2 B) and changes in FRET (Fig. 2 C), suggesting that nitrosylation of C364 is not critical to GK regulation. Thus, nitrosylation of cysteine 371 plays a key role in modulating GK association with secretory granules and conformational changes that correlate with GK activation.
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Since other proteins that react with NO are known to form complexes containing nNOS (Brenman et al., 1996; Fang et al., 2000; Nedvetsky et al., 2002), we examined the role of nNOS in determining GK localization. To detect interaction between GK and nNOS, we immunoprecipitated endogenous GK from cell lysates and probed for endogenous nNOS by Western blot (Fig. 3 A). We were able to detect nNOS in these precipitates and also from those of expressed GK-YFP and GK(C371S)-YFP. Incubation of GK precipitates with diethylamine nitric oxide (DEANO), a chemical that rapidly releases NO, resulted in elution of nNOS from endogenous GK and GK-YFP precipitates but not from GK(C371S)-YFP precipitates. These data show that GK association with nNOS is both consistent with GK association with secretory granules and also sensitive to the presence of NO.
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Overall, our data indicate that the GK localization and activity in the ß cell are determined by GK association with nNOS and that association is disrupted by GK nitrosylation at cysteine 371. Regulation of GK-NOS association by nitrosylation provides a sensitive means for modulating GK activity, thus affecting glucose-stimulated insulin secretion. Future work on the nature of the GK-NOS association will certainly increase our understanding of the signaling pathways that contribute to regulation of glucose-stimulated insulin secretion. The regulation of GK nitrosylation might also prove to be a useful target for pharmacological manipulation in the treatment of diabetes and other glycemic disorders. In addition, our data demonstrate that changes in protein nitrosylation can regulate protein localization, proteinprotein interactions, and protein function in a highly specific and rapid way that is similar to the role of protein phosphorylation.
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Materials and methods |
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Generation of constructs
Mutations to GK-YFP were made by 4 primer PCR (with Advantage 2 Polymerase Mix; BD Biosciences, CLONTECH Laboratories, Inc.) using the same set of end primers (sense, 5'-GGCACCAAAATCAACGGGAC-3' and antisense, 5'-CTCGCCCTTGCTCACCAT-3') along with primers containing the mutation (C220S sense, 5'-GACCGCCAATCTGAGGTCG-3' and antisense, 5'-CGACCTCAGATTGGCGGTCT-3'; C364S sense, 5'-CACCGACTCCGATATCGTGC-3' and antisense, 5'-CACGATATCGGAGTCGGTGAC-3'; C371S sense, 5'-CCGTGCCTCTGAAAGCGTG-3' and antisense, 5'-CACGCTTTCAGAGGCACGGC-3'; C434S sense, 5'-CACCCAACTCCGAAATCACCT-3' and antisense, 5'-GGTGATTTCGGAGTTGGGTG-3'). Mutated GK was then inserted into CFP-GK-YFP using restriction sites to replace wild-type GK. For bacterial expression, GK and GK(C371S) were excised from GK-YFP plasmids using BglII and XmaI and ligated into the BamHI and XmaI sites in pQE30 (QIAGEN). To create nNOS-CFP constructs, a silent mutation was introduced into the rat cDNA for nNOS in order to remove the AgeI restriction site using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) (sense primer, 5'-GGGATGACAACCGATACCACGAGGACATC-3' and antisense primer, 5'-GATGTCCTCGTGGTATCGGTTGTCATCCC-3'). The cDNA for the modified rat nNOS was then amplified by PCR (sense, 5'-AGCTAGCCACCATGGAAGAGAACACG-3' and antisense, 5'-TTAACCGGTGAGCTGAAAACCTCATCTGC-3') and inserted into the NheI and AgeI restriction sites in the pECFP-nuc vector (BD Biosciences, CLONTECH Laboratories, Inc.), and a derivative vector with the nuclear localization signal removed by BamHI and BglII digestion followed by religation. All primers were custom synthesized and purified by Integrated DNA Technologies. All plasmid constructs were purified using DNA preparation kits (QIAGEN), and the sequences of all constructs were verified by sequencing reactions performed by the Vanderbilt-Ingram Cancer Center DNA Sequencing Shared Resource.
Microscopy
ßTC3 cells were labeled with 5 µM DAF-FM diacetate (Molecular Probes) for 25 min at 25°C in BSA-free BMHH. Observation was with 488 nm excitation and 505530 nm collection by laser scanning confocal microscopy (LSM510; Carl Zeiss MicroImaging, Inc.). During observation, cells were heated to 32°C using a Bioptechs Delta T system. FRAP and intramolecular FRET measurements were performed on cultured ßTC3 cells as described previously (Rizzo et al., 2002). Statistical significance (P < 0.05 by ANOVA or t test as appropriate) is denoted by an asterisk and was determined from at least three granules in the same region or at least three cells for the FRET studies as compared with pretreatment FRET ratios. Data shown is representative of at least three independent trials. Observation of nNOS-CFP-nuc and GK-YFP were performed using the same filter settings as the FRAP measurements. FRET between nNOS-CFP and GK-YFP was observed using 864 nm two-photon excitation along with a KP700/514 main beam splitter and standard CFP and YFP collection using a 40x, 1.3NA F-FLUAR oil immersion objective lens. For the FRET measurements, the temperature was maintained at 37°C using an S-M incubator (Carl Zeiss MicroImaging, Inc.) controlled by the CTI temperature regulator along with humidification and an objective heater.
Detection of nitrosylated proteins
S-nitrosylated proteins were biotinylated and isolated from cell lysates using the protocol described (Jaffrey et al., 2001). Briefly, cells were lysed in HEN buffer (250 mM Hepes, pH 7.7, 1 mM EDTA, 0.1 mM neocuproine) using 0.5% Triton X-100 and 0.5% cholate before protection of free cysteines with methyl methanethiosulfonate (Pierce Chemical Co.) and derivatization of S-nitrosylated cysteines with ascorbic acid and biotin-HPDP (Pierce Chemical Co.). Biotinylated proteins were isolated by binding to neutravidin-agarose (Pierce Chemical Co.) followed by five washes with a high salt buffer (20 mM Hepes, pH 7.7, 600 mM NaCl, 1 mM EDTA, 0.5% Triton X-100) and elution in HNE buffer (20 mM Hepes, pH 7.7, 100 mM NaCl, 1 mM EDTA) plus 100 mM 2-mercaptoethanol. Alternatively, GK-YFPs were isolated by immunoprecipitation with A.v. Peptide Antibody (BD BioSciences, Clontech Laboratories, Inc.) preconjugated to IgG agarose (Sigma-Aldrich). Western blots were then performed on eluted proteins to detect GK and nitrosylated proteins.
Kinetic analysis of GK mutations
His-tagged recombinant GK and GK(C371S) were produced in M15 (pREP4) cells and purified by Ni:NTA affinity chromatography (QIAGEN) precisely as described (Tiedge et al., 1997). Protein concentrations were determined using the Advanced Protein Assay (Cytoskeleton Inc.) at A590 according to the manufacturer's protocol. Kinetic analysis was performed on 2 mU of purified protein at 37°C using a photometric assay containing 50 mM Hepes (pH 7.2), 100 mM KCl, 10 mM MgCl2, 2.5 mM dithiothreitol, 5 mM ATP, 2 mM NAD+, 4 U/ml glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides, and varying concentrations of glucose. 1 U was defined as the amount of protein required to produce 1 µmole NADH per minute. NADH production was assayed from stopped reactions (1 ml cold 500 mM NaHCO3 per 100 µl reaction mix) as described previously (Rizzo et al., 2002), and reaction velocities were calculated from reaction time courses over 2 min as a function of glucose concentration. Km and Vmax were calculated from linear regression analysis of Hanes-Woolf plots using Prism software (Graphpad).
Immunoprecipitation
BMHH-starved cells from one 60-mm dish were collected in cold PBS, pH 7.4 (PBS, prepared from 10x solution [GIBCO BRL]), and resuspended in lysis buffer (HNE buffer with 1% cholate, 1% Triton X-100, 1x Protease Inhibitor Cocktail for use with Mammalian Cells and Tissue Extracts [Sigma-Aldrich]) for 20 min. Following removal of insoluble material by centrifugation (5 min, 3000 g), normalized amounts of protein (determined by Advanced Protein Assay; A590) were incubated for 1 h with primary antibodies for GK (Jetton and Magnuson, 1992) or GFP preconjugated to 25 µl agarose IgG in 750 µl lysis buffer. Precipitates were washed twice in lysis buffer and three times in HNE buffer before analysis by Western blot or treatment with DEANO (Molecular Probes).
Figure preparation
Figures were prepared using Adobe Photoshop® 7.0 software.
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Acknowledgments |
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Funding for this work was provided by National Institutes of Health grants DK60275 (M.A. Rizzo), and DK53434 and CA86283 (both to D.W. Piston), and by National Science Foundation grant BBI-9871063 (to D.W. Piston).
Submitted: 17 January 2003
Revised: 13 March 2003
Accepted: 13 March 2003
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References |
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