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Regulation of beta cell glucokinase by S-nitrosylation and association with nitric oxide synthase.

Rizzo MA, Piston DW - J. Cell Biol. (2003)

Bottom Line: Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO).Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK.GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 735 Light Hall, Nashville, TN 37232, USA.

ABSTRACT
Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic beta cells. Insulin regulates GK activity by modulating its association with secretory granules, although little is known about the mechanisms involved in regulating this association. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO). Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK. Furthermore, inhibition of NO synthase (NOS) activity blocks insulin-stimulated changes in both GK association with secretory granules and GK conformation. Mutation of cysteine 371 to serine blocks S-nitrosylation of GK and causes GK to remain tightly bound to secretory granules. GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer. Finally, attachment of a nuclear localization signal sequence to NOS drives GK to the nucleus in addition to its normal cytoplasmic and granule targeting. Together, these data suggest that the regulation of GK localization and activity in pancreatic beta cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS.

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Nitrosylation of cysteine 371 is required for GK regulation. (A) Mutant GK-YFP constructs were expressed in βTC3 cells. Nitrosylated proteins were biotinylated before immunoprecipitation of GK-YFP proteins and analysis by Western blot for GK (anti-GK) or biotinylated proteins using peroxidase-conjugated streptavidin (Strept-HRP). (B) Association of the GK-YFP mutants with secretory granules was measured using FRAP in cells expressing the indicated GK-YFP construct and proinsulin-CFP. Fluorescence intensity of granule-associated GK-YFP is expressed as the percentage of prebleached intensity immediately after bleaching (white bars) and 2 s into recovery (black bars). Statistical significance from initial postbleach intensity (P < 0.05, t test) is denoted by an asterisk. (C) Mutations were made in CFP-GK-YFP and expressed in βTC3 cells. Cells were starved for 4 h in BMHH, and the normalized FRET ratio was calculated before (white bars) and after (black bars) stimulation with 100 nM insulin (5 min). Statistical significance from pretreatment FRET ratio (P < 0.05, t test) is denoted by an asterisk.
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fig2: Nitrosylation of cysteine 371 is required for GK regulation. (A) Mutant GK-YFP constructs were expressed in βTC3 cells. Nitrosylated proteins were biotinylated before immunoprecipitation of GK-YFP proteins and analysis by Western blot for GK (anti-GK) or biotinylated proteins using peroxidase-conjugated streptavidin (Strept-HRP). (B) Association of the GK-YFP mutants with secretory granules was measured using FRAP in cells expressing the indicated GK-YFP construct and proinsulin-CFP. Fluorescence intensity of granule-associated GK-YFP is expressed as the percentage of prebleached intensity immediately after bleaching (white bars) and 2 s into recovery (black bars). Statistical significance from initial postbleach intensity (P < 0.05, t test) is denoted by an asterisk. (C) Mutations were made in CFP-GK-YFP and expressed in βTC3 cells. Cells were starved for 4 h in BMHH, and the normalized FRET ratio was calculated before (white bars) and after (black bars) stimulation with 100 nM insulin (5 min). Statistical significance from pretreatment FRET ratio (P < 0.05, t test) is denoted by an asterisk.

Mentions: 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.


Regulation of beta cell glucokinase by S-nitrosylation and association with nitric oxide synthase.

Rizzo MA, Piston DW - J. Cell Biol. (2003)

Nitrosylation of cysteine 371 is required for GK regulation. (A) Mutant GK-YFP constructs were expressed in βTC3 cells. Nitrosylated proteins were biotinylated before immunoprecipitation of GK-YFP proteins and analysis by Western blot for GK (anti-GK) or biotinylated proteins using peroxidase-conjugated streptavidin (Strept-HRP). (B) Association of the GK-YFP mutants with secretory granules was measured using FRAP in cells expressing the indicated GK-YFP construct and proinsulin-CFP. Fluorescence intensity of granule-associated GK-YFP is expressed as the percentage of prebleached intensity immediately after bleaching (white bars) and 2 s into recovery (black bars). Statistical significance from initial postbleach intensity (P < 0.05, t test) is denoted by an asterisk. (C) Mutations were made in CFP-GK-YFP and expressed in βTC3 cells. Cells were starved for 4 h in BMHH, and the normalized FRET ratio was calculated before (white bars) and after (black bars) stimulation with 100 nM insulin (5 min). Statistical significance from pretreatment FRET ratio (P < 0.05, t test) is denoted by an asterisk.
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fig2: Nitrosylation of cysteine 371 is required for GK regulation. (A) Mutant GK-YFP constructs were expressed in βTC3 cells. Nitrosylated proteins were biotinylated before immunoprecipitation of GK-YFP proteins and analysis by Western blot for GK (anti-GK) or biotinylated proteins using peroxidase-conjugated streptavidin (Strept-HRP). (B) Association of the GK-YFP mutants with secretory granules was measured using FRAP in cells expressing the indicated GK-YFP construct and proinsulin-CFP. Fluorescence intensity of granule-associated GK-YFP is expressed as the percentage of prebleached intensity immediately after bleaching (white bars) and 2 s into recovery (black bars). Statistical significance from initial postbleach intensity (P < 0.05, t test) is denoted by an asterisk. (C) Mutations were made in CFP-GK-YFP and expressed in βTC3 cells. Cells were starved for 4 h in BMHH, and the normalized FRET ratio was calculated before (white bars) and after (black bars) stimulation with 100 nM insulin (5 min). Statistical significance from pretreatment FRET ratio (P < 0.05, t test) is denoted by an asterisk.
Mentions: 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.

Bottom Line: Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO).Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK.GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 735 Light Hall, Nashville, TN 37232, USA.

ABSTRACT
Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic beta cells. Insulin regulates GK activity by modulating its association with secretory granules, although little is known about the mechanisms involved in regulating this association. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO). Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK. Furthermore, inhibition of NO synthase (NOS) activity blocks insulin-stimulated changes in both GK association with secretory granules and GK conformation. Mutation of cysteine 371 to serine blocks S-nitrosylation of GK and causes GK to remain tightly bound to secretory granules. GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer. Finally, attachment of a nuclear localization signal sequence to NOS drives GK to the nucleus in addition to its normal cytoplasmic and granule targeting. Together, these data suggest that the regulation of GK localization and activity in pancreatic beta cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS.

Show MeSH