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Published online 21 April 2003. doi:10.1083/jcb.200301063
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© The Rockefeller University Press, 0021-9525/2003/4/243 $5.00
The Journal of Cell Biology, Volume 161, Number 2, 243-248

Report

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



Mark A. Rizzo and David W. Piston

Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232

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

Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic ß 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 ß 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 ß cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS.

Key Words: insulin; glucokinase; nitric oxide; nitric oxide synthase; fluorescence resonance energy transfer

* Abbreviations used in this paper: DAF-FM, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; DEANO, diethylamine nitric oxide; FRET, fluorescence resonance energy transfer; GK, glucokinase; L-NAME, NG-nitro-L-arginine-methyl ester; NO, nitric oxide; NOS, NO synthase; nNOS, neuronal NOS.


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