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

Rizzo MA, Piston DW
J Cell Biol. 2003 161 (2): 243-8

PMID: 12707306 · PMCID: PMC2172922 · DOI:10.1083/jcb.200301063

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.

MeSH Terms (19)

Active Transport, Cell Nucleus Animals Cells, Cultured Cysteine Fluorescence Resonance Energy Transfer Fluorescent Dyes Glucokinase Insulin Insulin Secretion Islets of Langerhans Mice Mutation Nitric Oxide Nitric Oxide Synthase Nitrosation Nitroso Compounds Recombinant Fusion Proteins Secretory Vesicles Sulfur

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