Journal
JOURNAL OF CELL BIOLOGY
Volume 161, Issue 2, Pages 243-248Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1083/jcb.200301063
Keywords
insulin; glucokinase; nitric oxide; nitric oxide synthase; fluorescence resonance energy transfer
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Funding
- NIDDK NIH HHS [F32 DK060275, R01 DK053434, DK53434, DK60275] Funding Source: Medline
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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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