Naturally Occurring Glucokinase Mutations Are Associated with Defects in Posttranslational S-Nitrosylation
| Autor: | N. D. Tribble, Michele L. Markwardt, Catherine A. Kraft, Mark A. Rizzo, Anna L. Gloyn, Shi-Ying Ding |
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| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: |
medicine.medical_treatment
Recombinant Fusion Proteins Biology Nicotinamide adenine dinucleotide Transfection Article chemistry.chemical_compound Enzyme activator Mice Endocrinology Cell Line Tumor Insulin-Secreting Cells Glucokinase medicine Fluorescence Resonance Energy Transfer Animals Humans Insulin Point Mutation Post-translational regulation Age of Onset Molecular Biology Wild type General Medicine S-Nitrosylation Molecular Imaging Enzyme Activation Kinetics Luminescent Proteins Glucose chemistry Biochemistry Diabetes Mellitus Type 2 Mutant Proteins Protein Processing Post-Translational Nicotinamide adenine dinucleotide phosphate |
| Popis: | Posttranslational activation of glucokinase (GCK) through S-nitrosylation has been recently observed in the insulin-secreting pancreatic β-cell; however, the function of this molecular mechanism in regulating the physiology of insulin secretion is not well understood. To more fully understand the function of posttranslational regulation of GCK, we examined two naturally occurring GCK mutations that map to residues proximal to the S-nitrosylated cysteine and cause mild fasting hyperglycemia (maturity-onset diabetes of the young; subtype glucokinase). The kinetics of recombinantly generated GCK-R369P and GCK-V367M were assessed in vitro. The GCK-R369P protein has greatly reduced catalytic activity (relative activity index 0.05 vs. 1.00 for wild type), whereas the GCK-V367M has near normal kinetics (relative activity index 1.26 vs. 1.00 for wild type). Quantitative imaging and biochemical assays were used to assess the effect of these mutants on the metabolic response to glucose, GCK activation, and S-nitrosylation of GCK in βTC3 insulinoma cells. Expression of either mutant in βTC3 cells did not affect the metabolic response to 5 mm glucose. However, expression of either mutant blocked the effects of insulin on glucose-stimulated nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate reduction, suggesting defects in posttranslational regulation of GCK. Each of these mutations blocked GCK activation, and prevented posttranslational cysteine S-nitrosylation. Our findings link defects in hormone-regulated GCK S-nitrosylation to hyperglycemia and support a role for posttranslational regulation of GCK S-nitrosylation as a vital regulatory mechanism for glucose-stimulated insulin secretion. |
| Databáze: | OpenAIRE |
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