Endogenous Activation of Glucokinase by 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase Is Glucose Dependent
| Autor: | Sara Langer, Sigurd Lenzen, Martin Tobias Kaminski, Simone Baltrusch |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Phosphofructokinase-2
Recombinant Fusion Proteins Endogeny Biology chemistry.chemical_compound Endocrinology Insulin-Secreting Cells Two-Hybrid System Techniques Chlorocebus aethiops Glucokinase Mole Animals Phosphofructokinase 2 Molecular Biology Cells Cultured Original Research chemistry.chemical_classification Activator (genetics) Fructose General Medicine Enzyme Activation Glucose Enzyme chemistry Biochemistry Mutagenesis Cell culture COS Cells |
| Zdroj: | Molecular Endocrinology. 24:1988-1997 |
| ISSN: | 1944-9917 0888-8809 |
| DOI: | 10.1210/me.2010-0115 |
| Popis: | Glucokinase (GK) plays a crucial role as glucose sensor in glucose-induced insulin secretion in pancreatic β-cells. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) acts as an endogenous GK activator. Therefore, the goal of this study was the analysis of GK-PFK-2/FBPase-2 complex formation and its effect on metabolic stimulus-secretion coupling in β-cells in dependence upon glucose. The interaction between GK and PFK-2/FBPase-2 was analyzed in insulin-secreting MIN6 cells with a new fluorescence-based mammalian two-hybrid system. In contrast to the commonly used mammalian two-hybrid systems that require sampling before detection, the system used allows monitoring of the effects of environmental changes on protein-protein interactions on the single-cell level. Increasing the glucose concentration in the cell culture medium from 3 to 10 and 25 mmol/liter amplified the interaction between the enzymes stepwise. Importantly, in line with these results, overexpression of PFK-2/FBPase-2 in MIN6 cells evoked only at 10 and 25 mmol/liter, an increase in insulin secretion. Furthermore, a PFK-2/FBPase-2 mutant with an abolished GK-binding motif neither showed a glucose-dependent GK binding nor was able to increase insulin secretion. The results obtained with the mammalian two-hybrid system could be confirmed by fluorescence resonance energy transfer experiments in COS cells. Furthermore, the established interaction between GK and the liver GRP served in all experiments as a control. Thus, this study clearly showed that binding and activation of GK by PFK-2/FBPase-2 in β-cells is promoted by glucose, resulting in an enhancement of insulin secretion at stimulatory glucose concentrations, without affecting basal insulin secretion. |
| Databáze: | OpenAIRE |
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