New aspects of hepatic glucokinase translocation by the glucokinase regulatory protein

BALTRUSCH S; FRANCINI F; TIEDGE M; LENZEN S

Paris

Congreso; 18th Congress of the International Diabetes Federation; 2003

Background and Aims: Glucokinase (GK) acts as a glucose sensor for coupling millimolar glucose concentrations to metabolism. In liver a glucokinase regulatory protein (GRP) modulates GK activity and mediates the nuclear-cytoplasmic translocation of GK in dependence of glucose and fructose metabolites. In our recent studies we investigated the molecular basis of the GK import-export mechanism and the nature of the GRP binding motif inside the GK. Through yeast two-hybrid analyses we could identify the GK amino acid residues Leu-58 and Asn-204 as most important for the GRP interaction. It was the aim of this study to characterize the molecular mechanisms of GK and GRP translocation by real time fluorescence microscopy using wild-type and mutant proteins. Materials and Methods: COS-1 and HeLa cells were transfected with EYFP-GRP and ECFP-GK wild-type protein or proteins with a mutation of the binding motif. Protein localization and colocalization were monitored by epifluorescence and laser scanning microscopy as well as fluorescence resonance energy transfer (FRET) in cells perifused with 5.5 mM or 25 mM glucose. Results: At low glucose concentration (5.5 mM) the ECFP-GK fusion protein was located in the nucleus together with EYFP-GRP. In contrast, cells co-transfected with GRP and the GK mutants L58R/N204Y, the ECFP-GK fusion protein remained predominantly in the cytoplasm at 5.5 mM glucose. In cells cultured at high (25 mM) glucose the ECFP-GK protein was in the cytoplasm whereas the EYFP-GRP protein showed a nuclear localization. However, after a pre-culture of the cells with low glucose the GK wild-type and GRP fusion proteins translocated together from the nucleus to the cytoplasm after perifusion with high glucose. The interaction of GK and GRP could be verified by FRET analyses. GK mutant proteins did not show any translocation from the nucleus. Conclusion: The amino acid Asn-204 plays a pivotal role for the regulation of GK because it confers the interaction with the GRP and is localized within the substrate binding site of the enzyme protein. Importantly, our data provide evidence that GK is shuttled as a complex with GRP from the nucleus to the cytoplasm. Background and Aims: Glucokinase (GK) acts as a glucose sensor for coupling millimolar glucose concentrations to metabolism. In liver a glucokinase regulatory protein (GRP) modulates GK activity and mediates the nuclear-cytoplasmic translocation of GK in dependence of glucose and fructose metabolites. In our recent studies we investigated the molecular basis of the GK import-export mechanism and the nature of the GRP binding motif inside the GK. Through yeast two-hybrid analyses we could identify the GK amino acid residues Leu-58 and Asn-204 as most important for the GRP interaction. It was the aim of this study to characterize the molecular mechanisms of GK and GRP translocation by real time fluorescence microscopy using wild-type and mutant proteins. Materials and Methods: COS-1 and HeLa cells were transfected with EYFP-GRP and ECFP-GK wild-type protein or proteins with a mutation of the binding motif. Protein localization and colocalization were monitored by epifluorescence and laser scanning microscopy as well as fluorescence resonance energy transfer (FRET) in cells perifused with 5.5 mM or 25 mM glucose. Results: At low glucose concentration (5.5 mM) the ECFP-GK fusion protein was located in the nucleus together with EYFP-GRP. In contrast, cells co-transfected with GRP and the GK mutants L58R/N204Y, the ECFP-GK fusion protein remained predominantly in the cytoplasm at 5.5 mM glucose. In cells cultured at high (25 mM) glucose the ECFP-GK protein was in the cytoplasm whereas the EYFP-GRP protein showed a nuclear localization. However, after a pre-culture of the cells with low glucose the GK wild-type and GRP fusion proteins translocated together from the nucleus to the cytoplasm after perifusion with high glucose. The interaction of GK and GRP could be verified by FRET analyses. GK mutant proteins did not show any translocation from the nucleus. Conclusion: The amino acid Asn-204 plays a pivotal role for the regulation of GK because it confers the interaction with the GRP and is localized within the substrate binding site of the enzyme protein. Importantly, our data provide evidence that GK is shuttled as a complex with GRP from the nucleus to the cytoplasm.Glucokinase (GK) acts as a glucose sensor for coupling millimolar glucose concentrations to metabolism. In liver a glucokinase regulatory protein (GRP) modulates GK activity and mediates the nuclear-cytoplasmic translocation of GK in dependence of glucose and fructose metabolites. In our recent studies we investigated the molecular basis of the GK import-export mechanism and the nature of the GRP binding motif inside the GK. Through yeast two-hybrid analyses we could identify the GK amino acid residues Leu-58 and Asn-204 as most important for the GRP interaction. It was the aim of this study to characterize the molecular mechanisms of GK and GRP translocation by real time fluorescence microscopy using wild-type and mutant proteins. Materials and Methods: COS-1 and HeLa cells were transfected with EYFP-GRP and ECFP-GK wild-type protein or proteins with a mutation of the binding motif. Protein localization and colocalization were monitored by epifluorescence and laser scanning microscopy as well as fluorescence resonance energy transfer (FRET) in cells perifused with 5.5 mM or 25 mM glucose. Results: At low glucose concentration (5.5 mM) the ECFP-GK fusion protein was located in the nucleus together with EYFP-GRP. In contrast, cells co-transfected with GRP and the GK mutants L58R/N204Y, the ECFP-GK fusion protein remained predominantly in the cytoplasm at 5.5 mM glucose. In cells cultured at high (25 mM) glucose the ECFP-GK protein was in the cytoplasm whereas the EYFP-GRP protein showed a nuclear localization. However, after a pre-culture of the cells with low glucose the GK wild-type and GRP fusion proteins translocated together from the nucleus to the cytoplasm after perifusion with high glucose. The interaction of GK and GRP could be verified by FRET analyses. GK mutant proteins did not show any translocation from the nucleus. Conclusion: The amino acid Asn-204 plays a pivotal role for the regulation of GK because it confers the interaction with the GRP and is localized within the substrate binding site of the enzyme protein. Importantly, our data provide evidence that GK is shuttled as a complex with GRP from the nucleus to the cytoplasm. Background and Aims: Glucokinase (GK) acts as a glucose sensor for coupling millimolar glucose concentrations to metabolism. In liver a glucokinase regulatory protein (GRP) modulates GK activity and mediates the nuclear-cytoplasmic translocation of GK in dependence of glucose and fructose metabolites. In our recent studies we investigated the molecular basis of the GK import-export mechanism and the nature of the GRP binding motif inside the GK. Through yeast two-hybrid analyses we could identify the GK amino acid residues Leu-58 and Asn-204 as most important for the GRP interaction. It was the aim of this study to characterize the molecular mechanisms of GK and GRP translocation by real time fluorescence microscopy using wild-type and mutant proteins. Materials and Methods: COS-1 and HeLa cells were transfected with EYFP-GRP and ECFP-GK wild-type protein or proteins with a mutation of the binding motif. Protein localization and colocalization were monitored by epifluorescence and laser scanning microscopy as well as fluorescence resonance energy transfer (FRET) in cells perifused with 5.5 mM or 25 mM glucose. Results: At low glucose concentration (5.5 mM) the ECFP-GK fusion protein was located in the nucleus together with EYFP-GRP. In contrast, cells co-transfected with GRP and the GK mutants L58R/N204Y, the ECFP-GK fusion protein remained predominantly in the cytoplasm at 5.5 mM glucose. In cells cultured at high (25 mM) glucose the ECFP-GK protein was in the cytoplasm whereas the EYFP-GRP protein showed a nuclear localization. However, after a pre-culture of the cells with low glucose the GK wild-type and GRP fusion proteins translocated together from the nucleus to the cytoplasm after perifusion with high glucose. The interaction of GK and GRP could be verified by FRET analyses. GK mutant proteins did not show any translocation from the nucleus. Conclusion: The amino acid Asn-204 plays a pivotal role for the regulation of GK because it confers the interaction with the GRP and is localized within the substrate binding site of the enzyme protein. Importantly, our data provide evidence that GK is shuttled as a complex with GRP from the nucleus to the cytoplasm. Background and Aims: Glucokinase (GK) acts as a glucose sensor for coupling millimolar glucose concentrations to metabolism. In liver a glucokinase regulatory protein (GRP) modulates GK activity and mediates the nuclear-cytoplasmic translocation of GK in dependence of glucose and fructose metabolites. In our recent studies we investigated the molecular basis of the GK import-export mechanism and the nature of the GRP binding motif inside the GK. Through yeast two-hybrid analyses we could identify the GK amino acid residues Leu-58 and Asn-204 as most important for the GRP interaction. It was the aim of this study to characterize the molecular mechanisms of GK and GRP translocation by real time fluorescence microscopy using wild-type and mutant proteins. Materials and Methods: COS-1 and HeLa cells were transfected with EYFP-GRP and ECFP-GK wild-type protein or proteins with a mutation of the binding motif. Protein localization and colocalization were monitored by epifluorescence and laser scanning microscopy as well as fluorescence resonance energy transfer (FRET) in cells perifused with 5.5 mM or 25 mM glucose. Results: At low glucose concentration (5.5 mM) the ECFP-GK fusion protein was located in the nucleus together with EYFP-GRP. In contrast, cells co-transfected with GRP and the GK mutants L58R/N204Y, the ECFP-GK fusion protein remained predominantly in the cytoplasm at 5.5 mM glucose. In cells cultured at high (25 mM) glucose the ECFP-GK protein was in the cytoplasm whereas the EYFP-GRP protein showed a nuclear localization. However, after a pre-culture of the cells with low glucose the GK wild-type and GRP fusion proteins translocated together from the nucleus to the cytoplasm after perifusion with high glucose. The interaction of GK and GRP could be verified by FRET analyses. GK mutant proteins did not show any translocation from the nucleus. Conclusion: The amino acid Asn-204 plays a pivotal role for the regulation of GK because it confers the interaction with the GRP and is localized within the substrate binding site of the enzyme protein. Importantly, our data provide evidence that GK is shuttled as a complex with GRP from the nucleus to the cytoplasm.Glucokinase (GK) acts as a glucose sensor for coupling millimolar glucose concentrations to metabolism. In liver a glucokinase regulatory protein (GRP) modulates GK activity and mediates the nuclear-cytoplasmic translocation of GK in dependence of glucose and fructose metabolites. In our recent studies we investigated the molecular basis of the GK import-export mechanism and the nature of the GRP binding motif inside the GK. Through yeast two-hybrid analyses we could identify the GK amino acid residues Leu-58 and Asn-204 as most important for the GRP interaction. It was the aim of this study to characterize the molecular mechanisms of GK and GRP translocation by real time fluorescence microscopy using wild-type and mutant proteins. Materials and Methods: COS-1 and HeLa cells were transfected with EYFP-GRP and ECFP-GK wild-type protein or proteins with a mutation of the binding motif. Protein localization and colocalization were monitored by epifluorescence and laser scanning microscopy as well as fluorescence resonance energy transfer (FRET) in cells perifused with 5.5 mM or 25 mM glucose. Results: At low glucose concentration (5.5 mM) the ECFP-GK fusion protein was located in the nucleus together with EYFP-GRP. In contrast, cells co-transfected with GRP and the GK mutants L58R/N204Y, the ECFP-GK fusion protein remained predominantly in the cytoplasm at 5.5 mM glucose. In cells cultured at high (25 mM) glucose the ECFP-GK protein was in the cytoplasm whereas the EYFP-GRP protein showed a nuclear localization. However, after a pre-culture of the cells with low glucose the GK wild-type and GRP fusion proteins translocated together from the nucleus to the cytoplasm after perifusion with high glucose. The interaction of GK and GRP could be verified by FRET analyses. GK mutant proteins did not show any translocation from the nucleus. Conclusion: The amino acid Asn-204 plays a pivotal role for the regulation of GK because it confers the interaction with the GRP and is localized within the substrate binding site of the enzyme protein. Importantly, our data provide evidence that GK is shuttled as a complex with GRP from the nucleus to the cytoplasm.