Interaction of Glucokinase With the Liver Regulatory Protein Is Conferred by Leucine-Asparagine Motifs of the Enzyme
- 1Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany
- 2Institute of Medical Biochemistry and Molecular Biology, University of Rostock, Rostock, Germany
- Address correspondence and reprint requests to Dr. Simone Baltrusch, Institute of Clinical Biochemistry, Hannover Medical School, D-30623 Hannover, Germany. E-mail: baltrusch.simone{at}mh-hannover.de
Abstract
The glucokinase regulatory protein (GRP) plays a pivotal role in the regulation of metabolic flux in liver by the glucose-phosphorylating enzyme glucokinase. Random peptide phage display library screening for binding partners of GRP allowed the identification of an asparagine-leucine consensus motif. Asparagine-leucine motifs of glucokinase located in the hinge region, as well as in the large domain, were changed by site-directed mutagenesis. The L58R/N204Y and the L309R/N313Y glucokinase mutants showed a significantly reduced interaction with GRP. The L355R/N350Y mutant had a fivefold-higher binding affinity for GRP than wild-type glucokinase. Imaging of glucokinase and GRP fluorescence fusion proteins revealed that the L58R/N204Y glucokinase mutant lacked glucose-dependent translocation by GRP, whereas the L355R/N350Y glucokinase mutant was trapped in the nucleus due to high affinity for GRP. The results indicate that the L58/N204 motif in the hinge region confers binding to GRP, while the L355/N350 motif may modulate the binding affinity for GRP. This latter motif is part of the α10 helix of glucokinase and accessible to GRP in the free and complex conformation.
- ECFP, enhanced cyan fluorescent protein
- ELISA, enzyme-linked immunosorbent assay
- EYFP, enhanced yellow fluorescent protein
- GRP, glucokinase regulatory protein
- GST, glutathione S-transferase
Footnotes
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- Accepted July 15, 2005.
- Received May 11, 2005.
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