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Characterization of Glucokinase-binding Protein Epitopes by a Phage-displayed Peptide Library

2001; Elsevier BV; Volume: 276; Issue: 47 Linguagem: Inglês

10.1074/jbc.m105470200

1083-351X

Simone Baltrusch, Sigurd Lenzen, David A. Okar, Alex J. Lange, Markus Tiedge,

Biochemical and Molecular Research

The low affinity glucose-phosphorylating enzyme glucokinase shows the phenomenon of intracellular translocation in beta cells of the pancreas and the liver. To identify potential binding partners of glucokinase by a systematic strategy, human beta cell glucokinase was screened by a 12-mer random peptide library displayed by the M13 phage. This panning procedure revealed two consensus motifs with a high binding affinity for glucokinase. The first consensus motif, LSA XX VAG, corresponded to the glucokinase regulatory protein of the liver. The second consensus motif, SLKVWT, showed a complete homology to the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which acts as a key regulator of glucose metabolism. Through yeast two-hybrid analysis it became evident that the binding of glucokinase to PFK-2/FBPase-2 is conferred by the bisphosphatase domain, whereas the kinase domain is responsible for dimerization. 5′-Rapid amplification of cDNA ends analysis and Northern blot analysis revealed that rat pancreatic islets express the brain isoform of PFK-2/FBPase-2. A minor portion of the islet PFK-2/FBPase-2 cDNA clones comprised a novel splice variant with 8 additional amino acids in the kinase domain. The binding of the islet/brain PFK-2/ FBPase-2 isoform to glucokinase was comparable with that of the liver isoform. The interaction between glucokinase and PFK-2/FBPase-2 may provide the rationale for recent observations of a fructose-2,6-bisphosphate level-dependent partial channeling of glycolytic intermediates between glucokinase and glycolytic enzymes. In pancreatic beta cells this interaction may have a regulatory function for the metabolic stimulus-secretion coupling. Changes in fructose-2,6-bisphosphate levels and modulation of PFK-2/FBPase-2 activities may participate in the physiological regulation of glucokinase-mediated glucose-induced insulin secretion.