Characterization of glucokinase-binding protein epitopes by a phage-displayed peptide library - Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel interaction partner

The low affinity glucose-phosphorylating enzyme glucokinase shows the pheno menon of intracellular translocation in beta cells of the pancreas and the liver. To identify potential binding partners of glucokinase by a systemati c strategy, human beta cell glucokinase was screened by a 12-mer random pep tide library displayed by the M13 phage. This panning procedure revealed tw o consensus motifs with a high binding affinity for glucokinase. The first consensus motif, LSAXXVAG, corresponded to the glucokinase regulatory prote in of the liver. The second consensus motif, SLKVWT, showed a complete homo logy to the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisph osphatase (PFK-2/FBPase-2), which acts as a key regulator of glucose metabo lism. 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 amplif ication of cDNA ends analysis and Northern blot analysis revealed that rat pancreatic islets express the brain isoform of PFK-2/FBPase-2. A minor port ion of the islet PFK-2/FBPase-2 cDNA clones comprised a novel splice varian t with 8 additional amino acids in the kinase domain. The binding of the is let/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-bisph osphate level-dependent partial channeling of glycolytic intermediates betw een glucokinase and glycolytic enzymes. In pancreatic beta cells this inter action 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 g lucokinase-mediated glucose-induced insulin secretion.