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 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, LSAXXVAG, 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.
Baltrusch
S
S
Institute of Clinical Biochemistry, Hannover Medical School, 30623 Hannover, Germany.
Lenzen
S
S
Okar
D A
DA
Lange
A J
AJ
Tiedge
M
M
D000595:Amino Acid Sequence; D000818:Animals; D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D018076:DNA, Complementary; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007515:Islets of Langerhans / Q000201:enzymology; D008969:Molecular Sequence Data; D019151:Peptide Library; D025481:Phosphofructokinase-2 / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D000939:Epitopes; D019151:Peptide Library; D018160:Receptors, Cytoplasmic and Nuclear; C105556:glucokinase receptor; D025481:Phosphofructokinase-2