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

Abstract

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 twohybrid 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 stimulussecretion 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.The low affinity glucose-phosphorylating enzyme glucokinase (hexokinase type IV) plays a pivotal role for the coupling of physiological millimolar glucose concentration changes to glycolysis in liver, pancreatic beta cells, and neuroendocrine cells (1-7). In pancreatic beta cells glucokinase, acting as a glucose sensor, catalyzes the rate-limiting step for glucosestimulated insulin secretion (1, 2, 4, 8). Studies in glucokinase knock-out mice, as well as the metabolic profile of diabetic patients with mutations of the glucokinase gene, provide evidence that the glucose sensor function of this enzyme in pancreatic beta cells can be fulfilled only within a narrow range of enzyme activity (5, 9 -12). Thus principles of posttranslational regulation of beta cell glucokinase have gained special interest in recent years (13-18). Liver glucokinase is regulated by insulin and glucagon on the transcriptional and translational level and by the glucokinase regulatory protein (GRP) 1 via translocation between cytosol and nucleus (6, 19 -23). In particular this translocation confers a short term adaptation of glucose metabolism to chang...

Glucokinase Is an Integral Component of the Insulin Granules in Glucose-Responsive Insulin Secretory Cells and Does Not Translocate During Glucose Stimulation

“…Two other proteins that colocalized with glucokinase and phogrin are GAPDH and PFK-2. The latter protein catalyzes the formation and degradation of fructose 2,6-bisphosphate and is a binding partner for glucokinase (35). Since a higher fraction of PFK-2 than glucokinase was recovered with both the insulin granules and the lowdensity organelles, it raises a question as to whether PFK2 acts as the binding protein or molecular scaffold for glucokinase or whether it has a role in stabilizing glucokinase, as was recently shown in insulin secretory cells (39).…”

“…Colocalization of GAPDH and PFK-2 with glucokinase immunoreactivity. Percoll fractions were immunoblotted for PFK-2, a glucokinase-binding protein (35), and for GAPDH, which has been implicated in granule movement and membrane fusion (37). Both the insulin granules and low-density organelles showed immunoreactivity to PFK-2 and GAPDH (Table 2).…”

“…We show that glucokinase is an integral component of the insulin secretory granule and that it does not translocate in response to glucose. (35). After washing, membranes were incubated for 1 h with the appropriate peroxidase-conjugated anti-IgG (Jackson Immunoresearch Laboratories).…”

Cell Biology Assessment of Glucokinase Mutations V62M and G72R in Pancreatic β-Cells

“…An increase in extracellular glucose concentration causes translocation of GK to the cytoplasm, enabling rapid stimulation of glucose phosphorylation. Translocation of GK between the nucleus and the cytoplasm is also dependent on other proteins that function as cytoplasmic receptors or shuttling proteins, such as the bifunctional protein phosphofructo-2-kinase (PFK2)/fructose-2,6-bisphosphate (FDP2), which catalyzes the formation and degradation of FDP2 (11,12).…”

“…Whether they express a truncated form of GKRP lacking the nuclear localizing signal (14) is not known. PFK2/FDP2 serves as a GK binding protein in ␤-cells and has a role in the regulation of GK activity (11,15,16). Additional binding proteins of GK have also been identified in ␤-cells, including neuronal nitric oxide and others (17)(18)(19).…”