Regulatory Protein Of Glucokinase Research Articles

Identification of Fructose 6-Phosphate- and Fructose 1-Phosphate-binding Residues in the Regulatory Protein of Glucokinase

Glucokinase is inhibited in the liver by a regulatory protein (GKRP) whose effects are increased by Fru-6-P and suppressed by Fru-1-P. To identify the binding site of these phosphate esters, we took advantage of the homology of GKRP to the isomerase domain of GlmS (glucosamine-6-phosphate synthase) and created 12 different mutants of rat GKRP. Mutations of three residues predicted to bind to Fru-6-P resulted in proteins that were approximately 5-fold (S110A) and 50-fold (S179A and K514A) less potent as inhibitors of glucokinase and had an at least 100-fold reduced affinity for the effectors. Mutation of another residue of the putative binding site (T109A) resulted in a 10-fold decrease in the inhibitory power and an inversion of the effect of sorbitol-6-P, a Fru-6-P analog. The replacement of Gly(107), a residue close to the binding site, by cysteine (as in GlmS and Xenopus GKRP) resulted in a protein that had 20 times more affinity for Fru-6-P and 30 times less affinity for Fru-1-P. These results are consistent with GKRP having one single binding site for phosphate esters. They also show that a missense mutation of GKRP can lead to a gain of function.

Evidence for glucose and sorbitol-induced nuclear export of glucokinase regulatory protein in hepatocytes

Glucokinase is rapidly exported from the nucleus of hepatocytes in response to a rise in glucose or fructose 1-P. We demonstrate using confocal microscopy and quantitative imaging that in contrast to previous findings, the regulatory protein of glucokinase (GKRP) also translocates from the nucleus during substrate-induced translocation of glucokinase. However, the fractional decrease in nuclear GKRP is smaller than for glucokinase and is determined by the metabolic state and not by the distribution of glucokinase. Translocation of glucokinase and GKRP is not inhibited by leptomycin B, an inhibitor of exportin-1 function. These findings highlight the importance of quantitative imaging for determining nuclear export of proteins and suggest that GKRP may have a role in nuclear export or import of glucokinase.

Mannitol 1-phosphate mediates an inhibitory effect of mannitol on the activity and the translocation of glucokinase in isolated rat hepatocytes.

When tested in the presence of an inhibitor of sorbitol dehydrogenase, both mannitol and sorbitol caused a progressive inhibition of the detritiation of [2-3H]glucose in isolated rat hepatocytes. The purpose of the present work was to investigate the possibility that this effect was mediated by the regulatory protein of glucokinase. When added to hepatocytes, mannitol decreased the apparent affinity of glucokinase for glucose and increased the concentration of fructose required to stimulate detritiation, without affecting the concentration of fructose 1-phosphate. Its effect could be attributed to the formation of mannitol 1-phosphate, a potent agonist of the regulatory protein, which, similarly to fructose 6-phosphate, reinforces its inhibitory action. Formation of mannitol 1-phosphate in hepatocytes was dependent on the presence of mannitol and was stimulated by compounds that increase the concentration of glucose 6-phosphate. Liver extracts catalysed the conversion of mannitol to mannitol 1-phosphate about 7 times more rapidly in the presence of glucose 6-phosphate than of ATP. The glucose 6-phosphate-dependent formation was entirely accounted for by a microsomal enzyme, glucose-6-phosphatase and was not due to a loss of latency of this enzyme. In hepatocytes in primary culture, mannitol decreased the detritiation rate and counteracted the effect of fructose to stimulate glucokinase translocation. Taken together, these results strongly support a central role played by the regulatory protein in the control of glucokinase activity and translocation in the liver, as well as a feedback control exerted by fructose 6-phosphate on this enzyme.

The regulatory protein of glucokinase.

The regulatory protein of glucokinase.

Evidence for a Role of Glucose-induced Translocation of Glucokinase in the Control of Hepatic Glycogen Synthesis

Glucokinase reversibly partitions between a bound and a free state in the hepatocyte in response to the metabolic status of the cell. Maximum binding occurs at low [glucose] (<5 mM) and minimum binding at high [glucose] or in the presence of sorbitol or fructose. In this study we determined the binding characteristics of glucokinase in the hepatocyte in situ, by adenovirus-mediated glucokinase overexpression combined with the digitonin-permeabilization technique. We also determined the sensitivity of glycogen synthesis to changes in either total glucokinase overexpression or in free glucokinase activity. Glucokinase overexpression is associated with an increase in both free and bound activity, with an overall decrease in the proportion of bound activity. In hepatocytes incubated at low [glucose] (0-5 mM), glucokinase binding involves a high-affinity binding site with a Kd of approximately 0.1 microM and a binding capacity of approximately 3 pmol/mg total cell protein and low-affinity binding with a Kd of approximately 1.6 microM. Increasing glucose concentration to 20 mM causes a dose-dependent increase in the Kd of the high- affinity site to approximately 0.6 microM, and this effect was mimicked by 50 microM sorbitol, a precursor of fructose 1-P, confirming that this site is the regulatory protein of glucokinase. Glycogen synthesis determined from the incorporation of [2-3H,U-14C]glucose into glycogen at 5 mM or 10 mM glucose was very sensitive to small increases in total glucokinase activity and correlated more closely with the increase in free glucokinase activity. The relation between glycogenic flux and glucokinase activity is sigmoidal. Expression of the sensitivity of glycogen synthesis to glucokinase activity as the control coefficient reveals that the coefficient is greater for the incorporation of 2-tritium (which occurs exclusively by the direct pathway) than for incorporation of 14C label (which involves direct and indirect pathways) and is greater at 5 mM glucose (when glucokinase is maximally sequestered at its high-affinity site) than at 10 mM glucose. The results support the hypothesis that compartmentation of glucokinase in the hepatocyte increases the sensitivity of glycogen synthesis to small changes in total glucokinase activity and that glucose-induced translocation of glucokinase has a major role in the acute control of glycogen synthesis.

Heterologous expression of an active rat regulatory protein of glucokinase

The cDNA presumed to encode the rat liver regulatory protein of glucokinase has been expressed in Escherichia coli and a partially soluble protein has been obtained. This recombinant protein was partially purified and found to have the same apparent molecular mass as the regulatory protein purified from rat liver. Like the latter, it inhibited rat liver glucokinase competitively with respect to glucose and its effect was sensitive to fructose 6-phosphate and fructose 1-phosphate.

Respective roles of glucose, fructose, and insulin in the regulation of the liver-specific pyruvate kinase gene promoter.

The L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway mainly expressed in the liver. Rat liver contains a regulatory protein that inhibits glucokinase (GK) activity. The effect of this protein is greatly reinforced by the fructose 6-phosphate and antagonized by the fructose 1-phosphate (Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179-184). In hepatocytes, fructose in low concentrations is phosphorylated into fructose 1-phosphate, and therefore is able to active GK in the absence of insulin via the regulatory protein in the liver. In primary culture of rat hepatocytes, 0.2 mM fructose in the presence of 20 or 40 mM glucose stimulated the activity of the L-PK gene promoter fused with the chloramphenicol acetyltransferase reporter gene, regardless of the addition of insulin, through the glucose/insulin response element. A constitutive GK expression vector co-transfected with the L-PK/chloramphenicol acetyltransferase construct is also able to confer an insulin-independent glucose responsiveness in hepatocytes. Thus, the insulin effect on glucose-dependent activation of the L-PK promoter is, under these experimental conditions, to permit glucose phosphorylation through the stimulation of the GK synthesis. In the presence of glucose, the L-PK promoter can also be activated by a post-translational GK activation, mediated by a low concentration of fructose acting via the regulatory protein of glucokinase.

Cloning and sequencing of rat liver cDNAs encoding the regulatory protein of glucokinase

cDNAs encoding the rat liver regulatory protein of glucokinase were cloned and sequenced. The deduced protein contains 568 amino acids for a molecular mass of 62,867 Da. Northern blot analysis showed the presence of a major RNA species of 2.35 kb in rat liver. No signal was observed with muscle, brain, heart, testis, intestine or spleen RNA. Recombinant regulatory protein expressed in Escherichia coli was insoluble and inactive, and was presumably contained in inclusion bodies. Western blot analysis showed that the recombinant protein was recognized by antibodies raised against regulatory protein purified from rat liver.