Regulation of 6-phosphofructo-2-kinase activity by cyclic AMP-dependent phosphorylation.

Abstract

Addition of glucagon to isolated rat hepatocytes resulted in inhibition of 6-phosphofructo-2-kinase (ATP:D-fructose-6-phosphate 2-phosphotransferase) activity in extracts of the cells and in a decrease in the intracellular level of fructose 2,6-bisphosphate. The effect on 6-phosphofructo-2-kinase was characterized by a decrease in the affinity of the enzyme for fructose 6-phosphate. To investigate the mechanism of action of glucagon, 6-phosphofructo-2-kinase from rat liver was partially purified by polyethylene glycol precipitation, DEAE-cellulose chromatography, (NH4)2SO4 fractionation, Sephacryl S-200 gel filtration, DEAE-Sephadex chromatography, and Sephadex G-100 gel filtration. Incubation of the purified enzyme with the catalytic subunit ofthe cyclic AMP-dependent protein kinase from rat liver and [y-32P]ATP resulted in 32p incorporation into a protein with a subunit Mr of 49,000 as determined by NaDodSO4 disc gel electrophoresis. Associated with this phosphorylation was an inhibition of 6-phosphofructo-2-kinase activity that was also characterized by a decrease in the affinity of the enzyme for fructose 6-phosphate. Both the phosphorylation and the inhibition of the purified 6-phosphofructo-2-kinase were blocked by addition of the heatstable protein kinase inhibitor. It is concluded that the glucagoninduced decrease in fructose 2,6-bisphosphate levels observed in isolated hepatocytes is due, at least in part, to cyclic AMP-dependent phosphorylation and inhibition of 6-phosphofructo-2-ldnase.A number of groups have presented evidence for the existence in liver of a novel effector of 6-phosphofructo-1-kinase (1-7). The structure of this compound was first put forth as fructose 2,6-bisphosphate by Van Schaftingen and Hers (1) and subsequently confirmed by us spectroscopically and by 13C NMR (5). Fructose 2,6-bisphosphate is an allosteric activator of 6-phosphofructo-l-kinase (1, 2, 4-7) and an inhibitor of fructose 1,6-bisphosphatase (8-10). The level of fructose 2,6-bisphosphate has been shown to vary widely depending on the hormonal and dietary state (2, 11-13). For example, glucagon addition to isolated hepatocytes results in a 90% decrease in its level within minutes (2,4,11,12). These results suggest that the enzyme responsible for the synthesis or degradation (or both) offructose 2,6-bisphosphate is regulated by a cyclic AMP (cAMP)-dependent phosphorylation mechanism (11). Recently, the enzyme responsible for fructose 2,6-bisphosphate synthesis was isolated and shown to catalyze the transfer of the -y phosphate of ATP to the C-2 position of fructose 6-phosphate (14-16). The goal of the present study was to determine whether rat liver 6-phosphofructo-2-kinase is regulated by cAMP-dependent protein kinase-catalyzed phosphorylation and to characterize this regulation. MATERIALS AND METHODSPreparation and Incubation of Isolated Hepatocytes. Isolated rat hepatocytes were prepared from fed rats (male, Sprague-Dawley, 175-225 g) as described (3). The cells were suspended (final concentration, 50 mg o...

Purification and Characterization of a Novel Xylulose 5-Phosphate-activated Protein Phosphatase Catalyzing Dephosphorylation of Fructose-6-phosphate,2-kinase:Fructose-2,6-bisphosphatase

“…Synthesis and degradation of Fru 2,6-P 2 are catalyzed by a bifunctional enzyme, Fru-6-P,2-kinase:Fru-2,6-bisphosphatase. Liver Fru-6-P,2-kinase:Fru-2,6-Pase is phosphorylated by cAMP-dependent protein kinase (2)(3)(4). When blood glucose level falls, glucagon level increases which raises the cAMP level in hepatic cells.…”

Insulin-like Effects of Vanadate on Rat Liver 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase mRNA and Protein Inductions in Diabetic Rats.

“…This bifunctional enzyme activity is ruled by the phosphorylation state and the amount of the enzyme protein [14,15,17,18]. Insulin is known to regulate both the phosphorylation state and the protein level of the liver 6PF-2-K/Fru-2,6-P2ase [16][17][18].…”

“…The Fru-2,6-P2 level is regulated by a bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase). This enzyme activity is regulated by several allosteric effectors and covalent modification through phosphorylation, mainly catalyzed by cyclic AMP dependent protein kinase (A-kinase) [14,15]. Insulin is proposed to inhibit A-kinase dependent phosphorylahon of the enzyme protein, increasing the 6PF-2-K activity in an acute manner [16].…”

Involvement of the chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase sequence His444-Arg-Glu-Arg in modulation of the bisphosphatase activity by its kinase domain

“…6PF-2K activity was measured by monitoring the production of Fru-2,6-P # [18]. Fru-2,6-P # ase activity was assayed by following the rate of release of [$#P]P i from [2-$#P]Fru-2,6-P # , as described previously [19].…”