Mechanism of action of glucagon on hepatocyte phosphofructokinase activity.

Claus, Thomas H.; Schlumpf, J.; el-Maghrabi, M R; Pilkis, J; Pilkis, S J

doi:10.1073/pnas.77.11.6501

Cited by 52 publications

(25 citation statements)

References 25 publications

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“…This is analogous to the results reported for 6-phosphofructo-1-kinase [27,28] where unphosphorylated enzyme is reported to show greater affinity for F2,6P2 which, in this case, results in a decreased sensitivity to ATP inhibition.…”

Section: Fraction Numbersupporting

confidence: 75%

The effect of fructose 2,6‐bisphosphate and AMP on the activity of phosphorylated and unphosphorylated fructose‐1,6‐bisphosphatase from rat liver

Ekdahl

Ekman

1984

FEBS Letters

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Rat liver fructose-1,6-bisphosphatase was partially phosphorylated in vitro and separated into unphosphorylated and fully phosphorylated enzyme. The effects of fructose 2,dbisphosphate and AMP on these two enzyme forms were examined. Unphosphorylated fructose-l ,dbisphosphatase was more easily inhibited by both effecters. Fructose 2,dbisphosphate affected both Ko.5 and V,,, while the main effect of AMP was to lower V,,. Fructose 2,dbisphosphate and AMP together acted synergistically to decrease the activity of fructose-1,6-bisphosphatase, and since unphosphorylated and phosphorylated enzyme forms are affected differently, this might be a way to amplify the effect of phosphorylation.

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Section: Fraction Numbersupporting

confidence: 75%

The effect of fructose 2,6‐bisphosphate and AMP on the activity of phosphorylated and unphosphorylated fructose‐1,6‐bisphosphatase from rat liver

Ekdahl

Ekman

1984

FEBS Letters

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“…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 of liver/ml) in Krebs-Henseleit bicarbonate buffer/0.5% bacitracin and incubated without any additions for 20 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

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

el-Maghrabi¹,

Claus²,

Pilkis³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…Nevertheless, PFlK and F1,6Pase are phosphorylated in isolated hepatocytes and in vivo, and the extent of phosphorylation of each enzyme increases in response to glucagon [7,10,111. Furthermore, peptide mapping studies indicate that glucagon increases the phosphorylation of the same tryptic peptide in PF1 K that is phosphorylated in vitro by cyclic-AMP-dependent protein kinase [12].…”

mentioning

confidence: 99%

The protein phosphatases involved in cellular regulation. Glycolysis, gluconeogenesis and aromatic amino acid breakdown in rat liver

et al. 1984

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The identities of the protein phosphatases involved in the regulation of hepatic glycolysis, gluconeogenesis and aromatic amino acid breakdown were investigated using 6-phosphofructo-1 -kinase, fructose-I ,6-bisphosphatase, L-pyruvate kinase, phenylalanine hydroxylase and the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates. Purified preparations of protein phosphatases-1, 2A, 2 B and 2 C exhibited activity towards all five substrates in vitro, although phosphatases-I and 2B were only weakly active. Studies in liver extracts using inhibitor-2 and trifluoperazine, which inhibit protein phosphatase-I and 2B, respectively, confirmed that these phosphatases are unlikely to be important in dephosphorylating these substrates in vivo. Sequential fractionation of rat liver extracts by anion-exchange chromatography and gel-filtration failed to resolve any protein phosphatases acting on each substrate, apart from protein phosphatases-2A and 2C. The present results, together with those described in the following paper (in this journal) indicate that under the assay conditions used, protein phosphatase-2A is the most powerful phosphatase acting on each substrate, although protein phosphatase-2 C contributes a significant percentage of the activity towards 6-phosphofructo-1 -kinase. No clear evidence was obtained for a role of metabolites in the regulation of dephosphorylation of the five substrates. This study reinforces our contention that only a few serine-specific and threonine-specific protein phosphatase catalytic subunits participate in cellular regulation.

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Mechanism of action of glucagon on hepatocyte phosphofructokinase activity.

Cited by 52 publications

References 25 publications

The effect of fructose 2,6‐bisphosphate and AMP on the activity of phosphorylated and unphosphorylated fructose‐1,6‐bisphosphatase from rat liver

The effect of fructose 2,6‐bisphosphate and AMP on the activity of phosphorylated and unphosphorylated fructose‐1,6‐bisphosphatase from rat liver

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

The protein phosphatases involved in cellular regulation. Glycolysis, gluconeogenesis and aromatic amino acid breakdown in rat liver

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