Aspects on the phosphorylation of muscle phosphofructokinase by protein kinase C--inhibition by phosphofructokinase stabilisers

Our report presents data on the phosphorylation of muscle phosphofructokinase by Ca2+-activated, phospholipid-dependent protein kinase. We have found a stoichiometrical phosphorylation (about 1.5 mol per mol subunit), and a low apparent Km (about 0.7 microM). These data speak in favor of a physiological role for the reaction, as does the fact that phosphofructokinase from a new species (rat) was successfully phosphorylated. On the other hand we present the hitherto unpublished circumstance that the phosphorylation is inhibited by conditions that stabilise the activity of phosphofructokinase. This fact makes us question the true significance of this reaction.     

Protein-induced inactivation and phosphorylation of rabbit muscle phosphofructokinase.

Several previously untested proteins promote the reversible inactivation of rabbit skeletal muscle phosphofructokinase. Grouped in decreasing order of effectiveness, they include the following: skeletal muscle troponin C greater than troponin, the two smooth muscle myosin light chains, alpha-actinin, and S-100 much greater than parvalbumin and soybean trypsin inhibitor. The efficiency of troponin C in this process may even exceed that previously reported for calmodulin. Sequences near calcium binding site III are apparently involved in the troponin C-phosphofructokinase interaction. Troponin C and calmodulin exert calcium-dependent effects on the physical and chemical properties of muscle phosphofructokinase. When calcium is present, comigration with either protein allows the enzyme to enter the stacking gel during urea-polyacrylamide gel electrophoresis. Both enhance the phosphorylation of phosphofructokinase catalyzed by the cAMP-dependent protein kinase, with phosphate incorporations approaching 2 mol of P/mol of protomer. Reaction occurs at Ser774 and at Ser376--a novel site whose phosphorylation is highly sensitive to troponin C and less so to calmodulin. Maximum phosphorylation has slight effect on the catalytic activity of the enzyme under standard assay conditions. The troponin C induced or calmodulin-induced phosphorylation of phosphofructokinase requires calcium and is strongly inhibited by either fructose 2,6-bisphosphate or fructose 1,6-bisphosphate. Inactivation occurs in the presence or absence of calcium, with generally higher concentrations of effectors required for protection in the latter case. Liver and yeast phosphofructokinases shows little activity loss in the presence of either calmodulin or troponin C. We have developed and tested a general mathematical model for the protein-induced inactivation of phosphofructokinase which may find application to other systems.     

Phosphorylation of phosphofructokinase from skeletal muscle: correlations between phosphorylation and muscle function.

The specific radioactivity of [³²P]-phosphate incorporated into muscle phosphofructokinase was in equilibrium with the specific radioactivity of the γ-phosphate group of ATP. The incorporation was independent of the presence of cycloheximide. The total content of covalently bound phosphate in phosphofructokinase was correlated with the functional state of the muscle from which the enzyme was purified. Muscle dissected post mortem led to phosphofructokinase containing less than 2 phosphate groups per tetramer. Muscle dissected in vivo gave phosphofructokinase with 4 phosphates per tetramer when kept at rest and 8 phosphates per tetramer when stimulated to contract.     

Significance of phosphorylation of phosphofructokinase

In order to understand the effect of phosphorylation on phosphofructokinase, the allosteric kinetic behavior, ligand binding at various pHs, and pH-dependent cold inactivation of phosphofructokinase phosphorylated to different extents were studied. A subtilisin-digested phosphofructokinase from which a COOH-terminal peptide containing a phosphorylation site has been cleaved (Riquelme, P. T., and Kemp, R. G. (1980) J. Biol. Chem. 255, 4367-4371) was also included in these studies in order to investigate the possible role of this region of the molecule. Allosteric kinetics and direct binding experiments have shown that increasing phosphorylation of phosphofructokinase results in increased sensitivity to ATP inhibition and stronger binding of ATP to the inhibitory site of the enzyme. Ths subtilisin-cleaved phosphofructokinase is the least sensitive to the inhibition and shows the weakest binding of ATP. The opposite effect is observed with the binding isotherms of fructose-6-P. There is no difference in the binding of fructose-2,6-P2 among these enzymes. Binding of ATP to the inhibitory site of these enzymes as determined by fluorescence quenching (Pettigrew, D. W., and Frieden, C. (1979) J. Biol. Chem. 254, 1887-1895) is affected by pH; the binding is greatly enhanced at lower pH. Moreover, there is little difference in the binding among the modified enzymes at pH 8, but at lower pHs the binding to the phosphorylated enzyme is much more enhanced than the dephosphoenzyme. A pH-dependent cold inactivation study has shown that the phosphorylation of the enzyme causes an increase in the pK value for the inactivation, and the extent of the pK shift depends upon the degree of phosphorylation. Based on these results, a model originally proposed by Frieden et al. (Frieden, C., Gilbert, H. R., and Bock, P. E. (1976) J. Biol. Chem. 251, 5644-5647) can be applied to explain a possible role for the phosphorylation and the peptide portion of phosphofructokinase in its complex allosteric kinetic behavior.     

Studies on specific elution of rabbit muscle phosphofructokinase with allosteric ligands]

The specific elution of rabbit skeletal muscle phosphofructokinase (PFK) from DEAE-cellulose is studied in the linear gradient of different allosteric ligands. Citrate and fructose-6-phosphate elute PFK at concentrations of 1.0 and 2.5 mM respectively, i.e. without increasing the ionic strength of the starting buffer (similar to 0.12). The specificity of elution is confirmed by comparison of the ionic strength of these solutions with that of buffer eluting PFK in buffer gradient (mu=0.17) as well as by comparison with the eluting ability of other ligands. Fructose-1,6-diphosphate elutes PFK only at the concentration of 5.5 mM which corresponds to the ionic strength 0.17. MgATP and AMP are inefficient as specific eluents whereas ATP and ADP elute only a small part of PFK with concomitant substantial increase of the ionic strength (up to 0.17--0.18). These results are discussed in terms of a charge compensation mechanism as a result of the displacement of PFK conformers equilibrium under the influence of the allosteric ligands.     

Studies on the structure of yeast phosphofructokinase

In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase. This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations. The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods. However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer. On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase. Obviously, some methods of molecular weight determination have led to erroneous results. In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution.     

Evidence for phosphorylation of yeast phosphofructokinase

Radioactively labelled material from yeast cells grown in the presence of [32P]phosphate was specifically recognized by antibodies raised against yeast phosphofructokinase. Purified yeast phosphofructokinase was phosphorylated in a cyclic AMP-independent manner by a protein kinase enriched from yeast extracts. This phosphorylation occurred specifically on the beta-subunit, and 0.56 mol of phosphate/mol of subunit was incorporated. The results indicate the phosphorylation of yeast phosphofructokinase both in vivo and in vitro. Phosphofructokinase phosphorylated in vitro was more stable against proteolytic degradation compared to the non-phosphorylated enzyme.     

Studies on glycolysis in vitro: role of glucose phosphorylation and phosphofructokinase activity on total velocity

An in vitro glycolysis system has been developed to study the regulation of glycolysis on kinetic structure basis, in order to determine the extent of regulatory effects on the whole system of individual enzymes according to their kinetic data, in rat liver and muscle. Hexokinase or glucose-6-phosphate addition to the system with glucose as substrate increases lactate production rate by 2.5 in liver and by 10 in muscle, which suggest glucose phosphorylation step is a limiting step in this system. Fructose 2,6-bisphosphate addition to the system increases lactate production rate in liver only when glucose is the substrate, but not with glucose-6-phosphate as substrate. There is a linear relationship between glycolytic activity, as lactate produced per min and protein quantity, which suggests that this system can also be used to assay glycolytic activity in tissue extracts. Specific glycolytic activity found, as mumol of L-lactate produced per min, per protein mg was 0.1 for muscle and 0.01 for liver.     

Identification of the phosphorylated sites of phosphofructokinase from skeletal muscle after in vivo and in vitro phosphorylation.

Phosphofructokinase from mice muscle was radioactively labelled either in vivo by the injection of [³²P]-phosphate or in vitro by the incubation with cAMP-dependent protein kinase and [γ-³²P]-ATP. Two labelled peptides were obtained after tryptic digestion in either case showing that at least two sites were phosphorylated. Independent of the labelling method, the labelled peptides showed an analogous pattern on the peptide maps, indicating that both methods led to the phosphorylation of the same sites.     

Hormone-stimulated phosphorylation of liver phosphofructokinase in vivo.

The effect of glucagon on the phosphorylation and the enzymic activity of phosphofructokinase in rat liver in vivo was investigated. Glucagon stimulated the phosphorylation of liver phosphofructokinase approximately 3- to 5-fold and increased cAMP levels 5-fold and blood glucose levels 2-fold over the values obtained for control animals. The specific radioactivity of ATP isolated from liver was the same in both control and hormone-treated animals. During the purification of the 32P-labeled enzyme from both animals, no difference was observed in the total or specific enzyme activities of the enzymes from the various fractions. Thus, phosphofructokinase appears to be phosphorylated in vivo by a cyclic AMP-dependent protein kinase. Although phosphorylation does not affect the maximum catalytic activity of the enzyme, it does render the enzyme significantly more sensitive to ATP inhibition. Thus, at a given concentration of ATP, the phosphorylated phosphofructokinase exhibits considerably lower activity than the unphosphorylated enzyme. The possible relationship between our observations and glucagon-mediated control of glycolysis is discussed.     

Regulation of rat liver phosphofructokinase by glucagon-induced phosphorylation

In perfused rat liver, the effects of various hormones on the stimulation of phosphorylation and allosteric properties of purified phosphorfructokinase were investigated. Rat livers were perfused with [³²P]phosphate followed with various hormones or cyclicAMP, and ³²P-labeled phosphofructokinase was isolated. ³²P incorporation into the enzyme and enzyme inhibition by ATP or citrate were determined. Only glucagon increased the ³²P incorporation into phosphofructokinase and this increase was approximately threefold. The cyclicAMP level was increased simultaneously approximately four- to fivefold compared to the control perfused liver. Similar results were obtained by perfusing the liver with cyclicAMP (0.1 mm). The phosphorylated phosphofructokinase showed a decrease in the K_i values for ATP (from 0.4 to 0.2 mm) and citrate (from 2 to 0.6 mm). Neither epinephrine nor insulin affected the extent of phosphorylation or the allosteric properties of the enzyme. The half-maximal concentration of glucagon required for phosphorylation of phosphofructokinase and modification of its allosteric properties was approximately 6 × 10^?11m. It is concluded that glucagon increases the inhibition of liver phosphofructokinase by ATP and citrate through phosphorylation of the enzyme involving a β-receptor-mediated cyclicAMP-dependent mechanism.