Fructose-2,6-bisphosphate in rat mesenteric lymph nodes

1. The fructose-2,6-bisphosphate (Fru-2,6-P2) content of mesenteric lymph nodes was measured in rats. 2. The effects of Fru-2,6-P2 on the activity of 6-phosphofructo-1-kinase (PFK-1) from rat mesenteric lymph nodes were also studied. 3. The affinity of the enzyme for fructose-6-phosphate was increased by Fru-2,6-P2 whereas the inhibition of the enzyme with high concentrations of ATP was released by Fru-2,6-P2. 4. The activity of lymphocyte PFK-1 was highly stimulated in a simultaneous presence of low concentrations of AMP and Fru-2,6-P2. 5. These results show that rat lymphocyte PFK-1 is highly regulated with Fru-2,6-P2 which means that glycolysis in rat lymphocytes is controlled by Fru-2,6-P2.     

The stimulation of heart glycolysis by increased workload does not require AMP-activated protein kinase but a wortmannin-sensitive mechanism

Increasing heart workload stimulates glycolysis by enhancing glucose transport and fructose-2,6-bisphosphate (Fru-2,6-P(2)), the latter resulting from 6-phosphofructo-2-kinase (PFK-2) activation. Here, we investigated whether adenosine monophosphate (AMP)-activated protein kinase (AMPK) mediates PFK-2 activation in hearts submitted to increased workload. When heart work was increased, PFK-2 activity, Fru-2,6-P(2) content and glycolysis increased, whereas the AMP:adenosine triphosphate (ATP) and phosphocreatine/creatine (PCr:Cr) ratios, and AMPK activity remained unchanged. Wortmannin, the well-known phosphatidylinositol-3-kinase inhibitor, blocked the activation of protein kinase B and the increase in glycolysis and Fru-2,6-P(2) content induced by increased work. Therefore, the control of heart glycolysis by contraction differs from that in skeletal muscle where AMPK is involved.     

Role of fructose 2,6-bisphosphate in mammary gland of fed, starved and re-fed lactating rats.

The fructose 2,6-bisphosphate (Fru-2,6-P2) content and intracellular concentration of lactating mammary gland was measured in fed, starved and re-fed rats. There was little or no change on starvation, and about 1.5-fold rise on re-feeding, contrasting with estimated glycolytic changes of about 10-fold. The 6-phosphofructokinase (PFK-1) activity of mammary extracts was highly sensitive to added Fru-2,6-P2 under all conditions examined, and appeared to approach saturation at physiological concentrations of this effector. The activity of mammary PFK-1 measured under optimal and 'physiological' conditions suggested that this enzyme operates in vivo at about 24% of maximal rate, and is likely to be an important rate-limiting factor in mammary glycolysis.     

Phosphorylation-activated 6-phosphofructo-2-kinase from mantle tissue of marine mussels.

PKF-2 from mussel mantle was phosphorylated by cAMP-dependent protein kinase. The phosphorylation does not change the enzyme activity at neutral pH values, but at acid pH the activity of the phosphorylated form is higher than the native PFK-2. With respect to the native enzyme, the activation consisted of a reduction in the Km for Fru-6-P and a decrease in the inhibitory effect of PEP. These results are in keeping with the stabilized concentration of Fru-2,6-P2 found in the mussel mantle during the physiological hypoxia caused by the closure of the valves.     

Contractile activity modifies Fru-2,6-P(2) metabolism in rabbit fast twitch skeletal muscle.

Modification of muscular contractile patterns by denervation and chronic low frequency stimulation induces structural, physiological, and biochemical alterations in fast twitch skeletal muscles. Fructose 2,6-bisphosphate is a potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. The concentration of Fru-2,6-P(2) depends on the activity of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which catalyzes the synthesis and degradation of this metabolite. This enzyme has several isoforms, the relative abundance of which depends on the tissue metabolic properties. Skeletal muscle expresses two of these isoforms; it mainly contains the muscle isozyme (M-type) and a small amount of the liver isozyme (L-type), whose expression is under hormonal control. Moreover, contractile activity regulates expression of muscular proteins related with glucose metabolism. Fast twitch rabbit skeletal muscle denervation or chronic low frequency stimulation can provide information about the regulation of this enzyme. Our results show an increase in Fru-2,6-P(2) concentration after 2 days of denervation or stimulation. In denervated muscle, this increase is mediated by a rise in liver PFK-2/FBPase-2 isozyme, while in stimulated muscle it is mediated by a rise in muscle PFK-2/FBPase-2 isozyme. In conclusion, our results show that contractile activity could alter the expression of PFK-2/FBPase-2.     

Amino-acids imitate the EDTA activation on the fructose-1,6-bisphosphatase of mantle tissue from the sea mussel (Mytilus galloprovincialis Lmk).

In the absence of AMP and Fru-2,6-P2, several amino-acids such as histidine, lysine, alanine, aspartic acid, and other molecules, as reduced glutathione or citrate, activate FBPase-1 from Mytilus galloprovincialis mantle. AMP decreases Vmax and Km for Fru-1,6-P2 both in the absence and in the presence of activators; but the addition of Fru-2,6-P2 decreases the affinity of the enzyme by its substrate. Na+ acts as a inhibitor reducing both Vmax and Km. The Km value is lower than the physiological level of Fru-1,6-P2, suggesting that the enzyme is operative but its activity is very reduced.     

Cardiac expression of kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase inhibits glycolysis, promotes hypertrophy, impairs myocyte function, and reduces insulin sensitivity

Glycolysis is important to cardiac metabolism and reduced glycolysis may contribute to diabetic cardiomyopathy. To understand its role independent of diabetes or hypoxic injury, we modulated glycolysis by cardiac-specific overexpression of kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (kd-PFK-2). PFK-2 controls the level of fructose 2,6-bisphosphate (Fru-2,6-P(2)), an important regulator of glycolysis. Transgenic mice had over 2-fold reduced levels of Fru-2,6-P(2). Heart weight/body weight ratio indicated mild hypertrophy. Sirius red staining for collagen was significantly increased. We observed a 2-fold elevation in glucose 6-phosphate and fructose 6-phosphate levels, whereas fructose 1,6-bisphosphate was reduced 2-fold. Pathways branching off of glycolysis above phosphofructokinase were activated as indicated by over 2-fold elevated UDP-N-acetylglucosamine and glycogen. The kd-PFK-2 transgene significantly inhibited glycolysis in perfused hearts. Insulin stimulation of metabolism and Akt phosphorylation were sharply reduced. In addition, contractility of isolated cardiomyocytes was impaired during basal and hypoxic incubations. The present study shows that cardiac overexpression of kinase-deficient PFK-2 reduces cardiac glycolysis that produced negative consequences to the heart including hypertrophy, fibrosis, and reduced cardiomyocyte function. In addition, metabolic and signaling responses to insulin were significantly decreased.     

Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases

The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6-phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo-2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP-dependent protein kinase, the synthetic peptide corresponding to the cAMP-dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP-dependent protein kinase.     

Active hepatic glycogen synthesis from gluconeogenic precursors despite high tissue levels of fructose 2,6-bisphosphate

When fasted rats ate regular lab chow there was a lag time of about 2 h before the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2) in liver began to rise from its low basal level. By contrast, in animals refed on a sucrose-based diet hepatic [Fru-2,6-P2] increased 20-fold (to a value of approximately 12 nmol/g wet weight) during the first hour. These responses correlated with differences in the ability of the two diets to increase the circulating [insulin]/[glucagon] ratio and thus to elevate the ratio of 6-phosphofructo-2-kinase to fructose-2, 6-bisphosphatase. Liver glycogen was deposited briskly in both groups of rats. To assess its mechanism of synthesis (directly from glucose versus indirectly via the gluconeogenic pathway), animals eating the chow or sucrose diets received intravenous infusions of [14C]bicarbonate, [1-14C] fructose, and 3H2O. After isolation, the glycogen was subjected to positional isotopic analysis of its glucose residues. The results established that regardless of the diet the bulk of liver glycogen was gluconeogenic in origin. The fact that with sucrose feeding carbon flow through hepatic fructose-1,6-bisphosphatase remained active despite high levels of Fru-2,6-P2 (a potent inhibitor of this enzyme in vitro) presents a metabolic paradox. Conceivably, the suppressive effect of Fru-2, 6-P2 on hepatic fructose-1,6-bisphosphatase is overridden in vivo by some unknown factor or factors generated in response to sucrose feeding. Alternatively, metabolic zonation in liver might result in the coexistence of hepatocytes rich in Fru-2,6-P2 (high glycolytic, low gluconeogenic, low glycogenic capacitites) with cells depleted of Fru-2,6-P2 (low glycolytic, high gluconeogenic, high glycogenic capacities).     

Oscillation in fructose 2,6-bisphosphate levels and in the phosphorylation states of fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase in ischemic rat liver.

In order to determine the role of fructose (Fru) 2,6-P2 in stimulation of phosphofructokinase in ischemic liver, tissue contents of Fru-2,6-P2, hexose-Ps, adenine nucleotides, and Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated during the first few minutes of ischemia. The Fru-2,6-P2 concentration in the liver changed in an oscillatory manner. Within 7 s after the initiation of ischemia, Fru-2,6-P2 increased from 6 to 21 nmol/g liver and decreased to 5 nmol/g liver within 30 s. Subsequently, it reached the maximum value at 50, 80, and 100 s and decreased to the basal concentration at 60, 90, and 120 s. Oscillatory patterns were also observed with Glc-6-P and Fru-6-P, but the ATP/ADP ratio decreased monotonically. Determination of Fru-6-P,2-kinase activity and the phosphorylation states of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase demonstrated that at 7 and 50 s, where Fru-2,6-P2 was the highest, the enzyme was activated and mostly in a dephosphorylated form. On the other hand, at 0, 30, and 300 s, the enzyme was predominantly in the phosphorylated form. The concentration of cAMP in the liver also changed in an oscillatory manner between 0.5 to 1.3 nmol/g with varying frequency of 10 to 40 s. These results indicated that: (a) Fru-2,6-P2 was important in rapid activation of phosphofructokinase in the first few seconds and up to 2-3 min, and (b) the oscillation of Fru-2,6-P2 concentration was the result of activation and inhibition of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase, which was caused by changes in the phosphorylation state of the enzyme.     

Variations of fructose-2,6-diphosphate levels in cultured HT29 human colon cancer cells: influence of hexoses and lactate concentrations

Under the standard conditions of culture, Fru-2,6-P2 level in HT29 cells is transitorily increased as a consequence of medium change; the peak value occurs after 2 hr, followed by a gradual return to a basal value (40 pmol/mg protein) which is maintained as long as medium glucose concentration stands above 2 mM. A 20 hr glucose deprivation lowers Fru-2,6-P2 level to trace value, but, when glucose is reintroduced, the peak value is much higher; large Fru-2,6-P2 accumulation is correlated with higher rates of glucose uptake and lactate release, which suggests an activation of glycolysis at the level of phosphofructokinase-1. Fru-2,6-P2 level depends on the glucose concentration within the range of 0 to 5 mM. At this concentration and above, maximal effect is reached. Previous glucose deprivation renders the Fru-2,6-P2 forming system more sensitive to glucose. When given instead of glucose, fructose enters the glycolytic pathway and produces same effect as glucose on the Fru-2,6-P2 level. Galactose turns it to almost zero which coincides with low glycolytic rate. Acidity of the culture medium favorishes the Fru-2,6-P2 formation; however, change in pH cannot explain the variations of Fru-2,6-P2 level observed under the standard culture conditions. Lactate concentrations over 10 mM in the medium are found to significantly inhibit the Fru-2,6-P2 producing system. Therefore, lactate accumulation in the medium could be an important factor controlling Fru-2,6-P2 level during standard cell culture.     

Structures of mammalian and bacterial fructose-1,6-bisphosphatase reveal the basis for synergism in AMP/fructose 2,6-bisphosphate inhibition

Fructose-1,6-bisphosphatase (FBPase) operates at a control point in mammalian gluconeogenesis, being inhibited synergistically by fructose 2,6-bisphosphate (Fru-2,6-P(2)) and AMP. AMP and Fru-2,6-P(2) bind to allosteric and active sites, respectively, but the mechanism responsible for AMP/Fru-2,6-P(2) synergy is unclear. Demonstrated here for the first time is a global conformational change in porcine FBPase induced by Fru-2,6-P(2) in the absence of AMP. The Fru-2,6-P(2) complex exhibits a subunit pair rotation of 13 degrees from the R-state (compared with the 15 degrees rotation of the T-state AMP complex) with active site loops in the disengaged conformation. A three-state thermodynamic model in which Fru-2,6-P(2) drives a conformational change to a T-like intermediate state can account for AMP/Fru-2,6-P(2) synergism in mammalian FBPases. AMP and Fru-2,6-P(2) are not synergistic inhibitors of the Type I FBPase from Escherichia coli, and consistent with that model, the complex of E. coli FBPase with Fru-2,6-P(2) remains in the R-state with dynamic loops in the engaged conformation. Evidently in porcine FBPase, the actions of AMP at the allosteric site and Fru-2,6-P(2) at the active site displace engaged dynamic loops by distinct mechanisms, resulting in similar quaternary end-states. Conceivably, Type I FBPases from all eukaryotes may undergo similar global conformational changes in response to Fru-2,6-P(2) ligation.     

Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase appears to be the only enzyme catalyzing the formation and hydrolysis of Fru-2,6-P2. The enzyme as we isolate it, contains a trace of tightly bound Fru-6-P. In this condition, it exhibited an ATPase activity comparable to its kinase activity. Inorganic phosphate stimulated all of its activities, by increasing the affinity for all substrates and increasing the Vmax of ATP and Fru-2,6-P2 hydrolysis. The enzyme catalyzed ADP/ATP and Fru-6-P/Fru-2,6-P2 exchanges at rates comparable to net reaction rates. It was phosphorylated by both [gamma-32P]ATP and [2-32P] Fru-2,6-P2, and the label from either donor was chased by either unlabeled donor, showing that the bound phosphate is hydrolyzed if not transferred to an acceptor ligand. The rate of labeling of the enzyme by [2-32P]Fru-2,6-P2 was 2 orders of magnitude greater than the maximal velocity of the bisphosphatase and therefore sufficiently fast to be a step in the hydrolysis. Both inorganic phosphate and Fru-6-P increased the rate and steady state of enzyme phosphorylation by ATP. Fru-2,6-P2 inhibited the ATPase and kinase reactions and Fru-6-P inhibited the Fru-2,6 bisphosphatase reaction while ATP and ADP had no effect. Removal of the trace of Fru-6-P by Glu-6-P isomerase and Glu-6-P dehydrogenase reduced enzyme phosphorylation by ATP to very low levels, greatly inhibited the ATPase, and rendered it insensitive to Pi, but did not affect ADP/ATP exchange. (alpha + beta)Methylfructofuranoside-6-P did not increase the rate or steady state labeling by ATP. These results suggest that labeling of the enzyme by ATP involved the production of [2-32P]Fru-2,6-P2 from the trace Fru-6-P. The 6-phosphofructo-2-kinase, fructose 2,6-bisphosphatase, and ATP/ADP exchange were all inhibited by diethylpyrocarbonate, suggesting the involvement of histidine residues in all three reactions. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a Fru-2,6 bisphosphatase site which is readily phosphorylated by Fru-2,6-P2.     

Cloning, expression and chromosomal localization of a human testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene

6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2) is a bifunctional enzyme responsible for the synthesis and breakdown of Fru-2,6-P2, a key metabolite in the regulation of glycolysis. Several genes encode distinct PFK-2/FBPase-2 isozymes that differ in their tissue distribution and enzyme regulation. In this paper, we present the isolation of a cDNA from a human testis cDNA library that encodes a PFK-2/FBPase-2 isozyme. Sequencing data show an open reading frame of 1407 nucleotides that codifies for a protein of 469 amino acids. This has a calculated molecular weight of 54kDa and 97% similarity with rat testis PFK-2/FBPase-2, with complete conservation of the amino acid residues involved in the catalytic mechanism. Fluorescence in-situ hybridization (FISH) localized testis PFK-2/FBPase-2 gene (PFKFB4) in human chromosome 3 at bands p21-p22. A Northern blot analysis of different rat tissues showed the presence of a 2.4-kb mRNA expressed specifically in testis. In mammalian COS-1 cells, the human testis cDNA drives expression of an isozyme with a molecular weight of 55kDa. This isozyme shows clear PFK-2 activity. Taken together, these results provide evidence for a new PFK-2/FBPase-2 gene coding for a human testis isozyme.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression is regulated by diet composition and ration size in liver of gilthead sea bream, Sparus aurata

Modulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P(2)ase) gene expression by diet composition and ration size was studied in the liver of gilthead sea bream, Sparus aurata. From five different types of diet supplied to fish, those with either high carbohydrate/low protein or high carbohydrate/low lipid content stimulated 6PF-2-K/Fru-2,6-P(2)ase expression at the levels of mRNA, immunodetectable protein and kinase activity as well as promoting higher fructose-2,6-bisphosphate (Fru-2,6-P(2)) values. The expression of the bifunctional enzyme and Fru-2,6-P(2) levels showed also direct dependence on the quantity of diet supplied. These findings demonstrate for the first time nutritional regulation of 6PF-2-K/Fru-2,6-P(2)ase at mRNA level by diet composition and ration size and suggest that the carnivorous fish S. aurata can adapt its metabolism, by stimulation of liver glycolysis, to partial substitution of protein by carbohydrate in the diet. In addition, the expression of 6PF-2-K/Fru-2,6-P(2)ase can be used as an indicator of nutritional condition.     

Phosphofructokinase from Fasciola hepatica: activation by phosphorylation and other regulatory properties distinct from the mammalian enzyme

Phosphofructokinase from the liver fluke, Fasciola hepatica, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase isolated from this organism. Phosphorylated fluke phosphofructokinase had a sevenfold lower apparent Km for its substrate, Fru-6-P, and an eightfold higher 0.5 Vopt for ATP, the enzyme's primary inhibitor, than native phosphofructokinase. Activation of fluke phosphofructokinase following phorphorylation by a mammalian protein kinase catalytic subunit was previously reported (E. S. Kamemoto and T. E. Mansour (1986) J. Biol. Chem. 261, 4346-4351). The catalytic subunit of protein kinase isolated from the liver fluke phosphorylated sites on fluke phosphofructokinase similar to those phosphorylated by the mammalian enzyme. Maximal phosphate incorporation was 0.3 mol P/mol of protomer. The native enzyme was found to contain 1.3 mol P/mol of protomer. In contrast to fluke phosphofructokinase, activity of the mammalian heart enzyme was slightly decreased following phosphorylation. The dependence of allosteric interaction on an acidic pH observed with the mammalian phosphofructokinase was not observed with the fluke enzyme. Unlike mammalian phosphofructokinase, allosteric kinetics of the fluke enzyme was observed at alkaline pH (8.0). Fluke phosphofructokinase was found to be relatively insensitive to inhibition by citrate, a known potent inhibitor of the mammalian enzyme. Fru-2,6-P2, a potent modifier of phosphofructokinase from a variety of sources, was found to activate both native and phosphorylated fluke phosphofructokinase. The most potent activators of fluke phosphofructokinase were found to be Fru-2,6-P2, AMP, and phosphorylation. The endogenous level of Fru-2,6-P2 in the flukes was determined to be 29 +/- 1.3 nmol/g wet wt, a level that may well modulate enzyme activity. Fru-6-P,2-kinase, the enzyme responsible for synthesis of Fru-2,6-P2, was found to be present in the flukes. Our results suggest physiological roles for phosphorylation and Fru-2,6-P2 in regulation of fluke phosphofructokinase.     

Phosphofructokinase 2 and glycolysis in HT29 human colon adenocarcinoma cell line. Regulation by insulin and phorbol esters.

Kinetic properties of phosphofructokinase 2 (PFK2) and regulation of glycolysis by phorbol 12-myristate 13-acetate (PMA) and insulin were investigated in highly glycolytic HT29 colon cancer cells. PFK2 was found to be inhibited by citrate and, to a lesser extent, by phosphoenolpyruvate and ADP, but to be insensitive to inhibition by sn-glycerol phosphate. From these kinetic data, PFK2 from HT29 cells appears different from the liver form, but resembles somewhat the heart isoenzyme. Fructose 2,6-bisphosphate (Fru-2,6-P2) levels, glucose consumption and lactate production are increased in a dose-dependent manner in HT29 cells treated with PMA or insulin. The increase in Fru-2,6-P2 can be related to an increase in the Vmax. of PFK2, persisting after the enzyme has been precipitated with poly(ethylene glycol), without change in the Km for fructose 6-phosphate. The most striking effects of PMA and insulin on Fru-2,6-P2 production are observed after long-term treatment (24 h) and are abolished by actinomycin, cycloheximide and puromycin, suggesting that protein synthesis is involved. Furthermore, the effects of insulin and PMA on glucose consumption, lactate production, Fru-2,6-P2 levels and PFK2 activity are additive, and the effect of insulin on Fru-2,6-P2 production is not altered by pre-treatment of the cells with the phorbol ester. This suggests that these effects are exerted by separate mechanisms.     

Increase in Fru-2,6-P(2) levels results in altered cell division in Schizosaccharomyces pombe

Mitogenic response to growth factors is concomitant with the modulation they exert on the levels of Fructose 2,6-bisphosphate (Fru-2,6-P2), an essential activator of the glycolytic flux. In mammalian cells, decreased Fru-2,6-P2 concentration causes cell cycle delay, whereas high levels of Fru-2,6-P2 sensitize cells to apoptosis. In order to analyze the cell cycle consequences due to changes in Fru-2,6-P2 levels, the bisphosphatase-dead mutant (H258A) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme was over-expressed in Schizosaccharomyces pombe cells and the variation in cell phenotype was studied. The results obtained demonstrate that the increase in Fru-2,6-P2 levels results in a defective division of S. pombe, as revealed by an altered multisepted phenotype. The H258A-expressing cells showed impairment of cytokinesis, but normal nuclear division. In order to identify cellular mediators responsible for this effect, we transformed different S. pombe strains and observed that the cytokinetic defect was absent in cells defective for Wee1 kinase function. Therefore, in S. pombe, Wee1 integrates the metabolic signal emerging from changes in Fru-2,6-P2 content, thus coupling metabolism with cell proliferation. As the key regulators of the cell cycle checkpoints are conserved throughout evolution, these results may help to understand the experimental evidences obtained by manipulation of Fru-2,6-P2 levels in mammalian cells.