Regulation of fructose 2,6-biphosphate levels in Neurospora crassa

Both wild type and cr-1 mutant (adenylate cyclase and cyclic AMP-deficient) strains of Neurospora crassa contain fructose 2,6-biphosphate at levels of 2t nmol/g dry tissue weight. This level decreases by about 50% in both strains upon depriving the cells of carbon or nitrogen sources for 3 h. An increase in cyclic AMP levels produced by addition of lysine to nitrogen-starved cells produced no increase in fructose 2,6-biphosphate levels. Both strains respond to short-term addition of salicylate, acetate, or 2,4-dinitrophenol with an increase in fructose 2,6-biphosphate. Thus, the above-described regulation of fructose 2,6-biphosphate levels is cyclic AMP-independent. A suspension of the wild type produces a transient increase of fructose 2,6-biphosphate in response to administration of glucose, whereas the mutant strain does not respond unless it is fed exogenous cyclic AMP. Substitution of acetate for sucrose as a sole carbon source for growth leads to a differential decrease in fructose 2,6-biphosphate levels between the two strains: the wild type strain has 63% and the cr-1 mutant strain has 37% of the levels of fructose 2,6-biphosphate on acetate as compared to sucrose-grown controls. This may be the basis for an advantage of cr-1 over wild type in growth on acetate. Thus, although most regulation of fructose 2,6-biphosphate is cyclic AMP-independent, the levels can be regulated by a combination of carbon source and cyclic AMP levels.     

Adenylyl cyclase deficient cr-1 (Crisp) mutant of Neurospora crassa: Cyclic AMP-dependent nutritional deficiencies

The inability to synthesize cyclic AMP drastically affects the nutritional metabolism of Neurospora crassa. The adenylyl cyclase-less mutant cr-1 (crisp) did not utilize several carbon sources, including glycerol, mannitol, arabinose, and casaminoacids. However, in glucose or acetate it grew as well as the wild type. The following evidence suggested that these nutritional deficiencies were a direct result of the cr-1 mutation: (i), in crosses to wild type they segregated together with the crisp morphological marker; (ii), cyclic AMP added to the cr-1 mutant growth medium overcame the nutritional deficiencies; (iii), the cyclic AMP effect was specific for the crisp mutant, for it was not observed with the wild type, nor with a spontaneous glycerol-utilizing cr-1 strain.     

In vivo control of gluconeogenesis in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp) mutant.

The rate of cycloheximide-resistant incorporation of carbon from [14C]alanine and [14C]acetate into polysaccharidic material was used to study gluconeogenic activity in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp-1) mutant. The wild-type efficiently utilized alanine and acetate as gluconeogenic substrates, whereas the mutant used acetate efficiently but was unable to use alanine. Cycloheximide-resistant 14C-incorporating activity was sensitive to carbon catabolite effects (repression and inactivation) in the two strains, which suggested that cyclic AMP metabolism was not involved in these regulatory responses. In the wild type, gluconeogenesis was induced by incubation of the cells in the absence of a carbon source. In contrast, cr-1 required supplementation with acetate. This finding suggested that induction of gluconeogenesis in N. crassa could be mediated by metabolites formed in carbon-starved cells. The cr-1 mutant seemed to be deficient in this process and to depend on an exogenous effector to induce gluconeogenesis. Incubation of cr-1 with cyclic AMP partially overcame the acetate requirement for induction of gluconeogenesis.     

Fructose 2,6-bisphosphate in Dictyostelium discoideum. Independence of cyclic AMP production and inhibition of fructose-1,6-bisphosphatase

The occurrence of fructose 2,6-bisphosphate was detected in Dictyostelium discoideum. The levels of this compound were compared with those of cyclic AMP and several glycolytic intermediates during the early stages of development. Removal of the growth medium and resuspension of the organism in the differentiation medium decreased the content of fructose 2,6-bisphosphate to about 20% within 1 h, remaining low when starvation-induced development was followed for 8 h. The content of cyclic AMP exhibited a transient increase that did not correlate with the change in fructose 2,6-bisphosphate. If after 1 h of development 2% glucose was added to the differentiation medium, fructose 2,6-bisphosphate rapidly rose to similar levels to those found in the vegetative state, while the increase in cyclic AMP was prevented. The contents of hexose 6-phosphates, fructose 1,6-bisphosphate and triose phosphates changed in a way that was parallel to that of fructose 2,6-bisphosphate, and addition of sugar resulted in a large increase in the levels of these metabolites. The content of fructose 2,6-bisphosphate was not significantly modified by the addition of the 8-bromo or dibutyryl derivatives of cyclic AMP to the differentiation medium. These results provide evidence that the changes in fructose 2,6-bisphosphate levels in D. discoideum development are not related to a cyclic-AMP-dependent mechanism but to the availability of substrate. Fructose 2,6-bisphosphate was found to inhibit fructose-1,6-bisphosphatase activity of this organism at nanomolar concentrations, while it does not affect the activity of phosphofructokinase in the micromolar range. The possible physiological implications of these phenomena are discussed.     

Role of fructose 2,6-bisphosphate in the regulation of glycolysis and gluconeogenesis in chicken liver

Glucagon and dibutyryl cyclic AMP inhibited glucose utilization and lowered fructose 2,6-bisphosphate levels of hepatocytes prepared from fed chickens. Partially purified preparations of chicken liver 6-phosphofructo-1-kinase and fructose 1,6-bisphosphatase were activated and inhibited by fructose 2,6-bisphosphate, respectively. The sensitivities of these enzymes and the changes observed in fructose 2,6-bisphosphate levels are consistent with an important role for this allosteric effector in hormonal regulation of carbohydrate metabolism in chicken liver. In contrast, oleate inhibition of glucose utilization by chicken hepatocytes occurred without change in fructose, 2,6-bisphosphate levels. Likewise, pyruvate inhibition of lactate gluconeogenesis in chicken hepatocytes cannot be explained by changes in fructose 2,6-bisphosphate levels. Exogenous glucose caused a marked increase in fructose 2,6-bisphosphate content of hepatocytes from fasted but not fed birds. Both glucagon and lactate prevented this glucose effect. Fasted chicken hepatocytes responded to lower glucose concentrations than fasted rat hepatocytes, perhaps reflecting the species difference in hexokinase isozymes.     

Adenylate cyclase stimulating agents and mitogens raise fructose 2,6-bisphosphate levels in human fibroblasts. Evidence for a dual control of the metabolite

Fructose 2,6-bisphosphate, the most potent activator of 6-phosphofructo-1-kinase, has been demonstrated to mediate the increase of glycolytic flux induced by mitogens human fibroblasts. In the present work the molecular basis of transmembrane control of fructose 2,6-bisphosphate has been investigated. Prostacyclin and isoprenaline, known to activate adenylate cyclase, are able to increase fructose 2,6-bisphosphate levels, indicating that in human fibroblasts cyclic AMP plays a positive role in the control of the metabolite concentration, opposite to that exerted in hepatocytes. Substances known to activate protein kinase C such as phorbol 12-myristate 13-acetate, or to stimulate phosphoinositide turnover such as thrombin and bradykinin are also effective in raising fructose 2,6-bisphosphate. Therefore, we conclude that cyclic AMP and protein kinase C are likely involved in the control of fructose 2,6-bisphosphate levels in human fibroblasts.     

Control of Neurospora crassa morphology by cyclic adenosine 3'', 5''-monophosphate and dibutyryl cyclic adenosine 3'', 5''-monophosphate.

The role of cyclic adenosine 3', 5'-monophosphate (cyclic AMP) in the control of the Neurospora asexual life cycle was studied. Endogenous cyclic AMP levels were 10 to 20 times higher in strains having the wild-type cr-1 allele than in those carrying the mutated allele. In a wild-type strain these levels remained constant throughtout the entire growth period in shaken liquid cultures, except during a short period at the beginning of the stationary growth phase. In this period a marked increase in the cyclic nucleotide level was observed. The culture of cr-1 mutant strains in the presence of cyclic AMP or its dibutyryl derivative restores some morphological properties characteristic of wild-type strains. Specifically these cyclic nucleotides stimulated the rate of mycelial elongation, as well as the differentiation of aerial hyphae.     

On the control mechanism of bacterial growth by cyclic adenosine 3',5'-monophosphate

Inhibition of E. coli growth by cyclic adenosine monophosphate is observed in wild type strains cultured in glucose as carbon source, but not in a cyclic AMP receptor protein deficient mutant. A deletion mutant of the adenylate cyclase gene requires cyclic adenosine monophosphate for optimal growth. Using glucose as carbon source, 2 mM cyclic AMP promotes maximal rates of cell multiplication in this mutant; however higher concentrations of the nucleotide inhibit growth. Cell multiplication of wild type strains grown in glycerol is not affected by cyclic adenosine monophosphate. Nevertheless, in this carbon source the growth rate of the adenylate cyclase mutant is strongly inhibited by concentrations of this nucleotide beyond 0.1 mM. This suggests that growth inhibition by exogenous cyclic adenosine monophosphate is highly dependent on the intracellular levels of the nucleotide.     

Cyclic AMP regulation of fructose metabolism in germinatingPilobolus longipes spores

DormantPilobolus longipes spores metabolized fructose primarily to ethanol, CO₂, and trehalose. Cyclic AMP-induced spore activation was accompanied by a large stimulation of glycolytic activity. Mobilization of reserves, which was cyclic AMP dependent, accounted for a portion of the glycolytic product. The remaining product was derived from exogenous fructose. Increases in both fructose transport activity and hexose 6-phosphate levels were associated with 6-deoxyglucose-induced spore activation. Phosphofructokinase-1 activity in spore extracts was almost totally dependent upon fructose, 2,6-bisphosphate. High fructose 2,6-bisphosphate levels were correlated with rapid fructose metabolism. However, fructose alone caused a rise in fructose 2,6-bisphosphate content (sufficient to fully stimulate phosphofructokinase-1 activity) but there was no concurrent stimulation of glycolysis. These results suggest that glycolytic rates are determined mainly by hexose 6-phosphate levels and that cyclic AMP regulation of transport is an important determinant of hexose 6-phosphate concentration.     

The mechanism by which glucose increases fructose 2,6-bisphosphate concentration in Saccharomyces cerevisiae. A cyclic-AMP-dependent activation of phosphofructokinase 2

When glucose was added to a suspension of Saccharomyces cerevisiae in stationary phase, it caused a transient increase in the concentration of cyclic AMP and a more persistent increase in the concentration of hexose 6-phosphate and of fructose 2,6-bisphosphate. These effects of glucose on cyclic AMP and fructose 2,6-bisphosphate but not that on hexose 6-phosphate were greatly decreased in the presence of 0.15 mM acridine orange or when a temperature-sensitive mutant deficient in adenylate cyclase was used at the restrictive temperature. Incubation of the cells in the presence of dinitrophenol and in the absence of glucose increased the concentration of both cyclic AMP and fructose 2,6-bisphosphate, but with a minimal change in that of hexose 6-phosphate. Glucose induced also in less than 3 min a severalfold increase in the activity of 6-phosphofructo-2-kinase and this effect was counteracted by the presence of acridine orange. When a cell-free extract of yeast in the stationary phase was incubated with ATP-Mg and cyclic AMP, there was a 10-fold activation of 6-phosphofructo-2-kinase. Finally, the latter enzyme was purified 150-fold and its activity could then be increased about 10-fold upon incubation with ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase. This activation resulted from a 4.3-fold increase in V and a 2-fold decrease in Km. Both forms of the enzyme were inhibited by sn-glycerol 3-phosphate. From these results it is concluded that the effect of glucose in increasing the concentration of fructose 2,6-bisphosphate in S. cerevisiae is mediated by the successive activation of adenylate cyclase and of cyclic-AMP-dependent protein kinase and by the phosphorylation of 6-phosphofructo-2-kinase by the latter enzyme. In deep contrast with what is known of the liver enzyme, yeast 6-phosphofructo-2-kinase is activated by phosphorylation instead of being inactivated.     

The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers

The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3-5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6-phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26 degrees C, but were much more pronounced at the nonpermissive temperature of 35 degrees C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyr1), able to incorporate exogenous cyclic AMP. When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic-AMP-dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6-bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6-bisphosphate. Uncouplers, but not a nitrogen source, also induced an activation of glycogen phosphorylase and an inactivation of glycogen synthase when added to the cdc35 mutant incubated at the restrictive temperature of 35 degrees C without affecting cyclic AMP concentration.     

Vanadate counteracts glucagon effects in isolated rat hepatocytes

The incubation of isolated rat hepatocytes with vanadate increased the concentration of fructose 2,6-bisphosphate without modifying 6-phosphofructo-2-kinase activity. Vanadate also reverted and prevented the decrease of fructose 2,6-bisphosphate levels, of the "active" form of the 6-phosphofructo 2-kinase and of the pyruvate kinase activity ratio produced by glucagon, by probably counteracting the increase in cyclic AMP concentration.     

Lysine 274 is essential for fructose 2,6-bisphosphate inhibition of fructose-1,6-bisphosphatase.

Lysine 274 is conserved in all known fructose-1,6-bisphosphatase sequences. It has been implicated in substrate binding and/or catalysis on the basis of reactivity with pyridoxal phosphate as well as by x-ray crystallographic analysis. Lys274 of rat liver fructose-1,6-bisphosphatase was mutated to alanine by the polymerase chain reaction, and the T7-RNA polymerase-transcribed construct containing the mutant sequence was expressed in Escherichia coli. The mutant and wild-type forms of the enzyme were purified to homogeneity, and their specific activity, substrate dependence, and inhibition by fructose 2,6-bisphosphate and AMP were compared. While the mutant exhibited no change in maximal velocity, its Km for fructose 1,6-bisphosphate was 20-fold higher than that of the wild-type, and its Ki for fructose 2,6-bisphosphate was increased 1000-fold. Consistent with the unaltered maximal velocity, there were no apparent difference between the secondary structure of the wild-type and mutant enzyme forms, as measured by circular dichroism and ultraviolet difference spectroscopy. The Ki for the allosteric inhibitor AMP was only slightly increased, indicating that Lys274 is not directly involved in AMP inhibition. Fructose 2,6-bisphosphate potentiated AMP inhibition of both forms, but 500-fold higher concentrations of fructose 2,6-bisphosphate were needed to reduce the Ki for AMP for the mutant compared to the wild-type. However, potentiation of AMP inhibition of the Lys274----Ala mutant was evident at fructose 2,6-bisphosphate concentrations (approximately 100 microM) well below those that inhibited the enzyme, which suggests that fructose 2,6-bisphosphate interacts either with the AMP site directly or with other residues involved in the active site-AMP synergy. The results also demonstrate that although Lys274 is an important binding site determinant for sugar bisphosphates, it plays a more significant role in binding fructose 2,6-bisphosphate than fructose 1,6-bisphosphate, probably because it binds the 2-phospho group of the former while other residues bind the 1-phospho group of the substrate. It is concluded that the enzyme utilizes Lys274 to discriminate between its substrate and fructose 2,6-bisphosphate.     

ACTH stimulates fructose 2,6-bisphosphate synthesis and glycolysis in Y-1 adrenal tumor cells

The effect of ACTH on glycolysis has been studied in Y-1 tumor adrenal cells. ACTH caused a sustained increase in the liberation of lactate as well as a stimulation of both basal and glucose-induced fructose 2,6-bisphosphate content. ACTH produces changes also in the activities of phosphofructokinase-1 and phosphofructokinase-2. The addition of Ca2+ or dibutyryl cyclic AMP did not modify neither lactate production nor fructose 2,6-bisphosphate levels. The results suggest that fructose 2,6-bisphosphate regulates ACTH-induced glycolysis at the phosphofructokinase-1 step, although the biochemical mechanism of phosphofructokinase-2 activation remains elusive.     

Properties of a germination mutant of Phycomyces blakesleeanus

Spores of the Phycomyces blakesleeanus strain S440 germinated only for some 4 to 7% when activated with a heat treatment or with ammonium acetate. Contrary to wild type spores, they showed no increase in trehalase activity during or after the activating treatment. This was not due to a variant trehalase or a defective protein kinase but rather to the absence of an increase in cellular cyclic AMP which normally occurs in the wild type. Phosphodiesterase activity in the mutant was comparable to wild type activity and in both strains phosphodiesterase was inactivated by a heat treatment. The phosphodiesterase inhibitor 1-isobutyl, 3-methyl xanthine caused germination and trehalase activation in the wild type but not in the mutant. The results corroborate the importance of cyclic AMP in the breaking of dormancy and the activation of trehalase in this fungus.     

Single point mutations in either gene encoding the subunits of the heterooctameric yeast phosphofructokinase abolish allosteric inhibition by ATP

Yeast phosphofructokinase is a heterooctameric enzyme subject to a complex allosteric regulation. A mutation in the PFK1 gene, encoding the larger -subunits, rendering the enzyme insensitive to allosteric inhibition by ATP was found to be caused by an exchange of proline 728 for a leucine residue. By in vitro mutagenesis, we introduced this mutation in either PFK1 or PFK2 and found that the exchange in either subunit drastically reduced the sensitivity of the holoenzyme to ATP inhibition. This was accompanied by a lack of allosteric activation by AMP, fructose 2,6-bisphosphate, or ammonium and an increased resistance to heat inactivation. Yeast cells carrying either one mutation or both in conjunction did not display a strong phenotype when grown on fermentable carbon sources and did not show any significant changes in intermediary metabolites. Growth on non-fermentable carbon sources was clearly impaired. The strain carrying both mutant alleles was more sensitive to Congo Red than the wild-type strain or the single mutants indicating differences in cell wall composition. In addition, we found single pfk null mutants to be less viable than wild type at different storage temperatures and a pfk2 null mutant to be temperature-sensitive for growth at 37 degrees C. The latter mutant was shown to be respiration-dependent for growth on glucose.     

Structural organization of the Z-line protein, alpha-actinin, in developing skeletal muscle cells

The vegetative growth of the adenylyl cyclase-deficient mutant cr-1 (crisp), of Neurospora crassa, resembled a conidiogenic microcycle. It was demonstrated that an enzyme which is exclusively confined to conidia in wild-type strains, i.e., nicotinamide adenine dinucleotide (phosphate) glycohydrolase (NAD(P)ase; EC 3.2.2.6), was continuously released in the culture medium by the mutant. NAD(P)ase activity of agitated cr-1 cultures was much lower than that of standing cultures; nevertheless, the enzyme was actively produced immediately after agitation was stopped. Supplementation of the growth medium with cyclic AMP normalized the morphological phenotype of the cr-1 mutant and drastically reduced NAD(P)ase production. These results suggest that NAD(P)ase regulation is somehow dependent on cyclic AMP metabolism. However, the effect of the nucleotide over the enzyme does not appear to be direct, since other crisp mutants, cr-2 and cr-3, which also overproduced NAD(P)ase, were completely unresponsive to cyclic AMP. These strains possess normal adenylyl cyclase activity.     

Fructose 2,6-bisphosphate, sugar phosphates and adenine nucleotides in the regulation of glucose metabolism in the lactating rat mammary gland

Fructose 2,6-bisphosphate is present in the rat mammary gland, rising from a value of 1.4 nmol/g in pregnancy to 4.3 nmol/g tissue at 14 days lactation; the equivalent values calculated/ml intracellular water are 5.2 and 11.6 nmol, respectively. The tissue content of fructose 6-phosphate, fructose 1,6-bisphosphate, ATP and phosphoenolpyruvate remain relatively constant in the transition from pregnancy to the height of lactation. The changes in AMP, cyclic AMP, and citrate content of the mammary gland during lactation are such as to promote an increase in fructose 2,6-bisphosphate formation and flux through phosphofructokinase.     

Yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase. Properties of phospho and dephospho forms and of two mutants in which serine 11 has been changed by site-directed mutagenesis

The properties of dephospho- and phosphofructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae and of two mutant enzymes in which the phosphorylatable Ser11 had been changed by site-directed mutagenesis (Ser----Ala and Ser----Asp) were studied to clarify the role of cyclic AMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase. The mutant enzymes and wild type Ser11 fructose-1,6-bisphosphatase were overexpressed and purified to homogeneity. Phosphofructose-1,6-bisphosphatase was prepared by in vitro phosphorylation. The comparison of the properties of the above enzymes demonstrated that all four had similar maximum activity. However, the phosphoenzyme was about 3-fold more sensitive to AMP and fructose 2,6-bisphosphate inhibition than the dephosphoenzyme, suggesting that regulation operates in vivo by this mechanism, leading to decreased enzyme activity. The purified mutant enzymes Ala11 and Asp11 exhibited properties closely similar to those of dephospho- and phosphofructose-1,6-bisphosphatase, respectively. These results indicate that the functional group at residue 11 is an important factor in the regulation of fructose-1,6-bisphosphatase activity and that Ser(P) can be functionally substituted by Asp in this enzyme.     

Fructose 2,6-bisphosphate and AMP increase the affinity of the Ascaris suum phosphofructokinase for fructose 6-phosphate in a process separate from the relief of ATP inhibition

Kinetic data have been collected suggesting that heterotropic activation by fructose 2,6-bisphosphate and AMP is a result not only of the relief of allosteric inhibition by ATP but is also the result of an increase in the affinity of phosphofructokinase for fructose 6-phosphate. Modification of the Ascaris suum phosphofructokinase at the ATP inhibitory site produces a form of the enzyme that no longer has hysteretic time courses or homotropic positive (fructose 6-phosphate) cooperativity or substrate inhibition (ATP) (Rao, G.S. J., Wariso, B.A., Cook, P.F., Hofer, H.W., and Harris, B.G. (1987a) J. Biol. Chem. 262, 14068-14073). This form of phosphofructokinase is Michaelis-Menten in its kinetic behavior but is still activated by fructose 2,6-bisphosphate and AMP and by phosphorylation using the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK). Fructose 2,6-bisphosphate activates by decreasing KF-6-P by about 15-fold and has an activation constant of 92 nM, while AMP decreases KF-6-P about 6-fold and has an activation constant of 93 microM. Double activation experiments suggest that fructose 2,6-bisphosphate and AMP are synergistic in their activation. The desensitized form of the enzyme is phosphorylated by cAPK and has an increased affinity for fructose 6-phosphate in the absence of MgATP. The increased affinity results in a change in the order of addition of reactants from that with MgATP adding first for the nonphosphorylated enzyme to addition of fructose 6-phosphate first for the phosphorylated enzyme. The phosphorylated form of the enzyme is also still activated by fructose 2,6-bisphosphate and AMP.