Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.

STUDY QUESTION: How do elastic matrisome components change during the lifetime of the human ovary? SUMMARY ANSWER: The deposition and remodeling of mechanical matrisome components (collagen, elastin, elastin microfibril interface-located protein 1 (EMILIN-1), fibrillin-1 and glycosaminoglycans (GAGs)) that play key roles in signaling pathways related to follicle activation and development evolve in an age- and follicle stage-related manner. WHAT IS KNOWN ALREADY: The mechanobiology of the human ovary and dynamic reciprocity that exists between ovarian cells and their microenvironment is of high importance. Indeed, while the localization of primordial follicles in the collagen-rich ovarian cortex offers a rigid physical environment that supports follicle architecture and probably plays a role in their survival, ovarian extracellular matrix (ECM) stiffness limits follicle expansion and hence oocyte maturation, maintaining follicles in their quiescent state. As growing follicles migrate to the medulla of the ovary, they encounter a softer, more pliant ECM, allowing expansion and development. Thus, changes in the rigidity of the ovarian ECM have a direct effect on follicle behavior. Evidence supporting a role for the physical environment in follicle activation was provided in clinical practice by ovarian tissue fragmentation, which promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth and generation of mature oocytes. STUDY DESIGN, SIZE, DURATION: We investigated quantitative spatiotemporal changes in collagen, elastin, EMILIN-1, fibrillin-1 and GAGs from prepuberty to menopause, before conducting a closer analysis of the ECM surrounding follicles, from primordial to secondary stages, in both prepubertal and tissue from women of reproductive age. The study included ovarian tissue (cortex) from 68 patients of different ages: prepubertal (n = 16; mean age [±SD]=8 ± 2 years); reproductive (n = 21; mean age [±SD]=27 ± 4 years); menopausal with estrogen-based HRT (n = 7; mean age [±SD]=58 ± 4 years); and menopausal without HRT (n = 24; mean age [±SD]=61 ± 5 years). PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative investigations of collagen and GAG deposition in ovarian tissue throughout a woman's lifetime were conducted by analyzing brightfield images. Characteristic features of collagen fiber content were based on polarized light microscopy, since polarized light changes with fiber thickness. To evaluate the deposition and distribution of elastin, fibrillin-1 and EMILIN-1, multiplex immunofluorescence was used on at least three sections from each patient. Image processing and tailored bioinformatic analysis were applied to enable spatiotemporal quantitative evaluation of elastic system component deposition in the human ovary over its lifetime. MAIN RESULTS AND THE ROLE OF CHANCE: While collagen levels increased with age, fibrillin-1 and EMILIN-1 declined. Interestingly, collagen and elastin reached their peak in reproductive-age women compared to prepubertal (P < 0.01; P = 0.262) and menopausal subjects with (P = 0.706; P < 0.01) and without (P = 0.987; P = 0.610) HRT, indicating a positive impact of secreted estrogen and hormone treatment on collagen and elastin preservation. Interestingly, HRT appears to affect elastin presence in ovarian tissue, since a significantly higher (P < 0.05) proportion of elastin was detected in biopsies from menopausal women taking HRT compared to those not. Higher GAG levels were found in adult ovaries compared to prepubertal ovaries (P < 0.05), suggesting changes in tissue ultrastructure and elasticity with age. In this context, elevated GAG values are suspected to participate in hampering formation of the fibrillin-1 network (r = -0.2475; P = 0.04687), which explains its decline over time. This decline partially accounts for the decrease in EMILIN-1 (r = 0.4149; P = 0.00059). Closer examination of the ECM surrounding follicles from the primordial to the secondary stage, both before and after puberty, points to high levels of mechanical stress placed on prepubertal follicles compared to the more compliant ECM around reproductive-age follicles, as suggested by the higher collagen levels and lower elastin content detected mainly around primordial (P < 0.0001; P < 0.0001, respectively) and primary (P < 0.0001; P < 0.001, respectively) follicles. Such a stiff niche is nonpermissive to prepubertal follicle activation and growth, and is more inclined to quiescence. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The duration and form of administered HRT were not considered when studying the menopausal patient group undergoing treatment. Moreover, we cannot exclude interference from other nongynecological medications taken by the study patients on ovarian ECM properties since there is no information in the literature describing the impact of each medication on the ECM. Finally, since the ECM is by definition a very heterogeneous meshwork of proteins, the use of two-dimensional histology could be a limitation. Single time points on fixed tissues could also present limitations, since following ovary dynamics from prepuberty to menopause in the same patient is not feasible. WIDER IMPLICATIONS OF THE FINDINGS: From a biomechanical perspective, our study revealed important changes to ECM properties dictating the mechanical features of ovarian tissue, in line with the existing literature. Our findings pave the way for possible therapeutic targets at the ECM level in the context of female fertility and ovarian rejuvenation, such as mechanical stimulation, antifibrotic treatments, and prevention or reversion of elastic ECM degradation. Our study also sheds light on the follicle-specific ECM composition that is dependent on follicle stage and age. These data will prove very useful in designing biomimetic scaffolds and tissue-engineered models like the artificial ovary. Indeed, they emphasize the importance of encapsulating each type of isolated follicle in an appropriate biomaterial that must replicate the corresponding functional perifollicular ECM and respect ovarian tissue heterogeneity in order to guarantee its biomimicry. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C.A.A. is an FRS-FNRS research associate; grant 5/4/150/5 awarded to M.M.D.) and the Université Catholique de Louvain (PhD grant 'Coopération au développement' awarded to E.O.). None of the authors have any competing interests to declare.

Direct and indirect activation of eukaryotic elongation factor 2 kinase by AMP-activated protein kinase.

BACKGROUND: Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a key regulator of protein synthesis in mammalian cells. It phosphorylates and inhibits eEF2, the translation factor necessary for peptide translocation during the elongation phase of protein synthesis. When cellular energy demand outweighs energy supply, AMP-activated protein kinase (AMPK) and eEF2K become activated, leading to eEF2 phosphorylation, which reduces the rate of protein synthesis, a process that consumes a large proportion of cellular energy under optimal conditions. AIM: The goal of the present study was to elucidate the mechanisms by which AMPK activation leads to increased eEF2 phosphorylation to decrease protein synthesis. METHODS: Using genetically modified mouse embryo fibroblasts (MEFs), effects of treatments with commonly used AMPK activators to increase eEF2 phosphorylation were compared with that of the novel compound 991. Bacterially expressed recombinant eEF2K was phosphorylated in vitro by recombinant activated AMPK for phosphorylation site-identification by mass spectrometry followed by site-directed mutagenesis of the identified sites to alanine residues to study effects on the kinetic properties of eEF2K. Wild-type eEF2K and a Ser491/Ser492 mutant were retrovirally re-introduced in eEF2K-deficient MEFs and effects of 991 treatment on eEF2 phosphorylation and protein synthesis rates were studied in these cells. RESULTS & CONCLUSIONS: AMPK activation leads to increased eEF2 phosphorylation in MEFs mainly by direct activation of eEF2K and partly by inhibition of mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment of MEFs with AMPK activators can also lead to eEF2K activation independently of AMPK probably via a rise in intracellular Ca2+. AMPK activates eEF2K by multi-site phosphorylation and the newly identified Ser491/Ser492 is important for activation, leading to mTOR-independent inhibition of protein synthesis. Our study provides new insights into the control of eEF2K by AMPK, with implications for linking metabolic stress to decreased protein synthesis to conserve energy reserves, a pathway that is of major importance in cancer cell survival.

AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B.

Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation.

First trimester anesthesia exposure and fetal outcome. A review.

Approximately 0.5-2% of all pregnant women undergo nonobstetric surgery during their pregnancy. This percentage does not include patients who are in the early phase of gestation and are not aware of it at the time of surgery. When pregnancy is diagnosed, the concern raises whether surgery and anesthesia during early gestation pose hazard to the developing fetus, by increasing the risk of congenital anomalies and spontaneous abortion. Literature review suggests that there is no increase in congenital anomalies at birth in women who underwent anesthesia during pregnancy. However, first trimester anesthesia exposure does increase the risk of spontaneous abortion and lower birth weight. This is more likely due to surgical manipulation and the medical condition that necessitates surgery than to the exposure to anesthesia.

Identification of phosphorylation sites on human deoxycytidine kinase after overexpression in eucaryotic cells.

Compelling evidence suggests that deoxycytidine kinase (dCK), a key enzyme in the salvage of deoxyribonucleosides and in the activation of clinically relevant nucleoside analogues, can be regulated by reversible phosphorylation. In this study, we show that dCK overexpressed in HEK-293T cells was labelled after incubation of the cells with [32P]orthophosphate. Tandem mass spectrometry allowed the identification of 4 in vivo phosphorylation sites, Thr3, Ser11, Ser15, and Ser74. These results provide the first evidence that dCK is constitutively multiphosphorylated in intact cells. In addition, site-directed mutagenesis demonstrated that phosphorylation of Ser74, the major in vivo phosphorylation site, is crucial for dCK activity.

Variability in erythrocyte fructosamine 3-kinase activity in humans correlates with polymorphisms in the FN3K gene and impacts on haemoglobin glycation at specific sites.

BACKGROUND: Part of the fructosamines that are bound to intracellular proteins are repaired by fructosamine 3-kinase (FN3K). Because subject-to-subject variations in erythrocyte FN3K activity could affect the level of glycated haemoglobin independently of differences in blood glucose level, we explored if such variability existed, if it was genetically determined by the FN3K locus on 17q25 and if the FN3K activity correlated inversely with the level of glycated haemoglobin. RESULTS: The mean erythrocyte FN3K activity did not differ between normoglycaemic subjects (n = 26) and type 1 diabetic patients (n = 31), but there was a wide interindividual variability in both groups (from about 1 to 4 mU/g haemoglobin). This variability was stable with time and associated (P < 0.0001) with two single nucleotide polymorphisms in the promoter region and exon 6 of the FN3K gene. There was no significant correlation between FN3K activity and the levels of HbA1c, total glycated haemoglobin (GHb) and haemoglobin fructoselysine residues, either in the normoglycaemic or diabetic group. However, detailed analysis of the glycation level at various sites in haemoglobin indicated that the glycation level of Lys-B-144 was about twice as high in normoglycaemic subjects with the lowest FN3K activities as compared to those with the highest FN3K activities. CONCLUSION: Interindividual variability of FN3K activity is substantial and impacts on the glycation level at specific sites of haemoglobin, but does not detectably affect the level of HbA1c or GHb. As FN3K opposes one of the chemical effects of hyperglycaemia, it would be of interest to test whether hypoactivity of this enzyme favours the development of diabetic complications.

New targets of AMP-activated protein kinase.

The discovery of the AMP-activated protein kinase (AMPK) more than a decade ago has shed much light on the cellular response to stresses characterized by a fall in the concentration of ATP and an increase in the AMP/ATP ratio. All conditions known to increase this ratio activate AMPK, whose major role is to act as an emergency signal to conserve ATP. It does so by inhibiting anabolic processes and by activating pathways producing ATP. In recent years, our laboratory has discovered new targets of AMPK. The purpose of this short review is to summarize our contribution to this field.

Regulation of protein kinase D by multisite phosphorylation. Identification of phosphorylation sites by mass spectrometry and characterization by site-directed mutagenesis.

Activation of the serine/threonine kinase, protein kinase D (PKD/PKC mu) via a phorbol ester/PKC-dependent pathway involves phosphorylation events. The present study identifies five in vivo phosphorylation sites by mass spectrometry, and the role of four of them was investigated by site-directed mutagenesis. Four sites are autophosphorylation sites, the first of which (Ser(916)) is located in the C terminus; its phosphorylation modifies the conformation of the kinase and influences duration of kinase activation but is not required for phorbol ester-mediated activation of PKD. The second autophosphorylation site (Ser(203)) lies in that region of the regulatory domain, which in PKC mu interacts with 14-3-3tau. The last two autophosphorylation sites (Ser(744) and Ser(748)) are located in the activation loop but are only phosphorylated in the isolated PKD-catalytic domain and not in the full-length PKD; they may affect enzyme catalysis but are not involved in the activation of wild-type PKD by phorbol ester. We also present evidence for proteolytic activation of PKD. The fifth site (Ser(255)) is transphosphorylated downstream of a PKC-dependent pathway after in vivo stimulation with phorbol ester. In vivo phorbol ester stimulation of an S255E mutant no longer requires PKC-mediated events. In conclusion, our results show that PKD is a multisite phosphorylated enzyme and suggest that its phosphorylation may be an intricate process that regulates its biological functions in very distinct ways.

Mutagenesis of the fructose-6-phosphate-binding site in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Multiple alignment of several isozyme sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed conserved residues in the 2-kinase domain. Among these residues, three asparagine residues (Asn76, Asn97 and Asn133; numbering refers to the liver isozyme sequence) and three threonine residues (Thr132, Thr134 and Thr135) are located near the fructose 6-phosphate-binding site in the crystal structure of the bifunctional enzyme. The role of these residues in substrate binding and catalysis in the 6-phosphofructo-2-kinase domain has been studied by mutagenesis to alanine. Since the crystal structure of 6-phosphofructo-2-kinase does not contain fructose 6-phosphate, this substrate was docked into the putative binding site by computer modelling, and its interactions with the protein were predicted. Analysis of the mutagenesis-induced changes in kinetic properties and of the substrate-docking model revealed that all these residues are directly or indirectly involved in fructose-6-phosphate binding. All the mutants displayed an increased Km for fructose 6-phosphate (10-200-fold). We propose that Asn133 stabilises Arg138, which itself makes a direct electrostatic bond with the 6-phosphate group of fructose 6-phosphate, that Asn76 interacts with the C3 hydroxyl group of fructose 6-phosphate, that Thr132 makes a hydrogen bond with the C6 oxygen of this substrate, and that Thr134 interacts with two residues involved in fructose-6-phosphate binding, Thr132 and Tyr199. On the other hand, Asn97 and Thr135 play structural roles, by maintaining the structure of the fructose-6-phosphate-binding pocket.

Phosphorylation and activation of heart 6-phosphofructo-2-kinase by protein kinase B and other protein kinases of the insulin signaling cascades.

To understand the insulin-induced activation of 6-phosphofructo-2-kinase (PFK-2) of the bifunctional enzyme PFK-2/fructose-2,6-bisphosphatase in heart, the effect of phosphorylation by protein kinases of the insulin signaling pathways on PFK-2 activity was studied. Purified PFK-2/fructose-2, 6-bisphosphatase from bovine heart is a mixture of two isoforms (Mr 58,000 and 54,000 on SDS-polyacrylamide gels). The Mr 54,000 protein is an alternatively spliced form, lacking phosphorylation sites for protein kinases. Recombinant enzymes corresponding to the Mr 58,000 (BH1) and Mr 54,000 (BH3) forms were expressed and used as substrates for phosphorylation. The recombinant BH1 isoform was phosphorylated by p70 ribosomal S6 kinase (p70(s6k)), mitogen-activated protein kinase-activated protein kinase-1, and protein kinase B (PKB), whereas the recombinant BH3 isoform was a poor substrate for these protein kinases. Treatment with all protein kinases activated PFK-2 in the recombinant BH1 preparation. Phosphorylation of the recombinant BH1 isoform correlated with PFK-2 activation and was reversed by treatment with protein phosphatase 2A. All the protein kinases phosphorylated Ser-466 and Ser-483 in the BH1 isoform, but to different extents: p70(s6k) preferentially phosphorylated Ser-466, whereas mitogen-activated protein kinase-activated protein kinase-1 and PKB phosphorylated Ser-466 and Ser-483 to a similar extent. We propose that PKB is part of the insulin signaling cascade for PFK-2 activation in heart.

Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis.

Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

In a structural model of the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase based on the analogy with adenylate kinase, Lys-174, Asp-179 and Asp-191 residues are located in the putative active site. Asp-179 and Asp-191 are conserved in all known 6-phosphofructo-2-kinase sequences. In contrast, Lys-174 is conserved except in a yeast isoenzyme, fbp26, where it is replaced by glycine. Yeast fbp26 possesses fructose-2,6-bisphosphatase activity, but is devoid of 6-phosphofructo-2-kinase activity. Mutation of Asp-179 and Asp-191 of the rat liver isoenzyme to alanine increased the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate 2000- and 1000-fold respectively, whereas mutation of Lys-174 to glycine decreased the Vmax of 6-phosphofructo-2-kinase more than 4000-fold. In contrast, none of the mutations affected the kinetic parameters of fructose-2,6-bisphosphatase. CD and fluorescence measurements indicated that the mutations had no effect on the structure and stability of the recombinant proteins. The results show that Asp-179 and Asp-191 participate in fructose 6-phosphate binding, whereas Lys-174 is important for catalysis. Therefore the natural mutation of Lys-174 to glycine in the fbp26 yeast isoenzyme could explain the lack of 6-phosphofructo-2-kinase activity. These results support a novel 6-phosphofructo-2-kinase structure model based on adenylate kinase.

Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase.

Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted.

The ATP-binding site in the 2-kinase domain of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the role of Lys-54 and Thr-55 by site-directed mutagenesis.

All known 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes contain a sequence (GX4GK(S/T)) in the 6-phosphofructo-2-kinase domain corresponding to the so-called nucleotide binding fold signature or Walker A motif. Mutagenesis and crystal structure data from several nucleotide binding proteins, which also contain this sequence, showed the importance of the lysine and serine/threonine residues in nucleotide binding. We have studied the role of Lys-54 and Thr-55 in MgATP binding in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis. Lys-54 was mutated to methionine, whereas Thr-55 was mutated to valine, serine, and cysteine. Three mutants, Lys-54 to Met and Thr-55 to Cys or Val, displayed more than a 5000-fold decrease in 6-phosphofructo-2-kinase activity compared with the wild type. The mutations had no effect on fructose-2, 6-bisphosphatase activity and did not affect the activation of fructose-2,6-bisphosphatase after phosphorylation by cyclic 3', 5'-AMP-dependent protein kinase. Binding experiments with ATP, ADP, and their analogs (3'-N-methylanthraniloyl derivatives) showed that these two residues do not play the same role. Lys-54 is involved in ATP binding, whereas Thr-55 is important for catalysis.

Study of the roles of Arg-104 and Arg-225 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis.

The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) have been studied by site-directed mutagenesis. In recombinant rat liver PFK-2/FBPase-2, mutation of Arg-225 to Ser increased the Km of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentrations between pH 6 and 9.5. The mutation had no effect on the Vmax of PFK-2 or on the Km of PFK-2 for MgATP. The mutation also increased the Vmax. of FBPase-2 4-fold without changing the Km for Fru-2,6-P2 or IC50 of Fru-6-P. These findings are in agreement with a previous study [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protection by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and suggest that Arg-225 participates in Fru-6-P binding. In recombinant rat muscle PFK-2/FBPase-2, mutation of Arg-104 to Ser increased the Km for Fru-6-P 60-fold, increased the IC50 of citrate, increased the Vmax. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the Km of PFK-2 for MgATP. The mutation also decreased the Vmax. of FBPase-2 3-fold, increased the Km for Fru-2,6-P2 70-fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity was more sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is involved in Fru-6-P binding in the PFK-2 domain and that it might also bind citrate. Structural changes resulting from the mutation might be responsible for the changes in kinetic properties of FBPase-2.

Rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the kinase domain by site-directed mutagenesis.

Sequence alignment and modeling of the 2-kinase domain of the liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, on 6-phosphofructo-1-kinase from Bacillus stearothermophilus and Escherichia coli (Bazan, J. F., Fletterick, R. J., and Pilkis, S. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646) suggested that Cys-160 of the 2-kinase would correspond to Asp-127 of the 1-kinase, which acts as a general base catalyst. We have studied the validity of this alignment by site-directed mutagenesis of residues in the 2-kinase domain of skeletal muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Cys-160 was mutated to Asp or Ser. Two adjacent residues, Glu-157 and Asp-162, either of which could act as a general base catalyst, were mutated to Ala. Asp-162 corresponds to Asp-129 in the bacterial 1-kinase, which is also essential for catalysis and might bind Mg2+. None of these mutations significantly decreased the Vmax of the 2-kinase, suggesting that the mutated amino acids are not essential for catalysis and therefore do not play the same role as Asp-127 and Asp-129 in the bacterial 1-kinase. Mutation of Glu-157 and Asp-162 to alanine had no effect on the kinetic parameters of the bifunctional enzyme, indicating that these two negatively charged residues are not involved in catalysis and substrate binding.

Molecular forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase expressed in rat skeletal muscle.

The rat cDNA for the muscle-type (M) isozyme of 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) contains two putative translation initiation sites. To determine whether the M isozyme expressed in rat skeletal muscle corresponds to the short (PFK2M-sf) or the long (PFK2M-lf) isoform, we have expressed them in Escherichia coli. A third construction was also expressed in which the second ATG codon was deleted (PFK2M-lf delta ATG) to ensure that initiation started at the first ATG. The properties of these recombinant proteins were compared with those of the PFK-2/FBPase-2 present in rat skeletal muscle and liver. The recombinant proteins displayed PFK-2 and FBPase-2 activities and the M(r) values of the subunits measured by SDS-polyacrylamide gel electrophoresis were compatible with the calculated ones. The purified recombinant lf form contained not only the expected lf band (54,500 M(r)) but also the sf band (52,000 M(r)), indicating that the expression system could synthesize the long and the short isoforms from the same mRNA. The kinetic properties of the recombinant sf form were not different from those of the rat muscle enzyme. By contrast, lf delta ATG PFK-2 displayed a higher Km for its substrates and a lower Vmax. Immunoblotting with an antibody directed against the long isoform revealed a 54,500 M(r) band both in the lf and the lf delta ATG recombinant, but no band in rat skeletal muscle extracts. In these extracts, one band of 52,000 and a minor one of 54,500 M(r) were detected by an anti PFK-2/FBPase-2 antibody. The 54,500 M(r) band was recognized by an antibody directed against the L isozyme, suggesting that a small amount of the latter is expressed in skeletal muscle. Thus, the M isozyme differs from the L isozyme by replacement of the first 32 amino acids of the L isozyme by an unrelated nonapeptide.

Evidence for new phosphorylation sites for protein kinase C and cyclic AMP-dependent protein kinase in bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was phosphorylated by incubation with [gamma-32P]MgATP and cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). After digestion with chymotrypsin, the phosphorylation sites for the two protein kinases were identified by peptide mapping, and microsequencing. Evidence for new phosphorylation sites for PKA (Ser-483) and PKC (Ser-84 and Ser-466) was obtained.

The two forms of bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase result from alternative splicing.

Purified bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) showed two bands with subunit M(r) of 58,000 and 54,000 when analysed by SDS/PAGE. Both the 58,000- and 54,000-M(r) forms were phosphorylated by cyclic AMP-dependent protein kinase (PKA) and by protein kinase C (PKC) in vitro. Phosphorylation by PKA decreased the apparent Km of PFK-2 for one of its substrates, fructose 6-phosphate, while phosphorylation by PKC did not correlate with any change in PFK-2 activity. The differences between the 58,000- and 54,000-M(r) forms were studied by electroblotting, peptide mapping and microsequencing. Residues 451-510, which correspond to exon 15 in the rat and contain phosphorylation sites for PKA (Ser-466) and PKC (Thr-475), were absent from the 54,000-M(r) form. Peptide mapping after phosphorylation by [gamma-32P]MgATP and PKC showed a phosphorylated peptide containing Thr-475, which was present in the 58,000-M(r) form but not in the 54,000-M(r) form. The fact that the latter form was phosphorylated by PKC and PKA suggests that other phosphorylation sites for PKA and PKC are located outside the region encoded by exon 15. Finally, analysis of RNA from bovine heart showed that the tissue contains two PFK-2/FBPase-2 mRNAs, only one of which was recognized by a probe specific to the region coding for Ser-466 and Thr-475. Taken together, these findings demonstrate that the 58,000- and 54,000-M(r) forms of bovine heart PFK-2/FBPase-2 result from alternative splicing of the same primary transcript.