Characterization of a Human Placental Fructose-6-Phosphate, 2-Kinase/Fructose- 2,6 - Bisphosphatase
Ryuzo SakakibaraMasanori KatoNobuyuki OkamuraTakayuki NakagawaYoshihiro KomadaNobuaki TominagaMasahito ShimojoMasashi Fukasawa
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Abstract:
A full-length cDNA, which encodes a human placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a molecular weight of 58,000 by gel electrophoresis under denaturing conditions, compared to the deduced molecular weight of 59,410. The N-terminal sequence of 15 amino acids coincided with that of the deduced sequence. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with Vmax values of 142 and 0.2 milliunits/mg for the kinase and phosphatase activities, respectively. The phosphatase activity was extremely low, because one phosphatase active site residue was mutated, and consequently the kinase/phosphatase ratio was the highest among the known isozymes. Furthermore, the enzyme was phosphorylated by cAMP-dependent protein kinase, protein kinase C and also by [2-32P]fructose-2,6-bisphosphate. Phosphorylation by cAMP-dependent protein kinase and protein kinase C increased the maximal Fru-6-P,2-kinase activities by 1.8- and 1.1-fold, respectively. These results suggested that placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase is important in maintaining and regulating a relatively high rate of glycolysis in placenta.Keywords:
Phosphofructokinase 2
Dephosphorylation
Phosphofructokinase 2
Fructose 2,6-bisphosphate
Fructolysis
Fructose 1,6-bisphosphatase
Hexose
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The finding of protein phosphorylation in prokaryotes (Wang, J. Y. J., and Koshland, D. E. (1978) J. Biol. Chem. 253, 7605-7608) has been pursued further. The prokaryotic organism Salmonella typhimurium is shown to contain at least 10 phosphorylated proteins with serine or threonine phosphates which are produced by the action of at lest four protein kinases. The protein kinases are distinguished by their substrate specificity, their chromatographic behavior, and their inhibition patterns. The phosphorylations are reversible, and more than one protein phosphatase activity exists in these cells. The presence of specific protein kinases and phosphatases suggests that this form of protein covalent modification is involved in the regulation of different cellular functions in prokaryotes as it is in eukaryotes.
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Hydrogen Peroxide: A Key Messenger That Modulates Protein Phosphorylation Through Cysteine Oxidation
Ligand-receptor interactions can generate the production of hydrogen peroxide (H 2 O 2 ) in cells, the implications of which are becoming appreciated. Fluctuations in H 2 O 2 levels can affect the intracellular activity of key signaling components including protein kinases and protein phosphatases. Rhee et al . discuss recent findings on the role of H 2 O 2 in signal transduction. Specifically, H 2 O 2 appears to oxidize active site cysteines in phosphatases, thereby inactivating them. H 2 O 2 also can activate protein kinases; however, although the mechanism of activation for some kinases appears to be similar to that of phosphatase inactivation (cysteine oxidation), it is unclear how H 2 O 2 promotes increased activation of other kinases. Thus, the higher levels of intracellular phosphoproteins observed in cells most likely occur because of the concomitant inhibition of protein phosphatases and activation of protein kinases.
Second messenger system
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The identities of the protein phosphatases involved in the regulation of hepatic glycolysis, gluconeogenesis and aromatic amino acid breakdown were investigated using 6-phosphofructo-1-kinase, fructose-1,6-bisphosphatase, l-pyruvate kinase, phenylalanine hydroxylase and the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates. Purified preparations of protein phosphatases-1, 2A, 2B and 2C exhibited activity towards all five substrates in vitro, although phosphatases-1 and 2B were only weakly active. Studies in liver extracts using inhibitor-2 and trifluoperazine, which inhibit protein phosphatase-1 and 2B, respectively, confirmed that these phosphatases are unlikely to be important in dephosphorylating these substrates in vivo. Sequential fractionation of rat liver extracts by anion-exchange chromatography and gel-filtration failed to resolve any protein phosphatases acting on each substrate, apart from protein phosphatases-2A and 2C. The present results, together with those described in the following paper (in this journal) indicate that under the assay conditions used, protein phosphatase-2A is the most powerful phosphatase acting on each substrate, although protein phosphatase-2C contributes a significant percentage of the activity towards 6-phosphofructo-1-kinase. No clear evidence was obtained for a role of metabolites in the regulation of dephosphorylation of the five substrates. This study reinforces our contention that only a few serine-specific and threonine-specific protein phosphatase catalytic subunits participate in cellular regulation.
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A unique bifunctional enzyme, isocitrate dehydrogenase kinase/phosphatase (AceK) regulates isocitrate dehydrogenase (IDH) by phosphorylation and dephosphorylation in response to nutrient availability. Herein we report the crystal structure of AceK in complex with ADP and Mn2+ ions. Although the overall structure is similar to the previously reported structures which contain only one Mg2+ ion, surprisingly, two Mn2+ ions are found in the catalytic center of the AceK-Mn2+ structure. Our enzymatic assays demonstrate that AceK-Mn2+ showed higher phosphatase activity than AceK-Mg2+, whereas the kinase activity was relatively unaffected. We created mutants of AceK for all metal-coordinating residues. The phosphatase activities of these mutants were significantly impaired, suggesting the pivotal role of the binuclear (M1-M2) core in AceK phosphatase catalysis. Moreover, we have studied the interactions of Mn2+ and Mg2+ with wild-type and mutant AceK and found that the number of metal ions bound to AceK is in full agreement with the crystal structures. Combined with the enzymatic results, we demonstrate that AceK exhibits phosphatase activity in the presence of two, but not one, Mn2+ ions, similar to PPM phosphatases. Taken together, we suggest that metal ions help AceK to balance and fine tune its kinase and phosphatase activities.
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Hydrogen Peroxide: A Key Messenger That Modulates Protein Phosphorylation Through Cysteine Oxidation
Ligand-receptor interactions can generate the production of hydrogen peroxide (H2O2) in cells, the implications of which are becoming appreciated. Fluctuations in H2O2 levels can affect the intracellular activity of key signaling components including protein kinases and protein phosphatases. Rhee et al. discuss recent findings on the role of H2O2 in signal transduction. Specifically, H2O2 appears to oxidize active site cysteines in phosphatases, thereby inactivating them. H2O2 also can activate protein kinases; however, although the mechanism of activation for some kinases appears to be similar to that of phosphatase inactivation (cysteine oxidation), it is unclear how H2O2 promotes increased activation of other kinases. Thus, the higher levels of intracellular phosphoproteins observed in cells most likely occur because of the concomitant inhibition of protein phosphatases and activation of protein kinases.
Second messenger system
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