Regulation of protein kinase cascades by protein phosphatase 2A.

Many protein kinases themselves are regulated by reversible phosphorylation. Upon cell stimulation, specific kinases are transiently phosphorylated and activated. Several of these protein kinases are substrates for protein phosphatase 2A (PP2A), and PP2A appears to be the major kinase phosphatase in eukaryotic cells that downregulates activated protein kinases. This idea is substantiated by the observation that some viral proteins and naturally occurring toxins target PP2A and modulate its activity. There is increasing evidence that PP2A activity is regulated by extracellular signals and during the cell cycle. Thus, PP2A is likely to play an important role in determining the activation kinetics of protein kinase cascades.

CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase.

Microscopic screening of a collection of cold-sensitive mutants of Saccharomyces cerevisiae led to the identification of a new gene, CDC55, which appears to be involved in the morphogenetic events of the cell cycle. CDC55 maps between CDC43 and CHC1 on the left arm of chromosome VII. At restrictive temperature, the original cdc55 mutant produces abnormally elongated buds and displays a delay or partial block of septation and/or cell separation. A cdc55 deletion mutant displays a cold-sensitive phenotype like that of the original isolate. Sequencing of CDC55 revealed that it encodes a protein of about 60 kDa, as confirmed by Western immunoblots using Cdc55p-specific antibodies. This protein has greater than 50% sequence identity to the B subunits of rabbit skeletal muscle type 2A protein phosphatase; the latter sequences were obtained by analysis of peptides derived from the purified protein, a polymerase chain reaction product, and cDNA clones. An extragenic suppressor of the cdc55 mutation lies in BEM2, a gene previously identified on the basis of an apparent role in bud emergence.

Characterisation of two protein phosphatase 2A holoenzymes from maize seedlings.

Two holoenzymes of protein phosphatase 2A (PP2A), designated PP2AI and PP2AII, were purified from maize seedlings. The subunit composition of maize holoenzymes generally resembled those of animal PP2A. Using SDS/PAGE and Western blots with antibodies generated against peptides derived from animal PP2A, we established the subunit composition of plant protein phosphatase 2A. In both maize holoenzymes, a 38000 catalytic (PP2Ac) and a 66000 constant regulatory subunit (A) constituting the core dimer of PP2A were present. In addition, PP2AI (180000-200000) contained a protein of 57000 which reacted with antibodies generated against the peptide (EFDYLKSLEIEE) conserved in all eukaryotic Balpha regulatory subunits. In contrast, none of the proteins visualised in PP2AII (140000-160000) by double staining reacted with these antibodies. The activity of PP2AI measured with (32)P-labelled phosphorylase a in the presence of protamine and ammonium sulfate is about two times higher than that of PP2AII. PP2AI and PP2AII displayed different patterns of activation by protamine, polylysine and histone H1 and exhibit high sensitivity toward inhibition by okadaic acid. The data obtained provide direct biochemical evidence for the existence in plants of PP2A holoenzymes composed of a catalytic subunit complexed with one or two regulatory subunits.

Type 2A protein phosphatase, the complex regulator of numerous signaling pathways.

Type 2A protein phosphatase (PP2A) comprises a diverse family of phosphoserine- and phosphothreonine-specific enzymes ubiquitously expressed in eukaryotic cells. Common to all forms of PP2A is a catalytic subunit (PP2Ac) which can form two distinct complexes, one with a structural subunit termed PR65/A and another with an alpha4 protein. The PR65/A-PP2Ac dimer may further associate with a regulatory subunit and form a trimeric holoenzyme. To date, three distinct families of regulatory subunits, which control substrate selectivity and phosphatase activity and target PP2A holoenzymes to their substrates, have been identified. Other molecular mechanisms that regulate PP2Ac function include phosphorylation, carboxyl methylation, inhibition by intracellular protein inhibitors (I(1)(PP2A) and I(2)(PP2A)), and stimulation by ceramide. PP2A dephosphorylates many proteins in vitro, but in vivo protein kinases and transcription factors appear to represent two major sets of substrates. Several natural compounds can inhibit PP2A activity and are used to study its function. Mutations in genes encoding PR65/A subunits have been identified in several different human cancers and the PP2A inhibitor, termed fostriecin, is being tested as an anticancer drug. Thus, a more thorough understanding of PP2A structure and function may lead to the development of novel strategies against human diseases.

Short communication: beta-hydroxybutyrate dehydrogenase activity in human placenta.

It has been shown that mitochondria isolated from human placenta contain beta-hydroxybutyrate dehydrogenase activity. The properties of the beta-hydroxybutyrate dehydrogenase from human early placenta are very similar to those of the enzyme isolated from other mammalian tissues.

Tightly coupled mitochondria from human early placenta.

1. A procedure for the isolation of tightly coupled mitochondria from human early placenta is described. 2. Mitochondria obtained by this method were able to oxidize Krebs cycle intermediates, pyruvate, glutamate, glutamine, palmitoyl-carnitine, alpha-glycerophosphate and beta-hydroxybutyrate. 3. These mitochondria incubated in the medium containing ethylene diamine tetraacetic acid and bovine serum albumin and no added Mg2+ ions exhibited a high respiratory control and adenosine diphosphate:oxygen (ADP:O) ratios corresponding to the theoretical values for all substrates tested. Addition of Mg2+ ions markedly reduced the respiratory control index and ADP:O ratio. 4. Adenosine triphosphatase (ATPase) activity in the obtained mitochondrial preparation was stimulated about tenfold by Mg2+. Oligomycin inhibited Mg2+-stimulated ATPase activity by about 25 per cent, but completely inhibited this activity in the absence of Mg2+ ions. 5. It is concluded that the effect of Mg2+ ions on the respiratory control and ADP:O ratio reported in this paper is exerted mainly through the Mg/+-stimulated oligomycin-insensitive ATPase activity.

Acetoacetate utilization by human placental mitochondria.

It has been shown that mitochondria from human placenta incubated in the presence of 2-oxoglutarate or its precursors utilize acetoacetate at the rate about I nmol/min/mg protein. Utilization of acetoacetate is completely inhibited by arsenite. Mitochondria from human placental tissue show activity of 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. It is proposed that acetoacetate utilization by placental mitochondria proceeds via the conversion to acetoacetyl-CoA catalysed by 3-oxoacid-CoA transferase, and then to acetyl-CoA, catalysed by acetoacetyl-CoA thiolase.

Isolation, properties and role in progesterone biosynthesis of cytosolic malic enzyme from human term placenta.

Cytosolic malic enzyme (L-malate: NADP oxidoreductase decarboxylating, EC I. I. I.40) has been isolated and purified from postmitochondrial supernatant of human term placenta by ammonium sulphate fractionation, chromatography on diethylaminoethyl- (DEAE-)cellulose, Sepharose 6B, ADP-Sepharose 4B and Ultrogel AcA-34 to apparent homogeneity as judged from polyacrylamide gel electrophoresis (PAGE). The specific activity of the purified enzyme was 24.0 mumol X min-1 X mg-1 protein, which corresponds to about 7500-fold purification. The molecular weight of the native enzyme was determined by gel filtration to be about 250,000. Sodium dodecyl sulphate- (SDS-)PAGE showed one polypeptide band of molecular weight 63,000. It appears that the native protein is a tetramer composed of subunits of identical molecular weight. The isoelectric point of the purified malic enzyme was pH 5.55. The enzyme was shown to carboxylate pyruvate in the presence of high concentrations of pyruvate and bicarbonate at about 80 per cent of the rate of the forward reaction. The optimum pH for the carboxylation reaction was pH 7.3, and that for the decarboxylation reaction varied with malate concentration. The Km values, determined at pH 7.2, for malate, NADP+, Mn2+, and Mg2+ were 81 microM, 10 microM, 2.5 microM and 0.6 mM, respectively. The Km values for pyruvate, NADPH and bicarbonate were 4 mM, 25 microM and 20 mM, respectively. The enzyme converted malate to pyruvate (at pH 6.3) in the presence of 5 mM NAD+ at approximately 80 per cent of the maximum rate with NADP+. It exhibited oxaloacetate decarboxylase activity at about 10 per cent of the rate of oxidative decarboxylation of malate (with NADP+ as coenzyme) and pyruvate reductase activity at about 4 per cent of the rate of oxidative decarboxylation of malate with NADP+. The oxidative decarboxylation of malate was inhibited by malate at lower values of pH of incubation medium. This inhibition gradually decreased as the pH of the incubation medium increased. No inhibition was observed at pH 8.2. The addition of purified cytoplasmic malic enzyme, pyruvate, bicarbonate and NADPH generating system (consisting of NADP+, glucose 6-phosphate, glucose 6-phosphate dehydrogenase) stimulated about twofold progesterone biosynthesis by the isolated human placental mitochondria. This stimulation was abolished by arsenite and fluorocitrate. A possible role for the cytosolic malic enzyme in the regulation of progesterone biosynthesis in human placenta is discussed.

Serine/threonine protein phosphatases in the control of cell function.

Reversible protein phosphorylation is a fundamental mechanism by which many biological functions are regulated. Achievement of such control requires the coordinated action of the interconverting enzymes, the protein kinases and protein phosphatases. By comparison with protein kinases, a limited number of protein phosphatase catalytic subunits are present in the cell, which raises the question of how such a small number of dephosphorylating enzymes can counterbalance the action of the more numerous protein kinases. In mammalian cells, four major classes of Ser/Thr-specific phosphatase catalytic subunits have been identified, comprising two distinct gene families. The high degree of homology among members of the same family, PP1, PP2A and PP2B, and the high degree of evolutionary conservation between organisms as divergent as mammals and yeast, implies that these enzymes are involved in fundamental cell functions. Type 1 enzymes appear to acquire specificity by association with targeting regulatory subunits which direct the enzymes to specific cellular compartments, confer substrate specificity and control enzyme activity. In spite of the progress made in determining the structure of the PP2A subunits, very little is known about the control of this activity and about substrate selection. Recent studies have unravelled a significant number of regulatory subunits. The potential existence of five distinct B or B-related polypeptides, some of which are present in multiple isoforms, two A and two C subunit isoforms, raises the possibility that a combinatorial association could generate a large number of specific PP2A forms with different substrate specificity and/or cellular localization. Moreover, biochemical, biological and genetic studies all concur in suggesting that the regulatory subunits may play an important role in determining the properties of the Ser/Thr protein phosphatases and hence their physiological functions.

Comparison of uncoupling activities of chlorophenoxy herbicides in rat liver mitochondria.

The effects of the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-chloro-2-methylphenoxyacetic acid (MCPA) and 2-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP) on respiration and oxidative phosphorylation in rat liver mitochondria were examined in vitro. Respiration rates of glutamate, malate and succinate were investigated in the presence of each herbicide (0.1-4.0 mM). At lower concentrations, all herbicides stimulated state 4 respiration, decreased the respiratory control ratio and the ADP/O ratio. The respiration rate in state 3 and uncoupled state was unaffected. At higher concentrations all bioenergetic parameters, respiration in state 4, 3 and uncoupled state, as well as respiratory control ratio and ADP/O, were inhibited in a concentration-dependent manner. These data indicate that these herbicides alter energy metabolism in rat liver mitochondria by uncoupling of oxidative phosphorylation. 2,4,5-TP possesses the strongest uncoupling properties followed by 2,4,5-T, MCPA and 2,4-D in that order.

Effects of actinomycin D and cycloheximide on the increase in tyrosinase activity of hamster amelanotic melanoma cells in vitro.

Tyrosinase activity in the Ab hamster amelanotic melanoma cells cultured in serum-free Eagle's MEM increased 3 times after 6 h of primary cell culture. This increase was inhibited completely by cycloheximide, while actinomycin D had no effect on this process. After 24 h of culture in MEM with calf serum, further increase of the tyrosinase activity was inhibited by both cycloheximide and actinomycin D. The data presented may indicate that the increase of tyrosinase activity in the primary cell culture of the Ab melanoma is due initially to the unblocking of translation and later to the activation of transcription of the gene controlling the enzyme.

Purification and properties of the NAD(P)-dependent malic enzyme from human placental mitochondria.

The NAD(P)-dependent malic enzyme from human term placental mitochondria was purified 108-fold with a final yield of 72% and specific activity of about 2 mumol per minute per milligram protein. The final preparation was completely free of fumarase, malic, and lactic dehydrogenases. Divalent cations were required for NAD(P)-dependent malic enzyme activity, Mn2+ and Co2+ were by far more effective activators than Mg2+ and Ni2+, whereas the reaction did not proceed in the presence of Ca2+. The optimum pH with NAD and NADP as coenzymes was at around 7.1 and 6.4, respectively. The ratio of the rate of NAD:NADP reduction was 7.4 and 1.3 at pH 7.1 and 6.4, respectively. The enzyme is activated by succinate and fumarate and inhibited by ATP. In the absence of fumarate the Michaelis constants for L-malate and NAD were 2.82 and 0.33 mM; and in the presence of fumarate 1.18 and 0.22 mM, respectively. This study presents the first report showing the purification and kinetic properties of NAD(P)-dependent malic enzyme from human tissue.

Isolation and properties of glycerol-3-phosphate oxidoreductase from human placenta.

Glycerol-3-phosphate oxidoreductase (sn-glycerol 3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) from human placenta has been purified by chromatography on 2,4,6-trinitrobenzenehexamethylenediamine-Sepharose, DEAE-Sephadex A-50 and 5'-AMP-Sepharose 4B approximately 15800-fold with an overall yield of about 19%. The final purified material displayed a specific activity of about 88 mumol NADH min-1 mg protein-1 and a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The native molecular mass, determined by Ultrogel AcA 44 filtration, was 62000 +/- 2000 whereas the subunit molecular mass, established on polyacrylamide gel in the presence of 0.1% sodium dodecyl sulphate, was 38000 +/- 500. The isoelectric point of the enzyme protein, determined by column isoelectric focusing, was found to be 5.29 +/- 0.09. The pH optimum of the placental enzyme was in the range 7.4-8.1 for dihydroxyacetone phosphate reduction and 8.7-9.2 for sn-glycerol 3-phosphate oxidation. The apparent Michaelis constants (Km) for dihydroxyacetone phosphate, NADH, sn-glycerol 3-phosphate and NAD+ were 26 microM, 5 microM, 143 microM and 36 microM respectively. The activity ratio of cytoplasmic glycerol-3-phosphate oxidoreductase to mitochondrial glycerol-3-phosphate dehydrogenase in human placental tissue was 1:2. The consumption of oxygen by human placental mitochondria incubated with the purified glycerol-3-phosphate oxidoreductase, NADH and dihydroxyacetone phosphate was similar to that observed in the presence of sn-glycerol 3-phosphate. The possible physiological role of glycerol-3-phosphate oxidoreductase in placental metabolism is discussed.

Leucine catabolism in human term placenta.

It has been shown that L-leucine is transaminated in the presence of 2-oxoglutarate and subsequently decarboxylated by human term placenta. About 60% of the transaminase activity was recovered in the cytoplasmic fraction and the remaining amount in the mitochondria. The dehydrogenase activity is localized almost exclusively in the mitochondrial fraction. The rate of the transamination of L-leucine is many times higher than the rate of decarboxylation of oxoacid. The possible physiological role of leucine degradation in human placenta is discussed.

Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein.

The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-[3H]ethylcarboxamidoadenosine [( 3H]NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the [3H]NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.

The catalytic subunit of protein phosphatase 2A is carboxyl-methylated in vivo.

We have used polyclonal antibodies against an internal peptide (residues 169 to 182; Ab169/182) and a peptide corresponding to the carboxyl terminus (residues 299 to 309; Ab299/309) to look for in vivo modifications of protein phosphatase 2A catalytic (PP2Ac) subunit. Treatment of extracts from human breast cancer (MCF7) cells with either alkali or ethanol increased immunoreactivity of PP2Ac subunit severalfold on Western blots with Ab299/309, but did not apparently change molecular weight or isoelectric point of the protein. In contrast, immunoreactivity with Ab169/182 was unchanged by these treatments. Subsequently, we demonstrated that the increase in PP2Ac subunit recognition by Ab299/309 coincides with the demethylation of this protein at the carboxyl-terminal leucine (Leu309). Methylation of PP2Ac subunit, in vitro, increases its activity toward both phosphorylase a and a phosphopeptide. The carboxyl-terminal sequence (TPDYFL) of PP2Ac subunit is completely conserved between mammals, yeast, fruit fly, and plants which suggests that regulation of this enzyme activity by carboxyl-terminal methylation has been conserved during evolution.

Subcellular distribution of isocitrate dehydrogenase in early and term human placenta.

The activities of NAD-specific and NADP-specific isocitrate dehydrogenases were measured in early and term human placenta. In both tissues the activity of NADP-specific isocitrate dehydrogenase was severalfold higher than that of the NAD-dependent enzyme. Subcellular distribution of these two enzymes in the placental tissue was estimated. About 60% of the total NADP-specific isocitrate dehydrogenase activity was found in the mitochondrial fraction and about 40% in the cytosol fraction. Insignificant amounts of the total activity were bound to the microsomal fraction. The whole of the NAD-specific isocitrate dehydrogenase activity was localized in the mitochondrial fraction. The total mitochondrial NADP-specific isocitrate dehydrogenase activity in both early and term placenta was also estimated from the mitochondrial specific activity of this enzyme and the amount of mitochondrial protein in wet tissue, calculated from the activities of citrate synthase or cytochrome c oxidase assayed in the isolated mitochondrial fraction and in the tissue of early and term human placenta.

Eukaryotic translation termination factor 1 associates with protein phosphatase 2A and targets it to ribosomes.

Purification of type 2A protein phosphatase (PP2A) from rabbit skeletal muscle resulted in the isolation of a trimeric phosphatase which is composed of a catalytic (PP2Ac), a structural (PR65alpha/Aalpha), and a regulatory (PR55alpha/Balpha) subunit, together with translation termination factor 1 (eRF1) and another protein of 55 kD (EMBO J., 15, 101-112). Yeast two-hybrid system analysis demonstrated that the eRF1 interacted with PP2Acalpha but not with PR65alpha/Aalpha or PR55alpha/Balpha. The N-terminal region of PP2Acalpha, comprising 50 amino acid residues, and the C-terminal part of eRF1, corresponding to an internal region between amino acids 338-381, were found to be necessary for eRF1--PP2Acalpha interaction in yeast. Immunoprecipitations using 12CA5 antibodies and extracts from COS1 cells transiently transfected with eRF1 tagged with 9-amino acid epitope from influenza hemagglutinin (HA) demonstrated the presence of eRF1--PP2Acalpha--PR65alpha/Aalpha complex in these cells. In addition, polysomes obtained from COS1 cells overexpressing HA--eRF1 displayed several-fold higher PP2A activity than control polysomes. No effect of either PP2Ac or dimeric and trimeric PP2A holoenzymes on the rate of translation termination was detected using an in vitro reconstituted translation termination assay. In summary, eRF1 appears to represent a novel PP2A-targeting subunit that brings this phosphatase in contact with putative ribosomal substrate(s). It remains to be established whether termination of translation requires dephosphorylation of participating protein factor(s).

Adenotin and adenotin-like proteins coexist with adenosine receptors in mammalian tissues.

The relationship of adenotin, a low-affinity adenosine-binding protein, to adenosine receptors was examined in two human tissues and two mammalian cultured cell lines. An adenosine A2 receptor exists in the membranes from platelets, PC-12 cells, and JAR cells as shown by a stimulation of adenylate cyclase related to 5'-N-ethylcarboxamidoadenosine (NECA) or a NECA-related increase in intracellular cAMP levels. In contrast, binding studies with tritiated NECA revealed typical adenotin-like low-affinity binding sites on the membranes from the sources studied with agonist potencies as follows: NECA greater than 2-chloroadenosine greater than R-PIA. No evidence was found of coupling to a guanine nucleotide regulatory protein. Solubilization of platelet and placental membranes and precipitation with polyethylene glycol separated adenotin or the adenotin-like protein from a second adenosine binding site in each tissue. The pharmacologic properties of the precipitated binding sites were compatible with an adenosine A2 receptor in platelets and an adenosine A1 receptor in placenta. Our observations indicate that adenotin-like proteins exist outside the placenta. In addition, adenotin and adenotin-like proteins coexist with the adenosine A1 or A2 receptor in a number of cells and tissues and do not couple to a guanine nucleotide regulatory protein and stimulate adenylate cyclase. Therefore, adenotin is pharmacologically distinct from adenosine receptors, and its function remains to be discovered.