Regulation of cyclic nucleotide phosphodiesterase activity in human lung fibroblasts.

Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.     

Phorbol ester prevents the thyroid-stimulating-hormone-induced but not the forskolin-induced decrease of cAMP-dependent protein kinase activity in thyroid cell cultures

The potent tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) affects several thyroid cell functions and interacts with thyroid-stimulating hormone (TSH) either by inhibiting or potentiating its action on different cellular parameters. Since phorbol ester acts mainly through the activation of protein kinase C, which is its receptor, we studied this activation and its interaction with TSH and forskolin in suspension cultures of porcine thyroid cells. In thyroid cell cultures, TPA has a dual effect on protein kinase C activity: immediately (2-5 min) after exposure of cells to TPA, it began to be translocated from the cytosol to the particulate fraction. The transfer of the cytosolic enzyme was total and could occur with or without a loss of activity. The translocated enzyme still needed Ca2+ and phospholipids for its activation. The basal activity increased transiently (2-4 h) in both the cytosol and particulate fractions during translocation. The peak activity in the particulate fraction was reached 10-30 min after exposure of cells to TPA, and was followed by down-regulation of protein kinase C and almost complete disappearance of its activity. The residual activity was about 13% of control after a 2-day exposure to TPA. It was unequally distributed between cytosol (4%) and particulate fraction (9%). Prolonged exposure of cells to TPA did not affect either the activity or the subcellular distribution of the cAMP-dependent protein kinase activity. TPA interacted with TSH and prevented the decrease of this activity induced by prolonged exposure of cells to the hormone not only when it was introduced simultaneously with TSH, but also when it was added 24 h after TSH. However, the forskolin-induced decrease in cAMP-dependent protein kinase activity was not prevented by the presence of TPA. TPA also affected the increases in cAMP accumulation mediated by TSH and forskolin. The TSH-induced increase was significantly stimulated by TPA after short contacts (5-15 min), while longer preincubations of cells with TPA provoked a very strong inhibition of the TSH action. However, the forskolin-induced stimulation of the cAMP accumulation was maintained and even further increased in the presence of TPA. Consequently, the actions of TSH and TPA are apparently interdependent, while those of forskolin and TPA seem to be parallel and independent. Neither TSH nor forskolin prevented the TPA-induced down regulation of protein kinase C. The biologically inactive phorbol ester analogue 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity, and did not interact with either TSH or forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic evidence that a phorbol ester tumor promoter stimulates ornithine decarboxylase activity by a pathway that is independent of cyclic AMP-dependent protein kinases in CHO cells

Ornithine decarboxylase (ODC) inductions by cholera toxin and by the phorbol ester tumor promoter, TPA, were compared in wild-type Chinese hamster ovary (CHO) cells and in mutant cells having altered cyclic AMP-dependent protein kinase activity. The aim of these studies was to determine whether cyclic AMP-dependent protein kinase is involved in these inductions. The time course and the magnitude of ODC inductions by either 100 ng/ml cholera toxin or 100 ng/ml TPA were similar in wild-type cells with a maximum at 3-4 hours after treatment and a return to unstimulated levels by 8 hours. Induction of ODC by cholera toxin was suppressed more than 80% in the four protein kinase mutants studied (10215, 10248, 10260, and 10265), strongly implicating a cyclic AMP-dependent kinase step in the mechanism of induction. Similar results were found with the cyclic AMP analog 8-Br-cyclic AMP and the phosphodiesterase inhibitor, methyl-isobutylxanthine. The induction of ODC by TPA, on the other hand, was only partially inhibited (approximately 50%) in three of four mutants. Lower ODC activity in two mutants stimulated by cholera toxin or TPA whose kinetics were studied in more detail could not be ascribed to a reduced affinity (Km) of ornithine for the enzyme, but appeared to be due to reduced catalytic activity (Vmax) in the extracts. These results suggest that the induction of ODC by TPA proceeds by a mechanism which is only partially dependent on an intact cyclic AMP-dependent protein kinase activity.     

The induction of ornithine decarboxylase by tumor promoting phorbol ester analogues in Reuber H35 hepatoma cells

The induction of ornithine decarboxylase activity was studied in a rat hepatoma cell line (Reuber H35) incubated with a group of structurally-related phorbol ester analogues. A single application of 1.6 μM of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to H35 cells caused a dramatic increase in the activity of ornithine decarboxylase. The stimulation of the enzyme activity was rapid but transient, peaking at 4 to 5 hr with a value which was 116-fold greater than control and then declining to the basal level after 8 hr. In addition, the increase in ODC activity was dependent upon the concentration of TPA added to the culture medium and the EC₅₀ was estimated to be about 2.63 × 10^?7 M. Our studies of the effect of various phorbol ester analogues on the H35 ODC activity indicated an apparent correlation between the ability of phorbol ester derivatives to induce ODC activity in the H35 cells and their activity to promote papilloma formation in the mouse skin in that the various derivatives possessed the following relative abilities to increase ODC activity: TPA > PDB > PDA > 4 α-P > 4 α-PDD. Concurrent addition of either actinomycin D or cycloheximide abolished the increase in ODC activity after TPA treatment. Changes of intracellular concentrations of polyamines, particularly putrescine, were in good agreement with the increase in ODC activity in response to TPA: a 10-fold increase in putrescine over the control level was observed at 6 hr. Our data suggest that cultured Reuber H35 hepatoma cells exhibit a marked and specific response to the phorbol ester tumor promoters and may be of great value in studying the biochemical mechanism of ODC induction by these agents.     

The phorbol ester TPA inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes

Treatment of intact hepatocytes with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) potentiated the ability of glucagon to increase intracellular cyclic AMP concentrations. This effect was dose-dependent upon TPA, exhibiting an EC50 of 0.39 ng/ml and such activation was observed at both saturating and sub-saturating concentrations of glucagon. However, this stimulatory effect of TPA was completely abolished by the presence of the cyclic AMP phosphodiesterase inhibitor 1-isobutyl-3-methylxanthine, when TPA now inhibited the glucagon-stimulated increase in intracellular cyclic AMP concentrations. It is suggested that, as well as inhibiting glucagon-stimulated adenylate cyclase activity, TPA also inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes. Treatment of either hepatocyte homogenates or purified cyclic AMP phosphodiesterase with TPA failed to show any direct inhibitory effect of TPA on activity showing that TPA did not exert any direct inhibitory action on phosphodiesterase activity. However, homogenates made from hepatocytes that had been pre-treated with TPA did show a reduced cyclic AMP phosphodiesterase activity. It is suggested that TPA might inhibit cyclic AMP phosphodiesterase activity through phosphorylation by C-kinase.     

Increased activity of cyclic AMP phosphodiesterase from frozen-thawed rat liver. A role of lysosomal protease in enzyme activation.

The activity of cyclic AMP phosphodiesterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) in 105 000 X g supernatant fraction from frozen-thawed rat liver was 2.5 times higher than the corresponding preparation from fresh liver. This increased activity of frozen liver enzyme was accompanied by a decreased sensitivity of the enzyme to known activators such as alpha-tocopheryl phosphate and trypsin. Neither membrane-bound cyclic AMP phosphodiesterase, nor supernatant cyclic GMP phosphodiesterase increased in frozen liver preparation. It is unlikely that the activator protein of phosphodiesterase participated in the observed change of enzyme activity. Among rat tissues so far tested, the increased level of cyclic AMP phosphodiesterase was noted only in tissues rich in lysosome content. In the recombination experiment where phosphodiesterase from fresh liver was incubated with lysosomal fraction, stimulation of the enzyme activity was observed with a concomitant loss of sensitivity to above-mentioned activators. Since the stimulation by lysosomal fraction was effectively inhibited by cathepsin B1 inhibitors, leupeptin and antipain, it was deduced cathepsin-B1 (EC 3.4.12.3) type protease(s) was the main causative of activating the cyclic AMP phosphodiesterase. The freezing-thawing process of rat liver made the lysosomal membrane more permeable, and hence lysosomal proteases were released into soluble fraction during phosphodiesterase preparation. These results provide a warning not to use frozen liver for phosphodiesterase preparation, otherwise altered properties of the enzymes will be seen.     

Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae.

Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity.     

Regulation of urocaninase activity in the liver: role of 3',5'-AMP]

A dependence of rat liver urocaninase activity on the agents affecting the adenylate cyclase system was studied in vitro and in vivo. Urocaninase is considerably activated after the injection of glucagone, NaF, theophylline and 3',5'-AMP. Under conditions optimal for the protein kinase activity of phosphorylase the urocaninase of liver extracts was activated 7-fold on the average. The nezyme retains its activity after gel-filtration through Sephadex G-25 and is capable of inactivation in the presence of Mg2+ and of reactivation after addition of ATP and 3',5'-AMP. These data suggest a possibility of regulation of mammalian liver urocaninase activity by 3',5'-AMP-dependent phosphorylation of the enzyme. Derivatives of hypoxanthine (theophylline and caffeine) in concentration 10(-4) M activate urocaninase in liver extracts 2--3 and 1.5-fold respectively. The activation is probably not due to the 3',5'-AMP phosphodiesterase inhibition, since another phosphodiesterase inhibitor--papaverine--has no activating effect on urocaninase.     

Determination of cyclic nucleotide phosphodiesterase activity by high-performance liquid chromatography

A new assay for cyclic nucleotide phosphodiesterase activity by high-performance liquid chromatography with on-line radiochemical detection has been developed. The method is based on the measurement of 3H-labeled nucleoside monophosphates formed from cyclic nucleotides by the action of 3',5'-cyclic-nucleotide phosphodiesterase (PDE). The reaction products are determined from the incubation mixture after removal of the protein by injection of an aliquot into the liquid chromatograph. The detection limit with counting efficiency of 30% is 20 fmol of 3H-labeled product, which makes the method suitable for detection of low PDE activities.     

Characterization of an 8-lipoxygenase activity induced by the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate in mouse skin in vivo

An enzymatic activity has been found in cytosolic preparations from mouse epidermis which catalyzes the formation of 8-hydroperoxyeicosatetraenoic acid/8-hydroxyeicosatetraenoic acid (8-HPETE/8-HETE) from arachidonate. In contrast to 12-lipoxygenase this enzyme activity was not detectable in normal (untreated) mouse skin but only after in vivo treatment with the phorbol ester tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate). The induction showed a maximum at 24 h after TPA treatment strictly depended on the age of the mice and the TPA dose and was prevented by cycloheximide. The primary product formed from arachidonic acid was 8-HPETE, and the enzyme seems not to possess a significant peroxidase activity. This result as well as studies with specific inhibitors and its cytosolic localization indicates this enzyme to be a member of the lipoxygenase family. Most of the 8-lipoxygenase activity is located in cells of the suprabasal compartment of the epidermis. In spite of being a cytosolic enzyme 8-lipoxygenase appeared to be lipophilic to some extent and was activated by lecithin. The enzyme did not require calcium ions or ATP and showed a pH optimum at 7.5-8.0. 8-HPETE/8-HETE levels in mouse epidermis in vivo were determined by gas chromatography-mass spectrometry and found to be strongly increased after phorbol ester treatment, in agreement with the induction of 8-lipoxygenase observed.     

Phorbol esters induce both intracellular translocation and down-regulation of protein kinase C in MCF-7 cells

Exposure of MCF-7 human breast cancer cells to phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) results in a complete inhibition of cell proliferation. We investigated the effects of TPA on protein kinase C activity when cells were exposed to phorbol ester for various lengths of time. TPA induces within 5 min a drastic dose-dependent decrease of the cytosolic protein kinase C activity. The enzyme apparently lost at the cytosolic level was only partially recovered in the particulate fraction. The apparent down-regulation of the translocated enzyme which was only 34% after 1 min reached 72% and 84% after respectively 10 min and 15 min. Moreover, when cells are treated with TPA for longer periods of time, the particulate protein kinase C activity continues to decrease, dropping below control after 1 hour. This progressive decline leads to an almost complete disappearance of protein kinase C activity in MCF-7 cells after 45 hours of TPA treatment. The apparent loss of protein kinase C activity upon short- as well as long-exposure of cells to TPA was not accompanied by a concomitant increase of Ca, PL-independent protein kinase activity. We discuss the implication of these biochemical events in the inhibition of cell proliferation with regard to the respective short- and long-term effects of TPA on protein kinase C activity.     

Cyclic nucleotide phosphodiesterases of Hyalophora cecropia silkmoth fat body.

Two soluble forms of 3':5'-cyclic-nucleotide phosphodiesterase (o':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) were found in the larval fat body of the silkmoth Hyalophora cecropia. These differ in elution profile on Sephadex G-200, solubility in ammonium sulfate, metal ion requirements and kinetic properties. Phosphodiesterase I has Km values of 11 muM and 1.8 muM for cyclic AMP and cyclic GMP, respectively, has 5-fold greater maximal activity with cyclic AMP than with cyclic GMP, and is activated by Mg2+ and Co2+, and inhibited by EDTA. phosphodiesterase II has Km values of 625 muM and 125 muM for cyclic AMP and cyclic GMP, respectively, has similar maximal activity with both substrates, and is not activated by divalent metal ions or inhibited by EDTA. Cyclic nucleotides and methylxanthines competitively inhibit both enzymes. Phosphodiesterase is found in both soluble and particulate fractions of homogenates. Total activity is highest during the larval stage of the insect, drops markedly following pupation, and rises again during pharate adult development.     

Mitogenic Activity of Snake Venom Lectins

Abstract. Five lactose-inhibitable lectins have been isolated from snake venoms. These five share certain biochemical properties but are not identical (Gartner, Stocker & Williams, 1980; Gartner & Ogilvie, 1984). In this study the lectins were tested for their ability to stimulate lymphocytes to undergo DNA synthesis. We found that three of the lectins were comparable in mitogenic activity to the T cell lectin, concanavalin A (Con A). the mitogenic activity was blocked by lactose, a sugar which also blocks the haemagglutination activity of these lectins. Although mitogenic response appeared to be due to T cells, it depended on the presence of accessory cells in the culture. This requirement for macrophages could be replaced by the phorbol ester tumour promoter, 12- o -tetradecanoylphorbol-13-acetate (TPA).     

Induction of the pyridine nucleotide synthesis pathway in mitogen-stimulated human T-lymphocytes

Mitogen stimulation of purified human T-lymphocytes with the phorbol ester 12-O-tetradecanoyl, phorbol-13-acetate (TPA) and a monoclonal antibody to the T3 cell surface antigen caused a 6-11-fold increase in cellular levels of poly(ADP-ribose) polymerase, a 6-20-fold amplification of cellular NAD+ levels and a 3-21-fold increase in NADP+ levels. Treatment of the cells with a combination of the two mitogenic signals also caused a 5-20-fold increase in NMN pyrophosphorylase activity, a 3-14-fold increase in ATP-NMN adenylyl transferase activity, and a 5-13-fold increase in NAD kinase activity. This is the first report showing induction of these three enzymes as part of the mitogenic response in purified human T-lymphocytes. Maximum increases in activity of each of these three enzymes required the combined presence of TPA and monoclonal antibody to human T-cell T3 antigen anti-T3. Analysis of the relative enzyme levels indicates that NMN pyrophosphorylase is the rate-limiting enzyme for NAD synthesis and NAD kinase is the rate-limiting enzyme for NADP synthesis.     

Mitogenic activity of snake venom lectins

Five lactose-inhibitable lectins have been isolated from snake venoms. These five share certain biochemical properties but are not identical (Gartner, Stocker & Williams, 1980; Gartner & Ogilvie, 1984). In this study the lectins were tested for their ability to stimulate lymphocytes to undergo DNA synthesis. We found that three of the lectins were comparable in mitogenic activity to the T cell lectin, concanavalin A (Con A). The mitogenic activity was blocked by lactose, a sugar which also blocks the haemagglutination activity of these lectins. Although mitogenic response appeared to be due to T cells, it depended on the presence of accessory cells in the culture. This requirement for macrophages could be replaced by the phorbol ester tumour promoter, 12-o-tetradecanoylphorbol-13-acetate (TPA).     

Phosphorylation of histones is stimulated by phorbol esters in quiescent Reuber H35 hepatoma cells

Histones isolated from Reuber H35 rat hepatoma cells treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined for possible alterations in phosphorylation. Incorporation of 32P orthophosphate into individual acid-extracted histones was monitored by autoradiography and scintillation counting of polyacrylamide gels or by reverse-phase high performance liquid chromatography. Treatment of quiescent H35 cells (arrested by serum starvation) with submicromolar doses of TPA resulted in a rapid and specific increase in phosphorylation of histones H2B and H1(0). Smaller increases in phosphorylation were observed for H4. No significant change in phosphorylation of the major H1 histones or H2A were observed after 1 h of treatment. The phosphorylation was TPA dose-dependent, with a maximum increase of approximately 14-fold for H2B, 11-fold for H1(0), and 2-fold for H4 achieved at 0.8 M TPA. The nonpromoting parent compound phorbol did not induce any of these changes. Furthermore, the mitogenic hormone insulin did not cause a similar pattern of histone phosphorylation, suggesting that the effect observed was not due to a general mitogenic response in the H35 hepatoma cells. Addition of 8-Br-cAMP also failed to reproduce the effect of TPA on histone phosphorylation, suggesting that cAMP-dependent protein kinases are not likely to be involved in mediating this response to TPA.     

Regulation of cyclic nucleotide phosphodiesterase forms by serum and insulin in cultured fibroblasts.

A rapid reduction of cyclic nucleotide phosphodiesterase activity occurs after the replating of confluent cultures of BHK 21 c/13 fibroblasts into fresh medium. This reduction in activity depends on the density to which the cultures are reseeded and the concentration of serum in the medium. Enzyme activity in BHK cells is restored after 24 to 48 hours if cells are diluted into medium containing 10% fetal calf serum or 0.5% fetal calf serum supplemented with insulin (10(-6)M), but not into 0.5% serum alone. The restoration in enzyme activity is blocked by cycloheximide or Actinomycin D. When BHK cells become quiescent by maintanance in 0.5% serum conditions for 48 hours, a rapid (15--60 minutes) increase in cyclic AMP phosphodiesterase activity occurs when 10% serum is added to the cultures. Enzyme activity is increased even further after 24 to 48 hours in the 10% serum. Cycloheximide or Actinomycin D do not affect the rapid increase in enzyme activity in response to serum, but completely inhibit the long term increase. In contrast to serum, insulin (10(-8) to 10(-6)M) has no short term effect, but does increase enzyme activity after 24 to 48 hours to levels comparable to those seen with addition of 10% serum. As is the case with serum, this long term effect of insulin on enzyme activity is prevented by inhibitors of protein and RNA synthesis. Kinetic analyses of cyclic AMP phosphodiesterase activity in homogenates of quiescent BHK cells indicate the presence of only high Km (congruent to 20 muM) enzyme activity. Addition of serum or insulin to quiescent cells results in the appearance of apparent low Km enzyme activity in homogenates. Sucrose gradient analysis of BHK cells displays two forms of cyclic AMP phosphodiesterase enzyme activity: a 3--4 S form and 5--6 S form. In quiescent cells, the 5--6 S form greatly predominates relative to the 3--4 S form. Addition of serum to quiescent cells results in a rapid appearance of increased 3--4 S form enzyme activity. Insulin also increases the activity of this higher affinity 3--4 S enzyme form after 24 to 48 hours in culture. The functional significance of short and long term regulation of cyclic nucleotide phosphodiesterase(s) in cells is discussed.     

Phorbol ester regulation of the human gamma-glutamyltransferase gene promoter

In the present study the molecular mechanisms underlying tetradecanoylphorbol-13-acetate (TPA) mediated regulation of the human gamma-glutamyltransferase (GGT) gene were examined. TPA challenge of HeLa cells resulted in an increase of GGT mRNA and enzyme activity. Deletion analysis of the promoter revealed that the -348 to +60 fragment was able to mediate TPA induced expression. Gel shift and supershift analyses showed that TPA treatment increased nuclear protein binding to a putative AP-1 site (-225 to -214) and that c-Jun was part of the complex. This AP-1 element, when cloned either in its native arrangement or as tandem repeat 5' of the minimal thymidine kinase promoter, mediated a significant increase of luciferase activity after TPA treatment of transfected HeLa cells, while its mutated counterpart abolished the induction. The same AP-1 element was able to mediate TPA induced expression in HepG2 cells. Collectively these results indicate that like other GSH metabolising enzymes, GGT too is a target for AP-1 mediated regulation.     

Separation and investigation of the regulatory properties of two forms of cyclic nucleotide phosphodiesterase from rabbit heart--sensitive and insensitive to Ca-dependent regulator protein]

Two forms of cyclic nucleotide phosphodiesterase (ES 3.1.4.17)--PDE-I and PDE-II--sensitive and resistant to Ca-dependent protein regulator, were isolated from the soluble fraction of rabbit heart by chromatography on DEAE-cellulose. Both forms of enzyme are inhibited by 30--50% by Ca2+ (10(-4) M). Addition of Ca-dependent protein regulator activates PDE-I and eliminates Ca2+-induced inhibition of PDE-II. In heart extract Ca2+ increases the phosphodiesterase activity 1.5-fold. The amount of PDE-I makes up to about 10% of total phosphodiesterase activity of the heart; that of PDE-II is about 90%. In the presence of Ca-dependent protein regulator the rate of 3', 5'-AMP hydrolysis by PDE-I is increased 5--15-fold, while that of 3', 5'-GMP hydrolysis only 2.5-fold. Both PDE-I and PDE-II have close Km values for substrates--(3.5--4.0).10(-6) M for 3', 5'-AMP and 14.10(-6) M for 3', 5'-GMP. Inhibition by Ca2+ and effect of Ca-dependent protein regulator manifest themselves in changes in V for cyclic nucleotide hydrolysis and do not alter the Km value for the enzyme.     

Identification of sequences within the murine granulocyte-macrophage colony-stimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells

Phorbol esters (TPA) and concanavalin A (ConA) are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF) production in murine thymoma EL-4 cells by mRNA stabilization. The role of the 3'-untranslated region (3'-UTR) in GM-CSF mRNA stabilization induced by TPA and ConA in EL-4 cells was examined by transfection studies using chloramphenicol acetyltransferase (CAT) constructions. The GM-CSF 3'-UTR contains a 63-nucleotide region at its 3' end with repeating ATTTA motifs which is responsible for mRNA degradation in a variety of cell types (Shaw, G., and Kamen, R. (1986) Cell 46, 659-666). We produced constructs containing most of the GM-CSF 3'-UTR (303 nucleotides, pRSV-CATgm) or the 3'-terminal AT-rich region (116 nucleotides, pRSV-CATau) and measured CAT enzyme activity and CAT mRNA after transient transfection into EL-4 and NIH 3T3 cells. Low levels of CAT activity were seen in both cells with either plasmid compared with levels of CAT activity obtained with pRSV-CAT. TPA treatment caused an approximately 10-fold increase in CAT activity and mRNA in EL-4 cells transfected with pRSV-CATgm. No increases were seen in EL-4 cells transfected with pRSV-CATau or pRSV-CAT. No response to TPA was detected in transfected NIH 3T3 cells, indicating that the response to TPA is relatively cell-specific. There was no increase in CAT activity after ConA treatment in EL-4 or NIH 3T3 cells transfected with any of the constructs suggesting that the GM-CSF 3'-UTR lacks elements that can respond alone to ConA. Nuclear run-on and actinomycin D chase experiments in EL-4 cells showed that TPA induces CAT activity via mRNA stabilization. By linker-substitution mutagenesis we show that TPA inducibility depends on a 60-nucleotide region of the 3'-UTR whose 5' end is located 160 nucleotides upstream of the 5' end of the AU-rich region.