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.     

Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Putative effect of C kinase on alpha s-GTP-catalytic subunit interaction

Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to S49 lymphoma cells (wild type and a cyclic AMP-dependent protein kinase-lacking clone) has little effect alone but doubles accumulation of cyclic AMP in response to isoproterenol. The effect is immediate and has an apparent affinity and order of potency characteristic of the activation of protein kinase C by phorbol esters. Enhancement does not reflect an altered time course of the beta-adrenergic response, enhanced affinity of the cellular beta-receptor for agonist, or decreased degradation and export of cellular cyclic AMP. Reduction of the beta-adrenergic response by somatostatin does not remove the effect of TPA nor does TPA abolish the effect of somatostatin. Phorbol ester enhances cyclic AMP accumulation in response to cholera toxin in wild type and UNC clones but not in H21a or cyc-. TPA also enhances cAMP accumulation in response to forskolin in wild type cells. The effect of TPA is stable to rapid preparation of membranes. In adenylate cyclase assays on membranes from cells treated with TPA, the activation by guanosine 5'-(beta, gamma-imino)triphosphate is enhanced by 40% with no change in lag time; the effect of beta-agonist plus Gpp(NH)p is similarly enhanced; activation by Mn2+ is unchanged. We conclude that phorbol ester facilitates the productive interaction of the alpha subunit of the transducer protein Gs with the catalytic unit of adenylate cyclase, hypothetically via an action of protein kinase C.     

Interactions between cyclic AMP- and phorbol ester-dependent phosphorylation systems in S49 mouse lymphoma cells

High-resolution two-dimensional gel electrophoresis of proteins labeled with either 32Pi or [35S]methionine was used to study interactions between cyclic AMP and tetradecanoyl phorbol acetate (TPA) at the level of intracellular protein phosphorylation. Cultured S49 mouse lymphoma cells were used as a model system, and mutant sublines lacking either the catalytic subunit of cyclic AMP-dependent protein kinase or the guanyl nucleotide-binding "Ns" factor of adenylate cyclase provided tools to probe mechanisms underlying the interactions observed. Three sets of phosphoproteins responded differently to TPA treatment of wild-type and mutant cells: Phosphorylations shown previously to be responsive to activation of intracellular cyclic AMP-dependent protein kinase were stimulated by TPA in wild-type cells but not in mutant cells, a subset of phosphorylations stimulated strongly by TPA in mutant cells was inhibited in wild-type cells, and two novel phosphoprotein species appeared in response to TPA only in wild-type cells. The latter two classes of TPA-mediated responses specific to wild-type cells could be evoked in adenylate cyclase-deficient cells by treating concomitantly with TPA and either forskolin or an analog of cyclic AMP. Three conclusions are drawn from our results: 1) TPA stimulates adenylate cyclase in wild-type cells causing increased phosphorylation of endogenous substrates by cyclic AMP-dependent protein kinase, 2) activated cyclic AMP-dependent protein kinase inhibits phosphorylation (or enhances dephosphorylation) of a specific subset of TPA-dependent phosphoproteins, and 3) cyclic AMP-dependent events facilitate TPA-dependent phosphorylation of some substrate proteins.     

Phorbol ester inhibits luteinizing hormone-, forskolin-, and dibutyryl cyclic AMP-induced progesterone production in chicken granulosa cells

The possible role of protein kinase C in avian granulosa cell steroidogenesis was studied in vitro by examining the effect of tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on progesterone synthesis in chicken granulosa cells in short-term (3h) incubations. TPA (1-100 nM) caused a marginal but nonsignificant increase in progesterone production in granulosa cells isolated from the largest preovulatory follicle. When incubated in combination with luteinizing hormone (5-100 ng/mL), TPA suppressed the stimulatory effects of submaximally and maximally effective doses of the gonadotropin in a concentration-related manner. Similarly, the phorbol ester inhibited the steroidogenic responses to forskolin and dibutyryl cyclic AMP. By comparison, TPA had no appreciable effect on the metabolism of exogenous pregnenolone substrate to progesterone. Our data indicate that the tumor-promoting phorbol ester influences steroidogenic steps distal to cyclic AMP generation but at or before pregnenolone formation, and that protein kinase C may be a negative regulator of steroid biosynthesis in chicken granulosa cells.     

Glucocorticoid stimulation of amnion cell prostaglandin synthesis: suppression by protein kinase C inhibitors and independence of phorbol ester-sensitive protein kinase C.

Glucocorticoids stimulate the prostaglandin E2 production of confluent amnion cell cultures, but have no stimulatory effect on the PGE2 output of freshly isolated human amnion cells. Since protein phosphorylation may modify the responsiveness of target cells to steroids, and activators of protein kinase C (PKC), as well as corticosteroids, promote amnion cell PGE2 output by stimulating the synthesis of prostaglandin endoperoxide H synthase (PGHS), we investigated the possibility that PKC is involved in the glucocorticoid-induction of PGE2 synthesis in cultured amnion cells. The dexamethasone-induced PGE2 output of arachidonate-stimulated cells was blocked by the protein kinase inhibitors staurosporine, K-252a, H7, HA1004, and sphinganine, in a manner consistent with their effect on PKC. However, dexamethasone increased the PGE2 production of cultures treated with maximally effective concentrations of the PKC-activator compound TPA. Moreover, dexamethasone stimulated PGE2 synthesis in cultures which were desensitized to TPA-stimulation by prolonged phorbol ester treatment. Concentration-dependence studies showed that staurosporine completely (greater than 95%) blocked glucocorticoid-provoked PGE2 synthesis at concentrations which did not inhibit TPA-stimulated prostaglandin output, and that K-252a inhibited the effect of TPA by more than 95% at concentrations which decreased the effect of dexamethasone only moderately (approximately 40%). Dibutyryl cyclic AMP had no influence on the basal- or dexamethasone-stimulated PGE2 production, and on the staurosporine inhibition of the steroid effect. These results show that glucocorticoids and phorbol esters control amnion PGE2 production by separate regulatory mechanisms. It is suggested that the response of human amnion cells to glucocorticoids is modulated by protein kinase(s) other than phorbol ester-sensitive PKC and cyclic AMP-dependent protein kinase.     

Evidence that the mitochondrial activator of phosphorylated branched-chain 2-oxoacid dehydrogenase complex is the dissociated E1 component of the complex

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate). The half-maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon-stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 microgram/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon-stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.     

Phorbol ester stimulates catecholamine synthesis in isolated bovine adrenal medullary cells

In isolated bovine adrenal medullary cells, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C, stimulated [14C]catecholamine synthesis from [14C]tyrosine, but not from [14C]DOPA. This stimulatory effect of TPA on [14C]catecholamine synthesis was not dependent upon extracellular Ca2+, and TPA did not affect the uptake of 45Ca2+ or the release of catecholamine by the cells. TPA also did not affect the intracellular cyclic AMP (cAMP) level. 4 alpha-Phorbol 12, 13-didecanoate, which is not an activator of protein kinase C, did not stimulate the synthesis of [14C]catecholamine from [14C]tyrosine. The stimulatory effect of TPA on [14C]catecholamine synthesis was additive with that of carbamylcholine, but not with that of dibutyryl cAMP (DB-cAMP). From these results, it was suggested that protein kinase C is involved in the regulation of tyrosine hydroxylase activity and that this regulatory mechanism might be similar to that involving cAMP.     

Stimulation by glucose of cyclic AMP accumulation in mouse pancreatic islets is mediated by protein kinase C.

The mechanism of glucose-stimulated cyclic AMP accumulation in mouse pancreatic islets was studied. In the presence of 3-isobutyl-1-methylxanthine, both glucose and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, enhanced cyclic AMP formation 2.5-fold during 60 min of incubation. Both TPA-stimulated and glucose-stimulated cyclic AMP accumulations were abolished by the omission of extracellular Ca2+. The Ca2+ ionophore A23187 did not affect cyclic AMP accumulation itself, but affected the time course of TPA-induced cyclic AMP accumulation, the effect of A23187 + TPA mimicking the time course for glucose-induced cyclic AMP accumulation. A 24 h exposure to TPA, which depletes islets of protein kinase C, abolished the effects of both TPA and glucose on cyclic AMP production. Both TPA-induced and glucose-induced cyclic AMP productions were inhibited by anti-glucagon antibody, and after pretreatment with this antibody glucose stimulation was dependent on addition of glucagon. Pretreatment of islets with TPA for 10 min potentiated glucagon stimulation and impaired somatostatin inhibition of adenylate cyclase activity in a particulate fraction of islets. Carbamoylcholine, which is supposed to activate protein kinase C in islets, likewise stimulated cyclic AMP accumulation in islets. These observations suggest that glucose stimulates islet adenylate cyclase by activation of protein kinase C, and thereby potentiates the effect of endogenous glucagon on adenylate cyclase.     

Activation of cyclic AMP-dependent protein kinase and stimulation of protein phosphorylation in response to adenosine in C-1300 murine neuroblastoma

DEAE-cellulose chromatography of the 20,000g supernatant fraction of homogenates of C-1300 murine neuroblastoma (clone N2a) yields one major and two minor peaks of cyclic AMP-dependent protein kinase activity. Assessment of the endogenous activation state of the enzyme(s) reveals that the enzyme is fully activated by the treatment of whole cells with adenosine (10 μM) in the presence of the phosphodiesterase inhibitor Ro 20 1724 (0.7 mM). This treatment produces a large elevation in the cyclic AMP content of the cells. The treatment of whole cells with adenosine alone (1–100 μM) or Ro 20 1724 alone (0.1–0.7 mM) produces minimal elevations in cyclic AMP but nevertheless causes significant activations of cyclic AMP-dependent protein kinase. The autophosphorylation of whole homogenates of treated and untreated cells was studied using [γ-³²P] ATP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Treatments which activate cyclic AMP-dependent protein kinase selectively stimulate the incorporation of ³²P into several proteins. This stimulation is most prominent in the 15,000-dalton protein band. The addition of cyclic AMP to phosphorylation reactions containing homogenate of untreated cells stimulates the phosphorylation of the same protein bands. These results indicate that adenosine may have regulatory functions through its effect on the cyclic AMP: cyclic AMP-dependent protein kinase system.     

Activation of protein kinase C sensitizes the cyclic AMP signalling system of T51B rat liver cells

Activation of protein kinase C (PKC) bu phorbol esters (TPA) results in a modification of the cyclic AMP system leading to either attenuation or amplification of the cyclic AMP signal. In the non-neoplastic T51B rat live cell line, TPA, when added to intact cells, had no effect on the basal level of cyclic AMP synthesis but caused a 1.5 fold amplification of the stimulation induced by β-adrenergic agents, cholera toxin and forskolin. The effect appeared to be mediated by PKC since diacylglycerols caused the same amplification as did TPA while inactive phorbol esters were without effect. Phosphorylation of Gs or the catalytic subunit of adenylate cyclase by PKC is likely to be responsible for the enhancement of cyclic AMP synthesis. TPA also caused translocation of PKC; however, the time course of the translocation was loner than the time course of the enhancement of adenylate cyclase activity. Thus, the ability of TPA to amplify cyclic AMP synthesis is probably mediated by activation of PKC that is already present in the membrane.     

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.     

Altered cyclic AMP-dependent protein kinase activity in a mutant adrenocortical tumor cell line

A somatic cell genetic approach has been used to evaluate the role of cyclic AMP-dependent protein kinase in ACTH action on adrenal steroidogenesis. A mutant clone, 8BrcAMP^r-1, previously was isolated from an ACTH-sensitive adrenocortical tumor cell line (clone Y1) following mutagenesis and selective growth in 8-bromoadenosine 3′, 5′-monophosphate. This study demonstrates that the 8BrcAMP⁴-1 cells have an altered cyclic AMP-dependent protein kinase. The protein kinase in the cytosol of the mutant characteristically requires, for half-maximal activity, concentrations of cyclic AMP 7-fold higher than those required by the enzyme in preparations from the parent. The cytosolic cyclic AMP-dependent protein kinases of Y1 and 8BrcAMP^r-1 cells chromatograph similarly on columns of DEAE-cellulose. From each cell line, a major peak of activity (≥ 70% of recovered activity), designated as Peak I, elutes with 0.04–0.06 M NaCl; a second peak of activity, designated as Peak II, elutes with 0.12–0.14 M NaCl. Protein kinase activity in the Peak I fraction of mutant cells has a decreased apparent affinity (4-fold) for cyclic AMP relative to the corresponding fraction of parental Y1 cells. The protein kinase activities present in Peak II fractions from Y1 and mutant cells are indistinguishable. The protein kinase mutant exhibits poor steroidogenic responses to added ACTH and cyclic AMP; and as shown previously does not display the growth arrest and morphological changes produced in Y1 by these agents. These results suggest that cyclic AMP-dependent protein kinase is important in the regulation of adrenal steroidogenesis, morphology and growth by ACTH.     

Regulation of protein kinases activity by quercetin in Ehrlich ascites tumor cells

Cyclic AMP-independent protein kinase activities from Ehrlich ascites tumor cells, partially purified by DEAE-cellulose and phosphocellulose chromatography were inhibited by quercetin. The cyclic AMP in the tumor ascites cells and the cyclic AMP-dependent protein kinase activity from this tumor and from bovine and mouse tissues were unaffected by this drug. Since we reported that quercetin elevates cyclic AMP level in Ehrlich ascites tumor cells, this bioflavonoid may have a dual effect on the protein kinae activities in these cells, thus, increasing the cyclic AMP-dependent and decreasing the cyclic AMP-independent protein kinase activities.     

An inhibitory role for the protein kinase C pathway in ovarian steroidogenesis. Studies with cultured swine granulosa cells.

We have used primary cultures of swine granulosa cells to investigate the regulatory role of the protein kinase C pathway in the ovary. In this system, we observed the following. Swine granulosa cells bound [3H]phorbol 12,13-dibutyrate [( 3H]PDB) specifically with high affinity [apparent Ki for 12-O-tetradecanoylphorbol 13-acetate (TPA) = 3.1 (2.1-4.7) nM] and low capacity [0.68 (0.34-0.99) pmol/10(7) cells]. The cytosol of granulosa cells contained functionally active protein kinase C capable of phosphorylating distinct proteins in response to stimulation with active phorbol ester. TPA and PDB induced dose-dependent inhibition (greater than 85%) of follicle-stimulating-hormone (FSH)-stimulated progesterone production. Half-maximally inhibitory concentrations were 0.10 and 0.75 nM for TPA and PDB respectively, whereas phorbol analogues that do not activate protein kinase C were not inhibitory. TPA did not impede cyclic AMP generation in response to FSH, cholera toxin or forskolin acutely (within 48 h), but did inhibit the stimulatory effects of 8-bromo cyclic AMP, insulin and oestradiol on progesterone biosynthesis. In the presence of maximally effective concentrations of 25-hydroxy-, 20 alpha-hydroxy- or 22R-hydroxy-cholesterol as exogenous sterol substrates for cholesterol side-chain cleavage, treatment with TPA suppressed pregnenolone, progesterone and 20 alpha-hydroxypregn-4-en-3-one biosynthesis by more than 80%. The inhibitory effects of phorbol esters were not attributable to non-specific cytotoxicity, since prostaglandin F2 alpha production increased in the same cultures and aromatization of exogenously supplied testosterone to oestradiol was not suppressed. In intact granulosa cells, the effects of phorbol esters were mimicked by a synthetic non-diterpene diacylglycerol, 1-octanoyl-2-acetylglycerol, and the tumour promoter, mezerein, which specifically activates protein kinase C. We conclude that swine granulosa cells contain specific high-affinity receptors for phorbol esters that are functionally coupled to protein phosphorylation. Moreover, treatment with phorbol esters or non-phorbol activators of protein kinase C results in selective inhibition of cholesterol side-chain cleavage activity without impairing cyclic AMP generation or oestrogen biosynthesis.     

Action of cyclic nucleotide analgoues in Chinese hamster ovary cells

Cyclic nucleotide analogues have been tested for their ability to cause the morphological conversion of Chinese hamster ovary cells in culture, as well as for effects on cyclic AMP-related enzymes. The ability of the analogues to inhibit the cyclic AMP phosphodiesterase activity and to activate the cyclic AMP-dependent protein kinase activity in cell extracts has been measured. Cell cultures were incubated with the analogues and the effects on morphology, intracellular level of cyclic AMP, and in vivo protein kinase activation were determined. All analogues which induced the morphological conversion also caused in vivo activation of the cyclic AMP-dependent protein kinase. Only N⁶,O^2′-dibutryl and N⁶-monobutyryl cyclic AMp caused caused on increase in intracellular cyclic AMP, presumably through inhibition of the intracellular cyclic AMP phosphodiesterase activity. The increase in cyclic AMP appears to cause the protein kinase activation. However, analogues such as 8-bromo and 8-benzylthio cyclic AMP do not cause any change in intracellular cyclic AMP level and appear to activate the intracellular cyclic AMP-dependent protein kinase directly.     

Multiple Pathways of N-Kinase Activation in PC12 Cells

Past work established a cell-free assay for a nerve growth factor (NGF)-activated protein kinase activity (designated N-kinase) that utilizes tyrosine hydroxylase and histone H1 as substrates and that is distinct from a variety of well-characterized kinases. This study explores the specificity and mechanistic pathway(s) by which N-kinase activity is regulated in PC12 rat pheochromocytoma cells. N-kinase is rapidly activated in these cells by treatment with NGF, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), phorbol ester, or dibutyryl cyclic AMP. Our data indicate that the stimulated activity is the same for each agent by several criteria: It exhibits the same characteristic biphasic elution pattern by Mono S fast protein liquid chromatography (FPLC), except for the case of dibutyryl cyclic AMP in which one of the activity peaks is somewhat shifted; it shows the same elution pattern by FPLC on a Superose 12 column; it possesses identical substrate specificity; and, except in the case of dibutyryl cyclic AMP, it does not show additivity when each agent is added simultaneously with NGF. The multiple forms of N-kinase are interconvertible in that rechromatography on a Mono S column yields a single peak of activity. Also, when NGF and dibutyryl cyclic AMP are simultaneously presented to cells, the chromatographic profile resembles that with NGF alone. Activation occurs through several independent initial pathways. Down-regulation of protein kinase C by phorbol ester pretreatment prevents N-kinase activation by phorbol ester, but not by the other agents. A PC12 cell-derived line deficient in cyclic AMP-dependent protein kinase II activity exhibits N-kinase activation by all treatments except dibutyryl cyclic AMP. The properties of N-kinase suggests that it is similar or identical to the ribosomal S6 protein kinase described by Blenis and Erikson. Additional experiments revealed that N-kinase activity can be stimulated in several cell lines in addition to PC12 cells. These findings indicate that the N-kinase can be activated via multiple second-messenger pathways and that it could therefore potentially play a significant role in mediating shared intracellular responses to various extracellular signals.     

Evidence against direct involvement of cyclic AMP-dependent protein phosphorylation in the exocytosis of amylase.

To examine whether or not the activation of cyclic AMP-dependent protein kinase is coupled to the exocytosis of amylase from rat parotid cells, the effect of protein kinase inhibitors on amylase release and protein phosphorylation was studied. A membrane-permeable inhibitor of cyclic AMP-dependent protein kinase, N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8), and peptide fragments of the heat-stable protein kinase inhibitor [PKI-(5-24)-peptide and PKI-(14-24)-amide] strongly inhibited cyclic AMP-dependent protein kinase activity in the cell homogenate. However, H-8 had no inhibitory effect on amylase release from either intact or saponin-permeabilized parotid cells stimulated by isoproterenol or cyclic AMP. Moreover, PKI-(5-24)-peptide and PKI-(14-24)-amide did not inhibit cyclic AMP-evoked amylase release from saponin-permeabilized cells, whereas cyclic AMP-dependent phosphorylations of 21 and 26 kDa proteins in intact or permeabilized cells were markedly inhibited by these inhibitors. These results suggest that cyclic AMP-dependent protein phosphorylation is not directly involved in the exocytosis of amylase regulated by cyclic AMP.     

G1 specific increases in cyclic AMP levels and protein kinase activity in Chinese hamster ovary cells.

Chinese hamster ovary cells were synchronized by selective detachment of cells in mitosis. The adenosine 3':5'-cyclic monophosphate (cyclic AMP) intracellular concentrations and cyclic AMP-dependent protein kinase activities were measured as these cells traversed G1 phase and entered S phase. Protein kinase activity, assayed in the presence or absence of saturating exogenous cyclic AMP in the reaction mixture, was lowest in early G1 phase (2 h after mitosis), increased 2-fold (plus exogenous cyclic AMP in reaction mixture) or 3.5-fold (minus cyclic AMP in reaction mixture) to maximum values in mid to late G1 phase (4-5 h after mitosis), and then decreased as cells entered S phase. Intracellular cyclic AMP concentrations were minimal 1 h after mitosis, increased 5-fold to maximum levels at 4-6 after mitosis, and decreased as cells entered S phase. Similar to the fluctuations in intracellular cyclic AMP, the cyclic AMP-dependent protein kinase activity ratio increased more than 40% in late G1 or early S phase. Puromycin (either 10 mug/ml or 50 mug/ml) administered 1 h after mitosis inhibited cyclic AMP-dependent protein kinase activity up to 50% by 5 h after mitosis, while similar treatment (10 mug/ml) had no effect on the increase in cyclic AMP formation. These data demonstrate that: (1) total protein kinase activity changed during G1 phase and this increase was dependent on new protein synthesis; (2) the increased intracellular concentrations of cyclic AMP were not dependent on new protein synthesis; and (3) the activation of cyclic AMP-dependent protein kinase was temporally coordinated with increased intracellular concentration of cycli AMP as Chinese hamster ovary cells traversed G1 phase and entered S phase. These results suggest that cyclic AMP acts during G1 phase to regulate the activation of cyclic AMP-dependent protein kinase.     

Acetylcholinesterase molecular forms from rat and human erythrocyte membrane

Summary Inhibition of growth of PY815 mouse mastocytoma cells in vitro by N⁶, O²-dibutyryladenosine 3,5 cyclic monophosphate (DB cyclic AMP) was accompanied by increases in intracellular cyclic AMP and histamine and minor changes in cytosolic cyclic AMP-dependent histone kinase activity. However, DEAE-cellulose chromatography revealed substantial changes in the relative proportions of the principal cyclic AMP-dependent protein kinases and in free cyclic AMP-binding protein after DB cyclic AMP treatment. The activity of cytosolic cyclic AMP-dependent protein kinase type I (PK_I) decreased relative to cyclic AMP-dependent protein kinase type II (PK_II) and there was an increase in a cytosol cyclic AMP-binding protein with little associated protein kinase activity. The relative changes in activity of PK_I, PK_II and cyclic AMP binding protein after DB cyclic AMP treatment may reflect events important in the regulation of growth and differentiation of mast cells.Abbreviations DB cyclic AMP N⁶,O²-dibutyryladenosine-3, 5-cyclic monophosphate - cyclic AMP adenosine 3,5-cyclic monophosphate - PK_I type I cyclic AMP-dependent protein kinase - PK_II type II cyclic AMP-dependent protein kinase     

Indirect evidence that protein kinase C plays a critical role in signal transduction of both vasopressin and corticotropin-releasing factor on pituitary cells in culture

The possible role of protein kinase C (PKC) in the cyclic AMP-dependent mechanism of action of corticotropin-releasing factor (CRF) on proopiomelanocortin cells of anterior and intermediate pituitary glands was examined after pretreatment of cells in culture with the PKC inhibitor retinal or the phorbol ester PMA, which depletes cell stores of the kinase. We found that these drugs not only abolished ACTH response to PMA and vasopressin, which both activate PKC, but unexpectably also dampened by 80-90% the stimulatory effect of CRF. Cell treatment with retinal failed to prevent CRF-induced accumulation of cyclic AMP. Retinal and PMA pretreatments of intermediate pituitary cells likewise inhibited alpha-MSH secretion stimulated by CRF. These data provide evidence to suggest that the mechanism of action of CRF on pituitary cells involves both cyclic AMP and PKC messenger systems.