Differential expression of ectoMg2+-ATPase and ectoCa2+-ATPase activities in human hepatoma cells

A human hepatoma cell line (Li-7A) possesses ectoATPase activity which is activated by either Mg2+ or Ca2+. Both ectoMg2+-ATPase and ectoCa2+-ATPase hydrolyze other nucleoside triphosphates, are inactive with ADP and AMP, and are inhibited by both p-chloromercuriphenyl sulfonate (pCMPS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Different Km values for ATP and pH curves are obtained for ectoMg2+-ATPase and ectoCa2+-ATPase. The specific activities of the two ATPases remain relatively constant through several days of cell growth after an initial decrease. In contrast, the specific activities of the two ATPases, especially the ectoCa2+-ATPase, increases continuously in Li-7A cells cultured in the presence of EGF, cholera toxin, and hydrocortisone. The ATPases of the factor-treated cells are also indiscriminate with respect to nucleoside triphosphate substrates; however, the kinetic constants for substrates are altered when compared to that of the untreated cells. Most strikingly, the sensitivity to inhibitors is greatly reduced. It is concluded that the long-term effect of EGF, cholera toxin, and hydrocortisone on the Li-7A cells is the induction or activation of a new or minor component of the ectoATPases, which is preferentially activated by Ca2+ and insensitive to pCMPS.     

cAMP-mediated modulation of signal transduction of epidermal growth factor (EGF) receptor systems in human epidermoid carcinoma A431 cells. Depression of EGF-dependent diacylglycerol production and EGF receptor phosphorylation.

While a cAMP-dependent protein kinase (protein kinase A) has been suggested to phosphorylate epidermal growth factor (EGF) receptor in vitro, both intrinsic and EGF- or potent phorbol tumor promoter-induced phosphorylation of EGF receptor were found to be depressed in human epidermoid carcinoma A431 cells by prior incubation of the cells with various protein kinase A activators (e.g. cholera toxin, forskolin, cAMP analogues, or a combination of prostaglandin E1 and 3-isobutyl-1-methylxanthine). Protein kinase A activators did not change significantly either the number of EGF receptors or their affinity for EGF. The tryptic phosphopeptide map of EGF receptors from cells treated with cholera toxin alone or cholera toxin followed by EGF revealed unique peptides whose serine phosphorylation was preferentially depressed. However, the catalytic subunit of protein kinase A phosphorylated no threonine and little serine in the EGF receptors in the plasma membranes of isolated A431 cells in vitro, while serine residues in an unidentified 170-kDa membrane protein(s) other than EGF receptor were heavily phosphorylated. Pretreatment of the cells with forskolin blocked 1,2-diacylglycerol induction by EGF; growth inhibition by nanomolar levels of EGF could be partially restored by the presence of forskolin. These results indicate that an increase in intracellular cAMP modulates the EGF receptor signal transduction system by reducing EGF-induced production of diacylglycerol without direct phosphorylation of EGF receptors by protein kinase A in A431 cells.     

Inhibition of growth and induction of enzyme activities in a clonal human hepatoma cell line (Li-7A): comparison of the effects of epidermal growth factor and an anti-epidermal growth factor receptor antibody

A cloned human hepatoma cell line (Li-7A), possessing epidermal growth factor (EGF) receptors numbering in the range of 10-20 pmol/10(6) cells, was inhibited in its growth by EGF as well as an antagonist monoclonal antibody (MoAb) to the EGF receptor. The mode of action of the two ligands of EGF receptors appeared to be different as indicated by the following results: 1) EGF induced marked alteration in cell morphology, whereas the antibody did not; 2) cellular protein accumulated in the EGF-treated cells but not in the antibody treated cells; and 3) ectoATPase activities were greatly enhanced in Li-7A cells treated with EGF and cholera toxin but were unaffected in cells treated with antibody and cholera toxin. The last result also suggests that expression of ectoATPase activities is under the regulation of both EGF and cholera toxin. Li-7A cells provide an additional valuable experimental system for the study of EGF action, as well as the interactive effects of EGF and cholera toxin. The enrichment of the ATPase activities in the EGF-cholera toxin-treated cells can be exploited for the detailed study and isolation of these enzymes and elucidation of their physiological functions.     

Coordinate regulation of adenylate cyclase, protein kinase, and specific enzyme synthesis by cholera toxin in hormonally unresponsive hepatoma cells

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H₁ histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED₅₀ values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.     

Synergistic stimulation of S-adenosylmethionine decarboxylase activity by Ca2+ ionophore A23187, cholera toxin and 1-oleoyl-2-acetylglycerol

The Ca2+ ionophore A23187 induced S-adenosylmethionine decarboxylase in guinea-pig lymphocytes, and cholera toxin stimulated the induction synergistically. The activator of protein kinase C, 1-oleoyl-2-acetylglycerol, did not induce S-adenosylmethionine decarboxylase activity but potentiated the enzyme activity induced by A23187 or by A23187 and cholera toxin. The addition of both A23187 and cholera toxin induced S-adenosylmethionine decarboxylase, but the further addition of 1-oleoyl-2-acetylglycerol or 12-O-tetradecanoylphorbol 13-acetate did not potentiate the enzyme induction in protein kinase-C-down-regulated cells that had been treated with 12-O-tetradecanoylphorbol 13-acetate for 18 h. These results suggest that a Ca2+-dependent pathway, other than that for protein kinase C, is essential for the induction of S-adenosylmethionine decarboxylase and that a cAMP-dependent pathway and also protein kinase C are involved in the potentiation of the induction.     

Epidermal growth factor stimulates the production of phosphatidylinositol monophosphate and the breakdown of polyphosphoinositides in A431 cells

The effects of epidermal growth factor (EGF) on the metabolism of phosphatidylinositol were examined using A431 cells labeled with either 32PO3(4)- or myo-[3H] inositol. EGF was found to increase the incorporation of phosphate into phosphatidic acid, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-diphosphate as early as 15 s after addition of hormone. These changes were found to be due to two effects of EGF on the phosphatidylinositol cycle. First, EGF stimulated the breakdown of phosphatidylinositol 4,5-diphosphate to diacylglycerol and an inositol triphosphate. In addition, EGF induced a rise in the levels of phosphatidylinositol 4-monophosphate. The EGF-dependent increases in both inositol triphosphate production and phosphatidylinositol 4-monophosphate levels were inhibited by pretreatment of the cells with 12-O-tetradecanoylphorbol-13-acetate. Treatment of the cells with pertussis toxin did not inhibit either of these responses. However, treatment of the cells with cholera toxin selectively abolished the ability of EGF to stimulate the rise in phosphatidylinositol monophosphate levels but did not alter the ability of the hormone to induce the breakdown of phosphatidylinositol diphosphate. The effects of cholera toxin were not mimicked by forskolin, cAMP analogs, or isobutyl-methylxanthine. These data demonstrate that EGF stimulates the production of inositol triphosphate. In addition, the findings are consistent with the hypothesis that EGF independently stimulates a phosphatidylinositol kinase. Based on the effects of cholera toxin and the inability of cyclic nucleotides to mimic this response, the effect of EGF on the phosphatidylinositol kinase may be mediated via a guanine nucleotide-binding protein that is not involved in cAMP production.     

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.     

Ouabain-insensitive Na(+)-ATPase activity is an effector protein for cAMP regulation in basolateral membranes of the proximal tubule

This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from proximal tubule basolateral membranes by cAMP. An increase in dibutyryl-cAMP (d-cAMP) concentration from 10(-8) to 5x10(-5) M stimulates the ouabain-insensitive Na(+)-ATPase activity. The ATPase activity increases from 6.0+/-0.4 to 10.1+/-0.7 nmol Pi mg(-1) min(-1), in the absence and presence of 5x10(-6) M d-cAMP, respectively. Similarly, the addition of cholera toxin (CTX), forskolin (FSK) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) also increases the Na(+)-ATPase activity in a dose-dependent manner, with maximal effect at 10(-8) M, 10(-6) M and 10(-7) M, respectively. The effect of 10(-8) M CTX is not additive to the effect of GTPgammaS, and is completely abolished by 200 microM guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of CTX and FSK on the Na(+)-ATPase activity are accompanied by an increase in cAMP formation by the basolateral membranes of the proximal tubule cells. Furthermore, 10(-8) M protein kinase A peptide inhibitor (PKAi) completely abolishes the stimulatory effect of 5x10(-6) M d-cAMP or 10(-4) M FSK on the Na(+)-ATPase activity. Incubation of the basolateral membranes with [gamma-(32)P]ATP in the presence of d-cAMP or FSK increases the global hydroxylamine-resistant phosphorylation and especially promotes an increase in phosphorylation of protein bands of approximately 100 and 200 kDa. This stimulation is not seen when 10(-8) M PKAi is added simultaneously. Taken together these data suggest that activation of a cAMP/PKA pathway modulates the Na(+)-ATPase activity in isolated basolateral membranes of the proximal tubule.     

Cyclic AMP agonists induce the phosphorylation of phospholipase C-tau and of a 76 kDa protein co-precipitated by anti-(phospholipase C-tau) monoclonal antibodies in BALB/c-3T3 cells. Relationship to inositol phosphate formation.

Previous studies have demonstrated enhanced phosphorylation of phospholipase C-tau (PLC-tau), a key regulatory enzyme in phosphoinositide metabolism, in cells treated with platelet-derived growth factor (PDGF) and epidermal growth factor, both of which act via specific receptor tyrosine kinases. Our studies on BALB/c-3T3 cells show that agents that promote cellular cyclic AMP accumulation also increase the phosphorylation, specifically the serine phosphorylation, of this enzyme. Increased phosphorylation of PLC-t (2-3-fold) was evident within 5-10 min of addition of isobutylmethylxanthine (IBMX) and either cholera toxin or forskolin to cells, and persisted for at least 3 h. Treatment of cells with cyclic AMP agonists also enhanced, with similar kinetics, the phosphorylation of a 76 kDa protein co-precipitated by anti-PLC-tau monoclonal antibodies. Brief exposure of cells to cholera toxin/IBMX or forskolin/IBMX decreased inositol phosphate formation induced by the GTP-binding protein (G-protein) activator aluminium fluoride by approx. 50%, but was without effect on PDGF-stimulated inositol phosphate formation. These findings suggest that PLC-tau, and perhaps the 76 kDa co-precipitated protein, are substrates of cyclic AMP-dependent protein kinase in BALB/c-3T3 cells: however, the lack of effect of cyclic AMP elevation on PDGF-stimulated inositol phosphate formation indicates that the intrinsic activity of PLC-tau is unaltered by cyclic AMP-mediated phosphorylation.     

Isoproterenol induces actin depolymerization in human airway smooth muscle cells via activation of an Src kinase and GS

In a previous study, we showed that isoproterenol induced actin depolymerization in human airway smooth muscle cells by both protein kinase A (PKA)-dependent and -independent signaling pathways. We now investigate the signaling pathway of PKA-independent actin depolymerization induced by isoproterenol in these cells. Cells were briefly exposed to isoproterenol or PGE(1) in the presence and absence of specific inhibitors of Src-family tyrosine kinases, phosphatidylinositol-3-kinase (PI3 kinase), or MAP kinase, and actin depolymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I. Isoproterenol, cholera toxin, and PGE(1) induced actin depolymerization, indicated by a decrease in the intensity of filamentous/globular fluorescent staining. Pretreatment with the Src kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriimidine (PP2) or geldanamycin or the PKA inhibitor Rp-cAMPS only partly inhibited isoproterenol- or PGE(1)-induced actin depolymerization. In contrast, PP2 and geldanamycin did not inhibit forskolin-induced actin depolymerization, and AG-213 (an EGF receptor tyrosine kinase inhibitor) did not inhibit isoproterenol- or PGE(1)-induced actin depolymerization. PI3 kinase or MAP kinase inhibition did not inhibit isoproterenol-induced actin depolymerization. Moreover, isoproterenol but not forskolin induced tyrosine phosphorylation of an Src family member at position 416. These results further confirm that both PKA-dependent and PKA-independent pathways mediate actin depolymerization in human airway smooth muscle cells and that the PKA-independent pathway by which isoproterenol induces actin depolymerization in human airway smooth muscle cells involves Src protein tyrosine kinases and the G(s) protein.     

Functional role of protein kinase B/Akt in gastric acid secretion

Epidermal growth factor (EGF) stimulates gastric acid secretion and H(+)/K(+)-ATPase alpha-subunit gene expression. Because EGF activates the serine-threonine protein kinase Akt, we explored the role of Akt in gastric acid secretion. Akt phosphorylation and activation were measured by kinase assays and by Western blots with an anti-phospho-Akt antibody, using lysates of purified (>95%) canine gastric parietal cells in primary culture. EGF induced Akt phosphorylation and activation, whereas carbachol had no effect. LY294002, an inhibitor of phosphoinositide 3-kinase, completely blocked EGF induction of Akt phosphorylation, whereas the MEK1 inhibitor PD98059 and the protein kinase C inhibitor GF109203X had no effect. We examined the role of Akt in H(+)/K(+)-ATPase gene expression by Northern blotting using a canine H(+)/K(+)-ATPase alpha-subunit cDNA probe. The parietal cells were transduced with a multiplicity of infection of 100 of the adenoviral vector Ad.Myr-Akt, which overexpresses a constitutively active Akt gene, or with the control vector Ad.CMV-beta-gal, which expresses beta-galactosidase. Ad.Myr-Akt induced H(+)/K(+)-ATPase alpha-subunit gene expression 3-fold, whereas it failed to stimulate the gene cyclooxygenase-2, which was potently induced by carbachol in the same parietal cells. Ad.Myr-Akt induced aminopyrine uptake 4-fold, and it potentiated the stimulatory action of carbachol 3-fold. In contrast, Ad.Myr-Akt failed to induce changes in either parietal cell actin content, measured by Western blots with an anti-actin antibody or in the organization of the actin cellular cytoskeleton, visualized by fluorescein phalloidin staining and confocal microscopy. Transduction of the parietal cells with a multiplicity of infection of 100 of the adenoviral vector Ad.dom.neg.Akt, which overexpresses an inhibitor of Akt, blocked the stimulatory effect of EGF on both aminopyrine uptake and H(+)/K(+)-ATPase production, measured by Western blots with an anti-H(+)/K(+)-ATPase alpha-subunit antibody. Thus, EGF induces a cascade of events in the parietal cells that results in the activation of Akt. The functional role of Akt appears to be stimulation of gastric acid secretion through induction of H(+)/K(+)-ATPase expression.     

Fibroblast growth factor potentiates receptor- and nonreceptor-mediated stimulation of adenylate cyclase in hamster fibroblasts

Basic fibroblast growth factor (FGF) has no effect alone on the basal cAMP synthesis in Chinese hamster fibroblasts (CCL39) but it potentiates (by up to 50%) the stimulation of adenylate cyclase by prostaglandin E1, cholera toxin or forskolin. This potentiating effect is not abolished by pretreatment of the cells with pertussis toxin, which indicates that it is not due to the withdrawal of a tonic inhibition of adenylate cyclase by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi). Therefore, we conclude that FGF enhances the activation of adenylate cyclase by the stimulatory GTP-binding protein (Gs). Although activation of protein kinase C in CCL39 cells results in a similar potentiation of cAMP production, we provide evidence that the effect of FGF is not mediated by protein kinase C, since (1) the potentiating effects of FGF and phorbol esters are additive and (2) FGF effect persists after down-regulation of protein kinase C. A role of FGF-induced rise in cytoplasmic Ca2+ can also be ruled out because the FGF effect is not mimicked by a Ca2+ ionophore and it persists in Ca2(+)-free medium. Since a similar potentiating effect on cAMP production is elicited by epidermal growth factor, a mitogen known to activate a receptor tyrosine kinase, we suggest that the FGF effect on adenylate cyclase might be mediated by the tyrosine kinase activity that is very likely to be associated with FGF receptors.     

Post-transcriptional regulation of cAMP-dependent protein kinase activity by cAMP in GH3 pituitary tumor cells. Evidence for increased degradation of catalytic subunit in the presence of cAMP

The effects of cyclic AMP treatment on total cAMP-dependent protein kinase activity in GH3 pituitary tumor cells have been studied. Incubation of cells for 24 h with 1 microM forskolin resulted in a 50% decrease in total cAMP-dependent protein kinase activity which was reversible upon removal of forskolin from culture media. A similar response was observed in GH3 cells treated with 5 ng/ml cholera toxin and 0.5 mM dibutyryl cAMP but not 0.5 mM dibutyryl cGMP. Northern blot analysis demonstrated that the steady-state level of the mRNA for each of the six kinase subunit isoforms studied was not detectably altered after treatment with 1 microM forskolin for 24 h. The concentration of catalytic subunit was also assessed by binding studies using a radiolabeled heat-stable protein kinase inhibitor. Treatment of GH3 cells with 1 microM forskolin for 24 h reduced protein kinase inhibitor binding activity by 50%, consistent with the observed forskolin-induced decrease in total kinase activity. Analysis of endogenous heat-stable protein kinase inhibitor activity in GH3 cell extracts showed no significant difference between forskolin-treated cells and cells maintained under control conditions. To assess possible effects on catalytic subunit degradation, pulse-chase experiments were performed and radiolabeled catalytic subunit was isolated by affinity chromatography. The results demonstrated that treatment of cells with chlorophenylthio-cAMP detectably increased the apparent degradation of radiolabeled catalytic subunit. The increased degradation of the catalytic subunit was sufficient to account for the observed decreases in kinase activity. These results suggest that relatively long term cAMP treatment can alter total cAMP-dependent protein kinase activity through effects to alter the degradation of the catalytic subunit of the enzyme.     

Involvement of second messenger systems in stimulation of angiotensin converting enzyme of bovine endothelial cells.

We have demonstrated previously that a variety of agents including corticosteroids, thyroid hormone, cationophores, methylxanthines, and analogues of cAMP--all of which have diversified functions in various tissues--elevate cellular angiotensin converting enzyme (ACE) activity of bovine endothelial cells in culture. In addition to these agents, we have now found that direct and receptor-mediated stimulators of adenylate cyclase, i.e., forskolin and cholera toxin, increase cellular ACE activity after 48 h incubation in culture. In an attempt to search out a more unifying concept of these stimulatory effects, we have further investigated the roles of second messengers in the stimulatory actions. Ca2+ ionophore A23187 produced significant increases in both intracellular Ca2+ and ACE of endothelial cells. In contrast to Ca2+ ionophore, agents that transiently mobilize Ca2+ from intracellular reserves such as bradykinin, acetylcholine, and ATP have no effect on the level of cellular ACE. Representative agents that elevate cellular cAMP (e.g., isobutyl methylxanthine [IBMX] and dibutyryl cAMP) elevated cellular ACE, but the slightly increased [Ca2+]i produced by these agents did not reach statistical significance. While IBMX, cholera toxin, and forskolin elevated cellular cAMP, other ACE stimulatory agents (hormones and cationophores) had no effect on cAMP. Ca2+ ionophore and the agents that elevated intracellular cAMP potentiated the effect of dexamethasone, thyroid hormone, and aldosterone in elevating cellular ACE activity. Increases in ACE activity produced by all stimulants were inhibited by the presence of 10-50 nM ouabain in the culture medium. Inhibition of ACE elevation by oubain was reversed by increasing the extracellular [K+], thereby implicating Na+, K(+)-ATPase in the ACE regulatory mechanism. These results support the presence of multiple independent mechanisms for the regulation of cellular ACE. In addition to possible involvement of intracellular Ca(2+)- and cAMP-dependent pathways, ACE is also increased by corticosteroids and thyroid hormone through mechanisms unrelated to Ca2+ and cAMP.     

Differential influence of cAMP on the expression of the three subtypes (ATA1, ATA2, and ATA3) of the amino acid transport system A

Treatment of HepG2 cells with forskolin led to 60-100% stimulation of system A activity, measured as the Na+-dependent uptake of alpha-(methylamino)isobutyric acid. The stimulation was reproducible with cholera toxin and dibutyryl cAMP, and inhibitable by H7, a non-specific protein kinase inhibitor. The stimulatory effect was eliminated by cycloheximide and actinomycin D. The forskolin effect was associated with an increase in the maximal velocity of the transport system, with no change in substrate affinity. These cells express three different subtypes of system A (ATA1, ATA2, and ATA3). Treatment with forskolin increased the steady-state levels of ATA1 and ATA2 mRNAs, but decreased that of ATA3 mRNA.     

Nerve Growth Factor-Induced Down-Regulation of Calmodulin-Dependent Protein Kinase III in PC12 Cells Involves Cyclic AMP-Dependent Protein Kinase

Treatment of PC12 cells with nerve growth factor (NGF), epidermal growth factor (EGF), or agents that raise intracellular cyclic AMP (cAMP) levels (e.g., forskolin) reduces the activity of calmodulin-dependent protein kinase III (CaM-PK III) over a period of 8 h. The mechanism of this effect of NGF has now been examined in more detail, making use of a mutant PC12 cell line (A126-1B2) that is deficient in cAMP-dependent protein kinase activity. Control experiments showed that A126-1B2 cells retain other NGF-mediated responses (e.g., the induction of ornithine decarboxylase, a cAMP-independent event) and contain a complement of CaM-PK III and its substrate, elongation factor-2, comparable to that of wild-type cells. The ability of NGF or forskolin, but not of EGF, to down-regulate CaM-PK III was markedly attenuated in A126-1B2 compared to wild-type cells. Treatment of wild-type cells with the cAMP phosphodiesterase inhibitor, isobutylmethylxanthine, enhanced the effects of NGF, but not of EGF. The possibility that NGF led to a stimulation of cAMP-dependent protein kinase activity in wild-type cells was assessed by measurement of the "activation ratio" (-cAMP/+cAMP) of this enzyme before and at various times after NGF addition. A small, but significant, increase in the activation ratio from 0.3 to 0.48 was observed, reaching a peak 5 min after NGF treatment. EGF had no effect on the activation ratio in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A tyrosine-specific protein kinase from Ehrlich ascites tumor cells

A protein tyrosine kinase that phosphorylates both alpha and beta subunits of inactivated (Na+,K+)-ATPase from dog kidney was purified about 500-fold from Ehrlich ascites tumor cell membranes. The enzyme required divalent cations Mn2+, Mg2+, or Fe2+ but was inhibited by Cu2+ or Zn2+. The purified enzyme phosphorylated the beta subunit about five times faster than the alpha subunit of the (Na+,K+)-ATPase. The random polymer poly(Glu80Tyr20) was an excellent substrate while casein was only marginally phosphorylated. In contrast, the purified transforming gene product of Rous sarcoma virus phosphorylated all three substrates and the (Na+,K+)-ATPase was preferentially phosphorylated on the alpha subunit. The transforming gene product of Fujinami sarcoma visue and EGF receptor kinase from A431 cells phosphorylated (Na+,K+)-ATPase poorly whereas casein was an excellent substrate. The molecular weight of the partially purified protein tyrosine kinase from Ehrlich ascites tumor cells determined by gel filtration was about 60,000. One of two major phosphorylated phosphopeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis had an Mr of 60 kDa, thus suggesting that it might be the autophosphorylated protein tyrosine kinase. A phosphatase that hydrolyzes phosphorylated histones or poly(Glu80Tyr20) was partially purified from the same membrane.     

Cholera toxin treatment stimulates tumorigenicity of Rous sarcoma virus-transformed cells.

Chinese hamster ovary cells transformed by Rous sarcoma virus form tumors poorly in nude mice. Tumorigenicity was markedly stimulated by pretreatment of the cells with cholera toxin, which raises cyclic AMP levels and activates cyclic AMP-dependent protein kinase. Increased tumorigenicity was manifested by a severalfold increase in the rate of tumor formation, as well as earlier appearance and more rapid growth of tumors. In contrast, spontaneously transformed Chinese hamster ovary cells showed decreased tumorigenicity after cholera toxin treatment. The activation of tumorigenic potential in Rous sarcoma virus-transformed Chinese hamster ovary cells by cholera toxin correlated with increased phosphorylation of the viral oncogene product pp60src and stimulation of its tyrosine kinase activity.     

Nerve growth factor and other agents mediate phosphorylation and activation of tyrosine hydroxylase. A convergence of multiple kinase activities

A rapid phosphorylation of tyrosine hydroxylase occurs in the PC12 nerve-like clonal cell line in response to nerve growth factor (NGF), epidermal growth factor (EGF), dibutyryl-cAMP, cholera toxin, phorbol- 12-myristate-13-acetate (PMA), or potassium depolarization in the presence of calcium ions. Complete tryptic digestion and two-dimensional peptide mapping reveals four available sites of phosphorylation in the enzyme. Phosphoamino acid analysis demonstrates that serine is the amino acid residue phosphorylated in each peptide. Specific phosphorylation of each of the four sites is achieved by different subsets of the above agents. One peptide site is phosphorylated in response to EGF alone. A second site is phosphorylated only in response to NGF, cholera toxin or dibutyryl-cAMP. A third site is phosphorylated only in response to potassium depolarization and requires the presence of extracellular Ca2+. The fourth site is the only site phosphorylated in response to PMA. These data indicate that at least 4 distinct kinase systems can act to phosphorylate tyrosine hydroxylase in PC12 cells. The PMA-stimulated peptide site is also phosphorylated in response to every one of the other agents. Further proteolytic digestions and phosphopeptide mapping of this common peptide, using Staphylococcus V8 protease and thermolysin, did not generate different phosphopeptides resulting from the different agents. These data suggest that the phosphorylation of this common peptide in response to all of the agents may be mediated by a common kinase, and, hence, that tyrosine hydroxylase phosphorylation by some agents may be mediated by two kinases. Although phosphopeptide maps of tyrosine hydroxylase resulting from cAMP elevation or NGF are qualitatively similar, quantitative differences exist, suggesting differential regulation of the same kinases by these agents. Tyrosine hydroxylase was found to be activated 2--4-fold in response to each phosphorylating agent. Thus, NGF and EGF present novel, natural means of regulating the activation state of tyrosine hydroxylase in responsive neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of human gastric H(+)-K(+)-ATPase alpha-subunit gene expression by Helicobacter pylori

Clinical studies and in vitro data from isolated parietal cells suggest that acute Helicobacter pylori infection inhibits acid secretion. Gastric acidification is mediated by H(+)-K(+)-ATPase, an integral protein of parietal cell apical membranes. To test the hypothesis that H. pylori downregulates H(+)-K(+)-ATPase alpha-subunit (HKalpha) gene expression and to identify potential intracellular signaling pathways mediating such regulation, we transfected human gastric adenocarcinoma (AGS) cells with human and rat HKalpha 5'-flanking DNA fused to a luciferase reporter plasmid. Histamine caused dose-dependent, cimetidine-sensitive (10(-4) M) increases in cAMP, free intracellular Ca(2+), and HKalpha promoter activation in AGS cells. H. pylori infection of transfected AGS cells dose dependently inhibited basal and histamine-stimulated HKalpha promoter activity by 80% and 66%, respectively. H. pylori dose dependently inhibited phorbol myristate acetate-induced (10(-7) M) and staurosporine- (10(-7) M) and calphostin C-sensitive (5 x 10(-8) M) activation of HKalpha promoter. Also, H. pylori inhibited epidermal growth factor (EGF) (10(-8) M), genistein-sensitive (5 x 10(-5) M) activation of HKalpha promoter, reducing activity to 60% of basal level. These data suggest that H. pylori inhibits HKalpha gene expression via intracellular pathways involving protein kinases A and C and protein tyrosine kinase, AGS cells have functional histamine H(2) and EGF receptors, and transiently transfected AGS cells are a useful model for studying regulation of HKalpha gene expression.