Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In an attempt to characterize the ras signaling pathway, we studied the effects of expression vectors encoding the valine 12 mutant ras oncogene on rat prolactin (rPRL) promoter activity. Using this approach we have been able to dissect the interplay between the ras and the protein kinase A (PKA) pathways as they relate to neuroendocrine gene activation. Here we show that the ras oncogene product induces rPRL promoter activity selectively from 5- to 14-fold in GH4 rat pituitary tumor cells, whereas it has a minimal effect on the SV40 early promoter and no effect on the Rous sarcoma virus (RSV) or rat growth hormone promoters. By contrast, an inactivated form of ras (N-17 ras) did not stimulate the rPRL promoter, but rather inhibited it to 40% of control. Of note, activation of the PKA pathway by two different methods decreased the fold activation mediated by ras by at least 50%, whereas inhibition of the PKA pathway accentuated ras activation of the rPRL promoter. Although rPRL promoter activity is consistently induced by PKA activation in control GH4 cells, acute ras oncogene expression inhibited forskolin induction of rPRL promoter activity. Moreover, this ras-mediated interference of the forskolin activation of rPRL promoter activity was also noted in GH4 cells stably expressing ras. Taken together, these data show that the valine 12 ras oncogene activates the rPRL promoter selectively and, more importantly, that the ras and PKA signaling pathways are mutually antagonistic with respect to specific transcriptional activation of a neuroendocrine gene.
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PMID:The ras and protein kinase A pathways are mutually antagonistic in regulating rat prolactin promoter activity. 162 May 44

In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRH-Rs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/calmodulin-dependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 microM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 microM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 microM TRH. H-7 (20 microM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thyrotropin-releasing hormone (TRH) and phorbol myristate acetate decrease TRH receptor messenger RNA in rat pituitary GH3 cells: evidence that protein kinase-C mediates the TRH effect. 172 45

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.
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PMID: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. 216 38

P19, a group of 19,000 mol wt cytosolic proteins, with apparent isoelectric points of pI 5.9, pI 5.7, and pI 5.4, respectively, was identified in three peptide hormone-producing cell types: AtT20 mouse pituitary tumor cells, RIN-1122 rat insulinoma cells, and hamster insulinoma cells. Secretagogue-dependent phosphorylation of P19 was analyzed in 32P-labeled cells by two-dimensional electrophoresis and autoradiography. The results were quantitated by computer-assisted densitometry. Cellular levels of cAMP and hormone release were measured in parallel incubations. In addition to stimulating ACTH release, CRF raised the cellular level of cAMP and increased the 32P labeling of all three 19,000 mol wt proteins in AtT20 cells. Other agents known to act through cAMP, which included isoproterenol, forskolin, and 8-bromo-cAMP, mimicked the effect of CRF on both ACTH release and phosphorylation of P19. 12-O-Tetra-decanoylphorbol-13-acetate, a tumor-promoting phorbol ester, also stimulated both ACTH release and phosphorylation of P19. In contrast, although 40 mM K+ promoted ACTH release, it did not affect the phosphorylation of P19. Analogous findings were observed in insulinoma cells. Glucagon stimulated insulin release, increased cellular cAMP and promoted phosphorylation of P19 in RIN 1122 cells. 12-O-Tetradecanoylphorbol-13-acetate also enhanced insulin release and the phosphorylation of P19 in these cells. The results obtained with hamster insulinoma cells closely resembled the observations in RIN-1122 cells. In conclusion, P19, an apparently homologous set of cytosolic proteins, undergoes phosphorylation in three peptide hormone-producing cells in response to two groups of secretagogues, the effect of which is probably mediated, in one case, by cAMP-dependent protein kinase and, in the other, by protein kinase C. The data suggest the possibility that P19 participates in a secretory pathway activated by these two effector systems.
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PMID:P19, a hormonally regulated phosphoprotein of peptide hormone-producing cells: secretagogue-induced phosphorylation in AtT-20 mouse pituitary tumor cells and in rat and hamster insulinoma cells. 242 97

Two classes of calcium channels were activated by membrane depolarization in cell-free membrane patches from GH3 cells, an electrically excitable cell line derived from a mammalian pituitary tumor. One class had a conductance of approximately 10 pS in 90 mM barium, had a threshold of activation near -40 mV, and was inactivated rapidly at holding potentials more positive than -80 mV. The other class, with a conductance of approximately 23 pS and a threshold nearer -20 mV, did not inactivate in barium but stopped responding to depolarization altogether when the cytoplasmic side of the patch was exposed to a standard physiological saline solution. Buffering the concentration of calcium ions to less than 10 nM on the cytoplasmic side did not prevent this loss of activity. However, activity was restored and maintained for the duration of the patch when the catalytic subunit of cAMP-dependent protein kinase was added with MgATP to the cytoplasmic side of the membrane. Cell-free patch formation in the presence of the dihydropyridine, BAY K 8644, also delayed the loss of activity, but unlike the catalytic subunit plus ATP, BAY K 8644 alone did not restore activity when it was added after the channels no longer responded to depolarization. Evidently the dihydropyridine-sensitive class of voltage-activated calcium channels must be phosphorylated in order to open when the membrane is depolarized. That hypothesis provides a simple framework for understanding the modulation of calcium channel gating by neurotransmitters, calcium ions, and dihydropyridines.
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PMID:Voltage-activated calcium channels that must be phosphorylated to respond to membrane depolarization. 243 33

To determine the rat PRL (rPRL) promoter sequences that mediate pituitary-specific and cAMP-induced gene expression in vivo, various lengths of the rPRL promoter were ligated to the luciferase reporter gene and introduced into pituitary and non-pituitary cell lines. A 30-fold increase in rPRL promoter activity was observed in GH4 rat pituitary tumor cells compared to nonpituitary Rat2 fibroblast and HeLa cervical carcinoma cells. About 45% of this cell-specific promoter activity was competed by a plasmid containing the -67 to -45 rPRL promoter region, which is the most proximal binding site for a lactotroph-specific factor. Compared to a -425 rPRL construct, transfection with rPRL 5'-end points of -212, -178, and -127 contained 23%, 45%, and 1%, respectively, of luciferase activity. Forskolin stimulation resulted in a 10-fold induction of all the rPRL promoter fragments tested. Of note, a -127 deletion which was devoid of any basal promoter activity was also induced 10-fold by forskolin. The forskolin effect was abolished when GH4 rat pituitary cells were cotransfected with a plasmid encoding a protein kinase A inhibitor, indicating protein kinase A is involved in the activation mechanism. These data document that both positive and negative effectors influence basal rPRL promoter activity. Furthermore, the minimum sequences required for pituitary-specific rPRL promoter activity are altered by intracellular cAMP levels. Taken together, the data indicate that hormone-activated and cell-specific factors may interact to establish a particular setpoint for rPRL gene expression.
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PMID:Analysis of rat prolactin promoter sequences that mediate pituitary-specific and 3',5'-cyclic adenosine monophosphate-regulated gene expression in vivo. 254 56

The effects of the protein kinase C activator, phorbol myristate acetate (PMA), on cytosolic calcium levels and adrenocorticotropin (ACTH) release from the mouse anterior pituitary tumor cell line, AtT-20, were compared to those induced by the hormone, corticotropin-releasing factor (CRF), a stimulant of cAMP-dependent protein kinase activity. Cytosolic calcium levels were measured using the fluorescence probe Quin 2. PMA induced a time- and concentration-dependent rise in cytosolic calcium levels and ACTH release from AtT-20 cells that was blocked by verapamil and nifedipine, antagonists of voltage-regulated calcium channels, and tetraethylammonium (TEA), a K+ channel antagonist. The inactive phorbol ester, 4-phorbol 12,13-didecanoate, did not alter cytosolic calcium levels or ACTH release. Several minutes after the initial stimulation of calcium influx by PMA, cytosolic calcium levels returned to basal levels despite the continued presence of the phorbol ester. A short pretreatment (2-4 min) of AtT-20 cells with PMA abolished the ability of K+, CRF, and forskolin to raise intracellular calcium levels. These findings indicate that phorbol esters induce a secondary inhibition of calcium influx after an initial stimulation. In contrast to the effects of PMA, CRF induced a sustained rise in cytosolic calcium levels and did not reduce the subsequent stimulation of calcium influx by K+ or PMA. CRF-stimulated calcium influx was blocked by verapamil but not TEA. The ability of CRF to elevate cytosolic calcium levels was mediated by cAMP-dependent protein kinase because the insertion of a synthetic peptide inhibitor of cAMP-dependent protein kinase activity into AtT-20 cells attenuated the ability of CRF and forskolin but not PMA to raise cytosolic calcium levels. The results suggest that activators of protein kinase C and cAMP-dependent protein kinase regulate intracellular calcium levels in AtT-20 cells through different mechanisms.
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PMID:Activators of protein kinase C and cyclic AMP-dependent protein kinase regulate intracellular calcium levels through distinct mechanisms in mouse anterior pituitary tumor cells. 282 94

The PRL gene is expressed at a high basal level in rat pituitary tumor GH3 cells, and this basal level enhancement of PRL gene expression is maintained through a Ca2+-calmodulin-dependent mechanism. We have now examined whether the enzyme, DNA topoisomerase II, which has been shown to be phosphorylated by a Ca2+-calmodulin-dependent protein kinase, plays a role in the Ca2+-calmodulin-dependent basal level enhancement of PRL gene expression. The topoisomerase II inhibitor, novobiocin, at concentrations in the range of 35-140 microM, effectively blocked the ability of Ca2+ to increase PRL mRNA levels. Examination of the effects of novobiocin on the levels of protein synthesis, glucose-regulated protein (GRP) 78 mRNA, histone 3 mRNA, and 18S ribosomal RNA indicated that the drug selectivity inhibited PRL gene expression. Two other topoisomerase II inhibitors, m-AMSA and VM26, also diminished the Ca2+-induced levels of PRL mRNA at concentrations (100-400 nM) that did not lower total mRNA levels. We then examined whether topoisomerase II interacted nonrandomly with DNA from the 5' transcribed and 5'-flanking region of the rat PRL gene by in vitro mapping of topoisomerase II DNA cleavage sites. In initial assays with a 10.5 kilobase (kb) PRL genomic DNA fragment containing 3.5 kb of 5'-transcribed DNA and 7 kb of 5'-flanking DNA, we detected 4 major cleavage sites in the following regions: site 1, +1500 to +1600; site 2, +1 to -100; site 3, -1200 to -1300; and site 4, -2900 to -3000.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evidence for a role of topoisomerase II in the Ca2+-dependent basal level expression of the rat prolactin gene. 284 May 67

Although dopamine inhibits PRL release from the normal anterior pituitary lactotroph, a conclusive demonstration of the mechanisms involved in this response has been impeded by the presence of other cell types in the anterior pituitary. To circumvent this problem, we have isolated a clonal cell line, designated MMQ, from the 7315a rat pituitary tumor. The MMQ cell is an exemplary model for our use because it only secretes PRL. Our studies show that dopamine inhibits secretagogue-induced PRL release from these cells. In addition, dopamine decreases the intracellular cAMP concentration in MMQ cells that have been exposed to forskolin, cholera toxin, or vasoactive intestinal polypeptide, each a stimulator of cAMP generation. This inhibition is, in turn, reversed by the dopamine antagonist haloperidol and by pertussis toxin, an inactivator of the GTP-binding coupling protein. Dopamine also decreases the uptake and fractional efflux of 45Ca2+ by MMQ cells that have been exposed to the calcium channel activator maitotoxin. It seems, therefore, that dopamine decreases PRL release from MMQ cells at least in part by decreasing intracellular cAMP levels and calcium uptake. In additional experiments, we have found that MMQ cells are responsive to somatostatin, estrogen, progesterone, and acetylcholine, but not to TRH, angiotensin II, neurotensin, or bombesin. Furthermore, these cells possess a functional protein kinase-C system, as evidenced by the increase in PRL release and decrease in stimulated intracellular cAMP levels that occur in response to treatment with phorbol diesters. We suggest that the MMQ cell line will prove a useful model system for study of the biochemical effects of dopamine and other factors that modify PRL release.
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PMID:Characterization of the MMQ cell, a prolactin-secreting clonal cell line that is responsive to dopamine. 284 8

The properties of the cAMP-dependent protein kinases in AtT20 mouse pituitary tumor cells were characterized by a combination of immunological and biochemical techniques. Ninety per cent of the total cAMP-dependent protein kinase was in the 40,000 X g supernatant fraction. Protein kinases I and II were immunoprecipitated with specific antisera directed against their regulatory subunits. The immunoprecipitated kinases bound [3H]cAMP and were catalytically active when incubated with [gamma-32P]ATP-Mg and protamine or histone H2B. Immunoprecipitated protein kinases I and II bound [3H]cAMP with apparent Kb values of 1.5 and 15 nM, respectively. Regulatory subunit concentrations in AtT20 cells were measured by immunoprecipitation of [3H]cAMP-R complexes. R-I and R-II levels were 2.7 and 3.0 pmol of [3H]cAMP binding activity per mg of cytosolic protein, respectively, however, the ratio of protein kinase II to protein kinase I was 2.5 indicating the presence of a significant amount of free R-I. This was confirmed by DEAE-cellulose chromatography and the isolation of immunoreactive R-I devoid of protein kinase activity. A significant amount of R-I also coeluted with protein kinase II when AtT20 cell extracts were subjected to DEAE-cellulose chromatography. In quantitative immunoprecipitation experiments, 0.1 microliter of anti-brain R-II serum complexed up to 0.5 pmol of the [3H]cAMP-binding activity of protein kinase II prepared from bovine and rat brain, and AtT20 cells while 2 microliter of anti-brain R-II serum was required to precipitate an equal amount of protein kinase II from bovine skeletal muscle showing that the protein kinase II in AtT20 cells contained the neural-specific R-II subunit.
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PMID:Immunological and molecular characterization of the cAMP-dependent protein kinases in AtT20 cells. 608 92


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