Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The induction of epidermal differentiation by Ca2+ in vitro is associated with enhanced activity of phosphatidylinositol-specific phospholipase C (PLC). Neoplastic keratinocyte cell lines expressing a mutant c-Ha-ras gene and normal keratinocytes transformed to the neoplastic phenotype by transduction with the v-Ha-ras gene (v-Ha-ras keratinocytes) have elevated constitutive activity of PLC that increases further in response to Ca2+, but the cells do not differentiate normally. PLC-gamma 1 (145 kDa) is the major isoform detected by immunoblotting of extracts from control, v-Ha-ras, and neoplastic keratinocyte cell lines cultured in 0.05 mM Ca2+ medium. The amount of PLC-gamma 1 protein was higher in neoplastic cell lines than in normal and v-Ha-ras keratinocytes that had similar PLC-gamma 1 protein levels. Thus, higher PLC-gamma 1 protein levels cannot account for the elevated constitutive activity PLC in v-Ha-ras keratinocytes. After induction of differentiation by Ca2+, the amount of PLC-gamma 1 protein increased in all cell types, and PLC-delta 1 (85 kDa), barely detectable in 0.05 mM Ca2+, increased. PLC-beta 1 was not detected at any Ca2+ concentration. PLC-gamma 1 and PLC-delta 1 mRNA did not increase after elevation of extracellular Ca2+, suggesting that posttranscriptional mechanisms can regulate PLC-gamma 1 and PLC-delta 1 protein levels in normal and neoplastic keratinocytes. Activation of protein kinase C by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited the stimulation of inositol phosphate (InsP) formation by Ca2+ but did not alter basal InsP levels in normal keratinocytes. In contrast, TPA treatment reduced both Ca(2+)-stimulated and basal InsP formation in neoplastic cells lines and v-Ha-ras keratinocytes. In both normal and v-Ha-ras keratinocytes labeled with [32P]orthophosphate, antibodies against PLC-gamma 1 immunoprecipitated a complex of 32P-labeled proteins. The relative labeling of the PLC-gamma 1 band was greater in normal than in v-Ha-ras keratinocytes. Furthermore, treatment with TPA specifically increased the relative phosphorylation of PLC-gamma 1 in v-Ha-ras keratinocytes but not in normal keratinocytes. These results suggest that the negative regulation of constitutive activity of PLC by protein kinase C differs in normal and neoplastic keratinocytes and that this could be the mechanism of increased PLC activity produced by an oncogenic ras gene in keratinocytes.
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PMID:Differences in the regulation of phosphatidylinositol-specific phospholipase C in normal and neoplastic keratinocytes. 806 82

A putative protein tyrosine phosphatase inhibitor, phenylarsine oxide (PAO), potentiated phospholipase D (PLD) activity concentration-dependently in [3H] oleic acid-labeled rat basophilic leukemia (RBL-2H3) cells without significant increase in phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Although PAO induced tyrosine phosphorylation of several proteins, both PAO-induced PLD activation and tyrosine phosphorylation were not affected by a protein tyrosine kinase inhibitor, genistein. Another tyrosine kinase inhibitor, herbimycin A, prevented the PAO-induced PLD stimulation but had no effect on protein tyrosine phosphorylation. However, depletion of protein kinase C (PKC) greatly reduced PAO-stimulated PLD activity. These results indicate that PKC but not tyrosine kinase may be involved in PAO-mediated PLD activation.
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PMID:Potent activation of phospholipase D by phenylarsine oxide in rat basophilic leukemia (RBL-2H3) cells. 813 25

Possible mechanisms of AlCl3-induced inhibition of agonist-stimulated inositol phosphate (IP) accumulation were investigated using rat brain cortex slices, synaptosomes or homogenates. Under conditions in which AlCl3 inhibits carbachol (CARB)-stimulated IP accumulation (Gp-mediated), AlCl3 did not affect CARB (100 microM)-induced decreases (Gi-mediated) in 30 microM forskolin-stimulated cAMP accumulation, suggesting that AlCl3 may be specific for Gp-mediated signal transduction. To determine whether AlCl3 interfered with Gp function and/or phosphatidylinositol-specific phospholipase C (PiPLC) activity, effects of AlCl3 on CARB- and Ca(2+)-stimulated IP accumulation were examined in cortical synaptosomes. AlCl3 (500 microM) decreased CARB (1 mM)- and Ca2+ (20 microM ionomycin)-stimulated IP accumulation to 77 and 75% of control, respectively, suggesting that AlCl3 may not directly affect Gp activity, but does inhibit PiPLC activity. In cortical homogenates, AlCl3 (10-500 microM) inhibited hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) by PiPLC in a concentration-dependent manner with an estimated IC50 of 100 microM. The effects of AlCl3 on modulation of IP accumulation by extracellular Ca2+ and PKC were also examined as potential mechanisms. Decreasing the extracellular Ca2+ concentration ([Ca2+]e) from 1.0 to 0.1 mM decreased CARB-stimulated IP accumulation in slices. AlCl3 (500 microM) decreased significantly 1 mM CARB-stimulated IP accumulation in 1.0 and 0.1 mM Ca2+ solutions; however, the effect of AlCl3 on IP accumulation did not depend on [Ca2+]e. In cortical slices, inhibition of 1 mM CARB-stimulated IP accumulation by 500 microM AlCl3 was not altered by the PKC activator phorbol 12,13-dibutyrate (PdBu, 1 microM), or the PKC inhibitor H-7 (10 microM), suggesting that AlCl3 does not interfere with IP accumulation by activation of PKC. Other studies found that AlCl3 (10-100 microM) inhibited PKC activity in a concentration-dependent manner in both cytosolic and membrane fractions of cortical homogenates with an estimated IC50 of 60 microM. These results support the hypothesis that AlCl3 inhibition of agonist-stimulated IP accumulation may be mediated by inhibition of PiPLC activity, rather than disruption of G-protein function or modulation of the IP signalling system by Ca2+ or PKC.
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PMID:Mechanisms underlying AlCl3 inhibition of agonist-stimulated inositol phosphate accumulation. Role of calcium, G-proteins, phospholipase C and protein kinase C. 818 49

In murine keratinocytes, Ca(++)-induced terminal differentiation is accompanied by a rapid and sustained increase of inositol phosphates and diacylglycerol. Based on Western blotting analysis, basal keratinocytes cultured in 0.05 mM Ca++ medium express phospholipase C (PLC)-gamma 1 predominantly and no detectable PLC-beta 1. Differentiating keratinocytes cultured in 1.4 mM Ca++ express two- to threefold more PLC-gamma 1 protein and PLC-delta 1, but no detectable PLC-beta 1. Although the amount of PLC-gamma 1 and -delta 1 protein increased, PLC-gamma 1 and -delta 1 mRNA decreased in differentiating cells. Thus the sustained rise of PLC activity induced by Ca++ in differentiating keratinocytes may be associated with higher amounts of both PLC-gamma 1 and -delta 1 in maturing cells, determined by a posttranscriptional mechanism. Tyrosine phosphate content in PLC-gamma 1 was low in basal cells and did not change in cells exposed to 1.4 mM Ca++. However, genistein inhibited the increase in PLC activity induced by 1.4 mM Ca++. In contrast, transforming growth factor (TGF)alpha, which stimulates both PLC activity and growth in basal keratinocytes, increased tyrosine phosphorylation of PLC-gamma 1. These results suggest that tyrosine phosphorylation of PLC-gamma 1 by the epidermal growth factor (EGF) receptor is linked to stimulated proliferation, whereas stimulation of PLC activity by Ca++ is linked to keratinocyte differentiation and involves the action of a tyrosine kinase but not tyrosine phosphorylation of PLC-gamma 1. Based on studies using the intracellular free Ca++ chelator BAPTA, a rise in intracellular free Ca++ was not required for stimulation of PLC activity by raising extracellular Ca++. Phorbol esters inhibited PLC stimulation by 1.4 mM Ca++ medium and increased serine phosphorylation of PLC-gamma 1. Exogenous phosphatidylinositol-specific and phosphatidylcholine-specific bacterial PLC also inhibited endogenous inositol phosphate formation and increased endogenous diacylglycerol (DAG). Thus, direct serine phosphorylation of PLC-gamma 1 by protein kinase C is associated with the inhibition of Ca(++)-mediated PLC stimulation. These results show that keratinocytes have multiple mechanisms to regulate PLC activity in response to a specific signal.
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PMID:Keratinocyte differentiation is associated with changes in the expression and regulation of phospholipase C isoenzymes. 822 34

The accumulation of inositol 1,4,5-trisphosphate (IP3) after hormonal stimulation has a physiological role, possibly by alteration of Ca2+ levels in cardiac myocyte. However, this accumulation has not been studied under pathophysiological conditions. In this report, we examine phosphatidylinositol metabolism during cellular response to norepinephrine in pressure-overloaded hypertrophic rat heart. After stimulation with norepinephrine, the accumulations of IP3 and diacylglyceride significantly increased in isolated myocytes from stroke-prone spontaneously hypertensive rat (SHRSP) heart, indicating phosphatidylinositol-specific phospholipase C activity increased in SHRSP heart cells. Protein kinase C activity was also enhanced in SHRSP, with a marked increase in particulate activity. We determined the intracellular calcium concentration and found it to be higher in SHRSP than in Wistar-Kyoto (WKY) rats at 30-40 weeks of age. Ca2+ influx was also elevated in SHRSP stimulated by norepinephrine. In SHRSP heart, cytosolic Ca2+ concentration may rise quickly in response to some stimuli, such as alpha 1-adrenergic stimulation, which is shown to be one of the pathways that increases cytosolic Ca2+ levels in hypertrophied rat heart. These data suggest that a part of the phosphatidylinositol-turnover pathway, such as the phosphatidylinositol 4,5-bisphosphate-IP3-Ca2+ pathway or the diacylglyceride-protein kinase C pathway, may play an important role in the development of hypertrophy in SHRSP heart.
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PMID:Phosphatidylinositol metabolism in hypertrophic rat heart. 847 30

In the present investigation, a hCG sensitive glycosyl-phosphatidylinositol (GPI) was isolated from cultured rat granulosa cells obtained from the ovaries of diethylstilbestrol (DES) implanted immature rats. The inositol-phosphoglycan (IPG) moiety of the GPI-lipid contains galactose, glucosamine, and myoinositol as demonstrated by metabolic labelling of granulosa cells for different time periods (5-96 h) with [3H]galactose, [3H]glucosamine, or [3H]myoinositol and treatment of the purified [3H]GPI with phosphatidylinositol-specific phospholipase C. Labelling equilibrium of the GPI-lipid was achieved after 24 h ([3H]galactose and [3H]myoinositol) or 72 h ([3H]glucosamine) incubation, whereas incorporation of other labelled carbohydrates tested ([3H]galactosamine, [3H]mannose, and [3H]sorbitol) was negligible throughout the time period studied. The glucosamine C-1 appears to be linked through a glycosidic bond to the myoinositol molecule of the IPG moiety as revealed by the generation of phosphatidylinositol (PtdIns) after nitrous acid deamination of dual labelled ([3H]glucosamine/[14C]palmitate or [3H]glucosamine/[14C]myristate) glycosyl-phosphatidylinositol. To investigate the fatty acid composition of the diacylglycerol (DAG) backbone of the GPI, granulosa cells were also labelled (5-72 hr) with [14C]linoleate, [3H]myristate, [3H]oleate, [3H]palmitate, or [3H]stearate and the radioactivity associated with the purified glycosyl-phosphatidylinositol determined. Incorporation of [3H]palmitate and [3H]myristate into the GPI-lipid peaked after 8 h and 24 h of labelling, respectively, and both fatty acids were partially released after PLA2 treatment of the dual labelled ([3H]glucosamine/[14C]palmitate or [3H]glucosamine/[14C]myristate) GPI. In parallel experiments no significant incorporation of labelled stearate, oleate, or linoleic acid into the DAG backbone of the glycosylphosphatidylinositol could be detected. Granulosa cells were also labelled with [3H]glucosamine in the presence of FSH (30 ng/ml), cholera toxin (1 microgram/ml), or the membrane permeable cAMP analog (but)2cAMP (1 mM). Time related increases in GPI-labelling were apparent after 48 h and reached a maximum level (3-, 5-, and 7-fold for FSH, CT, and (but)2cAMP, respectively) after 72 h in culture. In another set of experiments, granulosa cells were labelled for 72 h with [3H]glucosamine in the presence of (but)2cAMP (1 mM), TPA (10(-7) M), or combination thereof. The effect of treatment with the membrane permeable cAMP analog on GPI labelling was prevented in the presence of TPA, whereas no differences in [3H]GPI content could be observed in untreated granulosa cells or cells cultured in the presence of the protein kinase C-activating phorbol ester alone.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Follicle-stimulating hormone and human chorionic gonadotropin induced changes in granulosa cell glycosyl-phosphatidylinositol concentration. 848 20

The translocation of protein kinase C (PKC) from the cytosolic to the particulate fraction in IIC9 fibroblasts has been studied to define the functions of 1,2-diacylglycerol (DAG) derived from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylcholine (PC). alpha-Thrombin caused a biphasic change in DAG, with two peaks at 15-60 s and 5-15 min, derived from PIP2 and PC, respectively, while platelet-derived growth factor (PDGF) induced a monophasic DAG increase from PC at 5-15 min. alpha-Thrombin also induced a rapid, but transient, increase of inositol 1,4,5-trisphosphate and cytosolic Ca2+, whereas PDGF did not. Three PKC isozymes, alpha, epsilon, and zeta, were identified by Western blotting in IIC9 cells and were mainly localized in the cytosol. A fraction of cytosolic PKC alpha was rapidly translocated by alpha-thrombin at 15 s, but its membrane association was lost within 1 min. PKC epsilon was also rapidly translocated; however, its membrane association was sustained for almost 60 min. PKC zeta was not translocated by alpha-thrombin or phorbol 12-myristate 13-acetate. PDGF translocated PKC epsilon at 5 min but had little effect at 15 s and did not translocate PKC alpha or zeta. Incubation with Bacillus cereus PC- or phosphatidylinositol-specific phospholipase C, which increased DAG but not phosphatidic acid, stimulated translocation of PKC epsilon, but not PKC alpha or zeta. Addition of chelators to inhibit the rise in intracellular Ca2+ largely blocked PKC alpha translocation induced by alpha-thrombin but had no effect on PKC epsilon translocation. Addition of ionomycin allowed alpha-thrombin to induce PKC alpha translocation at 5 min. PKC alpha translocation was mimicked by 1,2-dioctanoylglycerol plus ionomycin, but not by either alone. On the other hand, PKC epsilon was translocated by the DAG alone. These results support the conclusion that PIP2 hydrolysis activates both PKC alpha and epsilon at 15 s, whereas PC hydrolysis activates only PKC epsilon at 5 min. The differential activation at 5 min can be attributed to the failure of PC hydrolysis to increase Ca2+ and not to a difference in the molecular species of DAG derived from the phospholipids.
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PMID:Differential translocation of protein kinase C isozymes by thrombin and platelet-derived growth factor. A possible function for phosphatidylcholine-derived diacylglycerol. 848 6

Previous work from this laboratory has identified an endothelin (ET) type A (ETA) receptor on cultured rat renal medullary interstitial cells (RMIC), coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), dihydropyridine-insensitive receptor-operated Ca2+ channels, and phospholipase A2. The current studies explored a role for ET stimulation of phosphatidylcholine-specific phospholipase D (PC-PLD) in intracellular signaling of this cell type. ET stimulated PLD activation, as measured by phosphatidic acid (PA) or phosphatidylethanol (PEt) accumulation, in a time- and concentration-dependent manner. Inhibition of diacylglycerol (DAG) kinase by ethylene glycol dioctanoate or 6-(2)4-[(4-fluorophenyl)-phenylmethylene]-1-piperadinyl]ethy l-7-methyl-5H - thiaxolo-[3,2-alpyrimidin]-5-one (R 59022) failed to blunt PA accumulation, indicating that PLD, and not DAG, was the source of PA. Inhibition of PA phosphohydrolase (PAP) by propranolol increased late accumulation of PA, suggesting that the prevailing metabolic flow was in the direction of PA to DAG. Phorbol 12-myristate 13-acetate (PMA) augmented ET-evoked PEt accumulation, whereas downregulation of protein kinase C (PKC) obviated agonist-induced PEt production. PMA augmentation of PLD activity proceeded independent of cytosolic free Ca2+ concentration. Ca2+ derived from either intracellular or extracellular sources enhanced ET-related PEt accumulation but was without effect in PKC-downregulated cells. Collectively, these observations indicate that ET stimulates PLD production in RMIC. PKC is the major regulator of this process, with Ca2+ playing a secondary, modulatory role. In addition, these data suggest that PC-PLD is coupled to the ETA receptor.
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PMID:Endothelin activation of phospholipase D: dual modulation by protein kinase C and Ca2+. 849 38

Epidemiological and laboratory animal model studies suggest that the effect of dietary fat in colon carcinogenesis depends not only on the amount but on its fatty acid composition. Animal model studies demonstrated that high dietary corn oil or safflower oil rich in omega-6 fatty acids increased the colon tumor promotion, whereas diets containing fish oil high in omega-3 fatty acids had no such enhancing effect. One of the mechanisms by which high dietary fat enhances colon carcinogenesis may be through the modulation of colonic mucosal phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase C (PI-PLC), which are dominant pathways for arachidonic acid release and formation of eicosanoids. PI-PLC is also responsible for diacylglycerol formation and protein kinase C-dependent signal transduction and cell proliferation. In the present study, we investigated the modulating effect of high fat diets rich in omega-3 and omega-6 fatty acids on colonic mucosal PLA2, PI-PLC activities, and eicosanoid (prostaglandins and thromboxane B2) formation from arachidonic acid via cyclooxygenase (COX) during different stages of azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. At 5 weeks of age, groups of animals were fed the low-fat diet containing 5% corn oil. Beginning at 7 weeks of age, all animals except those intended for vehicle treatment received AOM s.c. once weekly for 2 weeks at a dose rate of 15 mg/kg body weight. Vehicle-treated groups received an equal volume of normal saline. One day after the second AOM or vehicle treatment, groups of animals were transferred to experimental diets containing 23.5% corn oil and 20.5% fish oil + 3% corn oil, whereas one group continued on the low-fat diet containing 5% corn oil. Groups of animals were then sacrificed at weeks 1, 12, and 36 after the second AOM-or saline-treatment. Colonic mucosa harvested at weeks 1, 12, and 36 and colonic tumors obtained at week 36 were analyzed for PLA2, PI-PLC, and eicosanoid formation from arachidonic acid by the action of COX. The results demonstrate that colon carcinogen treatment increases the activities of colonic mucosal PLA2 and PI-PLC and the formation of prostaglandins and thromboxane A2 from arachidonic acid through COX throughout the study period compared to saline-treated animals fed similar diets. The activities of PLA2, PI-PLC, and COX were significantly higher in colon tumors compared to colonic mucosa. These results also demonstrate that a high-fat diet containing corn oil increases colonic mucosal and tumor PLA2 and PI-PLC and the formation of prostaglandins and thromboxane B2 by the action of COX as compared to low dietary corn oil or a diet high in fish oil. The results of our study offer one of the mechanisms by which the amount and types of dietary fat modulate colon carcinogenesis.
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PMID:Modulating effect of amount and types of dietary fat on colonic mucosal phospholipase A2, phosphatidylinositol-specific phospholipase C activities, and cyclooxygenase metabolite formation during different stages of colon tumor promotion in male F344 rats. 856 67

It is evident from many studies that the effect of dietary fat on colon tumor promotion depends not only on the amount of fat but especially on fatty acid composition. Animal model studies have shown that diets which are high in omega-6 fatty acids increase colon tumor promotion, whereas diets rich in omega-3 fatty acids have no such enhancing effect. The mechanisms by which the high fat content of the diet promotes colon carcinogenesis may include the production of secondary bile acids in the colon and the modulation of colonic luminal bacterial 7 alpha-dehydroxylase that is involved in generating secondary bile acids, phosphatidylinositol-specific phospholipase C (PI-PLC), and mucosal PI-PLC, as well as diacylglycerol (DAG) kinase and protein kinase C (PKC). In the present study, we investigated the effect of high-fat diets that are rich in omega-3 and omega-6 fatty acids on cecal bacterial 7 alpha-dehydroxylase and PI-PLC, fecal secondary bile acids, and colonic mucosal DAG kinase and PKC activities during different stages of colon carcinogenesis in male F344 rats. At 5 weeks of age, groups of animals were fed a low-fat diet containing 5% corn oil (LFCO). Beginning at 7 weeks of age, all animals, except those intended as vehicle controls, received azoxymethane (AOM) s.c. once weekly for 2 weeks at a dose rate of 15 mg/kg body weight. Vehicle-treated groups received s.c. injections of normal saline. One day after the second AOM or saline treatment, the experimental groups of animals were transferred to a high-fat diet containing 23.5% corn oil (HFCO) or 20.5% fish oil + 3% corn oil (HFFO). One group continued on the LFCO diet. Animals were sacrificed at weeks 1, 12, and 36 after the AOM or saline treatment. Colonic mucosa were harvested at weeks 1, 12, or 36, and the colonic tumor tissues were examined for PKC and DAG kinase activities. Contents of the cecum were analyzed for bacterial 7 alpha-dehydroxylase and PI-PLC activities. Stool samples collected at week 12 were analyzed for bile acids. High corn oil content of the diet significantly increased the cecal bacterial 7 alpha-dehydroxylase and PI-PLC activities as compared to the diets with high fish oil or low corn oil content. Animals fed the HFCO diet excreted higher levels of secondary bile acids, such as deoxycholic acid and lithocholic acid, than those fed the LFCO or HFFO diets. Carcinogen treatment significantly enhanced the activities of DAG kinase and total membrane PKC activities in colonic mucosa compared to saline treatment in all dietary groups. Animals treated with saline or AOM and fed HFCO showed increased levels of DAG kinase and membrane PKC activities in the colonic mucosa when compared to LFCO and HFFO groups. DAG kinase and membrane PKC activities were higher in colon tumors than in the surrounding colonic mucosa, and also increased levels of these enzyme activities were found in the HFCO diet group. These results indicate that the modifying effect of dietary fat on colonic bacterial enzymes, secondary bile acids, colonic mucosal and tumor DAG kinase, and PKC that may play a role in colon carcinogenesis depends on the types and amount of fat given. The colon tumor-enhancing effect of a HFCO diet in contrast to the high dietary fish oil may be, in part, explained on the basis of its modulating effect on these bacterial and colonic mucosal enzymes and colonic secondary bile acids relevant to colon tumor promotion.
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PMID:Effect of amount and types of dietary fat on intestinal bacterial 7 alpha-dehydroxylase and phosphatidylinositol-specific phospholipase C and colonic mucosal diacylglycerol kinase and PKC activities during stages of colon tumor promotion. 862 6


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