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

The role of calcium in control of HCl secretion by the gastric parietal cell was examined using a recently available intracellular calcium-releasing agent, thapsigargin, which has been shown, in some cell types, to induce sustained elevation of intracellular calcium ([Ca2+]i), an action that appears to be independent of inositol lipid breakdown and protein kinase C activation and to be mediated, at least partially, by selective inhibition of endoplasmic reticulum Ca2(+)-ATPase. Using the calcium-sensitive fluorescent probe, fura-2, in combination with digitized video image analysis of single cells as well as standard fluorimetric techniques, we found that thapsigargin induced sustained elevation of [Ca2+]i in single parietal cells and in parietal cells populations. Chelation of medium calcium led to a transient rise and fall in [Ca2+]i, indicating that the sustained elevation in [Ca2+]i in response to thapsigargin was due to both intracellular calcium release and influx. Although thapsigargin appeared to affect the same calcium pool(s) regulated by the cholinergic agonist, carbachol, and the pattern of thapsigargin-induced increases in [Ca2+]i were similar to the plateau phase of the cholinergic response, thapsigargin did not induce acid secretory responses of the same magnitude as those initiated by carbachol (28 vs 600% of basal). The protein kinase C activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) potentiated the secretory response to thapsigargin but this combined response also did not attain the same magnitude as the maximal cholinergic response. In the presence but not the absence of medium calcium, thapsigargin potentiated acid secretory responses to histamine, which elevate both cyclic AMP (cAMP) and [Ca2+]i in parietal cells, as well as forskolin and cAMP analogues but had no effect on submaximal and an inhibitory effect on maximal cholinergic stimulation. Furthermore, thapsigargin did not fully mimic potentiating interactions between histamine and carbachol, either in magnitude or in the pattern of temporal response. Assuming that the action of thapsigargin is specific for intracellular calcium release mechanisms, these data suggest that 1) sustained influx of calcium is necessary but not sufficient for cholinergic activation of parietal cell HCl secretion and for potentiating interactions between cAMP-dependent agonists and carbachol; 2) mechanisms in addition to elevated [Ca2+]i and protein kinase C activation may be involved in cholinergic regulation; and 3) increases in [Ca2+]i in response to histamine are not directly involved in the mechanism of histamine-stimulated secretion.
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PMID:Thapsigargin potentiates histamine-stimulated HCl secretion in gastric parietal cells but does not mimic cholinergic responses. 184 93

The asialoglycoprotein (ASGP) receptor undergoes constitutive endocytosis through the coated pit/coated vesicle pathway in hepatocytes. Studies on HepG2 cells have shown that the receptor is phosphorylated at serine under control conditions and following protein kinase C stimulation. This study examined whether the ASGP receptor could also serve as a substrate for a tyrosine kinase in HepG2 cells. 32P labeling was performed in membrane preparations, in permeabilized cells at 4 degrees C, and in intact cells at 37 degrees C. The phosphorylated ASGP receptor was isolated by immunoprecipitation, hydrolyzed in 6 N HCl at 110 degrees C, and analyzed by two-dimensional high voltage electrophoresis. The receptor isolated from a membrane preparation incubated in vitro with [gamma-32P]ATP incorporated radiolabel predominantly (greater than 90%) into phosphotyrosine. ASGP receptor phosphorylation at both tyrosine and serine was detected in intact cells incubated with phosphatase inhibitors for 60 min at 37 degrees C. The presence of both phenylarsine oxide (20 microM) and sodium orthovanadate (200 microM) was required for tyrosine phosphorylation. Use of these inhibitors together resulted in a 16.4-fold increase in phosphorylation of the immunoprecipitated ASGP receptor, whereas phosphorylation of total HepG2 membrane proteins was not significantly augmented by this procedure. Selective proteolytic digestion of ASGP receptors in isolated vesicles demonstrated that the phosphorylation site identified in these studies is located at tyrosine 5 in the cytoplasmic tail.
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PMID:Tyrosine phosphorylation of the asialoglycoprotein receptor. 215 77

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) regulates the expression of c-myc protooncogene in HL-60 promyelocytic leukemia cells (Reitzma, P. H., Rothberg, P. G., Astrin, S. M., Trial, J., Barshavit, Z., Hall, A., Teitelbaum, S. U., and Kahn, A. J. (1983) Nature 306, 492-494). The regulation of c-myc expression occurs at least in part at the transcriptional level (Simpson, R. U., Hsu, T., Begley, D. A., Mitchell, B. S., and Alizadeh, B. N. (1987) J. Biol. Chem. 262, 4104-4108). Also, 1,25-(OH)2D3 stimulates an increase in protein kinase C (PKC) levels and inhibitors of PKC block 1,25-(OH)2D3-induced differentiation of HL-60 cells (Martell, R. E., Simpson, R. U., and Taylor, J. M. (1987) J. Biol. Chem. 262, 5570-5575). In this report we demonstrated that sphinganine, an inhibitor of PKC that is mechanistically and structurally distinct from 1-(5-isoquinoline sulfonyl)-2-methylpiperazine-HCl (H-7), also blocks 1,25-(OH)2D3 induction of HL-60 cell differentiation. The effect of inhibitors of PKC on 1,25-(OH)2D3 regulation of c-myc transcription was examined. H-7 (18 microM) and sphinganine (3 and 6 microM) blunted 1,25-(OH)2D3-induced reduction of c-myc transcription as assessed by nuclear run-off assays. We showed that c-myc/beta-actin ratios (cpm/cpm, % of control mean +/- S.E.) were as follows: ethanol control, 100 +/- 14%; 50 nM 1,25-(OH)2D3, 17 +/- 5%; 50 nM 1,25-(OH)2D3 and 6 microM H-7, 13 +/- 6%; 50 nM 1,25-(OH)2D3 and 18 microM H-7, 53 +/- 6% 50 nM 1,25-(OH)2D3 and 18 microM N-[2-guanidinoethyl]-5-isoquinoline sulfonamide (HA-1004), 10 +/- 8%; 50 nM 1,25-(OH)2D3 and 6 microM sphinganine, 49 +/- 8%. No significant differences in c-myc transcription between control, 18 microM H-7, 18 microM HA-1004, and 3 or 6 microM sphinganine-treated cells were observed. The block in c-myc transcription was beyond exon 1, and regulation of exon 1 transcription by 1,25-(OH)2D3 was not detected. Furthermore, we demonstrated that expression of markers for HL-60 cell differentiation was more rapidly induced by 25 nM 12-O-tetradecanoylphorbol-13-acetate than 50 nM 1,25-(OH)2D3, suggesting that direct activation of PKC by phorbol esters may make processes required for 1,25-(OH)2D3 induction of differentiation unnecessary. In summary, these data suggest that a primary effect of 1,25-(OH)2D3 on HL-60 cells is to regulate PKC levels, and regulation of c-myc transcription by 1,25-(OH)2D3 is a result of this action.
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PMID:1,25-Dihydroxyvitamin D3 regulation of c-myc protooncogene transcription. Possible involvement of protein kinase C. 268 61

The activation of the protein kinase C (PKC) pathway plays an integral part in the proliferation of many cell types including lymphocytes. We report that the PKC inhibitor H-7 caused inhibition of three commonly studied blastogenic responses (Con A, LPS, and MLR) with the strongest suppression being detected in the MLR. In contrast, HA1004, a potent inhibitor of cyclic nucleotide-dependent protein kinases, did not alter the blastogenic response but occasionally caused augmentation. The phenothiazine compounds studied inhibited the Con A and, to a lesser extent, the LPS responses. One of the compounds, promethazine-HCl, was effective in vivo in inhibiting splenomegaly resulting from the induction of graft vs. host disease. Our studies support the involvement of PKC in lymphoid blastogenesis. They also suggest that agents that can inhibit PKC activity may be useful in inducing immunosuppression in vivo.
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PMID:The effects of protein kinase inhibitors on lymphocyte blastogenesis and GVHD-induced splenomegaly. 276 Apr 15

In human platelets, phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), induce morphological changes, including pseudopod formation and the swelling and fusion of intracellular granule membranes with those of the surface-connected canalicular system, effects which have been attributed to activation of protein kinase C. However, a novel intracellular histamine antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]-ethanamine. HCl (DPPE), previously has been shown to block PMA-induced aggregation independently of protein kinase C interaction, an effect reversible in permeabilized platelets by the addition of histamine. We now demonstrate that DPPE inhibits, in a concentration-dependent manner, the effects of PMA on human platelet ultrastructure. In permeabilized platelets, histamine reverses this inhibition, although it alone induces minimal effects on morphology. The results support a role for this amine to promote the labilization of platelet granules and pseudopod formation induced by PMA, presumably by acting in concert with additional PMA-activated pathways.
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PMID:A role for intracellular histamine in ultrastructural changes induced in platelets by phorbol esters. 278 81

Several synthetic peptides reproducing fragments of protamines have been used as model substrates for Ca2+/phospholipid-dependent protein kinase C, tested both in the absence of any effector (basal conditions) and upon activation by either Ca2+ and phosphatidylserine (or diacylglycerol) or limited proteolysis. Only the peptide Arg4-Tyr-Gly-Ser-Arg6-Tyr [Ga(52-65)] shares the unique property of protamines of being readily phosphorylated even under basal conditions. Optimal activity in the absence of effectors is observed with Tris/HCl buffer pH 7.5; Pipes and Hepes are less effective at pH 7.5, and at pH 6.5 basal phosphorylation is reduced. Under the best conditions for basal phosphorylation of Ga(52-65), its derivative with ornithine replaced for arginine and those corresponding to its C-terminal fragments Gly-Ser-Arg6-Tyr [Ga(57-65)] and Gly-Ser-Arg3 [Ga(57-61)], as well as the peptides Pro-Arg5-Ser2-Arg-Pro-Val-Arg [Th(1-12)], Arg4-Tyr-Arg2-Ser-Thr-Val-Ala [Th(13-23)] and Arg2-Leu-Ser2-Leu-Arg-Ala are not significantly affected though all of them, like histones, are more or less readily phosphorylated upon activation of protein kinase C by Ca2+/phosphatidylserine. The peptide Ser2-Arg-Pro-Val-Arg [Th(7-12)] however, corresponding to the C-terminal part of Th(1-12), is not phosphorylated even in the presence of activators. Limited proteolysis can roughly mimic the Ca2+/phosphatidylserine effect inducing however different extents of activation depending on the nature of the peptide substrates. Our results support the following two conclusions. Basal phosphorylation by protein kinase C in the absence of any effector requires peptide substrates whose target residue(s) are included between two extended arginyl blocks and is also dependent on pH and nature of the buffer. Peptides having extended clusters of either arginyl or ornithyl residues on the C-terminal side of serine are also readily phosphorylated, but they need activation of protein kinase by either Ca2+/phosphatidylserine or limited proteolysis. The same is true of peptides having basic residues only on the N-terminal side, or even on both sides but in limited number.
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PMID:Ca2+ phospholipid-dependent and independent phosphorylation of synthetic peptide substrates by protein kinase C. 383 Jan 67

The sodium-dependent glucose transporter SGLT1 is expressed on the apical plasma membrane of fully differentiated enterocytes. Recently, we have found that the cotransport function appears gradually during the process of differentiation of the human intestinal epithelial cell clone HT-29-D4. However, the SGLT1 protein was detected in both undifferentiated and differentiated HT-29-D4 cells suggesting that sodium-glucose cotransport was dependent on post-translational events controlling the efficient targeting of the protein in the plasma membrane. In the present study, we have analyzed the molecular mechanisms controlling the functional expression of the SGLT1 protein during the course of HT-29-D4 differentiation. We show that the appearance of the cotransport function in the apical membrane is blocked by 1-5-isoquinolinesulfonyl)-2-methylpiperazine-HCl (H-7), a potent inhibitor of protein kinase C activity. Moreover, H-7 treatment was associated with an inability of HT-29-D4 cells to organize into a polarized monolayer of differentiated cells. Reciprocally, short term treatment (15 min) of undifferentiated cells by 0.1 microM phorbol myristyl acetate resulted in the appearance of the cotransport function. In contrast, inhibition of cAMP and cGMP-dependent protein kinases by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide-HCl did not prevent the development of sodium-glucose cotransport during the differentiation of HT-29-D4 cells. In addition, stimulation of cAMP-dependent protein kinases by 8-Cl-cAMP did not induce the cotransport function in undifferentiated HT-29-D4 cells. By using immunogold labeling at the electron microscopy level, we demonstrated that phorbol myristyl acetate induced the redistribution of SGLT1 protein from intracellular sites to the plasma membrane. In conclusion, our data show that the appearance of a functional sodium-glucose cotransporter in HT-29-D4 cells is controlled, at least in part, by intracellular pathways regulated by the activity of protein kinase C.
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PMID:The development of Na(+)-dependent glucose transport during differentiation of an intestinal epithelial cell clone is regulated by protein kinase C. 775 99

cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.
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PMID:Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel. 784 Jan 47

Previous studies have shown that fibronectin (Fn) enhances phagocytosis and killing of antibody-coated bacteria by neutrophils and macrophages. In an attempt to understand the mechanism of this enhancement, we have investigated the effects of Fn on phagocytosis-related actin organization as well as respiratory burst activity in neutrophils, monocytes and culture-derived macrophages. Employing an NBD-phallacidin flow cytometric analysis of filamentous actin formation, we found that Fn promotes rapid actin polymerization within 30 seconds in neutrophils, monocytes, and macrophages, but not lymphocytes. Enhancement of actin polymerization by Fn was concentration-dependent and mediated by a pertussis toxin- but not cholera toxin-sensitive G protein. Inhibition of protein kinase C by sphingosine (20 microM), calcium influx by verapamil (0.1 mM), or intracellular calcium mobilization by 8-(N,N-diethyl-amino) octyl-3,4,5-trimethoxybenzoate HCl (TMB-8; 0.1 mM) did not block Fn-enhanced actin polymerization in phagocytes. Incubation of neutrophils and macrophages on microtiter plates precoated with Fn suppressed superoxide (O2-) production induced by IgG- and IgA- opsonized group B streptococci. In contrast, Fn significantly enhanced IgA- and IgG-mediated O2- production by freshly isolated monocytes. These data suggest that Fn enhances phagocytosis, presumably through G protein-coupled cytoskeleton reorganization and augments O2- production by circulating monocytes. In contrast, it appears to suppress O2- production by the active phagocytic cells, neutrophils and macrophages. This may result in enhanced phagocytosis and intracellular killing of microorganisms without damaging interstitial tissues.
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PMID:Effects of fibronectin on actin organization and respiratory burst activity in neutrophils, monocytes, and macrophages. 810 71

Recently, it was reported that muscarinic-type cholinergic receptors coupled to the phosphoinositide messenger system are present in the rabbit inner medullary collecting duct and Madin-Darby canine kidney (MDCK) cells. The receptor density in MDCK cells is 50 times more than that in inner medullary collecting duct cells. To examine if muscarinic receptor activation influences Na-K-ATPase, the effects of a cholinergic agonist, carbachol, on Na-K-ATPase activity in MDCK cells were measured. Carbachol inhibited Na-K-ATPase activity in a time- and concentration-dependent manner. A maximum of approximately 80% of the enzyme activity was inhibited in 160 min with an EC50 of 5 microM carbachol. The inhibition of Na-K-ATPase activity was reversible; up to 80% of the enzyme activity was recovered within 4 h after carbachol was removed. The inhibitory effect of carbachol was blocked by a muscarinic antagonist atropine and by inhibitors of protein kinase C (PKC), 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine HCl, and N-(2-(methylamino)ethyl)-5-isoquinoline sulfonamide HCl. Direct activators of PKC, phorbol 12-myristate 13-acetate, N(n-heptyl)-5-chloro-1-naphthalene sulfonamide, and phosphatidyl serine, also inhibited Na-K-ATPase activity in MDCK cells, and their effect was also blocked by PKC inhibitors. These results indicate that cholinergic agonists inhibit Na-K-ATPase activity in MDCK cells by the activation of PKC. It is concluded that the inhibition of Na-K-ATPase by PKC may, in part, be responsible for the natriuretic action of cholinergic agonists, which have been shown to stimulate phosphoinositide hydrolysis in renal collecting duct cells.
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PMID:Cholinergic inhibition of Na-K-ATPase via activation of protein kinase C in Madin-Darby canine kidney cells. 840 83


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