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)

We studied the activation and inactivation of recombinant guanylyl cyclase (GC) C stably expressed in human kidney 293 cells. Activation of GC-C by heat-stable enterotoxin (STa), Cd2+, hemin, or the detergent Triton X-100 was followed by a rapid inactivation of the enzyme. Adenine nucleotides were found to prevent the inactivation process in native membranes, as well as following enzyme solubilization and immunopurification. Inactivation of GC-C was not associated with dephosphorylation. However, the phosphorylation of GC-C was promoted by phorbol esters, known activators of protein kinase C. The activation of purified GC-C by STa required the presence of a nonspecific factor as exemplified by bovine serum albumin. When immunopurified GC-C, stabilized by ATP and bovine serum albumin, was analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, proteins with almost twice the molecular mass (220 and 245 kDa) of the monomer were observed. The mobility of these high M(r) GC-C forms was reduced by STa, suggesting that STa induces a conformation change in a preexisting GC-C dimer. These results indicate that ATP interacts directly with GC-C, stabilizing its active conformation and that the activation of GC-C may occur in the absence of other specific regulatory factors.
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PMID:Heat-stable enterotoxin activation of immunopurified guanylyl cyclase C. Modulation by adenine nucleotides. 810 20

The role of protein kinase C (PKC) in mediating up-regulation of macrophage 1 adhesion protein (Mac-1) and adhesion of neutrophils in response to physiological agonists is not clear. Previous studies have relied on use of phorbol esters to activate PKC directly or on results obtained with non-selective inhibitors of protein kinases. 3-[8-(Aminomethyl)-6,7,8,9-tetrahydropyridol[1,2-a]-indol-10-yl]-4 -(1- methyl-3-indolyl)-1H-pyrrole-2,5-dione hydrochloride (Ro 31-8425) is a potent and highly selective inhibitor of PKC (Bit et al. J. Med. Chem. 1993. 36: 21). In these studies Ro 31-8425 has been used to define, more definitively, the role of PKC in mediating complement fragment C5a (C5a)-stimulated up-regulation of Mac-1 and adhesion of neutrophils to endothelial cells and to bovine serum albumin (BSA)-coated plastic. Phorbol 12, 13 dibutyrate (PBu2) increased surface expression of Mac-1 and stimulated adhesion of neutrophils to endothelial cells and to BSA-coated plastic. This confirms previous reports that activation of PKC can stimulate these responses. The PKC inhibitor, Ro 31-8425, inhibited the PBu2-stimulated responses, which confirms that Ro 31-8425 was effective in inhibiting PKC in these neutrophils. A more physiological agonist, C5a, also increased surface expression of Mac-1 and adhesion of neutrophils to endothelial cells and BSA-coated plastic. However, the responses to C5a were unaffected by Ro 31-8425. These results suggest that, although activation of PKC can promote up-regulation of Mac-1 and adhesion of neutrophils, this does not appear to be the physiological pathway. A non-selective protein kinase inhibitor, staurosporine, inhibited both PBu2 and C5a-stimulated adhesion. This suggests that a protein kinase other than PKC, possibly a tyrosine protein kinase, is likely to be involved in mediating C5a-stimulated Mac-1 up-regulation and adhesion. These results emphasise the need for caution in interpreting experiments and assuming a role for PKC. Use of a potent and selective inhibitor of PKC, Ro 31-8425, provides more definitive information.
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PMID:Effect of a selective protein kinase C inhibitor, Ro 31-8425, on Mac-1 expression and adhesion of human neutrophils. 812 32

Serum albumin is the most abundant protein synthesized by liver cells, and its production is a reliable indicator of the differentiated state of hepatocytes. We have recently shown that fetal rat hepatocytes cultured under proliferative conditions, i.e., in the presence of EGF, responded to glucagon and noradrenaline increasing albumin protein and mRNA levels (de Juan et al., 1992. J. Cell. Physiol., 152:95-101). This effect was mimicked by agents that increase cyclic AMP levels. In this report, we show that in regenerating liver, noradrenaline modulation of albumin expression seems to be different. Hepatocytes from hepatectomized rats were cultured at low cell density and in the presence of EGF. Under these conditions, noradrenaline, which acted synergistically with EGF increasing DNA synthesis (de Juan et al., 1992. Exp. Cell. Res., 202:495-500), produced a decrease in albumin mRNA levels. This effect was dose-dependent, being maximum at 1 microM noradrenaline. Noradrenergic effect seemed to be mediated by alpha 1-receptors, because it was blocked by prazosin, but not by propranolol. Other Ca(2+)-increasing agents, as vasopressin, angiotensin II, or ATP, did not produce any effect. However, albumin mRNA levels decreased when the cells were incubated in the presence of tetradecanoyl phorbol-13-acetate (TPA). In addition, noradrenergic modulation of albumin expression was blocked by staurosporine, a protein kinase inhibitor with relative specificity for protein kinase C. Thus we can conclude that the role of noradrenaline on the regulation of liver growth and differentiation changes from fetal to adult life. This change is probably due to its action on different receptors: beta-receptors in fetal hepatocytes and alpha 1-receptors in the adult liver.
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PMID:Noradrenergic modulation of albumin expression in growth-stimulated adult rat hepatocytes in primary culture. 812 74

When N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl-phosphatidic acid (C6-NBD-PA) is inserted into the plasma membrane of fibroblasts, it is metabolized by the cells to C6-NBD-diacylglycerol (DG), -triacylglycerol, -phosphatidylcholine (PC), and -phosphatidylethanolamine (PE) (Pagano, R. E., Longmuir, K. J., and Martin, O. C. (1983) J. Biol. Chem. 258, 2034-2040). In Madin-Darby canine kidney (MDCK) cells incubated at 10 degrees C with C6-NBD-PA, up to 70% of the newly synthesized C6-NBD-PC but no C6-NBD-PE could be depleted from the basolateral cell surface by the addition of bovine serum albumin to the medium. Preincubation of the cells with [3H]choline for 2 h at 37 degrees C prior to C6-NBD-PA addition at 10 degrees C labeled non-depletable C6-NBD-PC with a specific activity of > 10 times that of the depletable C6-NBD-PC on the basolateral cell surface, indicating that the latter had not been synthesized by the CDP-choline pathway. C6-NBD-DG could substitute for C6-NBD-PA as substrate for both intracellular and surface C6-NBD-PC synthesis. In addition, C6-NBD-PC synthesis on the cell surface was independent of the location of the C6-NBD-chain on the 1- or 2-position, indicating that the reaction occurred by transfer of phosphorylcholine. Using C6-NBD-ceramide, C6-NBD-sphingomyelin (SM) synthesis also was discovered on the basolateral but not on the apical cell surface. The conversion of PC plus ceramide to DG and SM on the basolateral MDCK cell surface suggests that the synthesis of C6-NBD-PC on this surface occurred via the reverse reaction of a SM synthase. Indeed, the surface C6-NBD-PC synthesis was reduced to 40-50% by addition of C6-NBD-ceramide or hydrolysis of cell surface SM by exogenous neutral sphingomyelinase. Since DG activates protein kinase C and ceramide indirectly inhibits this kinase but activates other kinase(s) and phosphatase(s), the phosphocholine transferase at the cell surface may have a regulatory role in signal transduction.
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PMID:Conversion of diacylglycerol to phosphatidylcholine on the basolateral surface of epithelial (Madin-Darby canine kidney) cells. Evidence for the reverse action of a sphingomyelin synthase. 829 25

Although active transport of sodium plays an important role in the resolution of pulmonary edema, the biochemical regulation of this process is still under investigation. The purpose of this study was to evaluate the activity of protein kinase C during the process of lung liquid clearance. Alveolar flooding was induced by instilling 5% bovine serum albumin solution, saline, or heterologous serum in the air spaces of rats. The activity of protein kinase C was measured in both the instilled and control lungs at 10 min and 1 and 4 h after fluid instillation. Four hours after instillation of 5% bovine serum albumin, the ratio of protein kinase C activity in the instilled lung compared with the control lung was 2.2 +/- 0.3. Similar results were obtained following instillation with heterologous serum or saline. Since we measured a clearance rate of 0.8 ml/h in anesthetized rats, we can postulate that the activation of protein kinase C occurred when > 40% of the liquid had been cleared from the lung. This increased activity of protein kinase C was not due to an increase in kinase activity in the inflammatory cells or an increase in enzyme quantity but due to a decrease of protein kinase C inhibitory activity in the lung. These results suggest that protein kinase C second messenger system may play a regulatory role in lung liquid clearance.
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PMID:Protein kinase C activity during the process of lung liquid clearance. 833 83

Evidence is presented that inducing P815 murine mastocytoma cells to grow with serum activates a Ca(2+)-stimulated phospholipase A2 and the rapid release of arachidonic acid by the cells. Slower growth was also maintained by arachidonic acid or its immediate precursors or by diacylglycerols when bovine serum albumin replaced the serum. Together, arachidonic acid and 1-oleoyl-2-acetylglycerol stimulated growth at the same rate as 10% serum consistent with a role for both arachidonic acid and protein kinase C in the response to serum. Arresting cell growth with N6,O2'-dibutyryladenosine 3',5'-cyclic phosphate and theophylline inhibited the release of arachidonic acid in response to serum, suggesting that cyclic AMP prevents phospholipase activation as one of its pleiotypic effects on growth. Attempts to demonstrate metabolism of [3H]arachidonic acid to eicosanoids in serum-treated P815 cells by high-performance liquid chromatography or thin layer chromatography were unsuccessful, with the major products being phospholipids and triacylglycerol. Incubating digitonin-permeabilized P815 cells with [gamma-32P]ATP and arachidonic acid rapidly increased the phosphorylation of some proteins in the cells, especially the M(r) 135,000 and M(r) 44,000 proteins which were considerably more phosphorylated than the rest. Phosphorylation of these proteins was not prevented by several inhibitors of protein kinase C, nor was it increased by diacylglycerols or phorbol ester, suggesting that arachidonic acid activates a growth-related protein kinase other than protein kinase C in P815 cells. The possibility that some polyunsaturated fatty acids may promote tumor cell growth by stimulating protein phosphorylation is considered.
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PMID:Arachidonic acid, a growth signal in murine P815 mastocytoma cells. 839 26

It is believed that inotropic agents exert their effects in cardiac muscle via a modulation of Ca2+ cycling; however, the involvement of phospholipase activation and the biochemical pathways participating in inotropic responsiveness remain unclear. The aim of the current study was to determine whether arachidonic acid and/or eicosanoids participate in inotropic responses by modulating Ca2+ cycling in cardiac myocytes. Experiments were performed with populations of freshly isolated, fura-2-loaded adult rat ventricular myocytes. Arachidonic acid stimulated a transient increase in cytosolic free Ca2+, which was still present after addition of EGTA but was significantly reduced by pretreatment with caffeine. Addition of arachidonic acid to either electrically stimulated or quiescent myocytes enhanced the amplitude of the ATP-induced Ca2+ transient. This effect was still observed in the presence of inhibitors of cyclooxygenase, lipoxygenase, and epoxygenase pathways but was significantly diminished after pretreatment with inhibitors of protein kinase C. In contrast, arachidonic acid attenuated the amplitude of electrically induced Ca2+ transients. This effect was mimicked by eicosatetraynoic acid and by the K+ channel agonist pinacidil. The inhibitory effect of eicosatetraynoic acid and arachidonic acid was reversed by addition of fatty acid-free bovine serum albumin. Together, these results suggest that arachidonic acid may play a physiological role in cardiac muscle excitation-contraction coupling as a modulator of sarcolemmal ion channels and/or Ca2+ release from the sarcoplasmic reticulum.
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PMID:Modulation of Ca2+ cycling in cardiac myocytes by arachidonic acid. 841 90

We previously reported that both the nuclear import rate of large karyophilic gold particles and the functional size of the pores are significantly greater in simian virus 40-transformed fibroblasts (the SV-T2 cell line) than in nontransformed BALB/c 3T3 cells. In this study, we found that cytosolic fractions obtained from SV-T2 cultures can increase nuclear transport capacity (both import rate and pore size) when microinjected into BALB/c 3T3 cells. The transport-enhancing function of the extracts can be abolished by the protein kinase inhibitors staurosporine and K252a as well as 5'-p-fluorosulfonylbenzoyladenosine and protein phosphatase 2A, which, although less specific, also interfere with kinase activity. Increases in transport capacity of the same magnitude as that produced by the SV-T2 extracts were obtained by microinjecting protein kinase A or C or recombinant mitogen-activated protein kinase. These data provide further support for the interpretation that the enhancer is a protein kinase. From experiments performed with specific kinase inhibitor peptides, it appears likely that protein kinase C is the active factor in the SV-T2 cytosolic fractions; however, this will require further verification. It was also determined, by using gold particles coated with bovine serum albumin conjugated to synthetic nuclear localization signal peptides that lacked phosphorylation sites, that the enhancer affects the transport machinery rather than the activity of the nuclear localization signals.
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PMID:Stimulation of nuclear import by simian virus 40-transformed cell extracts is dependent on protein kinase activity. 852 71

Curcumin [diferuloylmethane; 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], a major bioactive secondary metabolite found in the rhizomes of turmeric (Curcuma longa), is an inhibitor of Ca(2+)- and phospholipid-dependent protein kinase C (PKC) and of the catalytic subunit (cAK) of cyclic AMP-dependent protein kinase (IC50 values 15 and 4.8 microM, respectively). Curcumin inhibits plant Ca(2+)-dependent protein kinase (CDPK) (IC50 41 microM), but does not inhibit myosin light chain kinase or a high affinity 3',5'-cyclic AMP-binding phosphatase. Curcumin inhibits cAK, PKC and CDPK in a fashion that is competitive with respect to both ATP and the synthetic peptide substrate employed. The IC50 values for inhibition of cAK by curcumin are very similar when measured with kemptide (LRRASLG) (in the presence or absence of ovalbumin) or with casein or histone III-S as substrates. However, the presence of bovine serum albumin (0.8 mg ml-1) largely overcomes inhibition of cAK by curcumin.
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PMID:Inhibition of cyclic AMP-dependent protein kinase by curcumin. 876 15

Neurogranin (Ng) is a prominent protein kinase C (PKC) substrate which binds calmodulin (CaM) in the absence of Ca2+. Rat brain Ng contains four cysteine residues that were readily oxidized by nitric oxide (NO) donors, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEANO) and sodium nitroprusside, and by oxidants, H2O2 and o-iodosobenzoic acid. NO oxidation of Ng resulted in a conformational change detectable by increased electrophoretic mobility upon SDS-polyacrylamide gel electrophoresis. The NO-mediated mobility shift was reversed by treatment with dithiothreitol and was blocked by modification of Ng sulfhydryl groups with 4-vinylpyridine. Both the nonphosphorylated and PKC-phosphorylated Ng were susceptible to NO oxidation. Modification of Ng by DEANO was blocked by CaM in the absence of Ca2+; while in the presence of Ca2+, CaM did not protect Ng from oxidation by DEANO. CaM also failed to protect DEANO-mediated oxidation of PKC-phosphorylated Ng with or without Ca2+. Oxidation of Ng by the various oxidants apparently resulted in the formation of intramolecular disulfide bond(s) as judged by a reduction of apparent Mr on SDS-polyacrylamide gel electrophoresis; this oxidized form, unlike the reduced form, did not bind to CaM-affinity column. The oxidized Ng was also a poorer substrate for PKC; both the reduced and oxidized forms had similar Km values, but the Vmax of the oxidized form was about one-fourth of the reduced one. When comparing the rate of DEANO-mediated nitrosation of Ng with other sulfhydryl-containing compounds, it became evident that Ng ranked as one of the best NO acceptors among those tested, including serum albumin, glutathione, and dithiothreitol. Ng present in the rat brain synaptosomal preparations was also oxidized by DEANO in a dose-dependent manner when analyzed by immunoblot with a polyclonal antibody against this protein. These results suggest that Ng is a likely target of NO and other oxidants and that oxidation/reduction may serve as a mechanism for controlling both the PKC phosphorylation and the CaM-binding affinity of this protein.
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PMID:Nitric oxide modification of rat brain neurogranin affects its phosphorylation by protein kinase C and affinity for calmodulin. 879 3


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