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)

Mitoxantrone, a new anthraquinone, showed inhibitory an effect on protein kinase C (PKC) activity. Its IC50 value was 4.4 micrograms/ml (8.5 microM), which is much lower than those of the well-known anthracyclines daunorubicin and doxorubicin, the IC50 values of which are more than 100 micrograms/ml (> 170 microM). Kinetic studies demonstrated that mitoxantrone inhibited PKC in a competitive manner with respect to histone H1, and its Ki value was 6.3 microM (Ki values of daunorubicin and doxorubicin were 0.89 and 0.15 mM, respectively), and in a non-competitive manner with respect to phosphatidylserine and ATP. Inhibition of phosphorylation by mitoxantrone was observed with various substrates including S6 peptide, myelin basic protein and its peptide substrate derived from the amino-terminal region. Their IC50 values were 0.49 microgram/ml (0.95 microM), 1.8 micrograms/ml (3.5 microM), and 0.82 microgram/ml (1.6 microM), respectively. Mitoxantrone did not markedly inhibit the activity of cyclic AMP-dependent protein kinase, casein kinase I or casein kinase II, at concentrations of less than 10 micrograms/ml. On the other hand, brief exposure (5 min) of HL60 cells to mitoxantrone caused the inhibition of cell growth with an IC50 value of 52 ng/ml (0.1 microM). In HL60 cells, most of the PKC activity (about 90%) was detected in the cytosolic fraction. When HL60 cells exposed to 10 micrograms/ml mitoxantrone for 5 min were observed with fluorescence microscopy, the fluorescence elicited from mitoxantrone was detected in the extranuclear area. These results indicated that mitoxantrone is a potent inhibitor of PKC, and this inhibition may be one of the mechanisms of antitumor activity of mitoxantrone.
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PMID:Inhibitory effect of mitoxantrone on activity of protein kinase C and growth of HL60 cells. 129 84

We have examined the presence of protein kinase C in oocytes of Chaetopterus pergamentaceus and its role in the initiation of germinal vesicle breakdown (GVBD). First, we demonstrated that the oocytes contain a phospholipid- and calcium-dependent protein kinase, protein kinase C (PKC). Since PKC is the primary intracellular receptor for phorbol esters, we tested the ability of phorbol 12,13-dibutyrate (PDBu) to induce GVBD and compared several critical events and processes involved in GVBD induced by PDBu to those induced normally (by seawater). Seawater and 100-200 nM PDBu induced chromosome condensation, spindle formation, and spindle migration over a similar time course. Both treatments induced similar alterations in the SDS-PAGE pattern of newly synthesized proteins. The synthesis of polypeptides of approximately 46 and 54 kDa increased specifically. Both treatments increased oocyte protein phosphorylation, especially of proteins of 22, 32, 46, 55, 64, and 84 kDa. Both treatments resulted in the activation of an M-phase-specific histone H1 kinase activity, which demonstrates the appearance of maturation-promoting factor. Staurosporine, a potent protein kinase C inhibitor, blocked GVBD and the activation of M-phase-specific H1 kinase, whereas HA1004, which preferentially antagonizes protein kinase A, had no effect. The results of this study demonstrate that protein kinase C can activate a wide spectrum of essential biochemical and morphological processes involved in GVBD. Further, these studies suggest that protein kinase C elicits GVBD by activating maturation-promoting factor and support the hypothesis that protein kinase C plays an essential role in oocyte maturation in this species.
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PMID:Regulation of M-phase progression in Chaetopterus oocytes by protein kinase C. 130 10

Twelve analogs of 1,2-di-O-octanoylglycerol modified at C-3 and three quaternary N-alkyl-ammonium derivatives of glycerol were synthesized. The compounds were tested in vitro as potential modulators of the calcium activated, phospholipid dependent protein kinase C (PKC) and diacylglycerol (DAG) kinase activities in order to understand the molecular interactions of these enzymes with their natural activators, inhibitors, or substrates. PKC activity was assayed by measuring histone H1 phosphorylation, and the compounds synthesized were tested either in the presence (inhibitors) or in the absence (activators) of 1,2-di-O-octanoylglycerol analogs with the phosphatidylserine/Ca2+ mixture. DAG kinase activity was measured by the incorporation of phosphate into 1,2-di-O-oleoyl-sn-glycerol in the presence of the various analogs synthesized. In regard to PKC activity, the assays revealed that 1,2-di-O-octanoylglycerol analogs are inactive when modified at C-3 with groups which do not permit hydrogen bonding. Under our conditions, di-O-octanoylthioglycerol, which has been reported as inactive, was able to activate PKC in the presence of phosphatidylserine. It has been shown to give a synergistic activation with diacylglycerol and had no affinity for the phorbol ester receptor binding site, suggesting that O-octanoylthioglycerol interacts with the enzyme at a different site from the phorbol ester receptor binding site. PKC and DAG kinase activities are inhibited by N-alkyl-ammonium compounds (IC50 24 microM) only when either two 8-carbon alkyl or acyl chains are present at the 1- and 2-positions of the glycerol backbone.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Derivatives of di-O-octanoylglycerol and mono-O-octylglycerol as modulators of protein kinase C and diacylglycerol kinase activities. 132 95

Rat mesangial cells contain both calcium-dependent protein kinase C (PKC) activity, which phosphorylates histone H1 and endogenous proteins, and calcium-independent, phospholipid-dependent PKC activity, which phosphorylates only endogenous proteins. The calcium-dependent PKC was identified as PKC alpha by immunoblot analysis and hydroxyapatite chromatography (HPLC). The calcium-insensitive, phospholipid-dependent isoform was identified as PKC delta using similar techniques. The inhibition of these two PKC isoforms by the protein kinase inhibitor H7 [1-(iso-quinolinyl sulphonyl)-2-methyl piperazine] was examined using both histone H1 and endogenous proteins as substrates. Phosphorylations catalyzed by the calcium-dependent PKC isoform alpha were almost 90% inhibited when histone H1 was used, and only 55% when endogenous proteins were the substrate. In contrast, the phosphorylation of endogenous proteins catalysed by the calcium-insensitive, phospholipid-dependent PKC delta was not significantly affected by the inhibitor.
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PMID:Immunological identification of protein kinase C-alpha and protein kinase C-delta in cultured rat mesangial cells: differential sensitivity of the two isoforms towards the protein kinase inhibitor H7. 141 92

Phorbol-12,13-dibutyrate and 1,2-dioctanoylglycerol, activators of protein kinase C (PKC) that stimulate DNA synthesis in serum-deprived Swiss 3T3 fibroblasts, induce histone H1 kinase activity associated with anti-cdc2 immunoprecipitates after a lag period of 15h, a time point close to G1/S boundary of the cell cycle in these cells. Downregulation of PKC does not affect the basal cdc2 kinase activity, but potently inhibits both phorbol dibutyrate- and dioctanoylglycerol-induced cdc2 kinase activation. Phorbol dibutyrate induces a dramatic increase in the p34cdc2 protein level as well as the appearance of p35-p36 forms of cdc2 on Western blot. In PKC-downregulated cells, the p34 form of cdc2 remains elevated but p35-p36 forms do not appear upon phorbol dibutyrate stimulation. These results demonstrate that PKC activation leads to cdc2 kinase activation in mitogenically responsive Swiss 3T3 cells, and strongly suggest that both expression of p34cdc2 protein and its posttranslational modification(s) are involved in this process. Western blot analysis of PKC isozymes suggests that either PKC alpha, PKC delta or PKC epsilon may be involved in p34cdc2 kinase activation and mitogenesis.
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PMID:Activators of protein kinase C induce p34cdc2 histone H1 kinase stimulation in Swiss 3T3 fibroblasts. 144 45

In the conventional approach protein kinase activity and phorbol ester binding associated with protein kinase C (PKC) are measured by initially incubating samples in either test tubes or multiwell plates, followed by filtration of the terminated reaction mixture using either a manifold filtration device or a cell harvester. Here we report a method in which both the incubations and filtrations necessary for the determination of either protein kinase activity or phorbol ester binding are carried out in the same multiwell plate with fitted filtration discs made of polyvinylidene difluoride (Durapore membrane). Due to the very low binding of protein to these filters, there is no interference caused by these filters during the incubation period of the assays. The drawback with these filters compared to commonly used cellulose acetate membrane filters is that they retain less of the phosphate acceptor substrate histone H1 (only 15%) if filtered and washed with standard 5% trichloroacetic acid. However, this can be overcome by increasing the trichloroacetic acid concentration to 25% during filtration. For phorbol ester binding determinations, the samples are incubated with [3H]phorbol 12,13-dibutyrate in the microwells, the ligand bound PKC is adsorbed onto DEAE-Sephadex beads, and the beads then are filtered and washed in the same microwells. Furthermore, this multiwell filtration approach can also be adopted to previously described cytosolic phorbol ester receptor assays, which have the broader conditions for optimal binding to receptors. Durapore membrane filters are found to work well for punching into scintillation vials and there is complete recovery of the radioactivity retained with the filters. In the protein kinase assay the background radioactivity is very low (< 200 cpm) and in the phorbol ester binding assay the nonspecific binding is less than 1%. Thus, these low background values result in at least a fourfold increase in sensitivity for these assays. Since the incubations and filtrations are carried out in the same well without any transfer of the sample, the coefficient of variation in multiple determinations is found to be low. Furthermore, this method is rapid and more convenient for analyzing a larger number of samples than conventional methods which use test tubes, and it is less expensive to set up compared to the automated methods that use a cell harvester.
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PMID:Rapid filtration assays for protein kinase C activity and phorbol ester binding using multiwell plates with fitted filtration discs. 145 38

Protein phosphorylation catalysed by rat brain protein kinase C (PKC) has been studied in nuclei isolated from normal and regenerating rat liver. Histone H1 and a 40,000 molecular weight protein were hyperphosphorylated at all the explored regeneration times, ranging from 3 to 22 h after partial hepatectomy. Phosphorylation of the two substrates was totally dependent on calcium and lipids and was abolished by low concentration of staurosporine. The observed early change of phosphate content of histone H1 and of the 40,000 molecular weight protein on the time scale of liver regeneration suggests that PKC might be involved in the initial nuclear events leading to cell proliferation.
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PMID:Increased phosphorylation of nuclear substrates for rat brain protein kinase C in regenerating rat liver nuclei. 151 Aug 79

Two forms of protein kinase C (PKC) activity in cytosol of cultured rat mesangial cells have been characterized in vitro by using histone H1 or endogenous proteins as substrates. Histones H1-phosphorylation was significantly increased only when calcium, phosphatidylserine (PS) and 1,2-diacylglycerol (DAG) or phorbol myristate acetate (PMA) were present together in the incubation medium. EGTA, a calcium chelator, completely inhibited this activity. Upon hydroxyapatite chromatography (HPLC), the PKC activity was eluted as a main peak at 150 mM potassium phosphate with a shoulder at 180 mM. Both peaks corresponded to the type III PKC from rat brain and were identified as PKC alpha isoform by immunoblot analysis. In contrast with what was observed using histone H1, the increased phosphorylation of endogenous proteins in the presence of a mixture of Ca2+/PS, plus either DAG or PMA, was only partly reduced by EGTA. Moreover, the level of the PKC activity detected in the presence of EGTA was comparable to the level of kinase activity, measured in the presence of PS alone or associated with DAG or PMA. This suggests that mesangial cells contain PKC activity which does not absolutely require calcium. Polyacrylamide gel electrophoresis revealed that patterns of phosphorylated mesangial cell proteins are different depending on whether calcium was added or not. In the presence of calcium, PKC strongly phosphorylated the proteins of 53,000 molecular weight, a doublet of 37,000-39,000, the 24,000 and the triplet of 17,000-20,000-22,000 molecular weight. The addition of EGTA to the assays suppressed completely the labelling of most proteins; only the 20,000 molecular weight protein remained strongly labelled, while the 39,000 molecular weight band was only faintly visible. The same patterns of phosphorylations were obtained after omission of calcium in the assays containing only PS and DAG (or PMA). So, the main substrates of calcium-dependent PKC are proteins of 53,000, 39,000, 37,000, 22,000, 24,000 and 17,000 molecular weight while the protein of 20,000 molecular weight appears to be the main substrate of calcium-independent PKC. The existence in mesangial cells of at least two forms of PKC, which phosphorylate specific endogenous proteins, emphasizes the complexity of the phospholipid-dependent regulatory cascade and raises the possibility that actions of different regulators may be transduced through distinct PKC isozymes.
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PMID:Heterogeneity of protein kinase C in cultured rat mesangial cells. 161 24

Comparison of protein kinase activity in normal and regenerating rat liver nuclei indicates that exogenous histone H1 is hyperphosphorylated in 22-h regenerating nuclei. The protein kinase involved is not sensitive to protein kinase A inhibitor, is inhibited by staurosporine and by an anti-PKC polyclonal antibody, utilizes only ATP, and also phosphorylates the C-terminal fragment of histone H1. These data suggest that protein kinase C is responsible for the observed effects, in agreement with the presence of this enzyme in normal and regenerating nuclei demonstrated by immunoblotting.
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PMID:Nuclear protein kinases in rat liver: evidence for increased histone H1 phosphorylating activity during liver regeneration. 164 25

Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity. Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Small-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone H1 phosphorylated by protein kinase C. With the exception of histone H1, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone H1, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone H1 as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity.
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PMID:Control of protein phosphatase 2A by simian virus 40 small-t antigen. 170 74


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