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 phosphorylation sites in the myristoylated alanine-rich C kinase substrate or MARCKS protein consist of four serines contained within a conserved, basic region of 25 amino acids, termed the phosphorylation site domain. A synthetic peptide comprising this domain was phosphorylated by both protein kinase C and its catalytic fragment with high affinity and apparent positive cooperativity. Tryptic phosphopeptides derived from the peptide appeared similar to phosphopeptides derived from the phosphorylated intact protein. The peptide was phosphorylated by cAMP- and cGMP-dependent protein kinases with markedly lower affinities. In peptides containing only one of the four serines, with the other three serines replaced by alanine, the affinities for protein kinase C ranged from 25 to 60 nM with Hill constants between 1.8 and 3.0. The potential pseudosubstrate peptide, in which all four serines were replaced by alanines, inhibited protein kinase C phosphorylation of histone or a peptide substrate with an IC50 of 100-200 nM with apparently non-competitive kinetics; it also inhibited the catalytic fragment of protein kinase C with a Ki of 20 nM, with kinetics of the mixed type. The peptide did not significantly inhibit the cAMP- and cGMP-dependent protein kinases. It inhibited Ca2+/calmodulin-dependent protein kinases I, II, and III by competing with the kinases for calmodulin. In addition, the peptide inhibited the Ca2+/calmodulin-independent activity of a proteolytic fragment of Ca2+/calmodulin protein kinase II, with an IC50 approximately 5 microM. Thus, the phosphorylation site domain peptide of the MARCKS protein is a high affinity substrate for protein kinase C in vitro; the cognate peptide containing no serines is a potent but not completely specific inhibitor of both protein kinase C and its catalytic fragment.
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PMID:Protein kinase C substrate and inhibitor characteristics of peptides derived from the myristoylated alanine-rich C kinase substrate (MARCKS) protein phosphorylation site domain. 165 Mar 59

Two main forms of protein kinase C (PKC) activity were found in rat hepatocytes using DEAE-cellulose chromatography: PKC 1 and PKC 2. Treatment of cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 15 min caused a marked loss of PKC 1 activity and only a small loss of PKC 2 activity. Hydroxyapatite column chromatography resolved PKC 1 into three distinct peaks 1a, 1b and 1c, and PKC 2 into four peaks 2a, 2b, 2c and 2d. Immunoblot analysis with isozyme-specific monoclonal antibodies identified peak 1a as PKC-beta and peak 1b as PKC-alpha; the other peaks of activity were not identified. Treatment with TPA provoked a loss of activity of peaks 1b (PKC-alpha) and 1c, whereas peak 1a (PKC-beta) activity was not affected. The peaks of activity corresponding to PCK 2 did not show any major change due to TPA treatment except peak 2d that decreased. The apparent disappearance of PKC histone-kinase activity induced by TPA was also observed using other substrates (protamine or vinculin). The TPA-induced decrease in activity occurs in a time-dependent and dose-dependent fashion. However, the time-courses, the extent of depletion and the potency order of phorbol esters in induction of an activity decrease in the two groups of isoforms exhibited substantial differences.
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PMID:Differences in phorbol ester-induced decrease of the activity of protein kinase C isozymes in rat hepatocytes. 165 25

Calcium/phosphatidylserine-dependent protein kinase C (PKC) is activated by phosphatidylinositol 4,5-bisphosphate (PIP2), as well as by diacylglycerol (DG) and phorbol esters. Here we report that PIP2, like DG, increases the affinity of PKC for Ca2+, and causes Ca(2+)-dependent translocation of the enzyme from the soluble to a particulate fraction (liposomes). Phosphatidylinositol 4-phosphate (PIP) also displaces phorbol ester from PKC and causes Ca(2+)-dependent translocation of the enzyme to liposomes, but is much less efficient than PIP2, and a much weaker activator, with a histone phosphorylation v(PIP)/v(PIP2) of approximately 0.15. Scatchard analysis indicates competitive inhibition between PIP and phorbol ester with Ki(PIP) = 0.26 mol% as compared with Ki(PIP2) = 0.043 mol%. No effect of phosphatidylinositol (PI) on phorbol ester binding to PKC, translocation of PKC, or activation of PKC was observed. These results suggest that both PIP and PIP2 can complex with PKC, but full activation of the enzyme takes place only when PIP is converted to PIP2. We suggest that an inositide interconversion shuttle has a role in the regulation of protein phosphorylation.
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PMID:Interaction of protein kinase C with phosphoinositides. 165 11

Hyperglycemia causes insulin-receptor kinase (IRK) resistance in fat cells. We characterized the mechanism of IRK inhibition and studied whether it is the consequence of a glucose-induced stimulation of protein kinase C (PKC). Fat cells were incubated for 1 or 12 h in culture medium containing either a low-(5-mM) or high- (25-mM) glucose concentration. IRK was isolated, insulin binding was determined, and autophosphorylation was studied in vitro with [gamma-32P]ATP or was determined by Western blotting with anti-phosphotyrosine antibodies. Substrate phosphorylation was investigated with the artificial substrate poly(Glu80-Tyr20). Partially purified insulin receptor from rat fat cells, which were cultured under high-glucose conditions for 1 or 12 h, showed no alteration of insulin binding but a reduced insulin effect on autophosphorylation (30 +/- 7% of control) and poly(Glu80-Tyr20) phosphorylation (55.5 +/- 9% of control). Lineweaver-Burk plots of the enzyme kinetics revealed, beside a reduced Vmax, and increased KM (from 30 microM to 80 microM) for ATP of IRK from high-glucose-treated cells. Because a similar inhibition pattern was earlier found for IRK from fat cells after acute phorbol ester stimulation, we investigated whether activation of PKC might be the cause of the reduced IRK activity. We isolated PKC from the cytosol and the membrane fraction of high- and low-glucose fat cells and determined the diacylglycerol- and phospholipid-stimulated PKC activity toward the substrate histone. There was no significant change of cytosolic PKC; however, membrane-associated PKC activity was increased in high-glucose-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Prevention by protein kinase C inhibitors of glucose-induced insulin-receptor tyrosine kinase resistance in rat fat cells. 165 68

The impaired Na(+)-K(+)-ATPase activity in peripheral nerve from diabetic rats is prevented by dietary myo-inositol (MI) supplementation in vivo and corrected by protein kinase C (PKC) agonists in vitro, suggesting that PKC may mediate the effects of nerve MI depletion on Na(+)-K(+)-ATPase activity. However, little is known about the effect of diabetes on PKC activity or peptide in rat peripheral nerve. Therefore, the effect of streptozocin-induced diabetes and dietary MI supplementation on the activity and distribution of PKC in rat sciatic nerve homogenates and cytosolic and particulate fractions was explored with histone phosphorylation assay and Western-blot analysis. PKC activity but not peptide was selectively decreased in the cytosolic fraction by streptozocin-induced diabetes, and this abnormality was partially corrected by dietary MI supplementation. These results suggest that altered MI metabolism may affect nerve PKC specific activity, and this alteration may play a role in reduced Na(+)-K(+)-ATPase activity and blunted regenerative response in diabetic nerve.
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PMID:Diminished specific activity of cytosolic protein kinase C in sciatic nerve of streptozocin-induced diabetic rats and its correction by dietary myo-inositol. 165 70

The actions of ethanol on kinase stimulated phosphorylation were examined using highly purified protein kinases and a variety of purified substrates. Ethanol (25-200 mM) failed to alter the phosphorylation of histone IIa and histone IIIs by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), respectively. Moreover, ethanol (25-200 mM) did not affect the phosphorylation of synapsin I by Ca(2+)-calmodulin-dependent protein kinase II (CAM kinase II). Finally, neither PKA nor PKC stimulated phosphorylation of the GABAA receptor (GABAA-R) was modulated by ethanol at any concentration of ethanol tested. These results suggest that ethanol, in pharmacological concentrations, has no direct actions on the ability of these kinases to catalyze the phosphorylation of specific substrate proteins. In particular, ethanol does not appear to directly influence GABAA-R phosphorylation by either PKA or PKC.
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PMID:Ethanol has no effect on cAMP-dependent protein kinase-, protein kinase C-, or Ca(2+)-calmodulin-dependent protein kinase II-stimulated phosphorylation of highly purified substrates in vitro. 166 14

It has been suggested that the maintenance of long-term potentiation (LTP) in the hippocampal mossy fiber (MF) synapse involves a presynaptic mechanism that does not require the activation of protein kinase C (PKC), since this enzyme appears to be absent in the MF presynaptic terminals. In the present study the authors evaluated this proposal by directly comparing the metabolic properties of hippocampal MF synaptosomes and a conventional P2B synaptosomal preparation prepared from the same hippocampal tissue. Protein kinase C-dependent histone phosphotranferase activity was found to be comparable in MF and P2B synaptosomes. Western blot analysis was performed using antisera prepared against four of the PKC isoforms, and the results demonstrate that the alpha, beta, and gamma PKC isoforms are present in relatively equivalent amounts in these two subcellular fractions. However, the cytosolic fraction derived from the hippocampal MF synaptosomes appeared to contain a greater amount of the PKC-epsilon isoform when compared to the P2B synaptosomal preparation. Four distinct endogenous substrates present in the MF synaptosomes are shown to be phosphorylated in response to PKC activation. A functional role for PKC in the hippocampal MF nerve endings seems to be indicated by the finding that 4 beta-phorbol 12,13-dibutyrate (PDBu) and 4 beta-phorbol 12,13-diacetate produce a dose-dependent potentiation of the K(+)-evoked release of endogenous glutamate and dynorphin B, while the inactive 4-alpha-phorbol was without effect. The PDBu-induced enhancement of transmitter release was blocked by the PKC inhibitor, staurosporine. In addition, PDBu significantly facilitated the rise in cytosolic free calcium that immediately followed depolarization of the MF synaptosomal membrane. It is concluded that hippocampal MF presynaptic terminals possess a variety of PKC isoforms and that their activation may have an important facilitory influence on MF synaptic transmission and plasticity.
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PMID:A presynaptic role for protein kinase C in hippocampal mossy fiber synaptic transmission. 168 79

Among various phosphate acceptor proteins and peptides so far tested, a synthetic peptide having the sequence surrounding Ser(8) of myelin basic protein, Gln-Lys-Arg-Pro-Ser(8)-Gln-Arg-Ser-Lys-Tyr-Leu, (MBP4-14), is the most specific and convenient substrate which can be used for selective assay of protein kinase C. This peptide is not phosphorylated by cyclic AMP-dependent protein kinase, casein kinases I and II, Ca2+/calmodulin-dependent protein kinase II, or phosphorylase kinase, and can be routinely used for the assay of protein kinase C with low background in the crude tissue extracts. The Km value is considerably low (7 microM) with a Vmax value of twice as much as that for H1 histone.
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PMID:A synthetic peptide substrate for selective assay of protein kinase C. 168 74

Suramin inhibited protein kinase C (PKC) type I-III activity in a concentration-dependent manner. Similar inhibitory effects were observed with M-kinase, the constitutively active catalytic fragment of PKC, and autophosphorylation of PKC types I-III. Kinetic experiments indicated that suramin competitively inhibits activity with respect to ATP (Ki = 17, 27, and 31 microM, respectively) and that it can also inhibit by interaction with the substrate histone III-S. With protamine as the Pi acceptor, suramin inhibition was dependent on lipid, being approximately 4-fold less sensitive to inhibition in the absence of phosphatidylserine and diacylglycerol than in their presence. Suramin at low concentrations (10-40 microM), in the presence of Ca2+ and absence of lipid, was able to stimulate kinase activity (approximately 200-400%) in a type-dependent manner and at higher concentrations inhibited activity with histone III-S as substrate. These results indicate that suramin, a hexa-anionic hydrophobic compound, can act as a negatively charged phospholipid analog in activating PKC in the presence of Ca2+ and absence of lipid and can inhibit Ca2+/phosphatidylserine/diacylglycerol-stimulated kinase activity at higher concentrations by competing with ATP or by interaction with the exogenous substrate. Suramin inhibited cAMP-dependent protein kinase much less potently (IC50 = 656 microM) than PKC. The ability of suramin to inhibit PKC-mediated processes in intact cells was tested using the phorbol ester-stimulated respiratory burst of neutrophils as a model system. The respiratory burst of human neutrophils, when preincubated with suramin and then stimulated with phorbol ester, was inhibited in a concentration-dependent manner, suggesting that suramin may also be able to inhibit PKC-mediated processes in intact cells.
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PMID:Effects of suramin, an anti-human immunodeficiency virus reverse transcriptase agent, on protein kinase C. Differential activation and inhibition of protein kinase C isozymes. 169 Jul 10

The myeloid interleukin-3 (IL-3) dependent cell line, FDC-P1, enters the G0 stage of the cell cycle after IL-3 deprivation for 24 hr. The expression of 13 protooncogenes and immediate-early genes was compared with 4 "control" genes after the addition of either IL-3 or phorbol myristate acetate (PMA) to IL-3-deprived cells. mRNA transcripts encoding c-myc and the T-cell receptor c-gamma gene were induced to high levels only after IL-3 addition, whereas c-fos, fos-B, c-jun, jun-B, Krox-20, and Krox-24 were induced transiently only after PMA addition. The remaining genes (fra-1, p53, jun-D, c-Ha-ras, c-Ki-ras, c-raf, beta-actin, ornithine decarboxylase, and histone 2B) were detected after culture with either IL-3 or PMA. When cells were serum- and IL-3-deprived, c-fos, fos-B, c-jun, jun-B, Krox-20, and Krox-24 were detected after exposure to either serum or PMA. Moreover, culture with cycloheximide and PMA resulted in superinduction of these genes, whereas cycloheximide and IL-3 addition did not. mRNAs encoding these genes had half-lives of 10-20 min after PMA treatment, whereas that of beta-actin was longer (greater than 2 hr), suggesting that short mRNA half-lives contributed to the transient nature of these genes. Although c-fos, fos-B, c-jun, jun-B, Krox-20, and Krox-24 expression can be detected in IL-3-dependent cells after exposure to either PMA or serum, these genes were not detected after IL-3 addition, which allows cell-cycle progression. These results document the existence of IL-3 and PMA-responsive genes and demonstrate that IL-3 and protein kinase C agonists can induce distinct patterns of gene expression.
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PMID:Interleukin-3 and phorbol esters induce different patterns of immediate-early gene expression in an interleukin-3 dependent cell line. 170 18


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