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 zeta isoform of protein kinase C (PKC zeta) was purified to near homogeneity from the cytosolic fraction of bovine kidney by successive chromatography on DEAE-Sephacel, heparin-Sepharose, phenyl-5PW, hydroxyapatite, and Mono Q. The purified enzyme had a molecular mass of 78 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was recognized by an antibody raised against a synthetic oligopeptide corresponding to the deduced amino acid sequence of rat PKC zeta. The enzymatic properties of PKC zeta were examined and compared with conventional protein kinase C purified from rat brain. The activity of PKC zeta was stimulated by phospholipid but was unaffected by phorbol ester, diacylglycerol, or Ca2+. PKC zeta did not bind phorbol ester, and autophosphorylation was not affected by phorbol ester. Unsaturated fatty acid activated PKC zeta, but this activation was neither additive nor synergistic with phospholipid. These results indicate that regulation of PKC zeta is distinct from that of other isoforms and suggest that hormone-stimulated increases in diacylglycerol and Ca2+ do not activate this isoform in cells. It is possible that PKC zeta belongs to another enzyme family, in which regulation is by a different mechanism from that for other isoforms of protein kinase C.
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PMID:Purification and characterization of the zeta isoform of protein kinase C from bovine kidney. 132 99

A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways.
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PMID:Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes. 150 83

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.
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PMID:Tissue and cellular distribution of the extended family of protein kinase C isoenzymes. 155 49

Murine epidermis contains PKC zeta and eta as evidenced by the application of specific antisera. PKC zeta predominates in the cytosol and PKC eta in the particulate fraction. PKC zeta is shown to be present also in other murine tissues, with large amounts found in lung. Whereas epidermal PKC eta is completely down-regulated by treatment of mouse skin with TPA or bryostatin 1 for 18 h, PKC zeta is neither translocated by treatment with TPA for 20 min, nor down-regulated by treatment with TPA or bryostatin 1 for 18 h. PKC zeta is activated by phosphatidyl serine alone and does neither respond to Ca2+ nor to TPA. It is inhibited by staurosporine with an IC50 of 16 nM, which is within the same range of other PKC isoenzymes. The sensitivity of PKC zeta towards the staurosporine derivative K252a is similar to that of PKC alpha,beta,gamma but much higher than that of PKC delta and epsilon.
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PMID:Protein kinase C zeta and eta in murine epidermis. TPA induces down-regulation of PKC eta but not PKC zeta. 164 68

Protein kinase C (PKC), the major receptor for tumor-promoting phorbol esters, consists of a family of at least 12 distinct lipid-regulated enzymes. We examined the expression and regulation of PKC isoforms in C6-glioma and NG 108-15 hybrid cells. Western blot analysis indicated that both cell lines express four PKC isoforms, PKC alpha, PKC delta, PKC epsilon and PKC zeta. The expression of PKC alpha and PKC delta in C6-glioma cells was more abundant than NG 108-15 cells, however, PKC epsilon in NG 108-15 was more abundant than C6-glioma cells in which PKC epsilon was almost undetectable. Treatment of both cells with TPA for 10 min resulted in the translocation of PKC alpha, PKC delta and PKC epsilon to the membrane fraction. When the intact cells were treated with Ca(2+)-free, EGTA containing physiological saline solution, the membrane bound conventional PKC alpha (cPKC alpha) was greatly reduced and cytosolic cPKC alpha was only slightly increased. However, neither membrane bound nor cytosolic new PKC delta (nPKC delta), nPKC epsilon and atypical PKC zeta (aPKC zeta) was affected by extracellular Ca2+ depletion. In this condition, the translocation of cPKC alpha, nPKC delta and nPKC epsilon induced by TPA still occurred, however, that of cPKC alpha was reduced more than in the normal condition. After long-term treatment (17 h) with TPA, cPKC alpha, nPKC delta and nPKC epsilon were down-regulated both in the cytosol and membrane. The phenomena of cPKC alpha were confirmed by measuring the PKC activity with histone as the substrate. From in vitro endogenous phosphorylation studies, a 31 kDa substrate protein phosphorylation in C6 glioma cell membrane and 31 and 26 kDa proteins in NG 108-15 cell membrane were increased in the translocation but disappeared in the down-regulation of PKC.
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PMID:Differential expression of protein kinase C isoforms in glial and neuronal cells. Translocation and down-regulation of PKC isoforms in C6 glioma and NG 108-15 hybrid cells: effects of extracellular Ca(2+)-depletion. 749 43

A human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A (PP1/PP2A), has been established. In wild type cells, the cytotoxicity of OA was associated with mitotic arrest and concentration- and time-dependent DNA fragmentation, a hallmark of apoptosis. The mutant was 100-fold more resistant to OA in terms of effects on these parameters. Although the synthesis of several proteins was altered, enzyme assay and immunoblot analysis indicated that the levels of PP1 and PP2A were unchanged in the mutant. Protein kinase C (PKC) assays and immunoblot analysis of calcium-dependent (cPKC) and calcium-independent (nPKC) isoforms revealed that nPKC-epsilon was strikingly absent in the mutant, which otherwise expressed in comparable amounts all other isotypes (cPKC-alpha, cPKC-beta, and nPKC-zeta) also present in the wild type. Northern blot analysis confirmed an absence of PKC-epsilon mRNA in the mutant cells. The OA200 cells were cross-resistant not only to another PP1/PP2A inhibitor, calyculin A, but also to structurally unrelated anticancer drugs (such as vinblastine and taxol) and furthermore, overexpressed the verapamil-sensitive drug pump P-glycoprotein at both the protein and mRNA levels. The mutant, however, was not cross-resistant to several PKC inhibitors tested including cardiotoxin, mastoparan, staurosporine, and an alkylphospholipid. Cardiotoxin, at a subtoxic concentration, enhanced by 6-fold vinblastine cytotoxicity in OA200 cells. These findings indicate that the multidrug resistance phenotype can be induced by cytotoxic agents other than conventional anticancer drugs, show that the development of multidrug resistance is not necessarily associated with increased cPKC activity, and identify certain PKC inhibitors that have potential as resistance modulators.
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PMID:Human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (protein phosphatase inhibitor) lacks protein kinase C-epsilon, exhibits multidrug resistance phenotype, and expresses drug pump P-glycoprotein. 751 66

Epidermal growth factor (EGF), 20 ng/ml, stimulated myelin basic protein (MBP) phosphorylation in crude extracts from human keratinocyte primary cultures. In order to identify the involved kinases, we separated by fast protein liquid chromatography proteins participating in MBP phosphorylation. We detected three MBP kinase activities in the keratinocyte crude extracts. The first MBP kinase activity was the only one stimulated by EGF and reacted with anti-mitogen-activated protein kinase (MAPK) antiserum recognising p42mapk and p44mapk isoforms. However, when protein kinase C (PKC) was either inhibited by the PKC inhibitor GF 109203X or depleted by a prolonged TPA treatment, the stimulation of MBP phosphorylation by EGF was strongly inhibited. The second MBP kinase activity eluted was due to a PKC isoform reacting with an anti-PKC zeta antibody, and the third was not identified. With this work, we have thus shown that, in human keratinocytes, EGF activates MAPK activity by a PKC-dependent pathway.
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PMID:Epidermal growth factor stimulates mitogen-activated protein kinase by a PKC-dependent pathway in human keratinocytes. 753 73

The effect of alkylglycerol supplementation on protein kinase C (PKC)-mediated signaling events has been studied in fibroblasts from Zellweger patients (SF 3271 cells). Western blotting analysis established that Zellweger fibroblasts express PKC alpha, epsilon, and zeta. Incubation with bradykinin induced a rapid transient translocation of PKC alpha and a more sustained translocation of PKC epsilon to the particulate fraction; translocation of PKC zeta was unaffected. Bradykinin-induced translocation and activation of PKC alpha, but not translocation of PKC epsilon, was blocked in SF 3271 cells which had been incubated with 1-O-hexadecylglycerol (1-O-HDG; 20 micrograms/ml) for 24 h and then incubated in the absence of 1-O-HDG and serum for a further 24 h. Supplementation with 1-O-HDG increased the mass of ether-linked phospholipid. Bradykinin initiated a transient increase in cytosolic Ca2+ concentration in both control and 1-O-HDG supplemented cells, indicating that the initial receptor linked events were not affected by 1-O-HDG supplementation. Bradykinin also caused a rapid activation of phospholipase D (PLD), measured by phosphatidylbutanol accumulation, and mitogen-activated protein kinase (MAPK) determined by myelin basic protein phosphorylation of Mono Q fractions. Both events were blocked by preincubation of the cells with 12-O-tetradecanoylphorbol-13-acetate for 24 h to deplete PKC protein. 1-O-HDG supplementation prevented the bradykinin-induced activation of PLD, but had no effect on the stimulation of MAPK activity. These results establish that modulation of the ether lipid composition of membranes can alter PKC isozyme translocation and indicate that a PKC isozyme other than PKC alpha, most likely PKC epsilon, is involved in MAPK activation.
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PMID:Evidence that the bradykinin-induced activation of phospholipase D and of the mitogen-activated protein kinase cascade involve different protein kinase C isoforms. 753 66

Cellular differentiation and proliferation are dependent upon phosphorylation by endogenous protein kinase C (PKC) isozymes in many cell types. Western blotting with a C-terminally directed rabbit polyclonal anti-PKC zeta antibody detected a doublet of approximately 81 kDa in normal hamster pancreatic tissue and hamster pancreatic carcinoma (PC-1) and human pancreatic carcinoma (PANC-1) cells. Preabsorption of the antibody with the specific peptide blocked the appearance of the 81-kDa band, indicating that the band was specifically recognized by the PKC zeta antibody. In contrast, antibodies for PKC alpha, beta, gamma, delta, and epsilon failed to show specific immunoreactivity for normal pancreatic tissue or PANC-1 or PC-1 cells. Immunocytochemical analysis identified PKC zeta in the cytoplasm of ductules and large ducts, to a lesser extent in the islets of the hamster pancreas, and in the normal cultured pancreatic duct epithelial cells and pancreatic carcinoma (PANC-1 and PC-1) cell lines. Specific reactivity was seen by electron microscopy in the ductal cells of the normal pancreatic tissue. In normal pancreatic ductal tissue and primary pancreatic ductal hyperplasia and carcinoma, the proportional labeling of PKC zeta in nuclei and cytoplasm was similar. Our results demonstrating the presence of PKC zeta isozyme in the normal pancreas, cultured normal pancreatic duct epithelial cells, and pancreatic carcinoma cells or carcinoma tissue suggests a role for this isozyme in the normal physiology of the pancreas and perhaps in pancreatic carcinoma.
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PMID:Identification of protein kinase C zeta isozyme in hamster pancreas and pancreatic carcinoma cell lines. 757 13

The myristoylated alanine-rich C-kinase substrate (MARCKS) is the major protein kinase C (PKC) substrate in many cell types including fibroblasts and brain cells. Here we describe the phosphorylation of MARCKS and the site specificity for different PKC isotypes. Conventional (c)PKC beta 1, novel (n)PKC delta and nPKC epsilon efficiently phosphorylated the MARCKS protein in vitro. The Km values were extremely low, reflecting a high affinity between kinases and substrate. The apparent affinity of nPKC delta (Km = 0.06 microM) was higher than that of nPKC epsilon and cPKC beta 1 (Km = 0.32 microM). The rate of substrate phosphorylation was inversely correlated with affinity and decreased in the order nPKC epsilon > cPKC beta 1 > nPKC delta. Atypical (a)PKC zeta did not phosphorylate the intact MARCKS protein. However, a 25-amino-acid peptide deduced from the MARCKS phosphorylation domain, was efficiently phosphorylated by aPKC zeta as well as by the other three PKC. Site analysis revealed that only serine residues S152, S156 and S163 were phosphorylated, with S163 phosphorylated highest, followed by S156 and S152; in contrast, S160 and S167 were not phosphorylated. No further PKC phosphorylation sites could be detected in MARCKS. The phosphorylation pattern was independent of the type of PKC isotype used. Kinetic analysis showed, that MARCKS is sequentially phosphorylated in the order S156 > S163 > S152 by cPKC, nPKC and aPKC. There was no dramatic difference in the sequential phosphorylation of MARCKS detectable when comparing the four PKC isotypes. The results are discussed in the context of the functional significance of MARCKS phosphorylation.
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PMID:The myristoylated alanine-rich C-kinase substrate (MARCKS) is sequentially phosphorylated by conventional, novel and atypical isotypes of protein kinase C. 758 87


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