EC:2.7.11.13 - FACTA Search

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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 have stably overexpressed the human protein kinase C alpha (hPKC alpha) in NIH 3T3 fibroblasts under the control of the interferon (IFN) type I inducible murine Mx promoter. These cells showed a 10-fold increase in the transcription of hPKC alpha mRNA after induction with interferon alpha. The increase in the amount and activity of protein kinase C (PKC)-protein in these cells was only about 3-fold after induction with interferon alpha. Compared to control cells which were transfected with the vector only, the NIH 3T3 fibroblasts transfected with the hPKC alpha cDNA showed already a slightly increased PKC-activity and amount of PKC-protein in the absence of interferon alpha. The hPKC alpha overexpressing cells had an altered, "transformed-like" morphology, which was reversed by staurosporine, an increased growth rate and a higher saturation density. The growth rate was further increased by treating the cells with interferon alpha. The hPKC alpha overexpressing cells were able to grow in soft agarose after treatment with phorbol ester such as TPA (12-O-tetradecanoylphorbol 13-acetate). After phorbol ester and interferon treatment a stronger expression of the protooncogene c-jun was detectable in the hPKC alpha overexpressing cells, whereas expression of c-fos and c-myc was not affected. Since these cells show a specific response pattern due to induced PKC alpha expression they might be useful as an assay system for the development of PKC isozyme-specific inhibitors and activators.
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PMID:Inducible overexpression of human protein kinase C alpha in NIH 3T3 fibroblasts results in growth abnormalities. 161 23

The effect of phorbol myristate acetate (PMA) on the hormonal responsiveness of hepatocytes from lean and obese Zucker rats was studied. Phenylephrine-stimulated phosphatydylinositol labeling and phosphorylase activation were antagonized by PMA in cells from obese and lean animals; bigger residual effects were observed in cells from obese animals even at high PMA concentrations. Cyclic AMP accumulation induced by isoproterenol, glucagon, forskolin and cholera toxin was higher in cells from lean animals than in those from obese rats. PMA diminished glucagon- and cholera toxin-induced cyclic AMP accumulation; cells from lean animals were more sensitive to PMA. Two groups of isoforms of protein kinase C (PKC) were observed in hepatocytes from Zucker rats using DEAE-cellulose column chromatography: PKC 1 and PKC 2. The PKC 1 isozymes were separated into four peaks using hydroxylapatite: aa, 1a (PKC-beta), 1b (PKC-alpha) and 1c. Short treatment with PMA decreased the activity of PKC 1 (peaks 1b (PKC-alpha) and 1c) and to a lesser extent of PKC 2; cells from lean animals were more sensitive to PMA than those obtained from obese rats. Our results indicate that cells from genetically obese Zucker rats are in general less sensitive to this activator of protein kinase C than those from their lean littermates. The possibility that alterations in the phosphorylation/dephosphorylation cycles, that control metabolism and hormonal responsiveness, may contribute to this obese state is suggested.
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PMID:Modulation by protein kinase C of the hormonal responsiveness of hepatocytes from lean (Fa/fa?) and obese (fa/fa) Zucker rats. 161 41

The localisation and immunochemical identification of 3 different forms of protein kinase C (PKC-alpha, PKC-beta and PKC-gamma) in retinas of different species were analysed by immunohistochemistry and SDS-PAGE-Western blotting, respectively. Only in some cases was there a correlation between the findings from each procedure. One reason for the lack of correlation could be the small amounts of PKC present in some retinas, which made detection possible only by first concentrating the antigen by SDS-PAGE and then carrying out Western blotting. Another possible reason is that an antibody recognises unknown antigens immunohistochemically, but, because of their specific characteristics, they are denatured when subjected to SDS-PAGE and Western blotting and therefore remain undetected. PKC-beta immunoreactivity is present in rabbit, frog and goldfish retinas but absent from the rat retina. However, SDS-PAGE and Western blotting experiments showed that the PKC-beta isoenzyme is absent from the fish retina but present in the rat retina. PKC-beta immunoreactivity in rabbit retina is present in ganglion and/or amacrine cells; in the frog retina the enzyme is associated with some bipolar cells. In the goldfish retina, PKC-beta is associated with a large population of cells in the ganglion cell layer as well as with some amacrine cell bodies. PKC-alpha is present primarily in bipolar cells of rat, fish and rabbit retinas and was not detected by immunohistochemistry or blotting experiments in the frog retina. SDS-PAGE and Western blotting of retinal extracts from different species showed that PKC-gamma occurs in the rabbit where it was associated with ganglion and/or amacrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The occurrence of three isoenzymes of protein kinase C (alpha, beta and gamma) in retinas of different species. 161 8

Differentiation of B16 mouse melanoma cells induced by retinoic acid (RA) is preceded by a large increase in protein kinase C alpha (PKC alpha) mRNA and protein. To determine the role of PKC alpha in the differentiation program, we stably transfected B16-F1 cells with a plasmid containing the full length PKC alpha cDNA driven by an SV40 promoter. Two out of thirty-two colonies screened were determined to overexpress PKC by 2-4-fold according to Western blot analysis and PKC enzyme activity. When compared to control cells (wild-type cells and cells transfected only with the neomycin resistance gene), PKC alpha overexpressing clones displayed longer doubling times, diminished anchorage-independent growth, and increased melanin production. RA treatment of control cells mimicked these phenotypic characteristics. When injected subcutaneously into syngeneic mice, PKC alpha overexpressing clones produced smaller tumors and had longer latencies than control cells. These findings, combined with the fact that phorbol esters down-regulate PKC and antagonize RA action suggest that PKC alpha plays a key role in the RA-induced melanoma differentiation.
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PMID:Increased expression of protein kinase C alpha plays a key role in retinoic acid-induced melanoma differentiation. 161 38

The levels of protein kinase C (PKC) activity, PKC isozymes, as well as the level of endogenous diacylglycerols (DAG) were examined in early emergence mouse skin papillomas and compared to the levels in the epidermis. The papillomas were derived from a two-stage carcinogenesis protocol in which mice were initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted twice weekly for only 12 weeks with 12-O-tetradecanoylphorbol-13-acetate (TPA). As expected, greater than 90% of these early emergence papillomas contained an activated Ha-ras gene with an A----T transversion in the 61st codon. There was a TPA-independent, irreversible decrease in total PKC activity (70%) in the early emergence papillomas compared to that in the epidermis. Immunoblot analysis of epidermis and papillomas taken 4 weeks following the cessation of TPA treatment, a time when PKC catalytic activity has completely recovered to control level in epidermis but not in papillomas, revealed that the levels of PKC-alpha and PKC-beta 2 were dramatically decreased in the cytosol of the papillomas, while the levels of these two isozymes in the particulate fraction were approximately equal to the epidermis. PKC-delta, -epsilon and -zeta immunoreactive proteins were present in both epidermis and papillomas and only minor changes were observed in the papillomas. PKC-delta and PKC-epsilon displayed a particulate fraction localization in both the epidermis and papillomas, while PKC-zeta was found in both subcellular fractions. We were unable to detect PKC-gamma in mouse epidermis or papillomas. Since the level of DAG has been shown to be elevated in some ras-transformed cells, we examined DAG levels in the papillomas, as an increased DAG level could explain the constitutive decreases in the levels of PKC. Measurements of cellular DAG indicated that there was no elevation in the total pool of DAG in the early emergence papillomas. These data demonstrate an irreversible decrease in and alteration of the subcellular distribution of PKC-alpha and beta 2 in DMBA-initiated/TPA-promoted papillomas. These changes are TPA-independent, and occur in the absence of an elevation in the total pool of endogenous DAG. These alterations of PKC isozymes may be important early events in multistage tumorigenesis.
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PMID:Alterations in protein kinase C isozymes alpha and beta 2 in activated Ha-ras containing papillomas in the absence of an increase in diacylglycerol. 163 76

Human platelets were found by immunoblot analysis to express protein kinase C (PKC) isozymes alpha, beta, delta, and zeta, but not gamma, epsilon, or eta. Exposure of platelets to thrombin, in the presence of 1 mM calcium, induced increased membrane association of PKC-alpha, -beta, and -zeta, while the subcellular distribution of PKC-delta remained unaltered. Maximal membrane association (2-fold) of PKC-alpha, -beta, and -zeta occurred within 1 min and was sustained for at least 10 min after the addition of thrombin. Similar results were obtained in the presence of the RGDS peptide, which blocks thrombin-induced binding of fibrinogen to its receptor, which indicates that PKC translocation was independent of fibrinogen binding. In the absence of added extracellular calcium, thrombin-induced translocation of PKC-alpha, -beta, and -zeta was transient, reaching a maximum at 1 min and returning to base line by 10 min. In the presence of calcium, thrombin induced a rapid (within 15 s) 8-fold rise in inositol 1,4,5-trisphosphate, which returned to baseline levels within 1 min, and a biphasic increase in sn-1,2-diacylglycerol (DAG), with peaks at 15 s and 2 min, which remained elevated for at least 5 min. Chelation of external calcium abolished the second phase of DAG formation but had no effect on the kinetics or magnitude of the increase in inositol 1,4,5-trisphosphate or the first phase of DAG formation. Two early PKC-dependent functions, serotonin release and 40-kDa protein phosphorylation, were independent of extracellular calcium and sustained DAG. These data demonstrate that in thrombin-stimulated human platelets the duration of the increased PKC membrane association closely parallels that of increased DAG content, and sustained elevations in DAG content and PKC translocation are dependent on extracellular calcium.
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PMID:Translocation of protein kinase C isozymes in thrombin-stimulated human platelets. Correlation with 1,2-diacylglycerol levels. 163 99

The expression of members of the Ca2+ and phospholipid-dependent protein kinase (PKC) family were studied in murine Swiss 3T3 cells. In addition to PKC-alpha, the presence of immunoreactive PKC-delta, -epsilon, and zeta was detected. Treatment with 500 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) led to the down-regulation of alpha, delta, and epsilon isoforms, but not that of zeta. Higher concentrations of TPA similarly had no effect on the level of PKC-zeta. In contrast to PKC-alpha, the membrane localization of PKC-delta, -epsilon, and -zeta was not enhanced by extraction in Ca(2+)-containing buffers, whereas acute TPA treatment increased membrane association of PKC-alpha, -delta, and -epsilon but not that of PKC-zeta.
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PMID:Identification of multiple PKC isoforms in Swiss 3T3 cells: differential down-regulation by phorbol ester. 163 59

Expression of rat protein kinase C-delta (PKC-delta) and PKC-zeta in insect cells using recombinant baculovirus resulted in the production of proteins with a molecular size of approximately 76 kD and 78 kD, respectively, as determined by immunoblotting with subtype-specific antisera. Although the PKC-zeta cDNA encoded for 592 amino acids, a 76 kD protein was also generated by in vitro transcription/translation. Extracts of cells expressing PKC-delta were able to bind phorbol ester to levels comparable to extracts of cells expressing PKC-alpha. No phorbol ester binding was, however, detected in insect cell extracts expressing PKC-zeta. However, similar levels of protein kinase activity were detected in lysates of cells expressing PKC-delta or PKC-zeta when protamine sulfate was used as exogenous substrate. Compared to protamine sulfate, both, myelin basic protein (MBP) or histone, were poor substrates for PKC-delta and PKC-zeta. In contrast to PKC-zeta, the PKC-delta enzyme activity phosphorylated MBP or histone in a phosphatidylserine-(PS)/diacylglycerol(DG)-dependent manner, albeit not to the same extent as PKC-alpha. Lack of stimulation of the enzyme activity of PKC-zeta by PS/DG, was confirmed by endogenous phosphorylation of insect cell proteins by PKC-zeta, whereas several insect cell proteins were phosphorylated by PKC-delta in a PS/DG-dependent manner, including a protein of 78 kD. Our data demonstrate that the 76 kD PKC-zeta, in contrast to PKC-delta, is unable to bind phorbol esters and displays a protein kinase activity that is independent of PS or PS/DG. In addition, staurosporine was about 2-4 order of magnitudes less effective in inhibiting the protein kinase activities of PKC-delta and PKC-zeta when compared to PKC-alpha.
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PMID:Expression and partial characterization of rat protein kinase C-delta and protein kinase C-zeta in insect cells using recombinant baculovirus. 164 61

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

The mechanism(s) by which the c-myc nuclear protein and the membrane-associated ras protein interact to mediate phenotypic changes is unknown. We now find that c-mcy gene expression is associated with alterations in the principal signal transduction pathway through which the ras protein is thought to function. We studied the transcript and protein expression of protein kinase C (PKC) isoforms in a culture line of human small cell lung cancer cells (NCI H209) in which expression of inserted c-myc and Ha-ras genes together, but not alone, causes a transition to a large cell phenotype. In control H209 cells, at the transcript and cell membrane protein levels, PKC-alpha is the dominant PKC species. In this cell line, the expression of an exogenous c-myc gene, but not of a viral Ha-ras gene, causes a 5- to 10-fold increase in the PKC-beta isoform transcript and protein. The insertion of ras into the exogenous myc-expressing 209 cells, in addition to causing phenotypic transition, results in the translocation of the PKC-beta protein from the cytosol to the membrane fraction and a decrease in membrane-associated PKC-alpha. Concomitant with these changes, the increased PKC isoform transcript levels induced by myc alone are completely reversed. These observations suggest that a complex set of PKC transcript and protein alterations, most prominently involving an increased PKC-beta protein level in the cell membrane, a decrease in PKC-alpha protein, and a decrease in all PKC isoform transcripts, may represent a fundamental event(s) for c-myc collaboration with Ha-ras to alter cell phenotype.
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PMID:c-myc gene-induced alterations in protein kinase C expression: a possible mechanism facilitating myc-ras gene complementation. 165 53

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