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)

The distribution of six isoforms of protein kinase C (PKC) in seromucous acinar cells of rat submandibular gland was examined and their translocation from the cytosolic- to the membrane fraction after different stimuli investigated. Western blotting, immunostaining with isoform-specific antibodies and scanning densitometry showed that PKC-alpha and epsilon were distributed fairly evenly between the cytosol and membranes in resting cells, while isoforms- beta, delta and zeta were all predominantly localized (over 80%) in membranes. PKC-gamma was not detected. PKC-alpha was mobilized to the membrane fraction by the phorbol ester, TPA, but not by the phosphoinositide-coupled agonists carbachol, methoxamine and substance P (SP). PKC-epsilon was translocated by TPA and carbachol but not by SP or methoxamine. Biochemical assay of total PKC confirmed that cytosolic enzyme activity was significantly reduced by TPA and carbachol to 29% and 75% respectively of control levels. These results suggest that muscarinic regulation of the mucosecretory response in the rat submandibular gland may be mediated by the PKC-epsilon isoform.
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PMID:Distribution and translocation of isoforms of protein kinase C in rat submandibular acinar cells. 747 51

We used rat pancreatic acini and measured the effects of various agents on digestive enzyme secretion, diacylglycerol (DAG) and the cellular distribution of protein kinase C (PKC) enzyme activity as well as isoforms of PKC determined by quantitative immunoblot analysis. TPA, but not CCK-8, caused translocation of PKC enzyme activity from the cytosol fraction to the membrane fraction. Immunoblot analysis detected PKC-alpha, PKC-delta, PKC-epsilon and PKC-zeta. PKC-beta, PKC-gamma and PKC-eta were not detected. TPA caused translocation of all isoforms from cytosol to membrane, whereas CCK-8 caused translocation of PKC-delta and PKC-epsilon, carbachol caused translocation of PKC-epsilon, and bombesin and secretin caused no detectable translocation of any isoform. Specific receptor antagonists could prevent, as well as reverse completely, the translocation of PKC isoforms caused by CCK-8 or carbachol. Agonists added in sequence with an interposed addition of a specific receptor antagonist caused cycling of PKC-epsilon between cytosol and membrane fractions. Each receptor-mediated agonist that caused translocation of PKC also increased DAG, and with CCK-8 and carbachol cycling of PKC-epsilon between cytosol and membrane was accompanied by corresponding cyclic changes in cellular DAG. CCK-JMV-180, bombesin and secretin increased DAG but did not cause translocation of any PKC isoform. Translocation of a PKC isoform could be accounted for by whether the increased DAG originated from PIP2 (accompanied by translocation) or from phosphatidylcholine (no accompanying translocation). Thus it appeared that DAG, in pancreatic acini, is functionally compartmentalized depending on the source of the lipid. Studies using CCK-8 and CCK-JMV-180 indicated that occupation of the low affinity state of the CCK receptor by either peptide increased DAG from phosphatidylcholine, whereas occupation of the very low affinity state by CCK-8 increased DAG from PIP2 and caused translocation of PKC-delta and PKC-epsilon. TPA stimulated amylase secretion, indicating that activation of PKC can stimulate enzyme secretion; however, with the various receptor-mediated secretagogues there was no consistent, unequivocal correlation between translocation of an isoform of PKC and accompanying changes in enzyme secretion.
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PMID:Effects of cholecystokinin (CCK) and other secretagogues on isoforms of protein kinase C (PKC) in pancreatic acini. 752 84

In this study we examined the effects of staurosporine, a potent inhibitor of protein kinase C (PKC), on the differentiation of C6 glial cells and on the expression and cellular distribution of specific PKC isoforms. Staurosporine reduced cell proliferation and induced distinctive changes in the morphological appearance of the cells to that characteristic of cells exhibiting astrocytic phenotypes. The differentiative effect of staurosporine was further indicated by the increased expression of two proteins related to astrocytic phenotypes, glial fibrillary acidic protein (GFAP) and glutamine synthetase. Thus, staurosporine induced a dose-dependent increase both in GFAP immunoreactivity and in the activity and protein levels of glutamine synthetase. Staurosporine also induced a decrease in the expression of PKC-beta 2 and an increase in that of PKC-gamma. In addition, it induced translocation of PKC-epsilon from the membrane to the cytosol, whereas no differences were observed in the distribution of the other PKC isoforms. The results of our study indicate that staurosporine induced astrocytic phenotypes in glial cells and that changes in the expression and cellular distribution of these PKC isoforms may be related to astrocytic differentiation.
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PMID:Staurosporine induces astrocytic phenotypes and differential expression of specific PKC isoforms in C6 glial cells. 756 44

The cAMP phosphodiesterase (PDE) activity of CHO cells was unaffected by the addition of Ca2+ +calmodulin (CaM), indicating the absence of any PDE1 (Ca2+/CaM-stimulated PDE) activity. Treatment with the tumour promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) led to the rapid transient induction of PDE1 activity which attained a maximum value after about 13 h before slowly decreasing. Such induction was attenuated by actinomycin D. PCR primers were designed to hybridize with two regions identified as being characteristic of PDE1 forms found in various species and predicted to amplify a 601 bp fragment. RT-PCR using degenerate primers allowed an approx. 600 bp fragment to be amplified from RNA preparations of rat brain but not from CHO cells unless they had been treated with PMA. CHO cells transfected to overexpress protein kinase C (PKC)-alpha and PKC-epsilon, but not those transfected to overexpress PKC-beta I or PKC-gamma, exhibited a twofold higher PDE activity. They also expressed a PDE1 activity, with Ca2+/CaM effecting a 1.8-2.8-fold increase in total PDE activity. RT-PCR, with PDE1-specific primers, identified an approx. 600 bp product in CHO cells transfected to overexpress PKC-alpha and PKC-epsilon, but not in those overexpressing PKC-beta I or PKC-gamma. Treatment of PKC-alpha transfected cells with PMA caused a rapid, albeit transient, increase in PDE1 activity, which reached a maximum some 1 h after PMA challenge, before returning to resting levels some 2 h later. The residual isobutylmethylxanthine (IBMX)-insensitive PDE activity was dramatically reduced (approx. 4-fold) in the PKC-gamma transfectants, suggesting that the activity of the cyclic AMP-specific IBMX-insensitive PDE7 activity was selectively reduced by overexpression of this particular PKC isoform. These data identify a novel point of 'cross-talk' between the lipid and cyclic AMP signalling systems where the action of specific PKC isoforms is shown to cause the induction of Ca2+/CaM-stimulated PDE (PDE1) activity. It is suggested that this protein kinase C-mediated process might involve regulation of PDE1 gene expression by the AP-1 (fos/jun) system.
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PMID:Induction of Ca2+/calmodulin-stimulated cyclic AMP phosphodiesterase (PDE1) activity in Chinese hamster ovary cells (CHO) by phorbol 12-myristate 13-acetate and by the selective overexpression of protein kinase C isoforms. 757 35

Varied patterns of immunoreactive bands of protein kinase C gamma (PKC gamma) and receptor for activated C-kinase-1 (RACK1) were detected by analysis of Western blots in crude extracts of wheat germ, lobster tail meat, and three strains of baker's yeast. Anti-PKC lambda also reacted with wheat germ and yeast extracts, but failed to react with the lobster extract. The findings may implicate a regulatory role and an evolutionary conservation of these PKC isoenzymes and their receptor proteins in eukaryotes.
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PMID:Immunoreactivity of PKC gammalambda and RACK1 in baker's yeast, lobster and wheat germ. 758 Oct 12

A 13-kilobase pair genomic DNA encoding a 78-amino acid brain-specific calmodulin-binding protein kinase C (PKC) substrate, neurogranin (Ng/RC3; also known as RC3 or p17), has been sequenced. The Ng/RC3 gene is composed of four exons and three introns, with the protein-coding region located in the first and second exons. This gene was found to have multiple transcriptional start sites clustered within 20 base pairs (bp); it lacks the TATA, GC, and CCAAT boxes in the proximal upstream region of the start sites. The promoter activity was characterized by transfection of 293 cells with nested deletion mutants of the 5'-flanking region fused to the luciferase reporter gene. A minimal construct containing bp +11 to +256 was nearly as active as that covering bp -1508 to +256, whereas a shorter one covering bp +40 to +256 had a greatly reduced activity. Between bp +11 and +40 lies a 12-nucleotide sequence (CCCCGCCCACCC) containing overlapping binding sites for AP2 (CCGCCCACCC) and SP1 (CCCGCC); this region may be important for conferring the basal transcriptional activity of the Ng/RC3 gene. The expression of a Ng/RC3-luciferase fusion construct (-1508/+256) in transfected 293 cells was stimulated by phorbol 12-myristate 13-acetate (PMA), but not by cAMP, arachidonic acid, vitamin D, retinoic acid, or thyroxines T3 and T4. PMA caused a 2-4-fold stimulation of all the reporter gene constructs ranging from +11/+256 to -1508/+256. The stimulatory effects of PMA could be magnified by cotransfection with both Ca(2+)-dependent and -independent phorbol ester-binding PKC-alpha, -beta I, -beta II, -gamma, -delta, and -epsilon cDNAs, but not by non-phorbol ester-binding PKC-zeta cDNA. The Ng/RC3 and PKC-gamma genes have a similar expression pattern in the brain during development. These two genes share at least four conserved sequence segments 1.5 kilobase pair upstream from their transcriptional start sites and a gross similarity in that they possess several AT-rich segments within bp -550 to -950. A near homogeneous 20-kDa DNA-binding protein purified from rat brain was able to bind to these AT-rich regions of both Ng/RC3 and PKC-gamma genes with footprints containing ATTA, ATAA, and AATA sequences.
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PMID:Structure and regulation of the gene encoding the neuron-specific protein kinase C substrate neurogranin (RC3 protein). 773 Mar 37

We have used immunocytochemical analyses to characterize the subcellular distribution of protein kinase C (PKC)-alpha, -beta I, -beta II, -gamma, -delta, -epsilon, -zeta, and -eta in NIH 3T3 fibroblasts that overexpress these different PKC isozymes. Immunofluorescence studies and Western blotting with antibodies specific for individual isoforms revealed that before activation the majority of the PKCs are not membrane-bound and are diffusely distributed throughout the cytoplasm. In addition, a fraction of PKC-delta and -eta appears membrane-bound and concentrated in the Golgi apparatus. Activation of each isozyme's kinase activity (with the exception of PKC-zeta) by treatment of these cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate results in isozyme-specific alterations of cell morphology, as well as in a rapid, selective redistribution of the different PKC isozymes to distinct subcellular structures. Within minutes after 12-O-tetradecanoylphorbol-13-acetate treatment, PKC-alpha and -epsilon concentrate at cell margins. In addition, PKC-alpha accumulates in the endoplasmic reticulum, PKC-beta II associates with actin-rich microfilaments of the cytoskeleton, PKC-gamma accumulates in Golgi organelles, and PKC-epsilon associates with nuclear membranes. Our results demonstrate that each activated PKC isozyme specifically associates with a particular cellular structure, presumably containing the substrate for that isozyme. These findings support the hypothesis that PKC substrate specificity in vivo is mediated, at least in part, by the restricted subcellular locale for each PKC isozyme and its target protein.
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PMID:Immunocytochemical localization of eight protein kinase C isozymes overexpressed in NIH 3T3 fibroblasts. Isoform-specific association with microfilaments, Golgi, endoplasmic reticulum, and nuclear and cell membranes. 773 Mar 83

The protein kinase C (PKC) family is composed of at least four conventional (alpha, beta I, beta II, and gamma) and several related novel (delta, epsilon, eta, and zeta) isoforms with different distribution and sensitivity to Ca2+ and phorbol esters. The enzyme is known to be present in cerebral endothelial cells. We have investigated the occurrence of seven isoforms (alpha, beta, gamma, delta, epsilon, eta, and zeta) by using reverse transcriptase-polymerase chain reaction in rat brain, in a freshly isolated brain microvessel fraction, in primary cultures of rat brain endothelial cells, in an immortalized rat brain endothelial cell line, and in aortic endothelial cell cultures. Brain tissue contained all seven investigated isoforms. A similar expression pattern was seen in freshly purified microvessels, but the PKC-gamma isoform could not be detected. Primary cultures of endothelial cells expressed PKC-alpha, -beta, -delta, -eta, and -epsilon isoenzymes, whereas the immortalized cell line expressed PKC-alpha, -delta, -epsilon, and -eta. The rat aortic endothelium contained only PKC-alpha and -delta isoforms. The variety of expression patterns of PKC family members in endothelial cells of different type may reflect differences in the functional responsiveness to environmental stimuli. Because PKC has been shown to be involved in the regulation of the blood-brain barrier permeability, the presence of different isoforms may confer a sophisticated intracellular regulatory mechanism to the brain endothelial cells.
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PMID:Expression of protein kinase C family members in the cerebral endothelial cells. 779 Aug 92

Mammalian protein kinase C (PKC) isotypes elicit a number of effects on expression in Schizosaccharomyces pombe. A small decrease in growth rate results from PKC-gamma expression, and treatment of these cells with phorbol esters leads to marked growth inhibition and vesicle formation. PKC-delta and -eta expression causes growth inhibition and vesiculation, and the magnitude of both of these effects is increased by phorbol esters. In contrast, PKC-epsilon expression produces growth inhibition but no vesicle accumulation, and this effect is not responsive to phorbol ester. Finally, PKC-zeta has no observable effect. Thus, isotype-specific biological effects are observed. The accumulation of vesicles correlates with phorbol ester-dependent growth inhibition and occurs only with expression of those isotypes that down-regulate in response to phorbol esters in these cells. Antibodies against mammalian clathrin light chain 1a identified clathrin-coated vesicles and up-regulation of clathrin expression in those cells where vesicles accumulate; the increased vesicular traffic includes an element of endocytosis. Thus expression of specific mammalian PKC isotypes up-regulates endocytosis in S. pombe, providing a likely explanation for PKC-mediated receptor internalization in higher eukaryotes.
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PMID:Expression of mammalian protein kinase C in Schizosaccharomyces pombe: isotype-specific induction of growth arrest, vesicle formation, and endocytosis. 780 58

The family of homologous enzymes known as protein kinase C (PKC) has been the object of intense interest because of its crucial role in cellular signal transduction. Although considerable information about the activation of PKC has been gained through structure-activity, molecular modeling, and synthetic studies of both natural and designed activators, information about the structure of PKC itself has been limited by its large size and requirement for phospholipid cofactors. Additionally, difficulties in the purification of truncated mutants of PKC have thus far prevented their analysis by nuclear magnetic resonance (NMR) or x-ray crystallographic methods. We describe the identification, synthesis, ligand-binding analysis, cofactor requirements, and preliminary NMR evaluation of two subdomains (peptides B and C) of the regulatory domain of PKC-gamma. Peptides B and C bind [3H]phorbol 12,13-dibutyrate with good affinity (Kd = 6.4 microM and 414 nM, respectively) in the presence of phosphatidylserine. In comparison, the binding affinity of [3H]phorbol 12,13-dibutyrate for PKC was found to be 2.6 nM. Like PKC itself, these peptides also recognize other PKC activators, including dioctanoylglycerol and teleocidin B-4, and exhibit an ability to differentiate phorbol ester from its C-4 epimer. NMR studies of PKC subdomains are also described, indicating that both peptides B and C are well behaved in solution and do not exhibit any concentration-dependent changes. Finally, these studies reveal that peptide B becomes conformationally ordered only in the presence of phospholipid, suggesting that the regulatory domain of PKC itself might be organized for activation only when associated with the lipid bilayer, where its activator (diacylglycerol) is encountered.
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PMID:Identification, activity, and structural studies of peptides incorporating the phorbol ester-binding domain of protein kinase C. 781 24

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