Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human platelets undergo a rapid, major reorganization of the cytoskeletal matrix upon exposure to thrombin, and accumulate 3-phosphorylated phosphoinositides in a protein kinase C (PKC)-dependent manner. These phosphoinositides have been suggested to be involved in actin polymerization/depolymerization. We reasoned that, if newly generated 3-phosphorylated phosphoinositide modulates cytoskeletal reorganization, a prerequisite for such action would be generation near cytoskeletal proteins. We have found that, after platelet activation, phosphatidylinositol 3-kinase and phosphatidylinositol(4)P 3-kinase activities, antibody-detectable phosphoinositide 3-kinase, and PKC become markedly and specifically enriched in a Triton X-100-insoluble cytoskeletal fraction that contains GPIIb/IIIa (integrin) and pp60c-src. The cytoskeletal fraction then accounts for up to 70% of total phosphoinositide 3-kinase activity, a function of recruited activated enzyme. These proteins are not occluded or directly associated with newly polymerized actin, since blockage by cytochalasin D of actin polymerization, and consequent inhibition of accumulation of about 40% of incremental protein and actin in this fraction, has no effect on its content of phosphoinositide 3-kinase, GPIIb/IIIa, pp60c-src, or PKC. Depolymerization of actin with DNase I, or inhibition of ligand binding to GPIIb/IIIa by RGDS, however, in combination with cytochalasin D, further depletes actin and significantly decreases sedimentability of GPIIb/IIIa as well as phosphoinositide 3-kinase, pp60c-src, and PKC, without inhibiting total 3-kinase activity. Our results suggest that, as a function of platelet activation, enzymes that regulate the synthesis of 3-phosphorylated phosphoinositides rapidly associate with the membrane skeleton and that skeletally associated phosphoinositide 3-kinase is more active than the Triton-soluble form.
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PMID:Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets. 131 17

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.
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PMID:Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin. 165 14

PMA treatment of human leukemic cells resulted in a significant increase in the phosphorylation of a 72-kDa protein, which was abrogated by treating the nuclear extracts with DNase I, but additionally stimulated by adding DNA. To be active, DNA must be double-stranded with an average size of 300 base pairs, but shows no apparent species- or sequence-specificity. NP-72 isolated from control or PMA-treated nuclei with 1 mM ATP lacked phosphorylating activity, suggesting it to be a substrate for a dsDNA-stimulated protein kinase(s). Simultaneous exposure of HL-60 cells to PMA and the protein kinase C inhibitor staurosporine diminished the phosphorylation of NP-72. These data suggest that leukemia cell differentiation is accompanied by the induction and/or activation of a dsDNA-stimulated protein kinase whose protein substrates include NP-72 and whose activity is directly or indirectly influenced by protein kinase C.
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PMID:dsDNA-stimulated phosphorylation of a 72-kDa nucleoprotein accompanies PMA-induced HL-60 leukemic cell differentiation. 177 55

Overexpression of the epidermal growth factor (EGF) receptor (c-erbB) proto-oncogene is a frequent occurrence in human carcinoma and appears to accompany autocrine or paracrine transforming growth factor-alpha expression, which in model systems can result in activation of EGF receptor tyrosine kinase activity and phenotypic transformation. Here we have investigated the transcriptional regulation of the EGF receptor gene, by run-on transcription in isolated nuclei derived from epithelioid tumor lines. The level of transcription was measured at various points on the 100-kilobase pair EGF receptor gene locus, on either sense or antisense DNA strands. We find the level of sense strand transcription along exon 1 is 8-fold higher than transcription in exons 2-26. Primary EGF receptor transcripts appear to pause or terminate prematurely between exons 1 and 2. Termination was mapped to a sequenced region approximately 2 kilobase pairs 3' of exon 1, proximal to a previously reported DNase I hypersensitive site and an enhancer-like activity. Transcription in the CpG-rich region surrounding exon 1 is bidirectional, with antisense transcripts initiating in intron 1 and extending through the coding first exon. Activation of protein kinase C results in a 5-fold induction of EGF receptor transcription, accompanied by a slow release in the block RNA elongation between exon 2 and exon 26, showing that EGF receptor RNA synthesis may be altered by changes in de novo transcription and by a block to RNA elongation.
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PMID:Contributory effects of de novo transcription and premature transcript termination in the regulation of human epidermal growth factor receptor proto-oncogene RNA synthesis. 198 48

Tissue-type plasminogen activator (t-PA) gene expression is regulated by the tumor-promoting phorbol ester, phorbol-12-myristate 13-acetate (PMA), by cyclic AMP analogues, and the cAMP agonist, forskolin. Based on nuclear "run-on" transcription assays, t-PA expression is modulated by PMA on the level of transcription. 8-Bromo-cyclic AMP and forskolin do not induce t-PA gene transcription alone but act synergistically with PMA. These effects are confirmed by transient expression assays in HeLa cells employing deletion mutants of the t-PA gene promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Constitutive expression and most of the PMA-mediated induction requires sequences downstream of position -145. DNase I protection ("footprint") analysis of this region reveals two protein-binding sites: one between position -102 and -115, differing from the consensus sequence of the cAMP-responsive element (CRE) by the substitution of an adenine for a guanine in the middle of the core motif (TGACATCA), and another, located in the first exon (between position +60 and +74), displaying homology to the consensus sequence of the activator protein 2- (AP-2) binding site (CCCCACCCCC). Base substitutions in the core of either the CRE-like element or the AP-2 site suppress constitutive CAT expression by over 80%, whereas the relative PMA- and PMA plus cAMP-mediated responses are retained. CAT expression is below the detection limit when both elements are mutagenized together. Hence, the CRE-like element and the exon-located AP-2-binding site have a cooperative impact on basal transcription, but each element can independently convey the effect of activators of the protein kinase C- and A-dependent pathways of signal transduction. The results of band-shift analysis and competition titration experiments demonstrate that the CRE-like element acts as a low affinity binding site for the same proteins which recognize the authentic CRE.
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PMID:A DNA motif related to the cAMP-responsive element and an exon-located activator protein-2 binding site in the human tissue-type plasminogen activator gene promoter cooperate in basal expression and convey activation by phorbol ester and cAMP. 216 21

The major part of mast cell actin is Triton-soluble and behaves as a monomer in the DNase I inhibition assay. Thus, actin exists predominantly in monomeric or short filament form, through filamentous actin is clearly apparent in the cortical region after rhodamine-phalloidin (RP) staining. The minimum actin content is estimated to be approximately 2.5 micrograms/10(6) cells (cytosolic concentration approximately 110 microM. After permeabilization of mast cells by the bacterial cytolysin streptolysin-O, approximately 60% of the Triton-soluble actin leaks out within 10 min. However, the staining of the cortical region by RP remains undiminished, and the cells are still capable of exocytosis when stimulated by GTP-gamma-S together with Ca2+. In the presence of cytochalasin E the requirement for Ca2+ is decreased, indicating that disassembly of the cytoskeleton may be a prerequisite for exocytosis. This disassembly is likely to be controlled by Ca2(+)-dependent actin regulatory proteins; their presence is indicated by a Ca2(+)-dependent inhibition of polymerization of extraneous pyrene-G-actin by a Triton extract of mast cells. The effect of cytochalasin E on secretion is similar to that of phorbol myristate acetate, an activator of protein kinase C; both agents enhance the apparent affinity for Ca2+ and cause variable extents of Ca2(+)-independent secretion. Exposing the permeabilized cells to increasing concentrations of Ca2+ caused a progressive decrease in F-actin levels as measured by flow cytometry of RP-stained cells. In this respect, both cytochalasin E and phorbol ester mimicked the effects of calcium. GTP-gamma-S was not required for the Ca2(+)-dependent cortical disassembly. Thus, since conditions have not yet been identified where secretion can occur in its absence, cortical disassembly may be essential (though it is not sufficient) for exocytosis to occur.
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PMID:Changes in the state of actin during the exocytotic reaction of permeabilized rat mast cells. 239 68

We previously demonstrated that down-regulation of protein kinase C (PKC) by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment leads to the specific repression of GnRH transcription in GT1-7 hypothalamic neurons. Here we have investigated the regulatory sequences and cognate DNA-binding proteins that mediate this transcriptional response. The promoter-proximal section of the GnRH gene contains an evolutionarily conserved sequence that is bound along its entire length by GT1-7 nuclear proteins in DNase I protection assays. Two distinct regions within this sequence are required for PKC regulation of the GnRH gene, as excision of either region results in loss of TPA repression of transcription. Excision of either of these regions also decreases basal transcription, demonstrating their role in GnRH promoter function. One region encompasses three AT-rich protein-binding sites; the other is an extended region of continuous DNase I protection, 50 nucleotides in length, that contains consensus recognition motifs for the CCAAT/EBP and helix-loop-helix families of transcription factors. Mobility shift analysis of binding to the latter region reveals that TPA treatment of GT1-7 neurons induces the formation of a specific DNA-protein complex with kinetics of appearance consistent with a role in repression of GnRH transcription. Thus, the sequences that mediate PKC regulation of GnRH are proximal to the promoter, evolutionarily conserved, and form TPA-inducible complexes with GT1-7 nuclear proteins.
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PMID:Regulation of gonadotropin-releasing hormone transcription by protein kinase C is mediated by evolutionarily conserved promoter-proximal elements. 747 68

Inhibins and activins are dimeric peptide hormones that regulate the circulating levels of follicle-stimulating hormone (FSH). In turn, FSH stimulates inhibin gene expression in the ovarian follicle; studies to date suggest that this effect is mediated by cAMP and that a cAMP-responsive element, identified in the 5'-flanking region of the alpha-inhibin gene, at least partially effects this response. To explore further the transcriptional regulation of the inhibin/activin genes, we have isolated and sequenced the 5'-flanking regions of the bovine alpha-, beta A- and beta B-inhibin/activin subunit genes and have analysed these regions by primer-extension analysis and DNase I footprinting with the transcription factor AP-2. Analyses indicated that all three gene promoter regions have a number of AP-2-binding sites that are resistant to competition by poly(dI-dC), suggesting that cAMP may control the inhibin/activin ratio by operating through alternative signal-transduction pathways or that inhibin/activin gene expression may be controlled by signals operating through the protein kinase C pathway. A comparison of the DNA sequences protected by AP-2 against DNase I digestion revealed a consensus AP-2-binding site of 5'-GSCCCDSS-3', where S represents a base pairing involving three (C or G) hydrogen bonds and D represents any base other than C. The nucleotide sequences of the bovine beta-subunit structural genes also are reported.
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PMID:Genomic cloning and sequence analyses of the bovine alpha-, beta A- and beta B-inhibin/activin genes. Identification of transcription factor AP-2-binding sites in the 5'-flanking regions by DNase I footprinting. 781 65

In LLC-PK1 cells, the urokinase-type plasminogen activator (uPA) gene is induced by two of the major signal transduction pathways, the protein kinase C (PKC) and the cAMP-dependent protein kinase (PKA) pathways. We have analyzed the chromatin structure of 26 kb of the uPA gene locus and have shown that PKA activation but not PKC activation induce major chromatin structural alterations in the uPA gene promoter. In uninduced cells, several DNase I hypersensitive (HS) sites were detected in the 5' and 3' flanking regions but not in the transcribed region. Two of the sites correspond to previously characterized regulatory sites: a cAMP responsive site at nucleotide position -3500 with respect to the initiation site, and the PEA3/AP1 site at -2100 that mediates PKC activation. After the activation of PKA but not PKC, a strong HS site was induced at -2600. Functional analysis of this region revealed cAMP responsive activity. Chromatin structural alterations again brought about specifically by PKA but not by PKC were were also detected in the upstream of the promoter by topoisomerase I cleavage site analysis, with two prominent sites appearing at -2800 and -3300. These results suggest that the strong cAMP induction of the uPA gene requires structural alterations that permit cooperative interactions between the multiple cAMP responsive sites.
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PMID:Activation of cAMP-dependent protein kinase alters the chromatin structure of the urokinase-type plasminogen activator gene promoter. 812 5

Protein kinase C gamma (PKC gamma) is a brain-specific isozyme expressed at a high level in the adult but not in the fetal or newborn rat. At least seventeen nuclear protein binding sites within the 5'-flanking region extending from -1612 to +243 had been identified by DNase I footprinting analysis and gel mobility shift assays. Among them, one site, GAATTAATAGG, at -669 to -679 is protected from DNase I digestion by nuclear protein from newborn but not from the adult rat brain. The levels of this binding protein, as determined by gel mobility shift assay, were found inversely related to the levels of PKC gamma in rat brain at different stages of development. These results suggest that this particular binding site may participate in the developmental regulation of PKC gamma gene.
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PMID:Identification of a nuclear protein binding element within the rat brain protein kinase C gamma promoter that is related to the developmental control of this gene. 831 5


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