Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism of interaction of phorbol esters with conventional protein kinase Cs was addressed by examining the direct binding of this class of activators to protein kinase C beta II. Binding measurements reveal that the major role of phorbol esters is to increase the affinity of protein kinase C for membranes by several orders of magnitude. The relative increase depends linearly on the mole fraction of phorbol esters in membranes, with the potency illustrated by the finding that 1 mol% phorbol 12-myristate 13-acetate (PMA) increases protein kinase C's membrane association by approximately 4 orders of magnitude. For comparison, diacylglycerol (DG), which also activates protein kinase C by increasing the enzyme's membrane affinity, is 2 orders of magnitude less effective than PMA in altering protein kinase C's membrane affinity. The remarkably high-affinity interaction with phorbol esters allowed us to measure the direct binding of protein kinase C to PMA in neutral membranes and, thus, to evaluate the effect of Ca2+ on the phorbol ester interaction in the absence of Ca2+ effects on the enzyme's interaction with acidic lipids. Changing the Ca2+ concentration over 5 orders of magnitude had no effect on the direct interaction of protein kinase C with PMA immobilized in phosphatidylcholine membranes. Thus, the Ca(2+)-binding site for membrane association and the phorbol ester-binding site do not interact allosterically. Lastly, a method that does not have the limitations of the Scatchard plot for analysis of amphitropic proteins was used to determine the dissociation constant of protein kinase C from phorbol esters: expressed relative to membrane lipids, the dissociation constant is 1.5 x 10(-5) mol %. In summary, our data reveal that (1) the direct binding of protein kinase C to phorbol esters, in the absence of interactions with acidic lipids, provides a major contribution to the free energy change involved in the association of protein kinase C with membranes and (2) this interaction is not regulated by Ca2+.
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PMID:Calcium-independent binding to interfacial phorbol esters causes protein kinase C to associate with membranes in the absence of acidic lipids. 863 93

Adenosine A1 receptor densities were increased in cultured LLC-PK1 and OK cells by chronic treatment with the adenosine receptor antagonists 1,3,7-trimethylxanthine (caffeine, 1 mM) and 1,3-dimethyl-8-cyclopentylxanthine [cyclopentyltheophylline (CPT), < or = 0.4 mM], respectively. The A1 receptor number per cell was increased twofold by 10-day treatments with 1 mM caffeine or 0.1 mM CPT, and the sodium-coupled glucose uptake was augmented twofold by 1 mM caffeine and sevenfold by 0.1 microM CPT (higher doses of CPT were progressively less stimulatory). Glucose uptake was blocked by acute (2-h) treatment with CPT, adenosine deaminase, or calphostin C. Caffeine (1 mM) or CPT (> or = 0.1 mM) inhibited cell proliferation for the first 10 days, then cell growth assumed a normal proliferative rate despite continued presence of antagonist. Cytosolic protein kinase C (PKC) beta-isoform immunoactivity and PKC-beta II mRNA were elevated at least twofold during 10 days of 0.1 mM CPT or 1 mM caffeine treatment. The sustained elevation in sodium-glucose symport and PKC activity observed with adenosine receptor antagonists was similar to acute (2-h) effects of the adenosine A1 agonist R(-)-N6-phenylisopropyladenosine (R-PIA, 0.1-1 microM). Moreover, cell proliferation was increased by adenosine (0.1 microM R-PIA), whereas Na-K-adenosinetriphosphatase activity was unaltered with chronic antagonist or acute adenosine treatments. Caffeine treatment may have some non-adenosine A1 receptor-mediated actions, because it slightly (30%) augmented protein kinase A activity. It is concluded that chronic exposure of proximal tubule cells to caffeine or CPT augments PKC and sodium-glucose transport but retards cell proliferation mainly via adenosine A1 receptor-mediated mechanisms.
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PMID:Upregulated renal adenosine A1 receptors augment PKC and glucose transport but inhibit proliferation. 877 86

Basophils and mast cells play a crucial role in immunological and allergic processes due to the release of inflammatory mediators such as histamine. It has been suggested for a long time that the histamine release (HR) from these cells is closely related to protein kinase (PKC) activity. However, the distinct role of PKC with its large variety of isozymes in different cell types and the actions of these isozymes in HR still remain unclear. Therefore, in the present study, we compared the effects of the two PKC inhibitors 7-O-methyl-UCN-01 (UCN-01-Me) and NPC 15437 as well as two PKC activators, bryostatin 1 and 2, on anti-IgE and Ca(2+)-ionophore-induced HR from human basophils and isolated human skin mast cells (HSMC). In both HSMC and basophils, anti-IgE-induced HR was inhibited by PKC inhibitor UCN-01-Me pre-incubation dose-dependently. In stark contrast, A23187-induced HR was unaffected by UCN-01-Me in both cell types. In our experiments, the inhibitory efficacy of the compound NPC 15437 on HR was much lower than that of UCN-01-Me and showed no statistical significance. Both bryostatins 1 and 2 produced good dose-dependent inhibition of HR from HSMC stimulated with anti-IgE, whereas HR from basophils was potentiated with these compounds. The same effects were observed with basophils stimulated with A23187, where potentiation of HR was up to fourfold of the control at the highest concentrations of bryostatins, while HSMC showed a slight decrease in HR compared to non-bryostatin-treated controls. Basophils and HSMC showed very clear differences in HR when directly stimulated with the bryostatins, since no HR was observed from HSMC while in basophils the HR increased up to 47% of total histamine at the highest concentrations of bryostatins (1 mumol/l). HR from basophils was observed to be strictly dose-dependent. The differences in the cell reactions of the two cell types incubated with these four compounds indicate distinct biochemical roles of PKC in the cascades leading to degranulation of the cells. Furthermore, the experiments with UCN-01-Me support the hypothesis of PKC-beta to play a substantial positive modulatory role for the degranulation of immunologically stimulated basophils.
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PMID:Heterogeneity of signal transduction mechanisms in human basophils and human skin mast cells. II. Effects of 7-O-methyl-UCN-01, NPC 15437 and bryostatin 1 and 2, four protein kinase C-modulatory agents, on mediator release. 909 18

Protein kinase C (PKC) was originally identified as a single serine/ threonine protein kinase with calcium- and phospholipid-dependent activity, but more recently PKC has been found to consist of a family of multiple isoenzymes with different biochemical characteristics, substrates, and cofactor requirements. PKC is particularly important in regulating airway smooth muscle (ASM) tone. Although a previous investigation has demonstrated PKC-beta, -delta, -epsilon, -theta and -zeta in canine trachealis muscle, additional PKC isoforms have not been characterized in ASM. Therefore, immunoblot analysis using nine isotype-specific antibodies was used to further characterize the expression of PKC isoforms in porcine ASM. In addition to the previously described beta-, delta-, epsilon-, and zeta-isoforms in ASM, the calcium-dependent alpha-isoform, and the calcium- and diacylglycerol-independent isoforms iota/lambda and mu were identified. This study demonstrates multiple PKC isoforms in porcine ASM that can participate in intracellular signaling pathways in this tissue.
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PMID:Qualitative immunoblot analysis of PKC isoforms expressed in airway smooth muscle. 914 31

Cycloheterophyllin, a prenylflavone, inhibited the superoxide anion (O2-) generation from formylmethionyl-leucyl-phenylalanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-stimulated rat neutrophils in a concentration-dependent manner with IC50 values of 47.0 +/- 5.0 and 1.7 +/- 0.4 microM, respectively. Cycloheterophyllin had no effect on O2- generation in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation. Cycloheterophyllin exerted a concentration-dependent inhibition of neutrophil cytosolic protein kinase C (PKC) and rat brain PKC, but had no effect on porcine heart protein kinase A (PKA). Unlike staurosporine, cycloheterophyllin did not affect the trypsin-treated rat brain PKC. [3H]Phorbol 12,13-dibutyrate ([3H]PDB) binding to neutrophil cytosolic PKC was significantly suppressed by cycloheterophyllin. However, cycloheterophyllin had negligible effect on the PMA-induced membrane translocation of PKC-beta and PKC-delta in neutrophils. Moreover, the fMLP-induced [Ca2+]i elevation and inositol trisphosphate (IP3) formation of neutrophils were not affected by cycloheterophyllin at concentrations which significantly suppressed the O2- generation. In cell-free system, addition of arachidonate (AA) into the mixture of cytosol and membrane fractions of the resting neutrophils to make NADPH oxidase assembly and activation. Cycloheterophyllin had no effect on O2- generation in AA-activated cell-free system. These results suggest that the suppression of PKC activity through the interaction with the regulatory region of PKC is involved in the inhibition by cycloheterophyllin of the O2- generation in rat neutrophils.
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PMID:Blockade of protein kinase C is involved in the inhibition by cycloheterophyllin of neutrophil superoxide anion generation. 915 Dec 91

We examined the question of whether insulin activates protein kinase C (PKC)-zeta in L6 myotubes, and the dependence of this activation on phosphatidylinositol (PI) 3-kinase. We also evaluated a number of issues that are relevant to the question of whether diacylglycerol (DAG)-dependent PKCs or DAG-insensitive PKCs, such as PKC-zeta, are more likely to play a role in insulin-stimulated glucose transport in L6 myotubes and other insulin-sensitive cell types. We found that insulin increased the enzyme activity of immunoprecipitable PKC-zeta in L6 myotubes, and this effect was blocked by PI 3-kinase inhibitors, wortmannin and LY294002; this suggested that PKC-zeta operates downstream of PI 3-kinase during insulin action. We also found that treatment of L6 myotubes with 5 microM tetradecanoyl phorbol-13-acetate (TPA) for 24 h led to 80-100% losses of all DAG-dependent PKCs (alpha, beta1, beta2, delta, epsilon) and TPA-stimulated glucose transport (2-deoxyglucose uptake); in contrast, there was full retention of PKC-zeta, as well as insulin-stimulated glucose transport and translocation of GLUT4 and GLUT1 to the plasma membrane. Unlike what has been reported in BC3H-1 myocytes, TPA treatment did not elicit increases in PKCbeta2 messenger RNA or protein in L6 myotubes, and selective retention of this PKC isoform could not explain the retention of insulin effects on glucose transport after prolonged TPA treatment. Of further interest, TPA acutely activated membrane-associated PI 3-kinase in L6 myotubes, and acute effects of TPA on glucose transport were inhibited, not only by the PKC inhibitor, LY379196, but also by both wortmannin and LY294002; this suggested that DAG-sensitive PKCs activate glucose transport through cross-talk with phosphatidylinositol (PI) 3-kinase, rather than directly through PKC. Also, the cell-permeable, myristoylated PKC-zeta pseudosubstrate inhibited insulin-stimulated glucose transport both in non-down-regulated and PKC-depleted (TPA-treated) L6 myotubes; thus, the PKC-zeta pseudosubstrate appeared to inhibit a protein kinase that is required for insulin-stimulated glucose transport but is distinct from DAG-sensitive PKCs. In keeping with the latter dissociation of DAG-sensitive PKCs and insulin-stimulated glucose transport, LY379196, which inhibits PKC-beta (preferentially) and other DAG-sensitive PKCs at relatively low concentrations, inhibited insulin-stimulated glucose transport only at much higher concentrations, not only in L6 myotubes, but also in rat adipocytes, BC3H-1 myocytes, 3T3/L1 adipocytes and rat soleus muscles. Finally, stable and transient expression of a kinase-inactive PKC-zeta inhibited basal and insulin-stimulated glucose transport in L6 myotubes. Collectively, our findings suggest that, whereas PKC-zeta is a reasonable candidate to participate in insulin stimulation of glucose transport, DAG-sensitive PKCs are unlikely participants.
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PMID:Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes. 934 99

The HIV-1 long terminal repeat (LTR) responds to a variety of cellular signal transduction pathways. We demonstrate that the cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) signaling pathways synergize to increase HIV-1 LTR-mediated transcription and viral replication in a latently infected promonocytic cell line (U1). The LTR-mediated synergy induced by cholera toxin (Ctx), a potent activator of the cAMP-dependent PKA pathway, and the PKC activator phorbol 12-myristate 13-acetate (PMA) was abrogated by a PKC-beta-specific inhibitor (LY333531). In contrast, the LTR-mediated synergy induced by Ctx and TNF alpha was not affected by LY333531. The synergy induced by Ctx and TNF alpha was also abrogated by mutation of the cAMP-responsive downstream sequence elements (DSE) in the 5' untranslated leader region, whereas the DSE mutations did not affect the synergy induced by Ctx and PMA. These distinctions indicate that Ctx cooperates differently with TNF alpha and PMA to activate the HIV-1 LTR. Ctx and PMA synergistically activated AP-1- and NF-kappa B-dependent transcription, even though no cooperative binding of AP-1 or NF-kappa B was observed in gel shift assays. An extensive mutational analysis of the HIV-1 LTR that included the NF-kappa B and AP-1 binding sites revealed no distinct cis-acting element or region within the HIV-1 LTR that was required for the transcriptional synergy. Ctx and PMA also synergistically interact to activate the HTLV-1 LTR. These results indicate that the transcriptional synergy elicited by Ctx and PMA targets multiple functional elements and promoters, requires a cooperative interaction between the PKA and PKC-beta pathways, and differs mechanistically from the transcriptional synergy induced by Ctx and TNF alpha.
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PMID:The cAMP-dependent protein kinase A and protein kinase C-beta pathways synergistically interact to activate HIV-1 transcription in latently infected cells of monocyte/macrophage lineage. 963 65

The transcription factor serum amyloid A (SAA)-activating factor (SAF), a family of zinc finger proteins, plays a significant role in the induced expression of the SAA gene. Activity of SAF is regulated by a phosphorylation event involving serine/threonine protein kinase (Ray, A., Schatten, H., and Ray, B. K. (1999) J. Biol. Chem. 274, 4300-4308; Ray, A., and Ray, B. K. (1998) Mol. Cell. Biol. 18, 7327-7335). However, the identity of the protein kinase has so far remained unknown. Induction of SAA by phorbol 12-myristate 13-acetate, a known agonist of protein kinase C (PKC), suggested a potential role of the PKC signaling pathway in the activation process. The DNA binding activity of endogenous SAF was increased by agonists of PKC. In vitro phosphorylation of SAF-1 by PKC-beta markedly increased its DNA binding ability. Consistent with these findings, treatment of cells with activators of PKC or overexpression of PKC-betaII in transfected cells increased expression of an SAF-regulated promoter. Further analysis with a GAL4 reporter system indicated that PKC-mediated phosphorylation mostly increases the DNA binding activity of SAF-1. Together these data indicated that the PKC signaling pathway plays a major role in controlling expression of SAF-regulated genes by increasing the interaction between promoter DNA and phosphorylated SAF.
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PMID:Activation of transcription factor SAF involves its phosphorylation by protein kinase C. 1099 78

Endothelial cells (ECs) are constantly subjected to hemodynamic forces including cyclic pressure-induced strain. The role of protein kinase C (PKC) in cyclic strain-treated ECs was studied. PKC activities were induced as cyclic strain was initiated. Cyclic strain to ECs caused activation of PKC-alpha and -epsilon. The translocation of PKC-alpha and -epsilon but not PKC-beta from the cytosolic to membrane fraction was observed. An early transient activation of PKC-alpha versus a late but sustained activation of PKC-epsilon was shown after the onset of cyclic strain. Consistently, a sequential association of PKC-alpha and -epsilon with the signaling molecule Raf-1 was shown. ECs treated with a PKC inhibitor (calphostin C) abolished the cyclic strain-induced Raf-1 activation. ECs under cyclic strain induced a sustained activation of extracellular signal-regulated protein kinases (ERK1/2), which was inhibited by treating ECs with calphostin C. ECs treated with a specific Ca(2+)-dependent PKC inhibitor (Go 6976) showed an inhibition in the early phase of ERK1/2 activation but not in the late and sustained phase. ECs transfected with the antisense to PKC-alpha, the antisense to PKC-epsilon, or the inhibition peptide to PKC-epsilon reduced strain-induced ERK1/2 phosphorylation in a temporal manner. PKC-alpha mediated mainly the early ERK1/2 activation, whereas PKC-epsilon was involved in the sustained ERK1/2 activation. Strained ECs increased transcriptional activity of Elk1 (an ERK1/2 substrate). ECs transfected with the antisense to each PKC isoform reduced Elk1 and monocyte chemotactic protein-1 promotor activity. Our findings conclude that a sequential activation of PKC isoform (alpha and epsilon) contribute to Raf/ERK1/2 activation, and PKC-epsilon appears to play a key role in endothelial adaptation to hemodynamic environment.
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PMID:Sequential activation of protein kinase C (PKC)-alpha and PKC-epsilon contributes to sustained Raf/ERK1/2 activation in endothelial cells under mechanical strain. 1139 52

Heat shock proteins play central roles in ensuring the correct folding and maturation of cellular proteins. Here we show that the heat shock protein Hsp70 has a novel role in prolonging the lifetime of activated protein kinase C. We identified Hsp70 in a screen for binding partners for the carboxyl terminus of protein kinase C. Co-immunoprecipitation experiments revealed that Hsp70 specifically binds the unphosphorylated turn motif (Thr(641) in protein kinase C beta II), one of three priming sites phosphorylated during the maturation of protein kinase C family members. The interaction of Hsp70 with protein kinase C can be abolished in vivo by co-expression of fusion proteins encoding the carboxyl terminus of protein kinase C or the carboxyl terminus of Hsp70. Pulse-chase experiments reveal that Hsp70 does not regulate the maturation of protein kinase C: the rate of processing by phosphorylation is the same in the presence or absence of disrupting constructs. Rather, Hsp70 prolongs the lifetime of mature protein kinase C; disruption of the interaction promotes the accumulation of matured and then dephosphorylated protein kinase C in the detergent-insoluble fraction of cells. Furthermore, studies with K562 cells reveal that disruption of the interaction with Hsp70 slows the protein kinase C beta II-mediated recovery of cells from PMA-induced growth arrest. Last, we show that other members of the AGC superfamily (Akt/protein kinase B and protein kinase A) also bind Hsp70 via their unphosphorylated turn motifs. Our data are consistent with a model in which Hsp70 binds the dephosphorylated carboxyl terminus of mature protein kinase C, thus stabilizing the protein and allowing re-phosphorylation of the enzyme. Disruption of this interaction prevents re-phosphorylation and targets the enzyme for down-regulation.
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PMID:The turn motif is a phosphorylation switch that regulates the binding of Hsp70 to protein kinase C. 1208 70


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