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)

Paraquat (methyl viologen, PQ) is a widely used herbicide that produces oxygen-derived free radicals and severely injures human lungs. In this study we examined the effects of PQ on the protein kinase C (PKC), ornithine decarboxylase (ODC) and c-jun oncogene expression in WI-38 human lung cells. Exposure of cells to 25-200 microM PQ resulted in an increase of [3H]phorbol dibutyrate (PDBu) binding and PKC redistribution in a dose-dependent manner. Interestingly, a superoxide dismutase mimic, 4-hydroxyl-2,2,6,6-tetramethyl-piperidine-1-oxyl (Tempol, 2.5 mM) and catalase (400 micrograms/ml) could significantly reduce the PQ-stimulated increase of phorbol ester binding and particular PKC phosphorylating activity, but dimethylsulfoxide (DMSO, 1.5%), an effective .OH trapping agent, failed to prevent this stimulation. In addition, an endogenous substrate of PKC, 80 kDa protein, was found to be highly phosphorylated in intact WI-38 cells treated with 50 microM PQ. The increase of phosphorylated proteins could be completely or partly abolished by Tempol or catalase, but only the phosphorylation of 80 kDa protein was diminished by protein kinase C inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7). A maximal peak of ODC activity was observed at 6 h of treatment with 50 microM PQ. PQ induced activity was reduced at the following rates, Tempol 85%, DMSO 80% and catalase 45%, but H-7 failed to do so. Furthermore, we found that the level of c-jun mRNA was transiently increased by PQ and the peak appeared at 1 h of treatment. When correlated with the PKC result, Tempol, catalase and H-7 all effectively blocked PQ-elicited c-jun transcript expression, but DMSO only exhibited a weakly inhibitory effect. We therefore propose that superoxide anion (O2- and H2O2 generated by PQ could activate PKC and lead to induction of c-jun gene expression; on the other hand, O2- and .OH might trigger other kinase pathways to elevate ODC activity. Finally, the sequential expression of c-jun oncogene and ODC may cooperate to relieve the oxidative damages elicited by PQ.
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PMID:Pronounced activation of protein kinase C, ornithine decarboxylase and c-jun proto-oncogene by paraquat-generated active oxygen species in WI-38 human lung cells. 766 13

Treatment of confluent monolayers of human umbilical vein endothelial cells with sublethal concentrations of hydrogen peroxide (H2O2) produces reversible cell retraction that opens gaps between adjacent cells. Despite the retraction, adjacent cells remain in contact through a network of dendrite-like processes. Retraction depends on cellular metabolism but not new protein synthesis or protein kinase C. Shape changes induced by H2O2 are accompanied by partial redistribution of actin filaments from the cell periphery in resting endothelial cells to a tangled network of centrally located filaments in H2O2-treated endothelial cells. This change in actin organization is associated with a loss of the normal distribution pattern of surface protein expression. Specifically, beta 1 and beta 3 integrins partly escape from focal adhesion plaques and migrate to the lateral and apical surface of the cell; PECAM-1 redistributes from the lateral borders to the basal surface; and ICAM-1 and ICAM-2 spread from apical caps to the basal surface and to the dendrite-like processes. The likely consequence of endothelial retraction accompanied by abnormal membrane protein distribution is a loss of normal endothelial cell functions. These changes are best considered manifestations of H2O2-induced sublethal injury that may cause endothelial dysfunction.
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PMID:Hydrogen peroxide-induced endothelial retraction is accompanied by a loss of the normal spatial organization of endothelial cell adhesion molecules. 767 77

Several inhibitors of tyrosine phosphatases, which included vanadate/H2O2, phenylarsine oxide, and diamide, blocked exocytosis in basophilic RBL-2H3 cells that had been transfected with the gene for the muscarinic m1 receptor. Because this block was observed whether the secretagogue acted through receptors (i.e. antigen and the muscarinic agonist, carbachol) or by direct activation of intracellular mechanisms (i.e. A23187, A23187 in combination with phorbol 12-myristate 13-acetate, and thapsigargin), the inhibitors appeared to act at a step distal to the mobilization of Ca2+ and activation of protein kinase C. All secretagogues caused the tyrosine phosphorylation of a 40-kDa protein, whereas the inhibitors caused a hyperphosphorylation of this protein. Therefore, both tyrosine kinase and phosphatase activities appear to regulate this phosphorylation which may, in turn, regulate secretion. The 40-kDa protein was identified as a mitogen-activated protein kinase-like protein on the basis of its reactivity to anti-mitogen-activated protein kinase antibodies. In addition, when cells were stimulated the tyrosine phosphorylated and the immunoreactive protein comigrated as a doublet on one-dimensional and as multiple phosphorylated forms on two-dimensional gel-electrophoretic systems.
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PMID:Tyrosine phosphorylation of a mitogen-activated protein kinase-like protein occurs at a late step in exocytosis. Studies with tyrosine phosphatase inhibitors and various secretagogues in rat RBL-2H3 cells. 769 76

We have previously shown that vanadate potentiates the activating effect of phorbol ester (TPA) on cellular phospholipase A2 (PLA2) in a pathway dependent on the formation of reactive oxygen species (ROS). Here we evaluate the chain of enzymes (protein kinases and phosphatases) that participate in this process. Treatment of macrophages with vanadate plus TPA led to activation of protein kinase C (PKC) and NADPH oxidase (O2- generation in intact cells), massive cellular protein tyrosine phosphorylation, suppression of protein tyrosine phosphatase (PTP) activity and a sustained activation of protein tyrosine kinase (PTK) and myelin basic protein kinase activity (the latter three enzyme activities were assessed in cell lysates). Inhibition of ROS formation by diphenyleneiodonium (DPI) prevented PTP inhibition, PTK activation and protein tyrosine phosphorylation by vanadate plus TPA. Vanadate plus H2O2 mimicked the effect of vanadate plus TPA on PKC activation, cellular protein tyrosine phosphorylation, PTP and PTK, but their effects were resistant to DPI. Suppression of PKC activity (down-regulation; selective inhibitors) prevented the above-mentioned effects of vanadate plus TPA, but not of vanadate plus H2O2. Collectively, the results show that ROS formation induced by TPA in association with vanadate is essential in the modulation of protein tyrosine phosphorylation and PLA2 activity.
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PMID:Reactive oxygen species mediate phorbol ester-regulated tyrosine phosphorylation and phospholipase A2 activation: potentiation by vanadate. 769 72

Prior studies have suggested that intracellular phosphorylation events and cellular redox mechanisms may interact in regulating a variety of cellular functions, including the transcriptional activation of gene expression. Increased activity of transcriptional factors NF kappa B and AP1 has been described in cells exposed to oxidative stress and following the direct stimulation of protein kinase C (PKC) by phorbol diesters. However, the mechanisms that may contribute to redox regulation of PKC are unknown. We studied the expression of PKC activity and several second messengers in human Jurkat T cells exposed to oxidative stress in the form of H2O2. Micromolar concentrations of H2O2 rapidly induced increased cytosolic PKC enzymatic activity in Jurkat T cells that was associated with a marked arrest of cellular proliferation. The increase in cytosolic PKC activity in cells treated with H2O2 was accompanied by elevations in intracellular free calcium ([Ca2+]i), generation of inositol phosphates, and release of arachidonic acid. Functional studies showed that H2O2 enhancement of cytosolic PKC activity required phospholipase C activity but was not primarily mediated by arachidonic acid. The response of PKC to oxidative stress displayed a lack of Ca2+ dependence and was uncoupled from the activity of protein tyrosine kinases (PTK). Furthermore, the reduced activation requirements of PKC from cells treated with H2O2 were associated with shifts in elution profiles of PKC enzymatic activity after Mono-Q chromatography. These shifts appeared to represent intrinsic changes in the conformation of PKC induced by oxidative stress because western blotting failed to reveal any PKC cleavage products or reductions in native PKC alpha or beta. These findings indicate that oxidative regulation of intracellular events can intersect phosphorylation events mediated by PKC through the release of second messengers as well as direct changes in PKC activation requirements. Moreover, redox regulation of PKC is distinct from T cell receptor signaling in that the activity of PKC is uncoupled from the regulatory influences of PTK.
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PMID:Regulation of protein kinase enzymatic activity in Jurkat T cells during oxidative stress uncoupled from protein tyrosine kinases: role of oxidative changes in protein kinase activation requirements and generation of second messengers. 770 13

We investigated the role of reactive oxygen intermediates and protein kinase C in the induction of expression of the c-jun gene in human ML-2 leukemic cells and normal human DET-551 fibroblasts by comparing the effects of exposure to either ionizing radiation or H2O2 in the presence or absence of appropriate inhibitors. In these cell types, the radiation- and H2O2-mediated increase in c-jun mRNA levels could be prevented by pretreatment of the cells with N-acetylcysteine, an antioxidant, or H7, an inhibitor of protein kinase C and protein kinase A, but not by HA1004, a specific inhibitor of protein kinase A and G. These results suggest a role for protein kinase C and reactive oxygen intermediates in the induction of c-jun gene expression in both normal and tumor cells. We also investigated potential differences in c-jun gene expression induced by radiation or H2O2 in normal and tumor cells by examining steady-state c-jun mRNA levels in a number of human fibroblast, leukemia, melanoma, sarcoma and carcinoma cell types. We observed heterogeneity in the steady-state level of c-jun mRNA in both the untreated normal and tumor cells and in such cells exposed to ionizing radiation or to H2O2. Exposure to radiation produced a varied response which ranged from little or no induction to an increase in the steady-state level of the c-jun mRNA of more than two orders of magnitude. Exposure to H2O2 gave a pattern similar to that of ionizing radiation. The basis for the differential induction in response to these agents may be attributable to either cell lineage or genetic heterogeneity or a combination of these two parameters.
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PMID:Heterogeneity in c-jun gene expression in normal and malignant cells exposed to either ionizing radiation or hydrogen peroxide. 772 34

Increasing evidence suggests that angiotensin II may act as a growth factor for several muscle cell types. Angiotensin II stimulation activates many immediate early response genes like c-Fos, c-Jun, c-Myc and Egr-1 in both vascular smooth muscle cells and cardiomyocytes, independently of whether a hyperplastic or hypertrophic response is taking place. In this study we report that angiotensin II significantly stimulates AP1-driven transcription in mouse skeletal muscle cells C2C12 stably transfected with a TRE-tk-CAT plasmid in a dose-dependent manner (peak stimulation at 10(-5) M of angiotensin II). Moreover, angiotensin II increases the binding of the AP1 complex to its DNA target in both quiescent C2C12 myoblasts and in differentiated C2C12 myotubes. Most of the TRE-bound complexes in both unstimulated and angiotensin II-treated cells consist of c-jun/c-fos heterodimers. Using a set of different protein kinase inhibitors, including HA1004, H7, tyrphostin, genistein and staurosporine, we could demonstrate that the angiotensin II-induced AP1 binding increase is not mediated by the cAMP-dependent pathway and that protein kinase C and tyrosine kinases are involved. Treatment of C2C12 cells with H2O2 induces a dose-dependent increase in c-jun/c-fos heterodimer binding, specifically reverted by the cysteine derivative and glutathione precursor N-acetyl-L-cysteine (NAC). The observation that the induction by angiotensin II of both the AP1 DNA binding activity and DNA synthesis in quiescent C2C12 myoblasts is abolished by NAC strongly suggests a role for reactive oxygen intermediates (ROIs) in the intracellular transduction of angiotensin II signals for immediate early gene induction and for cell proliferation.
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PMID:Reactive oxygen intermediates (ROIs) are involved in the intracellular transduction of angiotensin II signal in C2C12 cells. 775 83

Exposure of rabbit pulmonary vascular smooth muscle cells to H2O2 dose-dependently stimulates the cell membrane associated protein kinase C (PKC) activity, phospholipase A2 (PLA2) activity, phospholipase A2 (PLA2) activity, and arachidonic acid (AA0) release. Pretreatment of the cells with staurosporine (an inhibitor of PKC) prevents AA release and PLA2 activity caused by H2O2. Treatment of the cells with 4 beta-PMA (an active phorbol ester), or 4 alpha-PMA (an inactive phorbol ester) does not affect basal AA release. In contrast, 4 beta-PMA significantly stimulates the cell membrane associated PKC activity. Treatment of the cells with 4 beta-PMA for a short time (up-regulation of PKC) augments PLA2 activity and AA release caused by a sub-optimal dose of H2O2 (0.4 mM). Under this condition, staurosporine prevents the stimulatory effects of 4 beta-PMA on membrane PLA2 activity, and AA release. In contrast to the up-regulation, pretreatment with 4 beta-PMA for a longer time (down-regulation of PKC) does not appreciably augment PLA2 activity and AA release caused by 0.4 mM H2O2. Treatment of the cells with an intracellular Ca2+ antagonist TBM-8 prevents H2O2 induced membrane PLA2 activity and AA release without affecting membrane PKC activity. Treatment of the cells with TMB-8 before addition of 4 beta-PMA (up-regulation of PKC) followed by incubation with 0.4 mM H2O2 does not augment PLA2 activity and AA release, although membrane PKC activity increases under this condition.
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PMID:Down-regulation of protein kinase C attenuates the oxidant hydrogen peroxide-mediated activation of phospholipase A2 in pulmonary vascular smooth muscle cells. 775 14

Sublethal concentrations of reactive oxygen intermediates including H2O2 can alter human T cell function and inhibit proliferative responses but relatively little is known about the effects of low levels of oxidant stress on signaling pathways. In the present study, we investigated whether the exposure of Jurkat T cells to micromolar concentrations of H2O2 might influence the activity of certain serine/threonine kinases and protein phosphatases important for T cell signaling as well as initiation of nuclear events. Jurkat cells treated with 100-200 microM H2O2 exhibited rapid increases in cytosolic protein kinase C (PKC) activity without detectable translocation of PKC to the membrane/particulate compartment. The stimulation of PKC activity by H2O2 was associated with an increase in the activation of kinases phosphorylating myelin basic protein (MBP), a substrate for mitogen-activated protein (MAP) kinase and RRLSSLRA (S6 peptide; a substrate for the approximately 90-kDa ribosomal S6 kinases). Optimal activation of MAP kinase in cells treated with H2O2 was preceded by increases in protein tyrosine phosphorylations and occurred at sublethal concentrations of H2O2 which did not markedly deplete intracellular ATP. Pretreatment of cells with the PKC inhibitors sangivamycin and H7 suppressed but did not block the stimulation of MAP kinase activity in response to H2O2 or phytohemagglutinin. The activities of both protein tyrosine phosphatase (PTP) and protein phosphatase 2A (PP2A) were reduced after H2O2 treatment of intact cells. Furthermore, kinetic studies showed that H2O2 was capable of suppressing the activities of PTP and PP2A before inducing optimal increases in MAP kinase activity. These results demonstrate that the exposure of T cells to sublethal levels of oxidant stress acutely stimulates the MAP kinase cascade and suggest that this activation may involve PKC-dependent and -independent pathways as well as inhibition of certain protein phosphatases.
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PMID:Sublethal levels of oxidant stress stimulate multiple serine/threonine kinases and suppress protein phosphatases in Jurkat T cells. 777 89

It is known that older people are more sensitive to cancer and infectious agents and need more time to recover from such disorders. Can this difference in sensitivity to cancer and infections between elderly and younger people be a result of a difference in their immune systems and, more specifically, in the way monocytes react to infectious agents and cancer cells? To determine what happens after cells have aged, human monocytes were purified from young donors (approximately 25 years of age) and from older donors (65 years of age or older) and tested for their ability to respond to the polyclonal activator LPS. Our results showed that monocytes from aged donors (aged monocytes), when compared with monocytes from younger donors (young monocytes) did lose part of their cytotoxicity against tumor cells (A375 human melanoma cells and L929 murine fibroblast cells). In addition, aged monocytes displayed a sharp decrease in IL-1 secretion, but did display the intracellular 31 kDa IL-1 precursor. Moreover, aged monocytes displayed a decrease in the production of reactive oxygen intermediates such as NO2 and H2O2. Finally, aged monocytes stimulated by LPS displayed an increase in intracellular cyclic AMP and have lost their protein kinase C translocation from the cytosol to the plasma membranes. These results suggest that age affects the immunologic and antitumoral properties of human monocytes.
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PMID:Antitumoral properties of aged human monocytes. 781 87


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