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)

Clinically, nitric oxide (NO*) is widely used as a pulmonary vaso- and bronchodilator agent. However, the precise molecular mechanisms by which NO. induces smooth muscle relaxation are not well established. It has been suggested that NO. relaxes airway smooth muscle (ASM) via a 3',5'-cyclic guanosine monophosphate (cGMP)-dependent pathway, and our previous work has shown that Ca2+-activated K+ (KCa) channels are susceptible to cGMP-dependent protein kinase (PKG)-dependent phosphorylation (A. Alioua, J. P. Huggins, and E. Rousseau. Am. J. Physiol. 1995;268:L1057-L1063). To assess whether KCa channels are also directly activated by NO. or one of its derivatives such as peroxynitrite, the activity of these channels was measured upon fusion of sarcolemmal vesicles derived from bovine tracheal smooth muscle cells into planar lipid bilayers (PLB). It was found that in the absence of adenosine triphosphate (ATP), cGMP, and cGMP-dependent protein kinase, NO* donors such as 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA NONOate) or 3-morpholinosydnonimine hydrochloride (SIN-1) in the presence of superoxide dismutase (SOD), added on either side of the bilayer, caused a concentration- dependent increase in the open probability (Po) of KCa channels without altering their unitary conductance. Release of NO*, which was measured by chemiluminescence analysis in parallel experiments, affected the gating behavior of KCa channels in the presence of SOD and ethyleneglycol-bis-(beta-aminoethyl ether)- N,N'-tetraacetic acid (EGTA) by reducing the mean closed times and increasing the number and duration of short open events. PAPA NONOate, a true NO. donor, had similar effects in the presence of ethylenediaminetetraacetic acid (EDTA), a heavy-metal chelator, and K-urate, a peroxynitrite scavenger. Addition of either 5 mM dithiothreitol (DTT) or 5 mM reduced glutathione (GSH), as well as 5 mM N-ethylmaleimide (NEM)-an alkylating agent-to the trans (intracellular) side of an experimental chamber slightly increased channel Po but prevented further channel activation by NO* donors. However, neither DTT nor GSH was able to reverse the effect of NO*. In contrast to SIN-1, DTT had no effect when added to the cis (extracellular) side of the chamber. This suggests that the effect of NO* is most likely due to a chemical modification (nitrothiosylation) of intracellular sulfhydryl group(s). Neither PAPA NONOate (NO*), nor SIN-1 had any effect on sarcolemmal Cl- channels reconstituted from the same membrane preparations. Pharmacomechanical measurements made on epithelium-denuded rat bronchus showed that 100 nM charybdotoxin decreased the sensitivity of bronchial smooth muscle to SIN-1-induced relaxations. Altogether, our data suggest that NO-induced bronchorelaxation occurs partly via a direct activation of KCa channels, possibly through a covalent interaction with the cytoplasmic side of their alpha subunit.
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PMID:Direct activation of K(Ca) channel in airway smooth muscle by nitric oxide: involvement of a nitrothiosylation mechanism? 973 Aug 77

The nitric oxide (NO) donors S-nitrosopenicillamine or DetaNONOate, which release NO at a rate of 0-15 nM sec-1, were exposed to rat aortic vascular smooth muscle cells for a period of 0-24 h. This treatment resulted in an increase in total glutathione levels of two- to threefold under conditions where no cytotoxicity was detected. The signaling pathways do not involve activation of protein kinase G Ialpha nor are they cGMP dependent. Oxidation of reduced glutathione (GSH) was found after exposure to NO for 3-4 h at rates of formation at or above 8 nM sec-1. Increased intracellular GSH was due to enhanced expression of the rate-limiting enzyme for GSH synthesis, gamma-glutamylcysteine synthetase. Since NO has been shown previously to protect cells against oxidative stress, we propose that the increase in GSH by NO is a potential mechanism for enhancing the antioxidant defenses of the cell. This result also has important implications for identifying redox-sensitive cell signaling pathways that can be activated by NO.
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PMID:Nitric oxide-dependent induction of glutathione synthesis through increased expression of gamma-glutamylcysteine synthetase. 975 Jan 67

Since tumor promoter benzoyl peroxide (BPO) mimics phorbol esters in some aspects, its effects on protein kinase C (PKC) were previously studied. However, in those studies due to the presence of thiol agents in the PKC preparations, the sensitive reaction of BPO with redox-active cysteine residues in PKC was not observed. In this study, by excluding thiol agents present in the purified PKC preparation, low concentrations of BPO modified PKC, resulting in the loss of both kinase activity and phorbol ester binding (IC50 = 0. 2 to 0.5 microM). This modification, which was not dependent on transition metals, was totally blocked by a variety of thiol agents including GSH, which directly reacted with BPO. Substoichiometric amounts of BPO (0.4 mol/mol of PKC) oxidized two sulfhydryls in PKC and inactivated the enzyme which was readily reversed by dithiothreitol. The regulatory domain having zinc thiolate structures supporting the membrane-inserting region provided the specificity for PKC reaction with BPO, which partitioned into the membrane. Unlike H2O2, BPO did not induce the generation of the Ca2+/lipid-independent activated form of PKC. Other redox-sensitive enzymes such as protein kinase A, phosphorylase kinase, and protein phosphatase 2A required nearly 25- to 100-fold higher concentrations of BPO for inactivation. BPO also inactivated PKC in a variety of cell types. In the JB6 (30 P-) nonpromotable cell line and other normal cell lines, where BPO was more cytotoxic, it readily inactivated PKC due to a slow reversibility of this inactivation by the cell. However, in the JB6 (41 P+) promotable cell line, C3H10T1/2 and B16 melanoma cells, where BPO was less cytotoxic, it did not readily inactivate PKC due to a rapid reversibility of this inactivation by an endogenous mechanism. Nevertheless, BPO inactivated PKC at an equal rate in the homogenates prepared from all these cell types. Inclusion of NADPH reversed this inactivation in the homogenates to a different extent, presumably due to a difference in distribution of a protein disulfide reductase, which reverses this oxidative modification. BPO-induced modification of PKC occurred independent of the cellular status of GSH. However, externally added GSH and cell-impermeable thiol agents prevented the BPO-induced modification of PKC. Since BPO readily partitions into membranes, its reaction with redox-cycling thiols of membrane proteins such as PKC may trigger epigenetic events to prevent cytotoxicity, but favor tumor promotion.
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PMID:Tumor promoter benzoyl peroxide induces sulfhydryl oxidation in protein kinase C: its reversibility is related to the cellular resistance to peroxide-induced cytotoxicity. 1006 46

2',3'-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O2 and nitric oxide. Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.
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PMID:Programmed cell death in 2',3'-dideoxycytidine-resistant human monoblastoid U937 cells. 1081 77

The SmMAK16 gene from Schistosoma mansoni was cloned by chance when an adult worm cDNA library was probed with antiserum to affinity-purified S. mansoni GSH S-transferases. SmMAK16 encodes a hydrophilic protein of 259 amino acids with a molecular mass of 31 kDa. The protein shares 43% sequence identity and 66% similarity to the nuclear protein MAK16 of Saccharomyces cerevisiae that has been implicated both in cell cycle progression and biogenesis of 60S ribosomal subunits. Both proteins display a similar degree of sequence similar to the hypothetical protein CeMAK16 from Caenorhabditis elegans. These proteins share a number of apparent protein motifs, including two nuclear localization signals (NLS), multiple sites for phosphorylation by protein kinase CK2 and four conserved cysteine residues that resemble a zinc binding domain. SmMAK16 mRNA is more highly expressed in adult female worm than males. Recombinant SmMAK16 was phosphorylated by human protein kinase CK2. When chimeric constructs containing SmMAK16 fused the green fluorescent protein (GFP) were transiently transfected into COS-7s cells, the reporter was localized not in nuclei, but exclusively in nucleoli. The yeast and nematode homologues were likewise able to direct nucleolar accumulation of the fluorescent reporter. The high degree of sequence conservation together with the ability to direct nucleolar protein transport supports the hypothesis that MAK16 proteins play a key role in the biogenesis of 60S subunits.
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PMID:SmMAK16, the Schistosoma mansoni homologue of MAK16 from yeast, targets protein transport to the nucleolus. 1083 25

Patients suffering from the metabolic disease hereditary tyrosinemia type I (HT1), caused by fumarylacetoacetate hydrolase deficiency, have a high risk of developing liver cancer. We report that a sub-apoptogenic dose of fumarylacetoacetate (FAA), the mutagenic metabolite accumulating in HT1, induces spindle disturbances and segregational defects in both rodent and human cells. Mitotic abnormalities, such as distorted spindles, lagging chromosomes, anaphase/telophase chromatin bridges, aberrant karyokinesis/cytokinesis and multinucleation were observed. Some mitotic asters displayed a large pericentriolar material cloud and/or altered distribution of the spindle pole-associated protein NuMA. FAA-treated cells developed micronuclei which were predominantly CREST-positive, suggesting chromosomal instability. The Golgi complex was rapidly disrupted by FAA, without evident microtubules/tubulin alterations, and a sustained activation of the extracellular signal-regulated protein kinase (ERK) was also observed. Primary skin fibroblasts derived from HT1 patients, not exogenously treated with FAA, showed similar mitotic-derived alterations and ERK activation. Biochemical data suggest that FAA causes ERK activation through a thiol-regulated and tyrosine kinase-dependent, but growth factor receptor- and protein kinase C-independent pathway. Pre-treatment with the MEK inhibitor PD98059 and the Ras farnesylation inhibitor B581 decreased the formation of CREST-positive micronuclei by approximately 75%, confirming the partial contribution of the Ras/ERK effector pathway to the induction of chromosomal instability by FAA. Replenishment of intracellular glutathione (GSH) with GSH monoethylester abolished ERK activation and reduced the chromosomal instability induced by FAA by 80%. Together these results confirm and extend the previously reported genetic instability occurring in cells from HT1 patients and allow us to speculate that this tumorigenic-related phenomenon may rely on the biochemical/cellular effects of FAA as a thiol-reacting and organelle/mitotic spindle-disturbing agent.
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PMID:Fumarylacetoacetate, the metabolite accumulating in hereditary tyrosinemia, activates the ERK pathway and induces mitotic abnormalities and genomic instability. 1153 83

Previous studies using purified RNA polymerase from mustard (Sinapis alba) chloroplasts showed control of transcription by an associated protein kinase. This kinase was found to respond to reversible thiol/disulfide formation mediated by glutathione (GSH), although at concentrations exceeding those thought to exist in vivo. In the present study, several lines of evidence are presented to substantiate the functioning of this regulation mechanism, also in vivo: (a) Studies on the polymerase-associated transcription kinase revealed that at appropriate ATP levels, GSH concentrations similar to those in vivo are sufficient to modulate the kinase activity; (b) GSH measurements from isolated mustard chloroplasts showed considerable differences in response to light intensity; (c) this was reflected by run-on transcription rates in isolated chloroplasts that were generally higher if organelles were prepared from seedlings incubated under high-light as compared with growth-light conditions; (d) the notion of a general transcriptional switch was strengthened by in vitro experiments showing that the kinase not only affects the transcription of a photosynthetic gene (psbA) but also that of a non-photosynthetic gene (trnQ); and (e) the polymerase-kinase complex revealed specific differences in the phosphorylation state of polypeptides depending on the light intensity to which the seedlings had been exposed prior to chloroplast isolation. Taken together, these data are consistent with GSH and phosphorylation-dependent regulation of chloroplast transcription in vivo.
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PMID:Chloroplast transcription at different light intensities. Glutathione-mediated phosphorylation of the major RNA polymerase involved in redox-regulated organellar gene expression. 1170 85

The cytotoxic side effects of anti-neoplastic drugs are increased in patients with either type 1 or type 2 diabetes mellitus by a mechanism that is not clearly defined. We report that the circulating glucose metabolite, methylglyoxal (MGO), enhances cisplatin-induced apoptosis by activating protein kinase Cdelta (PKCdelta). We found that treatment of myeloma cells with the antioxidant N-acetylcysteine completely blocked cisplatin-dependent intracellular GSH oxidation, reactive oxygen species (ROS) generation, poly(ADP-ribose) polymerase cleavage, and apoptosis. Importantly, co-treatment of cells with the reactive carbonyl MGO and cisplatin increased apoptosis by 90% over the expected additive effect of combined MGO and cisplatin treatment. This same synergism was also observed when ROS generation was examined. MGO and cisplatin increased PKCdelta activity by 4-fold, and this effect was blocked by the PKCdelta inhibitor rottlerin but not by NAC. Furthermore, rottlerin blocked combined MGO and cisplatin-induced ROS generation and apoptosis. Finally, MGO and cisplatin induced c-Abl activation and c-Abl:PKCdelta association. Rottlerin blocked c-Abl activation, but the c-Abl inhibitor STI-571 increased MGO and cisplatin-induced apoptosis by 50%. Taken together these data indicate that MGO synergistically enhances cisplatin-induced apoptosis through activation of PKCdelta and that PKCdelta is critical to both cell death and cell survival pathways. These findings suggest that in the patient with diabetes mellitus heightened oxidative stress can enhance the cytotoxicity of agents that induce DNA damage.
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PMID:Methylglyoxal enhances cisplatin-induced cytotoxicity by activating protein kinase Cdelta. 1170 30

Nitric oxide (NO) exerts neurotrophic and neurotoxic effects on dopamine (DA) function in primary midbrain cultures. We investigate herein the role of glutathione (GSH) homeostasis in the neurotrophic effects of NO. Fetal midbrain cultures were pretreated with GSH synthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO), 24 h before the addition of NO donors (diethylamine/nitric oxide-complexed sodium and S-nitroso-N-acetylpenicillamine) at doses tested previously as neurotrophic. Under these conditions, the neurotrophic effects of NO disappeared and turned on highly toxic. Reduction of GSH levels to 50% of baseline induced cell death in response to neurotrophic doses of NO. Soluble guanylate cyclase (sGC) and cyclic GMP-dependent protein kinase (PKG) inhibitors protected from cell death for up to 10 h after NO addition; the antioxidant ascorbic acid also protected from cell death but its efficacy decreased when it was added after NO treatment (40% protection 2 h after NO addition). The pattern of cell death was characterized by an increase in chromatin condensed cells with no DNA fragmentation and with breakdown of plasmatic membrane. The inhibition of RNA and protein synthesis and of caspase activity also protected from cell death. This study shows that alterations in GSH levels change the neurotrophic effects of NO in midbrain cultures into neurotoxic. Under these conditions, NO triggers a programmed cell death with markers of both apoptosis and necrosis characterized by an early step of free radicals production followed by a late requirement for signalling on the sGC/cGMP/PKG pathway.
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PMID:Glutathione depletion switches nitric oxide neurotrophic effects to cell death in midbrain cultures: implications for Parkinson's disease. 1175 59

In mammalian cells, a growing body of evidence indicates a relationship between cellular redox balance and tyrosine kinase-mediated cell signalling. The phosphorylative cascade activated by extracellular signals is inhibited by reducing conditions and stimulated by oxidative stress, in particular at the level of mitogen activated protein kinase (MAPK) activation. The mussel Mytilus typically shows variations in antioxidant defence systems and decreases in glutathione content in response to both natural and contaminant environmental stressors. In isolated mussel digestive gland cells, both epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) have been recently demonstrated to activate tyrosine kinase receptors leading to multiple responses; among these, stimulation of the key glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK). The present study investigates the possible relationship between the tyrosine kinase-mediated metabolic effects of growth factors and cellular redox balance in mussel cells. The results demonstrate that the effects of growth factors on glycolytic enzymes were abolished by cell pretreatment with the antioxidant N-acetyl-cysteine (NAC). On the other hand, in cells where the glutathione content and synthesis were lowered either in vitro (by cell pretreatment with buthionine sulfoximine (BSO)), or in vivo (by mussel exposure to Cu(2+)) the metabolic effects of growth factors were unaffected. Moreover, the results show that, in both control and glutathione-depleted cells, growth factors can also regulate the level of glutathione apparently by modulating, via phosphorylative mechanisms involving MAPK activation, the activity of gamma-glutamylcysteine synthetase (GCS), the rate limiting enzyme in GSH biosynthesis. Overall, this study extends the hypothesis that cell signalling is intimately related to redox balance in marine invertebrate cells.
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PMID:Growth factor-mediated signal transduction and redox balance in isolated digestive gland cells from Mytilus galloprovincialis Lam. 1179 Mar 56


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