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 light-activated protein kinase C inhibitor, calphostin C, is shown to inhibit the ability of IL-3-dependent 32D cells to reduce the tetrazolium salt, MTT. To determine whether this inhibition was mediated through mitochondria which have been implicated in MTT reduction, isolated mitochondria were treated with calphostin C in the presence of various substrates for mitochondrial electron transport and EDTA (to exclude PKC involvement). Calphostin C extensively inhibited succinate-dependent MTT reduction (IC50 = 110nM) but had little effect on either NADH- or NADPH-dependent MTT reduction. An alternative protein kinase C inhibitor, H7, did not affect succinate-dependent mitochondrial MTT reduction, and the protein kinase A inhibitor, KT5720, had little effect on either cellular or mitochondrial MTT reduction. These results show that in addition to its role as a PKC inhibitor, calphostin C is also a potent inhibitor of succinate-dependent mitochondrial electron transport.
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PMID:The protein kinase C inhibitor, calphostin C, inhibits succinate-dependent mitochondrial reduction of MTT by a mechanism that does not involve protein kinase C. 137 66

A simple, improved procedure for the isolation of guanine-nucleotide-exchange factor (GEF) and for eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocyte lysates has been developed using ion-exchange chromatography on S-Sepharose, Q-Sepharose, Mono Q and Mono S. The majority of the eIF-2 is separated from GEF at an early stage in the procedure and the remaining small amount of eIF-2.GEF complex is separated from the bulk of the GEF by FPLC on Mono S. The procedure yields approximately 2 mg each of eIF-2 and GEF, of 90% and greater than 80% purity, respectively, from the blood of ten rabbits. All fractions of purified GEF contain four subunits of molecular masses 84, 66, 54 and 39 kDa, with various amounts of a fifth, 30-kDa subunit. The modulation of GEF activity was investigated using the highly purified factor in a guanine-nucleotide-exchange assay. The activity of GEF was stimulated by physiological concentrations of the polyamines, spermine and spermidine, but was unaffected by another polycationic compound, polylysine. Activity was also found to be inhibited by 1 mM NADP+ or NAD+, and this inhibition was overcome by the presence of 1 mM NADPH. Stoichiometric amounts of GEF were unable to release GDP from eIF-2.GDP complexes in the absence of free guanine nucleotides, suggesting that GEF operates by a ternary-complex mechanism. Casein kinase 1 or casein kinase 2 can each phosphorylate the largest subunit (84 kDa) of GEF. These enzymes both phosphorylate serine residues in GEF but they phosphorylate distinct sites, as demonstrated by phosphopeptide mapping following proteolytic or cyanogen bromide digestion. Neither of these kinases phosphorylated any of the other subunits of GEF to any significant extent and several other kinases were inactive against GEF. No effect of phosphorylation on activity could be demonstrated.
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PMID:Purification, phosphorylation and control of the guanine-nucleotide-exchange factor from rabbit reticulocyte lysates. 151 90

The NADPH-oxidase of human neutrophils can be activated in a cell-free system comprised of plasma membrane, cytosol, and an anionic amphiphile such as arachidonate or sodium dodecyl sulfate (SDS). Recently, we showed that diacylglycerol acts synergistically with SDS in the cell-free system to stimulate superoxide generation, with concurrent phosphorylation of a 47-kDa cytosolic protein which is thought to be a component of the oxidase (Burnham, D. N., Uhlinger, D. J., and Lambeth, J. D. (1990) J. Biol. Chem. 265, 17550-17559). We report herein that when undialyzed cytosol is used along with either SDS alone or SDS plus diacylglycerol as activators, adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) both stimulated superoxide generation several fold, yielding about the same maximal velocity. ATP and GTP showed lower levels of stimulation. Stimulation by ATP gamma S and GTP gamma S was nonadditive, and showed a 5-7-fold greater specificity for GTP gamma S. ATP gamma S stimulation was inhibited by the nucleoside diphosphate (NDP) kinase inhibitor UDP. In contrast, when extensively dialyzed cytosol was used, most of the stimulation by ATP gamma S was lost, while most of that by GTP gamma S was retained. Addition of GDP restored the ability of ATP gamma S to stimulate, consistent with NDP kinase-catalyzed formation of GTP gamma S from ATP gamma S plus GDP. This activity was demonstrated directly in both cytosol and plasma membrane. Using undialyzed cytosol, phosphorylation of p47 showed a similar nonspecificity for nucleoside triphosphates, due to NDP kinase activity, but revealed the expected ATP specificity when dialyzed cytosol was used. Neither ATP gamma S nor GTP gamma S were good substrates for protein phosphorylation. Under a variety of conditions, phosphorylation of p47 or other neutrophil proteins failed to correlate with oxidase activation. The present studies indicate that SDS and diacylglycerol stimulation of superoxide generation in the cell-free system is independent of protein kinase C or other protein kinase activity, and suggest a novel role for diacylglycerol in cell regulation.
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PMID:Nucleoside triphosphate requirements for superoxide generation and phosphorylation in a cell-free system from human neutrophils. Sodium dodecyl sulfate and diacylglycerol activate independently of protein kinase C. 165 41

Nitric oxide synthase was purified to apparent homogeneity from the cytosolic fractions obtained from rat and porcine cerebellum. Enzyme activity--measured as [3H]citrulline formation after incubation with [3H]arginine--was dependent on Ca2+/calmodulin, NADPH, and tetrahydro-L-biopterin. Specific activity varied between 450 to 780 nmol/min/mg protein. Purified nitric oxide synthases showed a single band on 8% SDS/PAGE gels and had an apparent molecular mass of 150,000 Da. The purified proteins were used as substrate for phosphorylation with different protein kinases. In the assays using two Ca2+/calmodulin-dependent protein kinases, CaM kinase II and CaM kinase-Gr, protein kinase C, and the catalytic subunit of protein kinase A, nitric oxide synthase was exclusively phosphorylated by protein kinase A. Such phosphorylation was linear over time for at least 60 min and resulted in nearly stoichiometric phosphate/protein incorporation. The serine in the protein kinase A-consensus sequence KRFGS is probably the site of phosphorylation in nitric oxide synthase. Kemptide, a known protein kinase A substrate, inhibited phosphorylation of nitric oxide synthase in a dose-dependent manner. No changes in nitric oxide synthase activity were observed upon phosphorylation by protein kinase A.
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PMID:Phosphorylation of nitric oxide synthase by protein kinase A. 172 13

A single i.p. injection of cisplatin (10 mg/kg body weight) into mice results in a significant increase in chemiluminescence and ATP contents of the peritoneal exudate cells (PEC) than that of PEC from untreated mice. It is also observed that in vitro treatment of macrophages with cisplatin, rIFN-gamma and LPS show increased activity of the protein kinase-C (PK-C). The activation of PK-C could result in stimulation of NADPH-oxidase resulting in increased levels of chemiluminescence. Increased contents of ATP in PEC after cisplatin treatment also suggests that this activation is energy dependent.
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PMID:Studies on chemiluminescence and protein kinase-C activity of cisplatin treated mouse peritoneal exudate cells. 177 Feb 12

We have investigated the possible role of plasma membrane oxidoreductases in the Ca2+ export mechanisms in rat brain synaptic membranes. Ca2+ efflux in nerve terminals is controlled both by a high-affinity/low capacity Mg-dependent ATP-stimulated Ca2+ pump and by a low affinity/high capacity ATP-independent Na(+)-Ca2+ exchanger. Both Ca2+ efflux mechanisms were strongly inhibited by pyridine nucleotides, in the order NADP greater than NAD greater than NADPH greater than NADH with IC50 values of ca. 10 mM for NADP and ca. 3 mM for the other agents in the case of the ATP-driven Ca2+ pump and with IC50 values between 8 and 10 mM for the Na(+)-Ca2+ exchanger. Oxidizing agents such as DCIP3 and ferricyanide inhibited the ATP-driven Ca2+ efflux mechanism but not the Na(+)-Ca2+ exchanger. In addition, full activation of plasma membrane oxidoreductases requires both an acceptor and an electron donor; therefore the combined effects of both substrates added together were also studied. When plasma membrane oxidoreductases of the synaptic plasma membrane were activated in the presence of both NADH (or NADPH) and DCIP or ferricyanide, the inhibition of the ATP-driven Ca2+ pump was optimal; by contrast, the pyridine nucleotide-mediated inhibition of the Na(+)-Ca2+ exchanger was partially released when both substrates of the plasma membrane oxidoreductases were present together. Furthermore, the activation of plasma membrane oxidoreductases also strongly inhibited intracellular protein phosphorylation in intact synaptosomes, mediated by either cAMP-dependent protein kinase, Ca2+ calmodulin-dependent protein kinases, or protein kinase C.
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PMID:Effects of plasma membrane oxidoreductases on Ca2+ mobilization and protein phosphorylation in rat brain synaptosomes. 224 77

Partially reduced oxygen species are toxic, yet sea urchin eggs synthesize H2O2 in a "respiratory burst" at fertilization, as an extracellular oxidant to crosslink their protective surface envelopes. To study the biochemical mechanism for H2O2 production, we have isolated an NADPH-specific oxidase fraction from homogenates of unfertilized Strongylocentrotus purpuratus eggs that produces H2O2 when stimulated with Ca2+ and MgATP2-. Concentrations of free Ca2+ previously implicated in regulation of egg activation modulate the activity of the oxidase. Inhibitors were used to test the relevance of this oxidase to the respiratory burst of fertilization. Procaine, two phenothiazines, and N-ethylmaleimide (but not iodoacetamide) inhibited H2O2 production by the oxidase fraction and oxygen consumption by activated eggs. The ATP requirement suggested that protein kinase activity might regulate the respiratory burst of fertilization; consonant with this hypothesis, H-7 and staurosporine were inhibitory. The respiratory burst oxidase of fertilization is an NADPH:O2 oxidoreductase that appears to be regulated by a protein kinase; although it bears a remarkable resemblance to the neutrophil oxidase, unlike the latter it does not form O2- as its initial product.
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PMID:Respiratory burst oxidase of fertilization. 253 93

We have previously reported that addition of Ca2+ and phospholipid (PL) inhibits translation in hemin-containing reticulocyte lysates through activation of a eukaryotic protein synthesis initiation factor (eIF-2) kinase. The possibility that this activation was mediated by a Ca2+-PL-dependent protein kinase (protein kinase C, PKC) appeared unlikely by the observation that it was prevented or reversed by NADPH-generating systems. Nevertheless, reticulocyte lysates contain a potent PKC activity and we deemed it desirable to isolate this enzyme to answer unequivocally the question whether it does or does not activate eIF-2 alpha kinase. We have purified reticulocyte PKC to near homogeneity with Mr 95,500 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme absolutely depended upon both Ca2+ and phosphatidylserine for activity on histone H1 or the beta-subunit of initiation factor eIF-2 and underwent autophosphorylation in a Ca2+- and PL-dependent manner. Mild treatment with trypsin yielded an Mr 82,000 polypeptide that still required Ca2+ and PL for activity. This Mr agrees with that reported for other PKCs, suggesting that these enzymes may undergo limited degradation during isolation. Further proteolytic treatment converted the reticulocyte enzyme into a Ca2+- and PL-dependent form, as is known for PKCs from other sources. The highly purified PKC had no effect on translation in hemin-supplemented reticulocyte lysates.
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PMID:Purification and properties of protein kinase C from rabbit reticulocyte lysates. 282 7

When human neutrophilic granulocytes are stimulated with chemoattractants or phorbol esters, these cells respond with a so-called respiratory burst: such stimuli induce the activation of a NADPH:O2 oxidoreductase, which converts oxygen into superoxide. This activation coincides with the phosphorylation of a number of proteins, amongst which a 47-kDa phosphoprotein. Neutrophils from patients with the autosomal form of chronic granulomatous disease (CGD) fail to mount a respiratory burst and concomitantly lack phosphorylation of the 47-kDa protein. We have shown this protein to be a substrate for protein kinase C. In the present paper we describe the phosphorylation of the 47-kDa phosphoprotein by cyclic AMP-dependent protein kinase. For these studies, we used neutrophil cytoplasts, i.e., neutrophils devoid of nucleus and granules, but with an intact NADPH:O2 oxidoreductase. Addition of dibutyryl cyclic AMP (Bt2cAMP) to intact human neutrophil cytoplasts resulted in an increase in protein phosphorylation. Among the phosphorylated proteins is a 47-kDa phosphoprotein. Increased protein phosphorylation was also observed upon addition of Bt2cAMP to neutrophil cytoplast lysates. In lysates of neutrophil cytoplasts from patients with the autosomal form of CGD, phosphorylation of the 47-kDa protein was absent. This finding (confirmed by analysis on two-dimensional gels) indicates that the 47-kDa phosphoprotein, relevant for the NADPH:O2 oxidoreductase, is a substrate for the cAMP-dependent protein kinase. Unlike phorbol ester-induced phosphorylation, Bt2cAMP-induced phosphorylation is not accompanied by initiation of a respiratory burst. This observation demonstrates that 47-kDa phosphoprotein phosphorylation can be uncoupled from respiratory burst activity and indicates that other modifications of the NADPH:O2 oxidoreductase are required for induction of activity.
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PMID:The 47-kDa protein involved in the NADPH:O2 oxidoreductase activity of human neutrophils is phosphorylated by cyclic AMP-dependent protein kinase without induction of a respiratory burst. 284 87

Phorbol ester treatment of intact neutrophils both stimulates protein kinase C (PK-C) and causes the rapid proteolytic conversion to a cytosolic, co-factor independent fragment, protein kinase M (PK-M). In intact neutrophils, phorbol ester treatment activates the NADPH-oxidase, the enzyme responsible for the oxidative burst. Addition of purified PK-M to resting neutrophil light density membranes activated the NADPH-oxidase in the presence of PS, ATP and Mg2+. A 3.5-fold greater stimulation of oxidase (ca. 25 nmoles O2-/min/mg membrane protein) was obtained with comparable PK-M concentrations to that observed with the reconstituted PK-C system, and approximately 1/3 that obtained with arachidonic acid (AA) or SDS. In contrast to the reconstituted system using PK-C, PMA and Ca++ were neither required nor affected activity. The effect of PS was unexpected, since PK-M does not require phospholipids for enzymatic activity, and likely represents the action of PS on the oxidase itself or on another component in the plasma membrane fraction. Our studies demonstrate for the first time that purified PK-M permits reconstitution of a physiologic phorbol ester response.
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PMID:Activation of human neutrophil NADPH-oxidase in vitro by the catalytic fragment of protein kinase-C. 292 44


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