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)

Purified cholesterol 7alpha-hydroxylases (C7alphaH) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma-32P] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. The amounts of 32P incorporation after separation of human and rat C7alphaH proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7alphaH catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7alphaH activities significantly decreased after dephosphorylation by AP (-57% - -72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP-dependent protein kinase. The increases in C7alphaH activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to 32P incorporation into the purified enzymes. Both the activation of C7alphaH and the amounts of 32P incorporation were time-dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP-dependent protein kinase. In a second set of experiments, purified human and rat liver C7alphaH were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7alphaH by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase. Rephosphorylation of the dephosphorylated C7alphaH proteins by cAMP-dependent protein kinase increased C7alphaH catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in 32P incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and 32P incorporation into the human C7alphaH protein. Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7alphaH (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/dephosphorylation mechanism in both the human and the rat enzymes.
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PMID:Cholesterol 7alpha-hydroxylase activities from human and rat liver are modulated in vitro posttranslationally by phosphorylation/dephosphorylation. 893 82

1. The possible mechanisms of action of the inhibitory effect of abruquinone A on the respiratory burst in rat neutrophils in vitro was investigated. 2. Abruquinone A caused an irreversible and a concentration-dependent inhibition of formylmethionylleucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2.-) generation with IC50 values of 0.33 +/- 0.05 microgram ml-1 and 0.49 +/- 0.04 microgram ml-1, respectively. 3. Abruquinone A also inhibited O2 consumption in neutrophils in response to fMLP/CB and PMA. However, abruquinone A did not scavenge the generated O2.- in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation. 4. Abruquinone A inhibited both the transient elevation of [Ca2+]i in the absence of [Ca2+]o (IC50 7.8 +/- 0.2 micrograms ml-1) and the generation of inositol trisphosphate (IP3) (IC50 10.6 +/- 2.0 micrograms ml-1) in response to fMLP. 5. Abruquinone A did not affect the enzyme activaties of neutrophil cytosolic protein kinase C (PKC) and porcine heart protein kinase A (PKA). 6. Abruquinone A had no effect on intracellular guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels but decreased the adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 7. The cellular formation of phosphatidic acid (PA) and phosphatidylethanol (PEt) induced by fMLP/ CB was inhibited by abruquinone A with IC50 values of 2.2 +/- 0.6 micrograms ml-1 and 2.5 +/- 0.3 micrograms ml-1, respectively. Abruquinone A did not inhibit the fMLP/CB-induced protein tyrosine phosphorylation but induced additional phosphotyrosine accumulation on proteins of 73-78 kDa in activated neutrophils. 8. Abruquinone A inhibited both the O2.- generation in PMA-activated neutrophil particulate NADPH oxidase (IC50 0.6 +/- 0.1 microgram ml-1) and the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free system (IC50 1.5 +/- 0.2 micrograms ml-1) 9. Collectively, these results indicate that the inhibition of respiratory burst in rat neutrophils by abruquinone A is mediated partly by the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways, and by suppressing the function of NADPH oxidase through the interruption of electron transport.
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PMID:Cellular localization of the inhibitory action of abruquinone A against respiratory burst in rat neutrophils. 913 99

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

1. The ability of acetylshikonin to inhibit the respiratory burst in rat neutrophils was characterized and the underlying mechanism of action was also assessed in the present study. 2. Acetylshikonin caused an irreversible and a concentration-dependent inhibition of formylmethionylleucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2.-) generation with IC50 values of 0.48 +/- 0.03 and 0.39 +/- 0.03 microM, respectively. Acetylshikonin also inhibited the O2 consumption in neutrophils in response to fMLP/CB as well as to PMA. 3. Acetylshikonin did not scavenge the generated O2.- in the xanthine-xanthine oxidase system or during dihydroxyfumaric acid (DHF) autoxidation but, on the contrary, acetylshikonin enhanced the O2.- generation in these cell-free oxygen radical generating systems. 4. Acetylshikonin inhibited the formation of inositol trisphosphate (IP3) (39.0 +/- 7.8% inhibition at 10 microM, P < 0.05) in neutrophils in response to fMLP. 5. Both the neutrophil cytosolic protein kinase C (PKC) activity and the PMA-induced PKC associated with the membrane were unaffected by acetylshikonin. 6. Acetylshikonin did not affect the porcine heart protein kinase A (PKA) activity. Upon exposure to acetylshikonin, the cellular cyclic AMP level was decreased in neutrophils in response to fMLP. 7. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP/CB were inhibited by acetylshikonin (60.1 +/- 7.3 and 63.2 +/- 10.5% inhibition, respectively, at 10 microM, both P < 0.05). Moreover, acetylshikonin attenuated the fMLP/CB-induced protein tyrosine phosphorylation (about 90% inhibition at 1 microM). 8. In PMA-activated neutrophil particulate NADPH oxidase preparations, acetylshikonin did not inhibit, but enhanced, the O2.- generation in the presence of NADPH. However, acetylshikonin decreased the membrane associated p47phox in PMA-activated neutrophils (about 60% inhibition at 1 microM). 9. Collectively, these results suggest that the attenuation of protein tyrosine phosphorylation and a failure in the assembly of a functional NADPH oxidase complex probably contribute predominantly to the inhibition of respiratory burst in neutrophils by acetylshikonin. In contrast, the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways play only a minor role in this respect.
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PMID:Investigation of the inhibition by acetylshikonin of the respiratory burst in rat neutrophils. 917 81

Saccharomyces cerevisiae pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HA-Tpk1.Bcy1) and to be phosphorylated in a cAMP-dependent process. Both glutathione S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GST-Pyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated in vivo, in intact cells overexpressing the protein, or in vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK gene with an attenuated mutation (tpk1(w1)). The effect of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels. Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mm. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6-bisphosphate it shows an n(H) value of 1.4, as compared with an n(H) of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity.
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PMID:In vivo and in vitro phosphorylation of two isoforms of yeast pyruvate kinase by protein kinase A. 1206 46

We demonstrate the occurrence of a cAMP-dependent protein kinase in Dictyostelium discoideum cells at the terminal stage of differentiation. A cAMP-binding component was purified to homogeneity by affinity chromatography. This subunit inhibits the activity of purified catalytic subunit from beef heart protein kinase; the inhibition is reversed upon addition of cAMP. The protein is highly specific for cAMP and has a dissociation constant of 4 nM. The isolated regulatory subunit is a monomer of 39 K, with a sedimentation coefficient of 3.5S and a frictional coefficient of 1.24. The differences between this regulatory subunit and regulatory subunits of protein kinases from other sources are discussed.
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PMID:A cAMP-dependent protein kinase is present in differentiating Dictyostelium discoideum cells. 1645 31

Cyclic adenosine 3':5'-monophosphate (cAMP) was extensively purified from rye grass (Lolium multiflorum) endosperm cells grown in axenic suspension culture. The cAMP was purified by neutral alumina and anion and cation exchange chromatography. The cAMP was quantitated by means of a radiochemical saturation assay using a beef heart cAMP-binding protein and also by an assay involving activation of beef heart protein kinase. The cAMP levels found (corrected for recovery of tracer cyclic 3',5'-[8-(3)H]AMP included from the point of sample extraction) ranged from 2 to 12 pmol/g fresh weight. The material purified from rye grass cultures was indistinguishable from authentic cAMP with respect to chromatography in two cellulose thin layer systems, behavior on dilution in both the saturation and protein kinase activation assays, and rates of degradation by a mammalian cAMP phosphodiesterase. The cAMP from rye grass cultures was completely degraded by a mammalian cAMP phosphodiesterase, and 1-methyl-3-isobutylxanthine inhibited such degradation. The protein kinase activation and saturation assays gave essentially the same values for the cAMP content of axenic rye grass culture extracts. Material satisfying the above criteria for identity with cAMP was also isolated from the culture medium. The increase observed in medium cAMP levels during culture growth provides evidence for the synthesis and secretion of cAMP by rye grass endosperm cells in suspension culture.
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PMID:Cyclic adenosine 3':5'-monophosphate in axenic rye grass endosperm cell cultures. 1666 Mar 72

We report the development of a peptide microarray based on previously determined phosphorylation sites in chloroplast proteins. Altogether, 905 peptides were spotted as 15mers in nine replicates onto glass slides. We used the microarray for in vitro phosphorylation experiments and specifically assessed the peptide substrate spectrum of chloroplast casein kinase II (pCKII). To this end, native pCKII from Arabidopsis thaliana and Sinapis alba chloroplasts was enriched by Heparin-Sepharose chromatography and its activity on the microarray was compared to the activity of a recombinant Arabidopsis pCKII. All three kinase preparations phosphorylated a similar set of peptides that were clearly distinct from those phosphorylated by bovine heart protein kinase A (PKA) in control experiments. The majority of the pCKII phosphorylation targets are involved in plastid gene expression, supporting the earlier denomination of pCKII as plastid transcription kinase (PTK). In addition we identified Alb3 as pCKII substrate that is essential for the integration of light-harvesting complex subunits (LHC) into the thylakoid membrane. Plastid CKII phosphorylation activity was characterized in greater detail in vitro with recombinant wildtype Alb3 and phosphorylation site mutants as substrates, establishing S424 as the pCKII phosphorylation site. Our data show that the peptide microarray ChloroPhos1.0 is a suitable tool for the identification of new kinase downstream targets in vitro that can be validated subsequently by in vivo experiments.
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PMID:The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana. 2529 73

The eukaryotic cell cycle is negatively regulated by cyclin-dependent kinase inhibitors (CKIs). p57^Kip2 is a member of the Cip/Kip family of CKIs and frequently inactivated by genomic mutations associated with human overgrowth disorders. There is increasing evidence for p57 to control cellular processes in addition to cell cycle and CDK regulation including transcription, apoptosis, migration or development. In order to obtain molecular insights to unknown functions of p57, we performed a protein interaction screen. We identified the transcription regulator four-and-a-half LIM-only protein 2 (FHL2) as a novel p57-binding protein. Co-immunoprecipitation and reporter gene assays were used to elucidate the physiological and functional relevance of p57/FHL2 interaction. We found in cancer cells that endogenous p57 and FHL2 are in a complex. We observed a substantial induction of established FHL2-regulated gene promoters by p57 in reporter gene experiments and detected strong induction of the intrinsic transactivation activity of FHL2. Treatment of cells with histone deacetylase (HDAC) inhibitors and binding of exogenous FHL2 to HDACs indicated repression of FHL2 transcription activity by HDACs. In the presence of the HDAC inhibitor sodium butyrate activation of FHL2 by p57 is abrogated suggesting that p57 shares a common pathway with HDAC inhibitors. p57 competes with HDACs for FHL2 binding which might partly explain the mechanism of FHL2 activation by p57. These results suggest a novel function of p57 in transcription regulation.
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PMID:The CDK inhibitor p57^Kip2 enhances the activity of the transcriptional coactivator FHL2. 3234 31


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