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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The addition of phorbol esters to U937 leukemic cells stimulates the phosphorylation of c-Jun on serines 63 and 73. To isolate the protein kinase which stimulates this phosphorylation, we have used heparin-Sepharose chromatography followed by affinity chromatography over glutathione-Sepharose beads bound with a fusion protein of glutathione S-transferase and amino acids 5-89 of c-Jun (GST-c-Jun). Using this procedure we purify a 67-kDa protein which is capable of phosphorylating GST-c-Jun as well as the complete c-Jun protein. By making mutations in serines 63 and 73 and then creating a fusion protein with GST (GST-c-Jun mut), we demonstrate that this protein kinase specifically phosphorylates these sites in the c-Jun amino terminus. Treatment of purified c-Jun amino-terminal protein kinase (cJAT-PK) with phosphatase 2A inhibits its ability to phosphorylate GST-c-Jun. This inactivated enzyme can be reactivated by phosphorylation with protein kinase C (PKC), although PKC is not capable of phosphorylating the GST-c-Jun substrate. Because v-Jun cannot be phosphorylated in vivo, we compared the ability of cJAT-PK to bind to GST-v-Jun or GST-c-Jun mut. The cJAT-PK bound 50-fold better to GST-c-Jun mut than GST-v-Jun suggesting that the delta domain which is missing in v-Jun plays a role in binding the cJAT-PK. These results suggest that there is a protein kinase cascade mediated by protein phosphatases and PKC which regulates c-Jun phosphorylation.
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PMID:Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity. 132 19

Calponin, a thin-filament protein of smooth muscle, has been implicated in the regulation of smooth-muscle contraction, since in vitro the isolated protein inhibits the actin-activated myosin MgATPase. This inhibitory effect, and the ability of calponin to bind to actin, is lost after its phosphorylation by protein kinase C or Ca2+/calmodulin-dependent protein kinase II [Winder & Walsh (1990) J. Biol. Chem. 265, 10148-10155]. If this phosphorylation reaction is of physiological significance, there must be a protein phosphatase in smooth muscle capable of dephosphorylating calponin and restoring its inhibitory effect on the actomyosin MgATPase. We demonstrate here the presence, in chicken gizzard smooth muscle, of a single major phosphatase activity directed towards calponin. This phosphatase was purified from the soluble fraction of chicken gizzard by (NH4)2SO4 fractionation and sequential chromatography on Sephacryl S-300, DEAE-Sephacel, omega-amino-octyl-agarose and thiophosphorylated myosin 20 kDa light-chain-Sepharose columns. The purified phosphatase contained three polypeptide chains of 60, 55 and 38 kDa which were shown to be identical with the subunits of SMP-I, a smooth-muscle phosphatase capable of dephosphorylating the isolated 20 kDa light chain of myosin but not intact myosin [Pato & Adelstein (1983) J. Biol. Chem. 258, 7047-7054]. Consistent with its identity with SMP-I, calponin phosphatase was classified as a type-2A protein phosphatase. Of several potential phosphoprotein substrates examined, calponin proved to be kinetically the best, suggesting that calponin may be a physiological substrate for this phosphatase. Finally, dephosphorylation of calponin which had been phosphorylated by protein kinase C restored completely its ability to inhibit the actin-activated MgATPase of smooth-muscle myosin. These observations support the hypothesis that calponin plays a role in regulating the contractile state of smooth muscle and that this function in turn is controlled by phosphorylation-dephosphorylation.
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PMID:Purification and characterization of calponin phosphatase from smooth muscle. Effect of dephosphorylation on calponin function. 132 79

The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) has been shown to potentiate the stimulatory effect of ethanol on the hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Following an initial 20-min period, the main product of PtdEtn degradation in cells treated with TPA plus ethanol was ethanolamine phosphate. Here, we have examined the regulatory role of PKC and the possible catalytic role of phospholipase C in the formation of ethanolamine phosphate. TPA, bryostatin, and bombesin, direct or indirect activators of PKC, had similar potentiating effects on ethanol-induced formation of [14C]ethanolamine phosphate from [14C]PtdEtn in [14C]ethanolamine-prelabelled NIH 3T3 fibroblasts. At lower concentrations of ethanol (40-80 mM), significant stimulation of ethanolamine phosphate formation required longer treatments (2 h or longer). The combined effects of TPA (100 nM) and ethanol (50-200 mM) on ethanolamine phosphate formation were not inhibited by the PKC inhibitors staurosporine or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7). In contrast, these inhibitors significantly inhibited TPA-induced formation of ethanolamine, catalyzed by a phospholipase-D-type enzyme. In membranes isolated from TPA+ethanol-treated cells, enhanced formation of ethanolamine phosphate was maintained for at least 20 min. Down-regulation of PKC by prolonged (24-h) treatment of NIH 3T3 fibroblasts by 300 nM TPA enhanced, while overexpression of alpha-PKC in Balb/c fibroblasts diminished, the stimulatory effect of ethanol on the formation of ethanolamine phosphate. Finally, addition of the protein phosphatase inhibitor okadaic acid (2 microM) to fibroblasts inhibited TPA+ethanol-induced formation of ethanolamine phosphate. These results suggest that alpha-PKC-mediated protein phosphorylation may negatively regulate PtdEtn hydrolysis and that the potentiating effect of TPA may result, at least partly, from increased degradation of this PKC isoform.
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PMID:The long-term combined stimulatory effects of ethanol and phorbol ester on phosphatidylethanolamine hydrolysis are mediated by a phospholipase C and prevented by overexpressed alpha-protein kinase C in fibroblasts. 132 80

Arachidonic acid (AA) increased, at constant Ca2+, the levels of force and 20-kDa myosin light chain (MLC20) phosphorylation in permeabilized smooth muscle, and slowed relaxation and MLC20 dephosphorylation. The Ca(2+)-sensitizing effect of AA was not inhibited by inhibitors of AA metabolism (indomethacin, nordihydroguaiaretic acid, or propyl gallate), of protein kinase C (pseudopeptide) or by guanosine-5'-O-(beta-thiodiphosphate) and was abolished by oxidation of AA in air. A non-metabolizable AA analog, 5,8,11,14-eicosatetraynoic acid) also had Ca(2+)-sensitizing effects. Extensive treatment with saponin abolished the Ca(2+)-sensitizing effects of phorbol 12,13-dibutyrate and guanosine-5'-O-(gamma-thiotriphosphate), but not that of AA. A purified, oligomeric MLC20 phosphatase isolated from gizzard smooth muscle was dissociated into subunits by AA, and its activity was inhibited toward heavy meromyosin but not phosphorylase. We conclude that AA may act as a messenger-promoting protein phosphorylation through direct inhibition of the form of protein phosphatase(s) that dephosphorylate MLC20 in vivo.
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PMID:Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium. 132 35

An endogenous protein which inhibits protein kinase C (PKC)-mediated effects has been detected in rat heart ventricular tissue. This functional PKC-inhibitory activity was completely abolished by okadaic acid, making it possible to measure PKC activity in non-purified cell fractions. This suggests that the PKC-inhibitory activity is a type 1 or 2A serine/threonine phosphatase. Confirming this, membrane and cytosolic PKC-inhibitory preparations were found to contain phosphatase activity which was suppressed by okadaic acid, exhibiting an IC50 (concn. required for 50% inhibition) of 1.5-2 nM. Furthermore, okadaic acid stimulated prostacyclin production in rat cardiomyocytes and aortic smooth-muscle cells and, like the PKC activator phorbol 12-myristate 13-acetate, it augmented the prostacyclin formation induced by the Ca2+ ionophore A23187. Our results strongly suggest that the endogenous PKC 'inhibitor' is the cellular phosphatase 2A, which plays an important role in regulating the phosphorylation level of PKC target proteins.
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PMID:Functional inhibition of protein kinase C-mediated effects in myocardial tissue is due to the phosphatase 2A. 132 18

The role of protein phosphatases in the regulation of insulin release from rat pancreatic islets was studied with protein phosphatase inhibitors, okadaic acid and calyculin A. Okadaic acid inhibited glucose- and glyceraldehyde-induced insulin release dose-dependently and also inhibited the potentiation of glucose-induced release either by adding forskolin, an activator of adenylate cyclase or by increasing K+ concentration to 25 mM. At a non-stimulatory concentration of 3 mM glucose, a high concentration (2 microM) of okadaic acid inhibited insulin release induced by high K+ or 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, but a low concentration (1 microM) of okadaic acid did not significantly inhibit TPA-induced insulin release. Calyculin A also inhibited glucose-induced insulin release, and the effect was greater than that of okadaic acid. The data suggest that protein phosphatases may play an important role in the regulation of insulin release.
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PMID:Effects of the protein phosphatase inhibitors okadaic acid and calyculin A on insulin release from rat pancreatic islets. 133 May 3

The phosphorylation and dephosphorylation of the 25 kDa mRNA cap binding protein eukaryotic initiation factor-4E (eIF-4E) is regulated during different physiologic and pathophysiologic states that include cell growth and the late phase of adenovirus infection. We have found that okadaic acid is much more effective in increasing the phosphorylated fraction of eIF-4E than phorbol 12-myristate 13-acetate in Hep G2 cells. Phosphoprotein phosphatase 2A dephosphorylated eIF-4E isolated from both phorbol 12-myristate 13-acetate- or okadaic acid-treated cells, whereas alkaline and acid phosphatase were relatively ineffective. The ability of purified [35S]eIF-4E isolated from okadaic acid-treated cells to bind mRNA caps was compared to phosphoprotein phosphatase 2A-treated [35S]eIF-4E and found to be no different. This suggests that alternative explanations for the previously observed effects of eIF-4E phosphorylation on protein synthesis must be considered. In addition, our results indicate that the in vivo phosphorylation of eIF-4E is not catalyzed solely by protein kinase C.
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PMID:Phosphoprotein phosphatase 2A dephosphorylates eIF-4E and does not alter binding to the mRNA cap. 133 9

Cytokines such as interleukin-1, which are found in the brain after trauma, regulate expression of nerve growth factor (NGF) mRNA and protein in hippocampal cultures. We have investigated possible mechanisms by which Il-1 beta regulates NGF in hippocampal cells. The induction of NGF mRNA by Il-1 beta was blocked by a receptor antagonist indicating that this effect is receptor mediated. Il-1 beta elicited a dramatic induction of c-fos mRNA and a slight elevation of c-jun mRNA in a time dependent manner which may allow for a role in the induction of NGF mRNA expression. We examined whether specific second messenger pathways were involved in mediating the action of Il-1 beta in the hippocampus. Activation of cAMP with forskolin or treatment with 8-Br-cAMP had no effect on NGF mRNA levels. Moreover, exposure of hippocampal cultures to Il-1 beta evoked no change in cAMP levels, indicating that this second messenger system played little or no role in the regulation of NGF expression by Il-1 beta in these cells. Further, interleukin-1 elicited no change in membrane inositol phosphate turnover, nor did it affect intracellular calcium levels. Treatment of cell cultures with the phorbol ester PMA elicited an increase in NGF mRNA, suggesting that activation of protein kinase C (PKC) may mediate NGF mRNA expression. However, prolonged treatment of cultures with PMA to desensitize PKC did not eliminate the Il-1 beta induction of NGF mRNA. Il-1 beta, therefore, did not appear to activate NGF expression via cAMP, Ca2+, or a PKC isoform that is downregulated by prolonged PMA treatment. However, a phosphorylation event may be involved in the signal transduction mechanism, as treatment with okadaic acid to inhibit protein phosphatase 2a potentiated the induction of NGF mRNA by Il-1 beta. The results presented indicate that Il-1 beta acts via its receptor to induce a rise in NGF expression. Identification of the specific second messenger pathway has remained elusive; however, a phosphorylation event appears to be intermediary. Moreover, the induction of c-fos and c-jun may represent a final common path in activation of NGF gene expression by different signals such as Il-1 beta and PMA.
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PMID:Mechanisms of nerve growth factor mRNA regulation by interleukin-1 beta in hippocampal cultures: role of second messengers. 133 37

The superoxide anion generation in Ehrlich ascites tumour (EAT) cells increased more than two-fold in the presence of the tumour promoter, tetradecanoyl phorbol myristate acetate (TPA). Epinephrine and dibutryl cAMP (Bt2 cAMP) inhibited in a dose-dependent manner, both basal and TPA-triggered superoxide generation in EAT cells. The kinetics of inhibition of superoxide generation showed a maximum inhibition between 30 and 40 min of preincubation with epinephrine or Bt2 cAMP of EAT cells and coincided with an increase in activity of a phosphoprotein phosphatase. In TPA-treated EAT cells, epinephrine or Bt2 cAMP increased the phosphatase activity in a dose-dependent manner. In vitro EGTA, EDTA and sodium fluoride inhibited phosphatase activity. Superoxide generation in response to TPA in Triton-permeabilized EAT cells was inhibited by inclusion of the phosphatase in the assay. Taken together, these results clearly suggest that the phosphatase activity in EAT cells develops as a result of protein kinase A (PKA) and protein kinase C (PKC)-mediated phosphorylation of the phosphatase which then mediates dephosphorylation of the PKC-triggered phosphorylation of proteins to inhibit respiratory burst. A cross-talk between PKA and PKC pathways negatively modulates superoxide generation in EAT cells.
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PMID:Mechanism of inhibition by cyclic AMP of protein kinase C-triggered respiratory burst in Ehrlich ascites tumour cells. 133 69

A rapid elevation of ribonucleotide reductase activity was observed with BALB c/3T3 fibroblasts treated with 10 nM okadaic acid, a nonphorbol ester tumor promoter and protein phosphatase inhibitor. Northern blot analysis of the two components of ribonucleotide reductase (R1 and R2) showed a marked elevation of R1 and R2 mRNA expression. Western blot analysis with R1 and R2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the elevation of the R2 rather than the R1 protein during treatment with okadaic acid. The okadaic acid induced elevations in R1 and R2 message levels occurred without a detectable change in the proportion of cells in S phase and were blocked by treatment of cells with actinomycin D, indicating the importance of the reductase transcriptional process in responding to the action of okadaic acid. Furthermore, down-regulation of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate pretreatment abrogated the okadaic acid mediated elevation of ribonucleotide reductase mRNAs, consistent with the involvement of this signal pathway in the regulation of ribonucleotide reductase and the effects of okadaic acid. Treatment of cells with 2.5 nM calyculin A, another non-phorbol ester tumor promoter and protein phosphatase inhibitor, resulted in a rapid elevation of both R1 and R2 mRNA levels within 10 min of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A. 133 11


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