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)

Many hormones regulate the rate of synthesis of phosphoenolpyruvate carboxykinase (PEPCK), the enzyme that governs the rate-limiting step in gluconeogenesis. In H4IIE rat hepatoma cells, glucocorticoids, retinoic acid and cyclic AMP (cAMP) increase PEPCK gene transcription whereas insulin and phorbol esters have the opposite effect. Insulin and phorbol esters are dominant as they prevent cAMP- and glucocorticoid-stimulated PEPCK gene transcription. In contrast, insulin and phorbol esters both stimulate transcription of gene 33 in the same H4IIE cells, with the same time course as seen for their inhibitory effect on PEPCK gene transcription. We now report that the protein phosphatase inhibitor, okadaic acid, mimics the action of insulin and phorbol esters on expression of both gene 33 and PEPCK gene in H4IIE cells. Okadaic acid stimulates gene 33 mRNA accumulation whereas it inhibits cAMP- and glucocorticoid-stimulated PEPCK mRNA accumulation. The effect of okadaic acid on the PEPCK gene is mediated through the PEPCK promoter as, in a cell line, HL1C, stably transfected with a PEPCK-chloramphenicol acetyltransferase (CAT) fusion gene, okadaic acid inhibits cAMP- and glucocorticoid-stimulated CAT expression. Desensitization of the protein kinase C pathway by exposure to phorbol 12-myristate 13-acetate for 16 h abolishes the subsequent action of the phorbol ester but does not markedly affect the inhibition of cAMP- and glucocorticoid-stimulated CAT expression by insulin or okadaic acid. Even though insulin and okadaic acid appear to repress PEPCK gene expression through a pathway initially distinct from that used by phorbol esters, transient-transfection studies show that the final target of the action of okadaic acid, insulin and phorbol ester is the same DNA element.
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PMID:Comparison of the effects of insulin and okadaic acid on phosphoenolpyruvate carboxykinase gene expression. 798 Apr 40

One-dimensional SDS-PAGE of cytosolic phosphopeptides confirms that glucagon promotes the phosphorylation of 11 phosphopeptides in isolated rat hepatocytes pre-equilibrated with 32PO4(3-). Nine of these phosphopeptides are tentatively identified, whereas two phosphopeptides (48 kDa and 46 kDa) remain unidentified. Transfer of the glucagon-challenged hepatocytes to medium free of 32PO4(3-) and glucagon led to the rapid net dephosphorylation of the phosphopeptides and to a rapid decline in the specific radioactivity of the [32P]ATP pool. There were profound differences between the post-glucagon rates of net dephosphorylation of the different hepatic phosphopeptides, consistent with net dephosphorylation being asynchronous during the recovery phase from acute glucagon challenge. On the basis of descending rates of dephosphorylation, four major groups of phosphopeptides were delineated. Okadaic acid, a potent inhibitor of protein phosphatase 2A and to a lesser extent protein phosphatase 1, inhibited the dephosphorylation of all of the phosphopeptides. A role for protein phosphatase 2A in protein dephosphorylation may be indicated by the observation that spermine, a specific activator of protein phosphatase 2A, stimulates the dephosphorylation of some, but not all, of the glucagon-stimulated phosphopeptides. Although phosphorylation during the recovery phase from glucagon challenge may be a complicating factor, the results suggest that post-glucagon dephosphorylation is a complex asynchronous process. The physiological consequences of this asynchrony may be that the suppression of glycogenolysis and gluconeogenesis and the activation of glycolysis are early events in the recovery process.
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PMID:Recovery from acute glucagon challenge in isolated rat hepatocytes: is protein dephosphorylation synchronous or asynchronous? 798 Dec 47

Ca(2+)-mobilizing and cAMP-dependent hormones rapidly increase sodium, potassium-dependent adenosine triphosphatase (Na+/K(+)-ATPase)-mediated transport in rat hepatocytes. To explore the possible role of protein phosphatases in these responses we used a protein phosphatase inhibitor, okadaic acid. Okadaic acid stimulation of ouabain-sensitive 86Rb(+)-uptake was maximal between two and three minutes and displayed an EC50 of 41 +/- 1 nM. Inhibition of Na+/H+ exchange with an amiloride analog abolished the response to insulin, but had no effect on okadaic acid-mediated stimulation of Na+/K(+)-ATPase transport. In hepatocytes metabolically-radiolabeled with 32Pi, okadaic acid stimulated the incorporation of radioactivity into several 95 kDa peptides, one of which reacted with anti-LEAVE peptide antisera, that recognizes Na+/K(+)-ATPase alpha-subunits. In other experiments Na+/K(+)-ATPase was immunoprecipitated from detergent-solubilized membrane fractions of metabolically-radiolabeled cells with an antisera to purified rat kidney Na+/K(+)-ATPase. A 95 kDa phosphoprotein was immunoprecipitated using anti-Na+/K(+)-ATPase antisera, but not by preimmune serum. Okadaic acid stimulated incorporation of radioactivity into this band by 220 +/- 28%. These findings provide support for the hypothesis that rapid stimulation of hepatic Na+/K(+)-ATPase by hormones may be related to protein kinase/phosphatase-mediated changes in the phosphorylation state of the Na+/K(+)-ATPase alpha-subunit.
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PMID:Okadaic acid stimulates ouabain-sensitive 86Rb(+)-uptake and phosphorylation of the Na+/K(+)-ATPase alpha-subunit in rat hepatocytes. 798 91

Treatment of rat dorsal root ganglion cultures with 1 microM okadaic acid leads to a fragmentation of neurofilaments and a reduction in the electrophoretic mobilities of the three subunits on SDS-polyacrylamide gels (Sacher, M. G., Athlan, E. S., and Mushynski, W. E. (1992) Biochem. Biophys. Res. Commun. 186, 524-530). Based on the observed response to varying concentrations of okadaic acid, fragmentation was inferred to be due to inhibition of protein phosphatase-2A activity and reduction in electrophoretic mobility to inhibition of protein phosphatase-1. Okadaic acid treatment led to an increase in amino-terminal, relative to carboxyl-terminal, domain phosphorylation in the low molecular weight (NF-L) subunit in the Triton X-100-soluble and -insoluble fractions. The purified catalytic subunit of protein phosphatase-2A dephosphorylated 32P-labeled NF-L and the middle molecular weight subunit from okadaic acid-treated cultures, whereas the catalytic subunit of protein phosphatase-1 had no effect. In the case of NF-L, phosphate moieties were preferentially removed from the amino-terminal domain. These results show that the amino-terminal domain of NF-L can be phosphorylated in situ and implicate protein phosphatase-2A in the turnover of phosphate moieties in this domain.
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PMID:Increased phosphorylation of the amino-terminal domain of the low molecular weight neurofilament subunit in okadaic acid-treated neurons. 803 96

Okadaic acid (OA) is a protein phosphatase inhibitor which has, among other properties, previously been shown to induce a fragmentation of the cisternae of the Golgi stack [for review, see Lucocq (1992) J. Cell Sci. 103, 875-880]. The effects of OA an reversible and mimic intracellular events which occur during mitosis. To date, due to a lack of endogenous marker proteins, the effects of OA on the trans-Golgi network (TGN) has not been studied. Certain drugs, e.g. Brefeldin A (BFA), have different effects on the morphology of the Golgi stack and the TGN; it is therefore relevant to ask what effect(s) OA has on the TGN. We now present data from a study in which we have used antibodies to TGN38, an integral membrane protein predominantly localized to the TGN of rat NRK cells [Luzio, Brake, Banting, Howell, Braghetta and Stanley (1990) Biochem. J. 270, 97-102], to investigate the effects of OA on this organelle. OA induces a reversible fragmentation of the TGN. This fragmentation occurs with similar kinetics to that observed within the Golgi stack, and is independent of protein synthesis. The sensitivity of the TGN to OA is similar to that of the Golgi stack. The fragmentation of the TGN induced by OA also leads to a 10-fold increase in the level of TGN38 expressed at the plasma membrane.
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PMID:Okadaic acid treatment leads to a fragmentation of the trans-Golgi network and an increase in expression of TGN38 at the cell surface. 803 93

Modulation by protein phosphorylation of the relation between acetylcholine (ACh)-activated current (IACh) and adenosine triphosphate-(ATP)-activated current (IATP) was investigated with the whole-cell voltage-clamp technique in rat sympathetic neurons. During simultaneous activation by 100 microM ATP of an inward current, the current evoked by 100 microM ACh was reduced to 60-70% of that in the absence of ATP. Effects of compounds that are known to modulate protein phosphorylation were tested by including them in the intracellular solution. The reduction of IACh by ATP was not observed when K252a (1 microM), a non-selective protein kinase inhibitor, adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma S], 1 mM) or alpha, beta-methylene ATP (1 mM) were included in the intracellular solution. Activators of protein kinases, adenosine 3',5'-cyclic monophosphate (cAMP, 100 microM), guanosine 3',5'-cyclic monophosphate (cGMP, 100 microM), phorbol 12-myristate 13-acetate (PMA, 1 microM), also abolished the reduction by ATP of IACh. The effects of okadaic acid, a protein phosphatase inhibitor, were paradoxical: okadaic acid (2 microM) itself abolished the reduction by ATP of IACh but it "antagonized" the abolishment by cAMP or cGMP of the reduction of IACh. Okadaic acid did not affect the disappearance of the reduction of IACh by ATP in the presence of intracellular PMA. The results suggest that the interaction between IACh and IATP is regulated by protein phosphorylation/dephosphorylation. Possible mechanisms underlying the effects of these modulators of protein phosphorylation are discussed.
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PMID:Modulation of the inhibitory action of ATP on acetylcholine-activated current by protein phosphorylation in rat sympathetic neurons. 805 61

To elucidate the mechanism causing the transient accumulation of intracellular cAMP in the FRTL-5 thyroid cell line, the short-term effect of thyroid-stimulating hormone (TSH) on phosphodiesterase (PDE) activity was studied. Together with an increase in cAMP levels, TSH produced a significant increase in total PDE activity as early as 3 min, with a maximal stimulation reached after 15 min. This short-term increase in PDE activity was dependent on the TSH concentration (ED50 = 4 x 10(-11) M TSH). Forskolin and dibutyryl cAMP produced an even larger stimulation than that produced by TSH, suggesting that the effect of TSH is mediated by cAMP. To determine the properties of the PDE forms activated by TSH, antibodies specific for the cAMP-PDEs were used to immunoprecipitate the PDEs present in control cells, and cells incubated for 15 min in the presence of 10 nM TSH. Comparison of the activity recovered in the immunoprecipitation pellets demonstrated that TSH produced more than a 2.5-fold increase in the cAMP-PDE form(s) recognized by this antibody. Conversely, the activity remaining in the supernatants was not affected by the TSH treatment. Most of the activity recovered in the immunoprecipitation pellets (90%) was inhibited by 10 microM Rolipram, an inhibitor specific for the high affinity cAMP-PDEs. No TSH stimulation of the Rolipram-insensitive PDE activity could be observed under these conditions. Western blot analyses with two different cAMP-PDE specific antibodies showed that a 15-min stimulation with TSH induced the appearance of a new band with electrophoretic mobility slower than the polypeptide present in unstimulated cells. The appearance of this band did not require ongoing protein synthesis because it occurred in the presence of cycloheximide. Metabolic [32P]orthophosphate labeling of intact FRTL-5 cells indicated that the TSH treatment caused an increased 32P incorporation into a polypeptide that co-purified with the stimulated PDE activity and had an electrophoretic mobility identical to that of the cAMP-PDE. Okadaic acid, a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A, elicited a potentiation of the TSH-stimulated PDE activity. The stimulating of a PDE with the same immunological properties and Rolipram sensitivity as the cAMP-PDE stimulated by TSH in the intact cells was reproduced, in a cell-free system, by incubating soluble extracts from FRTL-5 cells with the catalytic subunit of cAMP-dependent protein kinase. These data provide evidence that TSH produces a rapid activation of a cAMP-PDE in the FRTL-5 cells through a cAMP-dependent phosphorylation.
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PMID:The short-term activation of a rolipram-sensitive, cAMP-specific phosphodiesterase by thyroid-stimulating hormone in thyroid FRTL-5 cells is mediated by a cAMP-dependent phosphorylation. 813 62

Acetylcholine acting via muscarinic cholinoceptors decreased phosphorylation of phospholamban and troponin I without reducing adenosine 3',5'-cyclic monophosphate (cAMP) levels or cAMP-dependent protein kinase activity ratio in the presence of 10-100 nM isoproterenol in guinea pig ventricular myocytes. The effect of acetylcholine was more pronounced when adenosine deaminase (5 U/ml) was present and incubation period was short (10 s). Okadaic acid, an inhibitor of protein phosphatase activity, blocked the acetylcholine-mediated inhibition of isoproterenol-stimulated phosphorylation of phospholamban. It is suggested that acetylcholine reduces protein phosphorylation by a cAMP-independent mechanism in guinea pig ventricular myocytes.
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PMID:M2-specific muscarinic cholinergic receptor-mediated inhibition of cardiac regulatory protein phosphorylation. 816 Aug 16

We report that activators and inhibitors of protein kinase C (PKC) and protein phosphatases regulate the activity of a cloned rat brain gamma-aminobutyric acid (GABA) transporter (GAT1) expressed in Xenopus oocytes. Four compounds known to activate PKC increased GABA uptake 2-3.5-fold over basal control levels. Inhibition of PKC by bisindolylmaleimide reduced basal GABA uptake 80% and blocked the phorbol 12-myristate 13-acetate (PMA)-induced stimulation of transport. Okadaic acid, a protein phosphatase inhibitor, stimulated transport 2.5-fold; a 4-fold increase in GABA uptake occurred when oocytes were treated with cyclosporin A, a specific inhibitor of protein phosphatase 2B. Modulation resulted in changes to Vmax but not to Km and was influenced by the functional expression level of the transporter protein; as expression level increased, the ability to up-regulate transporter activity decreased. Down-regulation of transporter activity was independent of expression level. Modulation did not occur through phosphorylation of the three consensus PKC sites predicted by the primary protein sequence since their removal had no effect on the susceptibility of the transporter to modulation by PMA or bisindolylmaleimide. Subcellular fractionation of oocyte membranes demonstrated that under basal level conditions, the majority of GAT1 was targeted to a cytoplasmic compartment corresponding to the trans-Golgi or low density vesicles. Stimulation of PKC with PMA resulted in a translocation of transporters from this compartment to the plasma membrane. At higher expression levels of GAT1 protein, a larger portion of GAT1 was found on the plasma membrane during basal level conditions and treatment with bisindolylmaleimide resulted in removal of these transporters from the plasma membrane. At expression levels demonstrated to be resistant to modulation by PMA, PMA-treatment still resulted in translocation of transporters from the cytoplasm to the plasma membrane. Thus, the inability of PMA to increase uptake at high expression of the GAT1 protein is due to saturation at a step subsequent to translocation. These findings 1) demonstrate the presence of a novel regulated secretory pathway in oocytes and 2) suggest a modulatory mechanism for neurotransmitter transporters that could have significant effects upon synaptic function.
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PMID:Protein kinase C modulates the activity of a cloned gamma-aminobutyric acid transporter expressed in Xenopus oocytes via regulated subcellular redistribution of the transporter. 818 81

In bovine iris sphincter, myo-inositol 1,4,5-trisphosphate (IP3) 5-phosphatase and myo-inositol 1-phosphate (IP1) monophosphatase are mainly localized in the microsomal and soluble fractions, respectively. Studies on the properties of these enzymes can be summarized as follows. (1) The microsomal IP3 5-phosphatase hydrolyzed IP3 to myo-inositol 1,4-bisphosphate with an apparent Km of 28 microM and Vmax of 32 nmol/min per mg protein. The IP1 monophosphatase in the soluble fraction hydrolyzed IP1 into free inositol with an apparent Km of 89 microM and Vmax of 7 nmol/min per mg protein. (2) IP3 5-phosphatase and IP1 monophosphatase had optimal pH values at 8.0 and 7.0, respectively. (3) Both enzymes required Mg2+ and their highest specific activities were at a cation concentration of 2 mM. (4) Ca2+ (> 0.5 microM) exerted an inhibitory effect on IP3 5-phosphatase activity, and marked inhibition (47%) was observed at a concentration of 10 microM. Higher concentrations of the cation (> 100 microM) were required to inhibit IP1 monophosphatase. (5) IP1 monophosphatase, but not IP3 5-phosphatase, was inhibited by Li+. Li+ had no effect on the contractile response in this smooth muscle. (6) Both enzymes were inhibited by ATP and by the thiol-blocking agent, disulfiram. In addition, thimerosal, a thiol reagent, also inhibited the IP3 5-phosphatase activity. (7) Protein phosphorylation of the microsomal and soluble fractions with PKA or PKC had no effect on the activities of these enzymes. (8) Okadaic acid, a protein phosphatase inhibitor, had no effect on the activity of IP3 5-phosphatase. However, in the intact iris sphincter the toxin significantly reduced the carbachol-induced IP3 production, 1,2-diacylglycerol formation, measured as phosphatidic acid, and caused muscle relaxation.
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PMID:Studies on the properties of myo-inositol-1,4,5-trisphosphate 5-phosphatase and myo-inositol monophosphatase in bovine iris sphincter smooth muscle: effects of okadaic acid and protein phosphorylation. 818 62


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