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)

We investigated the inhibitory effects of intracellular cyclic adenosine monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrogenase (aldh3) gene expression using cultures of primary rat hepatocytes and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of many members of the Ah gene battery have been shown to be negatively regulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is transcriptionally inducible by polycyclic aromatic hydrocarbons (PAH), and this induction involving function of the arylhydrocarbon (Ah) receptor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine di-HCl (H7) and staurosporine. However, PAH induction of ALDH-3 activity, protein, and mRNA was potentiated 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhibitors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 and HA1004 in a concentration-dependent manner, but not by H7, and there was an inverse correlation observed between potentiation of PAH-induced aldh3 gene expression and inhibition of specific PKA activity by the PKA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cyclase activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction and H8 potentiation of PAH induction of aldh3 expression but had no effect on induction of cyp1A1 expression in cultured hepatocytes. Both basal and PAH-dependent expression of a chloramphenicol acetyltransferase expression plasmid containing approximately 3.5 kilobase pairs of the 5'-flanking region of aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA inhibitor H8 but not by the PKC inhibitor H7 (>20 microM). cAMP analogs, activators of PKA activity, or protein phosphatase inhibitors diminished expression of the reporter gene in a manner identical to the native gene in cultured rat hepatocytes. Using deletion analysis of the pALDH3.5CAT construct, we demonstrated the existence of a negative regulatory region in the 5'-flanking region between -1057 and -991 base pairs which appears to be responsible for the cAMP-dependent regulation of this gene under both basal and PAH-induced conditions. At least two apparently independent mechanisms which involve protein phosphorylation regulate aldh3 expression. One involves function of the Ah receptor which requires PKC protein phosphorylation to positively regulate both aldh3 and cyp1A1 gene expression and the other a cAMP-responsive process which allows PKA activity to negatively regulate expression of aldh3 under either basal or inducible conditions.
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PMID:cAMP-dependent negative regulation of rat aldehyde dehydrogenase class 3 gene expression. 901 60

The adherence of tumour cells to microvascular endothelium is believed to be a necessary step in their migration to sites of metastasis. It has been proposed that this process occurs when cell surface molecules on tumour cells bind to complementary sites on endothelial cells. The expression of these endothelial-derived cell adhesion molecules appears to be modulated by cytokines, a broad class of protein mediators which play important roles in immune and inflammatory reactions. It has been found by ourselves and others that exposure of endothelium to some cytokines augments the adhesion of inflammatory cells as well as tumour cells in in vitro assays. We used a murine model consisting of P815 mastocytoma cells and microvascular endothelium and found that pretreatment of endothelial monolayers with TNF-alpha, IL-1, LPS or PMA augmented the number of tumour cells that attach in a dose-dependent fashion. FACS analysis showed that the change in binding was due to an increase in the expression of VCAM-1 on the surface of the endothelial cell. Methylxanthines (caffeine and theophylline) as well as "classical" calcium-mobilizing agents (ionomycin and thapsigargin) inhibited the expression of VCAM-1 in MME. We also studied the possible mechanisms of TNF-alpha signal transduction in endothelial cells. We examined the involvement of protein kinases in the TNF-alpha effect. Although we found that inhibitors of PKC could inhibit the TNF-alpha effect, our studies suggest that the "classical" PKC pathway is not completely responsible for signaling since TNF-alpha did not cause translocation of PKC to the cell membrane and its effect could not be completely mimicked by PMA. We also studied the effect of TGF-beta on the binding of tumour cells to endothelium. Exposure of endothelium to TGF-beta led to the inhibition of both basal and TNF-alpha enhanced binding of P815 cells. Inhibitors of G-proteins do not abolish TGF-beta action, and PKC and PKA activators elicit an opposite effect. However, TGF-beta-mediated inhibition of both basal binding and TNF-alpha-enhanced P815 binding to endothelium is completely abolished in the presence of the protein phosphatase inhibitor okadaic acid suggesting that TGF-beta elicits its effect by stimulating protein phosphatase activity.
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PMID:Effect of cytokines on tumour cell-endothelial interactions. 934 51

The phosphorylation of rat cardiac microsomal proteins was investigated with special attention to the effects of okadaic acid (an inhibitor of protein phosphatases), inhibitor 2 of protein phosphatase 1 and inhibitor of cyclic AMP-dependent protein kinase (protein kinase A). The results showed that okadaic acid (5 microM) modestly but reproducibly augmented the protein kinase A-catalyzed phospholamban (PLN) phosphorylation, although exerted little effect on the calcium/calmodulin kinase-catalyzed PLN phosphorylation. Microsomes contained three other substrates (M(r) 23, 19 and 17 kDa) that were phosphorylated by protein kinase A but not by calcium/calmodulin kinase. The protein kinase A-catalyzed phosphorylation of these three substrates was markedly (2-3 fold) increased by 5 microM okadaic acid. Calmodulin was found to antagonize the action of okadaic acid on such phosphorylation. Protein kinase A inhibitor was found to decrease the protein kinase A-catalyzed phosphorylation of microsomal polypeptides. Unexpectedly, inhibitor 2 was also found to markedly decrease protein kinase A-catalyzed phosphorylation of phospholamban as well these other microsomal substrates. These results are consistent with the views that protein phosphatase 1 is capable of dephosphorylating membrane-associated phospholamban when it is phosphorylated by protein kinase A, but not by calcium/calmodulin kinase, and that under certain conditions, calcium/calmodulin-stimulated protein phosphatase (protein phosphatase 2B) is also able to dephosphorylate PLN phosphorylated by protein kinase A. Additionally, the observations show that protein phosphatase 1 is extremely active against the three protein kinase A substrates (M(r) 23, 19 and 17 kDa) that were present in the isolated microsomes and whose state of phosphorylation was particularly affected in the presence of dimethylsulfoxide. Protein phosphatase 2B is also capable of dephosphorylating these three substrates.
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PMID:Protein phosphorylation in rat cardiac microsomes: effects of inhibitors of protein kinase A and of phosphatases. 935 40

Since various secretory stimuli regulate not only secretion but also protein, RNA, and DNA syntheses in salivary glands, we evaluated the effect of secretory stimuli on the phosphorylation state of CREB (cAMP response element-binding protein). Isoproterenol, forskolin, and CPS-cAMP markedly stimulated the phosphorylation of CREB in parotid acinar cells, and PKA inhibitors H-8 and H-89 dose-dependently inhibited it. In contrast, carbachol (CCH) and A23187 decreased CREB phosphorylation, but CCH did not decrease it in the absence of extracellular Ca2+. Although protein phosphatase inhibitor calyculin A alone markedly increased the phosphorylation, it could not prevent CCH-induced dephosphorylation of CREB. CaM kinase IV, a putative protein kinase for CREB in response to Ca2+ elevation, was undetectable in parotid acinar cells.
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PMID:Regulation of CREB phosphorylation by cAMP and Ca2+ in parotid acinar cells. 935 75

Studies were designed to examine aspects of phosphorylation and dephosphorylation in rat lung cells in response to hyperoxic exposure. Protein kinase and phosphatase activities were measured in preparations of lungs from normoxic rats, hyperoxia-exposed rats (95% O2 for 60h), and rats recovering in room air for 1 and 3 days. Protein kinase C (PKC) activity immediately postexposure was significantly lower than in normoxic controls (normoxia 127.1 +/- 13 vs. hyperoxia 101.5 +/- 6 pmol/min mg-1) and continued to decline during the recovery period (85.3 +/- 4 and 78.2 +/- 6 pmol/min mg-1 at 1 and 3 days recovery, respectively). The PKC activity did not translocate from cytoplasm to the membranes. In contrast, PKA activity did not change in response to hyperoxia exposure or recovery. Protein phosphatase activity was decreased significantly by hyperoxia exposure (normoxia 30.7 +/- 3 vs. hyperoxia 21.9 +/- 1 pmol/min microgram-1) but returned to normoxic control levels by 1 and 3 days (24.1 +/- and 31.5 +/- 1 pmol/min microgram -1, respectively). Protein phosphatase activity was inhibited by okadaic acid (Ki = 1 nM) and calyculin A (Ki = 0.61 pM), indicating a type 2A protein phosphatase. Enzyme activities in cultured type II alveolar cells paralleled those observed in whole lung preparations. Decreased enzyme activities in the lung may be located to the development of acute lung injury during hyperoxic exposure.
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PMID:Protein kinase and phosphatase activity in the lungs of normoxic versus hyperoxic rats. 935 32

Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) are responsible for efficient synaptic clearance of extracellular 5HT. Previously (Qian, Y., Galli, A., Ramamoorthy, S., Risso, S., DeFelice, L. J., and Blakely, R. D. (1997) J. Neurosci. 17, 45-47), we demonstrated that protein kinase (PKC)-linked pathways in transfected HEK-293 cells lead to the internalization of cell-surface human (h) SERT protein and a reduction in 5HT uptake capacity. In the present study, we report that PKC activators rapidly, and in a concentration-dependent manner, elevate the basal level of hSERT phosphorylation 5-6-fold. Similarly, protein phosphatase (PP1/PP2A) inhibitors down-regulate 5HT transport and significantly elevate hSERT 32P incorporation, effects that are additive with those of PKC activators. Moreover, hSERT phosphorylation induced by beta-phorbol 12-myristate 13-acetate is abolished selectively by the PKC inhibitors staurosporine and bisindolylmaleimide I, whereas hSERT phosphorylation induced by phosphatase inhibitors is insensitive to these agents at comparable concentrations. Protein kinase A and protein kinase G activators fail to acutely down-regulate 5HT uptake but significantly enhance hSERT phosphorylation. Basal hSERT and okadaic acid-induced phosphorylation were insensitive to chelation of intracellular calcium and Ca2+/calmodulin-dependent protein kinase inhibitors. Together these results reveal hSERT to be a phosphoprotein whose phosphorylation state is likely to be tightly controlled by multiple kinase and phosphatase pathways that may also influence the transporter's regulated trafficking.
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PMID:Phosphorylation and regulation of antidepressant-sensitive serotonin transporters. 944 97

To investigate the role of phosphatases in synaptic plasticity using genetic approaches, we generated transgenic mice that overexpress a truncated form of calcineurin under the control of the CaMKIIalpha promoter. Mice expressing this transgene show increased calcium-dependent phosphatase activity in the hippocampus. Physiological studies of these mice and parallel pharmacological experiments in wild-type mice reveal a novel, intermediate phase of LTP (I-LTP) in the CA1 region of the hippocampus. This intermediate phase differs from E-LTP by requiring multiple trains for induction and in being dependent on PKA. It differs from L-LTP in not requiring new protein synthesis. These data suggest that calcineurin acts as an inhibitory constraint on I-LTP that is relieved by PKA. This inhibitory constraint acts as a gate to regulate the synaptic induction of L-LTP.
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PMID:Genetic and pharmacological evidence for a novel, intermediate phase of long-term potentiation suppressed by calcineurin. 948 97

The mechanisms through which changes in intracellular Ca2+ concentration ([Ca2+]i) might influence desensitization of neuronal nicotinic receptors (nAChRs) of rat chromaffin cells were investigated by simultaneous patch-clamp recording of membrane currents and confocal microscopy imaging of [Ca2+]i induced by nicotine. Increases in [Ca2+]i that were induced by membrane depolarization or occurred spontaneously did not influence inward currents elicited by focally applied test pulses (10 msec) of nicotine, indicating that raised [Ca2+]i per se did not trigger desensitization of nAChRs. Desensitization of nAChRs, evoked by 2 sec focal application of nicotine, which largely raised [Ca2+]i, was not affected by intracellular application of agents that activate or depress protein kinase C (PKC) or A (PKA) or inhibit phosphatase 1, 2 A and B. Conversely, recovery from desensitization was facilitated by the phorbol ester phorbol 12-myristate 13-acetate (PMA) or the phosphatase 2 B inhibiting complex of cyclosporin A-cyclophilin A, whereas it was impaired by the broad spectrum kinase inhibitor staurosporine. The effects of PMA or staurosporine were prevented by the intracellularly applied Ca2+ chelator BAPTA. The adenylate cyclase activator forskolin accelerated recovery, whereas the selective PKA antagonist Rp-cAMPS had an opposite effect. The action of staurosporine and Rp-cAMPS on recovery from desensitization was additive. It is proposed that when nAChRs are desensitized, they become susceptible to modulation by [Ca2+]i via intracellular second messengers such as serine/threonine kinases and calcineurin. Thus, the phosphorylation state of neuronal nAChRs appears to regulate their rate of recovery from desensitization.
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PMID:Recovery from desensitization of neuronal nicotinic acetylcholine receptors of rat chromaffin cells is modulated by intracellular calcium through distinct second messengers. 950 6

This laboratory reported previously that overexpressed heat shock protein 70 kDa (HSP-70) inhibited the activation of its transcriptional factor, HSF1. We had conducted experiments to understand the mechanisms whereby HSP-70 down-regulated the activation of HSF1. Genetically overexpressed HSP-70 had no effects on the HSF1 level in cytosol, but significantly inhibited phosphorylation of HSF1 in the nucleus. Transfection of cells with HSF1 cDNA resulted in increases in the unphosphorylated, but not phosphorylated, HSF1 levels in both the cytosol and nucleus. Because serine phosphorylation of various proteins was reduced in HSP-70 cDNA-transfected cells, we measured the activity of enzymes involved in serine phosphorylation. Overexpressed HSP-70 significantly inhibited the enzymatic activities of protein kinase A (PKA by 73 and 62% in the cytosol and membrane-bound fraction, respectively) and protein kinase C (PKC by 61% in membrane-bound fraction), whereas it activated that of protein phosphatase (PP by 33 and 86% in the cytosol and the membrane-bound fraction, respectively). Forskolin (a PKA stimulator), PMA (a PKC stimulator), and okadaic acid (an inhibitor of PP) were used to investigate whether HSP-70-induced changes in PKA, PKC, and PP were responsible for the HSF1 dephosphorylation. Forskolin did not change nuclear HSF1 phosphorylation, suggesting that decreases in PKA activity in HSP-70 overexpressing cells is not associated with HSF1 phosphorylation. PMA and okadaic acid induced an increase in HSF1 phosphorylation in both vector- and HSP-70 cDNA-transfected cells, although levels of phosphorylated HSF1 in HSP-70 cDNA-transfected cells were lower than those in vector-transfected cells. The PMA-induced increase in HSF1 phosphorylation in HSP-70 cDNA-transfected cells was blocked by pretreatment with staurosporine, a PKC inhibitor. These results suggest that overexpression of HSP-70 inhibits phosphorylation of HSF1 at serine residues by activating PP and inhibiting PKC activity.
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PMID:Overexpression of HSP-70 inhibits the phosphorylation of HSF1 by activating protein phosphatase and inhibiting protein kinase C activity. 953 17

Cysteine sulfinic acid decarboxylase (CSAD), the rate-limiting enzyme in taurine biosynthesis, was found to be activated under conditions that favor protein phosphorylation and inactivated under conditions favoring protein dephosphorylation. Direct incorporation of 32P into purified CSAD has been demonstrated with [gamma 32P]ATP and PKC, but not PKA. In addition, the 32P labeling of CSAD was inhibited by PKC inhibitors suggesting that PKC is responsible for phosphorylation of CSAD in the brain. Okadaic acid had no effect on CSAD activity at 10 microM suggesting that protein phosphatase-2C (PrP-2C) might be involved in the dephosphorylation of CSAD. Furthermore, it was found that either glutamate- or high K(+)-induced depolarization increased CSAD activity as well as 32P-incorporation into CSAD in neuronal cultures, supporting the notion that the CSAD activity is endogenously regulated by protein phosphorylation in the brain. A model to link neuronal excitation, phosphorylation of CSAD and increase in taurine biosynthesis is proposed.
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PMID:Regulation of taurine biosynthesis and its physiological significance in the brain. 963 49


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