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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of okadaic acid (OA), a protein phosphatase inhibitor, on transcriptional enhancement activity of rat glucocorticoid receptor (GR) were examined in transiently transfected cells. In the absence of hormone, GRs expressed in CV-1 and COS-1 fibroblasts were capable of enhancing transcription from cotransfected chloramphenicol acetyltransferase reporter plasmids in response to OA treatment. Synergistic enhancement resulted from combined hormone and OA treatment. The effects of OA on GR-mediated enhancement required the presence of linked glucocorticoid response elements and were observed with reporter plasmids that contained different promoters and glucocorticoid response elements. Since OA did not affect nuclear translocation of the receptor, enhancement mediated by unliganded GR was most likely accounted for by the accumulation of some unliganded GRs within nuclei of transfected CV-1 and COS-1 cells. Deletion of individual GR transactivation domains and point mutations within DNA- and hormone-binding domains severely reduced the response of receptors to OA, although some mutant receptors retained the capacity to elicit a synergistic response when exposed to OA and hormone. The effects of OA on transcriptional enhancement did not appear to correlate with major changes in GR phosphorylation, as visualized by two-dimensional tryptic mapping of in vivo 32P-labeled GRs. Thus, phosphorylation of various components of the GR signal transduction pathway, and not necessarily the receptor itself, may influence its transcriptional enhancement activity.
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PMID:Effects of okadaic acid, a protein phosphatase inhibitor, on glucocorticoid receptor-mediated enhancement. 131 Jul 97

Engagement of the T cell receptor for antigen activates phospholipase C resulting in an increase in intracellular free calcium concentration ([Ca2+]i) and activation of protein kinase C (PKC). Increased [Ca2+]i activates Ca2+/calmodulin-dependent kinases including the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM-K II), as well as calcineurin, a type 2B protein phosphatase. Recent studies have identified calcineurin as a key enzyme for interleukin (IL)-2 and IL-4 promoter activation. However, the role of CaM-K II remains unknown. We have used mutants of these kinases and phosphatases (gamma B*CaM-K and delta CaM-AI, respectively) to explore their relative role in cytokine gene transcription and their interactions with PKC-dependent signaling systems. gamma B*CaM-K and delta CaM-AI, known to exhibit constitutive Ca(2+)-independent activity, were cotransfected (alone or in combination) in Jurkat T cells with a plasmid containing the intact IL-2 promoter driving the expression of the chloramphenicol acetyltransferase reporter gene. Cotransfection of gamma B*CaM-K with the IL-2 promoter construct downregulated its transcription in response to stimulation with ionomycin and phorbol myristate acetate (PMA). The inhibitory effect of CaM-K II on IL-2 promoter was associated with decreased transcription of its AP-1 and NF-AT transactivating pathways. Under the same conditions, delta CaM-AI superinduced IL-2 promoter activity (approximately twofold increase). When both mutants were used in combination, gamma B*CaM-K inhibited the induction of the IL-2 promoter by delta CaM-AI. Similar results were obtained when a construct containing the IL-4 promoter also was used. gamma B*CaM-K also downregulated the activation of AP-1 in response to transfection with a constitutively active mutant of PKC or stimulation with PMA. These results suggest that CaM-K II may exert negative influences on cytokine gene transcription in human T cells, and provide preliminary evidence for negative cross-talk with the calcineurin- and PKC-dependent signaling systems.
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PMID:Calcium/calmodulin-dependent protein kinase II downregulates both calcineurin and protein kinase C-mediated pathways for cytokine gene transcription in human T cells. 786 38

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

The present work has examined the effects of okadaic acid, an inhibitor of serine/threonine protein phosphatase, PP-1 and PP-2A, on the regulation of EGR-1 gene expression in normal peripheral blood T- and Jurkat cells. The results demonstrate that okadaic acid treatment is associated with a transient induction of EGR-1 gene expression which was detectable by 30 min to 1 h and peaked at 3-6 h. EGR-1 mRNA was superinduced in cells treated with both okadaic acid and the protein synthesis inhibitor cycloheximide. The half-life of EGR-1 mRNA was similar in both control and okadaic acid-treated cells. In contrast, treatment with both okadaic acid and cycloheximide prolonged the half-life of EGR-1 transcripts. Nuclear run-on assays demonstrated that induction of EGR-1 gene expression by okadaic acid is controlled at least in part by a transcriptional mechanism. Transient expression assays with EGR-1 promotor fragments linked to the chloramphenicol acetyltransferase gene demonstrate that okadaic acid-induced EGR-1 transcription is conferred by the 5' most distal CArG box, CC (AT)6GG, in the EGR-1 promoter. Moreover, chloramphenicol acetyltransferase activity was induced by okadaic acid when the 5' most distal CArG element was linked to the heterologous herpes simplex virus thymidine kinase promoter, and not induced with a similar heterologous construct containing a mutated CArG sequence. These studies demonstrate that okadaic acid regulates EGR-1 gene expression at the transcriptional level via the CArG element and suggest that PP-1 and PP-2A play a role in T-cell activation.
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PMID:Involvement of serum response element in okadaic acid-induced EGR-1 transcription in human T-cells. 817 32

The effects of okadaic acid (OA), a potent and specific inhibitor of serine/threonine phosphatases 2A and 1, on the transient expression of a human hsp 70 promoter-linked chloramphenicol acetyltransferase gene transfected into N-18 mouse neuroblastoma cells were determined. Assays of reporter gene activity showed that nanomolar concentrations of OA markedly potentiated the heat-induced (but not the basal) expression of pHBCAT, a full-length hsp 70 promoter-driven chloramphenicol acetyltransferase gene construct. This effect of OA was dose-dependent and promoter-specific and appeared to be attributable to inhibition of protein phosphatase 2A as opposed to protein phosphatase 1. The ability of OA to potentiate the heat-induced expression of pHBCAT appeared to be a feature common to several different cell types examined. We propose that the heat-induced transcriptional activation of heat shock genes is associated with the phosphorylation of component(s) of the transcription complex and that OA enhances this phosphorylation, thereby potentiating the heat-induced hsp 70 promoter activity.
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PMID:Okadaic acid markedly potentiates the heat-induced hsp 70 promoter activity. 838 Apr 12

Phosphoenolpyruvate carboxykinase (PCK) is a key regulatory enzyme in renal ammoniagenesis and gluconeogenesis. LLC-PK1-F+ cells are porcine renal proximal tubule-like cells that express significant levels of the cytosolic PCK. Treatment of subconfluent LLC-PK1-F+ cells with 0.1 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) for 8 h causes a 21-fold increase in PCK mRNA. This response is very rapid and is not inhibited by 0.5 mM cycloheximide, indicating that ongoing protein synthesis is not required. Similarly, cells transfected with PCK(-490)CAT exhibit an 8- to 10-fold increase in chloramphenicol acetyltransferase (CAT) activity when treated with cAMP for 24 h. The addition of okadaic acid, a protein phosphatase inhibitor, both stimulated the CAT activity and potentiated the cAMP effect by twofold, suggesting that phosphorylation may contribute to the transcriptional activation. Assays using a series of PCK-CAT constructs containing specific deletions or block mutations established that the CRE-1 the P3(II) elements are required for the cAMP response. Cotransfection experiments using dominant negative expression vectors indicated that a CCAAT enhancer binding protein (C/EBP) transcription factor, and not CREB, mediates cAMP activation of transcription in LLC-PK1-F+ cells.
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PMID:cAMP activation of phosphoenolpyruvate carboxykinase transcription in renal LLC-PK1-F+ cells. 877 Jan 66

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

Nitric oxide produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of stroke and other neurodegenerative diseases. The present study underlines the importance of protein phosphatase (PP) 1 and 2A in the regulation of the differential expression of iNOS in rat primary astrocytes and macrophages. Compounds (calyculin A, microcystin, okadaic acid, and cantharidin) that inhibit PP 1 and 2A were found to stimulate the lipopolysaccharide (LPS)- and cytokine-mediated expression of iNOS and production of NO in rat primary astrocytes and C6 glial cells. However, these inhibitors inhibited the LPS- and cytokine-mediated expression of iNOS and production of NO in rat resident macrophages and RAW 264.7 cells. Similarly, okadaic acid, an inhibitor of PP 1/2A, stimulated the iNOS promoter-derived chloramphenicol acetyltransferase activity in astrocytes and inhibited the iNOS promoter-derived chloramphenicol acetyltransferase activity in macrophages, indicating that okadaic acid also differentially regulates the transcription of the iNOS gene in astrocytes and macrophages. The observed stimulation of the expression of iNOS in astrocytes and the inhibition of the expression of iNOS in macrophages with the inhibition of PP 1/2A activity clearly delineate a novel role of PP 1/2A in the differential regulation of iNOS in rat astrocytes and macrophages. Because the activation of NF-kappaB is necessary for the induction of iNOS and the expression of tumor necrosis factor (TNF)-alpha also depends on the activation of NF-kappaB, we examined the effect of okadaic acid on the LPS-mediated activation of NF-kappaB and production of TNF-alpha in rat primary astrocytes and macrophages. Interestingly, in both cell types, okadaic acid stimulated the LPS-mediated DNA binding as well as transcriptional activity of NF-kappaB and production of TNF-alpha. This study suggests that the stimulation of iNOS expression in astrocytes by inhibitors of PP 1/2A is possibly due to the stimulation of NF-kappaB activation; however, activation of NF-kappaB is not sufficient for the induction of iNOS in macrophages and that apart from NF-kappaB some other signaling pathway(s) sensitive to PP 1 and/or PP 2A is/are possibly involved in the regulation of iNOS in macrophages. This differential induction of iNOS as compared with similar activation of NF-kappaB by inhibitors of PP 1/2A indicates the involvement of different intracellular signaling events for the induction of iNOS in two cell types of the same animal species.
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PMID:Inhibitors of protein phosphatase 1 and 2A differentially regulate the expression of inducible nitric-oxide synthase in rat astrocytes and macrophages. 957 70

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