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)

Prolonged depolarization has been used as a model of adaptive changes in the expression of various proteins, such as ion channels and neurotransmitter biosynthetic enzymes, in response to increased trans-synaptic activity in the nervous system. In depolarized PC12 cells, tyrosine hydroxylase (TH) mRNA levels increased severalfold (Kilbourne, E. J., and Sabban, E. L. (1990) Mol. Brain Res. 8, 121-127). In this study, membrane depolarization caused an increase in the expression of the reporter gene chloramphenicol acetyltransferase (CAT), under transcriptional control of the 5' region of the rat TH gene. These results indicate that membrane depolarization leads to increased transcription of the TH gene. Protein kinase C inhibitors had no effect on the induction of TH mRNA by depolarization, as well as the increase in formation of CAT under control of the upstream region of the TH gene. The depolarization responsive element in the TH gene was mapped to the region containing the cAMP responsive element. This region of the TH gene also increased CAT activity in response to the calcium ionophore, ionomycin. Interestingly, combined treatment with cAMP analogs and membrane depolarization had a greater effect than either alone on TH mRNA levels, as well as on CAT activity in PC12 cells transfected with the plasmid containing the cAMP responsive element.
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PMID:Regulated expression of the tyrosine hydroxylase gene by membrane depolarization. Identification of the responsive element and possible second messengers. 134 5

To analyze the mechanism of the cell type-specific expression of protein kinase C beta (PKC beta), we isolated the 5'-portion of the human gene for PKC beta and identified multiple positive and negative regulatory sequences that regulate its transcription. S1 nuclease mapping as well as primer extension analysis of the 5'-end of the PKC beta mRNA identified a putative transcriptional initiation site (position +1) 484 base pairs (bp) upstream of the first ATG codon. The 5'-upstream sequence contains a CCAAT sequence at position -110, but no TATA box. The transcriptional activities of various 5'-deletion mutants of the PKC beta gene upstream region, fused to the chloramphenicol acetyltransferase structural gene, were examined in terms of chloramphenicol acetyltransferase expression after transfection into three kinds of rodent cell lines: P19 and GH4C1, which are positive for the expression of PKC beta mRNA; and 3Y1, which is negative. Mutants containing a 5'-flanking sequence longer than 1.9 kilobases (kb) showed chloramphenicol acetyltransferase activities of the same order as the expression of the endogenous gene. This indicates that this region contains sequences regulating the cell-type specificity of PKC beta gene expression and that the specificity is determined at least partially at the level of transcription. The 1.9-kb sequence contains at least three positive elements: P1 (-56 to -234 bp), P2 (-234 to -411 bp), and PN (-1.4 to -1.9 kb). PN is active only in P19 cells, P1 in GH4C1 and P19 cells, and P2 in all three cell lines. In addition to these positive elements, there are negative elements: N1 (-411 to -674 bp), which is active in all three cell lines; and PN, which is active only in GH4C1 cells. These results suggest the presence of multiple trans-acting factors that act on these positive and negative cis-acting elements and regulate the cell type-specific expression of the PKC beta gene.
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PMID:Positive and negative regulation of the transcription of the human protein kinase C beta gene. 155 24

Oncogene activation has been suggested to play some role in determining the hormone independency of tumors. In order to study the role of protein kinase C in mediating the inhibition of the glucocorticoid-dependent transcription from the Mouse Mammary Tumor Virus (MMTV)-Long Terminal Repeat induced by overexpressed activated ras oncogene, we studied the effects of protein kinase C activators [the tumor promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA)] and inhibitors [1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7)] on the dexamethasone (DEX)-mediated activation of a MMTV-Long Terminal Repeat-chloramphenicol acetyltransferase (pMMTV-CAT) chimeric reporter gene transiently transfected into NIH-3T3 cells and in Ha-ras-transformed fibroblasts (T24-NIH-3T3). TPA (30 ng/ml) together with DEX (0.1 microM) treatment of NIH-3T3 cells resulted in a significant decrease of CAT activity from pMMTV-CAT, compared to DEX treatment alone. The addition of H-7 (40 microM) was able to overcome the TPA-induced inhibition of DEX-dependent transcription from pMMTV-CAT. DEX-dependent expression of pMMTV-CAT was significantly reduced in T24-NIH-3T3 with respect to wild-type NIH-3T3 cells. Treatment of T24-NIH-3T3 cells with either H-7 or TPA significantly enhanced or decreased, respectively, the DEX-dependent expression of pMMTV-CAT. TPA and/or H-7 did not affect CAT activity from either pMMTV-CAT in the absence of DEX or from CAT gene under the control of the SV40 promoter. Similar glucocorticoid receptor sites and binding affinities were observed in T24-NIH-3T3 or TPA-treated NIH-3T3 cells compared to wild-type untreated cells. Our data suggest that activation of PKC is involved in the reduced transcriptional regulatory activity of glucocorticoid hormone induced by overexpressed Ha-ras oncogene in NIH-3T3 fibroblasts.
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PMID:Tumor-promoting phorbol ester and ras oncogene expression inhibit the glucocorticoid-dependent transcription from the mouse mammary tumor virus long terminal repeat. 255

Recent evidence demonstrates that the protein kinase C zeta (zeta PKC) isoform is required for the activation of nuclear factor kappa B (NF-kappa B) and mitogenic signaling in Xenopus oocytes and mammalian cells. The mechanism whereby zeta PKC regulates NF-kappa B most probably involves the activation of a putative I kappa B kinase of molecular mass approximately 50 kDa, which phosphorylates and inactivates I kappa B. Tumor necrosis factor alpha (TNF alpha) and interleukin-1, besides activating the phospholipase C-mediated breakdown of phosphatidylcholine, also generate ceramide, which is produced by stimulation of sphingomyelin hydrolysis. We show here that exogenous addition of sphingomyelinase (SMase) to NIH-3T3 fibroblasts transactivates a kappa B-dependent chloramphenicol acetyltransferase reporter plasmid, to an extent similar to that produced by TNF alpha or phosphatidylcholine/phospholipase C. More importantly, the ability of SMase to stimulate this parameter is severely impaired by transfection of a zeta PKC kinase-defective dominant negative mutant, which suggests a critical role of zeta PKC in SMase signaling. In keeping with this notion, we also demonstrate here that zeta PKC is activated in vitro by ceramide and in vivo by treatment of NIH-3T3 fibroblasts with SMase.
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PMID:Protein kinase C zeta isoform is critical for kappa B-dependent promoter activation by sphingomyelinase. 803 80

Parathyroid hormone (PTH) functions in part by regulating osteoblast cytokine expression. We recently demonstrated that PTH induced a rapid and transient increase in interleukin-6 (IL-6) mRNA expression in rat bones in vivo. To determine the molecular basis of this effect, we analyzed the human IL-6 promoter fused (-1,179 to +9) with the chloramphenicol acetyltransferase (CAT) reporter gene in stable transfections into human osteoblast-like osteosarcoma SaOS-2 cells. We compared the effects of PTH on IL-6 expression with adenylate cyclase activator forskolin, PKC activator phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187, interleukin-1 alpha (IL-1 alpha), prostaglandin E-2 (PGE-2), RS-66271 (a parathyroid hormone-related peptide analog), and platelet-derived growth factor-BB (PDGF-BB). Analyses of cell clones showed that IL-6 promoter expression was extremely low in the unstimulated state. Exposure to PTH (0.001-100 nM) for 12 h stimulated CAT expression in a dose-dependent manner (200-500% of control). Treatment with IL-1 alpha was more potent than PTH in inducing transcription of the IL-6 promoter (900-1,000%). Activation of the cAMP-PKA pathway by treatment with forskolin induced a comparable level of induction with PTH. Together, the effects of PTH and forskolin were additive. RS-66271, previously shown to have PTH-like effects, induced a comparable level of IL-6 promoter expression. When examined together, PTH+RS-66271 effects were comparable to PTH effects alone. Exposure to PGE-2, PMA, PDGF-BB, or A23187 for 12 h did not significantly alter IL-6 promoter expression. These results demonstrate PTH, forskolin, the PTHrP analog RS-66271, and IL-1 alpha stimulate IL-6 expression by stimulating gene transcription. The response to forskolin suggests that the messenger system mediated by PKA is sufficient to induce IL-6 expression.
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PMID:Parathyroid hormone (1-34)-mediated interleukin-6 induction. 932 32

Protein kinase C (PKC) activation after treatment of human neuroblastoma SK-N-BE(2)C cells with phorbol 12-myristate 13-acetate (PMA) was found to enhance transcription of the human dopamine beta-hydroxylase (DBH) in those cells. To identify which cis-acting element is responsive to the PMA treatment during DBH gene expression, we employed transient transfection assays with serially deleted constructs of the human DBH gene's 5' upstream region fused to the chloramphenicol acetyltransferase (CAT) gene. Treatment of transfected cells with PMA resulted in an approximate threefold increase in CAT expression for all deletion constructs ranging from -978 bp to -262 bp, while the enhancement did not occur with a construct shortened to -114 bp. The region between -262 and -114 bp from the initiation site of transcription contains several cis-regulatory elements including a cyclic AMP response element (CRE) and putative AP1 and YY1 sequences. Site-directed mutagenesis of those cis-acting elements were performed to identify which of the elements mediated the PMA-induced transcriptional enhancement. Substitution of bases in the putative AP1 site containing in part a putative YY1 sequence did not effect the PMA inducibility. However, specific mutations in the CRE sequence abolished the PMA-inducible effect. Changing the CRE sequence into an authentic AP1 sequence (TGACGTCC --> TGACTCA) did not affect the PMA inducibility, suggesting that AP1 factors might interact with the new AP1 site upon PKC activation. A specific PKC inhibitor, GF109203X, completely inhibited the stimulatory effect of PMA on the expression of the human DBH gene. PMA induced an increase in the DBH mRNA level as detected by Northern blot analysis. Gel retardation showed that the binding of nuclear factors to CRE, putative YY1, and AP1 was sequence specific. Our data suggest that the enhancement of the human DBH gene expression by PMA treatment is mediated by the CRE motif in the 5' upstream region of the gene, and occurs via a PKC-dependent pathway.
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PMID:A protein kinase C-activating phorbol ester enhances transcription of the human DBH gene through a cyclic AMP response element in SK-N-BE(2)C cells. 942 17

Low density lipoprotein (LDL) has been shown to perturb endothelial cells, with manifestations ranging from alterations in free radicals and arachidonate metabolism to stress fiber formation and monocyte recruitment. Some of these changes are regulated by LDL at the transcriptional level. Using mobility shift assays with consensus sequences for various transcription factors, we have detected an increase in activator protein 1 (AP-1), but not nuclear factor-kappaB (NF-kappaB), binding in human umbilical vein endothelial cells exposed to LDL. Following transfection, AP-1-driven chloramphenicol acetyltransferase and AP-1-driven-luciferase are upregulated by LDL. In contrast, there is no effect on NF-kappaB-driven chloramphenicol acetyltransferase. AP-1 increases in a biphasic fashion, with the first peak occurring 6 hours after and the second 48 hours after exposure to LDL. This AP-1 binding increase involves c-Jun, but not c-Fos, as shown by gel supershift, Northern hybridization, and Western blotting analyses. c-Jun mRNA levels are elevated by 9 hours after and remain so until at least 24 hours after exposure to LDL. c-Jun protein levels increase at 12 hours and continue to rise for 24 hours after exposure to LDL. Moreover, this LDL-increased AP-1 binding is suppressed by several protein kinase (PK) inhibitors: the PKC inhibitor calphostin C, the cAMP-dependent PK inhibitor H89, and the tyrosine PK inhibitors genistein and lavendustin A. This study demonstrates that (1) LDL is an endothelial agonist distinct from other cell stimulators, such as cytokines, endotoxin, and phorbol 12-myristate 13-acetate, because LDL appears to activate human umbilical vein endothelial cells predominantly through the transcription factor AP-1 and not NF-kappaB; and (2) LDL increases AP-1 via mechanisms involving multiple kinase activities and c-Jun transcription.
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PMID:LDL induces transcription factor activator protein-1 in human endothelial cells. 951 17

The purpose of this study was to analyze the mechanism of transcriptional activation of human chorionic gonadotropin-alpha (hCGalpha) gene by epidermal growth factor (EGF) in trophoblast cells. We stably transfected hCGalpha promoter-chloramphenicol acetyltransferase constructs into Rcho-1 trophoblast cells and monitored the promoter activities. -290-base pair hCGalpha promoter containing a tandem repeat of cAMP response element (CRE) was activated by EGF in a dose- and time-dependent manner. Deletion analysis of hCGalpha promoter suggested an involvement of CRE in EGF-induced hCGalpha transcriptional activation. Moreover, the hCGalpha promoter, of which both CREs were mutated, did not respond to EGF. These results indicate that EGF activates the hCGalpha gene transcription through CRE. Although EGF did not alter the amount of CRE-binding protein (CREB), EGF induced CREB phosphorylation. We next examined the mechanism of CREB phosphorylation by EGF. Protein kinase C inhibitors (H7, staurosporin, and chelerythrine) inhibited EGF-induced CREB phosphorylation, whereas either mitogen-activated protein kinase kinase-1 inhibitor (PD98059) or protein kinase A inhibitor (H8) showed no effect. Furthermore, H7 and staurosporin but not H8 inhibited hCGalpha promoter activation by EGF. In conclusion, EGF promotes hCGalpha gene transcription via the CRE region probably by phosphorylating CREB mainly through the protein kinase C pathway in trophoblast cells.
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PMID:Human chorionic gonadotropin-alpha gene is transcriptionally activated by epidermal growth factor through cAMP response element in trophoblast cells. 952 71

Our previous finding that insulin induces apolipoprotein AI (apoAI) transcription points to the participation of intracellular signaling. This finding prompted us to ask whether two classical G-protein-coupled signaling pathways requiring activated protein kinase A (PKA) or kinase C (PKC) may also regulate apoAI. Therefore, human hepatoma, Hep G2 cells stably transfected with pAI.474-CAT, a reporter construct spanning -474 to -7 of apoAI DNA fused to chloramphenicol acetyltransferase (CAT) were treated with 10 microm forskolin (FSK) or 50 nm phorbol dibutyrate (PDBu) to activate PKA and PKC, respectively. Results showed that the apoAI promoter activity increased 4-5-fold following 24 h of treatment with either FSK or PDBu. Induction by either agent was blocked with actinomycin D but not the protein synthesis inhibitor, cycloheximide. The PKA inhibitor, PKI 14-22 amide, abrogated induction by FSK, 100 microm 8-bromo-cAMP, or 100 ng/ml cholera toxin, but it had no effect on activation via PKC. Similarly, PDBu induction was attenuated by 2 microm of the PKC inhibitor, GF109203X, but it did not affect FSK activity. Next we used deletional constructs to show that the actions of FSK and PDBu required the insulin-responsive core element (IRCE). This motif matched the consensus binding site for the transcription factor, Sp1. The binding of Sp1 to the IRCE was confirmed by gel-retardation and supershift analysis. Site-directed mutagenesis of the IRCE eliminated Sp1 action and induction by FSK or PDBu. Whereas overexpression of Sp1 enhanced basal and FSK or PDBu induced promoter activity, transfection of an antisense oligomer against Sp1 mRNA attenuated both parameters. In summary, activation of PKA or PKC increases apoAI promoter activity. The activity of both signaling pathways is mediated by the IRCE, a motif that binds the transcription factor, Sp1.
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PMID:Activation of apolipoprotein AI gene expression by protein kinase A and kinase C through transcription factor, Sp1. 1082 13

The cAMP-dependent pathway up-regulates MITF (microphthalmia-associated transcription factor), important for key melanogenic proteins such as tyrosinase, TRP-1 (tyrosinase-related protein 1) and TRP-2. We asked whether MITF is also a key transcription factor for PKC-beta (protein kinase C-beta), required to phosphorylate otherwise inactive tyrosinase. When paired cultures of human melanocytes were treated with isobutylmethylxanthine, known to increase intracellular cAMP, both protein and mRNA levels of PKC-beta were induced by 24 h. To determine whether MITF modulates PKC-beta expression, paired cultures of human melanocytes were transfected with dn-MITF (dominant-negative MITF) or empty control vector. By immunoblotting, PKC-beta protein was reduced by 63+/-3.7% within 48 h. Co-transfection of an expression vector for MITF-M, the MITF isoform specific for pigment cells, or empty control vector with a full-length PKC-beta promoter-CAT (chloramphenicol acetyltransferase) reporter construct (PKC-beta/CAT) into Cos-7 cells showed >60-fold increase in CAT activity. Melanocytes abundantly also expressed MITF-A, as well as the MITF-B and MITF-H isoforms. However, in contrast with MITF-M, MITF-A failed to transactivate co-expressed PKC-beta/CAT or CAT constructs under the control of a full-length tyrosinase promoter. Together, these results demonstrate that MITF, specifically MITF-M, is a key transcription factor for PKC-beta, linking the PKC- and cAMP-dependent pathways in regulation of melanogenesis.
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PMID:MITF mediates cAMP-induced protein kinase C-beta expression in human melanocytes. 1641 96


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