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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent studies from this laboratory demonstrated that bradykinin transiently elevates intracellular Ca2+ and inhibits Cl-reabsorption in the in vitro microperfused medullary thick ascending limb (mTAL) of the rat. The present study was designed to identify the intracellular signaling mechanism(s) that mediate this response. Preincubation with the intracellular calcium chelator BAPTA (10(-5) M) completely eliminated the bradykinin-dependent increase in intracellular Ca2+ and the suppression of Cl- transport. Preincubation with the cGMP-dependent protein kinase inhibitor H-89 (10(-5) M) had no effect on the transport response to bradykinin. In contrast, 17-octadecynoic acid (17-ODYA; 10(-5) M), a suicide-substrate inhibitor of renal cytochrome P450 omega-hydroxylase, completely blocked the transport response to bradykinin, while the cyclooxygenase inhibitor sodium meclofenamate (10(-5) M) had no effect. Finally, addition of the cytochrome P450 omega-hydroxylase metabolite 20-hydroxyeicosatetraenoic acid (20-HETE; 10(-8) M) to the bathing medium significantly inhibited Cl- transport in the mTAL (delta -39 +/- 6.0%; p < 0.05), while the epoxygenase metabolite 5,6-epoxyeicosatrienoic acid (5,6-EET; 10(-8) M) had no effect. These data suggest that the bradykinin-dependent inhibition of Cl- transport in the mTAL of the rat is mediated by cytochrome P450 dependent metabolite(s) of arachidonic acid.
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PMID:P450 arachidonate metabolites mediate bradykinin-dependent inhibition of NaCl transport in the rat thick ascending limb. 911 29

Ligand- and second messenger-regulated expression of the gene for steroidogenic acute regulatory protein (StAR) was evaluated in luteinized porcine granulosa cells. For comparison, cytochrome P450 side-chain cleavage (P450scc) was examined. Northern hybridization with homologous cDNA probes demonstrated three StAR mRNA species, of 2.7, 1.6, and 0.8 kilobases (kb), with the smallest variably present, and a single P450scc band at 1.9 kb. FSH elevated both STAR and P450scc messages in a dose-dependent manner over 6 h and continually stimulated both over 24 h (p < 0.001). STAR message induction depended on transcription, as did that of P450scc. Over 6 h, actinomycin D eliminated constitutive StAR message and reduced that of P450scc by two thirds, indicating briefer persistence of StAR. Pretreatment with cycloheximide prevented FSH induction of StAR and P450scc mRNA, implicating intermediate protein synthesis in expression of both genes. Dibutyryl cAMP caused time-dependent increases in StAR and P450 mRNAs over 24 h (p < 0.001), indicating the importance of the protein kinase A (PKA) pathway in their gene expression. Activation of the protein kinase C (PKC) pathway by a phorbol ester eliminated FSH induction of STAR mRNA increases (p < 0.01) while only reducing P450scc induction (p < 0.05). Thus, StAR gene expression, as reflected in mRNA abundance, is regulated by FSH via the PKA pathway and is dependent on transcription and translation. Conversely, the PKC pathway inhibits induction of these important steroid synthetic genes in luteinized granulosa cells.
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PMID:Follicle-stimulating hormone and intracellular second messengers regulate steroidogenic acute regulatory protein messenger ribonucleic acid in luteinized porcine granulosa cells. 928 5

A CYP11B2 gene encoding cytochrome P450 aldosterone synthase (P450aldo) was isolated from a hamster genomic library. The gene, which contained 9 exons, was composed of 9,045 bp, of which 3,722 bp were located in the 5' untranslated region (5' UTR). A TATA box sequence (gataaa) and other putative cis elements, previously named Ad1 to Ad6, were identified in the 5' UTR of the hamster gene comparable to the CYP11B2 gene of other animal species. Footprint analysis showed protection by nuclear protein extracts from hamster adrenal zona glomerulosa (ZG) in the regions containing the above mentioned cis elements. In addition, a new protected cis element, between -143 and -161 bp, was demonstrated, and gel-shift assays revealed that the sequence of this new cis element was specifically retarded by factors in the nuclear extracts of hamster adrenal ZG. We then examined the transcriptional activity of the 5' UTR of the CYP11B2 gene, using chloramphenicol acyltransferase (CAT) as the reporter gene. Ten deletion plasmids were constructed using a modified pCAT vector. Transient transfections of the chimeric reporter constructs into Y1 cells showed that the highest basal promoter activity was obtained with the construct containing up to -134 bp. Increasing the length of the regulatory region of CYP11B2 gene to -167 bp resulted in less than two-thirds of the maximal activity, indicating the probability of putative inhibitory cis elements in this area of the gene. Forskolin stimulated the expression of the reporter gene of deletion plasmids excepting the construct containing only the TATA box, and the highest activity also occurred with the -134 bp construct. TPA had no stimulatory effects on any of the constructs, and interestingly it slightly inhibited CAT activity. In contrast to TPA, staurosporine, an inhibitor of the PKC pathway, stimulated CAT activity. To conclude, the promoter region of the hamster CYP11B2 gene transfected in Y1 cells is responsive to forskolin, indicating that the gene is controlled by the PKA signaling pathway. Paradoxically, staurosporine, but not TPA, stimulates the promoter activity of the CYP11B2 gene, indicating that PKC might, at least in Y1 cells, act as a negative regulator on the aldosterone synthase promoter. Moreover, a new cis element was shown to exert a negative effect on basal as well as on stimulated activities of the hamster promoter CYP11B2 gene.
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PMID:Characterization of the hamster CYP11B2 gene encoding adrenal cytochrome P450 aldosterone synthase. 930 41

In human placenta the cytochrome P450 side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase type 1 (3 beta-HSD-1) convert cholesterol and pregnenolone producing progesterone, whereas 17 beta-hydroxysteroid dehydrogenase type 1 (17 beta-HSD-1) mediates the interconversion of estrone and estradiol. We have examined the effects of calcium on phorbol ester- and cAMP-induced P450scc, 3 beta-HSD-1 and 17 beta-HSD-1 mRNAs in human JEG-3 cells. A23187 increased in a dose-dependent fashion in the 1.3 kb 17 beta-HSD-1 mRNA whereas a weaker increase followed by a gradual depletion effect of A23187 was observed on 3 beta-HSD-1 mRNA. No significant effect of A23187 on P450scc mRNA was observed. Using 0.50 microM of A23187 the induction of 3 beta-HSD-1 and 17 beta-HSD-1 mRNAs was maximum within about 6 h whereas P450scc mRNA levels stayed unaffected throughout the time-course period. The action of A23187 was synergistic on cAMP-stimulated 17 beta-HSD-1 mRNA levels, while in a dose-dependent manner A23187 progressively depleted 3 beta-HSD-1 and P450scc mRNA abundance probably by activation of a calcium-/calmodulin-dependent phosphodiesterase. On the phorbol 12-myristate, 13-acetate (PMA)-stimulated 3 beta-HSD-1, 17beta-HSD-1 and P450scc mRNA levels only the lowest concentration of A23187 potentialized the PMA effect on the 17 beta-HSD-1 mRNA levels. Using thapsigargin (TG), a cell-permeable sesquiterpene lactone that releases calcium by inhibiting sarco/endoplasmic reticular calcium-ATPase, our data indicated the presence in JEG-3 cells of TG-sensitive and TG-insensitive calcium-ATPases regulating 3 beta-HSD-1 and 17 beta-HSD-1 mRNA levels. These results emphasized the complexity of calcium contribution with the protein kinase A and C pathways in the regulation of P450scc, 3 beta-HSD-1 and 17 beta-HSD-1 mRNA levels. In addition, the different sensitivity of these genes to calcium suggest they could be activated by different subclasses of PKCs.
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PMID:Regulation of cytochrome P450 cholesterol side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase type 1 and estradiol-17 beta-hydroxysteroid dehydrogenase mRNA levels by calcium in human choriocarcinoma JEG-3 cells. 935 73

The physiological importance of adrenal 21-hydroxylase cytochrome P450 (CYP21) expression is clearly demonstrated by 21-hydroxylase deficiency, which results in adrenal hyperplasia and over-production of C19 steroids, leading to virilization. The mechanisms regulating normal expression of this key enzyme in human adrenocortical cells are ill defined. Herein we examine the role of the calcium, protein kinase C, and protein kinase A signaling pathways in the expression of CYP21 messenger ribonucleic acid (mRNA) using the H295R human adrenocortical cell model. Forskolin (10 mumol/L) treatment caused a progressive increase in CYP21 mRNA levels (maximum, 4-fold; P < 0.05) over 36 h of treatment, whereas angiotensin II (AII; 10 nmol/L) produced a smaller, biphasic rise (maximum, 1.8-fold at 12 h; P < 0.05). K+ (14 mmol/L) also induced a time-dependent (maximal, 1.5-fold at 12 h; P < 0.05) and dose-dependent (P < 0.05 12 mmol/L or above at 20 h) rise in CYP21 mRNA levels. The action of forskolin was reproduced by dibutyryl cAMP, confirming the involvement of cAMP in this response. The action of AII was greater than that of K+ or the calcium channel agonist BAYK8644, suggesting that AII action was not solely through the Ca2+ signaling pathway. The action of AII was reproduced and indeed exceeded by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA; 10 nmol/L; 5.5-fold increase; P < 0.05). The actions of forskolin alone were not significantly increased by combined treatment with AII, suggesting neither synergy nor attenuation of the effects of protein kinase A activation. This was further demonstrated at the level of mRNA and 21-hydroxylase activity by the observation that the effect of forskolin and TPA in combination did not exceed that of TPA alone. Inhibition of protein synthesis with cycloheximide blocked induction of CYP21 as well as type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSDII) mRNA expression in response to AII, forskolin, and dibutyryl cAMP, but had no effect on 17 alpha-hydroxylase cytochrome P450 (CYP17) or cholesterol side-chain cleavage cytochrome P450 (CYP11A) mRNA. Together, these findings were remarkably similar to those of our previous studies regarding mechanisms regulating 3 beta HSDII expression and underline the existence of a subset of steroidogenic enzymes regulated positively (CYP21 and 3 beta HSDII) as opposed to negatively (CYP17 and CYP11A) by the protein kinase C signaling pathway. The additional finding of a small induction of CYP21 expression in response to increased Ca2+, as previously reported for CYP17, but not 3 beta HSDII, expression, also demonstrates that the mechanisms of control of CYP21 and 3 beta HSDII are not identical. This latter finding may also relate to how CYP21 as well as CYP17 expression continues in the zona reticularis after adrenarche, whereas 3 beta HSD expression declines.
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PMID:Protein kinase A, protein kinase C, and Ca(2+)-regulated expression of 21-hydroxylase cytochrome P450 in H295R human adrenocortical cells. 958 61

17-(Allylamino)-17-demethoxygeldanamycin (17AAG), a compound that is proposed for clinical development, shares the ability of geldanamycin to bind to heat shock protein 90 and GRP94, thereby depleting cells of p185erbB2, mutant p53, and Raf-1. Urine and plasma from mice treated i.v. with 17AAG contained six materials with absorption spectra similar to that of 17AAG. Therefore, in vitro metabolism of 17AAG by mouse and human hepatic preparations was studied to characterize: (a) the enzymes responsible for 17AAG metabolism; and (b) the structures of the metabolites produced. These materials had retention times on high-performance liquid chromatography of approximately 2, 4, 5, 6, 7, and 9 min. When incubated in an aerobic environment with 17AAG, murine hepatic supernatant (9000 x g) produced each of these compounds; the 4-min metabolite was the major product. This metabolism required an electron donor, and NADPH was favored over NADH. Metabolic activity resided predominantly in the microsomal fraction. Metabolism was decreased by approximately 80% in anaerobic conditions and was essentially ablated by CO. Microsomes prepared from human livers produced essentially the same metabolites as produced by murine hepatic microsomes, but the 2-min metabolite was the major product, and the 4-min metabolite was next largest. There was no metabolism of 17AAG by human liver cytosol. Metabolism of 17AAG by human liver microsomes also required an electron donor, with NADPH being preferred over NADH, was inhibited by approximately 80% under anaerobic conditions, and was essentially ablated by CO. Liquid chromatography/mass spectrometry analysis of human and mouse in vitro reaction mixtures indicated the presence of materials with molecular weights of 545, 601, and 619, compatible with 17-(amino)-17-demethoxygeldanamycin (17AG), an epoxide, and a diol, respectively. The metabolite with retention time of 4 min was identified as 17AG by cochromatography and mass spectral concordance with authentic standard. Human microsomal metabolism of 17AAG was inhibited by ketoconazole, implying 3A4 as the responsible cytochrome P450 isoform. Incubation of 17AAG with cloned CYP3A4 produced metabolites 4 and 6. Incubation of 17AAG with cloned CYP3A4 and cloned microsomal epoxide hydrolase produced metabolites 2 and 4, with greatly decreased amounts of metabolite 6. Incubation of 17AAG with human hepatic microsomes and cyclohexene oxide, a known inhibitor of microsomal epoxide hydrolase, did not affect the production of metabolite 4 but decreased the production of metabolite 2 while increasing the production of metabolite 6. These data imply that metabolite 2 is a diol and metabolite 6 is an epoxide. Mass spectral fragmentation patterns and the fact that 17AG is not metabolized argue for the epoxide and diol being formed on the 17-allylamino portion of 17AAG and not on its ansamycin ring. These data have implications with regard to preclinical toxicology and activity testing of 17AAG as well as its proposed clinical development because: (a) production of 17AG requires concomitant production of acrolein from the cleaved allyl moiety; and (b) 17AG, which was not metabolized by microsomes, has been described as being as active as 17AAG in decreasing cellular p185erbB2.
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PMID:Metabolism of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) by murine and human hepatic preparations. 962 79

Many toxic compounds are activated by cytochrome P450 (CYP) 2E1 to reactive metabolites, which represents a potential hazard for cellular homeostasis. Therefore knowledge about CYP2E1 regulation could be of great biological importance. It has been shown that CYP2E1 is controlled transcriptionally and post-translationally by phosphorylation. In the present study we investigated the role of serine-129 (Ser129) in the protein kinase A (PKA) recognition sequence motif Arg-Arg-Phe-Ser129. To gain further insights into the possible relevance of Ser129 for CYP2E1 function, Ser129 was replaced by alanine (Ala) or glycine (Gly) by site-directed mutations of the cDNA coding for CYP2E1. The mutant cDNAs were transfected into Chinese hamster lung fibroblast V79 cells. Despite the mutation in the PKA phosphorylation motif, all strains produced catalytically active CYP2E1. However, there was a marked change in the substrate preference: The Gly129-containing strains hydroxylated p-nitrophenol (PNP) to a markedly higher extent than the wild-type cDNA-containing cells, while they demethylated N-nitrosodimethylamine (NDMA) to a markedly lower extent than the wild-type cells. All the strains activated NDMA to mutagenic products. Treatment with the membrane-permeating cAMP derivative db-cAMP reduced markedly both the PNP hydroxylase and the NDMA demethylase activities as well as the mutation frequency induced by NDMA in the Ser129-containing strain. This decrease in activity was not accompanied by a decrease in CYP2E1 content. In addition, the catalytic activities of CYP2E1 were decreased in microsomes from rat hepatocytes treated with db-cAMP. Also in this case, the decrease in activities was not accompanied by a decrease in enzyme protein. These findings argue that involvement of Ser129 and its phosphorylation is not in determining CYP2E1 protein level, but rather in controlling its catalytic activity. In contrast, in the strains containing Ala129 or Gly129, treatment with db-cAMP caused a marked increase in both PNP hydroxylase and NDMA demethylase. In these strains a similar db-cAMP-mediated increase was also observed in the mutation frequency, resulting from the treatment with the promutagen NDMA, which is activated by CYP2E1. Our results show that CYP2E1 in V79 cells responds in two separate ways to db-cAMP exposure depending on the amino acid residue present in the PKA recognition sequence. The enzyme is committed to a negative regulation by db-cAMP if Ser129 is the target amino acid for PKA, leading to a decrease in the metabolic activation to mutagenic and carcinogenic species. On the other hand, Ala129 or Gly129 substitution directed CYP2E1 toward a positive regulation by increasing its catalytic activities and metabolic activation to mutagenic intermediates in the presence of db-cAMP. We also obtained evidence that cAMP-mediated downregulation of wild-type (Ser129) CYP2E1 was not accompanied by its destruction but instead by its stabilization, which shows that Ser129 is not involved in CYP2E1 degradation but dictates requirements for its specific activities.
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PMID:Differential modulation of CYP2E1 activity by cAMP-dependent protein kinase upon Ser129 replacement. 966 27

The 25-hydroxyvitamin D3 1alpha-hydroxylase, also referred to as CYP27B1, is a mitochondrial cytochrome P450 enzyme that catalyzes the biosynthesis of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) from 25-hydroxyvitamin D3 in renal proximal tubular cells. Recently, human, mouse, and rat CYP27B1 cDNA have been cloned, however the gene regulation has not been fully elucidated. In the present study, porcine CYP27B cDNA was cloned, and the effects of cAMP and vitamin D3 on the regulation of CYP27B1 mRNA expression in LLC-PK1 cells were examined. PCR cloning revealed that porcine CYP27B1 cDNA consisted of 2316 bp, encoding a protein of 504 amino acids. The deduced amino acid sequence showed over 80% identity to the human, mouse, and rat enzyme. LLC-PK1 cells were incubated with humoral factors, and expression of CYP27B1 mRNA was measured by a quantitative reverse transcription-PCR. At the completion of 3-, 6-, 12-, and 24-h incubations, 500 micromol/L 8-bromo-cAMP had significantly increased CYP27B1 mRNA expression (260 to 340%). The adenylate cyclase activator forskolin at 50 micromol/L also had a stimulatory effect at 6 h (190%). Moreover, the protein kinase A inhibitor H-89 reduced the cAMP effect. On the other hand, 1alpha,25(OH)2D3 had no effect on CYP27B1 mRNA expression at 10 and 100 nmol/L, whereas expression of 25-hydroxyvitamin D3 24-hydroxylase (CYP24) mRNA was markedly increased by 1alpha,25(OH)2D3. These findings suggest that LLC-PK1 cells express CYP27B1 mRNA, and that cAMP is an upregulating factor of the CYP27B1 gene in vitro.
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PMID:Cloning of porcine 25-hydroxyvitamin D3 1alpha-hydroxylase and its regulation by cAMP in LLC-PK1 cells. 1023 81

Acetaminophen (APAP) is a widely used analgesic and antipyretic that can lead to severe liver damage when taken at excessive doses. APAP toxicity results when cytochrome P450-generated APAP metabolites trigger an oxidative stress and covalently modify target proteins. APAP has also been reported to inhibit cells from completing S-phase through a cytochrome P450-independent mechanism, raising the possibility that APAP may directly suppress liver regeneration and repair. Here we show that APAP also inhibits entrance of Hepa 1-6 cells into the cell cycle by blocking a number of events associated with the G0-G1 transition. We have found that APAP inhibits serum growth factor activation of c-myc expression, NF-kappaB DNA binding, and Raf kinase. Therefore, the ability of APAP to inhibit passage of cells through both G1 and S phases might interfere with organ regeneration and thus exacerbate acute liver damage caused by APAP.
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PMID:Modulation of serum growth factor signal transduction in Hepa 1-6 cells by acetaminophen: an inhibition of c-myc expression, NF-kappaB activation, and Raf-1 kinase activity. 1035 17

The pituitary peptide hormone ACTH regulates transcription of the cholesterol side chain cleavage cytochrome P450 (CYP11A) gene via cAMP and activation of cAMP-dependent protein kinase. A G-rich sequence element conferring cAMP-dependent regulation has been found to reside within region -118 to -100 of the bovine CYP11A promoter. Previous studies have suggested that it binds a protein antigenically related to the transcription factor Sp1. We now report that the -118/-100 element binds both Sp1 and Sp3, members of the Sp family of transcription factors. We have made use of Drosophila SL2 cells, which lack endogenous Sp factors, to dissect the possible functional roles of Sp1, Sp3, and Sp4. All factors stimulated the activity of cotransfected reporter constructs in which the promoter of the bovine CYP11A gene regulates luciferase expression. Sp3 did not repress Sp1-dependent activation, as has previously been shown for other G-rich promoters. Mutation of the -118/-100 element of CYP11A abolished Sp1-mediated activation of a CYP11A reporter gene in SL2 cells as well as cAMP responsiveness in human H295R cells. Furthermore, cotransfection of SL2 cells with the catalytic subunit of cAMP-dependent protein kinase together with Sp1 and a CYP11A reporter construct enhanced Sp1-dependent activation of the reporter 4.2-fold, demonstrating that Sp1 confers cAMP responsiveness in these cells. Thus, we show that introduction of Sp1 alone in an Sp-negative cell such as SL2 is sufficient to achieve the cAMP-dependent regulation observed using the -118/-100 element of CYP11A in adrenocortical cells.
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PMID:Role of Sp1 in cAMP-dependent transcriptional regulation of the bovine CYP11A gene. 1038 57


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