On the mechanism of the okadaic acid-induced inhibition of phosphatidylcholine biosynthesis in isolated rat hepatocytes.
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The mechanism of inhibition of phosphatidylcholine biosynthesis by okadaic acid was investigated in suspension cultures of isolated rat hepatocytes. Cells were pulsed with [methyl-3H]choline and chased in the absence or presence of 1 microM okadaic acid for up to 120 min. Phosphatidylcholine biosynthesis was inhibited after 15 min of chase. To see if okadaic acid altered the degree of phosphorylation of cytidylyltransferase (CT), hepatocytes were incubated with 32P(i) and chased in the absence or presence of okadaic acid. Okadaic acid caused a rapid (within 15 min) increase in the phosphorylation state of the cytosolic enzyme. Two-dimensional peptide map analysis revealed an increase in the phosphorylation of several peptides in okadaic acid-treated hepatocytes compared with controls. After 15 min of incubation of hepatocytes with okadaic acid, membrane CT activity was decreased and a corresponding increase in cytosolic CT activity was observed. In hepatocytes incubated with okadaic acid and oleate a correlation between membrane CT activity, diacylglycerol level, and phosphatidylcholine biosynthesis was observed. These data suggest that the concentration of diacylglycerol is responsible for the increase in membrane CT activity and subsequently phosphatidylcholine biosynthesis in oleate-treated cells. We postulate that the okadaic acid-induced decrease in phosphatidylcholine biosynthesis is due to an increase in the phosphorylation state of CT which promotes a translocation of CT activity from the membranes to the cytosol. (+info)
Comparison of the lipid regulation of yeast and rat CTP: phosphocholine cytidylyltransferase expressed in COS cells.
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The CTP: phosphocholine cytidylyltransferase (CT) gene from yeast and cDNA from rat liver were over-expressed 20-30-fold in COS cells. Most of the CT activities were found in the cytosolic fraction. The regulation of the yeast CT activity (Y-CT) by lipids was characterized for the first time in comparison with the regulation of the well-studied rat CT (R-CT). Sonicated vesicles composed of egg phosphatidylcholine (PC) or 1-stearoyl-2-oleoyl PC had no effect on Y-CT and only slightly stimulated R-CT activity. Both CTs were activated 10-50-fold by the anionic lipids cardiolipin, phosphatidyl-glycerol, phosphatidylinositol and oleic acid. The effects of varying the vesicle concentration and the mol% of anionic lipid in PC vesicles were tested. The concentration optima for the activation of Y-CT by oleic acid or anionic phospholipids were 5-10-fold lower than those for R-CT. For example, the stimulation of Y-CT activity by phosphatidylglycerol vesicles was optimal between 5 and 15 microM and declined at higher concentrations, but R-CT activation by these vesicles saturated at approximately 25 microM. The positively charged aminolipid sphingosine antagonized the stimulation by oleic acid of both Y-CT and R-CT. Y-CT activity was insensitive to PC vesicles containing the neutral lipids diacylglycerol, monoacylglycerol or oleyl alcohol. However, R-CT was stimulated 10-20-fold by vesicles containing these neutral lipids. Translocation of the CTs to microsomal membranes enriched with anionic or neutral lipids was compared. Oleic acid enrichment promoted translocation of Y-CT and R-CT, whereas diacylglycerol promoted only R-CT translocation. These data show that the activity of Y-CT is lipid-sensitive. Y-CT is affected only by charged lipids, whereas R-CT responds to charged and neutral lipid activators. The data are consistent with different modes of interaction of the two CTs with lipids. (+info)
Pre-translational regulation by glucocorticoid of fatty acid and phosphatidylcholine synthesis in type II cells from fetal rat lung.
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Exposure to fibroblast-conditioned cortisol-containing medium increased fatty acid synthase activity and fatty acid synthase, acetyl-CoA carboxylase and ATP citrate lyase mRNA abundance in fetal type II alveolar epithelial cells. Both fibroblast conditioning and cortisol in the medium were required for maximal effect on the mRNA levels, indicating involvement of mesenchymal-epithelial interaction in the cortisol effects. The observed effects provide evidence for an earlier hypothesis that increased activity of CTP:phosphocholine cytidylyltransferase in lung tissue caused by glucocorticoid is due to increased fatty acid synthesis. However, evidence suggesting pre-translational regulation of this enzyme by glucocorticoid was also found. (+info)
1-beta-D-arabinofuranosylcytosine-diphosphate-choline is formed by the reversal of cholinephosphotransferase and not via cytidylyltransferase.
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In an effort to identify the pathway leading to the formation of 1-beta-D-arabinofuranosylcytosine-diphosphate (ara-CDP)-choline from 1-beta-D-arabinofuranosylcytosine (ara-C) treatment of cultured cells, as well as of cells obtained from leukemia patients, we probed the enzymatic steps involved in the CDP-choline pathway for phosphatidylcholine biosynthesis. Ara-C-triphosphate was not a substrate for CTP:phosphocholine cytidylyltransferase activity under the conditions employed, whereas CTP and dCTP were utilized to form CDP-choline and dCDP-choline, respectively. When presented together, ara-C-triphosphate and CTP inhibited the enzymatic conversion of CTP to CDP-choline in the presence of phosphocholine, with a Ki of 6 mM. Since CTP:phosphocholine cytidylyltransferase did not appear to be responsible for the increased levels of ara-CDP-choline, we next studied the other enzyme in the pathway for phosphatidylcholine synthesis that could form ara-CDP-choline, CDP-choline:1,2-diacylglycerol cholinephosphotransferase. CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity present in microsomes isolated from L5178Y murine leukemia cells exhibited a reversal of its normal catalytic activity, using CMP and 1-beta-D-arabinofuranosylcytosine-monophosphate (ara-CMP) along with phosphatidylcholine to produce either CDP-choline or ara-CDP-choline, plus diradylglycerol. The Vmax and Km values for CMP were 0.78 +/- 0.04 nmol/min/mg and 340 +/- 20 microM, respectively, whereas the Vmax and Km for ara-CMP were 0.22 +/- 0.06 nmol/min/mg and 1410 +/- 540 microM, respectively. A Ki value of 3 mM was obtained for ara-CMP under the cell-free assay conditions used. These results indicate that ara-CDP-choline most likely arises from a reversal of the CDP-choline:1,2-diacylglycerol cholinephosphotransferase utilizing ara-CMP, rather than from the catalysis of ara-C-triphosphate plus phosphocholine to ara-CDP-choline by CTP:phosphocholine cytidylyltransferase. It is speculated that this mechanism may explain, in part, the rapid cellular lysis observed with high dose ara-C therapy. (+info)
Cct1, a phosphatidylcholine biosynthesis enzyme, is required for Drosophila oogenesis and ovarian morphogenesis.
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Patterning of the Drosophila egg requires cooperation between the germline cells and surrounding somatic follicle cells. In order to identify genes involved in follicle cell patterning, we analyzed enhancer trap lines expressed in specific subsets of follicle cells. Through this analysis, we have identified tandem Drosophila genes homologous to CTP: phosphocholine cytidylyltransferase (CCT), the second of three enzymes in the CDP-choline pathway, which is used to synthesize phosphatidylcholine. Drosophila Cct1 is expressed at high levels in three specific subsets of follicle cells, and this expression is regulated, at least in part, by the TGF-beta and Egfr signaling pathways. Mutations in Cct1 result in a number of defects, including a loss of germline stem cell maintenance, mispositioning of the oocyte, and a shortened operculum, suggesting that Cct1 plays multiple roles during oogenesis. In addition, Cct1 mutants display a novel branched ovariole phenotype, demonstrating a requirement for this gene during ovarian morphogenesis. These data provide the first evidence for a specific role for CCT, and thus for phosphatidylcholine, in patterning during development. (+info)
Membrane binding modulates the quaternary structure of CTP:phosphocholine cytidylyltransferase.
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CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids. Previous work demonstrated that CCT is a homodimer. In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT. Chemical cross-linking reactions were established which captured intradimeric interactions but not random CCT dimer collisions. The efficiency of capturing covalent cross-links with four different reagents was diminished markedly upon presentation of activating anionic lipid vesicles but not zwitterionic vesicles. Experiments were conducted to show that the anionic vesicles did not interfere with the chemistry of the cross-linking reactions and did not sequester available cysteine sites on CCT for reaction with the cysteine-directed cross-linking reagent. Thus, the loss of cross-linking efficiency suggested that contact sites at the dimer interface had increased distance or reduced flexibility upon binding of CCT to membranes. The regions of the enzyme involved in dimerization were mapped using three approaches: 1) limited proteolysis followed by cross-linking of fragments, 2) yeast two-hybrid analysis of interactions between select domains, and 3) disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines. We found that the N-terminal domain (amino acids 1-72) is an important participant in forming the dimer interface, in addition to the catalytic domain (amino acids 73-236). We mapped the intersubunit disulfide bond to the cystine 37 pair in domain N and showed that this disulfide is sensitive to anionic vesicles, implicating this specific region in the membrane-sensitive dimer interface. (+info)
Transcriptional repression of the CTP:phosphocholine cytidylyltransferase gene by sphingosine.
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We examined the effects of the bioactive lipid, sphingosine, on the expression of the rate-limiting enzyme involved in surfactant phosphatidylcholine synthesis, CCTalpha (CTP:phosphocholine cytidylyltransferase alpha). Sphingosine decreased phosphatidylcholine synthesis by inhibiting CCT activity in primary alveolar type II epithelia. Sphingosine decreased CCTalpha protein and mRNA levels by approx. 50% compared with control. The bioactive lipid did not alter CCTalpha mRNA stability, but significantly inhibited its transcriptional rate. In murine lung epithelia, sphingosine selectively reduced CCTalpha promoter-reporter activity when transfected with a 2 kb CCTalpha promoter/luciferase gene construct. Sphingosine also decreased transgene expression in murine type II epithelia isolated from CCTalpha promoter-reporter transgenic mice harbouring this 2 kb proximal 5'-flanking sequence. Deletional analysis revealed that sphingosine responsiveness was mapped to a negative regulatory element contained within 814 bp upstream of the coding region. The results indicate that bioactive sphingolipid metabolites suppress surfactant lipid synthesis by inhibiting gene transcription of a key surfactant biosynthetic enzyme. (+info)
Disruption of CCTbeta2 expression leads to gonadal dysfunction.
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There are two mammalian genes that encode isoforms of CTP:phosphocholine cytidylyltransferase (CCT), a key rate-controlling step in membrane phospholipid biogenesis. Quantitative determination of the CCT transcripts reveals that CCTalpha is ubiquitously expressed and is found at the highest levels in the testis and lung, with lower levels in the liver and ovary. CCTbeta2 is a very minor isoform in most tissues but is significantly expressed in the brain, lung, and gonads. CCTbeta3 is the third isoform recently discovered in mice and is expressed in the same tissues as CCTbeta2, with its highest level in testes. We investigated the role(s) of CCTbeta2 by generating knockout mice. The brains and lungs of mice lacking CCTbeta2 expression did not exhibit any overt defects. On the other hand, a large percentage of the CCTbeta2(-/-) females were sterile and their ovaries exhibited defective ovarian follicle development. The proportion of female CCTbeta2(-/-) mice with defective ovaries increased as the animals aged. The rare litters born from CCTbeta2(-/-) x CCTbeta2(-/0) matings had the normal number of pups. The abnormal ovarian histopathology was characterized by disorganization of the tissue in young adult mice and absence of follicles and ova in older mice, along with interstitial stromal cell hyperplasia which culminated in the emergence of tubulostromal ovarian tumors by 16 months of age. Grossly defective CCTbeta2(-/-) ovaries were associated with high follicle-stimulating (FSH) and luteinizing (LH) hormone levels. Male CCTbeta2(-/0) mice exhibited progressive multifocal testicular degeneration and reduced fertility but had normal FSH and LH levels. Thus, the most notable phenotype of CCTbeta2 knockout mice was gonad degeneration and reproductive deficiency. The results indicate that although CCTbeta2 is expressed at very low levels compared to the alpha-isoform, loss of CCTbeta2 expression causes a breakdown in the gonadal response to hormonal stimulation. (+info)