CTP:phosphocholine cytidylyltransferase in intestinal mucosa

CTP:phosphocholine cytidylyltransferase is thought to be a rate-limiting enzyme in phosphatidylcholine synthesis. This enzyme has not been well studied in intestine. We found that activity was greater in the non-lipid stimulated state (cytosolic form of the enzyme) than any previous tissue investigated (2.7 nM/min per mg protein). On addition of lysophosphatidylethanolamine, the enzyme only increased in activity 2.4-fold which is less than any previously reported tissue on lipid stimulation. As compared to liver, the enzyme was resistant to inhibition by chlorpromazine (gut, 100% activity remaining at 80 microM; 14% in liver). Tetracaine and propranolol were found to be impotent as inhibitors of the intestinal enzyme. Octanol-water partitioning showed that both chlorpromazine and tetracaine were hydrophobic, propranolol was not. pKa studies demonstrated that at the reaction pH, chlorpromazine would be uncharged. Physiologic experiments in which de novo phosphatidylcholine synthesis was either stimulated by bile duct fistulization and triacylglycerol infusion or suppressed by including phosphatidylcholine in a lipid infusion demonstrated that the enzyme (cytosolic enzyme) responded by decreasing Vmax but that the Km remained the same. In sum, these studies suggest that CTP:phosphocholine cytidylyltransferase in intestine is unique as compared to other tissues and that its response to a physiological stimulus is counter to that which would be adaptive.     

Substrate specificity of CTP:phosphocholine cytidylyltransferase.

The specificity of CTP:phosphocholine cytidylyltransferase from rat liver for phosphorylated bases has been investigated. The apparent Km for phosphocholine was 0.17 mM. As the number of methyl substituents on the phospho-base decreased, the apparent Km increased: 4.0 mM for phosphodimethylethanolamine, 6.9 for phosphomonomethylethanolamine and 68.4 for phosphoethanolamine. The Vmax for the reaction was similar for phosphocholine (12.6 mumol/min per mg protein), phosphomonomethylethanolamine (13.5 mumol/min per mg protein) and phosphoethanolamine (9.2 mumol/min per mg protein). When phosphodimethylethanolamine was the substrate, the Vmax was 3-fold higher (40.3 mumol/min per mg protein). Phosphoethanolamine, phosphomonomethylethanolamine and phosphodimethylethanolamine were competitive inhibitors of the cytidylyltransferase when phosphocholine was used as substrate with Ki values of 18.5 mM, 9.3 mM and 1.5 mM, respectively. The results show that the cytidylyltransferase is highly specific for phosphocholine.     

Feedback regulation of CTP:phosphocholine cytidylyltransferase translocation between cytosol and endoplasmic reticulum by phosphatidylcholine

The mechanism for the increased association of CTP:phosphocholine cytidylyltransferase (CT) with membranes of hepatocytes derived from choline-deficient, compared with choline-supplemented rats, has been investigated. The cells were maintained in culture for 4 h in a choline- and methionine-deficient medium. (Methionine is required for synthesis of phosphatidylcholine (PC) via methylation of phosphatidylethanolamine.) Afterward, the cells were incubated +/- choline for various times up to 4 h. In the presence, but not in the absence, of choline there was a translocation of CT activity from membranes to cytosol. During this time period there was no change in the amounts of unesterified fatty acids or diacylglycerol recovered from the hepatocytes. In addition, there was no evidence for a difference in the incorporation of 32P into CT or other cytosolic proteins isolated from hepatocytes +/- choline. In contrast, there was a highly significant correlation between the concentration of PC in the membranes and the increased activity of CT in the cytosol (R = 0.98) and the decreased activity in the membranes (R = 0.93). The concentration of PC could alternatively be altered by incubation of the choline-deficient hepatocytes with methionine or lyso-PC. With either of these supplementations highly significant correlation coefficients were observed between the concentration of PC in membranes and decreased activity of CT in membranes or increased activity in cytosol. The concentration of PC was reduced in the endoplasmic reticulum, but not the Golgi membranes, isolated from choline-deficient compared with choline-supplemented livers. The data suggest that the amount of PC in the endoplasmic reticulum feedback regulates the amount of CT associated with this membrane.     

Regulation of CTP:phosphocholine cytidylyltransferase by amphitropism and relocalization

Phosphatidylcholine (PC) synthesis in animal cells is generally controlled by cytidine 5'-triphosphate (CTP):phosphocholine cytidylyltransferase (CCT). This enzyme is amphitropic, that is, it can interconvert between a soluble inactive form and a membrane-bound active form. The membrane-binding domain of CCT is a long amphipathic alpha helix that responds to changes in the physical properties of PC-deficient membranes. Binding of this domain to membranes activates CCT by relieving an inhibitory constraint in the catalytic domain. This leads to stimulation of PC synthesis and maintenance of membrane PC content. Surprisingly, the major isoform, CCT alpha, is localized in the nucleus of many cells. Recently, a new level of its regulation has emerged with the discovery that signals that stimulate PC synthesis recruit CCT alpha from an inactive nuclear reservoir to a functional site on the endoplasmic reticulum.     

An increase in cytoplasmic CTP accelerates the reaction catalyzed by CTP:phosphocholine cytidylyltransferase in poliovirus-infected HeLa cells

Poliovirus increases phosphatidylcholine biosynthesis in HeLa cells by stimulation of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase (Vance, D.E., Trip, E.M., and Paddon, H.B. (1980) J. Biol. Chem. 255, 1064-1069). The mechanism for the virus effect has been investigated. An assay for the cytidylyltransferase which mimics the physiological conditions within the cell was developed. The enzyme activity was not changed at 3 h but was stimulated more than 2-fold at 4 and 5 h after infection with poliovirus. Enzyme activity was stimulated by addition of CTP to the assay. At 0.10 mM CTP the difference in activities from poliovirus- and mock-infected cells was abolished. Mg2+ inhibited the cytidylyltransferase activities and eliminated the differences between the two activities at a concentration of 0.05 mM. However, the endogenous amount of Mg2+ in the postmitochondrial supernatants was the same for infected and mock-infected cells. The addition of CDP-choline or PPi inhibited the cytidylyltransferase activity but had no effect on the relative differences in activities from infected and mock-infected cells. Measurement of CTP in the postmitochondrial fraction showed no differences at 3 h but was elevated 2- to 3-fold in poliovirus-infected cells at 4 and 5 h. It appears that the cytidylyltransferase reaction is faster in poliovirus-infected HeLa cells because of an increase of CTP in the cytoplasmic compartment. Moreover, it appears that the concentration of CTP in the cytoplasm can determine the rate of phosphatidylcholine biosynthesis in HeLa cells.     

Immunolocalization of membrane-associated CTP:phosphocholine cytidylyltransferase in phosphatidylcholine-deficient Chinese hamster ovary cells.

The location of CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary (CHO) cells made deficient in phosphatidylcholine was determined by immunofluorescence techniques. A rabbit polyclonal antibody was raised against a synthetic peptide corresponding to the amino-terminal 17 amino acid residues of rat liver cytidylyltransferase. The antibody recognized both native and denatured cytidylyltransferase from both rat liver and CHO cells. CHO cells were treated with phospholipase C to alter the lipid composition of the plasma membrane and to elicit translocation of cytidylyltransferase from the less active soluble pool to an activated membrane fraction. Visualization of cytidylyltransferase by indirect immunofluorescence revealed staining of the nuclear envelope in phospholipase C-treated cells but not in untreated cells. CHO cells were also starved for choline and supplemented with a choline analogue to provide an alternative technique of rendering the cells phosphatidylcholine-deficient. Although this treatment should affect different cellular membranes than those affected by phospholipase C treatment, cytidylyltransferase still translocated to the nuclear envelope, as shown by indirect immunofluorescence. These results indicate that activated, membrane-bound cytidylyltransferase is associated with the nuclear membrane and suggest that the nuclear membrane may be a site of de novo phosphatidylcholine synthesis.     

Activation of CTP : phosphocholine cytidylyltransferase in rat lung by fatty acids

CTP : phosphocholine cytidylyltransferase activity exists in both the microsome and cytosol fractions of adult lung, 36 and 59%, respectively. Although these enzyme activities are stimulated in vitro by added lipid activators (i.e. phosphatidylglycerol), there are significant levels of activity in the absence of added lipid. We have removed endogenous lipid material from microsome and cytosol preparations of rat lung by rapid extraction with isopropyl ether. The extraction procedure did not cause any loss of cytidylyltransferase activity in the cytosol. After the extraction the enzyme was almost completely dependent upon added lipid activator. Isopropyl ether extraction of microsome preparations produced a loss of 40% of the cytidylyltransferase activity, when measured in the presence of added phosphatidylglycerol. Lipid material extracted into isopropyl ether restored the cytidylyltransferase activity in cytosol. The predominant species of enzyme activator in the isopropyl ether extracts was fatty acid. A variety of naturally occurring unsaturated fatty acids stimulated the cytidylyltransferase to the same extent as phosphatidylglycerol. Saturated fatty acids were inactive.     

Oxysterol activation of phosphatidylcholine synthesis involves CTP:phosphocholine cytidylyltransferase alpha translocation to the nuclear envelope

In addition to suppressing cholesterol synthesis and uptake, oxysterols also activate glycerophospholipid and SM (sphingomyelin) synthesis, possibly to buffer cells from excess sterol accumulation. In the present study, we investigated the effects of oxysterols on the CDP-choline pathway for PtdCho (phosphatidylcholine) synthesis using wild-type and sterol-resistant CHO (Chinese-hamster ovary) cells expressing a mutant of SCAP [SREBP (sterol-regulatory-element-binding protein) cleavage-activating protein] (CHO-SCAP D443N). [(3)H]Choline-labelling experiments showed that 25OH (25-hydroxycholesterol), 22OH (22-hydroxycholesterol) and 27OH (27-hydroxycholesterol) increased PtdCho synthesis in CHO cells as a result of CCTalpha (CTP:phosphocholine cytidylyltransferase alpha) translocation and activation at the NE (nuclear envelope). These oxysterols also activate PtdCho synthesis in J774 macrophages. in vitro, CCTalpha activity was stimulated 2- to 2.5-fold by liposomes containing 5 mol% 25OH, 22OH or 27OH. Inclusion of up to 5 mol% cholesterol did not further activate CCTalpha. 25OH activated CCTalpha in CHO-SCAP D443N cells leading to a transient increase in PtdCho synthesis and accumulation of CDP-choline. CCTalpha translocation to the NE and intranuclear tubules in CHO-SCAP D443N cells was complete after 1 h exposure to 25OH compared with only partial translocation by 4-6 h in CHO-Mock cells. These enhanced responses in CHO-D443N cells were sterol-dependent since depletion with cyclodextrin or lovastatin resulted in reduced sensitivity to 25OH. However, the lack of effect of cholesterol on in vitro CCT activity indicates an indirect relationship or involvement of other sterols or oxysterol. We conclude that translocation and activation of CCTalpha at nuclear membranes by side-chain hydroxylated sterols are regulated by the cholesterol status of the cell.     

Colony-stimulating factor 1 regulates CTP: phosphocholine cytidylyltransferase mRNA levels.

Growth factor regulation of phosphatidylcholine (PtdCho) metabolism during the G1 stage of the cell cycle was investigated in the colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell-line BAC1.2F5. The transient removal of CSF-1 arrested the cells in G1. Incorporation of [3H]choline into PtdCho was stimulated significantly 1 h after growth factor addition to quiescent cells. Metabolic labeling experiments pointed to CTP:phosphocholine cytidylyltransferase (CT) as the rate-controlling enzyme for PtdCho biosynthesis in BAC1.2F5 cells. The amount of CT mRNA increased 4-fold within 15 min of CSF-1 addition and remained elevated for 2 h. The rise in CT mRNA levels was accompanied by a 50% increase in total CT specific activity in cell extracts within 4 h after the addition of CSF-1. CSF-1-dependent elevation of CT mRNA content was neither attenuated nor superinduced by the inhibition of protein synthesis with cycloheximide. The rate of CT mRNA turnover decreased in the presence of CSF-1 indicating that message stabilization was a key factor in determining the levels of CT mRNA. These data point to increased CT mRNA abundance as a component in growth factor-stimulated PtdCho synthesis.     

Regulation of CTP: phosphocholine cytidylyltransferase activity in type II pneumonocytes.

Phosphatidylcholine synthesis by rat type II pneumonocytes was altered either by depleting the cells of choline or by exposing the cells to extracellular lung surfactant. Effects of these experimental treatments on the activity of a regulatory enzyme, CTP:phosphocholine cytidylyltransferase, were investigated. Although choline depletion of type II pneumonocytes resulted in inhibition of phosphatidylcholine synthesis, cytidylyltransferase activity (measured in cell homogenates in either the absence or presence of added lipids) was greatly increased. Activation of cytidylyltransferase in choline-depleted cells was rapid and specific, and was quickly and completely reversed when choline-depleted cells were exposed to choline (but not ethanolamine). Choline-dependent changes in enzymic activity were apparently not a result of direct actions of choline on cytidylyltransferase and they were largely unaffected by cyclic AMP analogues, oleic acid, linoleic acid or cycloheximide. The Km value of cytidylyltransferase for CTP (but not phosphocholine) was lower in choline-depleted cells than in choline-repleted cells. Subcellular redistribution of cytidylyltransferase also was associated with activation of the enzyme in choline-depleted cells. When measured in the presence of added lipids, 66.5 +/- 5.0% of recovered cytidylyltransferase activity was particulate in choline-depleted cells but only 34.1 +/- 4.5% was particulate in choline-repleted cells. An increase in particulate cytidylyltransferase also occurred in type II pneumonocytes that were exposed to extracellular surfactant. This latter subcellular redistribution, however, was not accompanied by a change in cytidylyltransferase activity even though incorporation of [3H]choline into phosphatidylcholine was inhibited by approx. 50%. Subcellular redistribution of cytidylyltransferase, therefore, is associated with changes in enzymic activity under some conditions, but can also occur without a resultant alteration in enzymic activity.     

Trifluoperazine and chlorpromazine inhibit phosphatidylcholine biosynthesis and CTP:phosphocholine cytidylyltransferase in HeLa cells

The influence of chlorpromazine and trifluoperazine on phosphatidylcholine biosynthesis in HeLa cells was investigated. HeLa cells were prelabeled with [Me-3H]choline for 1 h. The cells were subsequently incubated with various concentrations of drugs. Both compounds were potent inhibitors of phosphatidylcholine biosynthesis, with 50% inhibition by 5 micron of either drug. Analysis of the radioactivity in the soluble precursors indicated a block in the conversion of phosphocholine to CDPcholine catalyzed by CTP:phosphocholine cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15). Inhibition by these drugs was slowly reversed after incubation for more than 2 h, or was immediately abolished when 0.4 mM oleate was included in the cell medium or when the drug-containing medium was removed. The subcellular location of the cytidylyltransferase was unaffected by either drug, nor did the drugs alter the rate of release of cytidylyltransferase from HeLa cells by digitonin treatment. The drugs had a direct inhibitory effect on cytidylyltransferase activity in HeLa cell postmitochondrial supernatants. Half-maximal inhibition was achieved with 30 microM trifluoperazine and 50 microM chlorpromazine. These drugs did not change the apparent Km of the cytidylyltransferase for CTP or phosphocholine. Inhibition of cytidylyltransferase by these compounds was reversible with exogenous phospholipid or oleate in the enzyme assay. The data indicate that both drugs inhibit phosphatidylcholine synthesis by an effect on the cytidylyltransferase. The mechanism of action remains unknown at this time.     

Sphingomyelin metabolites inhibit sphingomyelin synthase and CTP:phosphocholine cytidylyltransferase

Tissue injury in inflammation involves the release of several cytokines that activate sphingomyelinases and generate ceramide. In the lung, the impaired metabolism of surfactant phosphatidylcholine (PC) accompanies this acute and chronic injury. These effects are long-lived and extend beyond the time frame over which tumor necrosis factor (TNF)-alpha and interleukin-1beta are elevated. In this paper, we demonstrate that in H441 lung cells these two processes, cytokine-induced metabolism of sphingomyelin and the inhibition of PC metabolism, are directly interrelated. First, metabolites of sphingomyelin hydrolysis themselves inhibit key enzymes necessary for restoring homeostasis between sphingomyelin and its metabolites. Ceramide stimulates sphingomyelinases as effectively as TNF-alpha, thereby amplifying the sphingomyelinase activation, and TNF-alpha, ceramide, and sphingosine all inhibit PC:ceramide phosphocholine transferase (sphingomyelin synthase), the enzyme that restores homeostasis between sphingomyelin and ceramide pools. Second, ceramide inhibits PC synthesis, probably because of its effects on CTP:phosphocholine cytidylyltransferase, the rate-limiting enzymatic step in de novo PC synthesis. The data presented here suggest that TNF-alpha may be an inhibitor of phospholipid metabolism in inflammatory tissue injury. These actions may be amplified because of the ability of metabolites of sphingomyelin to inhibit the pathways that should restore the normal ceramide-sphingomyelin homeostasis.     

Choline deficiency causes translocation of CTP:phosphocholine cytidylyltransferase from cytosol to endoplasmic reticulum in rat liver

The choline-deficient rat liver has been chosen as a physiologically relevant model system in which to study the regulation of phosphatidylcholine biosynthesis. When 50-g rats were placed on a choline-deficient diet for 3 days, the activity of CTP:phosphocholine cytidylyltransferase (CT) was increased 2-fold in the microsomes and decreased proportionately in the cytosol. A low titer antibody to CT was obtained from chickens and used to identify the amount of CT protein in cytosol from rat liver. The amount of CT recovered from the choline-deficient cytosol was significantly less than in cytosol from choline-supplemented rats. When hepatocytes were prepared from choline-deficient livers, supplementation of the medium of the cells with choline caused CT to move from the membranes to cytosol within 1-2 h. The activity of another translocatable enzyme of glycerolipid metabolism, phosphatidate phosphohydrolase, was unchanged in cytosol from choline-deficient rat livers, and the microsomal activity of this enzyme was only minimally increased. When the livers were fractionated into endoplasmic reticulum and Golgi, there was a 2-fold increase in the activity on the endoplasmic reticulum from choline-deficient livers but no change in activity associated with Golgi. Thus, the increased association of CT with endoplasmic reticulum in choline-deficient livers appears to be specific to that subcellular fraction, and the subcellular location of other enzymes may not be affected.     

Membrane binding modulates the quaternary structure of CTP:phosphocholine cytidylyltransferase

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.     

Binding of CTP: phosphocholine cytidylyltransferase to large unilamellar vesicles

We have studied the binding of CTP: phosphocholine cytidylyltransferase from HeLa cell cytosol to large unilamellar vesicles of egg phosphatidylcholine (PC) or HeLa cell phospholipids that contain various amounts of oleic acid. A fatty acid/phospholipid molar ratio exceeding 10% was required for CTP: phosphocholine cytidylyltransferase binding to liposomes. At a fatty acid/phospholipid molar ratio of 1; 85% of the cytosolic CTP: phosphocholine cytidylyltransferase was bound. The enzyme also bound to liposomes with at least 20 mol% palmitic acid, monoolein, diolein or oleoylacetylglycerol. Oleoyl-CoA did not promote enzyme binding to liposomes. Binding to oleate-PC vesicles was blocked by Triton X-100 but not by 1 M KCl, and was reversed by incubation of the vesicles with bovine serum albumin. Cytidylyltransferase bound to egg PC vesicles that contained 33 mol% oleic acid equally well at 4 degrees C and 37 degrees C. The enzyme also bound to dimyristoyl- and dipalmitoylphosphatidylcholine vesicles containing oleic acid at temperatures below the phase transition for these liposomes. Binding of the cytidylyltransferase to egg PC vesicles containing oleic acid, monoolein, oleoylacetylglycerol or diolein resulted in enzyme activation, as did binding to dipalmitoylPC-oleic acid vesicles. However, binding to egg PC-palmitic acid vesicles did not fully activate the transferase. Various mechanisms for cytidylyltransferase interaction with membranes are discussed.     

Tumor necrosis factor-alpha inhibits expression of CTP:phosphocholine cytidylyltransferase

We investigated the effects of tumor necrosis factor alpha (TNFalpha), a key cytokine involved in inflammatory lung disease, on phosphatidylcholine (PtdCho) biosynthesis in a murine alveolar type II epithelial cell line (MLE-12). TNFalpha significantly inhibited [(3)H]choline incorporation into PtdCho after 24 h of exposure. TNFalpha reduced the activity of CTP:phosphocholine cytidylyltransferase (CCT), the rate-regulatory enzyme within the CDP-choline pathway, by 40% compared with control, but it did not alter activities of choline kinase or cholinephosphotransferase. Immunoblotting revealed that TNFalpha inhibition of CCT activity was associated with a uniform decrease in the mass of CCTalpha in total cell lysates, cytosolic, microsomal, and nuclear subfractions of MLE cells. Northern blotting revealed no effects of the cytokine on steady-state levels of CCTalpha mRNA, and CCTbeta mRNA was not detected. Incorporation of [(35)S]methionine into immunoprecipitable CCTalpha protein in pulse and pulse-chase studies revealed that TNFalpha did not alter de novo synthesis of enzyme, but it substantially accelerated turnover of CCTalpha. Addition of N-acetyl-Leu-Leu-Nle-CHO (ALLN), the calpain I inhibitor, or lactacystin, the 20 S proteasome inhibitor, blocked the inhibition of PtdCho biosynthesis mediated by TNFalpha. TNFalpha-induced degradation of CCTalpha protein was partially blocked by ALLN or lactacystin. CCT was ubiquitinated, and ubiquitination increased after TNFalpha exposure. m-Calpain degraded both purified CCT and CCT in cellular extracts. Thus, TNFalpha inhibits PtdCho synthesis by modulating CCT protein stability via the ubiquitin-proteasome and calpain-mediated proteolytic pathways.     

CTP:phosphocholine cytidylyltransferase alpha is a cytosolic protein in pulmonary epithelial cells and tissues

CTP:phosphocholine cytidylyltransferase (CCT) is a rate-determining enzyme in de novo synthesis of phosphatidylcholine (PC). The lung requires a steady synthesis of PC for lung surfactant of which disaturated PC is the essential active agent. Surfactant synthesis occurs in alveolar type II cells. Studies with non-pulmonary cells have suggested that CCT is both a nuclear and cytoplasmic protein. The unusual requirements of the lung for PC synthesis and, therefore, CCT activity suggest a unique mechanism of regulation and possibly localization of CCT. The localization of CCT alpha in lung epithelial cells and, of greater consequence, lung tissues are yet unknown. Three isoforms of CCT have been identified. Herein we investigated the localization of the ubiquitously expressed CCT alpha isoform. To ascertain CCT alpha localization in lungs and lung-related epithelial cells, we employed a number of localization methods. Immunogold electron microscopy using polyclonal antibodies raised to either the carboxyl terminus, catalytic domain, or amino terminus of CCT alpha localized CCT alpha mostly to the exterior plasma membrane or regions of the endoplasmic reticulum (ER) in both A549 and MLE-15 epithelial lung cell lines and primary cultures of fetal rat lung epithelial cells. In contrast to other studies, little or no nuclear labeling was observed. Indirect immunofluorescence of these cells with anti-CCT alpha antibodies resulted in a similar distribution. Indirect visualization of both hemagglutinin- and FLAG-tagged CCT alpha as well as direct visualization of enhanced green fluorescence protein-CCT alpha fusion protein corroborated a cytoplasmic localization of CCT alpha in pulmonary cells. Moreover, analysis of lung tissue from fetal and adult mouse by either immunogold electron microscopy or indirect immunofluorescence yielded a strong cytoplasmic CCT alpha signal with virtually no nuclear localization in epithelial cells lining the airways. The cytoplasmic localization of CCT alpha in type II cells was further substantiated with transgenic mice overexpressing FLAG-tagged CCT alpha using the lung-specific human surfactant protein C (SP-C) promoter. We conclude that CCT alpha does not localize to the nucleus in pulmonary tissues, and, therefore, nuclear localization of CCT alpha is not a universal event.     

Sphingosine inhibits the activity of rat liver CTP:phosphocholine cytidylyltransferase

We report CTP:phosphocholine cytidylyltransferase (CT) as another target enzyme of sphingosine actions in addition to the well-characterized protein kinase C. Effects of sphingosine and lysophingolipids were studied on the activity of purified cytidylyltransferase prepared by the method of Weinhold et al. (Weinhold, P. A., Rounsifer, M.E., and Feldman, D.A. (1986) J. Biol. Chem. 261, 5104-5110). The sphingolipids were tested as components of egg phosphatidylcholine (PC) vesicles, 25 mol% sphingosine inhibited the CT activity by about 50%. The inhibition of CT by sphingosine and lysosphingolipids was reversible. Sphingosine was found to be a reversible inhibitor of CT with respect to the activating lipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and fatty acid:phosphatidylcholine vesicles. Egg PC vesicles containing sphingosine, psychosine (galactosylsphingosine), glucopsychosine (glucosylsphingosine), and lysosphingomyelin (sphingosylphosphorylcholine) suppressed the activation by PC/oleic acid vesicles, whereas the parent sphingolipids did not. Egg PC vesicles containing oleylamine and hexadecyltrimethylamine inhibited CT activity, whereas egg PC-octylamine vesicles did not alter the enzyme activity. This indicates the importance of an amino group and long alkyl chain. LysoPC, a known detergent, did not inhibit the enzyme activity under the same assay conditions in which sphingosine inhibited. These results are the first report of a lipid inhibitor of purified CT.