Identification and characterization of the nuclear isoform of Drosophila melanogaster CTP:phosphocholine cytidylyltransferase

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the conversion of phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the eventual synthesis of phosphatidylcholine (PC). The enzyme is regulated by reversible association with cellular membranes, with the rate of catalysis increasing following membrane association. Two isoforms of CCT appear to be present in higher eukaryotes, including Drosophila melanogaster, which contains the tandem genes Cct1 and Cct2. Before this study, the CCT1 isoform had not been characterized and the cellular location of each enzyme was unknown. In this investigation, the cDNA encoding the CCT1 isoform from D. melanogaster has been cloned and the recombinant enzyme purified and characterized to determine catalytic properties and the effect of lipid vesicles on activity. CCT1 exhibited a V max of 23904 nmol of CDP-choline min (-1) mg (-1) and apparent K m values for phosphocholine and CTP of 2.29 and 1.21 mM, respectively, in the presence of 20 muM PC/oleate vesicles. Cytidylyltransferases require a divalent cation for catalysis, and the cation preference of CCT1 was found to be as follows: Mg (2+) > Mn (2+) = Co (2+) > Ca (2+) = Ni (2+) > Zn (2+). The activity of the enzyme is stimulated by a variety of lipids, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol, and the fatty acid oleate. Phosphatidylethanolamine and phosphatidic acid, however, did not have a significant effect on CCT1 activity. The cellular location of both CCT1 and CCT2 isoforms was elucidated by expressing green fluorescent fusion proteins in cultured D. melanogaster Schneider 2 cells. CCT1 was identified as the nuclear isoform, while CCT2 is cytoplasmic.     

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.     

c-Jun N-terminal kinase regulates CTP:phosphocholine cytidylyltransferase

CTP:phosphocholine cytidylyltransferase (CCTalpha) is a rate-regulatory enzyme required for phosphatidylcholine (PtdCho) synthesis. CCTalpha is also a phosphoenzyme, but the physiologic role of kinases on enzyme function remains unclear. We report high-level expression of two major isoforms of the c-Jun N-terminal kinase family (JNK1 and JNK2) in murine lung epithelia. Further, JNK1 and JNK2 phosphorylated purified CCTalpha in vitro, and this was associated with a dose-dependent decrease (approximately 40%) in CCT activity. To evaluate JNK in vivo, lung epithelial cells were infected with a replication defective adenoviral vector encoding murine JNK2 (Adv-JNK2) or an empty vector. Adv-JNK2 infection, unlike the empty vector, markedly increased JNK2 expression concomitant with increased incorporation of [32P]orthophosphate into endogenous CCTalpha. Although Adv-JNK2 infection only modestly reduced CCT activity, it reduced PtdCho synthesis by approximately 30% in cells. These observations suggest a role for JNK kinases as negative regulators of phospholipid synthesis in murine lung epithelia.     

Purification and kinetic characterization of CTP:phosphocholine cytidylyltransferase from Saccharomyces cerevisiae

CTP:phosphocholine cytidylyltransferase (CCT) regulates the biosynthesis of phosphatidylcholine in mammalian cells. In order to understand the mechanism by which this enzyme controls phosphatidylcholine synthesis, we have initiated studies of CCT from the model genetic system, the yeast Saccharomyces cerevisiae. The yeast CCT gene was isolated from genomic DNA using the polymerase chain reaction and was found to encode tyrosine at position 192 instead of histidine, as originally reported. Levels of expression of yeast CCT activity in Escherichia coli or in the yeast, Pichia pastoris, were somewhat low. Expression of yeast CCT in a baculovirus system as a 6x-His-tag fusion protein was higher and was used to purify yeast CCT by a procedure that included delipidation. Kinetic characterization revealed that yeast CCT was activated approximately 20-fold by 20 microM phosphatidylcholine:oleate vesicles, a level 5-fold lower than that necessary for maximal activation of rat CCT. The k(cat) value was 31.3 s(-1) in the presence of lipid and 1.5 s(-1) in the absence of lipid. The K(m) values for the substrates CTP and phosphocholine did not change significantly upon activation by lipids; K(m) values in the presence of lipid were 0.80 mM for phosphocholine and 1.4 mM for CTP while K(m) values in the absence of lipid were 1.2 mM for phosphocholine and 0.8 mM for CTP. Activation of yeast CCT, therefore, appears to be due to an increase in the k(cat) value upon lipid binding.     

Chemical cross-linking reveals a dimeric structure for CTP:phosphocholine cytidylyltransferase

CTP:phosphacholine cytidylyltransferase (EC 2.7.7.15) was purified from rat liver according to the method of Weinhold et al. (Weinhold, P. A., Rounsifer, M. E., and Feldman, D. A. (1986) J. Biol. Chem. 261, 5104-5110). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without beta-mercaptoethanol revealed a single major band of 42,000 daltons. This band corresponds to the 45-kDa catalytic subunit isolated by Feldman and Weinhold (Feldman, D. A., and Weinhold, P. A. (1987) J. Biol. Chem. 262, 9075-9081). A minor component of 84,000 daltons was intensified in nonreducing gels when the sulfhydryl reducing agent, dithiothreitol, was removed from the enzyme preparation by dialysis. Reduction with dithiothreitol and electrophoresis in the second dimension showed that this 84-kDa protein was derived from the 42-kDa protein. This result suggested that the 42 kDa protein can be converted to an 84-kDa protein by disulfide bond formation. Reaction with the thiol-cleavable cross-linking reagents, dithiobis(succimidyl propionate) or dimethyl-3,3'-dithiobispropionimidate, converted the 42-kDa cytidylyltransferase subunit into a diffuse band approximately twice its molecular mass. Disulfide reduction and electrophoresis in the second dimension showed that this band was derived exclusively from the 42-kDa subunit. This cross-linking pattern was observed when cytidylyltransferase was bound to a Triton X-100 micelle or when bound to a membrane vesicle containing phosphatidylcholine, oleic acid, and Triton X-100. Reaction of the fully reduced enzyme with glutaraldehyde also generated a cross-linked dimer. All three cross-linking reagents inactivated the enzyme. Reduction of the disulfide cross-linkers with dithiothreitol partially reactivated the transferase. When Triton was removed from the enzyme preparation by DEAE-Sepharose chromatography, reaction of the detergent-depleted enzyme with glutaraldehyde generated a band corresponding to a hexamer and higher molecular weight aggregates. The dimeric form was regenerated by addition of either Triton X-100 or phosphatidylcholine-oleic acid vesicles. We conclude that the purified, native cytidylyltransferase, when bound to a detergent micelle or membrane vesicle, is a dimer composed of two noncovalently linked 42-kDa subunits. In the absence of a membrane or micelle, the dimers self-aggregate in a reversible manner.     

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 binds anionic phospholipid vesicles in a cross-bridging mode

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the rate-limiting step in phosphatidylcholine (PC) synthesis, and its activity is regulated by reversible association with membranes, mediated by an amphipathic helical domain M. Here we describe a new feature of the CCTalpha isoform, vesicle tethering. We show, using dynamic light scattering and transmission electron microscopy, that dimers of CCTalpha can cross-bridge separate vesicles to promote vesicle aggregation. The vesicles contained either class I activators (anionic phospholipids) or the less potent class II activators, which favor nonlamellar phase formation. CCT increased the apparent hydrodynamic radius and polydispersity of anionic phospholipid vesicles even at low CCT concentrations corresponding to only one or two dimers per vesicle. Electron micrographs of negatively stained phosphatidylglycerol (PG) vesicles confirmed CCT-mediated vesicle aggregation. CCT conjugated to colloidal gold accumulated on the vesicle surfaces and in areas of vesicle-vesicle contact. PG vesicle aggregation required both the membrane-binding domain and the intact CCT dimer, suggesting binding of CCT to apposed membranes via the two M domains situated on opposite sides of the dimerization domain. In contrast to the effects on anionic phospholipid vesicles, CCT did not induce aggregation of PC vesicles containing the class II lipids, oleic acid, diacylglycerol, or phosphatidylethanolamine. The different behavior of the two lipid classes reflected differences in measured binding affinity, with only strongly binding phospholipid vesicles being susceptible to CCT-induced aggregation. Our findings suggest a new model for CCTalpha domain organization and membrane interaction, and a potential involvement of the enzyme in cellular events that implicate close apposition of membranes.     

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.     

The licC gene of Streptococcus pneumoniae encodes a CTP:phosphocholine cytidylyltransferase

The licC gene product of Streptococcus pneumoniae was expressed and characterized. LicC is a nucleoside triphosphate transferase family member and possesses CTP:phosphocholine cytidylyltransferase activity. Phosphoethanolamine is a poor substrate. The LicC protein plays a role in the biosynthesis of the phosphocholine-derivatized cell wall constituents that are critical for cell separation and pathogenesis.     

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.     

Phosphatidylcholine synthesis for lipid droplet expansion is mediated by localized activation of CTP:phosphocholine cytidylyltransferase

Lipid droplets (LDs) are cellular storage organelles for neutral lipids that vary in size and abundance according to cellular needs. Physiological conditions that promote lipid storage rapidly and markedly increase LD volume and surface. How the need for surface phospholipids is sensed and balanced during this process is unknown. Here, we show that phosphatidylcholine (PC) acts as a surfactant to prevent LD coalescence, which otherwise yields large, lipolysis-resistant LDs and triglyceride (TG) accumulation. The need for additional PC to coat the enlarging surface during LD expansion is provided by the Kennedy pathway, which is activated by reversible targeting of the rate-limiting enzyme, CTP:phosphocholine cytidylyltransferase (CCT), to growing LD surfaces. The requirement, targeting, and activation of CCT to growing LDs were similar in cells of Drosophila and mice. Our results reveal a mechanism to maintain PC homeostasis at the expanding LD monolayer through targeted activation of a key PC synthesis enzyme.     

The activation of CTP: phosphocholine cytidylyltransferase from rat liver by polyunsaturated phospholipid

Experimental evidence is reported that the addition in vitro of a polyunsaturated soybean phospholipid material (EPL) to a CTP:PC cytidylyltransferase preparation from rat liver (E.C. 2.7.7.15) produces noticeable stimulation of this enzymatic activity. Preincubation for different time intervals of EPL under air or oxygen further stimulates the activating effects. Little influence is exerted on the same enzyme by saturated lipids, such as dipalmitoyl-sn-glycero-3-phosphorylcholine and distearoyl-sn-glycero-3-phosphorylcholine. It is proposed that the lipid components of the EPL which exert the stimulatory action may be lyso-phospholipid moieties derived from EPL upon preincubation or directly present in the product. The biological significance of these activations in liver tissue is discussed.     

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.     

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.