Enhanced expression and activation of CTP:phosphocholine cytidylyltransferase beta2 during neurite outgrowth

During differentiation neurons increase phospholipid biosynthesis to provide new membrane for neurite growth. We studied the regulation of phosphatidylcholine (PC) biosynthesis during differentiation of two neuronal cell lines: PC12 cells and Neuro2a cells. We hypothesized that in PC12 cells nerve growth factor (NGF) would up-regulate the activity and expression of the rate-limiting enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase (CT). During neurite outgrowth, NGF doubled the amount of cellular PC and CT activity. CTbeta2 mRNA increased within 1 day of NGF application, prior to the formation of visible neurites, and continued to increase during neurite growth. When neurites retracted in response to NGF withdrawal, CTbeta2 mRNA, protein, and CT activity decreased. NGF specifically activated CTbeta2 by promoting its translocation from cytosol to membranes. In contrast, NGF did not alter CTalpha expression or translocation. The increase in both CTbeta2 mRNA and CT activity was inhibited by U0126, an inhibitor of mitogen-activated kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). In Neuro2a cells, retinoic acid significantly increased CT activity (by 54%) and increased CTbeta2 protein, coincident with neurite outgrowth but did not change CTalpha expression. Together, these data suggest that the CTbeta2 isoform of CT is specifically up-regulated and activated during neuronal differentiation to increase PC biosynthesis for growing neurites.     

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.     

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.     

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.     

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.     

Oxysterols inhibit phosphatidylcholine synthesis via ERK docking and phosphorylation of CTP:phosphocholine cytidylyltransferase

Surfactant deficiency contributes to acute lung injury and may result from the elaboration of bioactive lipids such as oxysterols. We observed that the oxysterol 22-hydroxycholesterol (22-HC) in combination with its obligate partner, 9-cis-retinoic acid (9-cis-RA), decreased surfactant phosphatidylcholine (PtdCho) synthesis by increasing phosphorylation of the regulatory enzyme CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha). Phosphorylation of CCTalpha decreased its activity. 22-HC/9-cis-RA inhibition of PtdCho synthesis was blocked by PD98059 or dominant-negative ERK (p42 kinase). Overexpression of constitutively active MEK1, the kinase upstream of p42 kinase, increased CCTalpha phosphorylation. Expression of truncated CCTalpha mutants lacking proline-directed sites within the C-terminal phosphorylation domain partially blocked oxysterol-mediated inhibition of PtdCho synthesis. Mutagenesis of Ser315 within CCTalpha was both required and sufficient to confer significant resistance to 22-HC/9-cis-RA inhibition of PtdCho synthesis. A novel putative ERK-docking domain N-terminal to this phosphoacceptor site was mapped within the CCTalpha membrane-binding domain (residues 287-300). The results are the first demonstration of a physiologically relevant phosphorylation site and docking domain within CCTalpha that serve as targets for ERKs, resulting in inhibition of surfactant synthesis.     

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.     

Oxidized lipoproteins inhibit surfactant phosphatidylcholine synthesis via calpain-mediated cleavage of CTP:phosphocholine cytidylyltransferase

We investigated effects of pro-atherogenic oxidized lipoproteins on phosphatidylcholine (PtdCho) biosynthesis in murine lung epithelial cells (MLE-12). Cells surface-bound, internalized, and degraded oxidized low density lipoproteins (Ox-LDL). Ox-LDL significantly reduced [3H]choline incorporation into PtdCho in cells by selectively inhibiting the activity of the rate-regulatory enzyme, CTP:phosphocholine cytdylyltransferase (CCT). Ox-LDL coordinately increased the cellular turnover of CCTalpha protein as determined by [35S]methionine pulse-chase studies by inducing the calcium-activated proteinase, calpain. Forced expression of calpain or exposure of cells to the calcium ionophore, A23187, increased CCTalpha degradation, whereas overexpression of the endogenous calpain inhibitor, calpastatin, attenuated Ox-LDL-induced CCTalpha degradation. The effects of Ox-LDL on CCTalpha breakdown were attenuated in calpain-deficient cells. In vitro calpain digestion of CCTalpha isolated from cells transfected with truncated or internal deletion mutants indicated multiple cleavage sites within the CCTalpha primary structure, leading to the generation of a 26-kDa (p26) fragment. Calpain hydrolysis of purified CCTalpha generated p26, which upon NH2-terminal sequencing localized a calpain attack site within the CCTalpha amino terminus. Expression of a CCTalpha mutant where the amino-terminal cleavage site and a putative carboxyl-terminal hydrolysis region were modified resulted in an enzyme that was significantly less sensitive to proteolytic cleavage and restored the ability of cells to synthesize surfactant PtdCho after Ox-LDL treatment. Thus, these results provide a critical link between proatherogenic lipoproteins and their metabolic target, CCTalpha, resulting in impaired surfactant metabolism.     

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.     

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.     

Trifluoperazine and other anaesthetics inhibit rat liver CTP: phosphocholine cytidylyltransferase

Chlorpromazine (25 microM) and trifluoperazine (25 microM) inhibited by 5-fold the activity of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme for phosphatidylcholine biosynthesis, in rat liver cytosol. Addition of saturating amounts of rat liver phospholipid to the enzyme assay rapidly reversed the drug-mediated inhibition. Three-fold or greater concentrations of these drugs were required to produce a 50% inhibition of the microsomal cytidylyltransferase. Incubation of rat hepatocytes with 20 microM trifluoperazine or chlorpromazine did not inhibit phosphatidylcholine biosynthesis. These results provide additional evidence for the hypothesis that the active form of cytidylyltransferase is on the endoplasmic reticulum and the enzyme in cytosol appears to be latent.     

Interdomain and membrane interactions of CTP:phosphocholine cytidylyltransferase revealed via limited proteolysis and mass spectrometry

CTP:phosphocholine cytidylyltransferase (CCT) is a multi-domain enzyme that regulates phosphatidylcholine synthesis. It converts to an active form upon binding cell membranes, and interdomain dissociations have been hypothesized to accompany this process. To identify these interdomain and membrane interactions, the tertiary structures of three forms of CCTalpha were probed by monitoring accessibility to proteases. Time-limited digestion with chymotrypsin or arginine C of soluble CCTalpha (CCT(sol)), phospholipid vesicle-bound CCT (CCT(mem)), and a soluble constitutively active CCT truncated at amino acid 236 generated complex mixtures of peptides that were resolved and identified by gel electrophoresis/immunoblotting and by matrix-assisted laser desorption/ionization-mass spectrometry, with or without coupling to capillary liquid chromatography. Identification of cleavage sites enabled assembly of peptide bond accessibility maps for each CCT form. Our results reveal a approximately 80-residue core within the catalytic domain (domain C) as the most inaccessible region in all three forms and the C-terminal phosphorylation domain as the most accessible. Membrane binding has little effect on the protease accessibility of these domains. To map the protease sites onto the catalytic domain, its three-dimensional structure was modeled from the atomic coordinates of glycerol-phosphate cytidylyltransferase (Protein Data Bank code 1COZ). The protease inaccessibility of most sites in domain C could be explained by burial or location within secondary structural elements. The accessibility of the N-terminal domain (domain N) was enhanced upon membrane binding. Residues Phe(234)-Leu(303) were inaccessible in CCT(mem), suggesting burial in the membrane. Surprisingly, residues Leu(274)-Leu(303) of this domain were also inaccessible in CCT(sol). We propose that this region is buried by interdomain contacts with domain N in CCT(sol). Membrane binding and burial of domain M in the lipid bilayer may disrupt this interaction, leading to increased exposure of sites in domain N.     

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.     

CTP:phosphocholine cytidylyltransferase: insights into regulatory mechanisms and novel functions

A key regulatory enzyme in phosphatidylcholine biosynthesis, CTP:cholinephosphate cytidylyltransferase (CCT), catalyzes the formation of CDP-choline. This review discusses the essential features of CCT and addresses intriguing new insights into the catalytic and regulatory properties of this complex enzyme. Characterization of a lipid-binding segment in rat CCT is described and the role of lipids in CCT activation is discussed. An analysis of the phosphorylation domain is presented and possible physiological rationales for reversible phosphorylation of CCT are discussed. The nuclear localization of CCT is examined in the context of multiple CCT isoforms, as is recent evidence establishing a potential link between CCT activity and vesicular transport.     

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.