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J. Biol. Chem., Vol. 280, Issue 2, 853-856, January 14, 2005
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Minireview
From the Protein Science Division, Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38105
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INTRODUCTION |
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There are three steps in the CDP-choline pathway. Choline is an essential nutrient in mammals and is transported into the cell and phosphorylated by choline kinase. CCT catalyzes the condensation of CTP and phosphocholine to form CDP-choline, which in turn donates the phosphocholine moiety to diacylglycerol to form PC. CCT mediates the rate-limiting step in this pathway (1) and is unique in that its enzymatic activity is regulated by association with membrane structures (14, 15). A current model posits that bilayer curvature elastic stress is sensed by CCT and governs the degree of membrane association, thus providing a mechanism for both positive and negative regulation of activity (16, 17). The PEMT is the only enzyme in the alternative pathway, and it performs all three of the methylation steps. The significance of PE methylation to PC production is controlled by the expression of PEMT, which is high in liver and testis (4, 18). The goal of this minireview is to highlight recent progress in understanding the regulation of CCT expression and a growing appreciation of its role in cell biology and organ function.
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Overview of CCT Biochemistry |
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) or not (CCT
). The catalytic domain contains two motifs, HXGH and RTEGISTS, characteristic of the cytidylyltransferase superfamily (23), which are involved in binding CTP (24, 25). A conserved lysine residue between the CTP binding motifs is implicated in phosphocholine binding (25). CCT is a dimer (26, 27). The high resolution structure of the homologous Bacillus subtilis G3PCT confirms a role for these features in the catalytic mechanism of the CCT protein family (28–31).
An amphipathic C-terminal -helix in the CCTs from mammals, plants, and Drosophila melanogaster (22, 32) clearly plays a major role in mediating the lipid interaction and regulation of the enzyme (1, 14, 15, 19, 32). The helical or M domain interacts with both neutral and anionic lipid mixtures and is the primary region of the protein that responds to the bilayer curvature elastic stress (6, 14, 27, 33, 34). Removal of the entire CCT C terminus results in an enzyme that retains catalytic activity at high CTP concentrations but cannot respond to lipid regulation (26, 35). The extreme C terminus, or the P domain in mammalian CCT, can activate the enzyme in response to anionic lipid mixtures when the internal M domain is deleted (35). The CCTs of lower eukaryotes do not have a domain homologous to the M domain but nonetheless are lipid-regulated (22). This group of CCTs includes S. cerevisiae (36, 37), Plasmodium falciparum (38), and Caenorhabditis elegans (39). Clearly more than one sequence motif is capable of forming a structure that can reversibly interact with membrane phospholipids and drive the protein conformational changes associated with catalytic regulation.
The mammalian CCTs are reversibly phosphorylated at multiple serine residues, representing about 25% of the P domain (1). Phosphorylation restricts CCT binding to select lipid vesicles (40) and reduces activity (40, 41). Evaluation of CCT membrane association in lysates or permeabilized cells or in situ localization does not reveal a clear relationship between phosphorylation and the cellular location of CCT. Rather, phosphorylation inhibits CCT activity in vitro even under optimum lipid activator conditions, consistent with the view that phosphorylation is a mechanism for kinase-regulated signaling (42, 43) to modulate the enzyme activity in addition to membrane lipid composition.
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CCT Genes and Isoforms |
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isoform (44, 45), and Pcyt1b encodes the CCT1 (in human only), -2, and -3 isoforms (45–47). CCT is located on mouse chromosome 16 (3q in human) and CCT is X-linked in both species. Total cellular CCT activity is the sum of CCT and CCT, and any of the four protein isoforms complement a conditional CCT-deficient cell line (45–47). The CCT and CCT proteins are virtually identical in the catalytic and M domains but differ significantly at both termini. CCT is found in the cytoplasm associated with membranes, but the majority is in the nucleus as directed by the N-terminal nuclear localization sequence. In contrast, CCT localizes exclusively outside the nucleus (46). Nuclear CCT is associated with chromatin, and a nuclear CDP-choline pathway is active in synthesizing highly saturated, medium chain diacyl- or alkylacyl-PC (48). This observation is consistent with the molecular species distribution of PC produced from the CDP-choline pathway in liver (49) indicating that nuclear CCT participates in PC synthesis. However, nuclear CCT is also thought of as a reserve for a prompt response to an extranuclear PC requirement, such as the membrane synthesis associated with mitogenic stimulation (50), or replacement of membrane due to pharmacological disruption of PC synthesis (51). Both hypotheses are possible and are not mutually exclusive. The majority of CCT protein is found outside the nucleus associated with glycogen pools (52) in pulmonary cells and tissues (53, 54), which may reflect a high demand for PC synthesis or suggest that the NLS may be cleaved. |
CCT in Cell Proliferation and Survival |
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mRNA level due to message stabilization in a macrophage cell line that is dependent on CSF-1 for both growth and survival (58). Following CSF-1 removal, the amount of CCT protein is reduced and located outside the nucleus, and then the mitogenic signal stimulates CCT protein synthesis and the new CCT accumulates and relocates to the cell nucleus (20). The re-addition of serum to lung epithelial cells results in early stimulation of PC synthesis soon after entry into G1 and is accompanied by increased CCT activity where all of the CCT protein is located in the extranuclear compartment and remains there (54). In IIC9 fibroblasts, which are dependent on serum for proliferation but not survival, CCT protein expression is abundant, and a portion of the endogenous nuclear CCT protein migrates to extranuclear membrane sites following serum stimulation (50). On the other hand, serum stimulation of C3H10T1/2 fibroblasts is not accompanied by an early increase in PC synthesis, early changes in CCT mRNA or CCT translocation (54). Thus, the data support the view that if and when prompt extranuclear PC synthesis is required, CCT responds either by gene expression or by translocation. The distribution of CCT remains constant during the cell cycle (54, 59, 60). Membrane PC accumulates during the S phase following increased CCT activity during G1 (54, 60–62), and the CCT activity remains elevated throughout the cell cycle (54, 60, 61) though it is dampened by progressive phosphorylation of the CCT during the approach to G2 and mitosis (54, 61). CCT transcription is stimulated 2-fold during the S phase through the phosphorylation (63) and binding (64) of the transcription factor Sp1 to the promoter region (60) in preparation for cell division. The amount of PC is not solely controlled by the expression of CCT. PC turnover is another key player in PC homeostasis during the cell cycle (61). Turnover is rapid during G1 and decreases during S phase to coordinate PC synthesis and membrane formation with the doubling of DNA and stable RNA. Although the membrane biogenesis program is coordinated with DNA synthesis, it is independent of DNA replication (61), meaning that inhibitors of DNA synthesis do not stop phospholipid accumulation during S phase and thus cannot be used to synchronize membrane biogenesis. In experiments where CCT overexpression is forced, the flux through the metabolic pathway increases, but the amount of PC mass does not (65, 66). This is due to the stimulation of PC turnover and the formation of GroPCho (65, 66). The enzyme responsible is suggested to be the iPLA2 (66–68) and/or the NTE (69). Overexpression of NTE increases GroPCho production, which is offset by reduced choline uptake and incorporation into CDP-choline, suggesting a homeostatic coupling with CCT. On the other hand, overexpression of iPLA2 does not alter PC synthesis but elevates the GroPCho as well as lyso-PC and free fatty acid levels (70), which may feed back and regulate CCT.
CCT in Apoptosis—CCT inhibition triggers apoptosis and it is a protease target in the apoptosis cascade initiated by other stimuli. PC synthesis is essential not only for cell growth and proliferation but also for cell survival (71). Depletion of cellular PC due to either a conditional defect in CCT (72, 73), chemical inhibition of CCT activity (19, 74–78), or bacterial infection (79) leads to cell death by apoptosis or paraptosis, a process that has characteristics of both apoptosis and necrosis (80). Reduction in cellular PC activates caspases (51, 80) and induces the endoplasmic reticulum stress-related protein CHOP without raising a general endoplasmic reticulum stress response (73). During the apoptotic response to reduced PC, CCT itself translocates from the nucleus and becomes a cytoplasmic target for caspase-mediated cleavage of the N-terminal NLS (51). The retention of CCT protein outside the nucleus is associated with enhanced CCT activity, suggesting a survival response that operates during cell death pathways initiated by other mechanisms such as tumor necrosis factor (81).
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Transcriptional Regulation of CCT Expression |
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is expressed in virtually every tissue, the CCT promoter is particularly active in pulmonary alveolar cells (82) where CCT is expressed at a high level compared with other tissues (4). The transcription factors Sp1, Sp2, and Sp3 activate CCT gene transcription through their interaction with three cisacting elements in the proximal promoter region (83). Sp1 activates expression during the cell cycle (63, 64), and Sp3 mediates enhanced expression in Ha-Ras-transformed fibroblasts following phosphorylation by the p42/44 kinase (84). Transcriptional enhancer-4 can regulate CCT expression either by suppression of transcription via its direct binding to the Eb promoter element or by activation through interaction with Ets-1 (85, 86). Lipid deprivation increases CCT mRNA synthesis (87), and direct assessment of promoter activity shows that a sterol response element located in the CCT promoter governs this response (88, 89). Expression and promoter activity are inhibited when sterols are added to the extracellular medium and induced in lipid-depleted medium. Evaluation of CCT metabolic activity in cell lines with defective sterol response element-binding protein processing, however, suggests that the regulation of CCT and the CDP-choline pathway may be indirect (90) due to the lack of correlation between CCT expression (as measured by mRNA or protein) and metabolic activity (as measured by radiolabeling). CCT2 isoform expression increases during nerve growth factor-induced differentiation of axonal membranes (91), but little is known about the CCT promoter and the factors that contribute to the differential expression of CCT1, -2, and -3. CCT1 is not expressed in mice (4) and may be an aberrant transcript in humans. Two distinct CCT isozymes are found in A. thaliana, and their expression increases differentially in response to cold acclimation (7, 92). A cold-response element and a gibberellin-response element are found in the AtCCT1 promoter region (93). The research on the transcriptional regulation of CCT is in its infancy, and continued work in this area will reveal how CCT expression responds to differentiation and disease. |
Tissue-specific Roles for CCT |
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and CCT expression are about equal in contrast to other tissues where CCT isoforms are found at roughly 10-fold lower levels. The amounts of CCT and CCT2 transcripts become equalized upon differentiation of cultured neuronal cells (4, 91), similar to the levels found in brain. Also, CCT expression is stimulated in hippocampal neurons treated with the neuropeptide arginine-vasopressin(4–8) (94). Lipodystrophies associated with some neuropathological disorders are linked to aberrant CCT activity. For example, Gaucher disease cells accumulate glucosylceramide, which directly activates CCT and stimulates PC synthesis (95). In Sandhoff disease, an accumulation of ganglioside GM2 is associated with reduced CCT activity and PC mass in the brain (96). In both diseases, altered PC synthesis may contribute to the pathology (97). Changes in CCT activity affect the utilization of the diacylglycerol intermediate, changing not only the rate of membrane PC synthesis but also the rate of triglyceride synthesis in an inverse manner (57, 98). Altered PC synthesis can also affect gene expression, as illustrated by the up-regulation of the genes involved in triglyceride formation when PC formation is deficient (99). The heightened expression of CCT in the brain suggests that it has a function in this tissue. However, the CCT2–/– mice do not have a brain phenotype (4).
CCT in the Lung—CCT has a regulatory role in the production of lung DSPC, a major surfactant component secreted into the alveolus. CCT mRNA stabilization during late gestational maturation of type II pneumocytes increases protein expression (100), glucocorticoid treatment of pulmonary alveolar cells elevates membrane-associated CCT activity (100, 101), and both processes are accompanied by stimulated DSPC synthesis. Signaling regulates CCT activity through a number of mechanisms. Calpain-mediated proteolysis in response to tumor necrosis factor (102) or oxidized LDL treatment (103) removes both the extreme N terminus and the regulatory domains of the protein and reduces activity. Altered sphingomyelin (101, 104, 105) or fatty acid metabolism (106, 107) changes the membrane composition and can influence the membrane association of CCT. Sphingomyelin hydrolysis generates sphingosine, an inhibitor of CCT (101, 108), and also reduces CCT expression in alveolar type II cells by a mechanism that is dependent on a negative regulatory element in the promoter region (109). Long chain ceramide is thought to inhibit CCT (104, 105, 110), but an in vitro assessment argues against a lipid-mediated mechanism (75). In contrast, the synthetic short chain ceramides are excellent inhibitors of CCT activity because of their structural similarity to lyso-PC (75, 105, 111).
CCT in the Liver—The PC in the liver is supplied by CCT and the PEMT (4). The expression of CCT is sufficient to support liver development and growth in animals that lack PEMT (112), but the mice develop hepatic steatosis (113). A choline-deficient diet upregulates CCT expression, and this correlates with increased flux through the CDP-choline pathway (114), suggesting that CCT expression responds to reduced cellular PC. Male and female mice express similar levels of liver CCT, but females have significantly more PEMT transcripts (4), suggesting that there is a gender bias in the reliance on CCT. In the absence of PEMT, only males have an inadequate amount of CCT and therefore produce insufficient PC to process triacylglycerol from a high fat/high cholesterol diet via VLDL secretion but can still produce HDL, albeit at reduced levels (115). These data are consistent with the view that PC from the CDP-choline pathway also contributes to lipoprotein secretion (18, 116). The existence of a separate PC pool governed by the PEMT is suggested by the localization of PEMT to the canalicular membrane where it may promote biliary excretion of PC (117). The data indicate that CCT and PEMT have overlapping roles and suggest that it is a quantitative difference in the capacity for PC production that has an impact on liver function.
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Lessons Learned from Genetically Modified Animals |
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 TOPINTRODUCTIONOverview of CCT BiochemistryCCT Genes and IsoformsCCT in Cell Proliferation...Transcriptional Regulation of...Tissue-specific Roles for CCT Lessons Learned from Genetically... PerspectivesREFERENCES |
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expression was anticipated to be lethal. This led to the development of genetic systems to direct lineage-specific CCT deletions (118). When CCT expression is selectively disrupted in mouse macrophages, there is no overt phenotype. This is attributed to CCT2 expression being up-regulated to support normal macrophage development (118). However, the macrophage adaptive response to free cholesterol loading is compromised because of reduced overall CCT activity, resulting in cell death. Similarly, development appears normal in mice where CCT expression is selectively ablated in liver. PEMT, which is a very significant contributor to PC synthesis in this tissue, is up-regulated 2-fold in response (18). PC as well as HDL, LDL, and apolipoprotein B output from the CCT knockout liver are reduced, indicating that CCT and the CDP-choline pathway do play an important role in regulating plasma levels of HDL and work together with the PEMT to regulate VLDL secretion (116). Overexpression of CCT activity in the lung and type II pneumocytes of transgenic mice results in only a modest increase in DSPC content (119). Elevated expression of CCT is often coupled with lipid feedback regulation and PC degradation (65, 66) that can be mediated at least in part by NTE (69).
Disruption of CCT2 expression results in a viable mouse but leads to gonadal dysfunction and reduced fertility, where females develop a defect in ovarian follicle development and the males exhibit progressive multifocal testicular degeneration (4). This reproductive phenotype was unanticipated considering that the highest level of CCT2 expression is in brain. The CCT2–/– mice have no obvious brain defect, but a caveat to these conclusions is that the CCT3 isoform is still expressed in this mouse model. Disruption of cct1, one of the two CCT genes in D. melanogaster, results in a reproductive phenotype where oogenesis and ovarian morphogenesis are defective (120). This phenotype is linked to aberrant membrane trafficking of ovarian growth and differentiation factor receptors (121). Therefore, a role for the isoforms in ovarian development seems to be a general finding, and further work will be required to firmly establish the specific function for this isoform. The idea that these defects may be in intracellular trafficking compartments that control hormonal signaling pathways is intriguing (120). The association of the CDP-choline pathway with vesicular trafficking in yeast (3, 122–124) suggests that CCT activity may have a similar function in mammalian cells.
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Perspectives |
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FOOTNOTES |
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To whom correspondence should be addressed. E-mail: suzanne.jackowski{at}stjude.org.
1 The abbreviations used are: PC, phosphatidylcholine; CCT, also known as CT, CTP:phosphocholine cytidylyltransferase; CSF-1, colony-stimulating factor 1; DSPC, disaturated phosphatidylcholine; G3PCT, CTP:glycerol-3-phosphate cytidylyltransferase; GroPCho, glycerol-3-phosphocholine; HDL, high density lipoprotein; LDL, low density lipoprotein; NLS, nuclear localization sequence; NTE, neuropathy target esterase; PEMT, phosphatidylethanolamine N-methyltransferase; PE, phosphatidylethanolamine; PI, phosphatidylinositol; VLDL, very low density lipoprotein.
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