ras-induced Up-regulation of CTP:Phosphocholine Cytidylyltransferase α Contributes to Malignant Transformation of Intestinal Epithelial Cells

Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.     

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.     

Cloning and characterization of a lipid-activated CTP:phosphocholine cytidylyltransferase from Caenorhabditis elegans: identification of a 21-residue segment critical for lipid activation1

The genome of the nematode Caenorhabditis elegans contains several genes that appear to encode proteins similar to CTP:phosphocholine cytidylyltransferase (CCT). We have isolated a 1044-nucleotide cDNA clone from a C. elegans cDNA library that encodes the 347-amino acid version of CCT that is most similar to previously-identified CCTs. Native and His-tagged forms were expressed and purified using a baculovirus expression system. The enzyme was maximally activated by 5 μM phosphatidylcholine:oleate (50:50) vesicles with a k_cat value in the presence of lipid 37-fold greater than the k_cat value in the absence of lipid. To localize the region of C. elegans CCT critical for lipid activation, a series of C-terminal truncation mutants was analyzed. CCT truncated after amino acids 225 or 245 was quite active in the absence of lipids and not further activated in the presence of lipids, supporting the concept that the lipid-activation segment is inhibitory to catalysis in the absence of lipids. CCT truncated after amino acids 266, 281, or 319 was activated by lipid similar to wild-type enzyme. Kinetic analysis in the absence of lipid revealed the lipid-independent CCT truncated after amino acid 245 to have a k_cat value 15-fold greater than either full-length CCT or CCT truncated after amino acid 266. We conclude that elements critical for activation of C. elegans CCT by lipids are contained within amino acids 246–266, that this region is inhibitory in the absence of lipids, and that the inhibition is relieved by the association of the enzyme with lipid.     

Isoform-specific and Protein Kinase C-mediated Regulation of CTP:Phosphoethanolamine Cytidylyltransferase Phosphorylation

CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme for de novo biosynthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. There are two isoforms of Pcyt2, -α and -β; however, very little is known about their specific roles in this important metabolic pathway. We previously demonstrated increased phosphatidylethanolamine biosynthesis subsequent to elevated activity and phosphorylation of Pcyt2α and -β in MCF-7 breast cancer cells grown under conditions of serum deficiency. Mass spectroscopy analyses of Pcyt2 provided evidence for isoform-specific as well as shared phosphorylations. Pcyt2β was specifically phosphorylated at the end of the first cytidylyltransferase domain. Pcyt2α was phosphorylated within the α-specific motif that is spliced out in Pcyt2β and on two PKC consensus serine residues, Ser-215 and Ser-223. Single and double mutations of PKC consensus sites reduced Pcyt2α phosphorylation, activity, and phosphatidylethanolamine synthesis by 50–90%. The phosphorylation and activity of endogenous Pcyt2 were dramatically increased with phorbol esters and reduced by specific PKC inhibitors. In vitro translated Pcyt2α was phosphorylated by PKCα, PKCβI, and PKCβII. Pcyt2α Ser-215 was also directly phosphorylated with PKCα. Mapping of the Pcyt2α- and -β-phosphorylated sites to the solved structure of a human Pcyt2β showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in other cytidylyltransferases. This study is the first to demonstrate differences in phosphorylation between Pcyt2 isoforms and to uncover the role of the PKC-regulated phosphorylation.     

The rate-limiting enzyme in phosphatidylcholine synthesis is associated with nuclear speckles under stress conditions

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in eukaryotic membranes and its biosynthetic pathway is generally controlled by CTP:Phosphocholine Cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCT is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum (ER) translocation; however, most of the enzyme is intranuclearly located. Here we demonstrate that CCTα is concentrated in the nucleoplasm of MDCK cells. Confocal immunofluorescence revealed that extracellular hypertonicity shifted the diffuse intranuclear distribution of the enzyme to intranuclear domains in a foci pattern. One population of CCTα foci colocalised and interacted with lamin A/C speckles, which also contained the pre-mRNA processing factor SC-35, and was resistant to detergent and salt extraction. The lamin A/C silencing allowed us to visualise a second more labile population of CCTα foci that consisted of lamin A/C-independent foci non-resistant to extraction. We demonstrated that CCTα translocation is not restricted to its redistribution from the nucleus to the ER and that intranuclear redistribution must thus be considered. We suggest that the intranuclear organelle distribution of CCTα is a novel mechanism for the regulation of enzyme activity.     

The ratio of phosphatidylcholine to phosphatidylethanolamine does not predict integrity of growing MT58 Chinese hamster ovary cells

Phosphatidylcholine (PC) homeostasis is important for maintaining cellular growth and survival. Cellular growth and apoptosis may also be influenced by the PC to phosphatidylethanolamine (PE) ratio as a reduction in this ratio can result in a loss of membrane integrity. To investigate whether a reduced PC:PE ratio influences cellular growth and apoptosis, we utilized the MT58 cell line, which contains a thermo-sensitive mutation in CTP:phosphocholine cytidylyltransferase-α, the rate-limiting enzyme for PC biosynthesis. Incubation of MT58 cells at the restrictive temperature of 41 °C results in a reduction of cellular PC and induces apoptosis. Furthermore, MT58 cells have a 50% reduction in the PC:PE ratio when incubated at 41 °C. In an attempt to normalize the PC:PE ratio, which may stabilize cellular membranes and rescue MT58 cells from apoptosis, the cells were treated with either silencing RNA to impair PE biosynthesis or lysophosphatidylcholine to increase PC mass. Impairing PE biosynthesis in MT58 cells reduced cellular PE and PC concentrations by 30% and 20%, but did not normalize the PC:PE ratio. Loss of both phospholipids enhanced the onset of apoptosis in MT58 cells. Lysophosphatidylcholine normalized cellular PC, increased PE mass by 10%, restored cellular growth and prevented apoptosis of MT58 cells without normalizing the PC:PE ratio. Furthermore, total amount of cellular PC and PE, but not the PC:PE ratio, correlated with cellular growth (R² = 0.76), and inversely with cellular apoptosis (R² = 0.97). These data suggest the total cellular amount of PC and PE, not the PC:PE ratio, influences growth and membrane integrity of MT58 cells.     

大鼠肝CTP:磷酸胆碱胞苷酰转移酶的部分纯化及其特性

 介绍一种部分纯化CTP:磷酸胆碱胞苷酰转移酶(CT)的方法,并对部分纯化CT的性质进行了研究。经盐析、DEAE-纤维素、磷酸-纤维素及CTP-聚琼脂糖柱层析可将大鼠肝胞液中的CT纯化100倍以上,结果重复性好。CT在胞液中及经初步纯化性质稳定,但进一步纯化则其活性很易丧失。鼠肝总磷脂及油酸可激活CT、使其聚合,磷脂酰絲氨酸是其中起主要作用的CT激活物;CT聚合物可被辛基-葡萄糖苷解聚,且保留80％酶活性。用CT常规底物CTP的类似物观察CT的特异性,发现dCTP是比CTP更好的底物,CDP、dCDP能抑制CT对CTP或dCTP的作用,而CMP、dCMP对酶活性无影响。结果提示分子中糖的2′-OH并非CT底物所必需,而和胞苷相连的三个磷酸则不可缺少。     

Characterization of cytidylyltransferase enzyme activity through high performance liquid chromatography

The cytidylyltransferases are a family of enzymes that utilize cytidine 5′-triphosphate (CTP) to synthesize molecules that are typically precursors to membrane phospholipids. The most extensively studied cytidylyltransferase is CTP:phosphocholine cytidylyltransferase (CCT), which catalyzes conversion of phosphocholine and CTP to cytidine diphosphocholine (CDP-choline), a step critical for synthesis of the membrane phospholipid phosphatidylcholine (PC). The current method used to determine catalytic activity of CCT measures production of radiolabeled CDP-choline from ¹⁴C-labeled phosphocholine. The goal of this research was to develop a CCT enzyme assay that employed separation of non-radioactive CDP-choline from CTP. A C18 reverse phase column with a mobile phase of 0.1 M ammonium bicarbonate (98%) and acetonitrile (2%) (pH 7.4) resulted in separation of solutions of the substrate CTP from the product CDP-choline. A previously characterized truncated version of rat CCTα (denoted CCTα236) was used to test the HPLC enzyme assay by measuring CDP-choline product formation. The V_max for CCTα236 was 3850 nmol/min/mg and K_0.5 values for CTP and phosphocholine were 4.07 mM and 2.49 mM, respectively. The HPLC method was applied to glycerol 3-phosphate cytidylyltransferase (GCT) and CTP:2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), members of the cytidylyltransferase family that produce CDP-glycerol and CDP-methylerythritol, respectively.     

Regulation of phosphatidylcholine homeostasis by calcium-independent phospholipase A2

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in mammalian cell membranes and is essential for cell viability. The levels of this lipid must be tightly controlled to maintain homeostasis. Therefore, changes in the rate of PtdCho synthesis are generally balanced by changes in PtdCho catabolism and vice versa. It is commonly accepted that the rate of PtdCho synthesis is regulated by CTP:phosphocholine cytidylyltransferase (CT). However, it is not certain if PtdCho mass is regulated by specific catabolic enzyme(s). Our goal is to determine if PtdCho homeostasis is regulated by a phospholipase A₂ (PLA₂). To this end, we have prepared Chinese hamster ovary (CHO) cell lines that overexpress CT. CT activity is 7–10-fold higher in the transfected cells than in parental CHO cells. This increase in CT activity is associated with increases in both PtdCho synthesis and PtdCho catabolism. Glycerophosphocholine is the PtdCho catabolite that accumulates in the transfected cells, which suggests that PtdCho turnover is mediated by a phospholipase A₂ (PLA₂). Indeed, higher levels of calcium-independent PLA₂ activity are measured in the cytosols of the CHO cells that overexpress CT, compared to parental CHO cells. The elevated calcium-independent PLA₂ activity is associated with increases in the expression of the 80-kDa calcium-independent PLA₂ (iPLA₂). Together, these data suggest that the 80-kDa iPLA₂ may be modulated in response to changes in PtdCho levels and therefore is involved in the regulation of PtdCho homeostasis in CHO cells.