Control of the CDPethanolamine pathway in mammalian cells: effect of CTP:phosphoethanolamine cytidylyltransferase overexpression and the amount of intracellular diacylglycerol

Abstract

For an insight regarding the control of PtdEtn (phosphatidylethanolamine) synthesis via the CDPethanolamine pathway, rat liver cDNA encoding ECT (CTP:phosphoethanolamine cytidylyltransferase) was transiently or stably transfected in Chinesehamster ovary cells and a rat liver-derived cell line (McA-RH7777), resulting in a maximum of 26-and 4-fold increase in specific activity of ECT respectively. However, no effect of ECT overexpression on the rate of [ 3 H]ethanolamine incorporation into PtdEtn was detected in both cell lines. This was explored further in cells overexpressing four times ECT activity (McA-ECT1). The rate of PtdEtn breakdown and PtdEtn mass were not changed in McA-ECT1 cells in comparison with controltransfected cells. Instead, an accumulation of CDPethanolamine (label and mass) was observed, suggesting that in McA-ECT1 cells the ethanolaminephosphotransferase-catalysed reaction became rate-limiting. However, overexpression of the human choline/ethanolaminephosphotransferase in McA-ECT1 and control-transfected cells had no effect on PtdEtn synthesis. To investigate whether the availability of DAG (diacylglycerol) limited PtdEtn synthesis in these cells, intracellular DAG levels were increased using PMA or phospholipase C. Exposure of cells to PMA or phospholipase C stimulated PtdEtn synthesis and this effect was much more pronounced in McA-ECT1 than in controltransfected cells. In line with this, the DAG produced after PMA exposure was consumed more rapidly in McA-ECT1 cells and the CDPethanolamine level decreased accordingly. In conclusion, our results suggest that the supply of CDPethanolamine, via the expression level of ECT, is an important factor governing the rate of PtdEtn biosynthesis in mammalian cells, under the condition that the amount of DAG is not limiting.

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

“…DAG and phorbol esters increase the rate of PE synthesis (7), and based on the metabolite pool sizes and the enzyme activity, the regulation of the CDP-ethanolamine pathway was predominantly mediated at the level of Pcyt2 (8,34,35). PMA stimulation of PE synthesis is accomplished through stimulation of Pcyt2 activity as well as by increases in the cellular content of the substrates ethanolamine (7) and DAG (8).…”

“…DAG and phorbol esters increase the rate of PE synthesis (7), and based on the metabolite pool sizes and the enzyme activity, the regulation of the CDP-ethanolamine pathway was predominantly mediated at the level of Pcyt2 (8,34,35). PMA stimulation of PE synthesis is accomplished through stimulation of Pcyt2 activity as well as by increases in the cellular content of the substrates ethanolamine (7) and DAG (8). PMA-resistant cells have an impaired PE synthesis and Pcyt2 activity (34,35), and phorbol esters target two different PKC isozymes in MCF-7 cells, PKC-␤I/II for the stimulation of PE synthesis and PKC␣ for the stimulation of PE degradation (8).…”

“…The de novo Kennedy pathway is quantitatively the most important route for the biosynthesis of PE in mammalian cells (6). In this pathway, ethanolamine is first phosphorylated by ethanolamine kinase to phosphoethanolamine (P-Etn), which is then converted to CDP-ethanolamine (CDP-Etn) by CTP:phosphoethanolamine cytidylyltransferase (Pcyt2; also known as ET (7,8) or ECT (9)). In the final step, CDPethanolamine:1,2-diacylglycerol ethanolamine phosphotransferase transfers P-Etn from CDP-Etn to DAG/alkylacylglycerol to produce PE/plasmalogens.…”

Alternative splicing of CTP:phosphoethanolamine cytidylyltransferase produces two isoforms that differ in catalytic properties

“…3D). This was likely because the reaction catalyzed by ethanolamine phosphotransferase, the last enzymatic step in the Kennedy pathway, is limited by the supply of diacylglycerol (25).…”

Developmental and Metabolic Effects of Disruption of the Mouse CTP:Phosphoethanolamine Cytidylyltransferase Gene (Pcyt2)

“…CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme in the CDP-ethanolamine pathway and catalyzes the formation of CDP-ethanolamine from phosphoethanolamine and CTP (3,17,30). Pcyt2 is a single gene that is alternatively spliced at exon 7 to produce two transcripts, Pcyt2␣ and Pcyt2␤ (23), which are differentially expressed among tissues.…”