Transcription of the CTP:Phosphocholine Cytidylyltransferase α Gene Is Enhanced during the S Phase of the Cell Cycle

Abstract

We have studied the transcription of the CTP:phosphocholine cytidylyltransferase ␣ (CT␣) gene in C3H10T1/2 fibroblasts as a function of the cell cycle. The cells were incubated for 48 h with 0.5% fetal bovine serum. The cells were induced into the G 1 phase of the cell cycle by the addition of medium with 10% fetal bovine serum. The cells began the synthesis of DNA after 12 h. At 16 and 20 h there was an increased amount of CT␣ mRNA that coincided with an increase in the expression of CT␣ proximal promoter-luciferase constructs (؊201/؉38 and ؊130/؉38). Luciferase constructs with the basal promoter (؊52/؉38) showed no change in activity during the cell cycle. Incorporation of [ 3 H]choline into phosphatidylcholine began to increase by 8 h after the addition of serum and peaked at 18 h. The mass of phosphatidylcholine nearly doubled between 8 and 26 h after addition of serum. CT activity increased by 6 h after serum addition and was maintained until 22 h. Thus, the increase of phosphatidylcholine biosynthesis in the G 1 phase of the cell cycle is not due to enhanced transcription of the CT␣ gene. Instead increased transcription of the CT␣ gene occurred during the S phase of the cell cycle in preparation for mitosis. Phosphatidylcholine (PC)1 represents ϳ50% of the membrane phospholipids in animal cells (1). PC is a substrate for the generation of bioactive lipid messengers (2), a precursor in the synthesis of sphingomyelin, phosphatidylethanolamine, and phosphatidylserine (3, 4) and is an important component of serum lipoproteins (5) and bile (6). PC biosynthesis occurs in all nucleated cells mainly via the CDP-choline/Kennedy pathway in which CTP:phosphocholine cytidylyltransferase (CT, EC 2.7.7.15) is considered to be the rate-limiting and regulated enzyme of this pathway (1,7,8). The first mammalian CT was purified from rat liver (9, 10) as a homodimer with a subunit molecular mass of 42 kDa and was the basis for the cloning of the corresponding cDNA encoding a 367-amino acid (CT␣) protein (11). To date, two additional isoforms of CT, CT␤1 and CT␤2, have been identified that are encoded by a second gene (12,13).While the primary mechanism by which CT activity is regulated in cells is activation by lipids after translocation to cellular membranes (1,8,14,15), it is now recognized that PC synthesis can also be regulated by changes in CT mRNA turnover and by CT gene transcription. Several reports indicate that CT can be regulated post-transcriptionally, apparently by reducing the rate of mRNA degradation (16,17). However, the precise mechanisms involved in regulating CT mRNA stability still have to be elucidated. Tessner et al. (16) provided the first evidence for increased CT mRNA during growth factor-stimulated PC synthesis in a murine macrophage cell line. Houweling et al. (18) demonstrated that CT is regulated at the level of its mRNA in rat liver after partial hepatectomy. The maximal increase in the CT mRNA coincided with maximal DNA synthesis 24 h after partial hepatectomy (19). CT is highly expressed dur...

Cell Cycle Dependence of Group VIA Calcium-independent Phospholipase A2 Activity

“…PC mass increased during G 1 and reached G 2 /M levels by early S phase. These results demonstrate that PC mass accumulates before cell division and are consistent with previous reports (16,26). As several reports have attributed glycerophospholipid accumulation to decreased turnover (16,18), we measured the half-life of PC to determine if this was true in the CHO cell system as well.…”

“…It is well established that PC accumulation during the cell cycle does not correlate with the kinetics of its synthesis (16,26,27). We obtained similar results in the CHO cell system as the cells exhibited a burst of PC synthesis but this occurred in early G 1 and not during late G 1 and S phase when PC mass accumulated (Fig.…”

Genomic organization, promoter activity, and expression of the human choline transporter-like protein 1

“…The production of CDP-choline with membrane-activated Pcyt1 is preceded by two steps, choline membrane transport and then choline phosphorylation with choline kinase, which is known to be a cytosolic protein (5,15,22,25). Traditional channeling of intermediates for PC synthesis from the plasma membrane to ER (8,19) assumes a unidirectional choline supply by a cell surface transporter, and such a role could be perhaps attributed to the CTL1a form of this family of transporters.…”

CTP:Phosphocholine Cytidylyltransferase: Paving the Way from Gene to Membrane

“…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 G 1 (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 G 2 and mitosis (54,61).…”

“…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 G 1 (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 G 2 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.…”