Oncogenic Ha-Ras Transformation Modulates the Transcription of the CTP:Phosphocholine Cytidylyltransferase α Gene via p42/44MAPK and Transcription Factor Sp3

Abstract

Phosphatidylcholine (PC)1 is the most abundant phospholipid in mammalian cellular membranes. Besides having a structural role in membranes and lipoproteins (1-3), PC plays an important role in signal transduction as a source of lipid second messengers (1-3). In all nucleated cells, PC is made primarily through the CDP-choline pathway in which the key enzyme is CTP:phosphocholine cytidylyltransferase (CT) (2-6).Two genes that encode CT activity have been identified and characterized. CT␣ is expressed in many cells and tissues (7-13) and has a predicted structure that contains catalytic, phosphorylation, and lipid binding domains as well as a nuclear localization sequence (8, 14 -21). Recently, CT␤ has been identified in human tissues and appears to exist as two splice variants, CT␤1 and CT␤2, differing at their C termini (22, 23). Like CT␣, CT␤1/2 contains catalytic and lipid binding domains. However, CT␤1 lacks the phosphorylation domain, and both CT␤1 and CT␤2 lack the nuclear localization sequence (23).In the last several years, studies have shown that CT can be regulated at both the transcriptional level and post-transcriptionally. CT mRNA is increased after partial hepatectomy in rats (13), after stimulation with colony-stimulating factor 1 in macrophages (24), and during development and growth (25)(26)(27)(28). It remains to be determined whether these increases in the message levels are the result of CT␣ and/or CT␤1/2 mRNA stability, an increase in gene transcription, or a combination of both.The murine CT␣ gene (Ctpct) was cloned and characterized by Tang and co-workers (29). The Ctpct promoter contains several putative elements for binding transcription factors, including Ap1, an overlapping site for nuclear factor-B, E2F, and Elk1, one sterol response element, as well as three elements for Sp-related factors (30). We have shown that Sp1, Sp2, and Sp3 bind competitively to three GC-rich elements and that relative promoter activity depends upon the abundance of these factors (31). We further established that transcription enhancer factor-4 can bind to an upstream regulatory element (Ϫ103/Ϫ82) and enhance the CT␣ gene expression through its interactions with the basal transcriptional machinery (32). In agreement with the finding that lipoprotein deficiency induces the expression of CT␣ mRNA and protein in alveolar type II epithelial cells (33), and our observation that the CT␣ promoter contains a putative sterol response element (30), studies by Kast et al. (34) indicate a role for cholesterol/sterol response element-binding protein and the functionality of the sterol response element in the regulation of CT␣ gene expression in Chinese hamster ovary cells and THP-1 cells. On the other hand, Lagace et al. (35) have shown that cholesterol/sterol response element-binding protein can stimulate PC biosynthe-

Oxysterols Inhibit Phosphatidylcholine Synthesis via ERK Docking and Phosphorylation of CTP:Phosphocholine Cytidylyltransferase

“…CCT␣ phosphorylation has been linked to membrane phospholipid synthesis with cell division (18,38,39), but to our knowledge, this study is the first showing the physiological relevance of specific phosphoacceptor sites. Our results mechanistically differ from the inhibitory effects of 25-HC on PtdCho synthesis that are regulated by cholesterol biosynthesis (40,41).…”

Sp3‐Mediated VEGF regulation is dependent on phosphorylation by extra‐cellular signals regulated kinases (Erk)

“…It is interesting to note that the Erk phosphorylation site is situated in the long Sp3 isoform, which behaves as an activator of transcription (Kennett et al, 1997) or a repressor depending on the promoter (Lambiase et al, 2005). Sp3 was also demonstrated to be a tyrosine phosphorylated protein and increased phosphorylation was already observed in Ha-Ras transformed cells (Bakovic et al, 2003). It is perhaps a reason why the mutation of the S73 to alanine is not sufficient to totally abolish Sp3 transcriptional activity.…”

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.…”

“…All promoters are lacking TATA or CAAT-like motifs but contain GC-rich sites in the vicinity of the initiation start site (designated as ϩ1 in Fig. 2), which could initiate transcription as typical for many ubiquitous promoters, for example, the cytomegalovirus promoter and the Pcyt1 promoter (4,5). All CTL1 promoters also contain conserved response elements for potential transcription factors common for housekeeping genes (Sp1 binding demonstrated for mouse and human muscle in Fig.…”