title: Oncogenic Ha-Ras transformation modulates the transcription of the CTP:phosphocholine cytidylyltransferase alpha gene via p42/44MAPK and transcription factor Sp3.
abstract: We have shown previously that expression of the murine CTP:phosphocholine cytidylyltransferase (CT) alpha gene is regulated during cell proliferation (Golfman, L. S., Bakovic, M., and Vance, D. E. (2001) J. Biol. Chem. 276, 43688-43692). We have now characterized the role of Ha-Ras in the transcriptional regulation of the CTalpha gene. The expression of CTalpha and CTbeta2 proteins and mRNAs was stimulated in C3H10T1/2 murine fibroblasts expressing oncogenic Ha-Ras. Incubation of cells with the specific inhibitor (PD98059) of p42/44(MAPK) decreased the expression of both CT isoforms. Transfection of fibroblasts with CTalpha promoter-luciferase constructs resulted in an approximately 2-fold enhanced luciferase expression in Ha-Ras-transformed, compared with nontransformed, fibroblasts. Electromobility shift assays indicated enhanced binding of the Sp3 transcription factor to the CTalpha promoter in Ha-Ras-transformed cells. Expression of several forms of Sp3 was increased in nuclear extracts of Ha-Ras-transformed fibroblasts compared with nontransformed cells. Tyrosine phosphorylation of one Sp3 form was decreased, whereas phosphorylation of two other forms of Sp3 was increased in nuclear extracts of Ha-Ras-transformed cells. When control fibroblasts were transfected with a Sp3-expressing plasmid, an enhanced expression of CTalpha and CTbeta was observed. However, the expression of CTalpha or CTbeta was not increased in Ha-Ras-transformed cells transfected with a Sp3 plasmid presumably because expression was already maximally enhanced. The results suggest that Sp3 is a downstream effector of a Ras/p42/44(MAPK) signaling pathway which increases CTalpha gene transcription.
authors.affiliation: Department of Biochemistry and Canadian Institutes of Health Research Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
authors.forename: Marica
authors.initials: M
authors.lastname: Bakovic
authors.forename: Kristin
authors.initials: K
authors.lastname: Waite
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Vance
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002461:Cell Line, Transformed; D019916:Choline-Phosphate Cytidylyltransferase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000502:physiology*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D015689:Oncogene Protein p21(ras) / Q000502:physiology*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D014170:Transformation, Genetic
chemical_list: D004268:DNA-Binding Proteins; D020033:Protein Isoforms; C497122:Sp3 protein, mouse; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019916:Choline-Phosphate Cytidylyltransferase; D015689:Oncogene Protein p21(ras)