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Originally published In Press as doi:10.1074/jbc.M300162200 on February 12, 2003

J. Biol. Chem., Vol. 278, Issue 17, 14753-14761, April 25, 2003
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Oncogenic Ha-Ras Transformation Modulates the Transcription of the CTP:Phosphocholine Cytidylyltransferase alpha  Gene via p42/44MAPK and Transcription Factor Sp3*

Marica BakovicDagger §, Kristin Waite§, and Dennis E. Vance||

From the 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

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 CTbeta 2 proteins and mRNAs was stimulated in C3H10T1/2 murine fibroblasts expressing oncogenic Ha-Ras. Incubation of cells with the specific inhibitor (PD98059) of p42/44MAPK decreased the expression of both CT isoforms. Transfection of fibroblasts with CTalpha promoter-luciferase constructs resulted in an ~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/p42/44MAPK signaling pathway which increases CTalpha gene transcription.

* This work was supported by a grant from the Canadian Institutes of Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Human Biology and Nutritional Sciences, University of Guelph, Animal Science/Nutrition Bldg., Rm. 346, Guelph, Ontario NIG2W1, Canada.

§ These authors contributed equally to this work.

Postdoctoral fellow of the Alberta Heritage Foundation for Medical Research. Present address: Human Cancer Genetics, The Ohio State University, 420 West 12th Ave., Columbus, OH 43210.

|| Medical scientist of the Alberta Heritage Foundation for Medical Research and Canada Research Chair in Molecular and Cell Biology of Lipids. To whom correspondence should be addressed: Dept. of Biochemistry, University of Alberta, 328 Heritage Medical Research Centre, Edmonton, Alberta T6G 2S2, Canada. Tel.: 780-492-8286; Fax: 780-492-3383; E-mail: dennis.vance@ualberta.ca.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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