Phosphorylation of Sp1 by cyclin-dependent kinase 2 modulates the role of Sp1 in CTP:phosphocholine cytidylyltransferase alpha regulation during the S phase of the cell cycle.
Phosphatidylcholine is the major lipid component in mammalian membranes. Phosphatidylcholine synthesis increases in C3H10T1/2 fibroblasts during the G(1) and S phases of the cell cycle. Previous studies demonstrated that the mRNA encoding CTP:phosphocholine cytidylyltransferase alpha (CTalpha) increases during S phase (Golfman, L. S., Bakovic, M., and Vance, D. E. (2001) J. Biol. Chem. 276, 43688-43692) and that this activation is driven by increased binding of Sp1 to the CTalpha promoter (Banchio, C., Schang, L. M., and Vance, D. E. (2003) J. Biol. Chem. 278, 32457-32464). We now demonstrate that cyclin-dependent kinase 2 (CDK2) phosphorylation of Sp1 activates CTalpha transcription during S phase. Sp1 binds in a phosphorylated state to the CTalpha promoter. Sp1 binding is enhanced by association with cyclin A/E and CDK2, both in vivo and in vitro. In cells that overexpress Sp1, co-expression of cyclin A and CDK2 induces a high and constant level of CTalpha expression, whereas reduction in the expression of cyclin A, cyclin E, and CDK2 eliminates the induction of CTalpha expression in S phase. Furthermore, CTalpha expression is decreased in cells overexpressing a dominant-negative form of CDK2 and in cells treated with the CDK2 kinase inhibitors roscovitine and olomoucine. These results enhance our understanding of the regulatory mechanisms involved in the expression of CTalpha in preparation for cell division.
Banchio
Claudia
C
Department of Biochemistry, Canadian Institutes of Health Research Group in Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
Schang
Luis M
LM
Vance
Dennis E
DE
D000818:Animals; D001798:Blood Proteins / Q000494:pharmacology; D042846:CDC2-CDC28 Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D019916:Choline-Phosphate Cytidylyltransferase / Q000235:genetics* / Q000378:metabolism; D019925:Cyclin A / Q000235:genetics / Q000378:metabolism; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D051379:Mice; D008809:Mice, Inbred C3H; D010766:Phosphorylation; D034741:RNA, Small Interfering; D016196:S Phase / Q000502:physiology*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic
D001798:Blood Proteins; D019925:Cyclin A; D019927:Cyclin E; D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D019916:Choline-Phosphate Cytidylyltransferase