Phosphorylation of Sp1 by Cyclin-dependent Kinase 2 Modulates the Role of Sp1 in CTP:Phosphocholine Cytidylyltransferase α Regulation during the S Phase of the Cell Cycle
Journal of Biological Chemistry
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 ␣ (CT␣) 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 CT␣ promoter (Banchio, C.,
... r (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 CT␣ transcription during S phase. Sp1 binds in a phosphorylated state to the CT␣ 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 CT␣ expression, whereas reduction in the expression of cyclin A, cyclin E, and CDK2 eliminates the induction of CT␣ expression in S phase. Furthermore, CT␣ 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 CT␣ in preparation for cell division. Sequential activation and inactivation of cyclin-dependent kinases (CDKs) 1 ensure an orderly progression through the cell cycle (1). CDK activities are regulated by a variety of mechanisms, such as a periodic accumulation of cyclins, degradation, nuclear localization, phosphorylation of CDKs, and association with different CDK inhibitors (1-3). Cyclin E, in combination with its associated cyclin-dependent kinase 2 (CDK2), is a positive cell cycle regulator controlling progression through G 1 and initiation of DNA replication (4 -7). Cyclin A binds both CDK2 and CDK1 (Cdc2), giving rise to two distinct cyclin A DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; LUC, luciferase; siRNA, small interfering RNA; CMV, cytomegalovirus.