Inhibition of Cyclin D1 Kinase Activity Is Associated with E2F-Mediated Inhibition of Cyclin D1 Promoter Activity through E2F and Sp1

ABSTRACT

Coordinated interactions between cyclin-dependent kinases (Cdks), their target “pocket proteins” (the retinoblastoma protein [pRB], p107, and p130), the pocket protein binding E2F-DP complexes, and the Cdk inhibitors regulate orderly cell cycle progression. The cyclin D1 gene encodes a regulatory subunit of the Cdk holoenzymes, which phosphorylate the tumor suppressor pRB, leading to the release of free E2F-1. Overexpression of E2F-1 can induce apoptosis and may either promote or inhibit cellular proliferation, depending upon the cell type. In these studies overexpression of E2F-1 inhibited cyclin D1-dependent kinase activity, cyclin D1 protein levels, and promoter activity. The DNA binding domain, the pRB pocket binding region, and the amino-terminal Sp1 binding domain of E2F-1 were required for full repression of cyclin D1. Overexpression of pRB activated the cyclin D1 promoter, and a dominant interfering pRB mutant was defective in cyclin D1 promoter activation. Two regions of the cyclin D1 promoter were required for full E2F-1-dependent repression. The region proximal to the transcription initiation site at −127 bound Sp1, Sp3, and Sp4, and the distal region at −143 bound E2F-4–DP-1–p107. In contrast with E2F-1, E2F-4 induced cyclin D1 promoter activity. Differential regulation of the cyclin D1 promoter by E2F-1 and E2F-4 suggests that E2Fs may serve distinguishable functions during cell cycle progression. Inhibition of cyclin D1 abundance by E2F-1 may contribute to an autoregulatory feedback loop to reduce pRB phosphorylation and E2F-1 levels in the cell.

ACKNOWLEDGMENTS

We are grateful to E. Harlow, D. Heimbrook, R. Weinberg, M. Pagano, G. Draetta, D. Livingston, W. Krek, D. Ginsberg, R. Watson, J. Wang, W. Kaelin, J. Nevins, L. Bandara, and N. La Thangue for plasmids and antibodies, and to D. Gebhard for assistance with flow cytometry analysis. We thank L. Yamasaki for helpful discussions and the MEF derived from the E2F-1 KO mice.

This work was supported in part by grant 94-27 from the American Cancer Society (Illinois Division, Inc.) and 1R29CA70897-01 and R01CA75503 from the National Cancer Institute (to R.G.P.). G.W. was supported in part by a Travel Fellowship from the Aichi Health Promotion Foundation, the Owari Kenyu-kai, and the Takasu Foundation. A.R. was supported by a P.F. Sobotka postgraduate scholarship from the University of Western Australia. G.V. was a recipient of a C. J. Martin postdoctoral fellowship from the Australian National Health and Medical Research Council and an AMRAD Corporation postdoctoral award. Work at the Albert Einstein College of Medicine was also supported by Cancer Center Core National Institutes of Health grant 5-P30-CA13330-26.

G. Watanabe and C. Albanese contributed equally to this work.