Adenovirus E1A-Regulated Transcription Factor p120^E4F Inhibits Cell Growth and Induces the Stabilization of the cdk Inhibitor p21^WAF1

ABSTRACT

Adenovirus E1A proteins influence cell growth and phenotype through physical interactions with cellular proteins that regulate basic processes such as cell cycle progression, DNA synthesis, and differentiation. p120^E4F is a low-abundance cellular transcription factor that represses the adenovirus E4 promoter and is regulated by E1A, through a phosphorylation-induced reduction of its DNA binding activity, to permit activation of the E4 promoter during early infection. To determine the normal biological role of p120^E4F, we assessed its ability to influence fibroblast cell growth and transformation. p120^E4F suppressed NIH 3T3 fibroblast colony formation but had little effect when coexpressed with E1A and/or activated ras. Cells that overexpressed p120^E4F were inhibited in their ability to enter S phase, had elevated levels of the cdk inhibitor p21^WAF1, and reduced cyclin D-cdk4/6 kinase activity. The increase of p21^WAF1 levels occurred through a p53-independent posttranscriptional mechanism that included a three- to fourfold increase in the half-life of p21^WAF1 protein. Coexpression of activatedras with p120^E4F stimulated cyclin D1 expression, elevated cyclin D-cdk4/6 kinase activity, and accelerated cell growth. These data suggest an important role for p120^E4F in normal cell division and demonstrate that p21^WAF1 can be regulated by protein turnover.

ACKNOWLEDGMENTS

We are grateful to Richard Ashmun, Sam Lucas, and Ed Wingfield for FACS analyses; Charles Sherr for antisera against cyclins D1, D2, and D3, cdk2, cdk4, and cdk6, and p21 and p27; Martine Roussel for NIH 3T3 clone 7 fibroblasts; Scott Hiebert for the construct expressing GST-Rb (379-928); Gerard Zambetti for CMV constructs expressing mutant p53 proteins and for performing p53 Western blot analyses; Jaideep Thottassery and John Schuetz for expressing p120^E4F in H35 hepatoma cells; and Ruby Tharp for excellent technical assistance. We also thank Gerard Zambetti, John Cleveland, Martine Roussel, John Schuetz, Jeffrey Sample, and Scott Hiebert for helpful discussions and critical reading of the manuscript.

This work was supported, in part, by National Institutes of Health (NIH) grant R01 GM51299 (R.J.R.), NIH/NCI Cancer Center Support CORE grant 5 P30 CA21765, and the American Lebanese Syrian Associated Charities of St. Jude Children’s Research Hospital (ALSAC).