TRANSFORMING P21(RAS) MUTANTS AND C-ETS-2 ACTIVATE THE CYCLIN D1 PROMOTER THROUGH DISTINGUISHABLE REGIONS

Several different oncogenes and growth factors promote G(1) phase progression, Cyclin D1, the regulatory subunit of several cyclin dependent kinases, is required for, and capable of shortening, the G(1) phase of the cell cycle. The present study demonstrates that transforming mutants of p21(ras) (Ras Val-12, Ras Leu-61) induce the cyclin D1 promoter in human trophoblasts (JEG-3), mink lung epithelial (Mv1.Lu), and in Chinese hamster ovary fibroblast cell lines, Site-directed mutagenesis of AP-l-like sequences at -954 abolished p21(ras)-dependent activation of cyclin D1 expression. The AP-l-like sequences were also required for activation of the cyclin D1 promoter by c-Jun. In electrophoretic mobility shift assays using nuclear extracts from cultured cells and primary tissues, several AP-1 proteins (c-Jun, JunB, JunD, and c-Fos) bound the cyclin D1 -954 region. Cyclin D1 promoter activity was stimulated by overexpression of mitogen-activated protein kinase (p41(MAPK)) or c-Ets-2 through the proximal 22 base pairs. Expression of plasmids encoding either dominant negative MAPK (p41(MAPKi)) Or dominant negatives of ETS activation (Ets-LacZ), antagonized MAPK dependent induction of cyclin D1 promoter activity. Epidermal growth factor induction of cyclin D1 transcription, through the proximal promoter region, was antagonized by either p41(MAPKi) or Ets-LacZ, suggesting that ETS functions downstream of epidermal growth factor and MAPK in the context of the cyclin D1 promoter. The activation of cyclin D1 transcription by p21(ras) provides evidence for cross-talk between the p21(ras) and cell cycle regulatory pathways.