A MUTANT E2F-1 TRANSCRIPTION FACTOR THAT AFFECTS THE PHENOTYPE OF NIH3T3 FIBROBLASTS INEFFICIENTLY ASSOCIATES WITH CYCLIN-A CDK2

The amino-terminal domain of the E2F1 transcription factor is the site of association with cyclin A - cdk2, mapping to residues 87-94. A mut ant of E2F1 lacking the first 87 amino acids (termed E2F1d87) has a nu mber of potent effects on cellular phenotype when constitutively expre ssed in NIH3T3 fibroblasts. For example, in these fibroblasts the dura tion of S phase and the sensitivity to S phase chemotherapeutic agents are both increased. Since E2F1d87 only partially truncates the cyclin A - cdk2 binding domain, it was important to determine the level of c yclin A - cdk2 association with this mutant to correlate any reduction in association with the observed effects on the cell cycle. It was fo und that cyclin A - cdk2 binds E2F1d87 in an in vitro assay but that t his binding is reduced approximately 8 fold compared with binding to f ull-length E2F1, whereas no detectable binding was seen to a mutant E2 F1 that lacks the first 117 amino acids. Correspondingly, H1 kinase ac tivity in E2F1d87 immunoprecipitates from E2F1d87-expressing cells was significantly reduced compared with that seen for full-length E2F1. F rom these data it appears that E2F1 with reduced cyclin A - cdk2 bindi ng activity mediates the alteration in cell cycle parameters seen in t hese cel:ls.