DIFFERENTIAL REGULATION OF E2F TRANSACTIVATION BY CYCLIN CDK2 COMPLEXES

The mammalian transcription factor E2F plays a critical role in the ex pression of genes required for cellular proliferation. To understand h ow E2F is regulated, we have developed a reconstituted in vitro transc ription assay. Using this E2F-responsive assay, we can demonstrate tha t E2F-mediated transcription can be directly repressed by the tumor su ppressor protein pRB. This inhibition is abolished by phosphorylation of pRB with either cyclin A/cdk2 or cyclin E/cdk2. However, these cycl in/kinase complexes exhibit differences in the ability to phosphorylat e E2F. Only cyclin A/cdk2 can phosphorylate E2F effectively, and this phosphorylation abolishes its ability to bind DNA and mediate trans-ac tivation. Thus, this in vitro transcriptional assay allows activation and inactivation of E2F transcription, and our findings demonstrate ho w transcriptional regulation of E2F can be linked to cell cycle-depend ent activation of kinases.