CYCLIN-A CDK2 BINDS DIRECTLY TO E2F-1 AND INHIBITS THE DNA-BINDING ACTIVITY OF E2F-1 DP-1 BY PHOSPHORYLATION/

E2F-1, a member of the E2F transcription factor family, contributes to the regulation of the G(1)-to-S phase transition in higher eukaryotic cells. E2F-1 forms a heterodimer with DP-1 and binds to several cell cycle regulatory proteins, including the retinoblastoma family (RB, p1 07, p130) and cyclin A/CDK2 complexes. We have analyzed E2F-1 phosphor ylation and its interaction with cyclin A/CDK2 complexes both in vivo and in vitro. In vitro, E2F-1 formed a stable complex with cyclin A/CD K2 but not with either subunit alone. DP-1 did not interact with cycli n A, CDK2, or the cyclin A/CDK2 complex. While the complex of cyclin A /CDK2 was required for stable complex formation with E2F-1, the kinase -active form of CDK2 was not required. However, E2F-1 was phosphorylat ed by cyclin A/CDK2 in vitro and was phosphorylated in vivo in HeLa ce lls. Two-dimensional tryptic phosphopeptide mapping studies demonstrat ed an overlap in the phosphopeptides derived from E2F-1 labeled in vit ro and in vivo, indicating that cyclin A/CDK2 may be responsible for t he majority of E2F-1 phosphorylation in vivo. Furthermore, an active D NA-binding complex could be reconstituted from purified E2F-1/DP-1 and cyclin A/CDK2. Binding studies conducted both in vitro and in vivo de monstrated that the cyclin A/CDK2-binding region resided within the N- terminal 124 amino acids of E2F-1. Because the stable association of E 2F-1 with cyclin A/CDK2 in vitro and in vivo did not require a DP-1- o r RB-binding domain and because the interactions could be reconstitute d from purified components in vitro, we conclude that the interactions between cyclin A/CDK2 and E2F-1 are direct. Finally, we report that t he DNA-binding activity of the E2F-1/DP-1 complex is inhibited followi ng phosphorylation by cyclin A/CDK2,