Xu M, Sheppard K A, Peng C Y, Yee A S, Piwnica-Worms H
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Mol Cell Biol. 1994 Dec;14(12):8420-31. doi: 10.1128/mcb.14.12.8420-8431.1994.
E2F-1, a member of the E2F transcription factor family, contributes to the regulation of the G1-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, p107, p130) and cyclin A/CDK2 complexes. We have analyzed E2F-1 phosphorylation 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/CDK2 but not with either subunit alone. DP-1 did not interact with cyclin 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 phosphorylated by cyclin A/CDK2 in vitro and was phosphorylated in vivo in HeLa cells. Two-dimensional tryptic phosphopeptide mapping studies demonstrated an overlap in the phosphopeptides derived from E2F-1 labeled in vitro and in vivo, indicating that cyclin A/CDK2 may be responsible for the majority of E2F-1 phosphorylation in vivo. Furthermore, an active DNA-binding complex could be reconstituted from purified E2F-1/DP-1 and cyclin A/CDK2. Binding studies conducted both in vitro and in vivo demonstrated that the cyclin A/CDK2-binding region resided within the N-terminal 124 amino acids of E2F-1. Because the stable association of E2F-1 with cyclin A/CDK2 in vitro and in vivo did not require a DP-1- or RB-binding domain and because the interactions could be reconstituted from purified components in vitro, we conclude that the interactions between cyclin A/CDK2 and E2F-1 are direct. Finally, we report that the DNA-binding activity of the E2F-1/DP-1 complex is inhibited following phosphorylation by cyclin A/CDK2.
E2F-1是E2F转录因子家族的成员之一，在高等真核细胞中参与调控G1期到S期的转换。E2F-1与DP-1形成异源二聚体，并与多种细胞周期调节蛋白结合，包括视网膜母细胞瘤家族（RB、p107、p130）以及细胞周期蛋白A/细胞周期蛋白依赖性激酶2（cyclin A/CDK2）复合物。我们已经在体内和体外分析了E2F-1的磷酸化及其与cyclin A/CDK2复合物的相互作用。在体外，E2F-1与cyclin A/CDK2形成稳定的复合物，但不与单独的任何一个亚基形成复合物。DP-1不与cyclin A、CDK2或cyclin A/CDK2复合物相互作用。虽然cyclin A/CDK2复合物是与E2F-1形成稳定复合物所必需的，但CDK2的激酶活性形式并非必需。然而，E2F-1在体外被cyclin A/CDK2磷酸化，并且在HeLa细胞的体内也被磷酸化。二维胰蛋白酶磷酸肽图谱研究表明，体外和体内标记的E2F-1衍生的磷酸肽存在重叠，这表明cyclin A/CDK2可能是体内E2F-1磷酸化的主要原因。此外，可以从纯化的E2F-1/DP-1和cyclin A/CDK2中重新构建有活性的DNA结合复合物。体内和体外进行的结合研究表明，cyclin A/CDK2结合区域位于E2F-1的N端124个氨基酸内。由于E2F-1在体内和体外与cyclin A/CDK2的稳定结合不需要DP-1或RB结合结构域，并且由于这些相互作用可以在体外由纯化的成分重新构建，我们得出结论，cyclin A/CDK2与E2F-1之间的相互作用是直接的。最后，我们报告cyclin A/CDK2磷酸化后，E2F-1/DP-1复合物的DNA结合活性受到抑制。
