He Yiwen, Cress W Douglas
Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa 33612, USA.
J Biol Chem. 2002 Jun 28;277(26):23493-9. doi: 10.1074/jbc.M202629200. Epub 2002 Apr 29.
The E2F family of transcription factors controls the expression of numerous genes that are required for the G(1)/S transition. Among the mechanisms that modulate the activity of the E2F proteins, cyclin A has been found to be important for the down-regulation of E2F-1, -2, and -3A activity after cells have progressed through G(1)/S. Specifically, phosphorylation of these E2F proteins by cyclin A/Cdk2 ultimately results in their necessary degradation as cells progress through S phase. E2F-3B was recently identified as an alternatively spliced form of E2F-3A that was predicted to lack a functional cyclin A binding domain. In this paper, we present considerable evidence that contradicts this prediction. First, we demonstrate binding of cyclin A to E2F-3B as bacterially expressed proteins in vitro. Second, we demonstrate binding of cyclin A to E2F-3B in mammalian cells in vivo. Third, we show that co-expression of cyclin A with E2F-3B significantly reduces E2F-3B-mediated transcriptional activity. Finally, in synchronized cells, we observe down-regulation of E2F-3B protein expression coincident with the up-regulation of cyclin A. We conclude that E2F-3B is a physiological target of cyclin A.
E2F转录因子家族控制着众多G(1)/S期转换所需基因的表达。在调节E2F蛋白活性的机制中，细胞周期蛋白A被发现对于细胞通过G(1)/S期后下调E2F-1、-2和-3A的活性很重要。具体而言，细胞周期蛋白A/Cdk2对这些E2F蛋白的磷酸化最终导致它们在细胞进入S期时被降解。E2F-3B最近被鉴定为E2F-3A的一种选择性剪接形式，预计缺乏功能性细胞周期蛋白A结合结构域。在本文中，我们提供了大量证据反驳这一预测。首先，我们证明了细胞周期蛋白A与体外细菌表达的E2F-3B蛋白结合。其次，我们证明了细胞周期蛋白A与体内哺乳动物细胞中的E2F-3B结合。第三，我们表明细胞周期蛋白A与E2F-3B共表达显著降低了E2F-3B介导的转录活性。最后，在同步化细胞中，我们观察到E2F-3B蛋白表达的下调与细胞周期蛋白A的上调同时发生。我们得出结论，E2F-3B是细胞周期蛋白A的一个生理靶点。
