Jordan-Sciutto K L, Hall D J
Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Biochem Cell Biol. 1998;76(1):37-44.
The amino-terminal domain of the E2F1 transcription factor is the site of association with cyclin A-cdk2, mapping to residues 87-94. A mutant of E2F1 lacking the first 87 amino acids (termed E2F1d87) has a number of potent effects on cellular phenotype when constitutively expressed in NIH3T3 fibroblasts. For example, in these fibroblasts the duration 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 cyclin A-cdk2 association with this mutant to correlate any reduction in association with the observed effects on the cell cycle. It was found that cyclin A-cdk2 binds E2F1d87 in an in vitro assay but that this binding is reduced approximately 8 fold compared with binding to full-length E2F1, whereas no detectable binding was seen to a mutant E2F1 that lacks the first 117 amino acids. Correspondingly, H1 kinase activity in E2F1d87 immunoprecipitates from E2F1d87-expressing cells was significantly reduced compared with that seen for full-length E2F1. From these data it appears that E2F1 with reduced cyclin A-cdk2 binding activity mediates the alteration in cell cycle parameters seen in these cells.
E2F1转录因子的氨基末端结构域是与细胞周期蛋白A-cdk2结合的位点，定位于第87 - 94位氨基酸残基。在NIH3T3成纤维细胞中组成性表达时，缺失前87个氨基酸的E2F1突变体（称为E2F1d87）对细胞表型有许多显著影响。例如，在这些成纤维细胞中，S期持续时间和对S期化疗药物的敏感性均增加。由于E2F1d87仅部分截断了细胞周期蛋白A-cdk2结合结构域，因此确定细胞周期蛋白A-cdk2与该突变体的结合水平，以将结合的任何减少与观察到的对细胞周期的影响相关联，这一点很重要。研究发现，在体外试验中细胞周期蛋白A-cdk2能与E2F1d87结合，但与结合全长E2F1相比，这种结合减少了约8倍，而与缺失前117个氨基酸的E2F1突变体未检测到结合。相应地，与全长E2F1相比，从表达E2F1d87的细胞中免疫沉淀得到的E2F1d87的H1激酶活性显著降低。从这些数据看来，细胞周期蛋白A-cdk2结合活性降低的E2F1介导了这些细胞中观察到的细胞周期参数的改变。
