Dynlacht B D, Flores O, Lees J A, Harlow E
Massachusetts General Hospital Cancer Center, Charlestown 02129.
Genes Dev. 1994 Aug 1;8(15):1772-86. doi: 10.1101/gad.8.15.1772.
The mammalian transcription factor E2F plays a critical role in the expression of genes required for cellular proliferation. To understand how E2F is regulated, we have developed a reconstituted in vitro transcription assay. Using this E2F-responsive assay, we can demonstrate that E2F-mediated transcription can be directly repressed by the tumor suppressor protein pRB. This inhibition is abolished by phosphorylation of pRB with either cyclin A/cdk2 or cyclin E/cdk2. However, these cyclin/kinase complexes exhibit differences in the ability to phosphorylate E2F. Only cyclin A/cdk2 can phosphorylate E2F effectively, and this phosphorylation abolishes its ability to bind DNA and mediate trans-activation. Thus, this in vitro transcriptional assay allows activation and inactivation of E2F transcription, and our findings demonstrate how transcriptional regulation of E2F can be linked to cell cycle-dependent activation of kinases.
哺乳动物转录因子E2F在细胞增殖所需基因的表达中起关键作用。为了解E2F是如何被调控的，我们开发了一种体外重组转录检测方法。利用这种E2F反应性检测方法，我们可以证明E2F介导的转录可被肿瘤抑制蛋白pRB直接抑制。用细胞周期蛋白A/cdk2或细胞周期蛋白E/cdk2对pRB进行磷酸化可消除这种抑制作用。然而，这些细胞周期蛋白/激酶复合物在磷酸化E2F的能力上存在差异。只有细胞周期蛋白A/cdk2能有效磷酸化E2F，这种磷酸化会消除其结合DNA和介导反式激活的能力。因此，这种体外转录检测方法可实现E2F转录的激活和失活，我们的研究结果证明了E2F的转录调控如何与细胞周期依赖性激酶激活相联系。
