Tanimoto A, Chen H, Kao C Y, Moran E, Sasaguri Y, Padmanabhan R
Department of Biochemistry and Molecular Biology, the University of Kansas Medical Center, Kansas City 66160-7421, USA.
Oncogene. 1998 Dec 17;17(24):3103-14. doi: 10.1038/sj.onc.1202236.
Cyclin-dependent protein kinases (Cdks) are key regulatory proteins of the eukaryotic cell cycle. Cdc2 is expressed in late G1/S phase and functions in the G2 to M phase transition. Adenovirus E1A proteins are known to induce the expression of p34cdc2 and DNA synthesis in normal quiescent cells. In this study, mutational analysis of the human cdc2 promoter revealed that transactivation of the promoter by the E1A proteins in cycling cells is mediated through the two CCAAT box binding motifs. A 110-kDa protein (CBF/cdc2) was identified in nuclear extracts from monkey kidney (CV-1) cells stably expressing E1A as well as from adenovirus-transformed human 293 cells. Further, we show that this EIA-inducible CBF/cdc2 is related to the CBF which was shown to activate the heat shock protein 70 promoter. Analyses of the functional domain(s) of E1A required for the induction of the CBF and transactivation of the cdc2 promoter in these conditions revealed that E1A mutants which were defective in binding the pRB family of proteins or the cellular p300 protein were still active in assays measuring the induction of the CBF and transactivation of the cdc2 promoter, albeit with reduced efficiencies. But the E1A mutant which lost both functional domains was inactive in these assays. These results suggest that E1A has redundant functional domains for the induction of the 110-kDa CBF and activation of human cdc2 gene expression.
细胞周期蛋白依赖性蛋白激酶（Cdks）是真核细胞周期的关键调节蛋白。Cdc2在G1/S期晚期表达，并在G2期向M期的转变中发挥作用。已知腺病毒E1A蛋白可诱导正常静止细胞中p34cdc2的表达和DNA合成。在本研究中，对人cdc2启动子的突变分析表明，E1A蛋白在循环细胞中对启动子的反式激活是通过两个CCAAT盒结合基序介导的。在稳定表达E1A的猴肾（CV-1）细胞以及腺病毒转化的人293细胞的核提取物中鉴定出一种110 kDa的蛋白（CBF/cdc2）。此外，我们表明这种EIA诱导的CBF/cdc2与已证明可激活热休克蛋白70启动子的CBF相关。对在这些条件下诱导CBF和反式激活cdc2启动子所需的E1A功能域的分析表明，在结合pRB蛋白家族或细胞p300蛋白方面存在缺陷的E1A突变体，在测量CBF诱导和cdc2启动子反式激活的试验中仍然具有活性，尽管效率有所降低。但失去两个功能域的E1A突变体在这些试验中无活性。这些结果表明，E1A在诱导110 kDa CBF和激活人cdc2基因表达方面具有冗余的功能域。
