MOLECULAR-CLONING AND CHARACTERIZATION OF A CELLULAR PHOSPHOPROTEIN THAT INTERACTS WITH A CONSERVED C-TERMINAL DOMAIN OF ADENOVIRUS E1A INVOLVED IN NEGATIVE MODULATION OF ONCOGENIC TRANSFORMATION
MOLECULAR-CLONING AND CHARACTERIZATION OF A CELLULAR PHOSPHOPROTEIN THAT INTERACTS WITH A CONSERVED C-TERMINAL DOMAIN OF ADENOVIRUS E1A INVOLVED IN NEGATIVE MODULATION OF ONCOGENIC TRANSFORMATION
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DOI：
10.1073/pnas.92.23.10467
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发表时间：
1995-11-07
影响因子：
11.1
通讯作者：
CHINADURAI, G
中科院分区：
文献类型：
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作者：
SCHAEPER, U;BOYD, JM;CHINADURAI, G
腺病毒 2/5 型 EIA 蛋白与激活的 Ras (T24 ras) 癌蛋白合作转化原代幼鼠肾 (BRK) 细胞。 E1A 的 N 末端一半（外显子 1）对于这种转化活动至关重要。虽然 E1A 的 C 端一半（外显子 2）是可有可无的，但位于 243R E1A 蛋白残基 225 和 238 之间的区域负向调节体外 T24 ras 协同转化以及 E1A/T24 ras 转化细胞的致瘤潜力。细胞 48 kDa 磷蛋白、C 端结合蛋白 (CtBP) 的结合也需要相同的 C 端结构域。我们通过酵母双杂交相互作用克隆克隆了 CtBP 的 cDNA。该 cDNA 编码 439 个氨基酸 (48 kDa) 的蛋白质，该蛋白质在酵母双杂交、体外蛋白质结合和体内共免疫沉淀分析中与外显子 2 特异性相互作用。该蛋白需要 243B E1A 蛋白的残基 225-238 进行相互作用。分离的 cDNA 的预测蛋白质序列与从生化纯化的 CtBP 制备的肽获得的氨基酸序列相同。 CtBP 结合结构域的精细定位表明，这种相互作用需要在各种人类和动物腺病毒的 ELA 蛋白中高度保守的 6 个氨基酸基序。这些结果表明，CtBP 与 E1A 蛋白的相互作用可能在腺病毒复制和致癌转化中发挥关键作用。
The adenovirus type 2/5 EIA proteins transform primary baby rat kidney (BRK) cells in cooperation with the activated Ras (T24 ras) oncoprotein. The N-terminal half of E1A (exon 1) is essential for this transformation activity. While the C-terminal half of E1A (exon 2) is dispensable, a region located between residues 225 and 238 of the 243R E1A protein negatively modulates in vitro T24 ras cooperative transformation as well as the tumorigenic potential of E1A/T24 ras-transformed cells. The same C-terminal domain is also required for binding of a cellular 48-kDa phosphoprotein, C-terminal binding protein (CtBP). We have cloned the cDNA for CtBP via yeast two-hybrid interaction cloning. The cDNA encodes a 439-amino acid (48 kDa) protein that specifically interacts with exon 2 in yeast two-hybrid, in vitro protein binding, and in vivo coimmunoprecipitation analyses. This protein requires residues 225-238 of the 243B E1A protein for interaction. The predicted protein sequence of the isolated cDNA is identical to amino acid sequences obtained from peptides prepared from biochemically purified CtBP. Fine mapping of the CtBP-binding domain revealed that a 6-amino acid motif highly conserved among the ELA proteins of various human and animal adenoviruses is required for this interaction. These results suggest that interaction of CtBP with the E1A proteins may play a critical role in adenovirus replication and oncogenic transformation.