A REGION IN THE C-TERMINUS OF ADENOVIRUS-2/5 E1A PROTEIN IS REQUIRED FOR ASSOCIATION WITH A CELLULAR PHOSPHOPROTEIN AND IMPORTANT FOR THE NEGATIVE MODULATION OF T24-RAS MEDIATED TRANSFORMATION, TUMORIGENESIS AND METASTASIS
A REGION IN THE C-TERMINUS OF ADENOVIRUS-2/5 E1A PROTEIN IS REQUIRED FOR ASSOCIATION WITH A CELLULAR PHOSPHOPROTEIN AND IMPORTANT FOR THE NEGATIVE MODULATION OF T24-RAS MEDIATED TRANSFORMATION, TUMORIGENESIS AND METASTASIS
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DOI：
10.1002/j.1460-2075.1993.tb05679.x
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发表时间：
1993-02-01
期刊：
影响因子：
11.4
通讯作者：
CHINNADURAI, G
中科院分区：
文献类型：
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作者：
BOYD, JM;SUBRAMANIAN, T;CHINNADURAI, G
我们研究了腺病毒2/5 E1A癌基因产物243R蛋白外显子2内的一系列小缺失突变，用于永生化、ras协同转化、肿瘤发生和转移。与野生型243R相比，位于残基193和残基243之间的多种缺失突变体更有效地与ras合作，诱导出致瘤性和转移性黏附较少的细胞的大转化灶。然而，在携带残基225-238缺失的突变体中观察到最大的转化增强（与缺失c端67个氨基酸相比）。这个突变体在永生方面也有更多的缺陷。这些结果表明，这14个氨基酸区域可能包含一个重要的功能，在永生化和肿瘤发生和转移的负调节。为了鉴定可能与E1A外显子2编码区（c端一半）相关并调节其转化潜力的细胞蛋白，我们构建了一个编码E1A c端68个氨基酸的嵌合基因，将其融合到细菌谷胱甘肽- s转移酶（GST）中。该融合蛋白用于纯化与E1a c端结合的细胞蛋白。发现一个48 kDa的细胞蛋白双链（指定CtBP）特异性结合GST-E1a c端融合蛋白以及细菌表达的全长E1a （243R）蛋白。它也与E1a特异性共免疫沉淀。对一组GST-E1a c端突变蛋白的分析表明，E1a和CtBP的关联需要残基225-238，这表明CtBP的关联与外显子2的永生化和转化调节活性之间存在相关性。CtBP是一种磷酸化蛋白，CtBP的磷酸化水平似乎在细胞周期中受到调节，这表明它可能在细胞增殖过程中发挥重要作用。
We have examined a series of small deletion mutants within exon 2 of the adenovirus 2/5 E1A oncogene product, the 243R protein, for immortalization, ras cooperative transformation, tumorigenesis and metastasis. Compared with wild-type 243R, various deletion mutants located between residues 193 and 243 cooperated more efficiently with ras to induce large transformed foci of less adherent cells that were tumorigenic and metastatic. However, the greatest enhancement of transformation (comparable to that obtained with a deletion of the C-terminal 67 amino acids) was observed with a mutant carrying a deletion of residues 225-238. This mutant was also more defective in immortalization. These results suggest that this 14 amino acid region may contain a function that is important for immortalization and negative modulation of tumorigenesis and metastasis. To identify cellular proteins that may associate with the exon 2-coded region of E1A (C-terminal half) and modulate its transformation potential, we constructed a chimeric gene coding for the C-terminal 68 amino acids of E1a fused to bacterial glutathione-S-transferase (GST). This fusion protein was used to purify cellular proteins that bind to the C-terminal region of E1a. A 48 kDa cellular protein doublet (designated CtBP) was found to bind specifically to the GST-E1a C-terminal fusion protein as well as to bacterially expressed full-length E1a (243R) protein. It also co-immunoprecipitated specifically with E1a. Analysis of a panel of GST-E1a C-terminal mutant proteins indicates that residues 225-238 are required for the association of E1a and CtBP, suggesting a correlation between the association of CtBP and the immortalization and transformation modulating activities of exon 2. CtBP is a phosphoprotein and the level of phosphorylation of CtBP appears to be regulated during the cell cycle, suggesting that it may play an important role during cellular proliferation.