The adenovirus type 2/5 E1A 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 243R 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 E1A 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.

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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.

Adenovirus E1A proteins immortalize primary animal cells and cooperate with several other oncogenes in oncogenic transformation. These activities are primarily determined by the N-terminal half (exon 1) of E1A. Although the C-terminal half (exon 2) is also essential for some of these activities, it is dispensable for cooperative transformation with the activated T24 ras oncogene. Exon 2 negatively modulates in vitro cooperative transformation with T24 ras as well as the tumorigenic and metastatic potentials of transformed cells. A short C-terminal sequence of E1A governs the oncogenesis-restraining activity of exon 2. This region of E1A binds with a cellular phosphoprotein, CtBP, through a 5-amino acid motif, PLDLS, conserved among the E1A proteins of human adenoviruses. To understand the mechanism by which interaction between E1A and CtBP results in tumorigenesis-restraining activity, we searched for cellular proteins that complex with CtBP. Here, we report the cloning and characterization of a 125-kDa protein, CtIP, that binds with CtBP through the PLDLS motif. E1A exon 2 peptides that contain the PLDLS motif disrupt the CtBP-CtIP complex. Our results suggest that the tumorigenesis-restraining activity of E1A exon 2 may be related to the disruption of the CtBP-CtIP complex through the PLDLS motif.Small DNA tumor viruses such as human adenoviruses and papilloma viruses encode powerful transforming genes. The products of these viral oncogenes subvert host cell cycle control by binding to specific cellular proteins. Among the transforming genes of various DNA tumor viruses, the E1a gene region of human adenoviruses has been studied most extensively and serves as a prototypical oncogene. The E1a gene of human adenovirus types 2 and 5 encodes two major proteins of 289 and 243 amino acids (289R and 243R). Both proteins contain two exons and are identical except for the presence of an internal 46-amino acid region unique to the 289R protein. While the 289R protein is required for productive viral infection, the 243R protein encodes all the transforming functions. Exon 1 plays a dominant role in controlling the cell proliferation and transforming activities governed by the E1A proteins. Exon 1 controls these activities by modulating cellular gene expression through interaction with cellular growth-regulatory proteins such as the retinoblastoma gene product (pRb) and related proteins (p107 and p130) as well as p300, a CREB binding protein-related transcription factor (reviewed in Refs. 1-3). One of the functional domains of exon 1 encompasses two regions, conserved regions 1 and 2 (CR1 and CR2). These regions are responsible for interactions between E1A and the cellular proteins pRb, p107, and p130, which cause these cellular proteins to release the E2F transcription factor, thus activating gene expression. A second functional domain, encompassing CR1 and the N terminus of E1A, interacts with a transcriptional adapter p300 implicated in transcriptional repression of certain genes. One of the modes by which interaction ...

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