Interaction between a Cellular Protein That Binds to the C-terminal Region of Adenovirus E1A (CtBP) and a Novel Cellular Protein Is Disrupted by E1A through a Conserved PLDLS Motif*

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 rasoncogene. Exon 2 negatively modulates in vitrocooperative 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][2][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 of E1A with p300 modulates transcription appears to involve disruption of a complex of p300 with a cellular acetyl transferase, P/CAF, which regulates transcription by chromatin remodeling (4).
Like exon 1, exon 2 is also required for immortalization of primary cells (5,6) and cooperative transformation with E1B (7,8). Although exon 2 is not essential for cooperative transformation with the activated T24 ras oncogene, it significantly influences the extent of oncogenic transformation. Deletions within the C-terminal 67 amino acids of the E1A 243R protein enhance E1A/T24 ras cooperative transformation (5,9) and tumorigenesis of transformed cells in syngeneic and athymic rodent models (5). Importantly, exon 2 also plays a role in tumor metastasis. Expression of wt E1A efficiently suppresses the metastatic potential of tumor cells (10 -12). In contrast, cells expressing E1A proteins that lack the C-terminal 67 amino acids are highly metastatic (5,13). Thus, exon 2 appears to negatively modulate in vitro transformation, tumorigenesis, and metastasis. We have localized these activities of exon 2 within a 14-amino acid region (residues 225 to 238) near the C terminus of the 243R protein (14). These transformation-restraining activities of the C-terminal region of E1A correlate with the interaction of a 48-kDa cellular phosphoprotein termed CtBP (14). CtBP binds to E1A via a 5-amino acid motif, PLDLS (residues 233-237 of Ad2/5 243R protein). Amino acid substitution mutants within this motif of E1A abolish complex formation with CtBP (15) and relieve the oncogenesis-restraining activities. 1 These results suggest that interaction of CtBP with E1A results in restraining of oncogenesis. To understand the mechanism by which the C terminus of E1A mediates the oncogenesis-restraining activity in concert with CtBP, we undertook a search for cellular proteins that complex with CtBP. Here, we report the cloning and characterization of a 125-kDa protein, CtIP, that binds to CtBP via the PLDLS motif. The * This work was supported by Research Grant CA-33616 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM  complex between CtBP and CtIP is disrupted by E1A C-terminal peptides that contain the PLDLS motif.

MATERIALS AND METHODS
Yeast Two-hybrid Screening-This screening was carried out using the yeast strain Y153 essentially as described in Ref. 16. A commercially available GAL4 activation domain tagged HeLa cell cDNA library (CLONTECH) was used as the prey.
5Ј Rapid Amplification of cDNA Ends (RACE) 2 -5Ј Sequences of CtIP cDNA were isolated by RACE using a commercially available kit (Life Technologies, Inc.). First strand cDNA synthesis was carried out with the CtIP specific primer AS2 (5Ј-gtgtcagctagaat-3Ј) using poly(A) RNA extracted from Raji cells as the template. Polymerase chain reactions were performed with CtIP-specific primers Cip-RACE1 (5Ј-ctggaataacgtcttcctcaca-3Ј) or Cip-RACE2 (5Ј-ctgctgttccctcagctgttg-3Ј) and anchor primers provided by the manufacturer (Life Technologies, Inc.). The polymerase chain reaction products were cloned into Bluescript KSϩ (Stratagene) and sequenced.
DNA Sequence Analysis-Both strands of various cDNA clones were sequenced by the dideoxy chain termination method using Sequenase version 2.0 (United States Biochemical Corp.) or the fmol TM sequencing system (Promega).
For competition binding studies, soluble E1A peptides containing the C-terminal 67 residues of E1A 243R or dl1135 were prepared from immobilized GST-Cter and GST-dl1135 by cleavage with factor Xa. Beads containing 1 mg of protein were resuspended in 400 l of factor Xa buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 2 mM CaCl 2 ) containing 8 units of factor Xa (New England Biolabs) and incubated for 6 -12 h at room temperature. The supernatants (containing cleaved E1A peptides) were mixed with 35 S-labeled CtBP and added to GST-Cter and GST-CtIP immobilized on glutathione beads.

RESULTS AND DISCUSSION
Isolation of cDNA Clones for CtBP-interacting Proteins-To identify and clone cDNAs for human cellular proteins that interact with human CtBP, we employed yeast two-hybrid screening. The yeast reporter strain Y153 (16) was transformed with the bait plasmid, pGB-CtBP (expressing the entire coding sequence of CtBP fused in-frame with the GAL4 DNA binding domain) together with a GAL4 activation domain tagged HeLa cell cDNA library. Yeast transformants that were positive for activation of two reporter genes, HIS3 and E. coli lacZ, were identified from 2 ϫ 10 5 independent transformants. From these screenings, six different cDNA clones that interacted with CtBP were isolated. Upon further testing of these clones for interaction with a battery of heterologous baits in yeast, three clones (8, 9, and 15) were found to specifically interact only with pGB-CtBP and not with a number of heterologous protein baits (not shown). DNA sequence analysis revealed that clone 8 encoded the C terminus of a known protein, the 70-kDa subunit of the KU autoantigen involved in DNA repair (18). Clones 9 and 15 were found to contain overlapping sequences of a cDNA for a novel protein. This protein, designated the CtBP-interacting protein, CtIP (CtBP-interacting protein) is characterized here. To confirm that CtIP could directly bind with CtBP, we carried out in vitro binding experiments. The open reading frame encoded by clone 15 (827 amino acids) was expressed as the GST-fusion protein (GST-CtIP). Bacterially expressed and affinity purified GST-CtIP as well as GST control proteins were immobilized on glutathione-agarose beads and tested for inter-action with in vitro synthesized 35 S-labeled CtBP. Bound proteins were eluted in SDS sample buffer and analyzed by SDS-PAGE (Fig. 1). CtBP interacted specifically with GST-CtIP but not with GST, confirming that it could directly associate with CtIP in vitro.
The DNA sequences of clone 9 and clone 15, were identical except for the presence of 153 bp of additional 5Ј sequences in clone 9. A Northern blot analysis of mRNA from several human cell lines using the cDNA in clone 15 as the probe revealed a single mRNA species of 3.7 kilobases (not shown). The observed size of the mRNA appeared to be about 800 bp larger than the cDNA sequences present in clone 9. The predicted open reading frame of clone 9 and clone 15, established in relation to the coding sequence of the GAL4 activation domain, contained several stop codons at the 3Ј end, suggesting that both cDNA clones encoded the carboxyl terminus of CtIP. To isolate the additional 5Ј-coding sequences of CtIP, we carried out 5ЈRACE reactions that resulted in isolation of 480 bp of 5Ј cDNA sequences beyond those present in clone 9. These 5Ј sequences encoded an additional 31 amino acids in-frame with the open reading frame present in clone 9. The complete cDNA sequence consists of 3247 bp and is presented in Fig. 2. Since the sequences upstream of the ATG contain several stop codons, we believe that synthesis of CtIP is initiated from the ATG codon at position 300. Thus, the complete cDNA sequence of CtIP codes for a protein of 897 amino acids (Fig. 2).
Role of PLDLS Motif on CtBP/CtIP Interaction-Data bank searches revealed that CtIP does not share significant homology with known proteins. However, upon close examination, we found that CtIP contains a 5-amino acid motif, PLDLS. This motif is highly conserved in the E1A proteins of all human adenoviruses (Fig. 3A). We have previously shown that CtBP binds to Ad2/5 E1A via the PLDLS motif. To determine whether the PLDLS motif of CtIP is also essential for its interaction with CtBP, we constructed a 5-amino acid substitution mutant, CtIP⌬, (PLDLS 3 LASQC). GST-CtIP and GST-CtIP⌬ fusion proteins were tested for interaction with in vitro synthesized CtBP (Fig. 3B). Binding of CtBP to GST-CtIP⌬ was significantly reduced compared with binding with GST-CtIP, suggesting that the PLDLS sequence constitutes the CtBP binding motif of CtIP. To further substantiate the requirement of the PLDLS motif of CtIP for heterodimerization with CtBP, we also carried out in vivo coimmunoprecipitation and Western blot analysis (Fig. 3C). We coexpressed CtBP (tagged with the T7 epitope) and full-length CtIP or CtIP⌬ in BSC40 cells using the recombinant vaccinia virus/T7 expression system. Cell lysates were immunoprecipitated with either T7 mAb (Novagen) or polyclonal CtIP antiserum. Immunoprecipitation with the T7 antibody revealed that wt CtIP but not CtIP⌬, which lacks the CtBP binding motif, coprecipitated with CtBP. Immunoprecipitation with the CtIP antibody also revealed coprecipitation of CtBP with CtIP wt and not with CtIP⌬. These results indicate that full-length CtIP interacts with CtBP in vivo and that the PLDLS motif is required for this interaction.
E1A Competes with CtIP for CtBP Interaction-Since CtIP contains the same CtBP binding motif as E1A, we investigated whether E1A would compete with CtIP for CtBP interaction. We have shown earlier that a GST fusion containing the Cterminal 67 residues of E1A (GST-Cter) can readily bind with CtBP (14). We prepared soluble E1A peptides (C-terminal 67 residues) by proteolytic cleavage of immobilized GST-Cter fusion protein and utilized them in competition binding experiments. CtBP was expressed by in vitro transcription/translation and then analyzed for binding to immobilized GST-CtIP or GST-Cter in the presence or absence of E1A C-terminal peptide competitors. As demonstrated in Fig. 4, CtBP interacted well with GST-CtIP or the E1A fusion protein, GST-Cter. However, these interactions were significantly reduced in the presence of a 200-fold molar concentration of wt E1A Cter peptide, but not the peptide from E1A mutant dl1135, which lacks the CtBP binding region (14). These results demonstrate that CtIP and E1A, which carry identical CtBP binding motifs, can compete for CtBP interaction.
The molecular mechanism by which the C-terminal region of E1A exerts the oncogenesis-restraining activity is not known. However, mutational analysis indicates that this activity of E1A correlates with its ability to complex with CtBP (14,15). Although it is not clear how this interaction leads to negative modulation of oncogenesis, identification of CtIP points to a potential molecular pathway. By analogy with the regulatory activities of exon 1, which result in disruption of protein complexes that sequester transcription factors, E2F (1-3) and P/CAF (4), it appears that the C-terminal region of E1A may also disrupt the CtBP-CtIP complex resulting in the release of CtIP. Characterization of the biochemical activities of CtIP would be necessary to further elucidate the mechanism of the oncogenesis-restraining activity of E1A. Since the CtBP-binding motif of E1A has been implicated in certain transcriptional regulatory activity by exon 1 (19), it is possible that CtIP may The PLDLS and related motifs are boxed. B, in vitro binding of CtIP or CtIP⌬ with CtBP. Immobilized GST or GST-CtIP proteins were incubated with 35 S-labeled CtBP, and the bound products were analyzed by 10% SDS-PAGE. Mutant CtIP⌬ contains a 5-aa substitution (PLDLS 3 LASQC) between residues 490 and 494 of CtIP. C, coimmunoprecipitation of CtBP and CtIP. T7 epitope-tagged CtBP was coexpressed with CtIP or CtIP⌬ in BSC40 cells using recombinant vaccinia virus expression system. Twenty hours postinfection, cell lysates were prepared and proteins were precipitated with T7 mAb (Novagen) or CtIP polyclonal antibody (anti-CtIP). The precipitates were analyzed by 10% SDS-PAGE and transferred to a nitrocellulose filter. The blots were probed with T7 mAb or anti-CtIP and detected with the ECL detection system (Amersham). possess a transcriptional regulatory activity. However, a data bank search based on sequence properties (PROPSEARCH, Ref. 20) has revealed that CtIP may share some similarities with certain mammalian proteins involved in DNA repair. It could be envisioned that potential transcriptional regulatory and/or DNA repair activities of CtIP may play a role in the tumorigenesis-restraining activity of E1A. Our studies also suggest a potential pharmacological use for peptides containing the PLDLS motif in suppression of certain neoplastic diseases.