From the Institute for Molecular Virology, St. Louis University Health Sciences Center, St. Louis, Missouri 63110
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ABSTRACT |
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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.
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INTRODUCTION |
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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 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 complex between CtBP and CtIP is disrupted by E1A C-terminal peptides that contain the PLDLS motif.
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MATERIALS AND METHODS |
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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.
5' Rapid Amplification of cDNA Ends (RACE)2-- 5' 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 (5'-gtgtcagctagaat-3') using poly(A) RNA extracted from Raji cells as the template. Polymerase chain reactions were performed with CtIP-specific primers Cip-RACE1 (5'-ctggaataacgtcttcctcaca-3') or Cip-RACE2 (5'-ctgctgttccctcagctgttg-3') and anchor primers provided by the manufacturer (Life Technologies, Inc.). The polymerase chain reaction products were cloned into Bluescript KS+ (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 fmolTM sequencing system (Promega).
GST Fusion Proteins-- GST and GST fusion proteins were expressed in Escherichia coli BL21 (DE3) cells (Promega) transformed with pGEX-5X3, pGST-CtBP, pGST-CtIP, GST-Cter (1), or GST-Cter (dl1135) essentially as described (14, 15). GST fusion proteins were purified by affinity chromatography on glutathione-agarose beads and used for protein binding studies with 35S-labeled proteins (prepared by in vitro transcription/translation) as described (14, 15). In vivo coimmunoprecipitation analyses using the vaccinia virus/T7 RNA polymerase system (17) were carried out as described (15).
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 µl of factor Xa buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 2 mM CaCl2) 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 35S-labeled CtBP and added to GST-Cter and GST-CtIP immobilized on glutathione beads. ![]() |
RESULTS AND DISCUSSION |
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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 × 105 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 interaction with in vitro synthesized 35S-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.
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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
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.
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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 C-terminal 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.
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ACKNOWLEDGEMENT |
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We thank B. Elangovan for his help in data bank searches.
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FOOTNOTES |
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* 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. The 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 GenBankTM/EMBL Data Bank with accession number(s) U72066 (CtIP).
To whom correspondence should be addressed: Institute for
Molecular Virology, St. Louis University Health Sciences Center, 3681 Park Ave., St. Louis, MO 63110. Tel.: 314-577-8416; Fax: 314-577-8406;
E-mail: chinnag{at}slu.edu.
1 U. Schaeper and G. Chinnadurai, unpublished data.
2 The abbreviations used are: RACE, rapid amplification of cDNA ends; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).
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REFERENCES |
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