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C-Terminal-Binding Protein Interacting Protein Binds Directly to Adenovirus Early Region 1A Through Its N-Terminal Region and Conserved Region 3

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C-Terminal-Binding Protein Interacting Protein Binds Directly to Adenovirus Early Region 1A Through Its N-Terminal Region and Conserved Region 3 Oncogene (2007) 26, 7467–7479 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3 RK Bruton1, M Rasti1, KL Mapp1, N Young1, RZ Carter1, IA Abramowicz1, GG Sedgwick1, DF Onion1, M Shuen2, JS Mymryk2, AS Turnell1 andRJA Grand 1 1Cancer Research UK Institute for Cancer Studies, The Medical School, University of Birmingham, Birmingham, UK and 2Departments of Oncology and Microbiology and Immunology, University of Western Ontario, Ontario, Canada C-terminal-binding protein interacting protein (CtIP) was Introduction first isolated as a binding partner of C-terminal-binding protein (CtBP). It is considered to contribute to the Adenovirus early region 1A (AdE1A) is the first protein transcriptional repression and cell cycle regulatory to be expressedfollowing adenovirus infection andis properties of the retinoblastoma (Rb) family of proteins essential for adenovirus E1-mediated transformation and to have a role in the cellular response to DNA (reviewedby Gallimore andTurnell, 2001; Berk, 2005). damage. Here, we have shown that CtIP is a novel target Two major E1A proteins are translatedfrom 13S and for the adenovirus oncoprotein early region 1A (AdE1A). 12S messenger RNAs differing only by the presence of AdE1A associates with CtIP in both Ad5E1-transformed a short amino-acidsequence locatedtowardsthe C cells and Ad5-infected cells and binds directly in terminus of the larger molecule. Comparison of the glutathione-S-transferase pull-down assays. Two binding amino-acid sequences of AdE1As from different viral sites have been mapped on Ad5E1A – the N-terminal serotypes indicates the presence of four highly conserved a-helical region (residues 1–30) and conserved region regions (CRs) locatedthroughout the molecule (Avva- 3(CR3)– the transcriptional activation domain. CtIP kumov et al., 2002, 2004). CR3 co-incides with the region can bind AdE1A and CtBP independently, raising the unique to the 13S mRNA product. AdE1A exerts its possibility that ternary complexes exist in Ad-transformed influence on infectedandtransformedcells through a and -infected cells. Significantly, reduction of CtIP complex series of protein–protein interactions (Galli- expression with small interfering RNAs results in reduc- more andTurnell, 2001). Most of the bindingsites for tion of the ability of a Gal4 DNA-binding domain-CR3 the cellular targets of AdE1A correspond to either the construct to transactivate a Gal 4-responsive luciferase CRs or to the N-terminal a-helical region. For example, reporter and this effect is reversed by reduction of CtBP the retinoblastoma (Rb) family of proteins bindto CR1 expression. Therefore, in this model, CtIP acts as a andCR2 (Dyson et al., 1992), p300 andCREB-binding transcriptional co-activator of AdE1A when dissociated protein (CBP) interact with the N-terminal region and from CtBP, through the action of AdE1A. These data CR1 (Eckner et al., 1994; Arany et al., 1995), while CtBP are consistent with observations that CtIP expression interacts with CR4 (Boyd et al., 1993). The modular is induced by AdE1A during viral infection and that nature of AdE1A has facilitated the mapping of protein- reduction of CtIP expression with RNA interference can binding sites and linking of biological functions to retard virus replication. In addition, AdE1A causes particular interactions andtherefore to certain regions of disruption of the CtIP/Rb complex during viral infection E1A. Binding of the Rb family or CBP/p300 is necessary by its interaction with CtIP, possibly contributing to for AdE1A to promote S phase entry, but binding of transcriptional derepression. both sets of proteins is required for AdE1A-mediated Oncogene (2007) 26, 7467–7479; doi:10.1038/sj.onc.1210551; transformation (Egan et al., 1988; Jelmsa et al., 1989; publishedonline 4 June 2007 Howe et al., 1990; Wang et al., 1993). CR3 contains a zinc (Zn2 þ )-finger motif andis the site of interaction with Keywords: adenovirus E1A; C-terminal-binding protein a number of proteins involvedin transcriptional regula- interacting protein; CtIP; C-terminal-binding protein; tion (Culp et al., 1988; Geisberg et al., 1994, 1995; Rasti CtBP et al., 2006). These interactions with proteins such as TATA-binding protein (TBP), associated transcription factor (ATFs), suppressor of ras (Sur2) mediator 23 (MED23), proteasomal components andtrans-acting factors are necessary for transcriptional activation and Correspondence: Dr R Grand, Cancer Research UK Institute for for the expression of other viral early region proteins Cancer Studies, The Medical School, University of Birmingham, (reviewedby Jones, 1995; Avvakumov et al., 2004). Edgbaston, Birmingham, West Midlands, B15 2TT, UK. E-mail: [email protected] CR1, CR2 andCR3 are encodedby exon 1 of AdE1A Received15 June 2006; revised24 April 2007; accepted26 April 2007; andthis area of the protein is responsible for most of the publishedonline 4 June 2007 observedinteractions. However, the bindingsite for the CtIP binds directly to adenovirus early region 1A RK Bruton et al 7468 ubiquitous corepressor CtBP has been mappedto a CtBP, we investigatedthe relationship between CtIP and highly conservedPXDLS motif in CR4 in exon 2 (Boyd AdE1A and examined the possibility of direct interac- et al., 1993; Schaeper et al., 1995). Loss of binding of tion between the two proteins. When Ad5E1A was CtBP by AdE1A results in retarded viral replication immunoprecipitatedfrom Ad5E1-transformedcells (Grand et al., 2006). Significantly, the effect of binding (293) or from MCF7 cells infectedwith wt Ad5, CtIP of CtBP on AdE1A-mediated transformation is context was identified by western blotting as a co-precipitating dependent. Deletion of the C-terminal region of AdE1A protein (Figure 1a andc). In complementary experi- reduces the frequency of transformation by AdE1A ments, CtIP was immunoprecipitatedfrom Ad5E1- together with AdE1B, but increases the frequency of transformedandAd5-infectedcell lysates and transformation by AdE1A and activated ras (Douglas co-precipitating Ad5E1A was identified by Western et al., 1991; Subramanian et al., 1989, 1991). CtBP has blotting following electrophoresis on ‘urea gels’ in the been shown to interact with a large number of absence of sodium dodecylsulphate (SDS) (Figure 1b mammalian transcriptional repressors, such as BKLF, and d). The observation that Ad5E1A with a deletion Ikaros, Net andthe Drosophila repressors Snail, Hairy encompassing the CtBP-binding site (dl 1135) co- andKnirps as well as histone deacetylases (HDACs) immunoprecipitates with CtIP suggests that the inter- (reviewedby Turner andCrossley, 2001; Chinnadurai, action is not mediated through CtBP (Figure 1d). CtIP 2002, 2004, 2006a, 2007). andAd12E1A were also co-immunoprecipitatedfrom The first mammalian CtBP-binding protein to be Ad12E1-transformed cells (Figure 1e). It is also notable isolatedwas C-terminal-bindingprotein interacting pro- that CtIP bound to Ad126f10E1A, which does not tein (CtIP). Like AdE1A, CtIP interacts with CtBP via a interact with CtBP (Grand et al., 2006) (Figure 1e). PXDLS motif andE1A may compete with CtIP for (Rather less CtIP appearedto be co-immunoprecipi- interaction with CtBP (Schaeper et al., 1998). The role of tatedfrom the rat cells than the human, possibly dueto CtIP is not clear – it binds to the Rb family and it has been reduced expression or the inability of the antibody to suggestedthat this is responsible for some of the recognize rat CtIP). In a further set of experiments, transcriptional repression properties of Rb through [35S]-labelledCtIP or CtBP1 were incubatedwith recruitment of CtBP andassociatedHDACs (Meloni glutathione-S-transferase (GST)-Ad512S and 13SE1A et al., 1999). CtIP also regulates Rb-dependent cell cycle or GST-Ad5E1A exons 1 and 2 (Figure 2). CtIP bound arrest in G1 through the modulation of Rb phosphoryla- directly to full-length Ad5E1A and Ad5E1A exon 1 tion (Chen et al., 2005). CtIP binds to breast cancer (expressedas GST-fusions), whereas CtBP1 interacted associated1 (BRCA1) andis involvedin the cellular with exon 2, confirming that the AdE1A/CtIP interac- response to DNA double strand breaks, being phosphory- tion is not mediated through CtBP. latedby ataxia telangiectasia mutated(ATM) (Wong et al., 1998; Yu et al., 1998; Li et al., 1999, 2000; Wu-Baer Dissociation constant for CtIP and Ad12E1A andBaer, 2001; Yu andChen, 2004). Significantly, Using enzyme-linkedimmunosorbant assays (ELISAs), BRCA1 catalyses CtIP ubiquitination andco-localizes the apparent K for AdE1A binding to CtIP has been with CtIP in DNA damaged-induced foci (Yu et al., 2006). d comparedwith values obtainedfor CtBP1 andCtBP2. Although the precise role of CtIP has yet to be determined PurifiedAd1213SE1A was coatedonto an ELISA plate in detail the protein must be of considerable importance to andGST-CtBP1, GST-CtBP2, GST-CtIP amino acids the functioning of the normal cell since CtIP knock-out 324–897 andGST-CtIP amino acids371–620 were mice die at an early stage in development (E4) largely serially diluted across the plate. Bound protein was through cell cycle arrest in G (Chen et al., 2005). 1 determined using an anti-GST antibody (Figure 3). Because of its close relationship to the Rb family of Using the Origin 7.5 software package apparent K s proteins andCtBP, we have consideredthe possibility d were determined. They were found to be 6, 2, 5 and that CtIP itself might be a novel target for AdE1A. Here, 79 nM for CtBP1, CtBP2, CtIP (324–897) andCtIP we have shown that these proteins interact independently (371–620), respectively. Thus, Ad12E1A has a similar of CtBP or Rb. CtIP binds to the N-terminal region and affinity for CtIP (324–897) as for CtBP 1 and2, CR3 of AdE1A regulating the ability of a Gal4 DNA- although binding to the shorter CtIP polypeptide is binding domain (DBD)-CR3 construct to transactivate a appreciably weaker. Gal-4-responsive luciferase reporter. This is consistent with our observation that knock down of CtIP expres- sion can retardthe rate of viral replication.
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