Identification of a second CtBP binding site in adenovirus type 5 E1A conserved region 3

Abstract

C-terminal binding protein (CtBP) binds to adenovirus early region 1A (AdE1A) through a highly conserved PXDLS motif close to the C terminus. We now have demonstrated that CtBP1 also interacts directly with the transcriptional activation domain (conserved region 3 [CR3]) of adenovirus type 5 E1A (Ad5E1A) and requires the integrity of the entire CR3 region for optimal binding. The interaction appears to be at least partially mediated through a sequence ((161)RRNTGDP(167)) very similar to a recently characterized novel CtBP binding motif in ZNF217 as well as other regions of CR3. Using reporter assays, we further demonstrated that CtBP1 represses Ad5E1A CR3-dependent transcriptional activation. Ad5E1A also appears to be recruited to the E-cadherin promoter through its interaction with CtBP. Significantly, Ad5E1A, CtBP1, and ZNF217 form a stable complex which requires CR3 and the PLDLS motif. It has been shown that Ad513SE1A, containing the CR3 region, is able to overcome the transcriptional repressor activity of a ZNF217 polypeptide fragment in a GAL4 reporter assay through recruitment of CtBP1. These results suggest a hitherto-unsuspected complexity in the association of Ad5E1A with CtBP, with the interaction resulting in transcriptional activation by recruitment of CR3-bound factors to CtBP1-containing complexes.

FIG. 1.

The interaction of CtBP1 with Ad5E1A polypeptides. (a) Dimensions of the Ad5 polypeptides used in pull-down experiments. (b) [³⁵S]methionine-labeled CtBP1 was incubated with the GST-Ad5E1A fusion proteins and polypeptides shown (20 μg). Complexes were isolated with glutathione agarose and fractionated by SDS-PAGE. Radiolabeled proteins were identified by fluorography and autoradiography. Input represents 10% of that used in the experiment. A Coomassie blue-stained version of the gel is shown in the lower panel. (c) GST-Ad5E1A proteins or polypeptides as shown (20 μg) were incubated with HCT116 or HT29 cell lysates. Protein complexes were isolated using glutathione agarose and bound CtBP1 and CtBP2 identified by Western blotting. A Ponceau S-stained Western blot, showing the amount of fusion proteins used in the pull down, is shown in the lower panel. (d) [³⁵S]-methionine-labeled CtBP1 was incubated with the GST-Ad5E1A fusion polypeptides shown. Complexes were isolated with glutathione agarose and fractionated by SDS-PAGE. Input represents 5% of that used in the experiment. A Coomassie blue-stained version of the gel is shown in the lower panel. (e) GST-Ad5E1A polypeptides as shown were incubated with HT29 and HCT116 cell lysates as for panel c. Bound CtBPs were identified by Western blotting. (f) HeLa cells were transfected with FLAG-tagged CtBP1 and Myc-tagged GFP or Myc-tagged GFP-CR3. After 24 h, CtBP1 was immunoprecipitated (IP) and coprecipitating GFP or GFP-CR3 identified by Western blotting.

FIG. 2.

Binding of CtBP1 to AdE1A conserved region 3. (a) [³⁵S]methionine-labeled CtBP1 was incubated with the CR3 regions of AdE1As from different viral serotypes, expressed as GST fusion proteins (20 μg). Ad5, Ad9, Ad4, Ad3, Ad40, and Ad12 are representatives of the group C, D, E, B:1, F and A human adenoviruses, respectively (2, 3). Protein complexes were isolated using glutathione agarose and fractionated by SDS-PAGE. Radiolabeled proteins were identified by fluorography and autoradiography. A Coomassie blue-stained version of the gel is shown in the lower panel. (b) Comparison of the amino acid sequences of portions of CR3s from different adenovirus serotypes. The sequence of the novel CtBP binding site from ZNF217 is also shown. (c) GST-Ad5CR3 polypeptides (with various deletions) were incubated with [³⁵S]methionine-labeled CtBP1. Protein complexes were isolated using glutathione agarose, fractionated by SDS-PAGE, and visualized by fluorography and autoradiography. Input represents 10% of that used in the experiment. A Coomassie blue-stained version of the gel is shown in the lower panel.

FIG. 3.

Ad5CR3 does not bind to the N-terminal region of CtBP1 normally associated with PXDLS-containing peptide interaction. GST-Ad5E1ACR3 and GST-Ad5E1A exon 2 were incubated with [³⁵S]methionine-labeled CtBP1 in the presence of various concentrations of a PXDLS-containing peptide (REQTVPVDLSVKRPR) (as shown). Protein complexes were isolated using glutathione agarose and fractionated by SDS-PAGE. Radiolabeled proteins were identified by fluorography and autoradiography. A Coomassie blue-stained version of the gel is shown in the lower panel.

FIG. 4.

Reduction of available CtBP enhances transcriptional activity of the CR3 domain of Ad5E1A. (a) CtBP1 and CtBP2 expression was reduced in A549 cells using appropriate siRNAs (shown by Western blotting in the inset). Control cells were transfected with a nonspecific control siRNA. After 3 days, cells were transfected with pcDNA3-GAL4-DBD and a GAL4-responsive luciferase reporter or pcDNA3-GAL4 DBD-Ad5CR3 and a GAL4-responsive luciferase reporter. After 24 h, luciferase activity was measured. (b) U2OS cells were cotransfected with a GAL-luciferase reporter plasmid and either with a plasmid expressing GAL4-DBD or the indicated GAL4-DBD fusion to Ad5E1A 1 to 82 or CR3 together with a plasmid expressing Ad5E1A exon 2 or the exon 2 ΔPLDLS mutant. Cells were harvested 24 h after transfection, and luciferase assays were performed and normalized using β-galactosidase.

FIG. 5.

Ad5E1A, CtBP1, and ZNF217 form a ternary complex. HeLa cells were transfected with constructs encoding ZNF217 (HA tagged), CtBP1 (FLAG tagged), and Ad5E1A as shown. (a) Samples were immunoprecipitated with an antibody against CtBP (FLAG tag) and Western blotted for Ad5E1A and ZNF217. Levels of expression of ZNF217, E1A, and CtBP are shown. (b) Samples cotransfected with E1A and CtBP were immunoprecipitated for CtBP (FLAG tag) and Western blotted for Ad5E1A. Levels of E1A and CtBP are shown. (c) HeLa cells were cotransfected with E1A, CtBP (FLAG tag), and ZNF217 (HA tag). Lysates were immunoprecipitated for CtBP (FLAG tag) and Western blotted for ZNF217. Levels of CtBP, ZNF217, and E1A are shown.

FIG. 6.

Ad513SE1A relieves transcriptional repression by ZNF217. U2OS cells were transfected with pM-GAL4-DBD and a GAL4-responsive luciferase reporter or pM-GAL4-ZNF217 and a GAL4-responsive luciferase reporter. In addition, various Ad5E1A constructs were cotransfected. Luciferase activity was measured after 48 h. Transfections were normalized for efficiency of transfection using β-galactosidase. (a) Cotransfection of Ad512S and 13S E1A and the RTP substitution mutant. (b) Cotransfection of Ad513S E1A and a novel mutant with deletion of just the PLDLS motif from Ad513S E1A. “Vector” refers to cotransfection of the reporter, the indicated GAL4 fusion, and an empty plasmid. The level of expression of Ad5E1A and actin in the cells is shown in the insert panels.

FIG. 7.

Ad513SE1A and Ad512SE1A differentially influence the E-cadherin promoter. HeLa cells were cotransfected with an E-cadherin reporter plasmid and either empty vector or vectors expressing the indicated E1As, as well as a β-galactosidase plasmid for normalization. Twenty-four hours after transfection, luciferase assays were performed and the results were normalized.