Recruitment of CBP/p300, TATA-binding protein, and S8 to distinct regions at the N terminus of adenovirus E1A

Abstract

The N-terminal region of the adenovirus (Ad) 12S E1A gene product targets several cellular proteins that are essential for the induction of S phase, cellular immortalization, cellular transformation, transcriptional repression, and transcriptional activation. The precise binding sites for these proteins, however, remain to be resolved. We therefore undertook an extensive site-directed mutagenesis approach to generate specific point mutants and to precisely map the binding sites for CBP, p300, TATA-binding protein (TBP), S4, S8, hGcn5, P/CAF, and Ran within the first 30 amino acids of the Ad5 12S E1A protein. We determined that although common residues within the N-terminal region can form partial binding sites for these proteins, point mutants were also generated that could discriminate between binding sites. These data indicate that AdE1A can target each of these proteins individually through distinct binding sites. It was evident, however, that the mutation of specific hydrophobic residues typically had the greatest effect upon AdE1A's ability to bind individual partners. Indeed, the mutation of L at positions 19 and 20 eliminated the ability of AdE1A to interact with any of the N-terminal binding proteins studied here. Interestingly, although TBP and S8 or CBP/p300 can exist as functional complexes, RNA interference revealed that the recruitment of either TBP, S8, or CBP/p300 to AdE1A was not dependent upon the expression of the other proteins. These data further indicate that AdE1A can target individual partner proteins in vivo and that it does not necessarily recruit these proteins indirectly as components of larger macromolecular complexes. Finally, we took advantage of the fine-mapping data to ascertain which proteins were targeted during the transformation process. Consistent with previous studies, CBP/p300 was found to be targeted by AdE1A during this process, although our data suggest that binding to other N-terminal proteins is also important for transformation.

FIG. 1.

(A) Comparative sequence alignment at the N terminus of AdE1A. The N termini of AdE1A proteins from the indicated human and simian serotypes were aligned with CLUSTAL W and imported into GeneDoc for shading of conserved residues with the BLOSUM 35 matrix. Other than the initiating M residue, only R2 and L20 (in Ad2/5) within the first 30 amino acids are absolutely conserved among serotypes. (B) Amino acid sequence of the Ad2/5 243-residue E1A protein. Boxed areas define conserved regions within the 12S gene product as well as the 30 amino acids at the N terminus under investigation in this study.

FIG. 2.

In vitro binding capacities of N-terminal AdE1A mutants for pRb and CtBP1. Ten micrograms of the appropriate GST-AdE1A fusion protein was incubated with 5 mg of A549 cell lysate. AdE1A-interacting proteins were precipitated with glutathione-Sepharose and selectively eluted with glutathione (see Materials and Methods). Proteins were subsequently separated by SDS-PAGE and transferred to nitrocellulose. The membranes were then Western blotted for pRb and CtBP1 to assess the binding capacities of these proteins for different N-terminal AdE1A mutants.

FIG. 3.

In vitro binding capacities of N-terminal AdE1A mutants for the acetyltransferases CBP, p300, hGcn5, and P/CAF. Ten micrograms of the appropriate GST-AdE1A fusion protein was incubated with 20 μl of l-[α-³⁵S]methionine-labeled CBP, p300, hGcn5, or P/CAF. Bound proteins were precipitated with glutathione-Sepharose and selectively eluted with glutathione (see Materials and Methods). Proteins were subsequently separated by SDS-PAGE and subjected to fluorography (Amersham Pharmacia). The gels were dried and then subjected to autoradiography. (A) Ability of CBP and p300 to bind N-terminal AdE1A mutants. (B) Ability of AdE1A mutants to bind hGcn5 and P/CAF.

FIG. 4.

In vitro binding capacities of N-terminal AdE1A mutants for TBP, S8, and Ran. Ten micrograms of the appropriate GST-AdE1A fusion protein was incubated with 5 mg of A549 cell lysate. AdE1A-interacting proteins were precipitated with glutathione-Sepharose and selectively eluted with glutathione (see Materials and Methods). Proteins were subsequently separated by SDS-PAGE and transferred to nitrocellulose. The membranes were then Western blotted for TBP and S8 (A) or Ran (B) to assess their relative affinities for different N-terminal AdE1A mutants. *, nonspecific band detected with the S8 Ab.

FIG. 5.

Binding of CBP/p300, TBP, and S8 to AdE1A N-terminal mutants in vivo. A549-derived cell lines stably expressing AdE1A mutants to similar levels (see Materials and Methods) were isolated in order to establish the binding capacity of each mutant for N-terminal binding proteins in vivo. Five micrograms of a CBP/p300 PAb, 10 μgof a TBP PAb, and 5 μg of an S8 PAb were incubated with 5 mg of the appropriate cell lysate and collected with protein G-Sepharose. Washed immunocomplexes were mixed with a sample buffer lacking SDS, separated in urea gels (see Materials and Methods), and subsequently transferred to nitrocellulose. The membranes were Western blotted for E1A by use of the M73 MAb. The abilities of CBP/p300 (A and D), TBP (B and D), and S8 (C and D) to bind specific AdE1A mutants are shown. *, nonspecific bands. WCE, whole-cell extract; I.P., immunoprecipitate.

FIG. 6.

(A) Secondary structure predictions for the N-terminal region of the Ad2/5 E1A protein by the Jpred consensus method (9). The Jpred program can be found at http://www.compbio.dundee.ac.uk/∼www-jpred/. The proposed structure for the first 30 amino acids of wt Ad5 12S E1A is compared with those of the N-terminal mutants used for this study. H, proposed α-helix; E, proposed β-pleated sheet; −, proposed random coil. For wt Ad2/5 E1A, a proposed β-pleated sheet extends from H3 to C6, and a proposed α-helix extends from I11 to A29. (B) Helical wheel depicting the spatial arrangement of amino acids (I11 to A29) comprising the proposed α-helix at the N terminus of wt Ad5 E1A. Residues are represented with shaded circles as follows: white, polar uncharged residues; light gray, nonpolar residues; dark gray, acidic residues. I11, top of the helix; A29, bottom of the helix.

FIG. 7.

Transforming potentials of N-terminal AdE1A mutants. HLREFs were transfected by electroporation with wt AdE1A or N-terminal AdE1A mutants in the presence of activated N-ras (see Materials and Methods). At 2 weeks posttransfection, bona fide AdE1A/ras-transformed colonies were counted by low-power microscopy. The data presented represent the averages of four independent experiments ± standard deviations. The transforming potential of each mutant is expressed relative to the mean ability of wt 12S AdE1A to transform HLREFs in combination with activated N-ras.

FIG. 8.

(A to E) N-terminal region of AdE1A independently targets TBP and S8 in vivo. (A and B) A549 cells were transfected with a siRNA targeted against either TBP or S8. Subsequent GST pull-down assays revealed that TBP and S8 can bind independently to AdE1A in vitro. (C to E) A549 cells were similarly transfected with a siRNA targeted against either TBP or S8. Anti-S8 and anti-TBP immunocomplexes were subsequently precipitated with protein G-Sepharose, separated by PAGE in the presence of urea, and transferred to nitrocellulose (see Materials and Methods). The membranes were then probed for AdE1A by use of the M73 MAb. The data presented indicate that both TBP (C) and S8 (D) associate independently with AdE1A in vivo. Whole-cell extracts separated by SDS-PAGE and transferred to nitrocellulose were probed for their levels of TBP, S8, and p53 (E) to gauge the efficiency of RNAi. (F to H) The N-terminal region of AdE1A targets CBP/p300 in vivo independent of TBP and S8. Anti-CBP/p300 immunocomplexes were precipitated with protein G-Sepharose, subsequently separated by PAGE in the presence of urea, and transferred to nitrocellulose (see Materials and Methods). The membranes were then probed for AdE1A by use of the M73 MAb. The data presented indicate that CBP/p300 can associate, independently of either S8 (F) or TBP (G), with AdE1A in vivo. Whole-cell extracts separated by SDS-PAGE and transferred to nitrocellulose were probed for their levels of CBP, p300, TBP, and S8 (H) to gauge the efficiency of RNAi. WCE, whole-cell extract; nonsil., nonsilencing RNA oligonucleotides. S8i and TBPi refer to situations in which S8 or TBP expression has been abolished by RNAi.