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. 2000 Dec 19;97(26):14323-8.
doi: 10.1073/pnas.011283598.

Acetylation of adenovirus E1A regulates binding of the transcriptional corepressor CtBP

Q Zhang  1 , H Yao, N Vo, R H Goodman
Affiliations

Acetylation of adenovirus E1A regulates binding of the transcriptional corepressor CtBP

Q Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

Adenovirus E1A mediates its effects on cellular transformation and transcription by interacting with critical cellular proteins involved in cell growth and differentiation. The amino terminus of E1A binds to CBP/p300 and associated histone acetyltransferases such as P/CAF. The carboxyl terminus binds to the carboxyl-terminal binding protein (CtBP), which associates with histone deacetylases. We show that 12S E1A can be acetylated by p300 and P/CAF and map one of the acetylation sites to Lys-239. This Lys residue is adjacent to the consensus CtBP binding motif, PXDLS. Mutation of Lys-239 to Gln or Ala blocks CtBP binding in vitro and disrupts the E1A-CtBP interaction in vivo. Peptide competition assays demonstrated that the interaction of E1A with CtBP is also blocked by Lys-239 acetylation. Supporting a functional role for Lys-239 in CtBP binding, mutation of this residue to Ala decreases the ability of E1A to block cAMP-regulated enhancer (CRE)-binding protein (CREB)-stimulated gene expression. Finally, we demonstrate that Lys-239 is acetylated in cells by using an antibody directed against an acetyl-Lys-239 E1A peptide. CtBP interacts with a wide variety of other transcriptional repressors through the PXDLS motif, and, in many instances, this motif is followed by a Lys residue. We suggest that acetylation of this residue by histone acetyltransferases, and the consequent disruption of repressor complexes, might be a general mechanism for gene activation.

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Figures

Figure 1
Figure 1
E1A is acetylated by p300, P/CAF, and Gcn5. (a) Recombinant E1A was incubated with full-length mouse p300 expressed in baculovirus-infected SF9 cells or human P/CAF catalytic domain expressed in bacteria in the presence of 3H-AcCoA for 30 min at 30°C. The reaction was subjected to SDS/PAGE analysis and fluorography. Autoacetylation of p300 was shown at the top of the gel. (b) Comparison of E1A and histone acetylation by different HATs. Recombinant E1A or core histones purified from chicken blood were incubated with full-length mouse p300, yeast Gcn5 catalytic domain, or P/CAF catalytic domain in the presence of 3H-AcCoA for 30 min at 30°C. The reaction was subjected to SDS/PAGE analysis and fluorography.
Figure 2
Figure 2
p300 and P/CAF acetylate E1A at Lys-239. GST-E1A fusion proteins were generated containing Lys-239 mutated to Arg (K239R), Gln (K239Q), or Ala (K239A). Wild-type (WT) and mutated GST-E1A proteins were incubated with full-length mouse p300 (Left) or P/CAF catalytic domain (Right) in the presence of 3H-AcCoA for 30 min at 30°C. Note that the mutations at Lys-239 abrogated the acetylation by P/CAF completely but only decreased the acetylation by p300. Similar sequences around Lys-14 of histone H3 (H3) and Lys-239 of 12S E1A (E1A) are aligned, with identical residues shaded and the other residues of interest denoted by an asterisk.
Figure 3
Figure 3
Alignment of PXDLS motifs in other transcriptional repressors. d, Drosophila; x, Xenopus. Identical residues are shaded.
Figure 4
Figure 4
Effects of E1A Lys-239 mutations on E1A-CtBP binding. (a) E1A Lys-239 mutations abolish the interaction between E1A and CtBP in vitro. GST-E1A fusion proteins, both wild-type (WT) and mutated [Lys-239 mutated to Arg (K239R), Gln (K239Q), or Ala (K239A)], were coupled to glutathione-Sepharose beads, blocked with BSA, and then incubated with His-tagged human CtBP for 1 h at 4°C. Equal amounts of GST and GST-fusion proteins were used in each pull-down assay. The bound material was dissolved in sample buffer and subjected to SDS/PAGE and Western blotting using an antibody against the His-tag. (b) E1A Lys-239 mutations abolish the interaction between E1A and CtBP in vivo. Gal4 fusion proteins (0.2 μg) containing the carboxyl-terminal 67 amino acids of E1A (Gal4-E1ACter Left) or the full-length E1A (Gal4-E1A Right) wild-type (WT) and Lys-239 mutations (K239A, K239Q, K239R) were cotransfected with 0.2 μg of VP16-CtBP and 1 μg of 5XGal-E1b-luciferase as indicated, and the luciferase activity was measured. The basal luciferase activity from 5XGal-E1b-luciferase alone was subtracted from all of the samples. Data are presented as mean value ± SD (n = 3 for Gal4-E1ACter; n = 6 for Gal4-E1A). The differences between the wild-type and K239A/K239Q mutations were significant in both experiments (P < 0.01). The difference between wild-type and K239R was significant (P < 0.01) in the Gal4-E1A experiment.
Figure 5
Figure 5
Contribution of CtBP-binding to E1A-mediated transcription repression. Four micrograms of CRE-luciferase, 8 μg of pRcRSV-CREB, 10 μg of pRcRSV-CBP, 4 μg of PKA, and various amounts of E1A expression vectors as indicated were transfected into F9 cells. Equal amounts of proteins from each sample were used for luciferase assay. Data are presented as mean value ± SD (n = 3).
Figure 6
Figure 6
Effects of E1A acetylation on CtBP binding. (a) GST-E1ACter fusion proteins were treated with p300 or P/CAF in the presence or absence of 3H-AcCoA for 60 min at 37°C and coupled to glutathione-Sepharose beads. After washing, the beads were blocked with BSA and incubated with His-tagged human CtBP for 1 h at 4oC. The bound material was dissolved in sample buffer and subjected to SDS/PAGE and Western blotting using an antibody against the His-tag. The stoichiometry of GST-E1ACter acetylation is approximately 30% for P/CAF and 23% for p300. (b) Peptide competition assay. CtBP was immobilized onto 96-well plates. Serially diluted wild-type (EPGQPLDLSCKRPR), mutated (EPGQPLDLSCQRPR, EPGQPLDLSCARPR), or acetylated (EPGQPLDLSC(Ac)KRPR) E1A peptides were mixed with GST-E1ACter and added to each well. GST-E1ACter bound to CtBP on the plate was determined by an ELISA using antibody against GST. Results from wild-type and acetylated E1A peptides are plotted in the figure. Data were curve-fitted by the Michaelis–Menton equation. All curves have chi-square values less than 0.05 and R values larger than 0.96. IC50 values for each peptide are presented in the Inset.
Figure 7
Figure 7
Acetylation of E1A Lys-239 in vivo. (a) Specificity of the anti-acetyl-E1A antibody. Serially diluted unacetylated and P/CAF-acetylated E1A protein was analyzed by Western blotting using the anti-acetyl-E1A antibody (Upper) or anti-E1A antibody (Lower). Arrow indicates E1A protein. (b) In vivo acetylation of E1A at Lys-239. Wild-type (WT, 0.5 μg) or Lys-239-mutated pRcRSV-E1A were transfected into COS 7 cells, along with 0.5 μg of P/CAF expression vector. E1A proteins were immunoprecipitated from the cell lysate and analyzed by Western blotting using the anti-acetyl-E1A antibody (Upper) or anti-E1A antibody (Lower). All of the samples, except for the vector alone, contained similar levels of E1A protein. Only extracts from the cells expressing the wild-type E1A stained positively with the anti-acetyl-Lys-239 antibody. Arrow indicates E1A protein.
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