Key to its function as a tumor suppressor, p53 regulates the expression of a specific set of genes involved in cell growth control. p53 activates transcription of genes containing specific DNA-binding sites for p53. Under normal physiological conditions, p53 exists in low abundance, apparently in an inactive, latent state with low sequence-specific DNA-binding activity (29
). Multiple types of modifications occur on p53 in response to DNA damage or other genotoxic stresses. Covalent modifications of p53, such as phosphorylation by kinases, dephosphorylation by phosphatases, and acetylation by acetylases, lead to its stabilization (53
), specific interaction with other regulatory proteins such as 14-3-3 (64
), and enhanced DNA-binding activity (17
). Consequently, any interference with covalent modifications of p53 could compromise its functions. The results presented here show that the E1B 55-kDa protein specifically inhibits p53 acetylation by PCAF both in vitro and in vivo (Fig. and ). Since acetylation of p53 has been shown to enhance its sequence-specific DNA-binding activity and also to be induced in response to DNA damage (17
), the E1B oncoprotein represses p53 functions at least in part by inhibiting its acetylation. Indeed, we have found that the DNA-binding activity of p53 in cells expressing E1B 55-kDa protein is greatly reduced (Fig. ).
How does E1B specifically inhibit acetylation of p53 by PCAF? We found that E1B interacts with both p53 and PCAF (Fig. to ). In addition, p53 also binds to PCAF (Fig. ). Interestingly, E1B can efficiently prevent the interaction between p53 and PCAF (Fig. ), suggesting that E1B may inhibit the acetylation of p53 by PCAF by blocking the enzyme-substrate interaction. Consistent with this, E1B does not affect the acetylation of histones by PCAF (Fig. ). Our data indicate that Ad2 E1B fragment aa 1 to 437 is able to prevent p53-PCAF interaction (Fig. ). Since this E1B fragment binds to p53 but not to PCAF (Fig. and ), binding of E1B to p53 may be sufficient for E1B to interfere with the p53-PCAF interaction. Nonetheless, E1B 55-kDa protein also binds to PCAF in vitro and in vivo (Fig. and ). Thus, by association with both p53 and PCAF, E1B protein might shift equilibrium of protein-protein complexes from p53-PCAF to p53-E1B and PCAF-E1B, thereby effectively sequestering p53 and PCAF and preventing PCAF from acetylating p53. It appears that PCAF and p53 bind to different sites in E1B (Fig. and ). This finding might permit an assessment of the relative importance of p53-E1B and PCAF-E1B interactions in inhibiting acetylation of p53 through extensive mutagenesis of the p53- and PCAF-binding sites in the E1B 55-kDa protein.
While the role of E1B-PCAF interaction in inhibiting acetylation of p53 is not clear at present, blocking the catalytic core of PCAF (the HAT domain) by E1B is unlikely to be the cause of inhibition; rather, binding of E1B to PCAF might induce conformational change of PCAF so that its substrate specificity is altered. We have found that a mutant PCAF lacking aa 62 to 464 can acetylate both p53 and histones because it still contains the HAT domain (44
). Interestingly, acetylation of p53 but not that of histones by this mutant PCAF is also inhibited by E1B (data not shown). Thus, binding of E1B to a domain in the C-terminal portion of PCAF may play a role in the observed inhibition of p53 acetylation, as this mutant PCAF lacks most of the sequence in its N-terminal region. Future study will address which domain of PCAF is responsible for binding to E1B 55-kDa protein.
The acetylation of p53 by PCAF is severely impaired in cells expressing E1B 55-kDa protein (Fig. ). Accumulation of acetylated p53 occurs normally in G401 cells upon treatment with the specific deacetylase inhibitor TSA (Fig. B). By contrast, acetylated p53 is virtually undetectable under the same conditions in cell line G401-CC3, derived from G401 cells by stable transfection with an Ad12 E1B 55-kDa protein-expressing vector (55
). The acetylation of p53 in 293 cells is also suppressed. While 293 cells express both E1A and E1B proteins, G401-CC3 cells express only E1B 55-kDa protein. Therefore, E1B 55-kDa protein is most likely to be responsible for the observed inhibition of p53 acetylation by PCAF, consistent with the in vitro studies (Fig. ). Effects of E1A oncoproteins on the activities of PCAF, p300, and CBP have been reported previously (1
). While E1A was found to bind to PCAF (44
), it does not appear to affect the HAT activity of PCAF. Instead, E1A protein was found to stimulate HAT activities of p300 and CBP under certain circumstances (1
). Conversely, recent studies demonstrated that the E1A oncoproteins may repress HAT activity of both PCAF and p300 in vitro (5
). Regardless of the potential role of E1A proteins in regulation acetylation, our results clearly indicate that E1B 55-kDa protein inhibits acetylation of p53 by PCAF both in vivo and in vitro.
Whereas acetylation of p53 enhances its sequence-specific DNA-binding activity (17
), a number of other modifications of the p53 C-terminal domain can also activate its DNA-binding function (20
). We found that anti-p53 antibody PAb421 can effectively enhance p53 DNA binding in G401-CC3 nuclear extracts (Fig. ), despite reduced p53 acetylation in this cell line (Fig. ). Thus, it is conceivable that binding of PAb421 to the C-terminal domain of p53 may have a similar effect on activating p53 DNA-binding activity as acetylation of the lysine residues within the p53 C-terminal domain. Interestingly, we found that PAb421 cannot activate p53 DNA binding in 293 nuclear extracts (Fig. ), suggesting that E1A may have additional inhibitory effects on p53 DNA-binding activity. Indeed, p53 appears to form high-molecular-weight oligomers when E1A proteins are expressed in G401 cells (54
). Such modified p53 protein may be less competent for DNA binding, as its transcriptional transactivation function was greatly repressed by E1A (54
The E1B 55-kDa protein represses transcriptional transactivation by p53 by binding directly to DNA-bound p53 without destabilizing p53-DNA complexes, thereby tethering the E1B transcriptional repression domain to promoters containing p53 binding sites (71
). Furthermore, E1B 55-kDa protein appears to enhance p53-DNA interaction and could repress transactivation mediated by p53 in an in vitro assay using purified RNA polymerase II components (39
). We show here that E1B 55-kDa protein inhibits effectively p53 acetylation. Inhibition of p53 acetylation (thus reducing its affinity to its binding sites [Fig. ]) and direct targeting of DNA-bound p53 might reflect two levels of repression of p53 functions by E1B. At the first level, E1B represses acetylation of p53 by PCAF, and possibly also by p300 and CBP. At the second level, E1B could still target any p53 that binds to specific sites within target promoters due to incomplete inhibition of acetylation or other means, thereby resulting in direct transcriptional repression. The two-level, fail-safe repression mechanisms on p53 suggest that E1B 55-kDa oncoprotein is a particularly powerful repressor of p53. Whether E1B also inhibits other types of covalent modifications occurring on p53 remains to be established. Our unpublished data indicated that phosphorylation of p53 at serine 392 was not affected by E1B 55-kDa protein in a Western blot analysis using a specific antibody against p53 phosphorylated at serine 392.
The Ad E1A and E1B proteins play important roles in cell transformation (3
). The E1A oncoproteins stimulate cell proliferation by binding to pRB, p300, and CBP (reviewed in reference 11
). The E1B 55-kDa protein acts in cell transformation by inactivating the p53 pathway. Although inactivation of pRB and p53 pathways is essential to the transformation of a normal cell into a tumor cell (18
), deregulation of other cellular regulatory circuitry may also be involved in cell transformation and development of cancer. As PCAF is implicated in regulation of cell differentiation, cell cycle progression, and transcriptional regulation (43
), deregulation of the PCAF pathway might also play a role in cell transformation. Intriguingly, just as E1A and E1B 55-kDa proteins inhibit p53 transactivation function, the same viral oncoproteins bind to PCAF, although it is likely that E1A and E1B affect different aspects of the PCAF functions. Furthermore, E1A appears to compete with PCAF for access to p300 (68
), which could affect critical functions of PCAF in regulating cellular pathways. Additionally, the Tax oncoprotein of the human T-cell leukemia virus type 1 recruits PCAF for transactivating viral promoters (23
). Such exploitation of PCAF by viral oncoproteins might perturb cellular physiology, thus contributing to cell transformation.
Increasing evidence supports critical roles for protein acetylation in cellular physiology, including regulation of protein-DNA and protein-protein interaction, as well as protein stability. Like phosphorylation, acetylation can regulate key cellular processes in response to extracellular signals. Thus, it has been proposed previously that acetylation as a biologically relevant modification may be as important as phosphorylation (26
). Consistent with this view, our data demonstrate that the Ad E1B 55-kDa oncoproteins specifically inhibit p53 acetylation by PCAF, while its histone acetylation and autoacetylation activities were not affected. These data suggest that the inhibition of p53 acetylation by viral proteins may represent an important mechanism of p53 inactivation. Future investigation on how E1B 55-kDa oncoprotein affects PCAF functions will provide insight into the biological functions mediated by PCAF, as well as how modulation of cellular acetylase activities by viral oncoproteins contributes to cell transformation and oncogenesis.