In this study we have identified a novel function for the acetyltransferase activity of the Gcn5 family member human PCAF. In addition to utilizing histones as a substrate, PCAF acetylates the transcription factor and tumor suppressor p53. The acetylation site in p53 is highly specific and, importantly, is distinct from the previously characterized sites acetylated by p300. The consequence of the acetylation by PCAF is greatly stimulated DNA binding by p53 in vitro. Most significant is that this study is the first to demonstrate that target lysines within a nonhistone substrate exhibit increased acetylation under physiological conditions that stimulate function. In particular, under in vivo conditions that cause DNA damage, which potentiate p53 activity, increased p53 acetylation is detected at lysines acetylated in vitro by PCAF and p300.
Previous studies have shown that CBP/p300 and PCAF form a coactivator complex to facilitate gene transcription by specific transcriptional activators (32
). In particular, we have shown that CBP and PCAF function synergistically to activate p53-dependent transcription of the endogenous p21/waf1/cip1
). The finding that CBP and PCAF both acetylate histones in vitro has raised the question of why multiple acetyltransferases function in complexes to potentiate transcription of target genes. One possibility, suggested by the fact that each coactivator has distinct histone target specificity (27
), is that they acetylate distinct sites within nucleosomes to achieve a synergistic effect on chromatin remodeling, as previously suggested (45
). Our results show that PCAF and CBP have clearly distinct specificities for acetylation of p53 and, hence, the potential for synergistic activation of p53 as well. Thus, at one level, CBP and PCAF acetylation of p53 enhances its binding affinity for promoters of target genes. At a second level, once bound to target genes, p53 recruitment of the CBP-PCAF coactivator complex acetylates nucleosomal histones, thereby promoting access to DNA of RNA polymerase II and other basal transcription factors. Another possible explanation for distinct acetylation by the different enzymes is that it allows p53 to respond to different activating signals.
We have compared the primary sequence of the PCAF-dependent acetylation site at K320 in p53 to the previously-determined yeast Gcn5-dependent acetylation sites in core histones H3 and H4 (32a). Interestingly, there is no apparent similarity between the sequences flanking K320 and the consensus histone acetylation site, suggesting great flexibility in the interaction of different acetylation substrates with the catalytic domain of the Gcn5 family. Structural studies of acetylation domains complexed with different substrates may reveal the basis of this sequence flexibility in the sites of acetylation.
The effects of p300 (22) and PCAF acetylation (Fig. ) on p53 DNA binding in vitro are clear. In support of a similar effect in vivo, we observed a modest reduction in transcriptional activity of p53 in transfection assays, using the K320R single substitution or the K(319/320/321)R triple substitution. We also tested substitution at K373 and found that arginine at this position exhibited less than a 10% reduction in transcription activity, while alanine showed a decrease of approximately 65% lowering (52a
). In general, these effects are qualitatively similar to reported results on mutation of other sites of posttranslational modification of p53. For example, phosphorylation at S15, S315, and S392 increases after irradiation, but substitutions at these residues have either a partial effect or no effect on p53 function, respectively (18
). There are several explanations for these rather modest effects. First, p300 and PCAF are likely to both acetylate histones and p53 to activate p53-responsive genes, so mutation of the acetylation sites within p53 may lower p53-mediated activation only partially. Second, the amount of exogenous p53 introduced by transfection may exceed the regulatory capacity of cells. Third, as mentioned above, there is evidence of several or many functionally redundant posttranslational modifications within the regulatory region and the activation domains of p53. In further support of physiological significance, we have screened a database of p53 gene mutations found in human tumors and cell lines (maintained by the International Agency for Research on Cancer; www.iarc.fr/p53/homepage.htm
) to determine the frequency of substitution mutations in RD1 and RD2. Among the reported potential regulatory sites (serine 315 [56
], serines 376 and 378 [29
], lysines 373, 381, and 382 [22
], serine 389 [44
], and serine 392 [44
]), substitutions are found only at lysine 320.
Abundant evidence indicates that p53’s ability to bind to DNA is tightly linked to its physiological functions in tumor suppression (35
). p53 is a tetramer (25
) and is postulated to assume two dynamic states, a high-affinity state and a low-affinity state for DNA binding (24
). Activation of p53 is thought to require a conformational switch from the low-to-high affinity states. In addition, both C-terminal regulatory domains (RD1 and RD2) negatively regulate p53’s DNA-binding activity in vitro, perhaps by maintaining p53 in the low-affinity state (25
). These results raise the possibility that RD1 and RD2 are targeted by agents, such as DNA damage, that regulate p53 function in vivo. Indeed, ionizing radiation leads to a specific dephosphorylation at serine 376 within RD2, which in turn leads to association of p53 with 14-3-3 proteins and enhanced sequence-specific DNA-binding activity (29
). In addition, phosphorylation of serine 392 within RD2, which activates p53 DNA binding in vitro (26
), has been observed in response to UV irradiation in vivo (18
The findings reported here and similar results from other laboratories (1
) suggest that RD1 and RD2 are also targeted by acetyltransferases in vivo. Lysine 320 within RD1 and lysine 373 within RD2 become acetylated after exposure of cells to UV or ionizing radiation. In vitro, the acetylation site at K320 is targeted by PCAF and the acetylation at K373 is targeted by p300, and both p300 and PCAF increase the affinity of p53 to bind its cognate DNA site. This suggests that there are multiple and perhaps redundant pathways to increase p53’s capacity for DNA binding in response to DNA damage. It will be important to establish the conditions under which each of these pathways contributes to the p53 DNA damage response and whether these modifications can occur independently of each other or only in a specific sequence.