p53 Sites Acetylated In Vitro by PCAF and p300 Are Acetylated In Vivo in Response to DNA Damage

Abstract

The p53 tumor suppressor protein is a sequence-specific transcription factor that modulates the response of cells to DNA damage. Recent studies suggest that full transcriptional activity of p53 requires the coactivators CREB binding protein (CBP)/p300 and PCAF. These coactivators interact with each other, and both possess intrinsic histone acetyltransferase activity. Furthermore, p300 acetylates p53 to activate its sequence-specific DNA binding activity in vitro. In this study, we demonstrate that PCAF also acetylates p53 in vitro at a lysine residue distinct from that acetylated by p300 and thereby increases p53’s ability to bind to its cognate DNA site. We have generated antibodies to acetylated p53 peptides at either of the two lysine residues that are targeted by PCAF or p300 and have demonstrated that these antibodies are highly specific for both acetylation and the particular site. Using these antibodies, we detect acetylation of these sites in vivo, and interestingly, acetylation at both sites increases in response to DNA-damaging agents. These data indicate that site-specific acetylation of p53 increases under physiological conditions that activate p53 and identify CBP/p300 and PCAF as the probable enzymes that modify p53 in vivo.

ACKNOWLEDGMENTS

We gratefully acknowledge the gifts of GST-PCAF and GST-p300 protein and expression plasmids from G. Blobel and E. Verdin, respectively. We thank H.-X. Zhang for assistance preparing GST-p53 fusion proteins, G. Simon for help using the p53 mutation database, and G. Moore, B. Lu, and members of the Berger lab for valuable discussions.

D.M.S. was supported by National Institutes of Health training grant to the Wistar Institute; R.C.T. was supported by a Howard Hughes Medical Institute predoctoral fellowship. The research was supported by grants from the National Cancer Institutes and the American Cancer Society (to T.D.H.) and from the National Institutes of General Medical Sciences, the National Science Foundation, the Council for Tobacco Research and the American Cancer Society (to S.L.B.).