DNA damage activates p53 through a phosphorylation–acetylation cascade

Abstract

The p53 tumor suppressor is a critical component of cellular mechanisms that respond to certain stresses to preserve genomic integrity by arresting cell-cycle progression or by inducing apoptosis (Levine 1997; Agarwal et al. 1998). p53 normally is a short-lived protein that is maintained at low levels, but in response to DNA-damaging agents, nucleotide depletion, or hypoxia, the p53 protein is transiently stabilized and accumulates in the nucleus in which it functions in part to induce or repress the transcription of several genes including WAF1 (El-Deiry et al. 1993), SFN (Hermeking et al. 1997), and MDM2 (Juven et al. 1993; Perry et al. 1993) that regulate cell-cycle progression. The Mdm2 protein binds the amino terminus of p53 and functions in an autoregulatory loop that attenuates p53 activity by directly sequestering its transactivation domain (Oliner et al. 1993; Wu et al. 1993). Mdm2 also targets p53 for degradation and, therefore, may play a critical role in regulating its accumulation in response to DNA damage (Haupt et al. 1997; Kubbutat et al. 1997). The segment of p53 that interacts with Mdm2, which encompasses the region between Glu-17 and Pro-27, includes two potential phosphorylation sites, Thr-18 and Ser-20, and is surrounded by several other potential phosphorylation sites including Ser-9, Ser-15, Ser-33, and Ser-37 (Meek 1998). Phosphorylation of p53 by DNA–PK, a DNA-activated protein kinase that phosphorylates p53 at Ser-15 and Ser-37 in vitro (Lees-Miller et al. 1992), was reported to inhibit the interaction with Mdm2 (Shieh et al. 1997), as does phosphorylation of the amino terminus of Mdm2 by DNA–PK (Mayo et al. 1997). Phosphorylation of Ser-15 in vivo is stimulated by DNA-damaging agents including ionizing radiation (IR) and UV. These recent findings suggest that amino-terminal phosphorylation may be one of the signals regulating the p53 response to DNA damage (Shieh et al. 1997; Siliciano et al. 1997).

In addition to causing p53 to accumulate, DNA damage is widely believed to activate p53 as a transcription factor through post-translational mechanisms. In support of this hypothesis, microinjection of damaged DNA or a monoclonal antibody to the carboxyl terminus of p53 activated p53-specific transcription and induced p53-dependent cell-cycle arrest under conditions in which no increase in p53 protein was detected (Hupp and Lane 1995; Huang et al. 1996). Furthermore, in mouse fibroblasts, UV-C activated p53’s DNA-binding activity in G₀ and G₁ without causing p53 accumulation (Haapajärvi et al. 1997; Pitkänen et al. 1998). p53 has two DNA-binding domains, a sequence-specific DNA-binding domain encompassing amino acid residues ∼100–300 that interacts with the consensus sequence element 5′-RRRCWWGYY-(N)_1-13-RRRCWWGYY-3′ (Funk et al. 1992), and a non-sequence-specific DNA-binding domain corresponding to the carboxy-terminal ∼100 amino acids (300–393) that include the tetramerization domain (Pavletich et al. 1993; Wang et al. 1993). Modifications to the carboxy-terminal domain, including deletion of the last 30 amino acids (Hupp et al. 1992), protein binding (Hupp et al. 1992), phosphorylation of Ser-315, Ser-378, and Ser-392 (Hupp and Lane 1995; Takenaka et al. 1995; Wang and Prives 1995), and most recently, acetylation by CREB-binding protein (CBP)/p300 (Gu and Roeder 1997) were shown to enhance sequence-specific DNA-binding of wild-type p53, possibly by inhibiting p53’s non-sequence-specific DNA-binding activity (Anderson et al. 1997). CBP and p300 are closely related histone acetyltransferases (HATs) that interact with p53 through its amino terminus and function as coactivators for p53-mediated transcription (Avantaggiati et al. 1997; Lill et al. 1997). CBP/p300 are complexed with another HAT, p300/CBP-associated factor PCAF (Yang et al. 1996). These recent findings suggest that activation of sequence-specific DNA-binding by p53 in response to DNA damage may be mediated through CBP/p300 and/or PCAF; however, a direct connection between DNA damage and activation of sequence-specific DNA-binding has not been made.

In this study, we present evidence that activation of sequence-specific DNA-binding by p53 after DNA damage depends on a signal transduction cascade involving phosphorylation-mediated p53 acetylation by the coactivator, p300. We confirm Gu and Roeder’s observation (1997) that p300 acetylates the carboxyl terminus of p53 in vitro, and show that PCAF acetylates p53 in vitro on Lys-320. Using antisera specific for each of the acetylated forms of p53, we show that Lys-382 is acetylated in response to both IR and UV light in vivo; p53 also became acetylated at Lys-320 after exposure of cells to UV. Acetylation by p300 and PCAF was strongly inhibited by phosphopeptides corresponding to the amino terminus of p53 phosphorylated at Ser-37 and/or Ser-33, and these residues were phosphorylated in response to UV and IR in vivo. These findings suggest that phosphorylations in response to DNA damage enhance the interaction of p300 and PCAF with p53, thereby driving p53 acetylation.

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