p53 C-Terminal Phosphorylation by CHK1 and CHK2 Participates in the Regulation of DNA-Damage-induced C-Terminal Acetylation

Abstract

The tumor suppressor protein p53 mediates stress-induced growth arrest or apoptosis and plays a major role in safeguarding genome integrity. In response to DNA damage, p53 can be modified at multiple sites by phosphorylation and acetylation. We report on the characterization of p53 C-terminal phosphorylation by CHK1 and CHK2, two serine/ threonine (Ser/Thr) protein kinases, previously implicated in the phosphorylation of the p53 N terminus. Using tryptic phosphopeptide mapping, we have identified six additional CHK1 and CHK2 sites residing in the final 100 amino acids of p53. Phosphorylation of at least three of these sites, Ser366, Ser378, and Thr387, was induced by DNA damage, and the induction at Ser366 and Thr387 was abrogated by small interfering RNA targeting chk1 and chk2. Furthermore, mutation of these phosphorylation sites has a different impact on p53 C-terminal acetylation and on the activation of p53-targeted promoters. Our results demonstrate a possible interplay between p53 C-terminal phosphorylation and acetylation, and they provide an additional mechanism for the control of the activity of p53 by CHK1 and CHK2. INTRODUCTIONThe protein p53 is often referred to as a "tumor suppressor," because it is frequently mutated in Ͼ50% of human cancers (Olivier et al., 2002). In response to stress, p53 undergoes extensive posttranslational modification, including phosphorylation and acetylation (Appella and Anderson, 2001;Brooks and Gu, 2003; Xu, 2003). Consequently, p53 is stabilized and activated (Vousden, 2002). As a transcription factor, p53 is composed of an N-terminal activation domain, a central specific DNA binding domain, and a C-terminal tetramerization domain, followed by a regulatory domain rich in basic amino acids (Ko and Prives, 1996). On activation, p53 binds to an array of gene promoters, some of which, such as p21/waf1, are responsible for stress-induced cell cycle arrest, whereas others, such as bax and puma, are responsible for driving cells into apoptosis (Vousden and Lu, 2002;Harms et al., 2004).Phosphorylation of p53 mostly occurs in the N-terminal activation domain at the Ser6, Ser9, Ser15, Thr18, Ser20, Ser33, Ser37, Ser46, Thr55, and Thr81 residues, with some phosphorylation occurring in the C-terminal linker and basic regions at Ser315, Ser371, Ser376, Ser378, and Ser392. Phosphorylation on most of these sites is induced by DNA damage, with some, such as Thr55 and Ser376, being repressed upon genotoxic stress (Appella and Anderson, 2001;Holmberg et al., 2002). How these individual residues contribute to p53 stabilization and activation is still not fully understood, and, at times, has been the subject of debate.Many protein kinases have been implicated in phosphorylating p53 (Holmberg et al., 2002). Notably, phosphorylation at Ser15 by ATM/ATR, either directly or through CHK1/ CHK2, or at Ser20 by CHK1/CHK2 has been shown to alleviate the inhibition or degradation of p53 by Mdm2, leading to p53 stabilization and activation (Shieh et al., 1997(Shieh et al., , 2000Banin et al., 1998;...

Phosphorylation of p53 by IκB kinase 2 promotes its degradation by β-TrCP

“…To further investigate the phosphorylation of serines 362 and 366 by IKK2 in vivo, we treated mouse embryonic fibroblast (MEF) cells expressing WT or AA mutant p53 with doxorubicin, and then immunoprecipitated the phosphorylated p53 proteins with antibody to phos-Ser-366 p53 (18) and immunoblotted them with p53 antibody. MEF cells infected with the lentiviral vector making p53 WT induced phosphorylation on doxorubicin treatment ( (Fig.…”

Acetylation of p53 at Lysine 373/382 by the Histone Deacetylase Inhibitor Depsipeptide Induces Expression of p21Waf1/Cip1

“…It is well known that in certain cases a stress-induced increase in p53 activity comes from a phosphorylation-acetylation cascade (12,16,43,63,66). There thus is a possibility that depsipeptide activates kinases and thereafter induces p53 acetylation in a postphosphorylation pattern.…”

“…In addition, phosphorylation of p53 at the Ser20 or Thr18 site plays a critical role in stabilizing the p300-p53 complex (12,53), and phosphorylation of p53 at Ser15 increases binding to CBP (43) and p300 (16). Recently, it was reported that p53 C-terminal phosphorylation induced by CHK1 and CHK2 also modulates Cterminal acetylation in responding to DNA damage (63). These data indicate that p53 modulation is a complex process, and the biological consequences of p53 activation induced by certain stimuli may be dependent on p53 posttranslational modifications at multiple sites.…”

p53 Basic C Terminus Regulates p53 Functions through DNA Binding Modulation of Subset of Target Genes

“…However, at least one of the amino acids present in p53⌬24 and not in p53⌬30 has been shown to be post-translationally modified. Serine 366 is in fact one of several residues phosphorylated by CHK2 after DNA damage, a modification that seemed only required for the activation of a subset of p53 target genes (46). Nevertheless, in all of the reports cited above, p53⌬30 seems to behave like p53⌬24, and in both cases, the p53 CTD serves as a positive regulator for p53 functions.…”