The p53 tumor suppressor protein preserves genome integrity by regulating growth arrest and apoptosis in response to DNA damage. In response to ionizing radiation (IR), ATM, the gene product mutated in ataxia telangiectasia, stabilizes and activates p53 through phosphorylation of Ser15 and, indirectly, Ser20. Recent studies have suggested that phosphorylation of human p53 at Ser20 is important for stabilizing p53. To examine the requirement for this DNA damage-induced phosphorylation in a physiological setting, we introduced a missense mutation into the endogenous p53 gene of mouse embryonic stem (ES) cells that changed serine 23, the murine equivalent of human serine 20, to alanine. Murine embryonic fibroblasts harboring the p53(S23A) mutation accumulated p53 as well as p21 and Mdm2 proteins to normal levels after DNA damage. Furthermore, ES cells and thymocytes harboring the p53(S23A) mutation also accumulated p53 protein to wild-type levels and underwent p53-dependent apoptosis similarly to wild-type cells after DNA damage. Therefore, phosphorylation of murine p53 at Ser23 is not required for p53 responses to DNA damage induced by ionizing radiation treatment. The mammalian Chk2 kinase is thought to mediate ATM-dependent signaling in response to DNA damage and to phosphorylate Ser23. We investigated the physiological role of Chk2 by generating Chk2-deficient mice. Chk2-/- mice, thymocytes and neurons of the developing brain were resistant to IR-induced apoptosis. The IR-induced G1/S cell cycle checkpoint, but not the G2/M or S phase checkpoints, was impaired in embryonic fibroblasts derived from Chk2-/- mice. IR-induced stabilization of p53 in Chk2-/- cells was 50 to 70% of that in wild-type cells and caffeine further reduced p53 accumulation, suggesting the existence of an ATM/ATR-dependent but Chk2-independent pathway for p53 stabilization. Despite p53 protein stabilization and phosphorylation of Ser23, p53-dependent transcriptional induction of target genes was not observed in Chk2-/- cells. Thus, Chk2 plays a pivotal role in the biological activity of p53 by regulating its transcriptional activation as well as its stabilization after IR-induced damage. Recently, we found in human lymphoblastoid cells that phosphorylation of p53 on Ser46, a residue important for p53 apoptotic activity, as well as on Ser9, in response to IR was dependent on the ATM protein kinase. IR-induced phosphorylation at Ser46 was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, but not by PD169316, a p38 MAPK inhibitor. p53 C-terminal acetylation at Lys320 and Lys382 required Ser15 phosphorylation by ATM. These observations suggest that ATM is involved in the initiation of p53-dependent apoptosis after IR in human lymphoblastoid cells. Presently, we are investigating mechanisms of p53 activation by the free radical, nitric oxide (NO). NO from donor drugs induced both ATM- and ATR-dependent p53 posttranslational modifications, leading to an increase in p53 transcriptional targets and G2/M cell cycle checkpoint impairment. In non-cancerous colon tissues from patients with ulcerative colitis, inducible nitric oxide synthase protein levels were positively correlated with p53 Ser15 phosphorylation levels and p21expression. Currently we are expanding these observations to animal models of chronic inflammation. The tumor suppressor p53 is stabilized and activated in response to cellular stress through acetylation of C-terminal lysines. Acetylation of p53 by p300/CBP is inhibited by its negative regulator, MDM2. Recently, we showed that MDM2 promotes p53 deacetylation by recruiting a complex containing the HDAC1 deacetylase. Ectopic expression of a dominant negative HDAC1 mutant restored p53 acetylation in the presence of MDM2, whereas wild-type HDAC1 and MDM2 deacetylated p53 synergistically. Fibroblasts overexpressing the dominant negative HDAC1 mutant displayed enhanced DNA damage-induced p53 acetylation, an increase of p53 levels and a more pronounced induction of p21. Our results suggest that the major function of p53 acetylation is to promote p53 stability by preventing MDM2-dependent ubiquitination, while recruitment of HDAC1 by MDM2 promotes p53 degradation by removing these acetyl groups.
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