Phosphorylation of Serine 18 Regulates Distinct p53 Functions in Mice

The p53 tumor suppressor is a transcription factor that functions to regulate cell proliferation and apoptosis. The importance of p53 in suppressing cancer is highlighted by the very high incidence of spontaneous tumor formation in p53-deficient mice and in mice bearing genetic alterations in other members of the p53 pathway (14, 27, 30, 42). The critical role of p53 in tumor suppression is further underscored by the high frequency of p53 mutations in human cancer, and malignancies that retain a wild-type p53 gene often have acquired other mutations that affect one or more of the pathways known to govern p53 function (53). Therefore, it is likely that inactivation of p53 is a common mechanistic step in the development of most human cancers. Cellular p53 activity is greatly increased following treatment of cells with DNA-damaging agents such as ionizing radiation and adriamycin (62). Induction of p53 activity can also occur in response to inappropriate growth-stimulatory signals, such as expression of an activated oncogene or exposure of the cells to other forms of stress (37, 38, 50, 69). Increased p53 activity resulting from DNA damage blocks the proliferation of cells in the G1 phase of the cell cycle by inducing the expression of genes such as the cyclin-dependent kinase inhibitor p21 (6, 13, 16, 20). In addition, increased p53 activity in the cell can initiate apoptosis by inducting the expression of proapoptotic BCL-2 family members involved in mitochondrion-associated cell death and by transactivating the expression of death receptor-associated genes (7, 41, 52, 66). Both p53-mediated inhibition of cell cycling and induction of apoptosis appear to be highly dependent upon the cell context. Induction of p53 has also been proposed to regulate passage of cells through the G2 phase of the cell cycle, chromosomal stability, and mitosis, although the precise molecular mechanisms used by p53 to perform these regulatory functions are less clear. Given the multiple roles of p53 in controlling cell cycling and cell death, proper regulation of p53 activity is obviously crucial to permit cells to undergo normal growth and to respond appropriately to stress. Regulation of cellular p53 activity has been reported to occur at the level of p53 transcription and translation (19, 49, 63). However, it is clear that Mdm2-mediated alteration of p53 protein stability is a major pathway used by many cells to govern p53 function. The Mdm2 oncoprotein binds to the amino-terminal portion of the p53 protein (11, 48), induces p53 monoubiquitination and nuclear export (35, 39), and targets p53 for proteosomal degradation (23, 34). The importance of Mdm2 as a negative regulator of p53 activity in cell growth and development has been demonstrated in mice by rescue of the embryo-lethal phenotype of Mdm2-deficient mice by deletion of p53 (28, 45). Numerous in vitro studies have determined that p53 is subject to posttranslational modification. Phosphorylation occurs in response to DNA damage on human p53 serine residues 15 and 20 (mouse Ser18 and Ser23, respectively) and has been found in several in vitro studies to upregulate p53 activity (15, 18, 36, 43, 60) and interfere with Mdm2-mediated degradation of p53 (55). The ATM (mutated in ataxia telangiectasia) kinase has been demonstrated to phosphorylate human p53 serine 15 (3, 8, 33, 51). Furthermore, both human p53Ser15 phosphorylation (8, 46, 56) and total p53 protein levels (31, 32, 46) are reduced in ATM-null cells following ionizing radiation treatment. These observations suggest that ATM-mediated phosphorylation of this residue increases the stability of p53. Expression of inactive forms of the ataxia telangiectasia-related kinase ATR also interferes with p53Ser15 phosphorylation (47, 58, 65). In addition, mouse embryonic fibroblasts (MEFs) deficient in both ATM and ATR fail to undergo G1 arrest in response to DNA damage and display greatly reduced phosphorylation of p53Ser18, the mouse homologue of human p53Ser15 (5). These data suggest that ATM and ATR functions may depend, in part, upon the ability of these related kinases to phosphorylate p53 at this serine residue. Recently mouse p53Ser18 was mutated in embryonic stem (ES) cells, and the p53 apoptotic response and cell cycle arrest following DNA damage of these cells were delayed (10). However, p53-mediated pathways of cell cycle inhibition and apoptosis are not fully functional in ES cells (1). Thus, the relationship between p53Ser18 phosphorylation and p53 stability and function remains unclear. In order to directly address the role of p53Ser15 phosphorylation in regulating p53 levels and p53 function in a genetically defined in vivo system, we used gene targeting in mouse ES cells to generate mice bearing p53 alleles that contain a p53Ser18 to Ala18 substitution. Our results indicate that the p53-dependent apoptosis induced by ionizing radiation is significantly reduced in the thymocytes and splenocytes of p53Ala18/Ala18 mice. Therefore, phosphorylation of p53Ser18 regulates p53-dependent apoptosis. However, phosphorylation of this residue does not alter the stability of the p53 protein. Wild-type mice and p53Ala18 mice display similar basal p53 levels and similar induced levels of p53 following DNA damage. Surprisingly, phosphorylation of p53Ser18 does not affect the rate of cell proliferation of embryonic fibroblasts. In addition, loss of Ser18 does not alter the p53-dependent G1 arrest of cell growth following DNA damage. Thus, the G1 arrest induced by ATM and ATR following DNA damage is not mediated by p53Ser18 phosphorylation. Furthermore, the reduced apoptotic activity of p53Ala18 does not rescue the embryonic lethality of Mdm2-null mice and does not alter p53-mediated tumor suppression. These results indicate that phosphorylation of the ATM/ATR target serine 18 regulates p53 apoptotic functions and not p53-mediated cell growth arrest and that phosphorylation of this site is not required for p53 suppression of spontaneous tumorigenesis.