Chapter 4 Lack of p 53 . S 389 phosphorylation predisposes mice to develop 2-acetylaminoflurene-induced bladder tumors but not ionizing radiation-induced lymphomas

Cellular activity of the tumor suppressor protein p53 is primarily regulated by post-translational modifications. Phosphorylation of the COOH-terminus, including Ser389, is thought to result in a conformational change of the p53 protein, enhancing DNA binding and transcriptional activity. In vitro studies presented here show that, in addition to UV radiation, Ser389 is phosphorylated upon exposure to 2-acetylaminofluorene (2-AAF). Both agents induce bulky DNA adducts repaired by nucleotide excision repair (NER). In contrast, ionizing radiation, known to induce DNA damage not repaired by NER, does not result in Ser389 phosphorylation. Previously, we have shown that p53.S389A mutant mice, lacking the Ser389 phosphorylation site, are sensitive to developing UV-induced skin tumors. Here, we show that p53.S389A mice are also prone to developing 2-AAF-induced urinary bladder tumors, whereas no increased tumor response was found upon ionizing irradiation. These results provide evidence for our hypothesis that phosphorylation of Ser389 is important for activation of p53 to exert its function as a tumor suppressor not exclusively upon the presence of UV-induced DNA damage, but also upon exposure to other bulky adduct-inducing agents. Analysis of 2-AAFand UV-induced tumors from p53.S389A mice revealed the presence of additional p53 mutations, indicating that lack of Ser389 phosphorylation by itself is not sufficient to abrogate p53 function in tumor suppression. In addition, analyses of skin tumors of p53.S389A mice revealed an interesting hotspot mutation previously found exclusively in NER-deficient mice and patients. Introduction Cancer is caused by loss of controlled cell growth, due to mutational inactivation or activation of genes involved in cell cycle regulation. Several mechanisms are known to be critical in the prevention of cells from becoming cancerous. Of these, cell cycle control and DNA repair result in the removal of DNA damage before mutations are formed, whereas the process of apoptosis eliminates cells with sustained DNA damage. These cellular responses are mediated, at least in part, by the p53 tumor suppressor protein[1-3]. The levels of p53 protein in normal unstressed cells are very low due to rapid protein degradation mediated by MDM2 [4]. However, in response to DNA damage or other sources of cellular stress, p53 protein levels increase and its transcriptional activity is greatly increased [5]. The key mechanism of p53 activation is post-translational modification, including (de)phosphorylation, acetylation, and sumoylation [1-3]. Several sites have been shown to be potential targets for modifications, and affect p53 functions in a variety of ways. For example, phosphorylation at its NH2-terminus disrupts the interaction of p53 with the MDM2 protein, thereby preventing degradation and thus stabilizing the p53 protein [6-8]. Modifications of the p53 COOHterminus are thought to result in a conformational change, thereby enhancing DNA-specific binding and transcriptional activity [9;10]. The specific patterns of p53 post-translational modifications seem to be dependent on the specific type of agent to which the cell is exposed to. At the COOH-terminal regulatory domain, Ser315 is phosphorylated after exposure to both UV-light and ionizing radiation [9;11]. This is in contrast with Ser392 (equivalent to mouse Ser389), which is specifically phosphorylated upon UV radiation [12;13]. Kinases responsible for Ser389 phosphorylation include CKII, p38 Map kinase, and PKR [13-16]. To enable the in vivo functional analysis of a specific p53 phosphorylation site, we recently generated mice with a single point mutation in the p53 gene, resulting in a serine to alanine