UC San Diego

p53 is the most commonly mutated tumor suppressor gene in human cancers. Activation of p53 maintains the genomic integrity and protects the organism against the propagation of cells that carry damaged DNA with potentially oncogenic mutations. While p53 protein is normally short-lived and kept at low levels in a relatively inactive form, upon genotoxic and cellular stresses p53 is transiently stabilized and activated as a transcription factor. The increase in protein level and transcriptional activity is attributed largely to the various posttranslational modifications of p53, including the N-terminal phosphorylation, and the multiple C- terminal modifications such as ubiquitination, acetylation, sumoylation, neddylation and methylation. Specifically, the six Lysine residues at p53 C-terminus can be posttranslationally modified by diverse mechanisms, among which the ubiquitination of those residues has been thought to be required for Mdm2-mediated ubiquitin- directed proteasomal degradation, and the acetylation is suggested to activate p53 transcriptional activity and contribute to its stabilization. To investigate the physiological functional outcome of the C-terminal modifications in regulating p53 stability and activity, we introduced Lysine to Arginine missense mutations at the six Lysine residues (K6R) into the endogenous p53 locus in mouse embryonic stem cells (ESCs). Unexpectedly, analysis of mouse ESCs, mouse embryonic fibroblast (MEFs) and thymocytes has concluded that ubiquitination of C-terminal Lysine residues is not required for efficient p53 degradation either before or after DNA damage. The outcome of loss of all potential posttranslational modifications is a modestly impaired p53 activity after DNA damage in a promoter-specific manner. Phosphorylation of Ser46 of human p53 is suggested to play an important role in activating p53-dependent apoptosis. To address the physiological role of Ser46 phosphorylation, we introduced Ser46 to Alanine mutation (S46A) into the human p53 knock- in (HUPKI) allele in mice. Consistent with the previous cell line studies, transactivation of p53 targeting apoptotic genes is preferentially affected by the mutation and p53-dependent apoptosis after DNA damage is partially impaired in mutant thymocytes and in E1A/Ras-overexpressed MEFs. In addition, Ser46 phosphorylation may contribute to preventing spontaneous immortalization of cultured MEFs and to oncogene Ras-induced premature senescence of MEFs