Serine 312 phosphorylation is dispensable for wild-type p53 functions in vivo

Regulation of p53 function is extremely crucial for the proper functioning of a cell, and thus the organism, as p53 activation restricts growth, whereas its functional loss leads to tumorigenesis.1, 2 Expectedly, p53 is found inactivated in most tumors,3 and activation of p53 is only noted in extreme conditions when cells are subjected to stress, as physiologically, cells cannot tolerate activated p53.4, 5 Deregulated p53 activation leads to embryonic death, as shown in the case of the Mdm2 and Mdm4 deficient mice,1 and also results in a dwarfed phenotype observed in mice expressing a mutant hyperactive p53.1 Collectively, all these findings highlight the need for precise regulation of p53 function for physiological processes to ensue. Not surprisingly, p53 is a labile protein whose levels are kept minimal primarily by the ubiquitin ligase Mdm2.6 Several post-translational modifications (PTMs) including acetylation and phosphorylation have also been suggested to regulate p53's turnover.7 Acetylation at the carboxyl-terminal lysines have been shown to be important for Mdm2-dependent degradation.8 Furthermore, phosphorylation at the amino-terminal sites have been shown to be crucial for disrupting Mdm2 binding, and hence, critical for p53 stabilization.9 Most of the phosphorylation sites on p53 are clustered in its amino- and carboxyl-terminals.7 Critical amino-terminal sites suggested to regulate p53 function include the serine (S) residues 15, 20 and 46.10 Among the carboxyl-terminal sites, S392 had been shown to be specifically regulated by ultra violet (UV) irradiation (but not by IR) – highlighting signal specificity – and seems to have a part in regulating optimal p53 functions.11 Other carboxyl-terminal sites, such as S315, S373 and S376 have also been implicated to varying extents.10, 12 Many of the phosphorylation sites are often modified in response to stress signals,7 consistent with the notion that phosphorylation regulates p53 functions during the SOS response. However, the S315 site has been suggested to be unique, being regulated during cell-cycle progression and also upon endoplasmic-reticulum (ER) stress, an event in which p53 is not upregulated, besides genotoxic stress.10, 12, 13, 14, 15, 16 Two seemingly opposite sets of results have emerged from the analysis of the S315 site in mouse and human cell lines – one which highlights the importance of this phosphorylation site for proper p53 functioning, and the other which highlights its role in leading to p53 degradation.12, 13 One possible explanation for these conflicting findings is that these observations have been made using various phospho-mutant cDNAs, and therefore, the results may not absolutely reflect the physiological significance of the phosphorylation events because of the unusually high p53 levels in the overexpression systems. Thus, the solution to faithfully recapitulate physiological conditions rely on the recent advances in knock-in mouse models whereby the phosphorylable serine residues have been substituted to alainine residues – to mimic an unphosphorylated state – in the germ-line of mice.1 We have therefore generated a knock-in S312A mutant mouse strain, in which the serine residue has been substituted with an unphosporylable alanine (A) residue, to evaluate the physiological importance of the S315 phosphorylation site. The detailed characterization of the p53S312A/S312A mice is presented.