The C-terminal lysines fine-tune P53 stress responses in a mouse model but are not required for stability control or transactivation

Abstract

P53 is an unstable transcription factor that is mutated in a majority of human cancers. With a significant role in initiating cell elimination programs, a network has evolved to fine-tune P53 transcriptional output and prevent errant activation. Modifications of the C terminus have long been viewed as critical binary determinants of P53 stability or activation. However, these conclusions are based on in vitro transfection or biochemical analyses where the stoichiometries between P53 and its regulators are perturbed. Therefore, we tested the importance of the C-terminal regulatory region for P53 control in mice where the seven C-terminal lysines were changed to arginine (Trp-53 7KR ). Surprisingly, the homozygous mutant mice are viable and phenotypically normal. We have functionally characterized the mutant protein in both MEFs and thymocytes, revealing the unexpected result that Trp-53 7KR exhibits a normal half-life and functions like WT P53 in cell cycle arrest and apoptosis, and in an E1A-ras xenograft tumor suppression assay. However, a significant difference is that P53 7KR is activated more easily by DNA damage in thymus than WT P53. Importantly, although MEFs encoding WT P53 spontaneously emerge from crisis to become immortal in a 3T3 growth protocol, we do not observe any such escape with the P53 7KR cells. We propose that the C-terminal modifications believed to be critical for proper P53 regulation are not essential, but contribute to a fine-tuning mechanism of homeostatic control in vivo.Mdm2 ͉ p53 ͉ tumor suppressor ͉ Trp-53 ͉ ubiquitination P 53 can respond to a variety of stimuli to either initiate cell cycle arrest or initiate apoptosis. Therefore, it is critical to control the output of this short-lived transcription factor (1). It is generally accepted that P53 regulation involves competition between the E3 ubiquitin ligase MDM2 and histone acetyl transferases (HATs). MDM2-mediated ubiquitination of the conserved C-terminal lysines of P53 initiates proteasome-dependent degradation, while the HATs acetylate the same lysines, resulting in P53 stabilization and activation (2, 3). Other studies report that C-terminal lysine methylation is required for P53 stabilization (4), neddylation inhibits transactivation (3), and sumoylation may have positive or negative roles (5). Regardless of the modification, the C terminus is thought to be critical for P53 stability or activation. However, these conclusions are often based on nonnative conditions, where the ratios between P53 and its regulators are altered. Therefore, we tested the importance of the C-terminal regulatory region for P53 control in mice where the seven C-terminal lysines were changed to arginine (Trp-53 7KR ). MethodsTargeting Construct. Using ET recombination (GeneBridges, Dresden Germany), we recovered the WT Trp-53 locus from a 129 strain mouse BAC (a generous gift from Manuel Serrano, Spanish National Cancer Center, Madrid) with the following primers 5Ј-GAT AAG CAA GAC ATT GTA CCA GCA ATT AAC CCC CCA CCC CCA ACT CCA TAG CGG CCG CTG AAG ...

Sirtuins: critical regulators at the crossroads between cancer and aging

“…MEFs from mice engineered to express p53 with seven C terminal lysines mutated to arginines (p53 7KR ), to mimic unacetylated p53, respond to DNA damage similarly to MEFs from wild-type mice, but show enhanced stability of p53 7KR that allows for enhanced induction of target genes in thymocytes after DNA damage (Krummel et al, 2005). Immortalized p53 7KR MEFs undergo senescence earlier, suggesting that regulation of p53 acetylation may fine-tune p53 activity over the lifespan of an organism (Krummel et al, 2005).…”

Transcriptional regulation by p53: one protein, many possibilities

“…56 Finally, it was recently shown that substitution of all p53 CTD lysines for arginines does not significantly affect the activities or stability of p53 in a mouse model. 57,58 Though controversial, the importance of the CTD in p53 functioning has been continuing to reveal itself in a number of more recent publications. McKinney and Prives, 59 Fojta et al, 60 and Palecek et al 61 have shown that the CTD is important for binding to various non-linear DNAs.…”

Crippling p53 activities via knock-in mutations in mouse models

“…The choice of which lysine is modified by what mechanism remains unclear. To further study the physiological roles of the post-translational modifications at the carboxyl-terminal lysine residues in regulation of p53 stability and activity, two groups generated mutations of all six lysines (K) to arginines (R) (Feng et al, 2005;Krummel et al, 2005). The K6R mutations at codons 367, 369, 370, 378, 379 and 383 of mouse p53 were introduced into ES cells by homologous recombination at the endogenous mouse locus (Feng et al, 2005).…”

“…These results suggest that ubiquitination of these six lysine residues are not required for efficient p53 degradation and that posttranslational modifications at the carboxyl-terminal lysines play a role in activating p53 activity on certain target genes after DNA damage. Mice with lysine-toarginine mutations at the same six lysines plus one additional lysine at codon 384 which does not have a corresponding lysine in human p53 (7KR) were also generated (Krummel et al, 2005). Mice homozygous for 7KR are born at the appropriate Mendelian ratio and are phenotypically normal.…”