Mouse mutants reveal that putative protein interaction sites in the p53 proline-rich domain are dispensable for tumor suppression.

The stability and activity of tumor suppressor p53 are tightly regulated and partially depend on the p53 proline-rich domain (PRD). We recently analyzed mice expressing p53 with a deletion of the PRD (p53(DeltaP)). p53(DeltaP), a weak transactivator hypersensitive to Mdm2-mediated degradation, is unable to suppress oncogene-induced tumors. This phenotype could result from the loss of two motifs: Pin1 sites proposed to influence p53 stabilization and PXXP motifs proposed to mediate protein interactions. We investigated the importance of these motifs by generating mice encoding point mutations in the PRD. p53(TTAA) contains mutations suppressing all putative Pin1 sites in the PRD, while p53(AXXA) lacks PXXP motifs but retains one intact Pin1 site. Both mutant proteins accumulated in response to DNA damage, although the accumulation of p53(TTAA) was partially impaired. Importantly, p53(TTAA) and p53(AXXA) are efficient transactivators and potent suppressors of oncogene-induced tumors. Thus, Pin1 sites in the PRD may modulate p53 stability but do not significantly affect function. In addition, PXXP motifs are not essential, but structure dictated by the presence of prolines, PXXXXP motifs that may mediate protein interactions, and/or the length of this region appears to be functionally significant. These results may explain why the sequence of the p53 PRD is so variable in evolution.

A mouse p53 mutant lacking the proline-rich domain rescues Mdm4 deficiency and provides insight into the Mdm2-Mdm4-p53 regulatory network.

The mechanisms by which Mdm2 and Mdm4 (MdmX) regulate p53 remain controversial. We generated a mouse encoding p53 lacking the proline-rich domain (p53DeltaP). p53DeltaP exhibited increased sensitivity to Mdm2-dependent degradation and decreased transactivation capacity, correlating with deficient cell cycle arrest and reduced apoptotic responses. p53DeltaP induced lethality in Mdm2-/- embryos, but not in Mdm4-/- embryos. Mdm4 loss did not alter Mdm2 stability but significantly increased p53DeltaP transactivation to partially restore cycle control. In contrast, decreasing Mdm2 levels increased p53DeltaP levels without altering p53DeltaP transactivation. Thus, Mdm4 regulates p53 activity, while Mdm2 mainly controls p53 stability. Furthermore, Mdm4 loss dramatically improved p53DeltaP-mediated suppression of oncogene-induced tumors, emphasizing the importance of targeting Mdm4 in chemotherapies designed to activate p53.

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

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.

The common fragile site FRA16D and its associated gene WWOX are highly conserved in the mouse at Fra8E1.

Recently, several common fragile sites (CFSs) have been cloned and characterized, including the two most frequently observed in the human population, FRA3B and FRA16D. In addition to their high frequency of breakage, FRA3B and FRA16D colocalize with genes crossing large regions of breakage. At FRA3B, the fragile histidine triad (FHIT) gene spans more than 1 Mb, and at FRA16D, the WWOX gene spans more than 750 kb. It has also been shown that in Mus musculus, a CFS Fra14A2 and the mouse Fhit gene are conserved in the orthologous region of the genome. In this study, we positioned the ortholog to WWOX (Wox1) at chromosome band 8E1 in the mouse genome. To determine whether, like Fra14A2 and Fhit, Fra8E1 and Wox1 colocalized in the mouse, we prepared bacterial and yeast artificial chromosome probes, and we hybridized them to aphidicolin-treated mouse metaphase chromosomes. Our data demonstrate that Wox1 colocalizes with Fra8E1. Furthermore, the sequence from this region, including introns, is highly conserved over at least a 100-kb region. This evolutionary conservation suggests that the two most active CFSs share many features, and that CFSs and their associated genes may be necessary for cell survival.