The tumor suppressor p53 is known to function in the prevention of genetic instability and in the activation of apoptosis. To mediate these functions, the constitutively expressed latent form of p53 is activated by a series of posttranslational modifications that occur at the N-and C-terminal regions. 1, 2 Among the post-translational modifications, phosphorylation of human p53 (hp53) at Ser46 was identified as a specific modification involved in apoptotic response. 3 Compared to other N-terminal phosphorylation sites, nearly 100% conserved in different species, the region encompassing Ser46 has low level of amino-acid (aa) identity questioning the existence of a Ser46 homologue site in mice. 4 Evidence indicates that some species-dependent differences exist in the post-translational modification of p53. However, the crucial role of hp53-Ser46 in apoptotic regulation and the widespread use of mouse models in biological and preclinical studies requires a direct evaluation of this issue, rather than relying only on sequence homology. The major bioinformatics tools currently available for proteome-wide identification of phosphorylation sites are NetPhos (http://www. cbs. dtu. dk/services/NetPhos) and its extension DISPHOS (http://www. ist. temple. edu/DISPHOS), two neural-network-based predictors, and Scansite (http://scansite. mit. edu), a motif-based service. Unfortunately, for p53 the prediction accuracy of these programs is largely unsatisfying as can be assessed by comparing the computationally predicted with the experimentally identified phospho-sites. Recently, we employed the recurrence quantification analysis (RQA), a nonlinear technique fruitfully applied to several different fields including protein sequence (http://homepages. luc. edu/~ cwebber and http://www2. phys. rush. edu/JZbilut/physiozbi. html), to successfully discriminate, on a purely sequence basis, between experimentally characterized DNA-contact defective and conformational p53 mutants. 5 Thus, we applied RQA to identify the putative ortholog of hp53-Ser46 (Supplementary Information 1S). To experimentally test whether the theoretically predicted Ser58 site of mouse p53 (mp53) is functionally homologous to hp53-Ser46, we first generated antibody (Ab) to specifically recognize phospho-Ser58 (Supplementary Information 2S). To determine whether mp53-Ser58 is phosphorylated in vivo and whether this phosphorylation is similar to that of hp53-Ser46, we irradiated the wtp53-carrying F9 murine cells with increasing doses of UV and tested p53 accumulation and phosphorylation by Western blotting, 24h after irradiation.

Accumulation of p53, phosphorylation of Ser18, and Ser389 (the mouse homologues of human Ser15 and Ser392) were detected in total cell extracts (TCEs) at each dose. In contrast, Ser58 was phosphorylated only at doses able to significantly promote cell death, as assessed by cell viability (Figure 1a). In addition, time course analysis of p53 expression and phosphorylation of F9 cells treated with apoptotic doses of UV or adriamycin (ADR)(Supplementary Information, Figure 2S-A and 2S-B) showed kinetics of Ser58 phosphorylation very similar to that previously reported for Ser46 in human cells subjected to similar stressing conditions. 3 Finally, the ALLN-stabilized p531 was detected by the anti-total p53 Ab but not by the phospho-specific Abs, supporting the specificity of our anti-Ser58P Ab (Supplementary Information Figure 2S-A).