Molecular basis of S100 proteins interacting with the p53 homologs p63 and p73.

S100 proteins modulate p53 activity by interacting with its tetramerization (p53TET, residues 325-355) and transactivation (residues 1-57) domains. In this study, we characterized biophysically the binding of S100A1, S100A2, S100A4, S100A6 and S100B to homologous domains of p63 and p73 in vitro by fluorescence anisotropy, analytical ultracentrifugation and analytical gel filtration. We found that S100A1, S100A2, S100A4, S100A6 and S100B proteins bound different p63 and p73 tetramerization domain variants and naturally occurring isoforms with varying affinities in a calcium-dependent manner. Additional interactions were observed with peptides derived from the p63 and p73 N-terminal transactivation domains. Importantly, S100 proteins bound p63 and p73 with different affinities in their different oligomeric states, similarly to the differential modes of binding to p53. On the basis of our data, we hypothesize that S100 proteins regulate the oligomerization state of all three p53 family members and their isoforms, with a potential physiological relevance in developmental and disease-related processes. The regulation of the p53 family by S100 is complicated and depends on the target preference of each individual S100 protein, the concentration of the proteins and calcium, as well as the splicing variation of p63 or p73. Our results outlining the complexity of the interaction should be considered when studying the functional effects of S100 proteins in their biological context.

Regulation by phosphorylation of the relative affinities of the N-terminal transactivation domains of p53 for p300 domains and Mdm2.

The transcriptional activity of the tumour suppressor, p53, requires direct binding between its transactivation domain (TAD, 1-57) and the transcriptional coactivator, p300. We systematically assessed the role of TAD phosphorylation on binding of the p300 domains CH3, Taz1, Kix and IBiD. Thr18 phosphorylation increased the affinity up to sevenfold for CH3 and Taz1, with smaller increases from phosphorylation of Ser20, Ser15, Ser37, Ser33, Ser46 and Thr55. Binding of Kix and IBiD was less sensitive to phosphorylation. Strikingly, hepta-phosphorylation of all Ser and Thr residues increased binding 40- and 80-fold with CH3 and Taz1, respectively, but not with Kix or IBiD. Substitution of all phospho-sites with aspartates partially mimicked the effects of hepta-phosphorylation. Mdm2, the main negative regulator of p53, competes with p300 for binding to TAD. Binding of Mdm2 to TAD was reduced significantly only on phosphorylation of Thr18 (sevenfold) or by hepta-phosphorylation (24-fold). The relative affinities of Mdm2 and p300 for p53 TAD can thus be changed by up to three orders of magnitude by phosphorylation. Accordingly, phosphorylation of Thr18 and hepta-phosphorylation dramatically shifts the balance towards favouring the binding of p300 with p53, and is thus likely to be an important factor in its regulation.