The human p53 protein is a homotetrameric, sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, cellular senescence, and DNA repair. It is maintained at low levels in unstressed cells, but is stabilized and activated following DNA damage through extensive post-translational modification (PTM). Our research has focused on identifying and exploring the biological roles of specific p53 PTMs to better understand how they modulate p53 function. We have identified a novel p53 species dimethylated at Lys382 (p53K382me2) and have shown that the tandem tudor domain of the DNA damage response mediator 53BP1 acts as an """"""""effector"""""""" for this modification. We found that the 53BP1 tudor domain preferentially recognizes p53K382me2 over other potential p53 and histone lysine methylation sites. p53K382me2 levels increase following DNA damage and this modificationfacilitates interaction between p53 and 53BP1. The generation of p53K382me2 promotes the accumulation of p53 protein that occurs upon DNA damage, and this increase in p53 levels requires 53BP1. The molecular mechanism of the nonhistone methyllysine recognition by the 53BP1 Tudor domain remained unknown. Recently, we obtained a 1.6 resolution crystal structure of the tandem Tudor domain of human 53BP1 in complex with a p53K382me2 peptide. In the complex, dimethylated Lys382 is bound in a cage formed by four aromatic residues and an aspartate of 53BP1. These results provide the basis for deciphering the role of this interaction in regulation of p53 and 53BP1 functions. Our work on the transcriptional activating function of p53 has focused in part on the recruitment of coactivators, such as CBP, p300 and PCAF, to the promoters of specific target genes. We have recently characterized the binding of the N-terminal transactivation domain of p53 to the Taz2 domain of the acetyltransferase p300. We used binding, thermodynamic, and structural studies to better understand the determinants of the interaction of the Taz2 domain with p531-40. We found that the full TAD1 is required for maximal binding and solved the NMR solution structure of p531-40 bound to Taz2. In the complex, p531-40 forms a short α-helix and interacts with Taz2 through an extended surface stabilized primarily by hydrophobic contacts. Phosphorylation of p53 at Ser15 or Thr18 increased its affinity for Taz2, possibly due to interaction of the phosphates with neighboring arginine residues in Taz2 and altered hydrophobic interactions. Furthermore, a second binding site for Taz2 was identified in p5335-59. The second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. Further investigation of Taz2 binding to these two sites by isothermal titration calorimetry indicated that upon complex formation the change in heat capacity at constant pressure, ΔCp, was negative for both sites, suggesting the importance of hydrophobic interactions. However, the more negative value of ΔCp for Taz2 binding to the p531-40 compared to the p5335-59 suggested that the importance of nonpolar and polar interactions differs between the two sites. Phosphorylation of p53 at Thr81 also mediates p53 protein-protein interaction, in this case, the interaction of p53 with Ubc13. Previous work had demonstrated that Ubc13, an E2 ubiquitin-conjugating enzyme, regulates p53 localization and transcriptional activity. We recently have demonstrated that association of p53 and Ubc13 on polysomes requires ongoing translation and results in p53 ubiquitination that interferes with its tetramerization. JNK phosphorylation of p53 at Thr81 results in dissociation of the Ubc13-p53 complex, which in turn leads to p53 multimerization and transcriptional activation. Inhibition of JNK activity or expression of a nonphosphorylatable mutant of p53 maintained an Ubc13-p53 complex that inhibits p53 multimerization. Our findings reveal that the regulation of p53 multimerization requires the action of JNK and Ubc13 on polysome-bound p53. Post-translational modification of p53 regulates the interactions with its negative regulators Mdm2 and MdmX;phosphorylation of p53 at Thr18 and Ser20 disrupts these interactions, allowing p53 to be stabilized and activated. To better understand the functions of these overlapping regulators of p53, we have used transgenic mice containing human p53 in place of the endogenous protein. In this model, p53 activity was reduced compared with wild type mice. We have recently observed that either the use of an Mdm2-p53 binding inhibitor, nutlin-3a, or the knockdown of Mdm2 partially restored p53-mediated apoptosis and G1 arrest in transgenic mouse embryo fibroblasts. Ablation of MdmX in combination with nutlin-3a treatment of the transgenic cells resulted in induction of apoptosis and cell cycle arrest comparable to wild type cells. Thus, the studies in the transgenic mouse model demonstrate the importance of inhibition of both Mdm2 and MdmX for activation of p53. Mouse models containing missense mutations at PTM sites of p53, developed in collaboration, continue to be a valuable resource for investigating the complex global effects of single or multiple PTMs of p53 in a physiological setting. Knock-in mice have been generated in which Ser18 (Ser15 in humans) was mutated to alanine in both alleles of endogenous p53, preventing phosphorylation. To better understand the role of this modification in the response of p53 to DNA damage, quantitative mass spectrometry was used to determine the global effects of ionizing radiation (IR) on the levels of proteins in thymocytes of the p53S18A knock-in mice and resulted in the identification of 73 proteins that were differently affected (p<0.05). The p53S18A mutation has widespread effects on specific protein levels following IR, affecting the levels of proteins involved in apoptosis, transcription, translation, and metabolism. Pathway analysis of the differently regulated proteins suggests that p53 activity and TGF-beta signaling are decreased in the p53S18A thymocytes. These results suggest that phosphorylation of Ser18 modulates both transcriptional and non-transcriptional functions of p53, influencing signaling of different pathways in the cell.
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