The p53 tumor suppressor 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 stabilized and activated following DNA damage through extensive post-translational modification (PTM). Our research has focused on identifying and exploring the biological roles of p53 PTMs to better understand how they modulate p53 function. Global effects of p53 PTM We have used mouse models containing missense mutations at p53 PTM sites to investigate the complex effects of p53 PTMs in a physiological setting. Knock-in mice were generated containing mutation of Ser18 (Ser15 in humans) to alanine in both alleles of endogenous p53, thereby preventing phosphorylation of this site. Quantitative mass spectrometry analysis of wild type or p53S18A thymocytes was performed to investigate the role of this modification in the response of p53 to ionizing radiation (IR). The primary effect of the p53S18A mutation was a loss of wild type response to IR. Among those proteins that were differently affected were several pro-apoptotic proteins, which increased in protein level after IR in the wild type and were either unaffected by IR in the mutant or showed less increase than in the wild type. A second group of proteins that was differently affected by the mutation contains proteins with roles in energy and metabolism pathways. For example, two proteins that are critical for oxidative phosphorylation, a process that is promoted by p53, were both found to increase after IR in the wild type but were unaffected by IR in the mutant. The role of p53 in energy pathways is only recently becoming known, and these studies critically highlight new functions for p53 regulation by post-translational modification. We are currently initiating studies to further understand the modulation of p53-dependent effects on metabolism in these knock-in mice. Effects of p53 N-terminal phosphorylation on its protein-protein interactions One of the naturally expressed isoforms of p53, deltaNp53, lacks the first transactivation domain (TAD1) of p53 but does contain the second transactivation domain (TAD2). The expression and stability of the two proteins are affected differently by cell type, cell cycle phase and exposure to various stresses. p53 and deltaNp53 form heterotetramers and the relative abundance of deltaNp53 influences the transactivation activity and target gene specificity of p53. Our characterization of the binding of TAD1 and TAD2 of p53 to the Taz2 domain of the transcriptional coactivator p300 demonstrated that although the two domains bound to Taz2 with equal affinity, the binding of TAD1 was affected by p53 phosphorylations, whereas the binding of TAD2 was unaffected. To better understand the differences between the complexes of Taz2 with TAD1 and TAD2, we are determining the solution structure of a p53 TAD2 peptide in complex with Taz2. p53 TAD2 binds to a similar region on Taz2 and also forms a short alpha-helix upon binding, exposing hydrophobic residues to form the primary stabilizing interactions with Taz2. Additionally, mutagenesis experiments within p53 TAD2 suggest that, although we did not previously see an effect on the binding affinity when Thr55 was phosphorylated, mutation of this site to alanine did substantially alter the conformation of the complex. We will continue to investigate these findings using biophysical methods. Furthermore, comparison of the structures of the two complexes is anticipated to shed light on how these two similar domains within p53 may function differently in co-activator recruitment after stress. Moreover, it may help elucidate some of the differences in transactivation between p53 and deltaNp53. Functional effects and interplay of p53 C-terminal modifications The C-terminus of p53 exhibits diverse post-translational modifications, including phosphorylation, methylation, acetylation, ubiquitinylation, sumoylation, and neddylation. We are interested in understanding the effects of these various site-specific modifications and the interplay between them. We have investigated the effects of mono- and dimethylation of p53 Lys382, a site that can be methylated, acetylated, or ubiquitinylated. We have continued to investigate the mechanism of p53 transcriptional repression by this modification. Following DNA damage, Lys382 becomes dimethylated, and we showed that this modification is critical for the interaction of p53 with the tandem Tudor domain (TD) of the DNA damage response mediator 53BP1. We obtained a 1.6 angstrum resolution crystal structure of the TD in complex with a p53 Lys382 dimethylated peptide. In the complex, dimethylated Lys382 is restrained by a set of hydrophobic and cation-pi interactions in a cage formed by four aromatic residues and an aspartate of the TD. However, in the complex, most of the p53 residues were not well resolved, suggesting that the peptide may bind in more than one conformation. As a means of understanding the determinants of binding in addition to the dimethylated lysine, we have been using biophysical means to investigate the binding of the C-terminal regulatory domain to 53BP1. We examined the binding of p53 377-386 to 53BP1 by isothermal titration calorimetry and observed that although mutation of His380 or Lys381 did not significantly affect the affinity of the interaction, it did significantly affect the enthalpy of complex formation. As these experiments were performed at pH 7.5 using Tris buffer, which has a large heat of ionization, we hypothesized that the histidine protonation state could be important for the interaction of p53 with 53BP1, with the observed large negative deltaH being indicative of the release of a proton upon complex formation. We next used isothermal titration calorimetry to analyze the binding of p53377-386 Lys382me2 to 53BP1 at different pH values. We found that the affinity of p53377-386 Lys382me2 binding to 53BP1 was similar at pH 5.9, 7.5, and 8.5. Thus, these results indicate that the protonation state of the His380 does not affect the equilibrium binding constant. However, the protonation state may modulate the kinetics of p53 binding to 53BP1. We are currently pursuing studies to measure the kon and koff rates for p53377 386 Lys382me2 binding to 53BP1 to better understand the determinants of the interaction of the p53 C-terminal regulatory domain with the Tudor domain of 53BP1. These experiments will provide insight into the interactions of 53BP1 with p53 and histones that facilitate repair of DNA damage.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Cooks, Tomer; Pateras, Ioannis S; Jenkins, Lisa M et al. (2018) Mutant p53 cancers reprogram macrophages to tumor supporting macrophages via exosomal miR-1246. Nat Commun 9:771
Veschi, Veronica; Liu, Zhihui; Voss, Ty C et al. (2017) Epigenetic siRNA and Chemical Screens Identify SETD8 Inhibition as a Therapeutic Strategy for p53 Activation in High-Risk Neuroblastoma. Cancer Cell 31:50-63
Mazur, Sharlyn J; Gallagher, Elyssia S; Debnath, Subrata et al. (2017) Conformational Changes in Active and Inactive States of Human PP2C? Characterized by Hydrogen/Deuterium Exchange-Mass Spectrometry. Biochemistry 56:2676-2689
Zhang, Zhan; Liu, Ling; Gomez-Casal, Roberto et al. (2016) Targeting cancer stem cells with p53 modulators. Oncotarget 7:45079-45093
Tong, Qiong; Mazur, Sharlyn J; Rincon-Arano, Hector et al. (2015) An acetyl-methyl switch drives a conformational change in p53. Structure 23:322-31
Tong, Qiong; Cui, Gaofeng; Botuyan, Maria Victoria et al. (2015) Structural plasticity of methyllysine recognition by the tandem tudor domain of 53BP1. Structure 23:312-21
Lu, X; Mazur, S J; Lin, T et al. (2014) The pluripotency factor nanog promotes breast cancer tumorigenesis and metastasis. Oncogene 33:2655-64
Jenkins, Lisa M Miller; Durell, Stewart R; Mazur, Sharlyn J et al. (2012) p53 N-terminal phosphorylation: a defining layer of complex regulation. Carcinogenesis 33:1441-9
Karamychev, Valeri N; Wang, Difei; Mazur, Sharlyn J et al. (2012) Radioprobing the conformation of DNA in a p53-DNA complex. Int J Radiat Biol 88:1039-45
Fujita, Kaori; Horikawa, Izumi; Mondal, Abdul M et al. (2010) Positive feedback between p53 and TRF2 during telomere-damage signalling and cellular senescence. Nat Cell Biol 12:1205-12

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