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). Among the proteins identified and quantified, we found that 70 had a significantly altered response to IR in the mutant thymocytes as compared with the wild type. The primary effect of the p53S18A mutation was a loss of wild type IR response. Among those proteins that were differently affected were six pro-apoptotic proteins and eleven proteins with roles in energy/metabolism pathways. Pathway analysis of the proteins differently affected by IR between the WT and p53S18A thymocytes suggested that TGFbeta signaling was decreased in the mutant. As for p53, TGFbeta signaling leads to apoptosis following DNA damage, suggesting that decreased apoptosis in thymocytes may be due to direct effects of p53 and indirect effects of p53 on TGFbeta signaling. These results highlight the importance of p53 post-translational modifications not only in transcription, but also in processes with far-reaching effects in the cell. p53 Ser20 phosphorylation. Phosphorylation of p53 at human Ser20 (Ser23 in mice) is activated by numerous types of stress, such as DNA damage, viral infection, and metabolic stress. The kinase that modifies Ser20 varies with the type of stress: CK1 modifies this site in response to DNA viral infection, AMPK in response to elevation of AMP/ATP ratio, and DAPK-1 in response to inappropriate oncogene activation. However, the kinase responsible for Ser20 phosphorylation in response to IR has not been identified. Knockdown of either or both Chk1 and Chk2 in human cells failed to abrogate the damage-dependent induction of p53 expression, and depletion of both Chk2 alleles in human colon cancer cells did not affect p53 phosphorylation at this site. However, we found that phosphorylation of mouse Ser23 following DNA damage was abrogated in primary Chk1+/-Chk2-/- MEFs. One potential explanation for the difference in these results is that our experiments were performed with primary knockout MEFs, whereas other studies used human cancer cells and did not analyze the phosphorylation status of p53 at human Ser20 in Chk1/Chk2 double-knockdown cells that were otherwise wild type. Therefore, although the contribution of Chk1 and Chk2 to the phosphorylation of p53 at Ser20 in response to DNA damage appears to differ between human and mouse cells, we propose that both enzymes are likely to function as damage-induced p53 Ser20 kinases in a redundant manner. Effects of p53 N-terminal phosphorylation on its protein-protein interactions. Recent work has identified a naturally expressed isoform of p53, deltaNp53, which 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 differently affected by cell type, cell cycle phase and exposure to various stresses. p53 and and DeltaNp53 form heterotetramers and the relative abundance of deltaNp53 influences the transactivation activity and target gene specificity of p53. We recently characterized the binding of TAD1 and TAD2 of p53 to the Taz2 domain of the transcriptional coactivator p300 and found 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. We also previously solved the solution structure of p53 TAD1 in complex with Taz2. Currently, we are determining the solution structure of a p53 TAD2 peptide in complex with Taz2. p53 TAD2 binds to a similar interface on Taz2 and similarly forms a short alpha-helix upon binding, exposing hydrophobic residues to form the primary stabilizing interactions with Taz2. As the effects of phosphorylation differ between the two subdomains, a comparison of the structures of the two complexes will 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 better 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 previously identified SET8/PR-Set7 as the enzyme responsible for monomethylation of Lys382 and showed that this modification repressed p53 transactivation of its target genes. We have since investigated the mechanism of p53 transcriptional repression by this modification. We found that p53 monomethylated at Lys382 is specifically recognized by the triple malignant brain tumor (MBT)-repeats of the chromatin compaction factor L3MBTL1. SET8-mediated methylation of p53 at Lys382 promotes the interaction between L3MBTL1 and p53 in cells as well as the chromatin occupancy of L3MBTL1 at p53 target promoters. In the absence of DNA damage, L3MBTL1 interacts with p53 mono-methylated at Lys382 and represses transcription from the p21 promoter. Activation of p53 by DNA damage is coupled to a decrease in the level of p53 monomethylated-Lys382, abrogation of the L3MBTL1-p53 interaction, and disassociation of L3MBTL1 from the p21 promoter. Also 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 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. Chromatin immunoprecipitation and DNA repair assays suggested that binding of the 53BP1 Tudor domain to p53 dimethylated at Lys382 may facilitate p53 accumulation at DNA damage sites and promote DNA repair.
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