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 stabilized and activated following DNA damage through extensive post-translational modification (PTM). Our research has focused on identifying and exploring the biological roles of post-translational modifications on p53 to better understand how they modulate p53 function. Our work on the transcriptional activating function of p53 has focused on its ability to recruit histone acetyl transferase co-activators such as CBP, p300 and PCAF to the promoters of specific target genes. We recently characterized the complex formed between the N-terminal transactivation domain (TAD) of p53 and the Taz2 domain of p300 and the effect of p53 phosphorylation on complex formation. We have determined the effect of various mono-, di-, and tri-phosphorylations on binding of p53(1-39) to Taz2, some of which significantly increased binding. Furthermore, we identified a second binding site for Taz2 within p53 residues 35-59. This second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. Mouse models containing missense mutations at sites of post-translational modification of p53, developed as part of a collaboration, continue to be a valuable resource for investigating the complex effects of single or multiple PTMs of p53 in a physiological setting. To elucidate functions of specific PTMs after DNA damage, knock-in mice were generated in which Ser18 (Ser15 in humans) was mutated to alanine in both alleles of endogenous p53, preventing phosphorylation. Additionally, knock-in mice were generated in which Lys317 (Lys320 in humans) was mutated to arginine, maintaining a positive charge but blocking PCAF acetylation at this site. Global proteomics analyses were performed using cleavable ICAT quantitative mass spectrometry to determine the change in the response of thymocytes to ionizing radiation (IR) in the mutant knock-in mice as compared with wild type (wt) mice. While many proteins were found to be significantly affected by IR in the wt thymocytes, there was a decrease in the number of affected proteins in the p53(S18A) thymocytes. Pro-apoptotic proteins that were increased in the wt after IR were not significantly affected in p53(S18A) mice, consistent with previous reports that thymocyte apoptosis is decreased in these mutant mice. When the effects of IR in the wt and p53(K317R) samples were compared, 46 proteins were found to be affected by the mutation (p less than 0.05), including proteins involved in apoptosis, transcription, and translation. Pathway analysis of the affected proteins suggests an increase in p53 activity in the p53(K317R) thymocytes, as well as a decrease in TNFalpha signaling. These results suggest that acetylation of Lys317 modulates the functions of p53 and influences the cross-talk between the DNA damage response and other signaling pathways. We have also studied the effects of p53 methylation on its function. Mediation of modular protein-protein interactions by lysine methylation of histone proteins regulates diverse chromatin signaling pathways. Lysines can be mono-, di- or tri- methylated, with the higher methylation states coupled to distinct functions by protein effectors that selectively recognize distinct lysine methylation events. Recently, lysine monomethylation of the tumor suppressor protein p53 has been shown to modulate its activity; however, the molecular mechanism that links monomethylated p53 to function is unclear. We have identified a novel p53 species dimethylated at Lys382 (p53K382me2) and shown that the tandem tudor domain (TD) of the DNA damage response mediator 53BP1 acts as an """"""""effector"""""""" for this modification. We demonstrated that 53BP1(TD) preferentially recognizes p53K382me2 over other potential p53 and histone lysine methylation sites. Wip1 is a conserved PP2C phosphatase expressed at low levels in most tissues and transcriptionally induced after DNA damage in a p53-dependent manner. Wip1 substrates discovered thus far are p53, p38MAPK, UNG2, Chk1, Chk2 and ATM. Wip1 dephosphorylates serine and threonine residues within pTXpY and pT/SQ motifs, and we have recently characterized its substrate specificity using biochemical studies. As many of the known pT/SQ Wip1 substrates identified thus far are phosphorylated by ATM, we searched for motifs compatible with Wip1 specificity among known ATM targets to identify novel substrates. Recently, we found that Wip1 dephosphorylated Ser112 of 4E-BP1 in vitro , and interacted with proteins involved in the initiation of translation. In addition, Wip1 was able to negatively modulate eIF4E activity and the rate of cap-dependent translation both in cellular models and in vivo . The use of specific kinase inhibitors showed that the effect of Wip1 knock-down was independent of mTOR and p38MAPK, but required ATM activity. Therefore, the modulation of eIF4E activity by Wip1 may be occurring at two levels: by inhibiting ATM activity through dephosphorylation of Ser1981, and by directly dephosphorylating Ser112 of 4E-BP1. In cells containing wt p53, the levels of Wip1 mRNA and protein increase following exposure to genotoxic stress, but the mechanism of regulation by p53 was unknown. Using HCT116 p53+/+ and p53-/- cells, we have characterized a conserved p53 response element located in the 5′ untranslated region (UTR) of the Wip1 gene that is required for the p53-dependent induction of transcription from the human Wip1 promoter. CREB binding to the CRE contributes to the basal expression of Wip1 and directs transcription initiation at upstream sites. Following exposure to ultraviolet (UV) or IR, the abundance of transcripts with short 5′ UTRs increased in cells containing wt p53, indicating increased utilization of downstream transcription initiation sites. In these cells, exposure to UV resulted in increased Wip1 protein levels even when Wip1 mRNA levels remained constant, indicating post-transcriptional regulation of Wip1 protein levels. As described above, the Wip1 gene is amplified and overexpressed in some human tumors with wt p53, and our results support the hypothesis that Wip1 phosphatase is a candidate proto-oncogene that may promote tumorigenesis through inactivation of p53. We have identified several specific Wip1 inhibitors through a combination of rational design and a screening assay using recombinant Wip1. Using a pyrrole based scaffold, we have synthesized a series of small molecules that mimic the three-dimensional arrangement of the polar and hydrophobic functional groups of a previously identified cyclic-peptide inhibitor. Iterative optimization cycles of design, solution-phase synthesis and activity testing have led to an effective inhibitor of Wip1 that is selective for this phosphatase over related enzymes both in vitro and in cellular assays. We have used solution-based synthesis of the best inhibitor to produce quantities sufficient for in vivo studies and crystallographic analysis, which will facilitate further optimization of the binding selectivity and aff [summary truncated at 7800 characters]
Showing the most recent 10 out of 31 publications
