Damage-mediated phosphorylation of human p53 threonine 18 through a cascade mediated by a casein 1-like kinase. Effect on Mdm2 binding

The p53 tumor suppressor protein is stabilized in response to ionizing radiation and accumulates in the nucleus. Stabilization is thought to involve disruption of the interaction between the p53 protein and Mdm2, which targets p53 for degradation. Here we show that the direct association between a p53 N-terminal peptide and Mdm2 is disrupted by phosphorylation of the peptide on Thr(18) but not by phosphorylation at other N-terminal sites, including Ser(15) and Ser(37). Thr(18) was phosphorylated in vitro by casein kinase (CK1); this process required the prior phosphorylation of Ser(15). Thr(18) was phosphorylated in vivo in response to DNA damage, and such phosphorylation required Ser(15). Our results suggest that stabilization of p53 after ionizing radiation may result, in part, from an inhibition of Mdm2 binding through a phosphorylation-phosphorylation cascade involving DNA damage-activated phosphorylation of p53 Ser(15) followed by phosphorylation of Thr(18).     

Phosphorylation of serine 18 regulates distinct p53 functions in mice

The p53 protein acts a tumor suppressor by inducing cell cycle arrest and apoptosis in response to DNA damage or oncogene activation. Recently, it has been proposed that phosphorylation of serine 15 in human p53 by ATM (mutated in ataxia telangiectasia) kinase induces p53 activity by interfering with the Mdm2-p53 complex formation and inhibiting Mdm2-mediated destabilization of p53. Serine 18 in murine p53 has been implicated in mediating an ATM- and ataxia telangiectasia-related kinase-dependent growth arrest. To explore further the physiological significance of phosphorylation of p53 on Ser18, we generated mice bearing a serine-to-alanine mutation in p53. Analysis of apoptosis in thymocytes and splenocytes following DNA damage revealed that phosphorylation of serine 18 was required for robust p53-mediated apoptosis. Surprisingly, p53Ser18 phosphorylation did not alter the proliferation rate of embryonic fibroblasts or the p53-mediated G(1) arrest induced by DNA damage. In addition, endogenous basal levels and DNA damage-induced levels of p53 were not affected by p53Ser18 phosphorylation. p53Ala18 mice developed normally and were not susceptible to spontaneous tumorigenesis, and the reduced apoptotic function of p53Ala18 did not rescue the embryo-lethal phenotype of Mdm2-null mice. These results indicate that phosphorylation of the ATM target site on p53 specifically regulates p53 apoptotic function and further reveal that phosphorylation of p53 serine 18 is not required for p53-mediated tumor suppression.     

Hypophosphorylation of Mdm2 augments p53 stability

The Mdm2 protein mediates ubiquitylation and degradation of p53 and is a key regulator of this tumor suppressor. More recently, it has been shown that Mdm2 is highly phosphorylated within its central acidic domain. In order to address the issue of how these modifications might regulate Mdm2 function, putative phosphorylation sites within this domain were substituted, individually or in pairs, with alanine residues. Mutants with serine-to-alanine substitutions between residues 244 and 260 abolished or at least reduced the capacity of Mdm2 to promote p53 degradation. In each case, loss of degradation function was independent of the ability to bind to p53 or p14ARF. Moreover, each of the Mdm2 mutants completely retained the capacity to act as a ubiquitin ligase in vivo. Thus, ubiquitylation and degradation can be uncoupled. Two-dimensional phosphopeptide mapping coupled with the use of phospho-specific antibodies revealed that Mdm2 is phosphorylated physiologically at several sites within this region, consistent with the idea that phosphorylation is important for Mdm2 activity. Strikingly, treatment of cells with ionizing radiation resulted in a significant decrease in the phosphorylation of residues that are important for p53 turnover. This hypophosphorylation preceded p53 accumulation. These findings indicate that Mdm2 contributes an additional function toward the degradation of p53 that is distinct from its ubiquitin ligase activity and is regulated by phosphorylation. Our model suggests that hypophosphorylation of Mdm2 in response to ionizing irradiation inactivates this novel function, thereby contributing to p53 stabilization.     

Synthesis of complex phosphopeptides as mimics of p53 functional domains.

A complete set of mono-, di- and triphosphorylated peptides comprising amino acids 10-27, the Mdm2 and p300 binding site(s) of p53, with and without a fluorescein label at the N-terminus, was synthesized by step-by-step solid phase synthesis. Fluorescence polarization analysis revealed that phosphorylation at Thr18 decreased binding to recombinant Mdm2 protein compared with the unphosphorylated and the two other single phosphorylated analogues. Unlabelled multiply phosphorylated peptides corresponding to this amino-terminal transactivation domain proved to be powerful tools in analysing the phosphate specificity of existing anti-p53 monoclonal and polyclonal antibodies using direct ELISA. The tetramerization domain of human p53 protein was modelled with a 53 residue-long unlabelled unphosphorylated and Ser315-phosphorylated peptide pair. CD analysis showed similar alpha-helical structures for both peptides and no major difference in the secondary structure could be observed upon phosphorylation. Size-exclusion HPLC indicated that these synthetic oligomerization domain mimics underwent a pH-dependent tetramerization process, but the presence of a phosphate group at Ser315 did not modify the oligomeric state of the 308-360 p53 fragments. Nevertheless, the fluorescein-labelled Ser315 phosphorylated peptide bound to the downstream signalling ligand DNA topoisomerase I protein with slightly higher affinity than did the unphosphorylated analogue.     

Mutation at Ser392 specifically sensitizes mutant p53H175 to mdm2-mediated degradation

Mdm2 is one of the main E3 ubiquitin ligases, which targets both wild type and mutant p53 for degradation. The ability of post-translational modifications, such as phosphorylation, to modulate the function and stability of wild type p53 has been extensively studied. However, their ability to modulate the functions and stability of mutant forms of p53 remains poorly documented. Here we show, for the first time, that the stability of mutant p53 can be regulated by phosphorylation. Mutation of serine 392 to alanine shortens the half life of p53H175, and renders p53H175A392 more sensitive to mdm2-mediated degradation than p53H175. This effect of Ser392 phosphorylation specifically affects p53H175, a misfolded mutant, and does not affect p53W248 which maintains a native conformation. Detailed analysis subsequently showed that the reduced stability of p53H175A392 is not due to an increase in mdm2/p300 binding or polyubiquitin chain formation, uncoupling the extent of polyubiquitin chain formation and the stability of mutant p53. This is supported by the observation that Ser392 mutation enhances polyubiquitin chain formation on p53W248, without reducing its stability. These results suggest that the inhibition of phosphorylation at Ser392 of p53, together with the use of an mdm2-enhancing agent such as nutlin, could present a new therapeutic strategy with which to treat tumors expressing mutant p53H175.     

The p53 oncoprotein is a substrate for tissue transglutaminase kinase activity

Increased expression and activity of the ubiquitous enzyme, tissue transglutaminase (TG2), is consistently seen in a variety of models of apoptosis. The p53 oncoprotein is also involved in apoptosis. Here we investigated the interaction of TG2 with p53 and show that the p53 is a substrate for the recently identified serine/threonine kinase activity of TG2. Phosphospecific antibodies indicated that TG2 phosphorylated p53 at Ser(15) and Ser(20), residues that are critically important in the interaction of p53 with Mdm2. The TG2-induced phosphorylation was abrogated by high Ca(2+) concentrations and inhibited by cystamine, a known inhibitor of TG2 cross-linking activity. Furthermore, we demonstrate that TG2-induced phosphorylation of p53 reduces the ability of p53 to interact with Mdm2. Although TG2 cross-linking activity has been clearly implicated in apoptosis, our observations reported here suggest TG2 modification of p53 could be an additional mechanism whereby TG2 could facilitate apoptosis.     

Mutation of mouse p53 Ser23 and the response to DNA damage

Recent studies have suggested that phosphorylation of human p53 at Ser20 is important for stabilizing p53 in response to DNA damage through disruption of the interaction between MDM2 and p53. To examine the requirement for this DNA damage-induced phosphorylation event in a more physiological setting, we introduced a missense mutation into the endogenous p53 gene of mouse embryonic stem (ES) cells that changes serine 23 (S23), the murine equivalent of human serine 20, to alanine (A). Murine embryonic fibroblasts harboring the p53(S23A) mutation accumulate p53 as well as p21 and Mdm2 proteins to normal levels after DNA damage. Furthermore, ES cells and thymocytes harboring the p53(S23A) mutation also accumulate p53 protein to wild-type levels and undergo p53-dependent apoptosis similarly to wild-type cells after DNA damage. Therefore, phosphorylation of murine p53 at Ser23 is not required for p53 responses to DNA damage induced by UV and ionizing radiation treatment.     

Regulation of Mdm2-Directed Degradation by the C Terminus of p53

The stability of the p53 tumor suppressor protein is regulated by interaction with Mdm2, the product of a p53-inducible gene. Mdm2-targeted degradation of p53 depends on the interaction between the two proteins and is mediated by the proteasome. We show here that in addition to the N-terminal Mdm2 binding domain, the C terminus of p53 participates in the ability of p53 to be degraded by Mdm2. In contrast, alterations in the central DNA binding domain of p53, which change the conformation of the p53 protein, do not abrogate the sensitivity of the protein to Mdm2-mediated degradation. The importance of the C-terminal oligomerization domain to Mdm2-targeted degradation of p53 is likely to reflect the importance of oligomerization of the full-length p53 protein for interaction with Mdm2, as previously shown in vitro. Interestingly, the extreme C-terminal region of p53, outside the oligomerization domain, was also shown to be necessary for efficient degradation, and deletion of this region stabilized the protein without abrogating its ability to bind to Mdm2. Mdm2-resistant p53 mutants were not further stabilized following DNA damage, supporting a role for Mdm2 as the principal regulator of p53 stability in cells. The extreme C terminus of the p53 protein has previously been shown to contain several regulatory elements, raising the possibility that either allosteric regulation of p53 by this domain or interaction between this region and a third protein plays a role in determining the sensitivity of p53 to Mdm2-directed degradation.