A Novel p53 Phosphorylation Site within the MDM2 Ubiquitination Signal

Abstract

Exposure to a wide variety of genotoxic and metabolic stresses leads to the activation of the p53 tumor suppressor protein, a sequence-specific DNA-binding protein and stressactivated transcription factor that controls and coordinates the expression of a battery of genes involved in transient growth arrest, DNA and cellular repair, and/or apoptosis (1). The function of the p53 protein is regulated post-translationally by enzymes that catalyze p53 ubiquitination, acetylation, and phosphorylation. In the absence of stress, the specific activity of p53 is suppressed by the action of E3 ubiquitin ligases like MDM2 that promote proteasomal degradation of the protein (2). In response to stress signals like DNA damage, this degradation program is suppressed, and sets of protein kinase pathways, notably ATM, trigger activation of the protein.The mechanisms of p53 activation by phosphorylation at the most evolutionarily conserved phosphoacceptor sites has been assigned both biochemically and genetically. The phosphoacceptor sites in the p53 transactivation domain are the most highly conserved between vertebrates and invertebrates: phosphorylation of p53 at Ser 20 stabilizes the interaction with acetyltransferases like p300 and in turn stimulates DNA-dependent acetylation (3, 4); phosphorylation at Ser 15 can stimulate CBP binding and p53 acetylation (5); and phosphorylation at Thr 18 can both stabilize p300 binding and reduce MDM2 binding (4, 6). The stabilization of p300 relates to the conversion of intrinsically unstructured activation motifs in p53 to a more helical character with a higher affinity for p300 (7). Mice with mutations at the equivalent Ser 20 residue develop spontaneous B-cell lymphomas (8), and Ser 15 mutant transgenes develop spontaneous late onset lymphoma (9). The Ser 392 phosphoacceptor site in the C-terminal domain of p53 is the second most highly conserved class of phosphoacceptor site but only within vertebrates. Increased phosphorylation of p53 at the Ser 392 site occurs in vivo after UV and ionizing radiation (10, 11), and this stimulates the sequence-specific DNA-binding function of p53 (12). Phosphorylation of p53 at Ser 392 enhances the stability of the p53 tetramerization domain (13), and phosphomimetic mutation at codon 392 results in enhanced thermostability of the p53 tetramer (14), providing biophysical evidence for conformational changes of this phosphorylation on p53. Genetic studies in mice have shown that mutation of the CK2 site results in enhanced skin or bladder cancer in response to UV damage or carcinogen exposure (15,16), and mouse embryo fibroblasts from such transgenic mice also have an attenuated p53 transcriptome (17). Further, the enhanced phosphorylation of p53 in the basal/ stem cells of UV-irradiated human skin (11) is attributable to the transcriptional activation of ATM by ⌬Np63 (18). Although these biochemical and genetic studies provide a paradigm for how phosphorylation can regulate p53 protein function at the most highly conserved phosphorylation sites, the effec...

A Novel p53 Phosphorylation Site within the MDM2 Ubiquitination Signal

“…Thus,p53 S269D is phenotypically equivalent to p53 R175H in that it is inactive (24) and highly sensitive to ubiquitin-like adducts in cells. By contrast, because transfected p53 S269A can be more active than WT p53 at inducing elevated levels of MDM2 protein (24), the observed ubiquitin-like adducts of p53 S269A can be attributed to enhanced induction of endogenous MDM2 protein. This phenomenon has been observed previously using the gain-of-function wild type p53 mutants in the S9-S10 loop: p53 S261A and p53 S264A (44) (highlighted in Fig.…”

“…S269A was also found to co-immunoprecipitate Hsp70, despite its wild type-like activity (24). This suggests that this mutant (S269A) is partially destabilized, despite being fully active.…”

“…S269D Is in an Unfolded or Mutant Conformation-A variety of mechanisms could account for inactivation of p53 following phosphorylation at Ser 269 (24). Inactivating p53 mutation of one neighboring residue within this MDM2-binding site in the S9-S10 linker region (p53 F270A ) has been shown to unfold p53 protein and promote "hyperubiquitination" of p53 in vivo (44).…”

“…protein exhibits a mutant conformation in vivo, thus explaining the inability of p53 S269D to act as a transcription factor (24). To test this, we used conformation-specific monoclonal antibodies to examine folding of the p53 isoforms.…”

Impact of the Adenoviral E4 Orf3 Protein on the Activity and Posttranslational Modification of p53

“…4B). Phosphorylation of residues within this domain has been demonstrated to alter its conformation, thereby impairing both DNA binding and the interaction of p53 with transcriptional coactivators (110,111). The tetramerization and C-terminal basic domains exhibited many differences in the extent and character of modifications at specific Ser residues, Arg residues, and Lys residues for all classes of PTM among the p53 populations.…”