Mutations in proline 82 of p53 impair its activation by Pin1 and Chk2 in response to DNA damage

Tumor suppression by the p53 protein largely depends on the elimination of damaged cells by apoptosis. Mutations in the polyproline region (PPR) of p53 impair its apoptotic function. Deletion of the PPR renders p53 more sensitive to inhibition by Mdm2 via an unknown mechanism. We have explored the mechanism by which the PPR modulates the p53/Mdm2 loop. Proline 82 of p53 was identified to be essential for its interaction with the checkpoint kinase 2 (Chk2) and consequent phosphorylation of p53 on serine 20, following DNA damage. These physical and functional interactions are regulated by Pin1 through cis-trans isomerization of proline 82. Our study unravels the pathway by which Pin1 activates p53 in response to DNA damage and explains how Pin1 protects p53 from Mdm2. Further, we propose a role for Pin1-dependent induction of p53 conformational change as a mechanism responsible for the enhanced interaction between p53 and Chk2 following DNA damage. Importantly, our findings elucidate the selection for mutations in the Pin1 target Thr81/Pro82 motif within the PPR of p53 in human cancer.     

The RNA surveillance protein SMG1 activates p53 in response to DNA double-strand breaks but not exogenously oxidized mRNA

DNA damage, stalled replication forks, errors in mRNA splicing and availability of nutrients activate specific phosphatidylinositiol-3-kinase-like kinases (PIKKs) that in turn phosphorylate downstream targets such as p53 on serine 15. While the PIKK proteins ATM and ATR respond to specific DNA lesions, SMG1 responds to errors in mRNA splicing and when cells are exposed to genotoxic stress. Yet, whether genotoxic stress activates SMG1 through specific types of DNA lesions or RNA damage remains poorly understood. Here, we demonstrate that siRNA oligonucleotides targeting the mRNA surveillance proteins SMG1, Upf1, Upf2 or the PIKK protein ATM attenuated p53 (ser15) phosphorylation in cells damaged by high oxygen (hyperoxia), a model of persistent oxidative stress that damages nucleotides. In contrast, loss of SMG1 or ATM, but not Upf1 or Upf2 reduced p53 (ser15) phosphorylation in response to DNA double strand breaks produced by expression of the endonuclease I-PpoI. To determine whether SMG1-dependent activation of p53 was in response to oxidative mRNA damage, mRNA encoding green fluorescence protein (GFP) transcribed in vitro was oxidized by Fenton chemistry and transfected into cells. Although oxidation of GFP mRNA resulted in dose-dependent fragmentation of the mRNA and reduced expression of GFP, it did not stimulate p53 or the p53-target gene p21. These findings establish SMG1 activates p53 in response to DNA double strand breaks independent of the RNA surveillance proteins Upf1 or Upf2; however, these proteins can stimulate p53 in response to oxidative stress but not necessarily oxidized RNA.Key words: DNA double strand breaks, nonsense-mediated mRNA decay (NMD), oxidative stress, phosphatidylinositiol-3-kinase-like kinases (PIKKs), RNA damage     

Downregulation of MDM2 stabilizes p53 by inhibiting p53 ubiquitination in response to specific alkylating agents

p53 is stabilized in response to DNA damaging stress. This stabilization is thought to result from phosphorylation in the N-terminus of p53, which inhibits p53:MDM2 binding, and prevents MDM2 from promoting p53 ubiquitination. In this report, the DNA alkylating agents mitomycin C (MMC) and methylmethane sulfonate (MMS), as well as UV radiation, stabilized p53 in a manner independent of phosphorylation in p53 N-terminus. This stabilization coincided with decreased levels of MDM2 mRNA and protein, and a corresponding decrease in p53 ubiquitination. Importantly, MDM2 overexpression inhibited the stabilization of p53 and decrease in ubiquitination following MMC, MMS, and UV treatment. This indicates that downregulation of MDM2 contributes to the stabilization of p53 in response to these agents.     

The RNA surveillance protein SMG1 activates p53 in response to DNA double-strand breaks but not exogenously oxidized mRNA

DNA damage, stalled replication forks, errors in mRNA splicing, and availability of nutrients activate specific phosphatidylinositiol-3 kinase-like kinases (PIKKs) that in turn phosphorylate downstream targets such as p53 on serine 15. While the PIKK proteins ATM and ATR respond to specific DNA lesions, SMG1 responds to errors in mRNA splicing and when cells are exposed to genotoxic stress. Yet, whether genotoxic stress activates SMG1 through specific types of DNA lesions or RNA damage remains poorly understood. Here, we demonstrate that siRNA oligonucleotides targeting the mRNA surveillance proteins SMG1, Upf1, Upf2, or the PIKK protein ATM attenuated p53 (ser15) phosphorylation in cells damaged by high oxygen (hyperoxia), a model of persistent oxidative stress that damages nucleotides. In contrast, loss of SMG1 or ATM, but not Upf1 or Upf2 reduced p53 (ser15) phosphorylation in response to DNA double strand breaks produced by expression of the endonuclease I-PpoI. To determine whether SMG1-dependent activation of p53 was in response to oxidative mRNA damage, mRNA encoding green fluorescence protein (GFP) transcribed in vitro was oxidized by Fenton chemistry and transfected into cells. Although oxidation of GFP mRNA resulted in dose-dependent fragmentation of the mRNA and reduced expression of GFP, it did not stimulate p53 or the p53-target gene p21. These findings establish SMG1 activates p53 in response to DNA double-strand breaks independent of the RNA surveillance proteins Upf1 or Upf2; however, these proteins can stimulate p53 in response to oxidative stress but not necessarily oxidized RNA.     

Phosphorylation and nuclear accumulation are distinct events contributing to the activation of p53

It has been recently shown that ionizing radiation (IR) and the mRNA synthesis inhibitor 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) act in synergy to induce p53-mediated transactivation of reporter plasmids in human cells [Oncogene 19 (2000) 3829]. We have extended these studies and show that ionizing radiation and DRB also act in synergy to induce ATM-mediated phosphorylation of the ser15 site of p53 and enhance the expression of endogenous p21 protein. Examination of the localization of p53 revealed that while DRB did not induce phosphorylation of the ser15 site of p53 but efficiently accumulated p53 in the nucleus, ionizing radiation induced phosphorylation of the ser15 site of p53 without prolonged nuclear accumulation. Importantly, the combination of DRB and IR resulted in a strong accumulation of phosphorylated p53 in the nucleus that was more persistent then p53 accumulation after IR alone. Furthermore, the nuclear export inhibitor leptomycin B showed a similar synergy with IR as did DRB regarding ser15 phosphorylation of p53 and p21 induction. These results suggest that the synergistic activation of the p53 response by the combination treatment is due to the activation of two distinct pathways where DRB causes the prolonged nuclear accumulation of p53 while ionizing radiation activates p53 by ATM-mediated phosphorylation.     

Acetylation of Lysine 382 and Phosphorylation of Serine 392 in p53 Modulate the Interaction between p53 and MDC1 In Vitro

Occurrence of DNA damage in a cell activates the DNA damage response, a survival mechanism that ensures genomics stability. Two key members of the DNA damage response are the tumor suppressor p53, which is the most frequently mutated gene in cancers, and MDC1, which is a central adaptor that recruits many proteins to sites of DNA damage. Here we characterize the in vitro interaction between p53 and MDC1 and demonstrate that p53 and MDC1 directly interact. The p53-MDC1 interaction is mediated by the tandem BRCT domain of MDC1 and the C-terminal domain of p53. We further show that both acetylation of lysine 382 and phosphorylation of serine 392 in p53 enhance the interaction between p53 and MDC1. Additionally, we demonstrate that the p53-MDC1 interaction is augmented upon the induction of DNA damage in human cells. Our data suggests a new role for acetylation of lysine 382 and phosphorylation of serine 392 in p53 in the cellular stress response and offers the first evidence for an interaction involving MDC1 that is modulated by acetylation.     

p53 negatively regulates Pin1 expression under ER stress

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a major role in the development of many diseases. A previous study indicated that the apoptotic regulator p53 is significantly increased in response to ER stress and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood. Here, we investigated whether p53 contributes to the impairment of Pin1 signaling under ER stress. We found that treatment with thapsigargin, a stimulator of p53 expression and an inducer of ER stress, decreased Pin1 expression in HCT116 cells. Also, we identified functional p53 response elements (p53REs) in the Pin1 promoter. Overexpression of p53 significantly decreased Pin1 expression in HCT116 cells while abolition of p53 gene expression induced Pin1 expression. Pin1 expression was significantly increased by treatment with the p53 inhibitor pifithrin-α or down-regulation of p53 expression. Taken together, ER stress decreased Pin1 expression through p53 activation, and this mechanism may be associated with ER stress-induced cell death. These data reported here support the importance of Pin1 as a potential target molecule mediating tumor development.     

High glucose induces mitochondrial p53 phosphorylation by p38 MAPK in pancreatic RINm5F cells

Pancreatic β-cell death in type 2 diabetes has been related to p53 subcellular localisation and phosphorylation. However, the mechanisms by which p53 is phosphorylated and its activation in response to oxidative stress remain poorly understood. Therefore, the aim of this study was to investigate mitochondrial p53 phosphorylation, its subcellular localisation and its relationship with apoptotic induction in RINm5F cells cultured under high glucose conditions. Our results show that p53 phosphorylation in the mitochondrial fraction was greater at ser392 than at ser15. This increased phosphorylation correlated with an increase in reactive oxygen species, a decrease in the Bcl-2/Bax ratio, a release of cytochrome c and an increase in the rate of apoptosis. We also observed a decline in ERK 1/2 phosphorylation over time, which is an indicator of cell proliferation. To identify the kinase responsible for phosphorylating p53, p38 mitogen-activated protein kinase (MAPK) activation was analysed. We found that high glucose induced an increase in p38 MAPK phosphorylation in the mitochondria after 24–72 h. Moreover, the phosphorylation of p53 (ser392) by p38 MAPK in mitochondria was confirmed by colocalisation studies with confocal microscopy. The addition of a specific p38 MAPK inhibitor (SB203580) to the culture medium during high glucose treatment blocked p53 mobilisation to the mitochondria and phosphorylation; thus, the release of cytochrome c and the apoptosis rate in RINm5F cells decreased. These results suggest that mitochondrial p53 phosphorylation by p38 MAPK plays an important role in RINm5F cell death under high glucose conditions.     

DNA damage-induced apoptosis requires the DNA-dependent protein kinase,and is mediated by the latent population of p53

Mouse embryo fibroblasts (MEFs) expressing the adenovirus E1A protein undergo apoptosis upon exposure to ionizing radiation. We show here that immediately following gamma-irradiation, latent p53 formed a complex with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK(CS)). The complex formation was DNase sensitive, suggesting that the proteins came together on the DNA, conceivably at strand breaks. This association was accompanied by phosphorylation of pre-existing, latent p53 at Ser18 (corresponding to Ser15 in human p53), which was not found in DNA-PK(CS)(-/-) cells. Most significantly, DNA damage-induced apoptosis was abolished in both DNA-PK(CS)(-/-) and p53(-/-) cells. In addition, blocking synthesis of inducible p53 by cycloheximide did not abrogate apoptosis, suggesting that the latent population of p53 is sufficient for executing the apoptotic program. Finally, E1A-expressing MEFs from a p53 "knock-in" mouse where Ser18 was mutated to an alanine had an attenuated apoptotic response, indicating that phosphorylation of this site by DNA-PK is a contributing factor for apoptosis.     

p53DINP1, a p53-inducible gene, regulates p53-dependent apoptosis

Using the differential display method combined with a cell line that carries a well-controlled expression system for wild-type p53, we isolated a p53-inducible gene, termed p53DINP1 (p53-dependent damage-inducible nuclear protein 1). Cell death induced by DNA double-strand breaks (DSBs), as well as Ser46 phosphorylation of p53 and induction of p53AIP1, were blocked when we inhibited expression of p53DINP1 by means of an antisense oligonucleotide. Overexpression of p53DINP1 and DNA damage by DSBs synergistically enhanced Ser46 phosphorylation of p53, induction of p53AIP1 expression, and apoptotic cell death. Furthermore, the protein complex interacting with p53DINP1 was shown to phosphorylate Ser46 of p53. Our results suggest that p53DINP1 may regulate p53-dependent apoptosis through phosphorylation of p53 at Ser46, serving as a cofactor for the putative p53-Ser46 kinase.     

Acetylation of mouse p53 at lysine 317 negatively regulates p53 apoptotic activities after DNA damage

Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53(K317R) mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53(K317R) MEFs after various types of DNA damage, increased p53-dependent apoptosis was observed in p53(K317R) thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53(K317R) thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.     

p73 and MDM2 confer the resistance of epidermoid carcinoma to cisplatin by blocking p53

p73 responds to DNA damage and exerts its pro-apoptotic function. However, p73 might contribute to the development of drug-resistance in certain tumor cells. In this study, we found that p73 and MDM2 correlate with cisplatin-resistant phenotype of human epidermoid carcinoma-derived cells. p73 and MDM2 were kept at low levels in the cisplatin-sensitive KB-3-1 cells, whereas p53 was induced to be phosphorylated at Ser-15 in response to cisplatin. In contrast, p73 and MDM2 were expressed at higher levels, and cisplatin-mediated p53 phosphorylation was undetectable in the cisplatin-resistant KCP-4 cells. Enforced expression of p73 in KB-3-1 cells caused an accumulation of unphosphorylated form of p53 and MDM2, and conferred the cisplatin resistance. Collectively, our results suggest that a loss of the cisplatin sensitivity is at least in part due to a lack of cisplatin-induced p53 phosphorylation, and p73 might cooperate with MDM2 to be involved in this process.