Shin'ichi Saito

Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice

SIRT1 is a mammalian homolog of the Saccharomyces cerevisiae chromatin silencing factor Sir2. Dominant-negative and overexpression studies have implicated a role for SIRT1 in deacetylating the p53 tumor suppressor protein to dampen apoptotic and cellular senescence pathways. To elucidate SIRT1 function in normal cells, we used gene-targeted mutation to generate mice that express either a mutant SIRT1 protein that lacks part of the catalytic domain or has no detectable SIRT1 protein at all. Both types of SIRT1 mutant mice and cells had essentially the same phenotypes. SIRT1 mutant mice were small, and exhibited notable developmental defects of the retina and heart, and only infrequently survived postnatally. Moreover, SIRT1-deficient cells exhibited p53 hyperacetylation after DNA damage and increased ionizing radiation-induced thymocyte apoptosis. In SIRT1-deficient embryonic fibroblasts, however, p53 hyperacetylation after DNA damage was not accompanied by increased p21 protein induction or DNA damage sensitivity. Together, our observations provide direct evidence that endogenous SIRT1 protein regulates p53 acetylation and p53-dependent apoptosis, and show that the function of this enzyme is required for specific developmental processes.

p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression

The tumour suppressor p53 becomes activated in response to upstream stress signals, such as DNA damage, and causes cell-cycle arrest or apoptosis. Here we report a novel role for p53 in the differentiation of mouse embryonic stem cells (ESCs). p53 binds to the promoter of Nanog, a gene required for ESC self-renewal, and suppresses Nanog expression after DNA damage. The rapid down-regulation of Nanog mRNA during ESC differentiation correlates with the induction of p53 transcriptional activity and Ser 315 phosphorylation. The importance of Ser 315 phosphorylation was revealed by the finding that induction of p53 activity is impaired in p53S315A knock-in ESCs during differentiation, leading to inefficient suppression of Nanog expression. The decreased inhibition of Nanog expression in p53S315A ESCs during differentiation is due to an impaired recruitment of the co-repressor mSin3a to the Nanog promoter. These findings indicate an alternative mechanism for p53 to maintain genetic stability in ESCs, by inducing the differentiation of ESCs into other cell types that undergo efficient p53-dependent cell-cycle arrest and apoptosis.

Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation

Components of the ras signaling pathway contribute to activation of cellular p53. In MCF‐7 cells, p38 kinase activated p53 more effectively than other members of the ras pathway. p53 and p38 kinase exist in the same physical complex, and co‐expression of p38 stabilized p53 protein. In vitro, p38 kinase phosphorylated p53 at Ser33 and Ser46, a newly identified site. Mutation of these sites decreased p53‐mediated and UV‐induced apoptosis, and the reduction correlated with total abrogation of UV‐induced phosphorylation on Ser37 and a significant decrease in Ser15 phosphorylation in mutant p53 containing alanine at Ser33 and Ser46. Inhibition of p38 activation after UV irradiation decreased phosphorylation of Ser33, Ser37 and Ser15, and also markedly reduced UV‐induced apoptosis in a p53‐dependent manner. These results suggest that p38 kinase plays a prominent role in an integrated regulation of N‐terminal phosphorylation that regulates p53‐mediated apoptosis after UV radiation.

Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis.

Phosphorylation of p53 at Ser 46 was shown to regulate p53 apoptotic activity. Here we demonstrate that homeodomain-interacting protein kinase-2 (HIPK2), a member of a novel family of nuclear serine/threonine kinases, binds to and activates p53 by directly phosphorylating it at Ser 46. HIPK2 localizes with p53 and PML-3 into the nuclear bodies and is activated after irradiation with ultraviolet. Antisense inhibition of HIPK2 expression reduces the ultraviolet-induced apoptosis. Furthermore, HIPK2 and p53 cooperate in the activation of p53-dependent transcription and apoptotic pathways. These data define a new functional interaction between p53 and HIPK2 that results in the targeted subcellular localization of p53 and initiation of apoptosis.

p300/CBP‐mediated p53 acetylation is commonly induced by p53‐activating agents and inhibited by MDM2

The tumor suppressor p53 is activated in response to many types of cellular and environmental insults via mechanisms involving post‐translational modification. Here we demonstrate that, unlike phosphorylation, p53 invariably undergoes acetylation in cells exposed to a variety of stress‐inducing agents including hypoxia, anti‐metabolites, nuclear export inhibitor and actinomycin D treatment. In vivo, p53 acetylation is mediated by the p300 and CBP acetyltransferases. Overexpression of either p300 or CBP, but not an acetyltransferase‐deficient mutant, efficiently induces specific p53 acetylation. In contrast, MDM2, a negative regulator of p53, actively suppresses p300/CBP‐mediated p53 acetylation in vivo and in vitro. This inhibitory activity of MDM2 on p53 acetylation is in turn abrogated by tumor suppressor p19ARF, indicating that regulation of acetylation is a central target of the p53–MDM2–p19ARF feedback loop. Functionally, inhibition of deacetylation promotes p53 stability, suggesting that acetylation plays a positive role in the accumulation of p53 protein in stress response. Our results provide evidence that p300/CBP‐mediated acetylation may be a universal and critical modifi cation for p53 function.

Chk2-deficient mice exhibit radioresistance and defective p53-mediated transcription

The mammalian Chk2 kinase is thought to mediate ATM‐dependent signaling in response to DNA damage. The physiological role of mammalian Chk2 has now been investigated by the generation of Chk2‐deficient mice. Although Chk2−/− mice appeared normal, they were resistant to ionizing radiation (IR) as a result of the preservation of splenic lymphocytes. Thymocytes and neurons of the developing brain were also resistant to IR‐induced apoptosis. The IR‐induced G1/S cell cycle checkpoint, but not the G2/M or S phase checkpoints, was impaired in embryonic fibroblasts derived from Chk2−/− mice. IR‐induced stabilization of p53 in Chk2−/− cells was 50–70% of that in wild‐type cells. Caffeine further reduced p53 accumulation, suggesting the existence of an ATM/ATR‐dependent but Chk2‐independent pathway for p53 stabilization. In spite of p53 protein stabilization and phosphorylation of Ser23, p53‐dependent transcriptional induction of target genes, such as p21 and Noxa, was not observed in Chk2−/− cells. Our results show that Chk2 plays a critical role in p53 function in response to IR by regulating its transcriptional activity as well as its stability.

Amplification of PPM1D in human tumors abrogates p53 tumor-suppressor activity

Expression of oncogenic Ras in primary human cells activates p53, thereby protecting cells from transformation. We show that in Ras-expressing IMR-90 cells, p53 is phosphorylated at Ser33 and Ser46 by the p38 mitogen-activated protein kinase (MAPK). Activity of p38 MAPK is regulated by the p53-inducible phosphatase PPM1D, creating a potential feedback loop. Expression of oncogenic Ras suppresses PPM1D mRNA induction, leaving p53 phosphorylated at Ser33 and Ser46 and in an active state. Retrovirus-mediated overexpression of PPM1D reduced p53 phosphorylation at these sites, abrogated Ras-induced apoptosis and partially rescued cells from cell-cycle arrest. Inactivation of p38 MAPK (the product of Mapk14) in vivo by gene targeting or by PPM1D overexpression expedited tumor formation after injection of mouse embryo fibroblasts (MEFs) expressing E1A+Ras into nude mice. The gene encoding PPM1D (PPM1D, at 17q22/q23) is amplified in human breast-tumor cell lines and in approximately 11% of primary breast tumors, most of which harbor wildtype p53. These findings suggest that inactivation of the p38 MAPK through PPM1D overexpression resulting from PPM1D amplification contributes to the development of human cancers by suppressing p53 activation.

Nitric oxide-induced cellular stress and p53 activation in chronic inflammation

Free radical-induced cellular stress contributes to cancer during chronic inflammation. Here, we investigated mechanisms of p53 activation by the free radical, NO. NO from donor drugs induced both ataxia-telangiectasia mutated (ATM)- and ataxia-telangiectasia mutated and Rad3-related-dependent p53 posttranslational modifications, leading to an increase in p53 transcriptional targets and a G2/M cell cycle checkpoint. Such modifications were also identified in cells cocultured with NO-releasing macrophages. In noncancerous colon tissues from patients with ulcerative colitis (a cancer-prone chronic inflammatory disease), inducible NO synthase protein levels were positively correlated with p53 serine 15 phosphorylation levels. Immunostaining of HDM-2 and p21WAF1 was consistent with transcriptionally active p53. Our study highlights a pivotal role of NO in the induction of cellular stress and the activation of a p53 response pathway during chronic inflammation.

p53 transcriptional activity is essential for p53‐dependent apoptosis following DNA damage

p53‐mediated transcription activity is essential for cell cycle arrest, but its importance for apoptosis remains controversial. To address this question, we employed homologous recombination and LoxP/Cre‐mediated deletion to produce mutant murine embryonic stem (ES) cells that express p53 with Gln and Ser in place of Leu25 and Trp26, respectively. p53Gln25Ser26 was stable but did not accumulate after DNA damage; the expression of p21/Waf1 and PERP was not induced, and p53‐dependent repression of MAP4 expression was abolished. Therefore, p53Gln25Ser26 is completely deficient in transcriptional activation and repression activities. After DNA damage by UV radiation, p53Gln25Ser26 was phosphorylated at Ser18 but was not acetylated at C‐terminal sites, and its DNA binding activity did not increase, further supporting a role for p53 acetylation in the activation of sequence‐specific DNA binding activity. Most importantly, p53Gln25Ser26 mouse thymocytes and ES cells, like p53−/− cells, did not undergo DNA damage‐induced apoptosis. We conclude that the transcriptional activities of p53 are required for p53‐dependent apoptosis.

ATM Mediates Phosphorylation at Multiple p53 Sites, Including Ser46, in Response to Ionizing Radiation

The p53 tumor suppressor protein preserves genome integrity by regulating growth arrest and apoptosis in response to DNA damage. In response to ionizing radiation (IR), ATM, the gene product mutated in ataxia telangiectasia, stabilizes and activates p53 through phosphorylation of Ser15 and (indirectly) Ser20. Here we show that phosphorylation of p53 on Ser46, a residue important for p53 apoptotic activity, as well as on Ser9, in response to IR also is dependent on the ATM protein kinase. IR-induced phosphorylation at Ser46 was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, but not PD169316, a p38 MAPK inhibitor. p53 C-terminal acetylation at Lys320 and Lys382, which may stabilize p53 and activate sequence-specific DNA binding, required Ser15phosphorylation by ATM and was enhanced by phosphorylation at nearby residues including Ser6, Ser9, and Thr18. These observations, together with the proposed role of Ser46 phosphorylation in mediating apoptosis, suggest that ATM is involved in the initiation of p53-dependent apoptosis after IR in human lymphoblastoid cells.