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p53/TAp63 and AKT regulate mammalian target of rapamycin complex 1 (mTORC1) signaling through two independent parallel pathways in the presence of DNA damage.

Cam M, Bid HK, Xiao L, Zambetti GP, Houghton PJ, Cam H - J. Biol. Chem. (2013)

Bottom Line: Under conditions of DNA damage, the mammalian target of rapamycin complex 1 (mTORC1) is inhibited, preventing cell cycle progression and conserving cellular energy by suppressing translation.We show that suppression of mTORC1 signaling to 4E-BP1 requires the coordinated activity of two tumor suppressors, p53 and p63.These data indicate that the negative regulation of cap-dependent translation by mTORC1 inhibition subsequent to DNA damage is abrogated in most human cancers.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Childhood Cancer and Blood Diseases, Nationwide Children's Hospital, Columbus, Ohio 43205.

ABSTRACT
Under conditions of DNA damage, the mammalian target of rapamycin complex 1 (mTORC1) is inhibited, preventing cell cycle progression and conserving cellular energy by suppressing translation. We show that suppression of mTORC1 signaling to 4E-BP1 requires the coordinated activity of two tumor suppressors, p53 and p63. In contrast, suppression of S6K1 and ribosomal protein S6 phosphorylation by DNA damage is Akt-dependent. We find that loss of either p53, required for the induction of Sestrin 1/2, or p63, required for the induction of REDD1 and activation of the tuberous sclerosis complex, prevents the DNA damage-induced suppression of mTORC1 signaling. These data indicate that the negative regulation of cap-dependent translation by mTORC1 inhibition subsequent to DNA damage is abrogated in most human cancers.

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p53−/− and p63−/− MEFs are not able to suppress 4E-BP1 phosphorylation in response to DNA damage.A, re-expression of p53 restores DNA damage induced dephosphorylation of 4E-BP1. p53-lox-STOP-lox MEFs were infected with the indicated adenoviruses (Adeno). 24 h after infection, MEFs were treated with the indicated DNA-damaging agents for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies as shown. Eto, etoposide; Cis, cisplatin; Top, topotecan. B, in contrast to p73−/− MEFs, p53−/− and p63−/− MEFs are not able to inhibit 4E-BP1 phosphorylation in response to DNA damage. MEFs of each genotype were treated with the indicated DNA-damaging agents for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies as shown. IR, ionizing radiation; Ctr, control. C, DNA damage-dependent Sestrin-2 induction is abolished in p53−/− MEFs, and REDD1 induction is abolished in p63−/− MEFs. MEFs of each genotype were treated with the indicated DNA-damaging agents for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies as shown. D, luciferase reporter assay of the REDD1 promoter activity in NHDF. A REDD1 promoter-luciferase construct was cotransfected (Lipofectamine 2000) with the indicated plasmids. 24 h after transfection, cells were treated with topotecan (10 μm), etoposide (20 μm), cisplatin (10 μm), or dimethyl sulfoxide (DMSO) as a control for 24 h. Luciferase assay was performed according to the instructions of the manufacturer (Promega). Error bars show the mean ± S.D. for triplicate wells in a representative experiment. mut, mutant; pcDNA, cells transfected with empty plasmid.
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Figure 5: p53−/− and p63−/− MEFs are not able to suppress 4E-BP1 phosphorylation in response to DNA damage.A, re-expression of p53 restores DNA damage induced dephosphorylation of 4E-BP1. p53-lox-STOP-lox MEFs were infected with the indicated adenoviruses (Adeno). 24 h after infection, MEFs were treated with the indicated DNA-damaging agents for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies as shown. Eto, etoposide; Cis, cisplatin; Top, topotecan. B, in contrast to p73−/− MEFs, p53−/− and p63−/− MEFs are not able to inhibit 4E-BP1 phosphorylation in response to DNA damage. MEFs of each genotype were treated with the indicated DNA-damaging agents for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies as shown. IR, ionizing radiation; Ctr, control. C, DNA damage-dependent Sestrin-2 induction is abolished in p53−/− MEFs, and REDD1 induction is abolished in p63−/− MEFs. MEFs of each genotype were treated with the indicated DNA-damaging agents for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies as shown. D, luciferase reporter assay of the REDD1 promoter activity in NHDF. A REDD1 promoter-luciferase construct was cotransfected (Lipofectamine 2000) with the indicated plasmids. 24 h after transfection, cells were treated with topotecan (10 μm), etoposide (20 μm), cisplatin (10 μm), or dimethyl sulfoxide (DMSO) as a control for 24 h. Luciferase assay was performed according to the instructions of the manufacturer (Promega). Error bars show the mean ± S.D. for triplicate wells in a representative experiment. mut, mutant; pcDNA, cells transfected with empty plasmid.

Mentions: Our data imply that a p53-dependent mechanism exists to control 4E-BP1 phosphorylation in the presence of DNA damage. We first tested whether p53 restoration might result in 4E-BP1 dephosphorylation in response to DNA damage. To restore p53 protein, we used a well established p53-lox-STOP-lox (LSL) MEF model (26). We infected p53-LSL MEFs with Adeno-empty as a control or Adeno-Cre for restoring p53 protein, and then MEFs were treated with DNA-damaging agents. As shown in Fig. 5A, restoring p53 resulted in dephosphorylation of 4E-BP1 only in the presence of DNA damage. Because overexpression of p53 alone did not result in hypophosphorylation of 4E-BP1, additional factors might be required for the complete regulation of 4E-BP1 phosphorylation. p53 belongs to the TP53 family, composed of the TP53, TP63, and TP73 genes, which exhibits strong structural homology and expression patterns. Importantly, a strong interplay has been described between p53 family members (27). For instance, all bind specifically to DNA response elements (p53RE), modulate gene expression, and, thus, determine cell fate outcome (28). Moreover, p53-mediated apoptosis is severely impaired in the absence of p63 and p73 in response to DNA damage (29). Thus, we explored the potential role of individual p53 family members in regulating mTORC1 signaling in the presence of DNA damage. MEFs deficient for one of the p53 family members and their control MEFs were treated with various DNA damage-inducing agents. As shown in Fig. 5B, in contrast to p73−/− MEFs, p53−/− and, surprisingly, p63−/− MEFs were not able to suppress 4E-BP1 phosphorylation under DNA damage conditions. These findings indicate that, even in the presence of p53, p63 is necessary for the regulation of 4E-BP1 phosphorylation. This suggests that these genes might act together or in an obligatory parallel pathway to suppress 4E-BP1 phosphorylation subsequent to DNA damage. To understand genotoxic stress-induced 4E-BP1 inhibition more precisely in p53−/− and p63−/− MEFs, we first analyzed Sestrin expression, which was suggested to suppress mTORC1 signaling (10). Consistent with previous data, DNA damage-induced Sestrin2 expression is strongly abolished in p53−/− but not p63−/−MEFs (Fig. 5C), indicating that induction of Sestrin2 alone is not adequate to suppress 4E-BP1 phosphorylation in the absence of p63. Another important stress-induced protein is REDD1, which was initially identified as a gene induced following stress stimuli that inhibit mTORC1 signaling (9, 30). Interestingly, previous data implicated a p53-independent up-regulation of REDD1 because TSC2−/−p53−/− MEFs showed REDD1 induction upon stress stimuli (9, 31). Surprisingly, in contrast to p53−/− and control MEFs, p63−/− MEFs were not able to induce REDD1 expression (Fig. 5C), suggesting that, in response to DNA damage, induction of both Sestrin2 by p53 and REDD1 by p63 are necessary to inhibit 4E-BP1 phosphorylation.


p53/TAp63 and AKT regulate mammalian target of rapamycin complex 1 (mTORC1) signaling through two independent parallel pathways in the presence of DNA damage.

Cam M, Bid HK, Xiao L, Zambetti GP, Houghton PJ, Cam H - J. Biol. Chem. (2013)

p53−/− and p63−/− MEFs are not able to suppress 4E-BP1 phosphorylation in response to DNA damage.A, re-expression of p53 restores DNA damage induced dephosphorylation of 4E-BP1. p53-lox-STOP-lox MEFs were infected with the indicated adenoviruses (Adeno). 24 h after infection, MEFs were treated with the indicated DNA-damaging agents for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies as shown. Eto, etoposide; Cis, cisplatin; Top, topotecan. B, in contrast to p73−/− MEFs, p53−/− and p63−/− MEFs are not able to inhibit 4E-BP1 phosphorylation in response to DNA damage. MEFs of each genotype were treated with the indicated DNA-damaging agents for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies as shown. IR, ionizing radiation; Ctr, control. C, DNA damage-dependent Sestrin-2 induction is abolished in p53−/− MEFs, and REDD1 induction is abolished in p63−/− MEFs. MEFs of each genotype were treated with the indicated DNA-damaging agents for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies as shown. D, luciferase reporter assay of the REDD1 promoter activity in NHDF. A REDD1 promoter-luciferase construct was cotransfected (Lipofectamine 2000) with the indicated plasmids. 24 h after transfection, cells were treated with topotecan (10 μm), etoposide (20 μm), cisplatin (10 μm), or dimethyl sulfoxide (DMSO) as a control for 24 h. Luciferase assay was performed according to the instructions of the manufacturer (Promega). Error bars show the mean ± S.D. for triplicate wells in a representative experiment. mut, mutant; pcDNA, cells transfected with empty plasmid.
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Figure 5: p53−/− and p63−/− MEFs are not able to suppress 4E-BP1 phosphorylation in response to DNA damage.A, re-expression of p53 restores DNA damage induced dephosphorylation of 4E-BP1. p53-lox-STOP-lox MEFs were infected with the indicated adenoviruses (Adeno). 24 h after infection, MEFs were treated with the indicated DNA-damaging agents for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies as shown. Eto, etoposide; Cis, cisplatin; Top, topotecan. B, in contrast to p73−/− MEFs, p53−/− and p63−/− MEFs are not able to inhibit 4E-BP1 phosphorylation in response to DNA damage. MEFs of each genotype were treated with the indicated DNA-damaging agents for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies as shown. IR, ionizing radiation; Ctr, control. C, DNA damage-dependent Sestrin-2 induction is abolished in p53−/− MEFs, and REDD1 induction is abolished in p63−/− MEFs. MEFs of each genotype were treated with the indicated DNA-damaging agents for 24 h. Cell extracts were analyzed by Western blot analysis with antibodies as shown. D, luciferase reporter assay of the REDD1 promoter activity in NHDF. A REDD1 promoter-luciferase construct was cotransfected (Lipofectamine 2000) with the indicated plasmids. 24 h after transfection, cells were treated with topotecan (10 μm), etoposide (20 μm), cisplatin (10 μm), or dimethyl sulfoxide (DMSO) as a control for 24 h. Luciferase assay was performed according to the instructions of the manufacturer (Promega). Error bars show the mean ± S.D. for triplicate wells in a representative experiment. mut, mutant; pcDNA, cells transfected with empty plasmid.
Mentions: Our data imply that a p53-dependent mechanism exists to control 4E-BP1 phosphorylation in the presence of DNA damage. We first tested whether p53 restoration might result in 4E-BP1 dephosphorylation in response to DNA damage. To restore p53 protein, we used a well established p53-lox-STOP-lox (LSL) MEF model (26). We infected p53-LSL MEFs with Adeno-empty as a control or Adeno-Cre for restoring p53 protein, and then MEFs were treated with DNA-damaging agents. As shown in Fig. 5A, restoring p53 resulted in dephosphorylation of 4E-BP1 only in the presence of DNA damage. Because overexpression of p53 alone did not result in hypophosphorylation of 4E-BP1, additional factors might be required for the complete regulation of 4E-BP1 phosphorylation. p53 belongs to the TP53 family, composed of the TP53, TP63, and TP73 genes, which exhibits strong structural homology and expression patterns. Importantly, a strong interplay has been described between p53 family members (27). For instance, all bind specifically to DNA response elements (p53RE), modulate gene expression, and, thus, determine cell fate outcome (28). Moreover, p53-mediated apoptosis is severely impaired in the absence of p63 and p73 in response to DNA damage (29). Thus, we explored the potential role of individual p53 family members in regulating mTORC1 signaling in the presence of DNA damage. MEFs deficient for one of the p53 family members and their control MEFs were treated with various DNA damage-inducing agents. As shown in Fig. 5B, in contrast to p73−/− MEFs, p53−/− and, surprisingly, p63−/− MEFs were not able to suppress 4E-BP1 phosphorylation under DNA damage conditions. These findings indicate that, even in the presence of p53, p63 is necessary for the regulation of 4E-BP1 phosphorylation. This suggests that these genes might act together or in an obligatory parallel pathway to suppress 4E-BP1 phosphorylation subsequent to DNA damage. To understand genotoxic stress-induced 4E-BP1 inhibition more precisely in p53−/− and p63−/− MEFs, we first analyzed Sestrin expression, which was suggested to suppress mTORC1 signaling (10). Consistent with previous data, DNA damage-induced Sestrin2 expression is strongly abolished in p53−/− but not p63−/−MEFs (Fig. 5C), indicating that induction of Sestrin2 alone is not adequate to suppress 4E-BP1 phosphorylation in the absence of p63. Another important stress-induced protein is REDD1, which was initially identified as a gene induced following stress stimuli that inhibit mTORC1 signaling (9, 30). Interestingly, previous data implicated a p53-independent up-regulation of REDD1 because TSC2−/−p53−/− MEFs showed REDD1 induction upon stress stimuli (9, 31). Surprisingly, in contrast to p53−/− and control MEFs, p63−/− MEFs were not able to induce REDD1 expression (Fig. 5C), suggesting that, in response to DNA damage, induction of both Sestrin2 by p53 and REDD1 by p63 are necessary to inhibit 4E-BP1 phosphorylation.

Bottom Line: Under conditions of DNA damage, the mammalian target of rapamycin complex 1 (mTORC1) is inhibited, preventing cell cycle progression and conserving cellular energy by suppressing translation.We show that suppression of mTORC1 signaling to 4E-BP1 requires the coordinated activity of two tumor suppressors, p53 and p63.These data indicate that the negative regulation of cap-dependent translation by mTORC1 inhibition subsequent to DNA damage is abrogated in most human cancers.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Childhood Cancer and Blood Diseases, Nationwide Children's Hospital, Columbus, Ohio 43205.

ABSTRACT
Under conditions of DNA damage, the mammalian target of rapamycin complex 1 (mTORC1) is inhibited, preventing cell cycle progression and conserving cellular energy by suppressing translation. We show that suppression of mTORC1 signaling to 4E-BP1 requires the coordinated activity of two tumor suppressors, p53 and p63. In contrast, suppression of S6K1 and ribosomal protein S6 phosphorylation by DNA damage is Akt-dependent. We find that loss of either p53, required for the induction of Sestrin 1/2, or p63, required for the induction of REDD1 and activation of the tuberous sclerosis complex, prevents the DNA damage-induced suppression of mTORC1 signaling. These data indicate that the negative regulation of cap-dependent translation by mTORC1 inhibition subsequent to DNA damage is abrogated in most human cancers.

Show MeSH
Related in: MedlinePlus