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The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway.

Maclaine NJ, Hupp TR - Aging (Albany NY) (2009)

Bottom Line: Mutations in p53 switch the cellular transcription program resulting in deregulation of the stress responses that normally maintain cell and tissue integrity.We demonstrate that distinct stress-activated kinases, including ataxia telangiectasia mutated (ATM), casein kinase 1 (CK1) and AMP-activated protein kinase (AMPK), mediate phosphorylation of a key phospho-acceptor site in the p53 transactivation domain in response to diverse stresses including ionizing radiation, DNA virus infection, and elevation in the intracellular AMP/ATP ratio.As diseases linked to aging can involve activation of p53-dependent changes in cellular protective pathways, the development of specific physiological models might further shed light on the role of p53 kinases in modifying age-related diseases.

View Article: PubMed Central - PubMed

Affiliation: University of Edinburgh, Institute of Genetics and Molecular Medicine, CRUK p53 Signal Transduction Laboratories, Edinburgh, EH4 2XR, Scotland, UK.

ABSTRACT
The tumour suppressor p53 is a transcription factor that has evolved the ability to integrate distinct environmental signals including DNA damage, virus infection, and cytokine signaling into a common biological outcome that maintains normal cellular control. Mutations in p53 switch the cellular transcription program resulting in deregulation of the stress responses that normally maintain cell and tissue integrity. Transgenic studies in mice have indicated that changes in the specific activity of p53 can have profound effects not only on cancer development, but also on organism aging. As the specific activity of p53 is regulated at a post-translational level by sets of enzymes that mediate phosphorylation, acetylation, methylation, and ubiquitin-like modifications, it is likely that physiological modifiers of the aging function of p53 would be enzymes that catalyze such covalent modifications. We demonstrate that distinct stress-activated kinases, including ataxia telangiectasia mutated (ATM), casein kinase 1 (CK1) and AMP-activated protein kinase (AMPK), mediate phosphorylation of a key phospho-acceptor site in the p53 transactivation domain in response to diverse stresses including ionizing radiation, DNA virus infection, and elevation in the intracellular AMP/ATP ratio. As diseases linked to aging can involve activation of p53-dependent changes in cellular protective pathways, the development of specific physiological models might further shed light on the role of p53 kinases in modifying age-related diseases.

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Activation of p53 by metabolic stress; effects of an AMPK inhibitor on p53 phosphorylation.(A) An AMPK inhibitor attenuates Ser20                                        site phosphorylation of p53 and p53 induction mediated by treatment with                                        AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without                                        (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour                                        pre-treatment with: increasing concentrations (2.5-20μM) of the AMPK                                        inhibitor Compound C (lanes 5-12), a DMSO solvent control (lanes 3-4), or a                                        culture medium control (lanes 1-2). Cell lysates were examined by Western                                        blotting with antibodies against the indicated proteins. (B) An AMPK                                        inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53                                        induction mediated by treatment with X-rays. MOLT-3 cells were treated with                                        (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and                                        cultured for 4 hours after an initial 44-hour pre-treatment with:                                        increasing concentrations (1.25-10μM) of the AMPK inhibitor Compound C                                        (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium                                        control (lanes 1-2). Cell lysates were examined by Western blotting with                                        antibodies against the indicated proteins. (C) A CK1                                        inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53                                        induction mediated by treatment with AICAR. MOLT-3 cells were treated with                                        (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24                                        hours after an initial 24-hour pre-treatment with: increasing                                        concentrations (10-60μM) of the CK1 inhibitor D4476 (lanes 5-12), a                                        DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2).                                        Cell lysates were examined by Western blotting with antibodies against the                                        indicated proteins. (D) An ATM inhibitor does not attenuate                                        Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment                                        with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without                                        (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment                                        with: increasing concentrations (1-10μM) of the ATM inhibitor KU-55933                                        (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium                                        control (lanes 1-2). Cell lysates were examined by Western blotting with                                        antibodies against the indicated proteins.
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Figure 6: Activation of p53 by metabolic stress; effects of an AMPK inhibitor on p53 phosphorylation.(A) An AMPK inhibitor attenuates Ser20 site phosphorylation of p53 and p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (2.5-20μM) of the AMPK inhibitor Compound C (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. (B) An AMPK inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations (1.25-10μM) of the AMPK inhibitor Compound C (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. (C) A CK1 inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (10-60μM) of the CK1 inhibitor D4476 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. (D) An ATM inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (1-10μM) of the ATM inhibitor KU-55933 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins.

Mentions: We subsequently screened cells for other signals, including hypoxia, glucose starvation, anoxia and perturbation of the AMP/ATP ratio, which could trigger p53 phosphorylation at Ser20. Of these signals, the most pronounced effect on Ser20 site phosphorylation was observed with the compound Acadesine (AICAR; Figure 6A; lane 2 vs 1), which is known to activate AMP-activated protein kinase (AMPK) by virtue of elevating the intracellular AMP levels. We had previously identified AMPK in a candidate kinase screen as an enzyme within the Calcium-Calmodulin kinase superfamily capable of targeting p53 at Ser20 in vitro [40]. The AICAR-mediated induction of Ser20 site phosphorylation was attenuated in a dose-dependent manner by the treatment of cells with the AMPK inhibitor Compound C (Figure 6A; lanes 6, 8, 10, 12 vs 2). On the other hand, the AMPK inhibitor was unable to prevent Ser20 site phosphorylation induced by X-rays (Figure 6B; lanes 6, 8, 10, 12 vs 5, 7, 9, 11), indicating that AMPK is not the Ser20 site enzyme induced by X-rays. Further, neither the CK1 inhibitor (Figure 6C), nor the ATM inhibitor (Figure 6D) abrogated the AICAR-induced p53 Ser20 phosphorylation (Figure 6C and D; lanes 6, 8, 10, 12 vs 5, 7, 9, 11). These data therefore confirm that p53 Ser20 phosphorylation is ATM-dependent after X-rays, CK1-dependent after virus infection, and AMPK-dependent after perturbation of AMP/ATP ratios (Figure 3).


The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway.

Maclaine NJ, Hupp TR - Aging (Albany NY) (2009)

Activation of p53 by metabolic stress; effects of an AMPK inhibitor on p53 phosphorylation.(A) An AMPK inhibitor attenuates Ser20                                        site phosphorylation of p53 and p53 induction mediated by treatment with                                        AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without                                        (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour                                        pre-treatment with: increasing concentrations (2.5-20μM) of the AMPK                                        inhibitor Compound C (lanes 5-12), a DMSO solvent control (lanes 3-4), or a                                        culture medium control (lanes 1-2). Cell lysates were examined by Western                                        blotting with antibodies against the indicated proteins. (B) An AMPK                                        inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53                                        induction mediated by treatment with X-rays. MOLT-3 cells were treated with                                        (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and                                        cultured for 4 hours after an initial 44-hour pre-treatment with:                                        increasing concentrations (1.25-10μM) of the AMPK inhibitor Compound C                                        (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium                                        control (lanes 1-2). Cell lysates were examined by Western blotting with                                        antibodies against the indicated proteins. (C) A CK1                                        inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53                                        induction mediated by treatment with AICAR. MOLT-3 cells were treated with                                        (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24                                        hours after an initial 24-hour pre-treatment with: increasing                                        concentrations (10-60μM) of the CK1 inhibitor D4476 (lanes 5-12), a                                        DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2).                                        Cell lysates were examined by Western blotting with antibodies against the                                        indicated proteins. (D) An ATM inhibitor does not attenuate                                        Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment                                        with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without                                        (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment                                        with: increasing concentrations (1-10μM) of the ATM inhibitor KU-55933                                        (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium                                        control (lanes 1-2). Cell lysates were examined by Western blotting with                                        antibodies against the indicated proteins.
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Figure 6: Activation of p53 by metabolic stress; effects of an AMPK inhibitor on p53 phosphorylation.(A) An AMPK inhibitor attenuates Ser20 site phosphorylation of p53 and p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (2.5-20μM) of the AMPK inhibitor Compound C (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. (B) An AMPK inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations (1.25-10μM) of the AMPK inhibitor Compound C (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. (C) A CK1 inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (10-60μM) of the CK1 inhibitor D4476 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. (D) An ATM inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (1-10μM) of the ATM inhibitor KU-55933 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins.
Mentions: We subsequently screened cells for other signals, including hypoxia, glucose starvation, anoxia and perturbation of the AMP/ATP ratio, which could trigger p53 phosphorylation at Ser20. Of these signals, the most pronounced effect on Ser20 site phosphorylation was observed with the compound Acadesine (AICAR; Figure 6A; lane 2 vs 1), which is known to activate AMP-activated protein kinase (AMPK) by virtue of elevating the intracellular AMP levels. We had previously identified AMPK in a candidate kinase screen as an enzyme within the Calcium-Calmodulin kinase superfamily capable of targeting p53 at Ser20 in vitro [40]. The AICAR-mediated induction of Ser20 site phosphorylation was attenuated in a dose-dependent manner by the treatment of cells with the AMPK inhibitor Compound C (Figure 6A; lanes 6, 8, 10, 12 vs 2). On the other hand, the AMPK inhibitor was unable to prevent Ser20 site phosphorylation induced by X-rays (Figure 6B; lanes 6, 8, 10, 12 vs 5, 7, 9, 11), indicating that AMPK is not the Ser20 site enzyme induced by X-rays. Further, neither the CK1 inhibitor (Figure 6C), nor the ATM inhibitor (Figure 6D) abrogated the AICAR-induced p53 Ser20 phosphorylation (Figure 6C and D; lanes 6, 8, 10, 12 vs 5, 7, 9, 11). These data therefore confirm that p53 Ser20 phosphorylation is ATM-dependent after X-rays, CK1-dependent after virus infection, and AMPK-dependent after perturbation of AMP/ATP ratios (Figure 3).

Bottom Line: Mutations in p53 switch the cellular transcription program resulting in deregulation of the stress responses that normally maintain cell and tissue integrity.We demonstrate that distinct stress-activated kinases, including ataxia telangiectasia mutated (ATM), casein kinase 1 (CK1) and AMP-activated protein kinase (AMPK), mediate phosphorylation of a key phospho-acceptor site in the p53 transactivation domain in response to diverse stresses including ionizing radiation, DNA virus infection, and elevation in the intracellular AMP/ATP ratio.As diseases linked to aging can involve activation of p53-dependent changes in cellular protective pathways, the development of specific physiological models might further shed light on the role of p53 kinases in modifying age-related diseases.

View Article: PubMed Central - PubMed

Affiliation: University of Edinburgh, Institute of Genetics and Molecular Medicine, CRUK p53 Signal Transduction Laboratories, Edinburgh, EH4 2XR, Scotland, UK.

ABSTRACT
The tumour suppressor p53 is a transcription factor that has evolved the ability to integrate distinct environmental signals including DNA damage, virus infection, and cytokine signaling into a common biological outcome that maintains normal cellular control. Mutations in p53 switch the cellular transcription program resulting in deregulation of the stress responses that normally maintain cell and tissue integrity. Transgenic studies in mice have indicated that changes in the specific activity of p53 can have profound effects not only on cancer development, but also on organism aging. As the specific activity of p53 is regulated at a post-translational level by sets of enzymes that mediate phosphorylation, acetylation, methylation, and ubiquitin-like modifications, it is likely that physiological modifiers of the aging function of p53 would be enzymes that catalyze such covalent modifications. We demonstrate that distinct stress-activated kinases, including ataxia telangiectasia mutated (ATM), casein kinase 1 (CK1) and AMP-activated protein kinase (AMPK), mediate phosphorylation of a key phospho-acceptor site in the p53 transactivation domain in response to diverse stresses including ionizing radiation, DNA virus infection, and elevation in the intracellular AMP/ATP ratio. As diseases linked to aging can involve activation of p53-dependent changes in cellular protective pathways, the development of specific physiological models might further shed light on the role of p53 kinases in modifying age-related diseases.

Show MeSH
Related in: MedlinePlus