Limits...
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.

Show MeSH

Related in: MedlinePlus

Activation of p53 by ionising radiation: effects of ATM-CHK pathway inhibitors on p53 phosphorylation. (A, B)                                    A CHK2 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 [1-10μM (A) or 10-100μM (B)] of the CHK2 inhibitor                                    (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, D) A CHK1 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                                    [1-10μM (C) or 5-50μM (D)] of the CHK1 inhibitor SB218078                                    (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.                                    (E) An ATM inhibitor attenuates Ser20 site phosphorylation of p53,                                    but not 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                                    (1-10μM) 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2806026&req=5

Figure 4: Activation of p53 by ionising radiation: effects of ATM-CHK pathway inhibitors on p53 phosphorylation. (A, B) A CHK2 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 [1-10μM (A) or 10-100μM (B)] of the CHK2 inhibitor (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, D) A CHK1 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 [1-10μM (C) or 5-50μM (D)] of the CHK1 inhibitor SB218078 (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. (E) An ATM inhibitor attenuates Ser20 site phosphorylation of p53, but not 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 (1-10μM) 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: In attempts to define whether the activation of p53 Ser20 site kinase(s) induced by different stresses is triggered by the same or different signaling pathways, we treated cells with specific kinase inhibitors in combination with distinct stresses known to activate p53. We performed all experiments using one cell culture model, namely the MOLT-3 cell line, which is a human acute lymphoblastic leukaemia T-cell line. The MOLT-3 cell line was first validated using ionizing radiation and kinase inhibitors specific for CHK2, CHK1 and ATM. As a control consistent with siRNA screens for CHK2 [43], the X-ray-induced Ser20 site phosphorylation of p53 was not attenuated by the CHK2 inhibitor (Figure 4A and B; lanes 6, 8, 10, 12 vs 5, 7, 9, 11). Further, the CHK1 inhibitor SB218078 was equally unable to prevent Ser20 site phosphorylation induced by X-rays (Figure 4C and D; lanes 6, 8, 10, 12 vs 5, 7, 9, 11). In fact, X-ray induced phosphorylation at Ser20 was elevated (Figure 4 C and D; lanes 6, 8, 10, 12 vs 4), and basal levels of p53 were stabilized by the CHK1 inhibitor in the absence X-ray treatment (Figure 4C and D; lanes 5, 7, 9, 11 vs 3). However, this stabilized form of p53 in undamaged cells was not phosphorylated at Ser20 (Figure 4C and D; lanes 5, 7, 9, 11).


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 ionising radiation: effects of ATM-CHK pathway inhibitors on p53 phosphorylation. (A, B)                                    A CHK2 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 [1-10μM (A) or 10-100μM (B)] of the CHK2 inhibitor                                    (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, D) A CHK1 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                                    [1-10μM (C) or 5-50μM (D)] of the CHK1 inhibitor SB218078                                    (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.                                    (E) An ATM inhibitor attenuates Ser20 site phosphorylation of p53,                                    but not 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                                    (1-10μM) 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2806026&req=5

Figure 4: Activation of p53 by ionising radiation: effects of ATM-CHK pathway inhibitors on p53 phosphorylation. (A, B) A CHK2 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 [1-10μM (A) or 10-100μM (B)] of the CHK2 inhibitor (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, D) A CHK1 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 [1-10μM (C) or 5-50μM (D)] of the CHK1 inhibitor SB218078 (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. (E) An ATM inhibitor attenuates Ser20 site phosphorylation of p53, but not 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 (1-10μM) 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: In attempts to define whether the activation of p53 Ser20 site kinase(s) induced by different stresses is triggered by the same or different signaling pathways, we treated cells with specific kinase inhibitors in combination with distinct stresses known to activate p53. We performed all experiments using one cell culture model, namely the MOLT-3 cell line, which is a human acute lymphoblastic leukaemia T-cell line. The MOLT-3 cell line was first validated using ionizing radiation and kinase inhibitors specific for CHK2, CHK1 and ATM. As a control consistent with siRNA screens for CHK2 [43], the X-ray-induced Ser20 site phosphorylation of p53 was not attenuated by the CHK2 inhibitor (Figure 4A and B; lanes 6, 8, 10, 12 vs 5, 7, 9, 11). Further, the CHK1 inhibitor SB218078 was equally unable to prevent Ser20 site phosphorylation induced by X-rays (Figure 4C and D; lanes 6, 8, 10, 12 vs 5, 7, 9, 11). In fact, X-ray induced phosphorylation at Ser20 was elevated (Figure 4 C and D; lanes 6, 8, 10, 12 vs 4), and basal levels of p53 were stabilized by the CHK1 inhibitor in the absence X-ray treatment (Figure 4C and D; lanes 5, 7, 9, 11 vs 3). However, this stabilized form of p53 in undamaged cells was not phosphorylated at Ser20 (Figure 4C and D; lanes 5, 7, 9, 11).

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