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Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions.

Cook PJ, Ju BG, Telese F, Wang X, Glass CK, Rosenfeld MG - Nature (2009)

Bottom Line: Life and death fate decisions allow cells to avoid massive apoptotic death in response to genotoxic stress.Here we report that a protein tyrosine phosphatase, EYA, is involved in promoting efficient DNA repair rather than apoptosis in response to genotoxic stress in mammalian embryonic kidney cells by executing a damage-signal-dependent dephosphorylation of an H2AX carboxy-terminal tyrosine phosphate (Y142).This post-translational modification determines the relative recruitment of either DNA repair or pro-apoptotic factors to the tail of serine phosphorylated histone H2AX (gamma-H2AX) and allows it to function as an active determinant of repair/survival versus apoptotic responses to DNA damage, revealing an additional phosphorylation-dependent mechanism that modulates survival/apoptotic decisions during mammalian organogenesis.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute School of Medicine, University of California, San Diego, California 92037, USA.

ABSTRACT
Life and death fate decisions allow cells to avoid massive apoptotic death in response to genotoxic stress. Although the regulatory mechanisms and signalling pathways controlling DNA repair and apoptosis are well characterized, the precise molecular strategies that determine the ultimate choice of DNA repair and survival or apoptotic cell death remain incompletely understood. Here we report that a protein tyrosine phosphatase, EYA, is involved in promoting efficient DNA repair rather than apoptosis in response to genotoxic stress in mammalian embryonic kidney cells by executing a damage-signal-dependent dephosphorylation of an H2AX carboxy-terminal tyrosine phosphate (Y142). This post-translational modification determines the relative recruitment of either DNA repair or pro-apoptotic factors to the tail of serine phosphorylated histone H2AX (gamma-H2AX) and allows it to function as an active determinant of repair/survival versus apoptotic responses to DNA damage, revealing an additional phosphorylation-dependent mechanism that modulates survival/apoptotic decisions during mammalian organogenesis.

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H2AX Y142 phosphorylation discriminates between apoptotic and repair responses to DNA-damage. (a) S139 phosphorylation of H2AX Y142F is present but reduced in comparison to wild type H2AX after 5Gy IR. (b) Affinity purification performed on nuclear extract from irradiated 293T cells using synthetic peptides representing the C-terminal tail of H2AX bearing S139 phosphorylation +/− Y142 phosphorylation followed by western blot analysis. Co-immunoprecipitaiton confirms interaction between wild-type H2AX and JNK1 (c) or Fe65 (d) but not H2AX Y142F in 293T cells exposed to high-dose IR (50Gy). (e) Endogenous Fe65 interacts with JNK1 in 293T cells treated with etoposide (30uM). Figures 5d and 5e show individual bands from a single western blot exposure. (f) siRNA knockdown of Fe65 in 293T cells blocks the damage-dependent (50Gy) interaction of JNK1 and γH2AX by co-immunoprecipitation in cells transfected with Fe65 siRNA or control siRNA 48 hours prior to harvest. Results were confirmed with two separate siRNA sets for Fe65. (g) H2AX−/− MEF cells were transfected with wild type or mutant H2AX (Y142F) expression constructs and exposed to high-dose IR (100Gy). Apoptotic response among transfectants was assessed by γH2AX staining and TUNEL. Bar graphs represent mean +/− SEM of fold apoptotic values for triplicate or greater cell counts of transfected (green) nuclei. Basal level of apoptosis for WT-H2AX transfected cells under these conditions was 25.7% TUNEL positive/total transfeced nuclei. Values were normalized to WT-H2AX-transfected samples. “**”p <.001. (h) Proposed model for Y142 phosphorylation status of H2AX in regulation of apoptotic versus repair response.
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Figure 5: H2AX Y142 phosphorylation discriminates between apoptotic and repair responses to DNA-damage. (a) S139 phosphorylation of H2AX Y142F is present but reduced in comparison to wild type H2AX after 5Gy IR. (b) Affinity purification performed on nuclear extract from irradiated 293T cells using synthetic peptides representing the C-terminal tail of H2AX bearing S139 phosphorylation +/− Y142 phosphorylation followed by western blot analysis. Co-immunoprecipitaiton confirms interaction between wild-type H2AX and JNK1 (c) or Fe65 (d) but not H2AX Y142F in 293T cells exposed to high-dose IR (50Gy). (e) Endogenous Fe65 interacts with JNK1 in 293T cells treated with etoposide (30uM). Figures 5d and 5e show individual bands from a single western blot exposure. (f) siRNA knockdown of Fe65 in 293T cells blocks the damage-dependent (50Gy) interaction of JNK1 and γH2AX by co-immunoprecipitation in cells transfected with Fe65 siRNA or control siRNA 48 hours prior to harvest. Results were confirmed with two separate siRNA sets for Fe65. (g) H2AX−/− MEF cells were transfected with wild type or mutant H2AX (Y142F) expression constructs and exposed to high-dose IR (100Gy). Apoptotic response among transfectants was assessed by γH2AX staining and TUNEL. Bar graphs represent mean +/− SEM of fold apoptotic values for triplicate or greater cell counts of transfected (green) nuclei. Basal level of apoptosis for WT-H2AX transfected cells under these conditions was 25.7% TUNEL positive/total transfeced nuclei. Values were normalized to WT-H2AX-transfected samples. “**”p <.001. (h) Proposed model for Y142 phosphorylation status of H2AX in regulation of apoptotic versus repair response.

Mentions: To begin to evaluate a possible connection between Eya-mediated tyrosine dephosphorylation of H2AX Y142 and modulation of the apoptotic response, we examined the function of this phosphotyrsoine mark in the context of the DNA damage response. FLAG-tagged H2AX Y142F mutant was phosphorylated on S139 in response to damage, although at levels significantly lower than FLAG-tagged wild-type H2AX. (Fig. 5a) Time course anlysis of S139 phoshorylation of H2AX Y142F in response to 10Gy IR in 293T human embryonic kidney cells revealed consistently reduced levels compared to wild type between 1 and 8 hours (Supplementary Fig. 8). Thus, while Y142 phosphorylation does not function as a pre-requisite for S139 phosphorylation in the DNA damage response [24], it may play a significant role in promoting or maintaining serine phosphorylation by DNA-damage response kinases.


Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions.

Cook PJ, Ju BG, Telese F, Wang X, Glass CK, Rosenfeld MG - Nature (2009)

H2AX Y142 phosphorylation discriminates between apoptotic and repair responses to DNA-damage. (a) S139 phosphorylation of H2AX Y142F is present but reduced in comparison to wild type H2AX after 5Gy IR. (b) Affinity purification performed on nuclear extract from irradiated 293T cells using synthetic peptides representing the C-terminal tail of H2AX bearing S139 phosphorylation +/− Y142 phosphorylation followed by western blot analysis. Co-immunoprecipitaiton confirms interaction between wild-type H2AX and JNK1 (c) or Fe65 (d) but not H2AX Y142F in 293T cells exposed to high-dose IR (50Gy). (e) Endogenous Fe65 interacts with JNK1 in 293T cells treated with etoposide (30uM). Figures 5d and 5e show individual bands from a single western blot exposure. (f) siRNA knockdown of Fe65 in 293T cells blocks the damage-dependent (50Gy) interaction of JNK1 and γH2AX by co-immunoprecipitation in cells transfected with Fe65 siRNA or control siRNA 48 hours prior to harvest. Results were confirmed with two separate siRNA sets for Fe65. (g) H2AX−/− MEF cells were transfected with wild type or mutant H2AX (Y142F) expression constructs and exposed to high-dose IR (100Gy). Apoptotic response among transfectants was assessed by γH2AX staining and TUNEL. Bar graphs represent mean +/− SEM of fold apoptotic values for triplicate or greater cell counts of transfected (green) nuclei. Basal level of apoptosis for WT-H2AX transfected cells under these conditions was 25.7% TUNEL positive/total transfeced nuclei. Values were normalized to WT-H2AX-transfected samples. “**”p <.001. (h) Proposed model for Y142 phosphorylation status of H2AX in regulation of apoptotic versus repair response.
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Figure 5: H2AX Y142 phosphorylation discriminates between apoptotic and repair responses to DNA-damage. (a) S139 phosphorylation of H2AX Y142F is present but reduced in comparison to wild type H2AX after 5Gy IR. (b) Affinity purification performed on nuclear extract from irradiated 293T cells using synthetic peptides representing the C-terminal tail of H2AX bearing S139 phosphorylation +/− Y142 phosphorylation followed by western blot analysis. Co-immunoprecipitaiton confirms interaction between wild-type H2AX and JNK1 (c) or Fe65 (d) but not H2AX Y142F in 293T cells exposed to high-dose IR (50Gy). (e) Endogenous Fe65 interacts with JNK1 in 293T cells treated with etoposide (30uM). Figures 5d and 5e show individual bands from a single western blot exposure. (f) siRNA knockdown of Fe65 in 293T cells blocks the damage-dependent (50Gy) interaction of JNK1 and γH2AX by co-immunoprecipitation in cells transfected with Fe65 siRNA or control siRNA 48 hours prior to harvest. Results were confirmed with two separate siRNA sets for Fe65. (g) H2AX−/− MEF cells were transfected with wild type or mutant H2AX (Y142F) expression constructs and exposed to high-dose IR (100Gy). Apoptotic response among transfectants was assessed by γH2AX staining and TUNEL. Bar graphs represent mean +/− SEM of fold apoptotic values for triplicate or greater cell counts of transfected (green) nuclei. Basal level of apoptosis for WT-H2AX transfected cells under these conditions was 25.7% TUNEL positive/total transfeced nuclei. Values were normalized to WT-H2AX-transfected samples. “**”p <.001. (h) Proposed model for Y142 phosphorylation status of H2AX in regulation of apoptotic versus repair response.
Mentions: To begin to evaluate a possible connection between Eya-mediated tyrosine dephosphorylation of H2AX Y142 and modulation of the apoptotic response, we examined the function of this phosphotyrsoine mark in the context of the DNA damage response. FLAG-tagged H2AX Y142F mutant was phosphorylated on S139 in response to damage, although at levels significantly lower than FLAG-tagged wild-type H2AX. (Fig. 5a) Time course anlysis of S139 phoshorylation of H2AX Y142F in response to 10Gy IR in 293T human embryonic kidney cells revealed consistently reduced levels compared to wild type between 1 and 8 hours (Supplementary Fig. 8). Thus, while Y142 phosphorylation does not function as a pre-requisite for S139 phosphorylation in the DNA damage response [24], it may play a significant role in promoting or maintaining serine phosphorylation by DNA-damage response kinases.

Bottom Line: Life and death fate decisions allow cells to avoid massive apoptotic death in response to genotoxic stress.Here we report that a protein tyrosine phosphatase, EYA, is involved in promoting efficient DNA repair rather than apoptosis in response to genotoxic stress in mammalian embryonic kidney cells by executing a damage-signal-dependent dephosphorylation of an H2AX carboxy-terminal tyrosine phosphate (Y142).This post-translational modification determines the relative recruitment of either DNA repair or pro-apoptotic factors to the tail of serine phosphorylated histone H2AX (gamma-H2AX) and allows it to function as an active determinant of repair/survival versus apoptotic responses to DNA damage, revealing an additional phosphorylation-dependent mechanism that modulates survival/apoptotic decisions during mammalian organogenesis.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute School of Medicine, University of California, San Diego, California 92037, USA.

ABSTRACT
Life and death fate decisions allow cells to avoid massive apoptotic death in response to genotoxic stress. Although the regulatory mechanisms and signalling pathways controlling DNA repair and apoptosis are well characterized, the precise molecular strategies that determine the ultimate choice of DNA repair and survival or apoptotic cell death remain incompletely understood. Here we report that a protein tyrosine phosphatase, EYA, is involved in promoting efficient DNA repair rather than apoptosis in response to genotoxic stress in mammalian embryonic kidney cells by executing a damage-signal-dependent dephosphorylation of an H2AX carboxy-terminal tyrosine phosphate (Y142). This post-translational modification determines the relative recruitment of either DNA repair or pro-apoptotic factors to the tail of serine phosphorylated histone H2AX (gamma-H2AX) and allows it to function as an active determinant of repair/survival versus apoptotic responses to DNA damage, revealing an additional phosphorylation-dependent mechanism that modulates survival/apoptotic decisions during mammalian organogenesis.

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Related in: MedlinePlus