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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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Tyrosine phosphorylated H2AX is a substrate for Eya phosphatase. (a) IP-western of tyrosine phosphorylated H2AX in response to DNA-damage signals. Bars represent quantified western blot signals normalized to untreated cells. (b) In-vitro phosphatase assay using immunopurified wild type Eya1/3 or enzymatically-inactive mutant proteins (Eya1 D323A, Eya3D246A) and bovine histone. Bars represent quantified western blot signals normalized to input. Mean values +/− SEM from triplicate western blot experiments are shown. “**”p-value <.001. (c) siRNA knockdown of endogenous Eya1/3 in 293T cells (48h) and subsequent IP-western for tyrosine phosphorylated H2AX. (d) Rescue of Eya function by co-transfection of human siRNA and murine wild type or enzymatically inactive mutant Eya3 constructs in 293T human embryonic kidney cells reveals loss of H2AX phosphotyrosine mark dependent on Eya phosphatase activity. (e) Individual substitution mutations of four H2AX tyrosine residues followed by IP-western to detect phosphotyrosine. (f) In-vitro phosphatase assay using bacterially expressed Eya3 EYA domain, wild-type or D246A, with purified peptides of the H2AX tail (AA 128–142) phosphorylated at S139 (CTpep pS) or Y142 (CTpep pY) demonstrates that Eya phosphatase activity is specific for phosphotyrosine. The Km value for Eya dephosphorylation of CTpep pY was 0.38mM with a corresponding Kcat/Km value of 0.96M−1min−1. Bar graphs represent mean +/− SEM of nM PO4 released from triplicate phosphatase reactions.
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Figure 4: Tyrosine phosphorylated H2AX is a substrate for Eya phosphatase. (a) IP-western of tyrosine phosphorylated H2AX in response to DNA-damage signals. Bars represent quantified western blot signals normalized to untreated cells. (b) In-vitro phosphatase assay using immunopurified wild type Eya1/3 or enzymatically-inactive mutant proteins (Eya1 D323A, Eya3D246A) and bovine histone. Bars represent quantified western blot signals normalized to input. Mean values +/− SEM from triplicate western blot experiments are shown. “**”p-value <.001. (c) siRNA knockdown of endogenous Eya1/3 in 293T cells (48h) and subsequent IP-western for tyrosine phosphorylated H2AX. (d) Rescue of Eya function by co-transfection of human siRNA and murine wild type or enzymatically inactive mutant Eya3 constructs in 293T human embryonic kidney cells reveals loss of H2AX phosphotyrosine mark dependent on Eya phosphatase activity. (e) Individual substitution mutations of four H2AX tyrosine residues followed by IP-western to detect phosphotyrosine. (f) In-vitro phosphatase assay using bacterially expressed Eya3 EYA domain, wild-type or D246A, with purified peptides of the H2AX tail (AA 128–142) phosphorylated at S139 (CTpep pS) or Y142 (CTpep pY) demonstrates that Eya phosphatase activity is specific for phosphotyrosine. The Km value for Eya dephosphorylation of CTpep pY was 0.38mM with a corresponding Kcat/Km value of 0.96M−1min−1. Bar graphs represent mean +/− SEM of nM PO4 released from triplicate phosphatase reactions.

Mentions: We next tested whether the interaction between H2AX and Eya could represent a substrate-enzyme relationship. Because current evidence suggests that Eya is a tyrosine-specific phosphatase [6–8], we assessed its activity as a tyrosine phosphatase on γH2AX. H2AX purified either from 293T cells or from bovine histone fraction possesses tyrosine phosphorylation as seen using a phosphotyrosine-specific antibody (Supplementary Fig. 5). This tyrosine phosphorylation mark on H2AX decreased in response to DNA damage induced by ionizing radiation, the topoisomerase I inhibitor CPT, or hypoxia (Fig. 4a). To determine whether this H2AX phosphorylation mark might be a target of Eya phosphatase activity, we utilized an in-vitro phosphatase assay, mixing immuno-purified HA-tagged Eya1 or Eya3 with H2AX protein. Wild-type Eya effectively removed the phosphotyrosine mark from H2AX, while the phosphatase-inactive mutant Eya proteins (Eya1 D323A or Eya3 D246A) had little or no effect (Fig. 4b).


Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions.

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

Tyrosine phosphorylated H2AX is a substrate for Eya phosphatase. (a) IP-western of tyrosine phosphorylated H2AX in response to DNA-damage signals. Bars represent quantified western blot signals normalized to untreated cells. (b) In-vitro phosphatase assay using immunopurified wild type Eya1/3 or enzymatically-inactive mutant proteins (Eya1 D323A, Eya3D246A) and bovine histone. Bars represent quantified western blot signals normalized to input. Mean values +/− SEM from triplicate western blot experiments are shown. “**”p-value <.001. (c) siRNA knockdown of endogenous Eya1/3 in 293T cells (48h) and subsequent IP-western for tyrosine phosphorylated H2AX. (d) Rescue of Eya function by co-transfection of human siRNA and murine wild type or enzymatically inactive mutant Eya3 constructs in 293T human embryonic kidney cells reveals loss of H2AX phosphotyrosine mark dependent on Eya phosphatase activity. (e) Individual substitution mutations of four H2AX tyrosine residues followed by IP-western to detect phosphotyrosine. (f) In-vitro phosphatase assay using bacterially expressed Eya3 EYA domain, wild-type or D246A, with purified peptides of the H2AX tail (AA 128–142) phosphorylated at S139 (CTpep pS) or Y142 (CTpep pY) demonstrates that Eya phosphatase activity is specific for phosphotyrosine. The Km value for Eya dephosphorylation of CTpep pY was 0.38mM with a corresponding Kcat/Km value of 0.96M−1min−1. Bar graphs represent mean +/− SEM of nM PO4 released from triplicate phosphatase reactions.
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Figure 4: Tyrosine phosphorylated H2AX is a substrate for Eya phosphatase. (a) IP-western of tyrosine phosphorylated H2AX in response to DNA-damage signals. Bars represent quantified western blot signals normalized to untreated cells. (b) In-vitro phosphatase assay using immunopurified wild type Eya1/3 or enzymatically-inactive mutant proteins (Eya1 D323A, Eya3D246A) and bovine histone. Bars represent quantified western blot signals normalized to input. Mean values +/− SEM from triplicate western blot experiments are shown. “**”p-value <.001. (c) siRNA knockdown of endogenous Eya1/3 in 293T cells (48h) and subsequent IP-western for tyrosine phosphorylated H2AX. (d) Rescue of Eya function by co-transfection of human siRNA and murine wild type or enzymatically inactive mutant Eya3 constructs in 293T human embryonic kidney cells reveals loss of H2AX phosphotyrosine mark dependent on Eya phosphatase activity. (e) Individual substitution mutations of four H2AX tyrosine residues followed by IP-western to detect phosphotyrosine. (f) In-vitro phosphatase assay using bacterially expressed Eya3 EYA domain, wild-type or D246A, with purified peptides of the H2AX tail (AA 128–142) phosphorylated at S139 (CTpep pS) or Y142 (CTpep pY) demonstrates that Eya phosphatase activity is specific for phosphotyrosine. The Km value for Eya dephosphorylation of CTpep pY was 0.38mM with a corresponding Kcat/Km value of 0.96M−1min−1. Bar graphs represent mean +/− SEM of nM PO4 released from triplicate phosphatase reactions.
Mentions: We next tested whether the interaction between H2AX and Eya could represent a substrate-enzyme relationship. Because current evidence suggests that Eya is a tyrosine-specific phosphatase [6–8], we assessed its activity as a tyrosine phosphatase on γH2AX. H2AX purified either from 293T cells or from bovine histone fraction possesses tyrosine phosphorylation as seen using a phosphotyrosine-specific antibody (Supplementary Fig. 5). This tyrosine phosphorylation mark on H2AX decreased in response to DNA damage induced by ionizing radiation, the topoisomerase I inhibitor CPT, or hypoxia (Fig. 4a). To determine whether this H2AX phosphorylation mark might be a target of Eya phosphatase activity, we utilized an in-vitro phosphatase assay, mixing immuno-purified HA-tagged Eya1 or Eya3 with H2AX protein. Wild-type Eya effectively removed the phosphotyrosine mark from H2AX, while the phosphatase-inactive mutant Eya proteins (Eya1 D323A or Eya3 D246A) had little or no effect (Fig. 4b).

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.

Show MeSH
Related in: MedlinePlus