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TIPRL Inhibits Protein Phosphatase 4 Activity and Promotes H2AX Phosphorylation in the DNA Damage Response.

Rosales KR, Reid MA, Yang Y, Tran TQ, Wang WI, Lowman X, Pan M, Kong M - PLoS ONE (2015)

Bottom Line: Unlike kinases, the activity and specificity of serine/threonine phosphatases is governed largely by their associated proteins.Knockdown of TIPRL resulted in increased PP4 phosphatase activity and formation of the active PP4-C/PP4R2 complex known to dephosphorylate γ-H2AX.In correlation with γ-H2AX levels, we found that TIPRL overexpression promotes cell death in response to genotoxic stress, and knockdown of TIPRL protects cells from genotoxic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Beckman Research Institute of City of Hope Cancer Center, Duarte, California, United States of America.

ABSTRACT
Despite advances in our understanding of protein kinase regulation in the DNA damage response, the mechanism that controls protein phosphatase activity in this pathway is unclear. Unlike kinases, the activity and specificity of serine/threonine phosphatases is governed largely by their associated proteins. Here we show that Tip41-like protein (TIPRL), an evolutionarily conserved binding protein for PP2A-family phosphatases, is a negative regulator of protein phosphatase 4 (PP4). Knockdown of TIPRL resulted in increased PP4 phosphatase activity and formation of the active PP4-C/PP4R2 complex known to dephosphorylate γ-H2AX. Thus, overexpression of TIPRL promotes phosphorylation of H2AX, and increases γ-H2AX positive foci in response to DNA damage, whereas knockdown of TIPRL inhibits γ-H2AX phosphorylation. In correlation with γ-H2AX levels, we found that TIPRL overexpression promotes cell death in response to genotoxic stress, and knockdown of TIPRL protects cells from genotoxic agents. Taken together, these data demonstrate that TIPRL inhibits PP4 activity to allow for H2AX phosphorylation and the subsequent DNA damage response.

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TIPRL promotes γ-H2AX phosphorylation in response to DNA damage.(A) HeLa cells were transiently transfected with vector control (VEC) or FLAG-TIPRL. 24hrs after transfection, cells were treated with CPT (2.5μM) or (B) doxorubicin (DOXO, 2μg/ml) for the indicated time points. Cells were lysed and immunoblots were probed with the indicated antibodies. (C) HeLa cells were transiently transfected with vector control (VEC) or FLAG-TIPRL. 24hrs after transfection, cells were treated with CPT (2.5μM) for 1.5h followed by washout for 6h. Cells were lysed and immunoblots were probed with the indicated antibodies. (D) 3T3 cells stably expressing LPC FLAG vector control (VEC) or LPC FLAG- TIPRL (TIPRL) were treated with 5μM CPT for 1.5hrs. Cells were fixed and stained for both DAPI and γ-H2AX and (E) images were quantified. Data represent ± standard deviation of the mean of three fields**p<0.01, Student’s t test.
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pone.0145938.g003: TIPRL promotes γ-H2AX phosphorylation in response to DNA damage.(A) HeLa cells were transiently transfected with vector control (VEC) or FLAG-TIPRL. 24hrs after transfection, cells were treated with CPT (2.5μM) or (B) doxorubicin (DOXO, 2μg/ml) for the indicated time points. Cells were lysed and immunoblots were probed with the indicated antibodies. (C) HeLa cells were transiently transfected with vector control (VEC) or FLAG-TIPRL. 24hrs after transfection, cells were treated with CPT (2.5μM) for 1.5h followed by washout for 6h. Cells were lysed and immunoblots were probed with the indicated antibodies. (D) 3T3 cells stably expressing LPC FLAG vector control (VEC) or LPC FLAG- TIPRL (TIPRL) were treated with 5μM CPT for 1.5hrs. Cells were fixed and stained for both DAPI and γ-H2AX and (E) images were quantified. Data represent ± standard deviation of the mean of three fields**p<0.01, Student’s t test.

Mentions: The PP4-C/PP4R2/PP4R3β complex has been shown to be a γ-H2AX phosphatase in mammalian cells [3,6] and the pph3 gene, which encodes the ortholog of PP4 catalytic subunit in budding yeast, serves as the sole γ-H2AX phosphatase in this organism [4,5,7]. We found that TIPRL decreases PP4 activity, therefore we wanted to determine if TIPRL expression also resulted in increased γ-H2AX levels following DNA damage. We transiently overexpressed TIPRL and assessed γ-H2AX levels in response to DNA damage. We found that TIPRL overexpression led to increased γ-H2AX phosphorylation in response to the DNA damaging agents CPT (Fig 3A) and doxorubicin (DOXO) (Fig 3B) compared to the vector control. To determine if TIPRL is involved in regulating γ-H2AX dephosphorylation during recovery from DNA damage, we performed a washout experiment and found, consistent with Fig 3A, TIRPL expression led to increased γ-H2AX compared to vector control cells; however, increased TIPRL levels impaired the ability of cells to reverse DNA damage-induced H2AX phosphorylation following washout of the drug, suggesting a delayed dephosphorylation due to decreased phosphatase acitivity in TIPRL overexpressing cells (Fig 3C). To further investigate the role of TIPRL in promoting γ-H2AX foci in response to DNA damage, TIPRL overexpressing cells were treated with CPT, and γ-H2AX staining was visualized by immunofluorescence microscopy (Fig 3D). TIPRL overexpression resulted in a twenty five percent increase in cells that contained γ-H2AX following CPT treatment (Fig 3E).


TIPRL Inhibits Protein Phosphatase 4 Activity and Promotes H2AX Phosphorylation in the DNA Damage Response.

Rosales KR, Reid MA, Yang Y, Tran TQ, Wang WI, Lowman X, Pan M, Kong M - PLoS ONE (2015)

TIPRL promotes γ-H2AX phosphorylation in response to DNA damage.(A) HeLa cells were transiently transfected with vector control (VEC) or FLAG-TIPRL. 24hrs after transfection, cells were treated with CPT (2.5μM) or (B) doxorubicin (DOXO, 2μg/ml) for the indicated time points. Cells were lysed and immunoblots were probed with the indicated antibodies. (C) HeLa cells were transiently transfected with vector control (VEC) or FLAG-TIPRL. 24hrs after transfection, cells were treated with CPT (2.5μM) for 1.5h followed by washout for 6h. Cells were lysed and immunoblots were probed with the indicated antibodies. (D) 3T3 cells stably expressing LPC FLAG vector control (VEC) or LPC FLAG- TIPRL (TIPRL) were treated with 5μM CPT for 1.5hrs. Cells were fixed and stained for both DAPI and γ-H2AX and (E) images were quantified. Data represent ± standard deviation of the mean of three fields**p<0.01, Student’s t test.
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Related In: Results  -  Collection

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pone.0145938.g003: TIPRL promotes γ-H2AX phosphorylation in response to DNA damage.(A) HeLa cells were transiently transfected with vector control (VEC) or FLAG-TIPRL. 24hrs after transfection, cells were treated with CPT (2.5μM) or (B) doxorubicin (DOXO, 2μg/ml) for the indicated time points. Cells were lysed and immunoblots were probed with the indicated antibodies. (C) HeLa cells were transiently transfected with vector control (VEC) or FLAG-TIPRL. 24hrs after transfection, cells were treated with CPT (2.5μM) for 1.5h followed by washout for 6h. Cells were lysed and immunoblots were probed with the indicated antibodies. (D) 3T3 cells stably expressing LPC FLAG vector control (VEC) or LPC FLAG- TIPRL (TIPRL) were treated with 5μM CPT for 1.5hrs. Cells were fixed and stained for both DAPI and γ-H2AX and (E) images were quantified. Data represent ± standard deviation of the mean of three fields**p<0.01, Student’s t test.
Mentions: The PP4-C/PP4R2/PP4R3β complex has been shown to be a γ-H2AX phosphatase in mammalian cells [3,6] and the pph3 gene, which encodes the ortholog of PP4 catalytic subunit in budding yeast, serves as the sole γ-H2AX phosphatase in this organism [4,5,7]. We found that TIPRL decreases PP4 activity, therefore we wanted to determine if TIPRL expression also resulted in increased γ-H2AX levels following DNA damage. We transiently overexpressed TIPRL and assessed γ-H2AX levels in response to DNA damage. We found that TIPRL overexpression led to increased γ-H2AX phosphorylation in response to the DNA damaging agents CPT (Fig 3A) and doxorubicin (DOXO) (Fig 3B) compared to the vector control. To determine if TIPRL is involved in regulating γ-H2AX dephosphorylation during recovery from DNA damage, we performed a washout experiment and found, consistent with Fig 3A, TIRPL expression led to increased γ-H2AX compared to vector control cells; however, increased TIPRL levels impaired the ability of cells to reverse DNA damage-induced H2AX phosphorylation following washout of the drug, suggesting a delayed dephosphorylation due to decreased phosphatase acitivity in TIPRL overexpressing cells (Fig 3C). To further investigate the role of TIPRL in promoting γ-H2AX foci in response to DNA damage, TIPRL overexpressing cells were treated with CPT, and γ-H2AX staining was visualized by immunofluorescence microscopy (Fig 3D). TIPRL overexpression resulted in a twenty five percent increase in cells that contained γ-H2AX following CPT treatment (Fig 3E).

Bottom Line: Unlike kinases, the activity and specificity of serine/threonine phosphatases is governed largely by their associated proteins.Knockdown of TIPRL resulted in increased PP4 phosphatase activity and formation of the active PP4-C/PP4R2 complex known to dephosphorylate γ-H2AX.In correlation with γ-H2AX levels, we found that TIPRL overexpression promotes cell death in response to genotoxic stress, and knockdown of TIPRL protects cells from genotoxic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Beckman Research Institute of City of Hope Cancer Center, Duarte, California, United States of America.

ABSTRACT
Despite advances in our understanding of protein kinase regulation in the DNA damage response, the mechanism that controls protein phosphatase activity in this pathway is unclear. Unlike kinases, the activity and specificity of serine/threonine phosphatases is governed largely by their associated proteins. Here we show that Tip41-like protein (TIPRL), an evolutionarily conserved binding protein for PP2A-family phosphatases, is a negative regulator of protein phosphatase 4 (PP4). Knockdown of TIPRL resulted in increased PP4 phosphatase activity and formation of the active PP4-C/PP4R2 complex known to dephosphorylate γ-H2AX. Thus, overexpression of TIPRL promotes phosphorylation of H2AX, and increases γ-H2AX positive foci in response to DNA damage, whereas knockdown of TIPRL inhibits γ-H2AX phosphorylation. In correlation with γ-H2AX levels, we found that TIPRL overexpression promotes cell death in response to genotoxic stress, and knockdown of TIPRL protects cells from genotoxic agents. Taken together, these data demonstrate that TIPRL inhibits PP4 activity to allow for H2AX phosphorylation and the subsequent DNA damage response.

Show MeSH
Related in: MedlinePlus