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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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Related in: MedlinePlus

TIPRL inhibits PP4 phosphatase activity and complex assembly.(A) HeLa cells were transiently transfected with LPC FLAG (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL). 24hrs after transfection, cells were lysed and immunoprecipitated with PP4-C antibody, upon which phosphatase activity was measured. Data represent ± standard deviation of the mean of three independent experiments. (B&C) HeLa cells were transfected with a scramble (siSCR) or TIPRL siRNA (siTIPRL). 48hrs after transfection, cell lysates were immunoprecipitated with PP4-C (B) or PP4R2 (C) antibody and phosphatase activity was measured. Data represent ± standard deviation of the mean of three independent experiments. (D) HeLa cells transfected with LPC FLAG-TIPRL (FLAG-TIPRL) were immunoprecipitated with the indicated antibodies and phosphatase activity was measured. (E) HeLa cells were transfected with a scramble (siSCR) or TIPRL siRNA (siTIPRL) and immunoprecipitated with PP4R2 antibody followed by immunoblotting with the indicated antibodies. (F) HeLa cells were transiently transfected with LPC FLAG (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL) and immunoprecipitated with PP4-C antibody followed by immunoblotting with the indicated antibodies. Long and short refer to the film exposure time. (G) 3T3 cells were treated with DMSO (CONT) or 5μM CPT for 1hr. Cells were lysed and immunoprecipitated with TIPRL followed by immunoblotting with the indicated antibodies. Band intensity was quantified using Image ***p<0.001, Student’s t test.
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pone.0145938.g002: TIPRL inhibits PP4 phosphatase activity and complex assembly.(A) HeLa cells were transiently transfected with LPC FLAG (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL). 24hrs after transfection, cells were lysed and immunoprecipitated with PP4-C antibody, upon which phosphatase activity was measured. Data represent ± standard deviation of the mean of three independent experiments. (B&C) HeLa cells were transfected with a scramble (siSCR) or TIPRL siRNA (siTIPRL). 48hrs after transfection, cell lysates were immunoprecipitated with PP4-C (B) or PP4R2 (C) antibody and phosphatase activity was measured. Data represent ± standard deviation of the mean of three independent experiments. (D) HeLa cells transfected with LPC FLAG-TIPRL (FLAG-TIPRL) were immunoprecipitated with the indicated antibodies and phosphatase activity was measured. (E) HeLa cells were transfected with a scramble (siSCR) or TIPRL siRNA (siTIPRL) and immunoprecipitated with PP4R2 antibody followed by immunoblotting with the indicated antibodies. (F) HeLa cells were transiently transfected with LPC FLAG (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL) and immunoprecipitated with PP4-C antibody followed by immunoblotting with the indicated antibodies. Long and short refer to the film exposure time. (G) 3T3 cells were treated with DMSO (CONT) or 5μM CPT for 1hr. Cells were lysed and immunoprecipitated with TIPRL followed by immunoblotting with the indicated antibodies. Band intensity was quantified using Image ***p<0.001, Student’s t test.

Mentions: To assess TIPRL’s ability to regulate PP4 protein phosphatase activity, TIPRL was transiently overexpressed in HeLa cells (Fig 2A). Vector and TIPRL overexpressing cell lysates were immunoprecipitated followed by phosphatase activity assays to evaluate the role of TIPRL in regulating phosphatase activity. Immunoprecipitation of the PP4 catalytic subunit in TIPRL overexpressing cells showed a dramatic decrease in phosphatase activity compared to the vector control; however, the total levels of PP4 proteins PP4-C and PP4R2 are unchanged (Fig 2A). To further confirm TIPRL’s role in regulating phosphatase activity, we transiently knocked down TIPRL (Fig 2B) and measured protein phosphatase activity. Immunoprecipitation of the PP4 catalytic subunit displayed a 2.5 fold increase in phosphatase activity when TIPRL was diminished compared to the scrambled control (Fig 2B). As PP4-C resides in both active (i.e. PP4R2/PP4-C complex) and inactive complexes (i.e. α4/PP4-C complex), we next asked if TIPRL preferably inhibits the active PP4 complexes. We immunoprecipitated PP4R2, a PP4-C interacting protein present in active complexes, and subjected the immunoprecipitates to a phosphatase assay and found that knockdown of TIPRL resulted in a 5 fold increase in PP4R2-associated phosphatase activity when PP4R2 was immunoprecipitated (Fig 2C). Compared to the 2.5 fold decrease in total PP4 activity (Fig 2A), this data indicates that TIPRL may target protein phosphatase active complexes. Because TIPRL inhibits phosphatase activity, we next asked whether TIPRL-associated PP4-C was inactive. To test this, we immunoprecipitated PP4-C or TIPRL followed by phosphatase assay. The phosphatase activity was further normalized to the PP4-C subunit detected by western blot in the PP4-C or TIPRL immunoprecipitates. Indeed, compared to total PP4-C activity, TIPRL bound PP4-C was inactive, suggesting TIPRL inhibits PP4-C activity due to direct binding (Fig 2D). Next, we wanted to determine if TIPRL inhibited phosphatase activity by promoting disassembly of the PP4 active complex. The PP4R2/PP4-C complex was evaluated by immunoprecipitating PP4R2 followed by a western blot using anti-PP4-C antibody. Cells expressing a siRNA to TIPRL displayed an increase in PP4-C bound to PP4R2 (Fig 2E), consistent with more phosphatase activity (Fig 2B & 2C). On the other hand, when TIPRL was overexpressed, the amount of PP4-C/PP4R2 complex dramatically decreased compared to vector control (Fig 2F), consistent with the decrease in phosphatase activity under the same conditions (Fig 2A). Furthermore, TIPRL association with PP4-C was increased upon treatment with DNA-damaging agent CPT, suggesting PP4-C is inhibited upon this stress condition via association with TIRPL (Fig 2G). Taken together, these results suggest that TIPRL may inhibit phosphatase activity through promoting disassembly of the active PP4 complex.


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 inhibits PP4 phosphatase activity and complex assembly.(A) HeLa cells were transiently transfected with LPC FLAG (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL). 24hrs after transfection, cells were lysed and immunoprecipitated with PP4-C antibody, upon which phosphatase activity was measured. Data represent ± standard deviation of the mean of three independent experiments. (B&C) HeLa cells were transfected with a scramble (siSCR) or TIPRL siRNA (siTIPRL). 48hrs after transfection, cell lysates were immunoprecipitated with PP4-C (B) or PP4R2 (C) antibody and phosphatase activity was measured. Data represent ± standard deviation of the mean of three independent experiments. (D) HeLa cells transfected with LPC FLAG-TIPRL (FLAG-TIPRL) were immunoprecipitated with the indicated antibodies and phosphatase activity was measured. (E) HeLa cells were transfected with a scramble (siSCR) or TIPRL siRNA (siTIPRL) and immunoprecipitated with PP4R2 antibody followed by immunoblotting with the indicated antibodies. (F) HeLa cells were transiently transfected with LPC FLAG (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL) and immunoprecipitated with PP4-C antibody followed by immunoblotting with the indicated antibodies. Long and short refer to the film exposure time. (G) 3T3 cells were treated with DMSO (CONT) or 5μM CPT for 1hr. Cells were lysed and immunoprecipitated with TIPRL followed by immunoblotting with the indicated antibodies. Band intensity was quantified using Image ***p<0.001, Student’s t test.
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Related In: Results  -  Collection

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pone.0145938.g002: TIPRL inhibits PP4 phosphatase activity and complex assembly.(A) HeLa cells were transiently transfected with LPC FLAG (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL). 24hrs after transfection, cells were lysed and immunoprecipitated with PP4-C antibody, upon which phosphatase activity was measured. Data represent ± standard deviation of the mean of three independent experiments. (B&C) HeLa cells were transfected with a scramble (siSCR) or TIPRL siRNA (siTIPRL). 48hrs after transfection, cell lysates were immunoprecipitated with PP4-C (B) or PP4R2 (C) antibody and phosphatase activity was measured. Data represent ± standard deviation of the mean of three independent experiments. (D) HeLa cells transfected with LPC FLAG-TIPRL (FLAG-TIPRL) were immunoprecipitated with the indicated antibodies and phosphatase activity was measured. (E) HeLa cells were transfected with a scramble (siSCR) or TIPRL siRNA (siTIPRL) and immunoprecipitated with PP4R2 antibody followed by immunoblotting with the indicated antibodies. (F) HeLa cells were transiently transfected with LPC FLAG (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL) and immunoprecipitated with PP4-C antibody followed by immunoblotting with the indicated antibodies. Long and short refer to the film exposure time. (G) 3T3 cells were treated with DMSO (CONT) or 5μM CPT for 1hr. Cells were lysed and immunoprecipitated with TIPRL followed by immunoblotting with the indicated antibodies. Band intensity was quantified using Image ***p<0.001, Student’s t test.
Mentions: To assess TIPRL’s ability to regulate PP4 protein phosphatase activity, TIPRL was transiently overexpressed in HeLa cells (Fig 2A). Vector and TIPRL overexpressing cell lysates were immunoprecipitated followed by phosphatase activity assays to evaluate the role of TIPRL in regulating phosphatase activity. Immunoprecipitation of the PP4 catalytic subunit in TIPRL overexpressing cells showed a dramatic decrease in phosphatase activity compared to the vector control; however, the total levels of PP4 proteins PP4-C and PP4R2 are unchanged (Fig 2A). To further confirm TIPRL’s role in regulating phosphatase activity, we transiently knocked down TIPRL (Fig 2B) and measured protein phosphatase activity. Immunoprecipitation of the PP4 catalytic subunit displayed a 2.5 fold increase in phosphatase activity when TIPRL was diminished compared to the scrambled control (Fig 2B). As PP4-C resides in both active (i.e. PP4R2/PP4-C complex) and inactive complexes (i.e. α4/PP4-C complex), we next asked if TIPRL preferably inhibits the active PP4 complexes. We immunoprecipitated PP4R2, a PP4-C interacting protein present in active complexes, and subjected the immunoprecipitates to a phosphatase assay and found that knockdown of TIPRL resulted in a 5 fold increase in PP4R2-associated phosphatase activity when PP4R2 was immunoprecipitated (Fig 2C). Compared to the 2.5 fold decrease in total PP4 activity (Fig 2A), this data indicates that TIPRL may target protein phosphatase active complexes. Because TIPRL inhibits phosphatase activity, we next asked whether TIPRL-associated PP4-C was inactive. To test this, we immunoprecipitated PP4-C or TIPRL followed by phosphatase assay. The phosphatase activity was further normalized to the PP4-C subunit detected by western blot in the PP4-C or TIPRL immunoprecipitates. Indeed, compared to total PP4-C activity, TIPRL bound PP4-C was inactive, suggesting TIPRL inhibits PP4-C activity due to direct binding (Fig 2D). Next, we wanted to determine if TIPRL inhibited phosphatase activity by promoting disassembly of the PP4 active complex. The PP4R2/PP4-C complex was evaluated by immunoprecipitating PP4R2 followed by a western blot using anti-PP4-C antibody. Cells expressing a siRNA to TIPRL displayed an increase in PP4-C bound to PP4R2 (Fig 2E), consistent with more phosphatase activity (Fig 2B & 2C). On the other hand, when TIPRL was overexpressed, the amount of PP4-C/PP4R2 complex dramatically decreased compared to vector control (Fig 2F), consistent with the decrease in phosphatase activity under the same conditions (Fig 2A). Furthermore, TIPRL association with PP4-C was increased upon treatment with DNA-damaging agent CPT, suggesting PP4-C is inhibited upon this stress condition via association with TIRPL (Fig 2G). Taken together, these results suggest that TIPRL may inhibit phosphatase activity through promoting disassembly of the active PP4 complex.

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