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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 interacts with the protein phosphatase 4 complex.(A) 293T cells transfected with LPC FLAG vector (FLAG-TIPRL (-)) or LPC FLAG-TIPRL (FLAG-TIPRL (+)) were immunoprecipitated with FLAG conjugated beads followed by immunoblotting with the indicated antibodies. (B) TIPRL was immunoprecipitated from HeLa cells followed by immunoblotting with the indicated antibodies to assess endogenous interaction with TIPRL.
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pone.0145938.g001: TIPRL interacts with the protein phosphatase 4 complex.(A) 293T cells transfected with LPC FLAG vector (FLAG-TIPRL (-)) or LPC FLAG-TIPRL (FLAG-TIPRL (+)) were immunoprecipitated with FLAG conjugated beads followed by immunoblotting with the indicated antibodies. (B) TIPRL was immunoprecipitated from HeLa cells followed by immunoblotting with the indicated antibodies to assess endogenous interaction with TIPRL.

Mentions: TIPRL is a highly conserved and ubiquitously expressed protein that interacts with multiple PP2A-family phosphatase catalytic subunits, including PP2A-C, PP4-C and PP6-C. Here, to further investigate if TIPRL also binds with PP4R2, a PP4-C interacting protein that has been found in active PP4 phosphatase complexes, we transiently overexpressed FLAG tagged vector or TIPRL and performed immunoprecipitation followed by immunoblotting. PP4-C and PP4R2 were found to interact with TIPRL (Fig 1A). The endogenous binding of TIPRL with PP4R2 and PP4-C was validated by co-IP experiments (Fig 1B).


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 interacts with the protein phosphatase 4 complex.(A) 293T cells transfected with LPC FLAG vector (FLAG-TIPRL (-)) or LPC FLAG-TIPRL (FLAG-TIPRL (+)) were immunoprecipitated with FLAG conjugated beads followed by immunoblotting with the indicated antibodies. (B) TIPRL was immunoprecipitated from HeLa cells followed by immunoblotting with the indicated antibodies to assess endogenous interaction with TIPRL.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696667&req=5

pone.0145938.g001: TIPRL interacts with the protein phosphatase 4 complex.(A) 293T cells transfected with LPC FLAG vector (FLAG-TIPRL (-)) or LPC FLAG-TIPRL (FLAG-TIPRL (+)) were immunoprecipitated with FLAG conjugated beads followed by immunoblotting with the indicated antibodies. (B) TIPRL was immunoprecipitated from HeLa cells followed by immunoblotting with the indicated antibodies to assess endogenous interaction with TIPRL.
Mentions: TIPRL is a highly conserved and ubiquitously expressed protein that interacts with multiple PP2A-family phosphatase catalytic subunits, including PP2A-C, PP4-C and PP6-C. Here, to further investigate if TIPRL also binds with PP4R2, a PP4-C interacting protein that has been found in active PP4 phosphatase complexes, we transiently overexpressed FLAG tagged vector or TIPRL and performed immunoprecipitation followed by immunoblotting. PP4-C and PP4R2 were found to interact with TIPRL (Fig 1A). The endogenous binding of TIPRL with PP4R2 and PP4-C was validated by co-IP experiments (Fig 1B).

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