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Phosphorylation of PNKP by ATM prevents its proteasomal degradation and enhances resistance to oxidative stress.

Parsons JL, Khoronenkova SV, Dianova II, Ternette N, Kessler BM, Datta PK, Dianov GL - Nucleic Acids Res. (2012)

Bottom Line: We have also purified a novel Cul4A-DDB1 ubiquitin ligase complex responsible for PNKP ubiquitylation and identify serine-threonine kinase receptor associated protein (STRAP) as the adaptor protein that provides specificity of the complex to PNKP.Strap(-/-) mouse embryonic fibroblasts subsequently contain elevated cellular levels of PNKP, and show elevated resistance to oxidative DNA damage.These data demonstrate an important role for ATM and the Cul4A-DDB1-STRAP ubiquitin ligase in the regulation of the cellular levels of PNKP, and consequently in the repair of oxidative DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Roosevelt Drive, Oxford OX3 7DQ, UK. jason.parsons@oncology.ox.ac.uk

ABSTRACT
We examined the mechanism regulating the cellular levels of PNKP, the major kinase/phosphatase involved in the repair of oxidative DNA damage, and find that it is controlled by ATM phosphorylation and ubiquitylation-dependent proteasomal degradation. We discovered that ATM-dependent phosphorylation of PNKP at serines 114 and 126 in response to oxidative DNA damage inhibits ubiquitylation-dependent proteasomal degradation of PNKP, and consequently increases PNKP stability that is required for DNA repair. We have also purified a novel Cul4A-DDB1 ubiquitin ligase complex responsible for PNKP ubiquitylation and identify serine-threonine kinase receptor associated protein (STRAP) as the adaptor protein that provides specificity of the complex to PNKP. Strap(-/-) mouse embryonic fibroblasts subsequently contain elevated cellular levels of PNKP, and show elevated resistance to oxidative DNA damage. These data demonstrate an important role for ATM and the Cul4A-DDB1-STRAP ubiquitin ligase in the regulation of the cellular levels of PNKP, and consequently in the repair of oxidative DNA damage.

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Phosphorylation of PNKP by ATM modulates its ubiquitylation-dependent degradation. (A and B) HCT116p53+/+ cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine transfection reagent (10 µl) in the presence of 200 pmol PNKP siRNA for 48 h, followed by a further treatment with Lipofectamine transfection reagent (10 µl) in the presence of a mammalian expression plasmid expressing siRNA-resistant Flag-tagged WT, K414/417/484R triple mutant PNKP or S114/126A or S114/126E double mutant PNKP (100 ng) for a further 24 h. Cells were pelleted by centrifugation, whole cell extracts were prepared and analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. The ratio of expression levels of mutant proteins compared to the WT protein, as determined using Flag antibodies and normalized against the tubulin loading control, were calculated from at least three independent experiments. (C) Recombinant WT PNKP (WT) or S114/126A or S114/126E double PNKP mutants (3.5 pmol) were in vitro ubiquitylated using active fraction containing Cul4A-DDB1-STRAP purified from HeLa whole cell extracts in the presence of E1 activating enzyme (0.7 pmol), H5a E2 conjugating enzyme (6 pmol) and ubiquitin (0.6 nmol) and analysed by 10% SDS-PAGE and immunoblotting using PNKP antibodies. (D) HCT116p53+/+ cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine (10 µl) in the presence of 200 pmol PNKP siRNA for 48 h, followed by a further treatment with Lipofectamine transfection reagent (10 µl) in the presence of a mammalian expression plasmid expressing siRNA-resistant Flag-tagged WT or S114/126E double mutant PNK (100 ng) for a further 24 h. Following incubation of the cells with MG-132 (10 µM) for 6 h, the cells were pelleted by centrifugation, whole cell extracts were prepared and incubated with anti-Flag magnetic beads (10 µl) for 2 h at 4°C with rotation. The beads were separated from the extract using a magnetic separation rack, washed several times with buffer containing 150 mM KCl prior to the addition of SDS loading dye and analysis by 10% SDS-PAGE and immunoblotting with HA (upper panel) or Flag (lower panel) antibodies. The ratio of expression levels of ubiquitylated PNKP compared to the unmodified protein, as determined using Flag antibodies, were calculated from at least three independent experiments and normalized to the levels observed with the WT protein which was set to 1.0. Molecular weight markers are indicated on the left-hand side of appropriate figures and the positions of ubiquitylated PNKP (PNKPub) are shown.
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gks909-F4: Phosphorylation of PNKP by ATM modulates its ubiquitylation-dependent degradation. (A and B) HCT116p53+/+ cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine transfection reagent (10 µl) in the presence of 200 pmol PNKP siRNA for 48 h, followed by a further treatment with Lipofectamine transfection reagent (10 µl) in the presence of a mammalian expression plasmid expressing siRNA-resistant Flag-tagged WT, K414/417/484R triple mutant PNKP or S114/126A or S114/126E double mutant PNKP (100 ng) for a further 24 h. Cells were pelleted by centrifugation, whole cell extracts were prepared and analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. The ratio of expression levels of mutant proteins compared to the WT protein, as determined using Flag antibodies and normalized against the tubulin loading control, were calculated from at least three independent experiments. (C) Recombinant WT PNKP (WT) or S114/126A or S114/126E double PNKP mutants (3.5 pmol) were in vitro ubiquitylated using active fraction containing Cul4A-DDB1-STRAP purified from HeLa whole cell extracts in the presence of E1 activating enzyme (0.7 pmol), H5a E2 conjugating enzyme (6 pmol) and ubiquitin (0.6 nmol) and analysed by 10% SDS-PAGE and immunoblotting using PNKP antibodies. (D) HCT116p53+/+ cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine (10 µl) in the presence of 200 pmol PNKP siRNA for 48 h, followed by a further treatment with Lipofectamine transfection reagent (10 µl) in the presence of a mammalian expression plasmid expressing siRNA-resistant Flag-tagged WT or S114/126E double mutant PNK (100 ng) for a further 24 h. Following incubation of the cells with MG-132 (10 µM) for 6 h, the cells were pelleted by centrifugation, whole cell extracts were prepared and incubated with anti-Flag magnetic beads (10 µl) for 2 h at 4°C with rotation. The beads were separated from the extract using a magnetic separation rack, washed several times with buffer containing 150 mM KCl prior to the addition of SDS loading dye and analysis by 10% SDS-PAGE and immunoblotting with HA (upper panel) or Flag (lower panel) antibodies. The ratio of expression levels of ubiquitylated PNKP compared to the unmodified protein, as determined using Flag antibodies, were calculated from at least three independent experiments and normalized to the levels observed with the WT protein which was set to 1.0. Molecular weight markers are indicated on the left-hand side of appropriate figures and the positions of ubiquitylated PNKP (PNKPub) are shown.

Mentions: The most common consequence of cellular ubiquitylation of proteins is to target the proteins for degradation by the 26S proteasome. Therefore, to analyse whether lysines 414, 417 and 484 are promoting ubiquitylation-dependent degradation of PNKP in vivo, we firstly down-regulated endogenous PNKP by siRNA and then transfected cells with mammalian expression plasmids encoding WT or the ubiquitylation-deficient K414/417/484 R triple mutant PNKP, each of which also contained silent mutations to ensure resistance against the siRNA. Even though in vitro ubiquitylation was not fully ablated (Figure 3E), the triple mutant of PNKP was found to be almost 1.5-fold more stable than the WT protein, indicating that lysines 414, 417 and 484 are the major sites that promote PNKP ubiquitylation and degradation in vivo (Figure 4A). Since we demonstrated that ATM-dependent phosphorylation of PNKP in response to oxidative DNA damage prevents its ubiquitylation resulting in an increase in the cellular protein levels of PNKP (Figure 1C and D), we analysed this further by generating site-specific mutants of PNKP at serines 114 and 126, the major ATM-dependent phosphorylation sites. We created a serine to alanine PNKP double mutant that is unable to be phosphorylated (S114/126A) as well as a serine to glutamic acid PNKP mutant that mimics ATM-dependent phosphorylation (S114/126E). We show that when the S114/126A PNKP mutant is transfected into HCT116p53+/+ cells, the protein has a similar level of stability compared to the WT protein, whereas the phosphomimetic S114/126E protein was ∼1.4-fold more stable (Figure 4B). We also find that the S114/126E phosphomimetic double mutant of PNKP is less susceptible (∼50% reduction) to in vitro ubiquitylation by purified Cul4A-DDB1-STRAP, in comparison to the WT protein (Figure 4C, compare lanes 2 and 6). In contrast, the S114/126A mutant is ubiquitylated as efficiently as the WT protein in vitro (Figure 4C, compare lanes 2 and 4). We also found that the S114/126E phosphomimetic double mutant of PNKP was less efficiently ubiquitylated in vivo (approximate reduction of 45%) following transfection into HCT116p53+/+ cells, in comparison to the WT protein (Figure 4D). Cumulatively, these data demonstrate that ATM-dependent phosphorylation of PNKP at serines 114 and 126 promotes protein stability by inhibiting ubiquitylation-dependent proteasomal degradation.Figure 4.


Phosphorylation of PNKP by ATM prevents its proteasomal degradation and enhances resistance to oxidative stress.

Parsons JL, Khoronenkova SV, Dianova II, Ternette N, Kessler BM, Datta PK, Dianov GL - Nucleic Acids Res. (2012)

Phosphorylation of PNKP by ATM modulates its ubiquitylation-dependent degradation. (A and B) HCT116p53+/+ cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine transfection reagent (10 µl) in the presence of 200 pmol PNKP siRNA for 48 h, followed by a further treatment with Lipofectamine transfection reagent (10 µl) in the presence of a mammalian expression plasmid expressing siRNA-resistant Flag-tagged WT, K414/417/484R triple mutant PNKP or S114/126A or S114/126E double mutant PNKP (100 ng) for a further 24 h. Cells were pelleted by centrifugation, whole cell extracts were prepared and analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. The ratio of expression levels of mutant proteins compared to the WT protein, as determined using Flag antibodies and normalized against the tubulin loading control, were calculated from at least three independent experiments. (C) Recombinant WT PNKP (WT) or S114/126A or S114/126E double PNKP mutants (3.5 pmol) were in vitro ubiquitylated using active fraction containing Cul4A-DDB1-STRAP purified from HeLa whole cell extracts in the presence of E1 activating enzyme (0.7 pmol), H5a E2 conjugating enzyme (6 pmol) and ubiquitin (0.6 nmol) and analysed by 10% SDS-PAGE and immunoblotting using PNKP antibodies. (D) HCT116p53+/+ cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine (10 µl) in the presence of 200 pmol PNKP siRNA for 48 h, followed by a further treatment with Lipofectamine transfection reagent (10 µl) in the presence of a mammalian expression plasmid expressing siRNA-resistant Flag-tagged WT or S114/126E double mutant PNK (100 ng) for a further 24 h. Following incubation of the cells with MG-132 (10 µM) for 6 h, the cells were pelleted by centrifugation, whole cell extracts were prepared and incubated with anti-Flag magnetic beads (10 µl) for 2 h at 4°C with rotation. The beads were separated from the extract using a magnetic separation rack, washed several times with buffer containing 150 mM KCl prior to the addition of SDS loading dye and analysis by 10% SDS-PAGE and immunoblotting with HA (upper panel) or Flag (lower panel) antibodies. The ratio of expression levels of ubiquitylated PNKP compared to the unmodified protein, as determined using Flag antibodies, were calculated from at least three independent experiments and normalized to the levels observed with the WT protein which was set to 1.0. Molecular weight markers are indicated on the left-hand side of appropriate figures and the positions of ubiquitylated PNKP (PNKPub) are shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gks909-F4: Phosphorylation of PNKP by ATM modulates its ubiquitylation-dependent degradation. (A and B) HCT116p53+/+ cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine transfection reagent (10 µl) in the presence of 200 pmol PNKP siRNA for 48 h, followed by a further treatment with Lipofectamine transfection reagent (10 µl) in the presence of a mammalian expression plasmid expressing siRNA-resistant Flag-tagged WT, K414/417/484R triple mutant PNKP or S114/126A or S114/126E double mutant PNKP (100 ng) for a further 24 h. Cells were pelleted by centrifugation, whole cell extracts were prepared and analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. The ratio of expression levels of mutant proteins compared to the WT protein, as determined using Flag antibodies and normalized against the tubulin loading control, were calculated from at least three independent experiments. (C) Recombinant WT PNKP (WT) or S114/126A or S114/126E double PNKP mutants (3.5 pmol) were in vitro ubiquitylated using active fraction containing Cul4A-DDB1-STRAP purified from HeLa whole cell extracts in the presence of E1 activating enzyme (0.7 pmol), H5a E2 conjugating enzyme (6 pmol) and ubiquitin (0.6 nmol) and analysed by 10% SDS-PAGE and immunoblotting using PNKP antibodies. (D) HCT116p53+/+ cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine (10 µl) in the presence of 200 pmol PNKP siRNA for 48 h, followed by a further treatment with Lipofectamine transfection reagent (10 µl) in the presence of a mammalian expression plasmid expressing siRNA-resistant Flag-tagged WT or S114/126E double mutant PNK (100 ng) for a further 24 h. Following incubation of the cells with MG-132 (10 µM) for 6 h, the cells were pelleted by centrifugation, whole cell extracts were prepared and incubated with anti-Flag magnetic beads (10 µl) for 2 h at 4°C with rotation. The beads were separated from the extract using a magnetic separation rack, washed several times with buffer containing 150 mM KCl prior to the addition of SDS loading dye and analysis by 10% SDS-PAGE and immunoblotting with HA (upper panel) or Flag (lower panel) antibodies. The ratio of expression levels of ubiquitylated PNKP compared to the unmodified protein, as determined using Flag antibodies, were calculated from at least three independent experiments and normalized to the levels observed with the WT protein which was set to 1.0. Molecular weight markers are indicated on the left-hand side of appropriate figures and the positions of ubiquitylated PNKP (PNKPub) are shown.
Mentions: The most common consequence of cellular ubiquitylation of proteins is to target the proteins for degradation by the 26S proteasome. Therefore, to analyse whether lysines 414, 417 and 484 are promoting ubiquitylation-dependent degradation of PNKP in vivo, we firstly down-regulated endogenous PNKP by siRNA and then transfected cells with mammalian expression plasmids encoding WT or the ubiquitylation-deficient K414/417/484 R triple mutant PNKP, each of which also contained silent mutations to ensure resistance against the siRNA. Even though in vitro ubiquitylation was not fully ablated (Figure 3E), the triple mutant of PNKP was found to be almost 1.5-fold more stable than the WT protein, indicating that lysines 414, 417 and 484 are the major sites that promote PNKP ubiquitylation and degradation in vivo (Figure 4A). Since we demonstrated that ATM-dependent phosphorylation of PNKP in response to oxidative DNA damage prevents its ubiquitylation resulting in an increase in the cellular protein levels of PNKP (Figure 1C and D), we analysed this further by generating site-specific mutants of PNKP at serines 114 and 126, the major ATM-dependent phosphorylation sites. We created a serine to alanine PNKP double mutant that is unable to be phosphorylated (S114/126A) as well as a serine to glutamic acid PNKP mutant that mimics ATM-dependent phosphorylation (S114/126E). We show that when the S114/126A PNKP mutant is transfected into HCT116p53+/+ cells, the protein has a similar level of stability compared to the WT protein, whereas the phosphomimetic S114/126E protein was ∼1.4-fold more stable (Figure 4B). We also find that the S114/126E phosphomimetic double mutant of PNKP is less susceptible (∼50% reduction) to in vitro ubiquitylation by purified Cul4A-DDB1-STRAP, in comparison to the WT protein (Figure 4C, compare lanes 2 and 6). In contrast, the S114/126A mutant is ubiquitylated as efficiently as the WT protein in vitro (Figure 4C, compare lanes 2 and 4). We also found that the S114/126E phosphomimetic double mutant of PNKP was less efficiently ubiquitylated in vivo (approximate reduction of 45%) following transfection into HCT116p53+/+ cells, in comparison to the WT protein (Figure 4D). Cumulatively, these data demonstrate that ATM-dependent phosphorylation of PNKP at serines 114 and 126 promotes protein stability by inhibiting ubiquitylation-dependent proteasomal degradation.Figure 4.

Bottom Line: We have also purified a novel Cul4A-DDB1 ubiquitin ligase complex responsible for PNKP ubiquitylation and identify serine-threonine kinase receptor associated protein (STRAP) as the adaptor protein that provides specificity of the complex to PNKP.Strap(-/-) mouse embryonic fibroblasts subsequently contain elevated cellular levels of PNKP, and show elevated resistance to oxidative DNA damage.These data demonstrate an important role for ATM and the Cul4A-DDB1-STRAP ubiquitin ligase in the regulation of the cellular levels of PNKP, and consequently in the repair of oxidative DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Roosevelt Drive, Oxford OX3 7DQ, UK. jason.parsons@oncology.ox.ac.uk

ABSTRACT
We examined the mechanism regulating the cellular levels of PNKP, the major kinase/phosphatase involved in the repair of oxidative DNA damage, and find that it is controlled by ATM phosphorylation and ubiquitylation-dependent proteasomal degradation. We discovered that ATM-dependent phosphorylation of PNKP at serines 114 and 126 in response to oxidative DNA damage inhibits ubiquitylation-dependent proteasomal degradation of PNKP, and consequently increases PNKP stability that is required for DNA repair. We have also purified a novel Cul4A-DDB1 ubiquitin ligase complex responsible for PNKP ubiquitylation and identify serine-threonine kinase receptor associated protein (STRAP) as the adaptor protein that provides specificity of the complex to PNKP. Strap(-/-) mouse embryonic fibroblasts subsequently contain elevated cellular levels of PNKP, and show elevated resistance to oxidative DNA damage. These data demonstrate an important role for ATM and the Cul4A-DDB1-STRAP ubiquitin ligase in the regulation of the cellular levels of PNKP, and consequently in the repair of oxidative DNA damage.

Show MeSH
Related in: MedlinePlus