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ATM-dependent downregulation of USP7/HAUSP by PPM1G activates p53 response to DNA damage.

Khoronenkova SV, Dianova II, Ternette N, Kessler BM, Parsons JL, Dianov GL - Mol. Cell (2012)

Bottom Line: We find that stabilization of Mdm2, and correspondingly p53 downregulation in unstressed cells, is accomplished by a specific isoform of USP7 (USP7S), which is phosphorylated at serine 18 by the protein kinase CK2.Phosphorylation stabilizes USP7S and thus contributes to Mdm2 stabilization and downregulation of p53.After ionizing radiation, dephosphorylation of USP7S by the ATM-dependent protein phosphatase PPM1G leads to USP7S downregulation, followed by Mdm2 downregulation and accumulation of p53.

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Affiliation: Department of Oncology, Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford OX3 7DQ, UK.

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Purification and Identification of CK2 as the Major Protein Kinase Phosphorylating USP7S at Serine 18(A) HeLa whole-cell extract (40 μg) was separated by 10% SDS-PAGE and analyzed by western blotting using antibodies specific to USP7S or phosphorylated USP7S (pUSP7S).(B) HeLa whole-cell extracts were immunoprecipitated using USP7S or pUSP7S antibodies and analyzed by western blotting. The quantification of bands is shown.(C) In vitro phosphorylation of dephosphorylated recombinant USP7S (1 pmol) by the final hydroxyapatite chromatography fractions purified from HeLa cells, analyzed using pUSP7S (top panel). Western blot analysis of the same fractions with CK2α and CK2α′ antibodies (lower panel).(D) In vitro phosphorylation of dephosphorylated recombinant USP7S (2.8 pmol) by recombinant CK2 protein (2 pmol) was performed at 30°C for 30 min and analyzed using pUSP7S antibodies.(E) In vitro phosphorylation of dephosphorylated recombinant USP7S (1.5 pmol) and dephosphorylated USP7S18A (1.5 pmol) by recombinant CK2 (2 pmol) was performed at 30°C for 60 min in the presence of γ32P-ATP and analyzed by 10% SDS-PAGE and phosphorimaging. Equal loading of the substrate is demonstrated using USP7S antibodies (lower panel).See also Figure S1 and Table S1.
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fig1: Purification and Identification of CK2 as the Major Protein Kinase Phosphorylating USP7S at Serine 18(A) HeLa whole-cell extract (40 μg) was separated by 10% SDS-PAGE and analyzed by western blotting using antibodies specific to USP7S or phosphorylated USP7S (pUSP7S).(B) HeLa whole-cell extracts were immunoprecipitated using USP7S or pUSP7S antibodies and analyzed by western blotting. The quantification of bands is shown.(C) In vitro phosphorylation of dephosphorylated recombinant USP7S (1 pmol) by the final hydroxyapatite chromatography fractions purified from HeLa cells, analyzed using pUSP7S (top panel). Western blot analysis of the same fractions with CK2α and CK2α′ antibodies (lower panel).(D) In vitro phosphorylation of dephosphorylated recombinant USP7S (2.8 pmol) by recombinant CK2 protein (2 pmol) was performed at 30°C for 30 min and analyzed using pUSP7S antibodies.(E) In vitro phosphorylation of dephosphorylated recombinant USP7S (1.5 pmol) and dephosphorylated USP7S18A (1.5 pmol) by recombinant CK2 (2 pmol) was performed at 30°C for 60 min in the presence of γ32P-ATP and analyzed by 10% SDS-PAGE and phosphorimaging. Equal loading of the substrate is demonstrated using USP7S antibodies (lower panel).See also Figure S1 and Table S1.

Mentions: It was recently reported that USP7 is phosphorylated at serine eighteen (further referred to as USP7S). However, the biological role of this phosphorylation, and the protein kinase and phosphatase involved in regulation of the serine 18 (Ser18) phosphorylation status of USP7S were not identified (Fernández-Montalván et al., 2007). We hypothesized that phosphorylation of USP7S in response to DNA damage regulates its stability and thus coordinates Mdm2 and p53 activated cellular responses. To uncover the role of Ser18 in USP7S regulation and the proteins involved in Ser18 phosphorylation/dephosphorylation, we generated an antibody specific to phosphorylated and unphosphorylated USP7S (Figures S1A and S1D available online). Both antibodies cross-reacted with USP7S protein in HeLa whole-cell extracts, confirming that phosphorylation of USP7S at Ser18 occurs in human cells (Figure 1A). We further estimated the amount of phosphorylated USP7S existing in human cells by immunoprecipitation of cell extracts using both sets of antibodies. An equal amount of phosphorylated USP7S protein was observed following immunoprecipitation with either USP7S or pUSP7S antibodies specific to phosphorylated protein (Figure 1B and Figure S1E, upper panel). However, pUSP7S antibodies were able to precipitate only 60%–80% of the total USP7S protein (Figure 1B and Figure S1E, lower panel), suggesting that approximately 60%–80% of USP7S is phosphorylated in human cells. We next wished to purify the cellular protein kinase involved in Ser18 phosphorylation. To achieve this, we established an in vitro kinase assay employing recombinant (dephosphorylated) USP7S (Figures S1B and S1C) and used this assay to detect, by western blotting, Ser18 phosphorylation activity in individual fractions after cellular extract fractionation. We observed robust phosphorylation of USP7S at Ser18 that was readily detectable following fractionation of HeLa whole-cell extracts over a series of chromatographic columns (Figures S1F–S1I). Fractions from the final hydroxyapatite column displaying substantial kinase activity against USP7S (Figure 1C, upper panel) were analyzed by mass spectrometry. The major protein kinase found in these fractions was CK2, of which all subunits (CK2α, CK2α′, and CK2β) were detected (Table S1 and data not shown). Western blot analysis of these fractions further confirmed co-purification of CK2 with USP7S Ser18 phosphorylation activity (Figure 1C, lower panel). In fact, USP7S possesses a high score consensus site for CK2 that includes Ser18 (Figure S1A). In accordance with this, we found that recombinant CK2 protein efficiently phosphorylated USP7S (Figure 1D) and that this phosphorylation was specific to Ser18, since when γ-32P-ATP was used in an in vitro phosphorylation assay we only observed efficient phosphorylation of previously dephosphorylated wild-type USP7S and not of dephosphorylated USP7S18A mutated protein where Ser18 was substituted for alanine (Figure 1E).


ATM-dependent downregulation of USP7/HAUSP by PPM1G activates p53 response to DNA damage.

Khoronenkova SV, Dianova II, Ternette N, Kessler BM, Parsons JL, Dianov GL - Mol. Cell (2012)

Purification and Identification of CK2 as the Major Protein Kinase Phosphorylating USP7S at Serine 18(A) HeLa whole-cell extract (40 μg) was separated by 10% SDS-PAGE and analyzed by western blotting using antibodies specific to USP7S or phosphorylated USP7S (pUSP7S).(B) HeLa whole-cell extracts were immunoprecipitated using USP7S or pUSP7S antibodies and analyzed by western blotting. The quantification of bands is shown.(C) In vitro phosphorylation of dephosphorylated recombinant USP7S (1 pmol) by the final hydroxyapatite chromatography fractions purified from HeLa cells, analyzed using pUSP7S (top panel). Western blot analysis of the same fractions with CK2α and CK2α′ antibodies (lower panel).(D) In vitro phosphorylation of dephosphorylated recombinant USP7S (2.8 pmol) by recombinant CK2 protein (2 pmol) was performed at 30°C for 30 min and analyzed using pUSP7S antibodies.(E) In vitro phosphorylation of dephosphorylated recombinant USP7S (1.5 pmol) and dephosphorylated USP7S18A (1.5 pmol) by recombinant CK2 (2 pmol) was performed at 30°C for 60 min in the presence of γ32P-ATP and analyzed by 10% SDS-PAGE and phosphorimaging. Equal loading of the substrate is demonstrated using USP7S antibodies (lower panel).See also Figure S1 and Table S1.
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fig1: Purification and Identification of CK2 as the Major Protein Kinase Phosphorylating USP7S at Serine 18(A) HeLa whole-cell extract (40 μg) was separated by 10% SDS-PAGE and analyzed by western blotting using antibodies specific to USP7S or phosphorylated USP7S (pUSP7S).(B) HeLa whole-cell extracts were immunoprecipitated using USP7S or pUSP7S antibodies and analyzed by western blotting. The quantification of bands is shown.(C) In vitro phosphorylation of dephosphorylated recombinant USP7S (1 pmol) by the final hydroxyapatite chromatography fractions purified from HeLa cells, analyzed using pUSP7S (top panel). Western blot analysis of the same fractions with CK2α and CK2α′ antibodies (lower panel).(D) In vitro phosphorylation of dephosphorylated recombinant USP7S (2.8 pmol) by recombinant CK2 protein (2 pmol) was performed at 30°C for 30 min and analyzed using pUSP7S antibodies.(E) In vitro phosphorylation of dephosphorylated recombinant USP7S (1.5 pmol) and dephosphorylated USP7S18A (1.5 pmol) by recombinant CK2 (2 pmol) was performed at 30°C for 60 min in the presence of γ32P-ATP and analyzed by 10% SDS-PAGE and phosphorimaging. Equal loading of the substrate is demonstrated using USP7S antibodies (lower panel).See also Figure S1 and Table S1.
Mentions: It was recently reported that USP7 is phosphorylated at serine eighteen (further referred to as USP7S). However, the biological role of this phosphorylation, and the protein kinase and phosphatase involved in regulation of the serine 18 (Ser18) phosphorylation status of USP7S were not identified (Fernández-Montalván et al., 2007). We hypothesized that phosphorylation of USP7S in response to DNA damage regulates its stability and thus coordinates Mdm2 and p53 activated cellular responses. To uncover the role of Ser18 in USP7S regulation and the proteins involved in Ser18 phosphorylation/dephosphorylation, we generated an antibody specific to phosphorylated and unphosphorylated USP7S (Figures S1A and S1D available online). Both antibodies cross-reacted with USP7S protein in HeLa whole-cell extracts, confirming that phosphorylation of USP7S at Ser18 occurs in human cells (Figure 1A). We further estimated the amount of phosphorylated USP7S existing in human cells by immunoprecipitation of cell extracts using both sets of antibodies. An equal amount of phosphorylated USP7S protein was observed following immunoprecipitation with either USP7S or pUSP7S antibodies specific to phosphorylated protein (Figure 1B and Figure S1E, upper panel). However, pUSP7S antibodies were able to precipitate only 60%–80% of the total USP7S protein (Figure 1B and Figure S1E, lower panel), suggesting that approximately 60%–80% of USP7S is phosphorylated in human cells. We next wished to purify the cellular protein kinase involved in Ser18 phosphorylation. To achieve this, we established an in vitro kinase assay employing recombinant (dephosphorylated) USP7S (Figures S1B and S1C) and used this assay to detect, by western blotting, Ser18 phosphorylation activity in individual fractions after cellular extract fractionation. We observed robust phosphorylation of USP7S at Ser18 that was readily detectable following fractionation of HeLa whole-cell extracts over a series of chromatographic columns (Figures S1F–S1I). Fractions from the final hydroxyapatite column displaying substantial kinase activity against USP7S (Figure 1C, upper panel) were analyzed by mass spectrometry. The major protein kinase found in these fractions was CK2, of which all subunits (CK2α, CK2α′, and CK2β) were detected (Table S1 and data not shown). Western blot analysis of these fractions further confirmed co-purification of CK2 with USP7S Ser18 phosphorylation activity (Figure 1C, lower panel). In fact, USP7S possesses a high score consensus site for CK2 that includes Ser18 (Figure S1A). In accordance with this, we found that recombinant CK2 protein efficiently phosphorylated USP7S (Figure 1D) and that this phosphorylation was specific to Ser18, since when γ-32P-ATP was used in an in vitro phosphorylation assay we only observed efficient phosphorylation of previously dephosphorylated wild-type USP7S and not of dephosphorylated USP7S18A mutated protein where Ser18 was substituted for alanine (Figure 1E).

Bottom Line: We find that stabilization of Mdm2, and correspondingly p53 downregulation in unstressed cells, is accomplished by a specific isoform of USP7 (USP7S), which is phosphorylated at serine 18 by the protein kinase CK2.Phosphorylation stabilizes USP7S and thus contributes to Mdm2 stabilization and downregulation of p53.After ionizing radiation, dephosphorylation of USP7S by the ATM-dependent protein phosphatase PPM1G leads to USP7S downregulation, followed by Mdm2 downregulation and accumulation of p53.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford OX3 7DQ, UK.

Show MeSH
Related in: MedlinePlus