ATM-dependent downregulation of USP7/HAUSP by PPM1G activates p53 response to DNA damage.
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.
Affiliation: Department of Oncology, Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford OX3 7DQ, UK.Show MeSH
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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).
Affiliation: Department of Oncology, Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford OX3 7DQ, UK.