doi: 10.1038/emboj.2011.419.
Regulation of p53 stability and function by the deubiquitinating enzyme USP42
Affiliations
- PMID:22085928
- PMCID: PMC3243628
- DOI: 10.1038/emboj.2011.419
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Regulation of p53 stability and function by the deubiquitinating enzyme USP42
Andreas K Hock et al. EMBO J..
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Abstract
The p53 tumour suppressor protein is a transcription factor that prevents oncogenic progression by activating the expression of apoptosis and cell-cycle arrest genes in stressed cells. The stability of p53 is tightly regulated by ubiquitin-dependent degradation, driven mainly by the ubiquitin ligase MDM2. In this study, we have identified USP42 as a DUB that interacts with and deubiquitinates p53. USP42 forms a direct complex with p53 and controls level of ubiquitination during the early phase of the response to a range of stress signals. Although we do not find a clear role for USP42 in controlling either the basal or fully activated levels of p53, the function of USP42 is required to allow the rapid activation of p53-dependent transcription and a p53-dependent cell-cycle arrest in response to stress. These functions of USP42 are likely to contribute to the repair and recovery of cells from mild or transient damage.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Figures
Identification of USP42 as a regulator of p53 stability. (A) p53-inducible Saos-2 cells stably expressing PG13-luciferase reporter were transfected with control or a pool of USP42 siRNA oligonucleotides. Forty-eight hours post transfection, p53 was induced by treatment with 1 μg/ml doxycycline. Cells were lysed 24 h after p53 induction and luciferase activity was determined using a luciferase assay system (Promega). (B) p53-inducible Saos-2 cells were transfected with control or individual USP42 siRNA oligonucleotides. Forty-eight hours post transfection, p53 was induced by treatment with 1 μg/ml doxycycline. Total cell lysates were prepared 24 h after p53 induction. Lysate proteins were resolved by SDS–PAGE and analysed by western blotting with anti-p53 DO1 and Actin antibodies. (C) p53-inducible Saos-2 cells were transfected with control or USP42 siRNA oligonucleotides. Forty-eight hours post transfection, p53 was induced by treatment with 1 μg/ml doxycycline, then 24 h after p53 induction, cells were treated with 100 μg/ml cyclohexamide for the indicated times. Total cell lysates were resolved by SDS–PAGE and analysed by western blotting with anti-p53 and anti-Actin antibodies. Quantification of three independent experiments is shown in the graph below.
USP42 binds p53. (A) Schematic showing the DUB domain and amino acids, which are predicted to be important for catalytic activity within USP42. (B) Flag-tagged USP42 and p53 constructs werein-vitro translated and allowed to interact for 1 h. Immunoprecipitations were performed using anti-Flag M2. Precipitated proteins were resolved by SDS–PAGE and analysed by western blotting with anti-Flag M2 and anti-p53 CM1 antibodies. (C) U2OS cells were transfected with p53 and with wt (wild-type) or C120A GFP–Flag-tagged USP42 plasmids where indicated. Cells were lysed and immunoprecipitations were performed using anti-Flag M2. Precipitated and input proteins were resolved by SDS–PAGE and analysed by western blotting with anti-GFP and anti-p53 CM1 antibodies. (D) Western blots of cytoplasmic and nuclear fractions of U2OS cell, using polyclonal antibodies against USP42, Lamin C (a nuclear protein) and α-tubulin (a cytoplasmic protein). (E) Immunoprecipitation of p53, MDM2 and USP42 from U2OS cells, followed by western blotting of the precipitated proteins with anti-USP42, anti-p53 and anti-MDM2 antibodies. (F) Immunoprecipitation of p53 and USP42 from U2OS cells untreated or treated with actinomycin D. Figure source data can be found with the Supplementary data.
USP42 deubiquitininates p53. (A)In vitro translated p53 was pre-incubated for 90 min with a bacterially expressed MDM2 and subsequently immunoprecipitated with an anti-p53 antibody. Immunoprecipitated p53 was then incubated within vitro translated wild-type (wt) or C120A Flag-tagged USP42, followed by western blotting with anti-p53 and anti-USP42. (B) U2OS cells were co-transfected with p53, MDM2, wild-type or C120A GFP–Flag–USP42 and HIS–ubiquitin, as indicated. Twenty-four hours after transfection, lysates were precipitated with HIS-tag isolation Dynabeads and western blotted for p53. The input level of each protein was assessed by western blotting (below). (C) U2OS cells were infected with wild-type GFP–Flag-tagged USP42 or control vector. Seventy-two hours after transfection, cells were selected with puromycin for 2 weeks to establish USP42 overexpressing or control cell pools. These cells were treated for the indicated time with 100 μg/ml of cyclohexamide to inhibit the translation. Cell lysates were prepared at these times and probed for USP42, p53 and cMyc protein level by western blot. Cdk2 was used as loading control. (D) Quantification of (C). Figure source data can be found with the Supplementary data.
USP42 regulates the stabilization and activation of the p53 response in response to several stress signals. (A) CHX assay U2OS cell extracts with antibodies against USP42, MDM2 p53, p21 and actin as a loading control. Cells were treated with non-targeting siRNA or a pool of siRNA against USP42 for 48 h before being treated for the indicated time with 100 μg/ml of cyclohexamide to inhibit the translation. Cell lysates were prepared at these times and analysed for the indicated proteins level by western blot. Actin was used as loading control. (B) Quantification of (A). (C) Western blot of U2OS cell extracts with antibodies against USP42, p53 and actin as a loading control. Cells were treated with non-targeting siRNA, a pool of siRNAs against exon 13 of USP42 or an siRNA against p53 and then incubated for 24 h with the indicated drugs to induce p53 expression. (D) Western blot of U2OS cell extracts with antibodies against USP42, MDM2 p53, p21 and actin as a loading control. U2OS were treated as described in (C) for the indicated time. (E) Ubiquitination assay of p53 in U2OS cells after knockdown of USP42. U2OS cells were treated with non-targeting siRNA or a pool of siRNA against USP42 and transfected with HIS–ubiquitin 12 h later. After 36 h, cells were incubated for the indicated times with actinomycin D and MG132 then harvested for HIS pulldown as described in Figure 3A. (F) Western blot of U2OS cell extracts with antibodies against USP42, MDM2 p53, p21, p27, β-catenin and actin as a loading control. U2OS were treated with the indicated siRNAs and then incubated for 10 h with vehicle or actinomycin D. Figure source data can be found with the Supplementary data.
USP42 depletion delays activation of several p53 target genes in response to stress. (A) U2OS cells were transfected with siRNAs against USP42 or non-targeting siRNA. Cells were treated for the indicated time with 5 nM actinomycin D to activate p53. mRNA expression of the indicated genes was then determined by qRT–PCR with specific primers; mRNA variation rate indicates the percentage activation or repression of expression of each gene compared with control. The results were normalized against two different standard genes and the graphs represent the mean of three independent experiments. (B) U2OS cells treated as in (A) were fixed and chromatin was immunoprecipitated with an antibody against p53. p53 bound DNA was then analysed by qPCR with specific primer amplifying the indicated region of thep21 gene. The results are expressed as a percentage of input and the mean of three experiments.
USP42 is required for efficient p53-mediated cell-cycle arrest. (A) Cell-cycle profile of U2OS cells treated with a non-targeting siRNA or a pool of siRNAs against USP42, followed by vehicle or actinomycin D treatment for 16 h. (B) U2OS cells overexpressing a control vector or a vector coding for an shRNA against p53 treated with a non-targeting siRNA or a pool of siRNAs against USP42 were treated 16 h with 5 nM of actinomycin D. During the last 90 min, cells were pulsed with BrdU, then analysed by flow cytometry for BrdU incorporation and DNA content. (B) A graph representative of three independent experiments performed as in (A) showing the percentage of cell in S-phase. (C) Cells depleted for USP42 as in (A) were incubated with Nutlin and analysed for BrdU incorporation by flow cytometry. The graph is representative of three independent experiments and shows the percentage of cells in S-phase. The western blot shows the extent of p53 depletion in the U2OS cells overexpressing the shRNA against p53. (D) HCT116 and HCT116 p53−/− cells were treated as in (B) and analysed for BrdU incorporation by flow cytometry. The graph is representative of three independent experiments and shows the percentage of cells in the subG1 fraction. (E) RPE cells were treated as in (B) and analysed for overall DNA content by flow cytometry. The graph is representative of three independent experiments and shows the percentage of cells in S-phase. (F) Growth curve of same cells as in (B) treated with vehicle or 5 nM of actinomycin D. Cells were counted at the indicated time with an automatic cell counter.
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