doi: 10.1016/j.cell.2009.12.032. Epub 2010 Jan 21.
USP10 regulates p53 localization and stability by deubiquitinating p53
Affiliations
- PMID:20096447
- PMCID: PMC2820153
- DOI: 10.1016/j.cell.2009.12.032
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USP10 regulates p53 localization and stability by deubiquitinating p53
Jian Yuan et al. Cell..
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Abstract
Stability and localization of p53 is essential for its tumor suppressor function. Ubiquitination by the E3 ubiquitin ligase Mdm2 is the major regulatory mechanism of p53, which induces p53 nuclear export and degradation. However, it is unclear whether ubiquitinated cytoplasmic p53 can be recycled. Here, we report that USP10, a cytoplasmic ubiquitin-specific protease, deubiquitinates p53, reversing Mdm2-induced p53 nuclear export and degradation. After DNA damage, USP10 is stabilized, and a fraction of USP10 translocates to the nucleus to activate p53. The translocation and stabilization of USP10 is regulated by ATM -mediated phosphorylation of USP10 at Thr42 and Ser337. Finally, USP10 suppresses tumor cell growth in cells with wild-type p53, with USP10 expression downregulated in a high percentage of clear cell carcinomas, known to have few p53 mutations. These findings reveal USP10 to be a novel regulator of p53, providing an alternative mechanism of p53 inhibition in cancers with wild-type p53.
2010 Elsevier Inc. All rights reserved.
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(A–D) HCT116 p53+/+ and p53−/− cell lysates were subject to immunoprecipitation with control IgG, anti-p53 (A–B), anti-USP10 (C) or anti-Mdm2 (D) antibodies. The immunoprecipitates were then blotted with indicated antibodies. (E) Purified USP10 was incubated with GST or GST-p53 coupled to GSH-Sepharose. Proteins retained on Sepharose were then blotted with indicated antibodies. (F) H1299 cells transfected with indicated constructs were lysed and lysates incubated with GST or GST-p53-GSH-Sepharose. Proteins retained on Sepharose were blotted with the indicated antibody.
(A) HCT116 cells were transfected with indicated constructs. 48hrs later, proteins and mRNA were extracted and subjected to Western Blot or QRT-PCR. (B) HCT116 cells infected with lentivirus encoding indicated shRNAs were left untreated or treated with MG132 for 4hrs, then proteins and mRNA were extracted and subjected to Western Blot or QRT-PCR. (A and B) Lower panel: quantification of p53, p21 or BAX transcript levels. (C) HCT116 cells stably expressing control shRNA, USP10 shRNA, or USP10 shRNA together with shRNA-resistant USP10 were treated with cycloheximide (0.1 mg/ml), and harvested at the indicated times. The left panels show immunoblots of p53 and USP10. (LE: long exposure; SE: short exposure) Right panel: quantification of the p53 protein levels relative to β-actin. (A–C) Error bars represent SEM of triplicate experiments. (D) HCT116 p53+/+ and p53−/− cells infected with lentivirus encoding indicated shRNAs were lysed and lysates blotted with indicated antibody. (E) HCT116 cells transfected with indicated constructs were lysed and lysates blotted with indicated antibody. (F–G) Regulation of p53 ubiquitination levelsin vivo by USP10. HCT116 cells transfected with indicated constructs (F) or stably expressing Ctrl or USP10 shRNA (G) were treated with MG132 for 4hrs before harvest. p53 was immunoprecipitated with anti-p53 polyclonal antibodies and immunoblotted with monoclonal anti-p53(DO-1) antibodies or anti-ub antibodies. (H) Deubiquitination of p53in vitro by USP10. Ubiquitinated p53 was incubated with purified USP10 or USP10CAin vitro, and then blotted with anti-p53 antibodies. “See also Figure S1”
(A) Subcellular localization of USP10 and HAUSP. U2OS cells were fixed and stained as indicated. Bars, 10 μm. (B) HCT116 cells transfected with indicated constructs were treated with MG132 for 4hrs, then fixed and stained as indicated. Bars, 10μm. (C) HCT116 cells transfected with indicated constructs were treated with MG132 and then left untreated or treated with 25ng/ml leptomycin B (LMB), after 4hrs, cells were harvested and fractionated as described in Methods. Cellular fractions were then blotted with indicated antibodies. (C, cytoplasmic; N, nuclear). A cytoplasmic marker protein, Tubulin and a nuclear marker protein, MAX were used as controls to confirm the quality of fractions. LE: long exposure; SE: short exposure. (D) HCT116 cells infected with lentivirus encoding indicated shRNA were treated with MG132 and then left untreated or treated with LMB. Additional 4hrs later, cells were fixed and stained as indicated. Bars, 10μm. (B&D) Lower and Right panels: Quantification of cells with different p53 subcellular localization. Nuc: Nucleus only; Cyto + Nuc: both cytoplasm and nucleus. The data represent the average of three experiments and 150 cells were monitored in each experiment. (E) HCT116 cells stably expressing indicated shRNA were treated with MG132 for 4 hrs. Then cells were harvested and fractionated, cytoplasmic or nuclear p53 was immunoprecipitated with anti-p53 polyclonal antibodies and immunoblotted with monoclonal anti-p53 (DO-1) antibodies. “See also Figure S2”
(A) p53 reporter constructs for the p21 promoter were co-transfected with indicated constructs into HCT116 p53+/+ and HCT116 p53−/− cells. Reporter activity was then determined as described in materials and methods. (B) p53 reporter assay was performed in HCT116 p53+/+ and HCT116 p53−/− cells stably expressing indicated shRNA. (C) H1299 cells were transfected with indicated constructs. 48hrs later, apoptotic cells were determined. (D) HCT116 p53+/+ and HCT116 p53−/− cells stably expressing indicated shRNA were plated and cell proliferation was then quantified at indicated time. (E) Soft agar colony-formation assay was performed using HCT116 p53+/+ and HCT116 p53−/− cells stably expressing control shRNA, USP10shRNA or USP10shRNA together with shRNA-resistant USP10. Right panel: quantification of colonies formed in soft agar. Bars, 400μm. (A–E) Error bars represent SEM of triplicate experiments. **, P < 0.01 two tailed student's t test.
(A) HCT116 cells stably expressing indicated shRNA were irradiated (10Gy) and harvested at indicated time, cell lysates were then blotted with indicated antibodies. (B) HCT116 cells were left untreated, treated with 10Gy radiation, with LMB, with both or treated with Ku55933 (ATM specific inhibitor) for 1hr then irradiated. An additional 1hr later cells were fixed and stained as indicated. Bars, 10μm. (C) HCT116 p53+/+ or HCT116 p53−/− cells were irradiated (10Gy) or left untreated, after 1hr cells were harvested and fractionated. Cellular fractions were then blotted with indicated antibodies. (D) HCT116 p53+/+ or p53−/− cells stably expressing indicated shRNA were left untreated or treated with 10Gy radiation, after 48hrs, apoptotic cells were determined. Error bar represent SEM of triplicate experiments. **, P < 0.01 two tailed student's t test. (E) The same cells in (D) were treated with 10Gy radiation, then harvested at indicated time. Cell cycle profiles were determined by FACS. “See also Figure S3”
(A) HCT116 cells were irradiated (10Gy) and harvested at indicated times. Protein and mRNA were extracted and analyzed by Western blot or QRT-PCR respectively. (B) HCT116 cells left untreated or irradiated were treated with CHX and harvested at indicated time. Cell lysates were then blotted with indicated antibodies. (C) HCT116 cells transfected with FLAG-USP10 were pretreated with DMSO, 25 mM Ku55933 or 3mM caffeine. After 2hr incubation, cells were left untreated or irradiated (10Gy). After additional 1hr, FLAG-USP10 was immunoprecipitated with anti-FLAG antibody and immunoblotted with phospho-SQ/TQ (pSQ/TQ) antibody. (D) ATM+/+ or ATM−/− cells were irradiated (10 Gy) or left untreated. After 1hr, USP10 was immunoprecipitated and immunoblot with indicated antibody. (E) ATM+/+ and ATM−/− cells were irradiated (10Gy) and harvested at indicated time and lysates blotted with indicated antibodies. (F) HCT116 cells stably expressing USP10 shRNA were reconstituted with shRNA resistant FLAG-USP10 WT, T42A, S337A or TS/AA (T42A and S337A double mutant). Cells were irradiated (10Gy) and harvested at indicated time and lysates blotted with indicated antibodies. (G) HCT116 cells stably expressing USP10 shRNA were reconstituted with shRNA resistant FLAG-USP10 WT or TS/AA. Cells were left untreated or irradiated (10Gy), USP10 phosphorylation was examined with pSQ/TQ antibody. (H) HCT116 cells were left untreated or treated with Ku55933 for 2hr then irradiated. The phosphorylation of USP10 was examined by site specific phospho antibodies:p-T42 and p-S337.(I) Cells as in (G) and cells expressing a phospho-mimetic mutant of USP10-T42E/S337D (TS/ED), lower panel, were irradiated (10Gy) or left untreated, and then harvested and fractionated. Fractions were then blotted with indicated antibodies. (J) Cells as in (G) were irradiated (10Gy) and harvested at indicated time, cell lysates were blotted with indicated antibodies. (K) Cells as in (G) were left untreated or irradiated, apoptotic cells were determined 48hrs later. Error bar represent SEM of triplicate experiments. **, P < 0.01 two tailed student's t test. “See also Figure S4”
(A) Expression of USP10 and p53 in Human renal tubular epithelial cell line (HK-2) and renal cell carcinoma (RCC) cell lines. Right panel shows p53 transcript level of HK-2 and RCC cells. Error bar represent SEM of triplicate experiments. (B) 11 pairs of fresh frozen RCC tissues and corresponding normal tissues were lysed, and cell lysates were blotted with indicated antibodies. (N: normal tissue; T: tumor tissue) (C) Immunohistochemical staining of USP10 in normal renal tissues and renal cell carcinoma. Lower table: quantification of USP10-positive or USP10-negative renal cell carcinoma cases. (ccRCC: clear cell Renal Cell Carcinoma). (D–E) Soft agar colony-formation assay was performed using CAKI-1 CAKI-2, or 786-O cells stably expressing indicated constructs. Right panels: quantification of colonies formed in soft agar. Error bar represent SEM of triplicate experiments. **, P< 0.01 two tailed student's t test. (F) The working model of p53 regulation by USP10. “See also Figure S5”
Comment in
- USP10: friend and foe.Jochemsen AG, Shiloh Y.Jochemsen AG, et al.Cell. 2010 Feb 5;140(3):308-10. doi: 10.1016/j.cell.2010.01.034.Cell. 2010.PMID:20144755
- Ubiquitin-specific Peptidase 10 (USP10) Deubiquitinates and Stabilizes MutS Homolog 2 (MSH2) to Regulate Cellular Sensitivity to DNA Damage.Zhang M, Hu C, Tong D, Xiang S, Williams K, Bai W, Li GM, Bepler G, Zhang X.Zhang M, et al.J Biol Chem. 2016 May 13;291(20):10783-91. doi: 10.1074/jbc.M115.700047. Epub 2016 Mar 14.J Biol Chem. 2016.PMID:26975374Free PMC article.
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