Suppression of the Deubiquitinating Enzyme USP5 Causes the Accumulation of Unanchored Polyubiquitin and the Activation of p53
2008; Elsevier BV; Volume: 284; Issue: 8 Linguagem: Inglês
10.1074/jbc.m805871200
ISSN1083-351X
AutoresSaurabh Dayal, Alison Sparks, Jimmy Jacob, Nerea Allende-Vega, David P. Lane, Mark K. Saville,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoBoth p53 and its repressor Mdm2 are subject to ubiquitination and proteasomal degradation. We show that knockdown of the deubiquitinating enzyme USP5 (isopeptidase T) results in an increase in the level and transcriptional activity of p53. Suppression of USP5 stabilizes p53, whereas it has little or no effect on the stability of Mdm2. This provides a mechanism for transcriptional activation of p53. USP5 knockdown interferes with the degradation of ubiquitinated p53 rather than attenuating p53 ubiquitination. In vitro studies have shown that a preferred substrate for USP5 is unanchored polyubiquitin. Consistent with this, we observed for the first time in a mammalian system that USP5 makes a major contribution to Lys-48-linked polyubiquitin disassembly and that suppression of USP5 results in the accumulation of unanchored polyubiquitin chains. Ectopic expression of a C-terminal mutant of ubiquitin (G75A/G76A), which also causes the accumulation of free polyubiquitin, recapitulates the effects of USP5 knockdown on the p53 pathway. We propose a model in which p53 is selectively stabilized because the unanchored polyubiquitin that accumulates after USP5 knockdown is able to compete with ubiquitinated p53 but not with Mdm2 for proteasomal recognition. This raises the possibility that there are significant differences in proteasomal recognition of p53 and Mdm2. These differences could be exploited therapeutically. Our study reveals a novel mechanism for regulation of p53 and identifies USP5 as a potential target for p53 activating therapeutic agents for the treatment of cancer. Both p53 and its repressor Mdm2 are subject to ubiquitination and proteasomal degradation. We show that knockdown of the deubiquitinating enzyme USP5 (isopeptidase T) results in an increase in the level and transcriptional activity of p53. Suppression of USP5 stabilizes p53, whereas it has little or no effect on the stability of Mdm2. This provides a mechanism for transcriptional activation of p53. USP5 knockdown interferes with the degradation of ubiquitinated p53 rather than attenuating p53 ubiquitination. In vitro studies have shown that a preferred substrate for USP5 is unanchored polyubiquitin. Consistent with this, we observed for the first time in a mammalian system that USP5 makes a major contribution to Lys-48-linked polyubiquitin disassembly and that suppression of USP5 results in the accumulation of unanchored polyubiquitin chains. Ectopic expression of a C-terminal mutant of ubiquitin (G75A/G76A), which also causes the accumulation of free polyubiquitin, recapitulates the effects of USP5 knockdown on the p53 pathway. We propose a model in which p53 is selectively stabilized because the unanchored polyubiquitin that accumulates after USP5 knockdown is able to compete with ubiquitinated p53 but not with Mdm2 for proteasomal recognition. This raises the possibility that there are significant differences in proteasomal recognition of p53 and Mdm2. These differences could be exploited therapeutically. Our study reveals a novel mechanism for regulation of p53 and identifies USP5 as a potential target for p53 activating therapeutic agents for the treatment of cancer. Ubiquitination of proteins plays a key role in the regulation of many important pathways in the cell (1Ciechanover A. Exp. Biol. Med. (Maywood). 2006; 231: 1197-1211Crossref PubMed Scopus (60) Google Scholar). It can act as a signal which targets proteins for degradation by the 26 S proteasome and can also control protein activity and localization (2Wilkinson K.D. Ventii K.H. Friedrich K.L. Mullally J.E. EMBO Rep. 2005; 6: 815-820Crossref PubMed Scopus (27) Google Scholar). Alterations in the ubiquitin-proteasome system have been implicated in a range of diseases including cancer, and there is considerable interest in components of this pathway as targets for therapeutic intervention. Bortezomib, a direct inhibitor of the protease activity of the proteasome, is used in cancer therapy. It is a standard treatment for multiple myeloma. However, it is not effective as a single agent for the treatment of a number of other types of cancers, and trials are under way to test its efficacy in combination with other therapeutic agents (3Dicato M. Boccadoro M. Cavenagh J. Harousseau J.L. Ludwig H. San Miguel J. Sonneveld P. Oncology. 2006; 70: 474-482Crossref PubMed Scopus (20) Google Scholar, 4Orlowski R.Z. Kuhn D.J. Clin. Cancer Res. 2008; 14: 1649-1657Crossref PubMed Scopus (483) Google Scholar).The 26 S proteasome is a large protein complex composed of one or two 19 S regulatory cap complexes and a 20 S core. The 19 S cap participates in ubiquitin recognition and mediates the unfolding of proteins targeted for degradation. The 20 S core carries out protein degradation (5Wolf D.H. Hilt W. Biochim. Biophys. Acta. 2004; 1695: 19-31Crossref PubMed Scopus (216) Google Scholar, 6Nandi D. Tahiliani P. Kumar A. Chandu D. J. Biosci. 2006; 31: 137-155Crossref PubMed Scopus (390) Google Scholar). The sequential action of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3) mediates the conjugation of ubiquitin to target proteins (7Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2887) Google Scholar). Proteins can be conjugated to one molecule of ubiquitin (monoubiquitinated) to multiple single ubiquitin molecules at different sites (multiply monoubiquitinated) and to chains of ubiquitin (polyubiquitinated). It is generally thought that a chain of at least four ubiquitin molecules is required for efficient recognition by the proteasome (8Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. EMBO J. 2000; 19: 94-102Crossref PubMed Scopus (1298) Google Scholar). However, there are examples where monoubiquitination is sufficient to target proteins for degradation by the proteasome (9Lam Y.A. Xu W. DeMartino G.N. Cohen R.E. Nature. 1997; 385: 737-740Crossref PubMed Scopus (369) Google Scholar, 10Guterman A. Glickman M.H. J. Biol. Chem. 2004; 279: 1729-1738Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 11Boutet S.C. Disatnik M.H. Chan L.S. Iori K. Rando T.A. Cell. 2007; 130: 349-362Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Ubiquitin is conjugated to proteins through the formation of an isopeptide bond between its C terminus (Gly-76) and most frequently the ∊-side chain amino group of a lysine residue in the target protein. Polyubiquitin chains are similarly generated by isopeptide bond formation between the C terminus of one ubiquitin and the ∊-side chain amino group of a lysine residue in the next ubiquitin in the chain. In free/unanchored polyubiquitin (polyubiquitin not conjugated to a target protein), one end of the chain, which is referred to as the proximal end, has a ubiquitin with a free C terminus. Any of seven lysines in ubiquitin can be used to form an isopeptide bond with another ubiquitin (12Pickart C.M. Fushman D. Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (821) Google Scholar, 13Ikeda F. Dikic I. EMBO Rep. 2008; 9: 536-542Crossref PubMed Scopus (648) Google Scholar). Lys-48-linked chains are predominantly involved in targeting proteins for proteasomal degradation.Ubiquitin-ubiquitin and ubiquitin-protein bonds can be cleaved by the action of deubiquitinating enzymes (DUBs). 4The abbreviations used are: DUB, deubiquitinating enzymes; USP, ubiquitin-specific protease; shRNA, short hairpin RNA; siRNA, small interfering RNA; DTT, dithiothreitol.4The abbreviations used are: DUB, deubiquitinating enzymes; USP, ubiquitin-specific protease; shRNA, short hairpin RNA; siRNA, small interfering RNA; DTT, dithiothreitol. There are five subclasses of DUBs, the largest of which is the ubiquitin-specific protease (USP) family (14Nijman S.M. Luna-Vargas M.P. Velds A. Brummelkamp T.R. Dirac A.M. Sixma T.K. Bernards R. Cell. 2005; 123: 773-786Abstract Full Text Full Text PDF PubMed Scopus (1385) Google Scholar). Some DUBs remove ubiquitin from substrates before proteasomal recognition, resulting in inhibition of substrate degradation. Another role of DUBs is to regulate the pools of unanchored ubiquitin and polyubiquitin. Unanchored isopeptide bond-linked polyubiquitin is generated as a result of deubiquitination of proteins and by de novo synthesis (15Hadari T. Warms J.V. Rose I.A. Hershko A. J. Biol. Chem. 1992; 267: 719-727Abstract Full Text PDF PubMed Google Scholar, 16Shabek N. Iwai K. Ciechanover A. Biochem. Biophys. Res. Commun. 2007; 363: 425-431Crossref PubMed Scopus (19) Google Scholar). One source of free polyubiquitin is the deubiquitination of proteins at the proteasome. After recognition of the ubiquitinated protein by the proteasome, the ubiquitin is released. This is necessary for entry of proteins into the proteasome (17Verma R. Aravind L. Oania R. McDonald W.H. Yates III, J.R. Koonin E.V. Deshaies R.J. Science. 2002; 298: 611-615Crossref PubMed Scopus (827) Google Scholar). Unanchored polyubiquitin is disassembled to regenerate free ubiquitin. The in vitro substrate specificity of USP5 (isopeptidase T) is consistent with an involvement of this enzyme in disassembly of free polyubiquitin. USP5 sequentially removes ubiquitin from the proximal end of unanchored polyubiquitin chains (15Hadari T. Warms J.V. Rose I.A. Hershko A. J. Biol. Chem. 1992; 267: 719-727Abstract Full Text PDF PubMed Google Scholar, 18Wilkinson K.D. Tashayev V.L. O'Connor L.B. Larsen C.N. Kasperek E. Pickart C.M. Biochemistry. 1995; 34: 14535-14546Crossref PubMed Scopus (260) Google Scholar, 19Stein R.L. Chen Z. Melandri F. Biochemistry. 1995; 34: 12616-12623Crossref PubMed Scopus (60) Google Scholar, 20Falquet L. Paquet N. Frutiger S. Hughes G.J. Hoang-Van K. Jaton J.C. FEBS Lett. 1995; 359: 73-77Crossref PubMed Scopus (43) Google Scholar). Homologues of USP5 are required for the dissociation of free polyubiquitin in Saccharomyces cerevisiae and Arabidopsis thaliana (Ubp14) (21Amerik A. Swaminathan S. Krantz B.A. Wilkinson K.D. Hochstrasser M. EMBO J. 1997; 16: 4826-4838Crossref PubMed Scopus (196) Google Scholar, 22Doelling J.H. Yan N. Kurepa J. Walker J. Vierstra R.D. Plant J. 2001; 27: 393-405Crossref PubMed Scopus (105) Google Scholar) and Dictyostelium discoideum (UbpA) (23Lindsey D.F. Amerik A. Deery W.J. Bishop J.D. Hochstrasser M. Gomer R.H. J. Biol. Chem. 1998; 273: 29178-29187Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The consequences of USP5 suppression have not been previously investigated in a mammalian system.The ubiquitin-proteasome system plays a major role in regulation of the p53 pathway. In most cases tumor progression requires loss of p53 function because of the protective role of p53 against tumor development. This can occur through inactivating mutations in p53. However, 50% of tumors retain wild-type p53, whose function is at least partially attenuated by other mechanisms. The activation of p53 by non-genotoxic agents is a therapeutic approach for the treatment of those cancers which express wild-type p53 (24Woods Y.L. Lane D.P. Hematol. J. 2003; 4: 233-247Crossref PubMed Scopus (24) Google Scholar, 25Dey A. Verma C.S. Lane D.P. Br. J. Cancer. 2008; 98: 4-8Crossref PubMed Scopus (61) Google Scholar). Recent studies with animal models of cancer have clearly highlighted the therapeutic potential of restoring p53 function (26Ventura A. Kirsch D.G. McLaughlin M.E. Tuveson D.A. Grimm J. Lintault L. Newman J. Reczek E.E. Weissleder R. Jacks T. Nature. 2007; 445: 661-665Crossref PubMed Scopus (1359) Google Scholar, 27Beraza N. Trautwein C. Hepatology. 2007; 45: 1578-1579Crossref PubMed Scopus (11) Google Scholar). p53 is regulated by ubiquitination at several levels. p53 is ubiquitinated and undergoes proteasomal degradation. In addition, repressors of p53 including Mdm2 are also ubiquitinated and are degraded by the proteasome. Changes in the stability of p53 and Mdm2 participate in p53 activation after cellular stress (28Stommel J.M. Wahl G.M. EMBO J. 2004; 23: 1547-1556Crossref PubMed Scopus (312) Google Scholar, 29Meulmeester E. Maurice M.M. Boutell C. Teunisse A.F. Ovaa H. Abraham T.E. Dirks R.W. Jochemsen A.G. Mol. Cell. 2005; 18: 565-576Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Mdm2 can repress the transcriptional activity of p53 by binding to its transactivation domain. Mdm2 is also an E3 ubiquitin ligase for p53. Mdm2 undergoes "autoubiquitination" in vitro or when overexpressed; however, studies using mouse embryo fibroblasts expressing a knockin mutant of Mdm2 without intrinsic E3 ligase activity indicate that another E3 is involved in ubiquitination of Mdm2 in these cells (30Itahana K. Mao H. Jin A. Itahana Y. Clegg H.V. Lindstrom M.S. Bhat K.P. Godfrey V.L. Evan G.I. Zhang Y. Cancer Cell. 2007; 12: 355-366Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The effect of direct inhibitors of the proteolytic activity of the proteasome on the activity of wild-type p53 is complex. In some studies inhibitors have been reported to block p53 degradation and increase p53 transactivation activity (31Williams S.A. McConkey D.J. Cancer Res. 2003; 63: 7338-7344PubMed Google Scholar, 32Concannon C.G. Koehler B.F. Reimertz C. Murphy B.M. Bonner C. Thurow N. Ward M.W. Villunger A. Strasser A. Kogel D. Prehn J.H. Oncogene. 2007; 26: 1681-1692Crossref PubMed Scopus (79) Google Scholar). In other studies they have been shown to stabilize wild-type p53 without increasing its transcriptional activity (28Stommel J.M. Wahl G.M. EMBO J. 2004; 23: 1547-1556Crossref PubMed Scopus (312) Google Scholar, 33Zhu Q. Wani G. Yao J. Patnaik S. Wang Q.E. El-Mahdy M.A. Praetorius-Ibba M. Wani A.A. Oncogene. 2007; 26: 4199-4208Crossref PubMed Scopus (56) Google Scholar, 34Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (709) Google Scholar). A likely reason for this is that by stabilizing Mdm2, direct inhibitors of the protease activity of the proteasome cause accumulation of sufficient Mdm2 to repress p53 by direct binding (28Stommel J.M. Wahl G.M. EMBO J. 2004; 23: 1547-1556Crossref PubMed Scopus (312) Google Scholar). In addition, a transcription-independent direct effect of p53 at the mitochondria has been implicated in cell death resulting from proteasome inhibition (35Nair V.D. McNaught K.S. Gonzalez-Maeso J. Sealfon S.C. Olanow C.W. J. Biol. Chem. 2006; 281: 39550-39560Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Sensitivity to proteasome inhibition has been reported to be partially dependent on the p53 status in some cancer cells and to be independent of p53 in others (36Boccadoro M. Morgan G. Cavenagh J. Cancer Cell Int. 2005; 5: 18Crossref PubMed Scopus (171) Google Scholar).p53 has been shown to be regulated by the DUBs herpesvirus associated ubiquitin specific protease (HAUSP) /USP7 (29Meulmeester E. Maurice M.M. Boutell C. Teunisse A.F. Ovaa H. Abraham T.E. Dirks R.W. Jochemsen A.G. Mol. Cell. 2005; 18: 565-576Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 37Brooks C.L. Gu W. Cell Cycle. 2004; 3: 895-899PubMed Google Scholar) and USP2a (38Stevenson L.F. Sparks A. Allende-Vega N. Xirodimas D.P. Lane D.P. Saville M.K. EMBO J. 2007; 26: 976-986Crossref PubMed Scopus (221) Google Scholar), which selectively deubiquitinate components of the p53 pathway. In this study we carried out a screen using an shRNA library targeting members of the DUB family (39Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (826) Google Scholar) with the aim of identifying additional DUBs whose suppression results in p53 activation. USP5 was identified as a regulator of p53 in this screen. Knockdown of USP5 stabilizes p53 while having little or no effect on the stability of Mdm2. This results in an increase in the levels and transcriptional activity of p53. USP5 can make a major contribution to the disassembly of free polyubiquitin in mammalian cells. Our data indicate that the effects of USP5 knockdown on the p53 pathway are mediated by the accumulation of unanchored polyubiquitin. The differential effect of USP5 knockdown on the stability of Mdm2 and p53 can be explained by differences in sensitivity to competition with free polyubiquitin for recognition by the proteasome.EXPERIMENTAL PROCEDURESCell Culture—ARN8 cells were derived from wild-type p53-expressing A375 human melanoma cells by stable transfection with a p53-responsive reporter construct (RGCΔFos-LacZ) (40Blaydes J.P. Hupp T.R. Oncogene. 1998; 17: 1045-1052Crossref PubMed Scopus (94) Google Scholar). ARN8 cells were cultured in Dulbecco's modified Eagle's medium, HCT116 p53+/+ and p53-/- cells were cultured in McCoy's 5A medium, and H1299 cells were cultured in RPMI, each supplemented with 10% fetal calf serum and 50 μg/ml gentamycin. Cells were grown at 37 °C, 5% CO2 in a humidified atmosphere. HCT116 p53-/- cells were generated from the parental cell line by homologous recombination (41Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2517) Google Scholar).Plasmids and Synthetic siRNA Duplexes—For the His6-ubiquitin construct, a single copy of human ubiquitin was cloned into the HindIII and XhoI sites of pcDNA3. The G75A/G76A ubiquitin mutant was generated from wild-type pcDNA3 His6-ubiquitin using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The shRNA library targeting 50 deubiquitinating enzymes was constructed as described previously using the pSUPER vector (39Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (826) Google Scholar) and was generously provided by Professor René Bernards (The Netherlands Cancer Institute). Non-targeting control duplex #2 and synthetic siRNA duplexes A (D-006095-02) and B (D-006095-03) complementary to USP5 were purchased from Dharmacon. There are two known alternatively spliced forms of USP5 which differ by an insertion of 23 amino acids. The substrate specificity of the isoforms appears to be identical in vitro (42Gabriel J.M. Lacombe T. Carobbio S. Paquet N. Bisig R. Cox J.A. Jaton J.C. Biochemistry. 2002; 41: 13755-13766Crossref PubMed Scopus (13) Google Scholar). The siRNA USP5 (A) and (B) used in this study target both splice forms. The sequences of p53 siRNA (A) and (B) were GACTCCAGTGGTAATCTAC (43Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3942) Google Scholar) and GCATGAACCGGAGGCCCAT (44Martinez L.A. Naguibneva I. Lehrmann H. Vervisch A. Tchenio T. Lozano G. Harel-Bellan A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14849-14854Crossref PubMed Scopus (171) Google Scholar), respectively.Transfections—For the screen with the shRNA library, ARN8 cells were seeded onto 24-well tissue culture plates (1.5 × 104 cells/well) and transfected in duplicate with 0.8 μg of empty pSUPER vector or each pool of DUB shRNA using FuGENE 6 transfection reagent (Roche Applied Science) following the manufacturer's instructions. β-Galactosidase activity was assayed 72 h after transfection. For transfection with synthetic siRNA ARN8 or H1299 cells, well were seeded onto 6-well plates (6 × 104 cells/well) or 24-well plates (1.5 × 104 cells/well). HCT116 cells were seeded onto 6-well plates (0.5–1 × 105 cells/well). Transfection was carried out using Oligofectamine (Invitrogen) according to the manufacturer's instructions. The final concentration of each siRNA was 30 nm. Cells were harvested 72 h post-transfection. For transfection with wild-type or mutant ubiquitin, cells were trypsinized, and 1 × 106 ARN8 cells in suspension were nucleofected with 10 μg of plasmid using nucleofection kit V (Amaxa) and program X-001 according to the manufacturer's instructions. Cells were seeded onto 6-well plates (1.5 to 3 × 105 cells/well) or 24-well plates (3 × 104 cells/well) and harvested 24 h after transfection.Quantitative Detection of β-Galactosidase—Cells in 24-well plates were washed twice with phosphate-buffered saline and lysed with 100 μl of passive lysis buffer (Promega). 150 μl of substrate solution containing 80 μg/ml chlorophenol red β-d-galactopyranoside (Roche Applied Science), 0.5 mm MgCl2, 23 mm β-mercaptoethanol in 0.1 m sodium phosphate buffer, pH 7.5, was added to 40 μl of extract in a 96-well plate. The assay was quantified by absorbance measurements at a wavelength of 590 nm in a plate reader.Gel Electrophoresis and Western Blotting—Cells were washed twice with phosphate-buffered saline at 4 °C. Cell extracts were prepared by direct lysis into SDS-urea electrophoresis sample buffer: 100 mm Tris, pH 6.8, 4% SDS, 8 m urea, 20% glycerol, 20 mm EDTA, 0.014% bromphenol blue. DNA was sheared by passage through a 25-gauge needle, and protein concentrations were measured using the BCA protein assay (Pierce). DTT was added to a final concentration of 100 mm, samples were heated for 5 min at 95 °C, and proteins were resolved by SDS-PAGE. Gels were transferred onto nitrocellulose for 16 h at 25 mA, and membranes were processed as described previously (45Saville M.K. Sparks A. Xirodimas D.P. Wardrop J. Stevenson L.F. Bourdon J.C. Woods Y.L. Lane D.P. J. Biol. Chem. 2004; 279: 42169-42181Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). To expose epitopes in ubiquitin, membranes to be probed for ubiquitin were boiled in deionized water for 30 min before blocking. Peroxidase-conjugated secondary anti-mouse antibodies (Jackson ImmunoResearch Laboratories) were used at a dilution of 1/10,000 and anti-rabbit antibodies (Pierce) at a dilution of 1/2,000. Bound antibodies were detected by enhanced chemiluminescence (Amersham Biosciences) or using Supersignal West Dura extended duration substrate (Pierce). The primary antibodies used were 4B2 for Mdm2, DO-1 for p53, Ab-1 for β-actin (Calbiochem), and Ubi-1 (Novus) and P4D1 (Upstate) for ubiquitin. Rabbit anti-USP5 and anti-Ser-15-phosphorylated p53 polyclonals were obtained from Proteintech Group Inc. and Cell Signaling Technology, respectively. A mouse monoclonal anti-His6 antibody was obtained from Clontech.RNA Preparation and Real Time PCR—RNA was extracted using RNeasy columns (Qiagen), and real time PCR was carried out as described previously (45Saville M.K. Sparks A. Xirodimas D.P. Wardrop J. Stevenson L.F. Bourdon J.C. Woods Y.L. Lane D.P. J. Biol. Chem. 2004; 279: 42169-42181Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Probes and primers for p53, Mdm2 P1 and P2, Bax, p21 (Waf1/Cip1), and β-actin were as described previously (38Stevenson L.F. Sparks A. Allende-Vega N. Xirodimas D.P. Lane D.P. Saville M.K. EMBO J. 2007; 26: 976-986Crossref PubMed Scopus (221) Google Scholar, 45Saville M.K. Sparks A. Xirodimas D.P. Wardrop J. Stevenson L.F. Bourdon J.C. Woods Y.L. Lane D.P. J. Biol. Chem. 2004; 279: 42169-42181Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The USP5-specific primers and probes were as follows: 5′-CGGGACCAGGCCTTGAA-3′, 5′-TCGTCAATGTGACTGAAGATCCA-3′; probe, 6-FAM-CGCTGCGGGCCACGAACAATA-TAMRA.Immunofluorescence—Cells were seeded on glass slides. After transfection with synthetic siRNA duplexes, cells were fixed with ice-cold methanol-acetone and incubated with primary antibodies followed by Alexa Fluor 488 dye-conjugated anti-mouse and Alexa Fluor 594 dye-conjugated anti-rabbit secondary antibodies (Invitrogen) as described previously (45Saville M.K. Sparks A. Xirodimas D.P. Wardrop J. Stevenson L.F. Bourdon J.C. Woods Y.L. Lane D.P. J. Biol. Chem. 2004; 279: 42169-42181Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Monoclonal antibody 4B2 was used to detect Mdm2 and rabbit polyclonal CM1 for p53.Immunoprecipitations—To preserve ubiquitination after lysis immunoprecipitation was carried out essentially as described previously (46Xirodimas D.P. Saville M.K. Bourdon J.C. Hay R.T. Lane D.P. Cell. 2004; 118: 83-97Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). After transfection with siRNA duplexes, cells were lysed in SDS immunoprecipitation lysis buffer: phosphate-buffered saline containing 1% Nonidet P-40, 1% SDS, 5 mm EDTA, 10 mm iodoacetamide, 1 mm DTT, and protease inhibitor mixture (Roche Applied Science). The lysates were incubated at 60 °C for 10 min. DNA was sheared by passage through a 25-guage needle, and protein concentrations were measured using the BCA protein assay (Pierce). The pooled lysates from one 6-well plate were used for each immunoprecipitation (total volume 300 μl). The samples normalized for protein were diluted 10-fold with immunoprecipitation wash buffer: phosphate-buffered saline containing 1% Nonidet P-40, 5 mm EDTA, 1 mm DTT, and protease inhibitor mixture (Roche Applied Science). The diluted lysates were pre-cleared with a 200-μl packed volume of Sepharose beads by incubation for 45 min at 4 °C. The lysates were then incubated with 10 μl of CM1 or 10 μl of preimmune rabbit serum for 2 h at 4 °C. 20 μl packed volume of protein G-Sepharose beads were added, and the samples incubated for a further hour. The beads were washed five times with immunoprecipitation wash buffer and eluted in SDS-urea sample buffer. The immunoprecipitates were analyzed by Western blotting for p53 and ubiquitin using antibodies DO-1 and P4D1, respectively.In Vitro Deubiquitination Assay—After transfection with siRNA, cells were lysed in Nonidet P-40 buffer: 0.1% Nonidet P-40, 50 mm Tris, pH 7.5, 150 mm NaCl, 5% glycerol, 1 mm DTT protease inhibitor mixture (Roche Applied Science). The lysates were centrifuged (14,000 × g, 10 min, 4 °C). The indicated amount of the extracts was added to 15 μl of assay buffer: 50 mm Tris, pH 7.5, 150 mm NaCl, 2 mm DTT. Reactions were initiated by the addition of 0.75 μg of recombinant Lys-48-linked polyubiquitin (Biomol). Samples were incubated at 37 °C for the indicated time, and the reactions were terminated by the addition of 15 μl of SDS-urea electrophoresis sample buffer. Polyubiquitin levels were determined by Western blotting for ubiquitin.RESULTSUSP5 Knockdown Activates p53—To identify DUBs whose suppression activates p53, we carried out a screen using a library consisting of pools of 4 shRNA targeting 50 members of the DUB family (39Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (826) Google Scholar). The screen was carried out in a derivative of the wild-type p53 expressing A375 melanoma cell line (ARN8), generated by stable transfection with a p53-responsive reporter construct (RGCΔFos-LacZ) that drives expression of β-galactosidase (40Blaydes J.P. Hupp T.R. Oncogene. 1998; 17: 1045-1052Crossref PubMed Scopus (94) Google Scholar). In this screen transfection with the pool of shRNA complementary to USP5 was observed to result in an increase in β-galactosidase reporter activity (Fig. 1A). To validate USP5 as a target we looked at the effects of two synthetic siRNA complementary to different sequences in USP5. Transfection with either of these siRNA resulted in an increase in β-galactosidase activity (Fig. 1B). This confirms that USP5 knockdown genuinely causes an increase in reporter activity. We also looked at the effect of USP5 knockdown on the level of p53 and Mdm2. Suppression of USP5 resulted in an increase in p53 and Mdm2 protein expression (Fig. 1C).To investigate whether USP5 knockdown regulates the expression of endogenous p53 target genes, ARN8 cells were transfected with siRNA complementary to USP5, and mRNA levels of p53-responsive genes were assayed by real-time PCR. Mdm2 is itself a transcriptional target of p53. The Mdm2 gene has two promoters. One of these is p53-independent (P1), and the other is p53-responsive (P2). These give rise to different mRNA due to variations in the 5′-untranslated region. Consistent with specific transcriptional activation of p53, suppression of USP5 increased the level of Mdm2 P2 mRNA but had no affect on the level of Mdm2 P1 mRNA (Fig. 1D). In addition, USP5 knockdown increased mRNA expression of the p53-responsive genes p21 and Bax. Suppression of USP5 did not affect p53 mRNA levels (Fig. 1E), confirming that USP5 knockdown does not have a general effect on transcription and showing that changes in p53 protein expression are not due to changes in p53 mRNA levels.To determine whether these effects of USP5 suppression are p53-dependent, ARN8 cells were co-transfected with siRNA targeting p53 and USP5. Transfection with either of two siRNA complementary to different sequences in p53 resulted in a reduction in p53 protein expression without affecting USP5 knockdown by co-transfected USP5 siRNA (Fig. 2A). p53 knockdown inhibited the increase in β-galactosidase reporter activity (Fig. 2B) and mRNA levels of the endogenous p53 target genes Mdm2 and p21 resulting from suppression of USP5 (Figs. 2, C and D). The increase in Mdm2 protein expression was also attenuated by suppression of p53 (Fig. 2A). To further investigate the p53 dependence of the affects of USP5 knockdown, HCT116 p53+/+ and p53-/- cells were transfected with USP5 siRNA. In HCT116 p53+/+ cells USP5 knockdown resulted in an increase in p53 and Mdm2 protein levels (Fig. 2E) and an increase in Mdm2 P2 mRNA expression (Fig. 2F). No increase in Mdm2 protein or mRNA levels was observed after USP5 knockdown in HCT116 p53-/- cells. The effects of USP5 knockdown in p53-null H1299 cells were also determined. Again in the absence of p53, siRNA-mediated knockdown of USP5 did not increase Mdm2 protein levels and did not affect the mRNA levels of p53 target genes (supplemental Figs. 1, A and B). These data indicate that siRNA-mediated suppression of USP5 results in an increase in p5
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