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The Chaperone-associated Ubiquitin Ligase CHIP Is Able to Target p53 for Proteasomal Degradation

2005; Elsevier BV; Volume: 280; Issue: 29 Linguagem: Inglês

10.1074/jbc.m501574200

ISSN

1083-351X

Autores

Claudia Esser, Martin Scheffner, Jörg Höhfeld,

Tópico(s)

Cancer Research and Treatments

Resumo

The cellular level of the tumor suppressor p53 is tightly regulated through induced degradation via the ubiquitin/proteasome system. The ubiquitin ligase Mdm2 plays a pivotal role in stimulating p53 turnover. However, recently additional ubiquitin ligases have been identified that participate in the degradation of the tumor suppressor. Apparently, multiple degradation pathways are employed to ensure proper destruction of p53. Here we show that the chaperone-associated ubiquitin ligase CHIP is able to induce the proteasomal degradation of p53. CHIP-induced degradation was observed for mutant p53, which was previously shown to associate with the chaperones Hsc70 and Hsp90, and for the wild-type form of the tumor suppressor. Our data reveal that mutant and wild-type p53 transiently associate with molecular chaperones and can be diverted onto a degradation pathway through this association. The cellular level of the tumor suppressor p53 is tightly regulated through induced degradation via the ubiquitin/proteasome system. The ubiquitin ligase Mdm2 plays a pivotal role in stimulating p53 turnover. However, recently additional ubiquitin ligases have been identified that participate in the degradation of the tumor suppressor. Apparently, multiple degradation pathways are employed to ensure proper destruction of p53. Here we show that the chaperone-associated ubiquitin ligase CHIP is able to induce the proteasomal degradation of p53. CHIP-induced degradation was observed for mutant p53, which was previously shown to associate with the chaperones Hsc70 and Hsp90, and for the wild-type form of the tumor suppressor. Our data reveal that mutant and wild-type p53 transiently associate with molecular chaperones and can be diverted onto a degradation pathway through this association. The p53 tumor suppressor has been termed "the guardian of the genome" (1Lane D.P. Nature. 1992; 358: 15-16Crossref PubMed Scopus (4402) Google Scholar). In normal cells p53 is present at low concentration. DNA damage and other stresses such as hypoxia cause an accumulation of p53, which leads to cell cycle arrest or apoptosis (2Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6673) Google Scholar, 3Vousden K.H. Lu X. Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2673) Google Scholar). p53 acts as a transcription factor to activate target genes that are involved in these responses to prevent damaged cells from proliferating and passing mutations on to the next generation (4Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5707) Google Scholar). Cells that lack functional p53 are unable to respond appropriately to stress and are prone to oncogenic transformation. In fact, missense mutations that inactivate p53 are found in ∼50% of all human tumors making them the most frequent genetic alterations in cancer (5Hainaut P. Hollstein M. Adv. Cancer Res. 2000; 77: 81-137Crossref PubMed Scopus (833) Google Scholar, 6Woods Y.L. Lane D.P. Hematol. J. 2003; 4: 233-247Crossref PubMed Scopus (24) Google Scholar). p53 is regulated through a variety of posttranslational modifications, including phosphorylation, acetylation, and attachment of ubiquitin, the small ubiquitin-like modifier SUMO and the ubiquitin-like protein Nedd8 (4Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5707) Google Scholar, 7Xirodimas 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 (425) Google Scholar, 8Harper J.W. Cell. 2004; 118: 2-4Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Central to the regulation of p53 is the ubiquitin ligase Mdm2 (8Harper J.W. 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Ubiquitin ligases (E3(s)) 1The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; E2, ubiquitin carrier protein; E1, ubiquitin-activating enzyme; MOPS, 3-(N-morpholino)propanesulfonic acid; siRNA, small interfering RNA; GA, geldanamycin. 1The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; E2, ubiquitin carrier protein; E1, ubiquitin-activating enzyme; MOPS, 3-(N-morpholino)propanesulfonic acid; siRNA, small interfering RNA; GA, geldanamycin.provide specificity to ubiquitin conjugation as they mediate the final step in the conjugation process, following the activation of ubiquitin by the E1 enzyme and its transfer onto a ubiquitin-conjugating (E2) enzyme (11Hershko A. Ciechanover A. Varshavsky A. Nat. Med. 2000; 10: 1073-1081Crossref Scopus (556) Google Scholar). Mdm2 belongs to the RING finger E3s, which facilitate ubiquitylation by tethering the E2-ubiquitin complex to the substrate protein (12Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar). Mdm2-mediated ubiquitylation targets p53 for degradation by the 26 S proteasome and is of central importance for establishing low p53 levels in normal cells (13Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2798) Google Scholar, 14Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3629) Google Scholar). The functional interplay between Mdm2 and p53 was elegantly demonstrated in gene knock-out studies, in which the embryonic lethality of mdm2 null mice was rescued by simultaneous deletion of the p53 gene (15Jones S.N. Roe A.E. Donehower L.A. Bradley A. Nature. 1995; 378: 206-208Crossref PubMed Scopus (1051) Google Scholar, 16Montes de Oca Luna R. Wagner D.S. Lozano G. Nature. 1995; 378: 203-206Crossref PubMed Scopus (1190) Google Scholar). Stress-induced phosphorylation of p53 attenuates the interaction with Mdm2, leading to stabilization and activation of the transcription factor (17Prives C. Hall P.A. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1220) Google Scholar). Intriguingly, Mdm2 is itself a transcriptional target of p53, which establishes a negative feedback loop to terminate p53-mediated stress responses (18Barak Y. Juven T. Haffner R. Oren M. EMBO J. 1993; 12: 461-468Crossref PubMed Scopus (1165) Google Scholar). Additional mechanisms that regulate the Mdm2-p53 interplay include autoubiquitylation of Mdm2, the association of Mdm2 with diverse binding partners, and alterations of the intracellular localization of Mdm2 and p53 (8Harper J.W. Cell. 2004; 118: 2-4Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 10Michael D. Oren M. Semin. Cancer Biol. 2003; 13: 49-58Crossref PubMed Scopus (633) Google Scholar, 19Linares L.K. Hengstermann A. Ciechanover A. Müller S. Scheffner M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12009-12014Crossref PubMed Scopus (274) Google Scholar, 20Sui G. Affar el B. Shi Y. Brignone C. Wall N.R. Yin P. Donohoe M. Luke M.P. Calvo D. Grossman S.R. Shi Y. Cell. 2004; 117: 859-872Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Furthermore, Mdm2 not only mediates ubiquitylation of p53 but can also conjugate Nedd8 to the tumor suppressor to inactivate its transcriptional activity (7Xirodimas 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 (425) Google Scholar). Despite the central role of Mdm2 in proteasomal targeting of p53, additional ubiquitin ligases were recently shown to participate in the degradation of the tumor suppressor in normal cells, including p300, Pirh2, and COP1 (21Grossman S.R. Deato M.E. Brignone C. Chan H.M. Kung A.L. Tagami H. Nakatani Y. Livingston D.M. Science. 2003; 300: 342-344Crossref PubMed Scopus (381) Google Scholar, 22Leng R.P. Lin Y. Ma W. Wu H. Lemmers B. Chung S. Parant J.M. Lozano G. Hakern R. Benchimol S. Cell. 2003; 112: 779-791Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar, 23Dornan D. Wertz I. Schimizu H. Arnott D. Frantz G.D. Dowd P. O'Rourke K. Koeppen H. Dixit V.M. Nature. 2004; 429: 86-92Crossref PubMed Scopus (583) Google Scholar). Although p300 seems to cooperate with Mdm2 during ubiquitylation, Pirh2 and COP1 trigger the destruction of p53 independent of Mdm2. Multiple degradation pathways apparently exist to maintain low levels of p53 in normal cells. It is unclear whether these degradation pathways are truly redundant or whether they are selectively engaged in p53 destruction dependent on cell lineage, developmental stage, or physiological situation. In any case, the complexity of p53 degradation may allow to integrate diverse signaling events through which p53 can be regulated. We have recently identified a pathway for protein degradation in the mammalian cytoplasm and nucleus that involves a close cooperation of the molecular chaperones Hsc70 and Hsp90 with the ubiquitin-proteasome system (24Esser C. Alberti S. Höhfeld J. Biochim. Biophys. Acta. 2004; 1695: 171-188Crossref PubMed Scopus (189) Google Scholar). Of central importance on this degradation pathway is the chaperone-associated ubiquitin ligase CHIP (25Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.-Y. Patterson C. Mol. Cell. Biol. 1999; 19: 4535-4545Crossref PubMed Scopus (736) Google Scholar). Through binding to the carboxyl termini of Hsc70 and Hsp90, CHIP mediates the ubiquitylation of chaperone-bound client proteins in conjunction with E2 enzymes of the Ubc4/5 family and induces client degradation by the 26 S proteasome (26Demand J. Alberti S. Patterson C. Höhfeld J. Curr. Biol. 2001; 11: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 27Murata S. Minami Y. Minami M. Chiba T. Tanaka K. EMBO Rep. 2001; 2: 1133-1138Crossref PubMed Scopus (454) Google Scholar, 28Jiang J. Ballinger C.A. Wu Y. Dai Q. Cyr D.M. Höhfeld J. Patterson C. J. Biol. Chem. 2001; 276: 42938-42944Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). Affected chaperone clients can be broadly divided into two subclasses: (i) Hsc70- and Hsp90-associated signaling proteins, for example the glucocorticoid hormone receptor (26Demand J. Alberti S. Patterson C. Höhfeld J. Curr. Biol. 2001; 11: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 29Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Höhfeld J. Patterson C. Nat. Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar) and the oncogenic receptor tyrosine kinase ErbB2 (30Xu W. Marcu M. Yuan X. Mimnaugh E. Patterson C. Neckers L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12847-12852Crossref PubMed Scopus (351) Google Scholar) and (ii) aggregation-prone proteins that are subjected to chaperone-assisted quality control, such as misfolded cystic fibrosis transmembrane conductance regulator (31Meacham G.C. Patterson C. Zhang W. Younger J.M. Cyr D.M. Nat. Cell Biol. 2001; 3: 100-105Crossref PubMed Scopus (695) Google Scholar, 32Alberti S. Böhse K. Arndt V. Schmitz A. Höhfeld J. Mol. Biol. Cell. 2004; 15: 4003-4010Crossref PubMed Scopus (141) Google Scholar) and hyperphosphorylated tau (33Shimura H. Schwartz D. Gygi S.P. Kosik K.S. J. Biol. Chem. 2004; 279: 4869-4876Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 34Petrucelli L. Dickson D. Kehoe K. Taylor J. Snyder H. Grover A. De Lucia M. McGowan E. Lewis J. Prihar G. Kim J. Dillmann W.H. Browne S.E. Hall A. Voellmy R. Tsuboi Y. Dawson T.M. Wolozin B. Hardy J. Hutton M. Hum. Mol. Genet. 2004; 13: 703-714Crossref PubMed Scopus (565) Google Scholar). However, the full range of cellular substrates of CHIP remains to be explored. Remarkably, mice that lack CHIP develop apoptosis in multiple organs after environmental challenge (35Dai Q. Zhang C. Wu Y. McDonough H. Whaley R.A. Godfrey V. Li H.H. Madamanchi N. Xu W. Neckers L. Cyr D. Patterson C. EMBO J. 2003; 22: 5446-5458Crossref PubMed Scopus (247) Google Scholar). This seems to reflect the role of CHIP in the conformational regulation of the heat shock transcription factor but may also mirror altered associations between the chaperone machinery and diverse apoptosis regulators in the absence of CHIP (36Mosser D.D. Morimoto R.I. Oncogene. 2004; 23: 2907-2918Crossref PubMed Scopus (434) Google Scholar). Many transforming mutants of p53 display structural defects and were previously found associated with the molecular chaperones Hsc70 and Hsp90 in tissue culture cells and in human tumor specimens (37Davidoff A.M. Iglehart J.D. Marks J.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3439-3442Crossref PubMed Scopus (312) Google Scholar, 38Blagosklonny M.V. Toretsky J. Neckers L. Oncogene. 1995; 11: 933-939PubMed Google Scholar, 39Whitesell L. Sutphin P.D. Pulcini E.J. Martinez J.D. Cook P.H. Mol. Cell. Biol. 1998; 18: 1517-1524Crossref PubMed Scopus (189) Google Scholar). For example, Arg175 mutations destabilize loop regions within the DNA binding domain, leading to partial unfolding and association with Hsc70 (40Hinds P.W. Finlay C.A. Quartin R.S. Baker S.J. Fearon E.R. Vogelstein B. Levine A.J. Cell Growth & Differ. 1990; 1: 571-580PubMed Google Scholar, 41Gannon J.V. Greaves R. Iggo R. Lane D.P. EMBO J. 1990; 9: 1595-1602Crossref PubMed Scopus (932) Google Scholar, 42Stephen C.W. Lane D.P. J. Mol. Biol. 1992; 225: 577-583Crossref PubMed Scopus (220) Google Scholar, 43Bargonetti J. Manfredi J.J. Chen X. Marshak D.R. Prives C. Genes Dev. 1993; 7: 2565-2574Crossref PubMed Scopus (247) Google Scholar, 44Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2123) Google Scholar). In contrast to the findings for mutant p53, an interaction of wild-type p53 with molecular chaperones could not be detected by coimmunoprecipitation (39Whitesell L. Sutphin P.D. Pulcini E.J. Martinez J.D. Cook P.H. Mol. Cell. Biol. 1998; 18: 1517-1524Crossref PubMed Scopus (189) Google Scholar, 45Finlay C.A. Hinds P.W. Tan T.H. Eliyahu D. Oren M. Levine A.J. Mol. Cell. Biol. 1988; 8: 531-539Crossref PubMed Scopus (1037) Google Scholar). However, this does not exclude that the extended chaperone interactions observed for mutant p53 actually represent a pathological exaggeration of transient interactions between wild-type p53 and the cellular chaperone machinery, similar to observations made for other signaling proteins (46Xu Y. Singer M.A. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 109-114Crossref PubMed Scopus (163) Google Scholar, 47Cheung J. Smith D.F. Mol. Endocrinol. 2000; 14: 939-946Crossref PubMed Scopus (150) Google Scholar). Wild-type p53 possesses an intrinsic conformational lability (48Milner J. Watson J.V. Oncogene. 1990; 5: 1683-1690PubMed Google Scholar, 49Hainaut P. Butcher S. Milner J. Br. J. Cancer. 1995; 71: 227-231Crossref PubMed Scopus (52) Google Scholar), and may therefore undergo dynamic associations with molecular chaperones at a posttranslational stage. Such associations may modulate the presentation of p53 for degradation and could serve as sensors of cell stress. Here we show that wild-type p53 and an Arg175 mutant form of the tumor suppressor (p53R175H) can be targeted for proteasomal degradation by the CHIP ubiquitin ligase. Our data thus reveal a novel degradation pathway for the tumor suppressor that is entered through a transient association of wild-type and mutant p53 with molecular chaperones. Purified Proteins and Antibodies—The following proteins were expressed recombinantly and purified as described previously: rat Hsc70, human Hsp40, human UbcH5b, human CHIP, and wheat E1 (26Demand J. Alberti S. Patterson C. Höhfeld J. Curr. Biol. 2001; 11: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 50Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (335) Google Scholar, 51Alberti S. Demand J. Esser C. Emmerich N. Schild H. Höhfeld J. J. Biol. Chem. 2002; 277: 45920-45927Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Purified bovine ubiquitin was purchased from Sigma. For immunoblotting anti-p53 (DO-1; Oncogene Research Products, San Diego, CA), polyclonal anti-Hsc/Hsp70 (F. U. Hartl, MPI for Biochemistry), monoclonal anti-Hsc/Hsp70 (SPA-820; StressGen Biotechnologies, San Diego, CA), and anti-CHIP antibodies (25Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.-Y. Patterson C. Mol. Cell. Biol. 1999; 19: 4535-4545Crossref PubMed Scopus (736) Google Scholar) were used. Cell Culture and Transfection—H1299 cells were grown in RPMI media (Sigma) supplemented with 10% fetal calf serum, penicillin, and streptomycin. Transfection of H1299 cells was performed using DOTAP liposomal transfection reagent according to the protocol of the manufacturer (Roche Diagnostics). U2OS cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum, penicillin, and streptomycin. For transfection of U2OS cells Effectene transfection reagent (Qiagen, Valencia, CA) was used. Cell extracts were prepared 24 h post-transfection. Degradation Assays—To analyze the degradation kinetics of p53, H1299 cells were seeded in six-well plates and were transfected with 0.1 μg of pRC/CMV-p53wt or 0.1 μg of pcDNA3.1-p53R175H and 1.2 μg of pcDNA3.1-CHIP as indicated. The total amount of added DNA was kept constant at 2.5 μg/well by the addition of pcDNA3.1. Protein lysates were prepared at indicated time points after addition of cycloheximide (60 μg/ml). Cells were washed once with phosphate-buffered saline and lysed in radioimmune precipitation assay buffer (25 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 10% glycerol, 2 mm EDTA) supplemented with Complete protease inhibitor (Roche Diagnostics). The lysate was centrifuged at 20,000 × g for 30 min at 4 °C, and the supernatant was used as a soluble extract. Equal amounts of protein were separated by SDS-PAGE. Levels of p53 and p53R175H were determined by immunoblotting and quantified at indicated time points. To analyze the effect of geldanamycin on the degradation of p53, H1299 cells were treated with geldanamycin (1 μm) and dimethyl sulfoxide, respectively, 12 h after transfection. In Vitro Ubiquitylation Assay—In vitro transcription of p53 and p53R175H was performed with pRC/CMV-p53wt and pcDNA3.1-p53R175H using the T7 RiboMax system according to the manufacturer's instructions (Promega). The obtained RNA was used for in vitro translation of radiolabeled p53 and p53R175H with nuclease-treated rabbit reticulocyte lysate (Promega). For ubiquitylation of p53 and p53R175H, 8 μl of the translation reactions was incubated with 0.1 μm E1, 4 μm UbcH5b, 6 μm CHIP, 6 μm Hsc70, and 0.6 μm Hsp40 as indicated. Each sample received 2.5 μg/μl ubiquitin, 1 μg/μl ubiquitin-aldehyde, 10 mm ATP, 10 mm MgCl2, 10 mm dithiothreitol, 10 mm phosphocreatine, and 10 mm creatine kinase. The total volume of the samples was adjusted to 20 μl with 25 mm MOPS, pH 7.2, 100 mm KCl, and 1 mm phenylmethylsulfonyl fluoride. Samples were incubated for 2 h at 30 °C and then analyzed by SDS-PAGE and phosphorimaging. Reporter Gene Assays—H1299 cells were seeded in six-well plates and were transfected with 0.6 μg of a firefly luciferase reporter gene plasmid (pp53-TA-Luc; BD Biosciences Clontech), 0.2 μg pRC/CMV-p53wt, and 1.2 μg pcDNA3.1-CHIP as indicated. The total amount of added DNA was kept constant at 2.5 μg/well by addition of pcDNA3.1. U2OS cells were seeded in six-well plates and were transfected with 0.2 μg of ppluc-p53 and increasing amounts of pcDNA3.1-CHIP from 0 to 0.5 μg as indicated. The total amount of DNA was kept constant at 0.8 μg/well by the addition of pcDNA3.1. Cells were washed once with phosphate-buffered saline and lysed in 100 μl of lysis buffer (50 mm MOPS, pH 7.2, 100 mm KCl, 0.5% Tween 20) containing Complete protease inhibitor. The lysate was centrifuged at 20,000 × g for 30 min at 4 °C, and the supernatant was used as a soluble extract. 5 μl of cell extract were analyzed with luciferase assay reagent (Promega). Levels of p53 and CHIP were determined after SDS-PAGE and immunoblotting. RNA Interference—Endogenous CHIP was depleted in U2OS cells using siRNA oligonucleotides (Dharmacon, Lafayette, CO). The oligonucleotide CHIP1 is directed against the sequence GAAGAAGCGCTGGAACAGC, and CHIP2 targets the sequence ACCACGAGGGTGATGAGGA of the human CHIP gene. Green fluorescent protein siRNA (GGCTACGTCCAGGAGCGCACC) served as the control. U2OS cells were seeded in six-well plates and were transfected twice at a 72-h interval with siRNA oligonucleotides using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. 72 h after the second transfection cells were washed once with phosphate-buffered saline and lysed in 100 μl of radioimmune precipitation assay buffer supplemented with Complete protease inhibitor. The lysate was centrifuged at 20,000 × g for 30 min at 4 °C, and the supernatant was used as a soluble extract. Equal amounts of protein were separated by SDS-PAGE. Levels of CHIP and p53 were determined by immunoblotting. p53R175H but Not Wild-type p53 Forms Stable Complexes with Hsc70 in H1299 Cells—To investigate a potential influence of the chaperone-associated ubiquitin ligase CHIP on the turnover of p53, the human lung cancer cell line H1299 was used. In an initial experiment we analyzed the association of wild-type p53 and the conformational mutant p53R175H with Hsc70 following transient transfection of the p53-deficient cell line with corresponding expression plasmids. Although wild-type p53 was not found in association with Hsc70, complexes between p53R175H and Hsc70 were readily detectable after immunoprecipitation (Fig. 1). Transient transfection of H1299 cells thus recapitulates the findings obtained in p53-expressing cell lines and tumor specimens that conformational mutants but not wild-type p53 form stable complexes with molecular chaperones (37Davidoff A.M. Iglehart J.D. Marks J.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3439-3442Crossref PubMed Scopus (312) Google Scholar, 38Blagosklonny M.V. Toretsky J. Neckers L. Oncogene. 1995; 11: 933-939PubMed Google Scholar, 39Whitesell L. Sutphin P.D. Pulcini E.J. Martinez J.D. Cook P.H. Mol. Cell. Biol. 1998; 18: 1517-1524Crossref PubMed Scopus (189) Google Scholar). p53R175H and Wild-type p53 Can Be Targeted for Degradation by the CHIP Ubiquitin Ligase—As the p53R175H mutant of p53 was found associated with Hsc70, we investigated whether the turnover of the mutant form is affected by CHIP. Upon elevation of the cellular levels of CHIP in H1299 cells a significant decline in the levels of coexpressed p53R175H was observed (Fig. 2A). This decline could be attributed to an accelerated degradation of p53R175H induced by the CHIP ubiquitin ligase (Fig. 2, B and C). Apparently, the oncogenic mutant protein can be turned over by a chaperone-assisted degradation pathway. Despite the fact that complexes between wild-type p53 and Hsc70 were not detectable by immunoprecipitation, it was previously speculated that wild-type p53 may undergo highly transient interactions with molecular chaperones (39Whitesell L. Sutphin P.D. Pulcini E.J. Martinez J.D. Cook P.H. Mol. Cell. Biol. 1998; 18: 1517-1524Crossref PubMed Scopus (189) Google Scholar). We reasoned that such transient interactions might become detectable upon CHIP overexpression, when even those proteins that associate with Hsc70 in a highly transient manner would be irreversibly diverted onto a degradation pathway (29Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Höhfeld J. Patterson C. Nat. Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar, 31Meacham G.C. Patterson C. Zhang W. Younger J.M. Cyr D.M. Nat. Cell Biol. 2001; 3: 100-105Crossref PubMed Scopus (695) Google Scholar, 32Alberti S. Böhse K. Arndt V. Schmitz A. Höhfeld J. Mol. Biol. Cell. 2004; 15: 4003-4010Crossref PubMed Scopus (141) Google Scholar). In fact, CHIP was able to induce the degradation of wild-type p53, albeit with a slightly reduced efficiency when compared with the findings for p53R175H (Fig. 3). Our data reveal that wild-type p53 associates transiently with molecular chaperones and can be diverted onto a degradation pathway through this association. Geldanamycin Induces the Degradation of Wild-type and Mutant p53 in H1299 Cells—We investigated how the ansamycin antibiotic geldanamycin (GA) affects p53 degradation. GA specifically inhibits Hsp90, which usually results in the proteasomal degradation of client proteins that depend on the activity of the chaperone (52Neckers L. Trends Mol. Med. 2002; 8: S55-S61Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar). The turnover of wild-type and mutant p53 was stimulated upon GA treatment, consistent with an association of both forms with Hsp90 (Fig. 4). However, only in the case of p53R175H was a synergistic effect of GA treatment and CHIP elevation observed. The prolonged and more stable association of mutant p53 with Hsp90 and Hsc70 may provide the molecular basis for this synergism. CHIP Mediates Ubiquitylation of p53 and p53R175H in Cooperation with UbcH5b and Hsc70—To verify that CHIP acts as an E3 ubiquitin ligase during p53 degradation, p53 and p53R175H were in vitro translated in rabbit reticulocyte lysate. Upon addition of purified CHIP to translation reactions, ubiquitylated forms of wild-type and mutant p53 accumulated (Fig. 5). An increase in the amount of ubiquitylated p53 was also observed when UbcH5b and Hsc70 were added, suggesting a close cooperation of CHIP, UbcH5b, and Hsc70 during the ubiquitylation of p53, similar to recent observations for other CHIP substrates (26Demand J. Alberti S. Patterson C. Höhfeld J. Curr. Biol. 2001; 11: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 32Alberti S. Böhse K. Arndt V. Schmitz A. Höhfeld J. Mol. Biol. Cell. 2004; 15: 4003-4010Crossref PubMed Scopus (141) Google Scholar). CHIP Affects p53-mediated Transcription—We analyzed how CHIP influences p53-mediated transcription using a reporter construct that contains firefly luciferase under the control of a p53-response element. Overexpression of CHIP led to a significant reduction of luciferase activity in corresponding cell extracts (Fig. 6A). Conceivably, such a reduction may be caused by a CHIP-induced degradation of luciferase itself. However, it was previously shown that CHIP does not target luciferase for degradation but stimulates the folding of luciferase in tissue culture cells (53Kampinga H.H. Kanon B. Salomons F.A. Kabakov A.E. Patterson C. Mol. Cell. Biol. 2003; 23: 4948-4958Crossref PubMed Scopus (72) Google Scholar). In this regard the observed reduction of luciferase activity rather appears to be an under-estimate of the effect of CHIP on p53-mediated transcription. Notably, the observed reduction in luciferase activity was comparable with the reduction of p53 levels (Fig. 6B). CHIP-induced attenuation of p53-mediated transcription was also observed in U2OS cells that endogenously express p53 (Fig. 6C). It seems that CHIP alters p53-dependent transcriptional responses through an induced degradation of p53. Depletion of Endogenous CHIP Stabilizes p53—Endogenous levels of CHIP were depleted in U2OS cells following transfection with siRNAs (Fig. 7). Intriguingly, depletion of the chaperone-associated ubiquitin ligase caused a significant increase of p53 levels. The findings emphasize the role of chaperone-assisted degradation in maintaining low concentrations of p53 under physiological conditions. Here we identify a novel degradation pathway for the tumor suppressor p53. This pathway is entered through a transient association of wild-type and mutant p53 with molecular chaperones and involves the chaperone-associated ubiquitin ligase CHIP. CHIP cooperates with E2 enzymes of the Ubc4/5 family to mediate the attachment of an ubiquitin-derived degradation signal to chaperone-bound p53, which leads to the proteasomal destruction of the tumor suppressor. CHIP-mediated degradation critically depends on the interaction of the ubiquitin ligase with the chaperones Hsc70 and Hsp90, which were shown to present chaperone clients to the ubiquitin ligase (26Demand J. Alberti S. Patterson C. Höhfeld J. Curr. Biol. 2001; 11: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 29Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Höhfeld J. Patterson C. Nat. Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar, 31Meacham G.C. Patterson C. Zhang W. Younger J.M. Cyr D.M. Nat. Cell Biol. 2001; 3: 100-105Crossref PubMed Scopus (695) Google Scholar, 32Alberti S. Böhse K. Arndt V. Schmitz A. Höhfeld J. Mol. Biol. Cell. 2004; 15: 4003-4010Crossref PubMed Scopus (141) Google Scholar). It has long been appreciated that oncogenic mutant forms of p53 associate with Hsc70 and Hsp90 in tissue culture cells and in tumor specimens (37Davidoff A.M. Iglehart J.D. Marks J.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3439-3442Crossref PubMed Scopus (312) Google Scholar, 39Whitesell L. Sutphin P.D. Pulcini E.J. Martinez J.D. Cook P.H. Mol. Cell. Biol. 1998; 18: 1517-1524Crossref PubMed Scopus (189) Google Scholar). Because of conformational alterations mutant p53s are retained in complexes with the two chaperones and with several of their regulatory cochaperones (39Whitesell L. Sutphin P.D. Pulcini E.J. Martinez J.D. Cook P.H. Mol. Cell. Biol. 1998; 18: 1517-1524Crossref PubMed Scopus (189) Google Scholar, 54King F.W. Wawrzynow A. Höhfeld J. Zylicz M. EMBO J. 2001; 20: 6297-6305Crossref PubMed Scopus (139) Google Scholar). Therefore, oncogenic mutants are amenable to a regulation through chaperone-assisted degradation. A similar association with molecular chaperones was not yet unequivocally demonstrated for wild-type p53, as complexes between the tumor suppressor and Hsc70 or Hsp90 could not be isolated (39Whitesell L. Sutphin P.D. Pulcini E.J. Martinez J.D. Cook P.H. Mol. Cell. Biol. 1998; 18: 1517-1524Crossref PubMed Scopus (189) Google Scholar). It was speculated, however, that such complexes may escape detection because of their highly transient nature (39Whitesell L. Sutphin P.D. Pulcini E.J. Martinez J.D. Cook P.H. Mol. Cell. Biol. 1998; 18: 1517-1524Crossref PubMed Scopus (189) Google Scholar). Such transient associations with Hsc70 or Hsp90 may become detectable when the chaperone client is irreversibly diverted onto a degradation pathway through the action of the CHIP ubiquitin ligase. In fact, we observed that wild-type p53 is sensitive to an elevation of the cellular levels of the chaperone-associated ubiquitin ligase. Furthermore, depletion of endogenous CHIP stabilized wild-type p53 in U2OS cells. Taken together, our data establish a role of molecular chaperones in the regulation of wild-type p53. Evidence suggests that chaperones associate with p53 at multiple stages and influence the oligomeric state, the nucleocytoplasmic transport and the transcriptional activity of the tumor suppressor (reviewed in Refs. 55Zylicz M. King F.W. Wawrzynow A. EMBO J. 2001; 20: 4634-4638Crossref PubMed Scopus (183) Google Scholar, 56Akakura S. Yoshida M. Yoneda Y. Horinouchi S. J. Biol. Chem. 2001; 276: 14649-14657Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 57Müller L. Schaupp A. Walerych D. Wegele H. Buchner J. J. Biol. Chem. 2004; 279: 48846-48854Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 58Walerych D. Kudla G. Gutkowska M. Wawrzynow B. Müller L. King F.W. Helwak A. Boros J. Zylicz A. Zylicz M. J. Biol. Chem. 2004; 279: 48836-48845Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 59Burch L. Shimizu H. Smith A. Patterson C. Hupp T.R. J. Mol. Biol. 2004; 337: 129-145Crossref PubMed Scopus (27) Google Scholar). For example, Hsc70 was shown to sequester a mutant form of p53 in the cytoplasm by masking a nuclear localization signal present at the carboxyl terminus (56Akakura S. Yoshida M. Yoneda Y. Horinouchi S. J. Biol. Chem. 2001; 276: 14649-14657Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). p53 displays multiple binding sites for Hsc70, located in the amino-terminal transactivation domain, the DNA-binding core domain, and the carboxyl terminus (60Stürzbecher H.W. Addison C. Jenkins J.R. Mol. Cell. Biol. 1988; 8: 3740-3747Crossref PubMed Scopus (51) Google Scholar, 61Lam K.T. Calderwood S.K. Biochem. Biophys. Res. 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Biol. 2003; 332: 1131-1141Crossref PubMed Scopus (195) Google Scholar). Binding sites within these regions may remain accessible for Hsc70 when the protein is largely in a native conformation. Notably, studies using conformational antibodies indicate that even the core domain is a metastable structure that is easily perturbed upon treatment with chelating or oxidizing agents or by raising the temperature (49Hainaut P. Butcher S. Milner J. Br. J. Cancer. 1995; 71: 227-231Crossref PubMed Scopus (52) Google Scholar, 67Hainaut P. Milner J. Cancer Res. 1993; 53: 4469-4473PubMed Google Scholar). The core domain is also recognized by Hsp90 (57Müller L. Schaupp A. Walerych D. Wegele H. Buchner J. J. Biol. Chem. 2004; 279: 48846-48854Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 68Rüdiger S. Freund S.M. Veprintsev D.B. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11085-11090Crossref PubMed Scopus (87) Google Scholar), and Walerych et al. (58Walerych D. Kudla G. Gutkowska M. Wawrzynow B. Müller L. King F.W. Helwak A. Boros J. Zylicz A. Zylicz M. J. Biol. Chem. 2004; 279: 48836-48845Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) recently demonstrated that Hsp90 is required to maintain the DNA binding activity of the core domain under physiological conditions. Apparently, p53 is a protein of large conformational flexibility and seems to be in a conformational equilibrium between native and less structured states. This flexibility may provide the means for complex intra- and intermolecular interactions and for the association of p53 with a multitude of regulatory proteins. The accompanying conformational changes are apparently assisted by Hsc70 and Hsp90. In this regard the extended chaperone interactions observed for mutant p53 seem to represent a pathologic exaggeration of physiologic interactions of wild-type p53 with the chaperone machinery. Conceivably, the CHIP-mediated degradation pathway might be entered through an association of a chaperone client with either Hsc70 or Hsp90, as CHIP is able to bind both chaperones. Treatment of tissue culture cells with small molecular inhibitors of Hsp90, such as GA, has helped to verify this hypothesis. Geldanamycin blocks the interaction of Hsp90 with chaperone clients. Remarkably, the Hsp90 inhibitor stimulates the CHIP-mediated degradation of signaling proteins that rely on an association with Hsc70 and Hsp90, i.e. the oncogenic receptor tyrosine kinase ErbB2 (30Xu W. Marcu M. Yuan X. Mimnaugh E. Patterson C. Neckers L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12847-12852Crossref PubMed Scopus (351) Google Scholar). GA treatment dissociates Hsp90 while increasing the association of Hsc70 with ErbB2. Redistribution into Hsc70 complexes followed by proteasomal degradation was also observed for oncogenic mutants of p53 upon treatment of tumor cell lines with GA (39Whitesell L. Sutphin P.D. Pulcini E.J. Martinez J.D. Cook P.H. Mol. Cell. Biol. 1998; 18: 1517-1524Crossref PubMed Scopus (189) Google Scholar). Here we show that GA-mediated inhibition of Hsp90 induces the degradation of mutant and wild-type p53 in H1299 cells. This provides additional evidence for an association of both forms with molecular chaperones. GA-induced degradation of mutant but not wild-type p53 was further stimulated by the overexpression of CHIP. The highly transient association of wild-type p53 with Hsc70 and Hsp90 may limit the synergistic effects of GA treatment and CHIP overexpression. In any case, the diversion of chaperone clients, including p53, onto a proteasomal degradation pathway seems to be preferentially mediated by an Hsc70/CHIP chaperone machinery. The observation that elevated CHIP levels increase the sensitivity of oncogenic mutant forms of p53 against Hsp90 inhibitors might be of profound relevance for cancer treatment. Several Hsp90-inhibitors are currently tested in clinical trials as anti-tumor agents (52Neckers L. Trends Mol. Med. 2002; 8: S55-S61Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar). The expression level of CHIP might be an important determinant of the therapeutic success of such pharmacological interventions. Notably, siRNA-mediated depletion of CHIP significantly stabilized p53. This suggests a critical role of chaperone-assisted degradation in maintaining low concentrations of the tumor suppressor under physiological conditions and points to a novel link between the cellular chaperone machinery and apoptosis regulation. Intriguingly, recent evidence also suggests a cross-talk between CHIP and Mdm2 in p53 regulation (59Burch L. Shimizu H. Smith A. Patterson C. Hupp T.R. J. Mol. Biol. 2004; 337: 129-145Crossref PubMed Scopus (27) Google Scholar). The molecular basis for this cross-talk is provided by the formation of a heterocomplex comprising Mdm2, p53, and Hsp90 (59Burch L. Shimizu H. Smith A. Patterson C. Hupp T.R. J. Mol. Biol. 2004; 337: 129-145Crossref PubMed Scopus (27) Google Scholar, 69Peng Y. Chen L. Li C. Lu W. Chen J. J. Biol. Chem. 2001; 276: 40583-40590Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). In the heterocomplex the activity of Mdm2 to ubiquitylate p53 is inhibited by Hsp90 (64Pavletich N.P. Chambers K.A. Pabo C.O. Genes Dev. 1993; 7: 2556-2564Crossref PubMed Scopus (440) Google Scholar). At the same time Mdm2 and Hsp90 cooperate to stimulate the unfolding of native p53 tetramers (59Burch L. Shimizu H. Smith A. Patterson C. Hupp T.R. J. Mol. Biol. 2004; 337: 129-145Crossref PubMed Scopus (27) Google Scholar). Because of its ability to bind Hsp90, CHIP can enter the heterocomplex and then further promote p53 unfolding (59Burch L. Shimizu H. Smith A. Patterson C. Hupp T.R. J. Mol. Biol. 2004; 337: 129-145Crossref PubMed Scopus (27) Google Scholar). This may represent an initial step in diverting p53 from Mdm2-mediated regulation onto a CHIP-induced degradation pathway. In line with a role of CHIP in apoptosis regulation, an anti-apoptotic activity of CHIP was recently demonstrated based on the analysis of CHIP knock-out mice (35Dai Q. Zhang C. Wu Y. McDonough H. Whaley R.A. Godfrey V. Li H.H. Madamanchi N. Xu W. Neckers L. Cyr D. Patterson C. EMBO J. 2003; 22: 5446-5458Crossref PubMed Scopus (247) Google Scholar). Although the anti-apoptotic activity was largely attributed to the role of CHIP in the conformational regulation of the heat shock transcription factor, the ability of the chaperone-associated ubiquitin ligase to induce p53 degradation may contribute to this activity.

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