Regulation of p53 by the Ubiquitin-conjugating Enzymes UbcH5B/C in Vivo
2004; Elsevier BV; Volume: 279; Issue: 40 Linguagem: Inglês
10.1074/jbc.m403362200
ISSN1083-351X
AutoresMark K. Saville, Alison Sparks, Dimitris P. Xirodimas, Julie Wardrop, Lauren Stevenson, Jean‐Christophe Bourdon, Yvonne L. Woods, David P. Lane,
Tópico(s)Epigenetics and DNA Methylation
Resumop53 levels are regulated by ubiquitination and 26 S proteasome-mediated degradation. p53 is a substrate for the E3 ligase Mdm2, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination in intact cells have not been defined previously. To investigate the E2 specificity of Mdm2 we carried out an in vitro screen using a panel of ubiquitin E2s. Of the E2s tested only UbcH5A, -B, and -C and E2-25K support Mdm2-mediated ubiquitination of p53. The same E2s also support Mdm2 auto-ubiquitination. Small interfering RNA-mediated knockdown of UbcH5B/C causes accumulation of Mdm2 and p53 in unstressed cells. We show that suppression of UbcH5B/C inhibits p53 ubiquitination and degradation. Despite up-regulating the level of nuclear p53, UbcH5B/C knockdown does not on its own result in an increase in p53 transcriptional activity or sensitize p53 to activation by the therapeutic drugs doxorubicin and actinomycin D. We provide evidence that Mdm2 is responsible, at least in part, for repression of the transcriptional activity of the accumulated p53. In MCF7 cells levels of UbcH5B/C are reduced by doxorubicin and actinomycin D. This observation and the sensitivity of p53 expression to levels of UbcH5B/C raise the possibility that E2 regulation could be involved in signaling pathways that control the stability of p53. Our data indicate that UbcH5B/C are physiological E2s for Mdm2, which make a significant contribution to the maintenance of low levels of p53 and Mdm2 in unstressed cells and that inhibition of p53 ubiquitination and degradation by targeting UbcH5B/C is not sufficient to up-regulate p53 transcriptional activity. p53 levels are regulated by ubiquitination and 26 S proteasome-mediated degradation. p53 is a substrate for the E3 ligase Mdm2, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination in intact cells have not been defined previously. To investigate the E2 specificity of Mdm2 we carried out an in vitro screen using a panel of ubiquitin E2s. Of the E2s tested only UbcH5A, -B, and -C and E2-25K support Mdm2-mediated ubiquitination of p53. The same E2s also support Mdm2 auto-ubiquitination. Small interfering RNA-mediated knockdown of UbcH5B/C causes accumulation of Mdm2 and p53 in unstressed cells. We show that suppression of UbcH5B/C inhibits p53 ubiquitination and degradation. Despite up-regulating the level of nuclear p53, UbcH5B/C knockdown does not on its own result in an increase in p53 transcriptional activity or sensitize p53 to activation by the therapeutic drugs doxorubicin and actinomycin D. We provide evidence that Mdm2 is responsible, at least in part, for repression of the transcriptional activity of the accumulated p53. In MCF7 cells levels of UbcH5B/C are reduced by doxorubicin and actinomycin D. This observation and the sensitivity of p53 expression to levels of UbcH5B/C raise the possibility that E2 regulation could be involved in signaling pathways that control the stability of p53. Our data indicate that UbcH5B/C are physiological E2s for Mdm2, which make a significant contribution to the maintenance of low levels of p53 and Mdm2 in unstressed cells and that inhibition of p53 ubiquitination and degradation by targeting UbcH5B/C is not sufficient to up-regulate p53 transcriptional activity. In normal unstressed cells the levels and activity of the p53 tumor suppressor are kept low. p53 is stabilized, and its transcriptional activity is up-regulated following diverse stresses, including ionizing and UV radiation, genotoxic drugs, and the inappropriate activation of oncogenes. This regulates the expression of multiple target genes and leads to cell cycle arrest or apoptosis, thus preventing damaged cells from proliferating (1Ryan K.M. Phillips A.C. Vousden K.H. Curr. Opin. Cell Biol. 2001; 13: 332-337Crossref PubMed Scopus (570) Google Scholar, 2Oren M. Cell Death Differ. 2003; 10: 431-442Crossref PubMed Scopus (898) Google Scholar). Most tumor cells have escaped this p53 surveillance. p53 is frequently inactivated by mutation. However, there are also many tumors expressing wild-type p53, which is inactivated by other mechanisms, including overexpression of Mdm2 and loss of p14ARF. Activation of the endogenous p53 pathway is an attractive approach to treatment of tumors expressing wild-type p53 (3Lain S. Lane D. Eur. J. Cancer. 2003; 39: 1053-1060Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 4Woods Y.L. Lane D.P. Hematol. J. 2003; 4: 233-247Crossref PubMed Scopus (24) Google Scholar). The precise mechanisms by which the activity of p53 is regulated are thus the focus of much interest. The maintenance of low levels of p53 in cells involves its ubiquitination and consequent targeting for degradation by the 26 S proteasome (5Yang Y. Li C.C. Weissman A.M. Oncogene. 2004; 23: 2096-2106Crossref PubMed Scopus (166) Google Scholar). Because ubiquitination is involved in many key processes, there is also considerable general interest in components of the ubiquitination pathway as therapeutic targets for treatment of diverse diseases. Clinical trials have been carried out with the proteasome inhibitor PS-341. This displays anti-tumor activity and has been approved for therapy of patients with multiple myeloma (6Richardson P.G. Barlogie B. Berenson J. Singhal S. Jagannath S. Irwin D. Rajkumar S.V. Srkalovic G. Alsina M. Alexanian R. Siegel D. Orlowski R.Z. Kuter D. Limentani S.A. Lee S. Hideshima T. Esseltine D.L. Kauffman M. Adams J. Schenkein D.P. Anderson K.C. N. Engl. J. Med. 2003; 348: 2609-2617Crossref PubMed Scopus (2411) Google Scholar, 7Voorhees P.M. Dees E.C. O'Neil B. Orlowski R.Z. Clin. Cancer Res. 2003; 9: 6316-6325PubMed Google Scholar, 8Lara Jr., P.N. Davies A.M. Mack P.C. Mortenson M.M. Bold R.J. Gumerlock P.H. Gandara D.R. Semin. Oncol. 2004; 31: 40-46Crossref PubMed Scopus (39) Google Scholar).Ubiquitination of proteins occurs through the sequential actions of three enzymes (9Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6788) Google Scholar, 10Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2885) Google Scholar). Initially ubiquitin is activated by the ubiquitin-activating enzyme (E1). 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; PKA, protein kinase A; RNAi, RNA interference; RT, reverse transcriptase; siRNA, small interfering RNA; STIP, super thioredoxin insert protein; TRX, thioredoxin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; Ni-NTA, nickel-nitrilotriacetic acid; DMEM, Dulbecco's modified Eagle's medium; 6-FAM, 6-carboxyfluorescein; TAMRA, carboxytetramethylrhodamine.1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; PKA, protein kinase A; RNAi, RNA interference; RT, reverse transcriptase; siRNA, small interfering RNA; STIP, super thioredoxin insert protein; TRX, thioredoxin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; Ni-NTA, nickel-nitrilotriacetic acid; DMEM, Dulbecco's modified Eagle's medium; 6-FAM, 6-carboxyfluorescein; TAMRA, carboxytetramethylrhodamine. Ubiquitin is then transferred from the E1 to a ubiquitin-conjugating enzyme (E2). A ubiquitin ligase (E3) then facilitates transfer of ubiquitin from an E2 to the substrate. There is one human ubiquitin E1 and multiple ubiquitin E2s (10Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2885) Google Scholar, 11Jones D. Crowe E. Stevens T.A. Candido E.P. Genome Biology. 2002; (http://genomebiology.com/2001/3/1/RESEARCH/0002)Google Scholar). Mdm2 acts as an E3 ligase for p53 (12Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1587) Google Scholar) and can promote its ubiquitination and degradation in vivo (13Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3656) Google Scholar, 14Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2812) Google Scholar). Mdm2 also "auto-ubiquitinates" and is itself targeted for degradation by the proteasome (15Xirodimas D. Saville M.K. Edling C. Lane D.P. Lain S. Oncogene. 2001; 20: 4972-4983Crossref PubMed Scopus (155) Google Scholar). In addition to regulating p53 protein expression Mdm2 can inhibit p53 transcriptional activity by binding to its transactivation domain (16Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. Cell. 1992; 69: 1237-1245Abstract Full Text PDF PubMed Scopus (2776) Google Scholar, 17Chen J. Marechal V. Levine A.J. Mol. Cell. Biol. 1993; 13: 4107-4114Crossref PubMed Scopus (620) Google Scholar), and it may directly repress basal transcription from p53-responsive promoters (18Thut C.J. Goodrich J.A. Tjian R. Genes Dev. 1997; 11: 1974-1986Crossref PubMed Scopus (230) Google Scholar). We have recently observed that Mdm2 can promote conjugation of the ubiquitin-like protein NEDD8 (neural precursor cell-expressed developmentally down-regulated) to p53 (19Xirodimas 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 (427) Google Scholar). This modification also represses the transcriptional activity of p53. The relative contribution to inhibition of p53 of Mdm2-dependent ubiquitin and NEDD8 conjugation and transcriptional repression through direct binding to p53 are not clear. Loss of Mdm2 in mice is embryonic lethal in a p53-dependent manner (20Jones S.N. Roe A.E. Donehower L.A. Bradley A. Nature. 1995; 378: 206-208Crossref PubMed Scopus (1056) Google Scholar, 21Montes de Oca Luna R. Wagner D.S. Lozano G. Nature. 1995; 378: 203-206Crossref PubMed Scopus (1196) Google Scholar). In transgenic adult mice, which express lower levels of Mdm2 than wild-type mice, the transactivation and apoptotic activities of p53 are enhanced (22Mendrysa S.M. McElwee M.K. Michalowski J. O'Leary K.A. Young K.M. Perry M.E. Mol. Cell. Biol. 2003; 23: 462-472Crossref PubMed Scopus (191) Google Scholar). Agents that bind to Mdm2 and block its association with p53 increase both the levels and transcriptional activity of p53 in vivo. These include high affinity Mdm2 binding peptides, the Mdm2-specific antibody 3G5 (23Bottger A. Bottger V. Sparks A. Liu W.L. Howard S.F. Lane D.P. Curr. Biol. 1997; 7: 860-869Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar), and the Nutlin family of small molecules (24Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E.A. Science. 2004; 303: 844-848Crossref PubMed Scopus (3732) Google Scholar). These and other observations indicate that Mdm2 is a key negative regulator of p53 (25Freedman D.A. Wu L. Levine A.J. Cell Mol. Life Sci. 1999; 55: 96-107Crossref PubMed Scopus (479) Google Scholar, 26Michael D. Oren M. Semin. Cancer Biol. 2003; 13: 49-58Crossref PubMed Scopus (640) Google Scholar). Mdm2 is itself a target for p53 transcriptional regulation. Mdm2 is induced by p53 resulting in a negative feedback loop (27Lane D.P. Hall P.A. Trends Biochem. Sci. 1997; 22: 372-374Abstract Full Text PDF PubMed Scopus (135) Google Scholar). The Mdm2 structural homologue MdmX is also an important regulator of p53. MdmX binds to p53 and inhibits its transcriptional activity but does not itself ubiquitinate p53 in cells (28Stad R. Little N.A. Xirodimas D.P. Frenk R. van der Eb A.J. Lane D.P. Saville M.K. Jochemsen A.G. EMBO Rep. 2001; 2: 1029-1034Crossref PubMed Scopus (186) Google Scholar), although a low level of E3 ligase activity has been observed in vitro (29Badciong J.C. Haas A.L. J. Biol. Chem. 2002; 277: 49668-49675Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Mdm2 and MdmX heterodimerize through their ring fingers (30Tanimura S. Ohtsuka S. Mitsui K. Shirouzu K. Yoshimura A. Ohtsubo M. FEBS Lett. 1999; 447: 5-9Crossref PubMed Scopus (266) Google Scholar). Strikingly, ablation of MdmX also results in p53-dependent embryonic lethality in mice (31Parant J. Chavez-Reyes A. Little N.A. Yan W. Reinke V. Jochemsen A.G. Lozano G. Nat. Genet. 2001; 29: 92-95Crossref PubMed Scopus (406) Google Scholar, 32Finch R.A. Donoviel D.B. Potter D. Shi M. Fan A. Freed D.D. Wang C.Y. Zambrowicz B.P. Ramirez-Solis R. Sands A.T. Zhang N. Cancer Res. 2002; 62: 3221-3225PubMed Google Scholar).Although auto-ubiquitination of Mdm2 and Mdm2-mediated ubiquitination of p53 have been reconstituted in vitro using purified E2s (12Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1587) Google Scholar, 33Fang 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 (862) Google Scholar, 34Midgley C.A. Desterro J.M. Saville M.K. Howard S. Sparks A. Hay R.T. Lane D.P. Oncogene. 2000; 19: 2312-2323Crossref PubMed Scopus (229) Google Scholar), the E2 specificity of Mdm2 has not been investigated in detail, and the one or more ubiquitin E2s involved in regulating the Mdm2/p53 pathway in intact cells have not been determined previously. Ubiquitin E2s consist of a conserved core domain of ∼150 amino acids. They have an active site cysteine residue, which forms a thiolester bond with ubiquitin transferred from the E1. Ubiquitination, and by inference ubiquitin E2s, are involved in regulating many cellular processes. However, the precise physiological roles of many of the ubiquitin E2s are not well defined, particularly in mammalian cells. There is evidence that E2s have unique functions, presumably arising from their specificity for particular E3s. Genetic studies in Saccharomyces cerevisiae indicate that Ubc2 (RAD6) is central to DNA repair, Ubc3 (CDC34) is required for the G1-S transition, and Ubc4/5 are required for viability (10Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2885) Google Scholar).Regulation of E2 levels or activity could provide a mechanism to alter the stability of key proteins. Studies using rat tissue extracts indicate that E2s can be rate-limiting for ubiquitination (35Rajapurohitam V. Bedard N. Wing S.S. Am. J. Physiol. 2002; 282: E739-E745Crossref PubMed Scopus (39) Google Scholar). The expression of S. cerevisiae Ubc4 and -5 is growth- and heat shock-regulated (36Seufert W. Jentsch S. EMBO J. 1990; 9: 543-550Crossref PubMed Scopus (402) Google Scholar). The levels of specific mammalian E2s have also been reported to be regulated by the cell cycle and by agents, including interferons α and γ, insulin, insulin-like growth factor 1, amyloid-β, and herpes simplex virus (37Wing S.S. Banville D. Am. J. Physiol. 1994; 267: E39-E48Crossref PubMed Google Scholar, 38Wing S.S. Bedard N. Biochem. J. 1996; 319: 455-461Crossref PubMed Scopus (46) Google Scholar, 39Shang F. Gong X. Taylor A. 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Eye Res. 2004; 78: 197-205Crossref PubMed Scopus (23) Google Scholar). E2 activity can be influenced by phosphorylation. CDC34 and its homologue Ubc3b are substrates for protein kinase CK2 (46Semplici F. Meggio F. Pinna L.A. Oliviero S. Oncogene. 2002; 21: 3978-3987Crossref PubMed Scopus (44) Google Scholar), and UbcH1 (HHR6) is phosphorylated by CDK1 and -2 (47Sarcevic B. Mawson A. Baker R.T. Sutherland R.L. EMBO J. 2002; 21: 2009-2018Crossref PubMed Scopus (60) Google Scholar).In this study we identified ubiquitin E2s involved in the Mdm2/p53 pathway in intact cells. A panel of E2s were tested for their ability to support the ubiquitin ligase activity of Mdm2 in vitro. We show that there is specificity in E2 usage by Mdm2. Of the E2s tested only members of the UbcH5 family (A-C) and E2-25K are able to support the ubiquitin ligase activity of Mdm2. We observed that the pattern of Mdm2-mediated ubiquitination of p53 in vitro is dependent on the E2 used. Using siRNA-mediated knockdown we show that UbcH5B/C make a significant contribution to the maintenance of low levels of Mdm2 and p53 in unstressed cells. In contrast, suppression of UbcH5A or E2-25K has little effect on expression of Mdm2 and p53 in the cell lines examined. Levels of UbcH5B/C protein expression were found to be higher than those of UbcH5A, possibly accounting for the preferential usage of UbcH5B/C in intact cells. The balance of activities of the Mdm2/p53 pathway is such that the p53 accumulated following UbcH5B/C knockdown is not transcriptionally active, the level of Mdm2 being sufficient to maintain transcriptional repression of p53. We find that expression of UbcH5B/C is reduced by the p53 stabilizing therapeutic drugs doxorubicin and actinomycin D. Because p53 expression is sensitive to the levels of UbcH5B/C, it is conceivable that E2 regulation could play some part in signaling to the p53 pathway.EXPERIMENTAL PROCEDURESAntibodies—The antibodies used were 4B2 for Mdm2; DO-1 for p53 unless otherwise indicated; H-81 for CDC34 (Santa Cruz Biotechnology); UBC4 (C-15) for UbcH5B/C (Santa Cruz Biotechnology), which was precleared by absorption with GST-UbcH5A; Ab-1 for p21 (Oncogene); L27 for CD20 (BD Biosciences); and Ab-1 for actin (Calbiochem). A rabbit polyclonal anti-E2-25K antibody was purchased from Affiniti Research, a rabbit anti-ubiquitin antiserum was purchased from Sigma and a sheep anti-UbcH5A polyclonal antiserum raised against purified full-length UbcH5A was provided by Dr. R. T. Hay. This was affinity-purified and then precleared by absorption with GST-UbcH5B and -C. Where indicated, cross-reactivity was removed by incubating antibodies (15-30 μg) three times for 4 h with GST-E2s (∼50 μg) bound to glutathione-Sepharose beads in 0.5 ml of 5% dried milk, 0.1% Tween 20 in PBS.Plasmids and Synthetic siRNA Duplexes—To generate constructs for bacterial expression of GST-E2s, the following IMAGE clones were obtained from the UK MRC Human Genome Mapping Project resource center: HHR6A (3854960), HHR6B (2178494), UbcH2 (1908854), UbcH5B (2169015), UbcH5C (3854578), and UbcH8 (3838534). UbcH5A cDNA was donated by Dr. R. T. Hay. These were used as templates for PCR, and the products were cloned into the BamHI and EcoRI sites of pGEX-2T with the exception of HHR6A, which was cloned into the SmaI and EcoRI sites. pT7-7-Mdm2 used for bacterial expression of human Mdm2 was described previously (34Midgley C.A. Desterro J.M. Saville M.K. Howard S. Sparks A. Hay R.T. Lane D.P. Oncogene. 2000; 19: 2312-2323Crossref PubMed Scopus (229) Google Scholar). For expression of His6-tagged human p53 in bacteria, a PCR product was cloned into the SphI site of pT7-7-SphI, which was generated as described previously (48Midgley C.A. Owens B. Briscoe C.V. Thomas D.B. Lane D.P. Hall P.A. J. Cell Sci. 1995; 108: 1843-1848Crossref PubMed Google Scholar). pGEX-2TK-ubiquitin used for bacterial expression of GST-ubiquitin was donated by Dr. K. Madura. For expression in cell lines, a transcriptionally inactive mutant of human p53 (His-273) was cloned into the EcoRI and XhoI sites of pcDNA3. Plasmids used to express human Mdm2 and His6-ubiquitin have been described previously (15Xirodimas D. Saville M.K. Edling C. Lane D.P. Lain S. Oncogene. 2001; 20: 4972-4983Crossref PubMed Scopus (155) Google Scholar). pCMVCD20 was donated by Dr. R. Watson. siRNA target sequences were chosen according to the criteria of Elbashir et al. (49Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8075) Google Scholar). The following target sequences were inserted into the pSUPER vector (50Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3941) Google Scholar), which was obtained from Dr. R. Bernards: CDC34, CACCTACTACGAGGGCGGC; UbcH5A, GGTGGAGTCTTCTTTCTCA; UbcH5B, CAGATTACCCCTTCAAACC; UbcH5C, CAGACAGAGATAAGTACAA; UbcH5B/C, CAGTAATGGCAGCATTTGT; UbcH5B/C (2Oren M. Cell Death Differ. 2003; 10: 431-442Crossref PubMed Scopus (898) Google Scholar), GATCACAGTGGTCGCCTGC; UbcH5B/C (3Lain S. Lane D. Eur. J. Cancer. 2003; 39: 1053-1060Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), TGGACTCAGAAGTATGCCA; and E2-25K, TAGTGGCCTTGTCTTCAAA. pSUPER-UbcH5B/C targets identical sequences in UbcH5B and -C. pSUPER-UbcH5B/C (2Oren M. Cell Death Differ. 2003; 10: 431-442Crossref PubMed Scopus (898) Google Scholar) and UbcH5B/C (3Lain S. Lane D. Eur. J. Cancer. 2003; 39: 1053-1060Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) target sequences in UbcH5C, which differ from those in UbcH5B, by two and one nucleotide, respectively, and were found to suppress both UbcH5B and -C mRNA. Nonspecific control duplex IX, Mdm2 SMART pool (M-003279-00), and synthetic siRNA duplexes, which target the same sequences as pSUPER-UbcH5A, UbcH5B/C, and E2-25K, were purchased from Dharmacon.Gel Electrophoresis and Western Blotting—Cells were washed twice with PBS 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). Dithiothreitol 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, or for 1 h at 150 mA, and membranes were processed as described in Midgley et al. (34Midgley C.A. Desterro J.M. Saville M.K. Howard S. Sparks A. Hay R.T. Lane D.P. Oncogene. 2000; 19: 2312-2323Crossref PubMed Scopus (229) Google Scholar). Membranes probed for ubiquitin were boiled in de-ionized water prior to blocking to expose epitopes in ubiquitin. Peroxidase-conjugated secondary antibodies were supplied by Jackson ImmunoResearch Laboratories and used at a dilution of 1/10,000. Bound antibodies were detected by enhanced chemiluminescence (Amersham Biosciences) or using SuperSignal West Dura Extended Duration Substrate (Pierce).Expression and Purification of Recombinant Proteins—Untagged human Mdm2 was prepared from bacterial inclusion bodies as described previously (34Midgley C.A. Desterro J.M. Saville M.K. Howard S. Sparks A. Hay R.T. Lane D.P. Oncogene. 2000; 19: 2312-2323Crossref PubMed Scopus (229) Google Scholar). Human E1 was purified from recombinant baculovirus-infected insect cells by affinity chromatography on ubiquitin-Sepharose as described in Desterro et al. (51Desterro J.M. Rodriguez M.S. Kemp G.D. Hay R.T. J. Biol. Chem. 1999; 274: 10618-10624Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Human GST-HHR6A, HHR6B, UbcH2 (52Kaiser P. Seufert W. Hofferer L. Kofler B. Sachsenmaier C. Herzog H. Jentsch S. Schweiger M. Schneider R. J. Biol. Chem. 1994; 269: 8797-8802Abstract Full Text PDF PubMed Google Scholar), UbcH5A, UbcH5B, UbcH5C, and UbcH8 were expressed in bacteria and purified as described previously (53Desterro J.M. Thomson J. Hay R.T. FEBS Lett. 1997; 417: 297-300Crossref PubMed Scopus (303) Google Scholar). GST-ubiquitin containing a protein kinase A (PKA) consensus site between the ubiquitin and the tag was expressed in bacterial, purified, and phosphorylated by PKA as described in Tongaonkar et al. (54Tongaonkar P. Madura K. Anal. Biochem. 1998; 260: 135-141Crossref PubMed Scopus (15) Google Scholar). GST was cleaved from E2s and ubiquitin by thrombin digestion prior to use in ubiquitination assays, and free thrombin was removed by incubation with benzamidine-Sepharose. UbcH6, -7, and -10 and E2-25K proteins were supplied by Affiniti Research, and CDC34 was donated by Dr. R. T. Hay. His6-p53 was expressed in bacteria. Following induction with isopropyl-1-thio-β-d-galactopyranoside at room temperature p53 in the soluble fraction was partially purified on a heparin-Sepharose column as described previously (55Hansen S. Hupp T.R. Lane D.P. J. Biol. Chem. 1996; 271: 3917-3924Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Fractions containing full-length p53 were further purified on Ni2+-NTA-agarose.In Vitro Ubiquitination Assays—Reactions contained 50 mm Tris, pH 7.6, 5 mm MgCl2, 2 mm ATP, 10 mm creatine phosphate, 3.5 units/ml creatine kinase, and 0.6 unit/ml inorganic pyrophosphatase, 20 μm [32P]ubiquitin, and human E1 (50 nm), E2s (1.5 μm), and where indicated p53 (1 μm). Reactions were initiated by the addition of human Mdm2 (50-100 nm). Following a 2-h incubation at 37 °C, reactions were terminated by the addition of SDS-urea sample buffer. Products were resolved by SDS-PAGE. [32P]Ubiquitin was detected by phosphorimaging of dried gels, and p53 was detected by Western blotting using DO-1.Cell Culture and Transfection—MCF7 breast tumor cells and U2-OS osteosarcoma cells were cultured in DMEM and H1299 lung carcinoma cells in RPMI. Media were supplemented with 10% fetal calf serum and gentamycin (50 μg/ml), and cells were kept at 37 °C, 5% CO2 in a humidified atmosphere. Unless otherwise indicated MCF7 and U2-OS cells were transfected with 1.5 μg of pSUPER construct per 10-cm dish. Transfection of these cells lines with plasmids was performed with FuGENE 6 transfection reagent (Roche Applied Science) following the manufacturer's instructions. Cells were washed after 16 h, and fresh medium was added. Unless otherwise indicated H1299 cells were transfected with 7 μg of pSUPER constructs per 10-cm dish. Transfection of this cell line was performed using the calcium phosphate method essentially as described in Xirodimas et al. (15Xirodimas D. Saville M.K. Edling C. Lane D.P. Lain S. Oncogene. 2001; 20: 4972-4983Crossref PubMed Scopus (155) Google Scholar). Transfection with single siRNA synthetic duplexes (100 nm) or SMART pools (200 nm) was carried out using Oligofectamine (Invitrogen) according to the manufacturer's instructions.CD20 Enrichment of Transfected Cells—MCF7 cells in 10-cm dishes were transfected with pSUPER constructs and pCMVCD20 (1 μg). At the indicated time after transfection, cells were detached by washing with 3 mm EDTA in PBS. Cells were resuspended thoroughly in DMEM, 10% FCS, and incubated for 20 min at 4 °C with magnetic Dynabeads Pan Mouse IgG (Dynal Biotech) coated with anti-CD20 antibody (1 × 107 beads and 1 μg of antibody per dish). The beads were isolated with a magnet, and the selected cells were washed twice with PBS before lysis for RNA extraction or Western blotting.Immunofluorescence—Cells were seeded onto NUNC Permanox slides. 36 h after transfection with synthetic siRNA duplexes, cells were fixed with ice-cold methanol-acetone and incubated with primary antibodies followed by fluorescence isothiocyanate-conjugated donkey anti-mouse secondary antibodies (Jackson ImmunoResearch) as described previously (15Xirodimas D. Saville M.K. Edling C. Lane D.P. Lain S. Oncogene. 2001; 20: 4972-4983Crossref PubMed Scopus (155) Google Scholar).Purification of His6-tagged Ubiquitin Conjugates—MCF7 cells were seeded onto 10-cm dishes and transfected with pSUPER constructs and His6-ubiquitin (1 μg) using FuGENE 6. 64 h later, cells were harvested by direct lysis into 8 m urea, 100 mm Na2HPO4/NaH2PO4, 10 mm Tris, pH 8.0, 10 mm β-mercaptoethanol, 5 mm imidazole. Purification of His6-ubiquitin conjugates using Ni2+-NTA-agarose beads was carried out as described in Xirodimas et al. (15Xirodimas D. Saville M.K. Edling C. Lane D.P. Lain S. Oncogene. 2001; 20: 4972-4983Crossref PubMed Scopus (155) Google Scholar).Quantitative Detection of β-Galactosidase—Cells in 24-well plates were washed twice with PBS and lysed on the plate with 150 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 15 μl of extract in a 96-well plate. The assay was quantitated by absorbance measurements at a wavelength of 590 nm in a plate reader. Luciferase activities were measured using the Berthold microplate luminometer LB 96V and used to control for transfection efficiency.RNA Preparation and Real-time RT-PCR—Total RNA was extracted using RNeasy columns (Qiagen) according to the manufacturer's instructions, including an on-column DNase treatment step. RNA (300-600 ng) was incubated with random hexamers and Superscript II reverse transcriptase (Invitrogen) to generate cDNA. Real-time PCR was carried out with an ABI Prism 7700 sequence detector using the following protocol: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. For ubiquitin E2s, primers and 6-FAM/TAMRA-labeled probes were as used in Okamoto et al. (56Okamoto Y. Ozaki T. Miyazaki K. Aoyama M. Miyazaki M. Nakagawara A. Cancer Res. 2003; 63: 4167-4173PubMed Google Scholar). Other specific probes and primers were as follows: p53 primers: 5′-CAGCCAAGTCTGTGACTTGCA-3′, 5′-GTGTGGAATCAACCCACAGCT-3′, probe: 6-FAM-TCCCCTGCCC
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