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• W2100041351 abstract "The last step in the activation of the transcription factor NF-κB is signal-induced, ubiquitin- and proteasome-mediated degradation of the inhibitor IκBα. Although most of the components involved in the activation and degradation pathways have been identified, the ubiquitin carrier proteins (E2) have remained elusive. Here we show that the two highly homologous members of the UBCH5 family, UBCH5b and UBCH5c, and CDC34/UBC3, the mammalian homolog of yeast Cdc34/Ubc3, are the E2 enzymes involved in the process. The conjugation reaction they catalyze in vitro is specific, as they do not recognize the S32A,S36A mutant species of IκBα that cannot be phosphorylated and conjugated following an extracellular signal. Furthermore, the reaction is specifically inhibited by a doubly phosphorylated peptide that spans the ubiquitin ligase recognition domain of the inhibitor. Cys-to-Ala mutant species of the enzymes that cannot bind ubiquitin inhibit tumor necrosis factor α-induced degradation of the inhibitor in vivo. Not surprisingly, they have a similar effect in a cell-free system as well. Although it is clear that the E2 enzymes are not entirely specific to IκBα, they are also not involved in the conjugation and degradation of the bulk of cellular proteins, thus exhibiting some degree of specificity that is mediated probably via their association with a defined subset of ubiquitin-protein ligases. The mechanisms that underlie the involvement of two different E2 species in IκBα conjugation are not clear at present. It is possible that different conjugating machineries operate under different physiological conditions or in different cells. The last step in the activation of the transcription factor NF-κB is signal-induced, ubiquitin- and proteasome-mediated degradation of the inhibitor IκBα. Although most of the components involved in the activation and degradation pathways have been identified, the ubiquitin carrier proteins (E2) have remained elusive. Here we show that the two highly homologous members of the UBCH5 family, UBCH5b and UBCH5c, and CDC34/UBC3, the mammalian homolog of yeast Cdc34/Ubc3, are the E2 enzymes involved in the process. The conjugation reaction they catalyze in vitro is specific, as they do not recognize the S32A,S36A mutant species of IκBα that cannot be phosphorylated and conjugated following an extracellular signal. Furthermore, the reaction is specifically inhibited by a doubly phosphorylated peptide that spans the ubiquitin ligase recognition domain of the inhibitor. Cys-to-Ala mutant species of the enzymes that cannot bind ubiquitin inhibit tumor necrosis factor α-induced degradation of the inhibitor in vivo. Not surprisingly, they have a similar effect in a cell-free system as well. Although it is clear that the E2 enzymes are not entirely specific to IκBα, they are also not involved in the conjugation and degradation of the bulk of cellular proteins, thus exhibiting some degree of specificity that is mediated probably via their association with a defined subset of ubiquitin-protein ligases. The mechanisms that underlie the involvement of two different E2 species in IκBα conjugation are not clear at present. It is possible that different conjugating machineries operate under different physiological conditions or in different cells. Generation and activation of the transcription factor NF-κB involve two successive ubiquitin- and proteasome-mediated proteolytic steps: (i) processing of the precursor protein p105 to the active subunit p50 and (ii) signal-induced degradation of the inhibitor IκBα. Degradation of IκBα is triggered by a broad array of stimuli, such as binding of cytokines or viral products to their appropriate receptors. The receptors then recruit adaptor proteins such as TRADD and TRAF. Consequently, activated NF-κB-inducing kinase (1Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1165) Google Scholar) is released and sequestered into a large complex that contains, among other proteins, IκB kinases α and β (see, for example, Ref. 2Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Ly J.W. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar), NEMO (NF-κB essential modulator) (3Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar) or IκB kinase γ (4Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar), and IκB kinase complex-associated protein (5Cohen L. Henzel J.W. Baeuerle P.A. Nature. 1998; 395: 292-296Crossref PubMed Scopus (270) Google Scholar). Complex formation leads, most probably, to activation of the IκB kinases that phosphorylate NF-κB-complexed IκBα on serines 32 and 36. This modification leads to its targeting and rapid degradation by the ubiquitin-proteasome pathway (6Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1491Crossref PubMed Scopus (1315) Google Scholar, 7Chen Z. Hagler J. Palombella V. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1170) Google Scholar, 8Traenckner E.B.-M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (933) Google Scholar, 9Alkalay I. Yaron A. Hatzubai A. Orian A. Ciechanover A. Ben Neriah Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10599-10603Crossref PubMed Scopus (391) Google Scholar, 10Yaron A. Gonen H. Alkalay I. Hatzubai A. Jung S. Beyth S. Mercurio F. Manning A.M. Ciechanover A. Ben Neriah Y. EMBO J. 1997; 16: 6486-6494Crossref PubMed Scopus (202) Google Scholar). Most of the upstream components of the signaling pathway have been identified. Also, recent studies have identified an SCF complex that contains Skp1p, Cullin1, and the F-box protein β-TrCP as the ubiquitin-IκBα ligase complex (see for example Refs. 11Yaron A. Hatzubai A. Davis M. Lavon I. Amit S. Manning A.M. Andersen A.S. Mann M. Mercurio F. Ben Neriah Y. Nature. 1998; 396: 590-594Crossref PubMed Scopus (571) Google Scholar, 12Winston J.T. Strack P. Beer-Romero P. Chu C. Elledge S.J. Harper J.W. Genes Dev. 1999; 13: 270-283Crossref PubMed Scopus (811) Google Scholar, 13Spencer E. Jiang J. Chen Z.J. Genes Dev. 1999; 13: 284-294Crossref PubMed Scopus (374) Google Scholar). However, all these studies have not identified the ubiquitin carrier protein(s) involved in targeting of phosphorylated IκBα (pIκBα), 1The abbreviations used are: pIκBα, phosphorylated IκBα; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein ligase; TNF-α, tumor necrosis factor α; PAGE, polyacrylamide gel electrophoresis. and the identity of the enzyme(s) has remained elusive. The ubiquitin system targets a wide array of short-lived regulatory proteins such as transcriptional activators, tumor suppressors and growth modulators, cell cycle regulators, and signal transduction pathway components. Consequently, it is involved in the regulation of many basic cellular processes. Among these are cell cycle and division, differentiation, and development; response to stress and extracellular stimuli; modulation of cell-surface receptors; DNA repair; regulation of the immune and inflammatory responses; biogenesis of organelles; and apoptosis. Degradation of a protein by the ubiquitin system involves two distinct and successive steps: (i) covalent attachment of multiple ubiquitin molecules to the target protein and (ii) degradation of the tagged substrate by the 26 S proteasome. Conjugation proceeds via a three-step mechanism involving three enzymes. Initially, ubiquitin is activated by the ubiquitin-activating enzyme (E1). One of several E2 enzymes (ubiquitin carrier proteins or ubiquitin-conjugating enzymes (designated UBCs)) transfers ubiquitin from E1 to the substrate, either directly or via a member of the ubiquitin-protein ligase family of enzymes (E3) to which the substrate protein is specifically bound. The first ubiquitin moiety typically binds via its C-terminal Gly and generates an isopeptide with an ε-NH2 group of a Lys residue of the protein substrate. In successive reactions, a polyubiquitin chain is synthesized by transfer of additional activated ubiquitin moieties to Lys48 of the previously conjugated molecule. The structure of the ubiquitin system appears to be hierarchical: a single E1 carries out activation of ubiquitin required for all modifications. It can transfer ubiquitin to several species of E2 enzymes. Each E2 acts in concert with either one or several E3 enzymes. Following conjugation, the protein moiety of the adduct is recognized, most probably via its polyubiquitin chain, and degraded by the 26 S proteasome complex. Free and reutilizable ubiquitin is released via the activity of isopeptidases (for recent reviews and a monograph on the ubiquitin system, see, for example, Refs. 14Baumeister W. Walz J. Zuhl F. Seemüller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1306) Google Scholar, 15$$Google Scholar, 16Ciechanover A. EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1196) Google Scholar, 17Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6907) Google Scholar). A few E2 enzymes, such as E2-C, which is involved in the targeting cyclins (18Aristarkhov A. Eytan E. Moghe A. Admon A. Hershko A. Ruderman J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4294-4298Crossref PubMed Scopus (121) Google Scholar), appear to be E3- and substrate group-specific. Other E2 enzymes are involved in transfer of ubiquitin to several E3 enzymes and in the targeting of different groups of substrates. Three homologous human enzymes (UBCH5a, UBCH5b, and UBCH5c) have been described (19Jensen J.P. Bates P.W. Yang M. Vierstra R.D. Weissman A.M. J. Biol. Chem. 1995; 270: 30408-30414Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) that are closely related to Saccharomyces cerevisiae Ubc4 and Ubc5, which are involved in targeting many cellular proteins, particularly under stress. They are also homologous to Drosophila melanogaster UBCD1 and to Arabidopsis thalianaUBC8–12. UBCH5b and UBCH5c are ∼98% identical, whereas UBCH5a is ∼90% similar to the b and c species. Members of the family have been shown to be involved in cell-free conjugation of several proteins (e.g. p53 (20Scheffner M. Huibregtse J.M. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8797-8801Crossref PubMed Scopus (235) Google Scholar) and p105 (21Coux O. Goldberg A.L. J. Biol. Chem. 1998; 273: 8820-8828Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar)) and to interact with certain E3 enzymes such as members of the HECT domain family of ligases (22Kumar S. Kao W.H. Howley P.M. J. Biol. Chem. 1997; 272: 13548-13554Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The cellular substrates of these enzymes have, however, remained obscure. S. cerevisiae cdc34/ubc3, which is involved in G1 → S transition, encodes a 295-amino acid E2 (23Goebl M.G. Yochem J. Jentsch S. McGrath J.P. Varshavsky A. Byers B. Science. 1988; 241: 1331-1335Crossref PubMed Scopus (321) Google Scholar). The human homolog of this enzyme has been cloned (24Plon S.E. Leppig K.A. Do H.-N. Groundine M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10484-10488Crossref PubMed Scopus (82) Google Scholar). Recent evidence indicates that CDC34/UBC3 acts in concert with different SCF ubiquitin ligase complexes that target, among other substrates, phosphorylated G1 regulatory proteins (reviewed recently in Refs. 16Ciechanover A. EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1196) Google Scholar, 17Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6907) Google Scholar, and 25Maniatis T. Genes Dev. 1999; 13: 505-510Crossref PubMed Scopus (369) Google Scholar, 26Krek W. Curr. Opin. Genet. Dev. 1998; 8: 36-42Crossref PubMed Scopus (143) Google Scholar, 27Peters J.-M. Curr. Opin. Cell Biol. 1998; 10: 759-768Crossref PubMed Scopus (225) Google Scholar). It is also involved, along with SCF complexes, in other processes such as down-regulation of methionine biosynthesis gene products (28Patton E.E. Willems A.R. Sa D. Kuras L. Thomas D. Craig K.L. Tyers M. Genes Dev. 1998; 12: 692-705Crossref PubMed Scopus (235) Google Scholar). High pressure liquid chromatography-purified synthetic peptides were purchased from SynPep (Dublin, CA). cDNAs coding for wild-type and S32A,S36A IκBα proteins were as described (9Alkalay I. Yaron A. Hatzubai A. Orian A. Ciechanover A. Ben Neriah Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10599-10603Crossref PubMed Scopus (391) Google Scholar, 10Yaron A. Gonen H. Alkalay I. Hatzubai A. Jung S. Beyth S. Mercurio F. Manning A.M. Ciechanover A. Ben Neriah Y. EMBO J. 1997; 16: 6486-6494Crossref PubMed Scopus (202) Google Scholar, 11Yaron A. Hatzubai A. Davis M. Lavon I. Amit S. Manning A.M. Andersen A.S. Mann M. Mercurio F. Ben Neriah Y. Nature. 1998; 396: 590-594Crossref PubMed Scopus (571) Google Scholar). Antibodies to IκBα, p65, and MyoD were from Santa Cruz Biotechnology, whereas an antibody to human CDC34/UBC3 was from Transduction Laboratories. The wheat germ-based coupled transcription-translation kit and TNF-α were from Promega. Ni2+-nitrilotriacetic acid-agarose and anti-RGS-His antibody were from QIAGEN Inc. Materials for SDS-PAGE were from Bio-Rad. Hexokinase and okadaic acid were from Roche Molecular Biochemicals. l-[35S]Methionine and Na125I were obtained from NEN Life Science Products. Ubiquitin, ATP, phosphocreatine kinase, phosphocreatine, 2-deoxyglucose, bestatin, doxycycline, Tris buffer, and isopropyl-β-d-thiogalactopyranoside were from Sigma. HEPES was from Calbiochem. DEAE-cellulose (DE52) was from Whatman. Tissue culture sera and media were purchased from Biological Industries (Kibbutz Bet Haemek, Israel) or from Sigma. Restriction and modifying enzymes were from New England Biolabs Inc. Immobilized protein A was from Amersham Pharmacia Biotech. Reagents for ECL were from Pierce. Centricons and Centripreps (10-kDa molecular mass cutoff) for rapid concentration and dialysis by centrifugation were from Amicon. Nitrocellulose paper was from Schleicher & Schüll. Oligonucleotides were synthesized by the local facility in the Department of Immunology and Cell Biology at the University of Kyoto and by Biotechnology General (Rehovot, Israel). All other reagents used were of high analytical grade. Preparation and Fractionation of Crude Cell Lysates—HeLa cells grown in suspension in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum were washed twice in a buffer containing 20 mm HEPES (pH 7.4), 1 mm dithiothreitol, and 150 mm NaCl. Following resuspension (10 ml of 108 cells/ml) in a similar buffer without NaCl, the cells were exposed, for two cycles of 15 min each, to 1500 p.s.i. of N2 in a high pressure cell (Parr Instrument Co., Moline, IL). The disrupted cells were centrifuged successively for 30 min at 1500, 12,000, and 50,000 ×g. The supernatant from the last centrifugation step was collected and either used as a crude extract or further fractionated over DEAE-cellulose onto unadsorbed material (Fraction I) and a high salt eluate (Fraction II) as described (29Hershko A. Heller H. Elias S. Ciechanover A. J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar). Fraction II was further fractionated by (NH4)2SO4 into Fraction IIA (0–38%) and Fraction IIB (42–80%) as described (30Blumenfeld N. Gonen H. Mayer A. Smith C.E. Siegel N.R. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1994; 269: 9574-9581Abstract Full Text PDF PubMed Google Scholar). E1 was purified from human erythrocytes as described (29Hershko A. Heller H. Elias S. Ciechanover A. J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar). Crude Fraction II or Fraction IIA was used as a source of E3 (30Blumenfeld N. Gonen H. Mayer A. Smith C.E. Siegel N.R. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1994; 269: 9574-9581Abstract Full Text PDF PubMed Google Scholar). E2-14K was purified from rabbit reticulocytes as described (31Abu Hatoum O. Gross-Mesilaty S. Breitschopf K. Hoffman A. Gonen H. Ciechanover A. Bengal E. Mol. Cell. Biol. 1998; 18: 5670-5677Crossref PubMed Google Scholar). Recombinant E2-C was as described (18Aristarkhov A. Eytan E. Moghe A. Admon A. Hershko A. Ruderman J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4294-4298Crossref PubMed Scopus (121) Google Scholar). cDNA coding for UBCH7 was as described (32Nuber U. Schwarz S. Kaiser P. Schneider R. Scheffner M. J. Biol. Chem. 1996; 271: 2795-2800Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Following induction and cell disruption, the bacterial cell extract was resolved on DEAE-cellulose, and the enzyme that was contained in Fraction I was further purified via gel filtration chromatography on a Superdex 75 HiLoad column (16 × 600 mm; Amersham Pharmacia Biotech). Recombinant E2-8A (33Wing S.S. Bedard N. Morales C. Hingamp P. Trasler J. Mol. Cell. Biol. 1996; 16: 4064-4072Crossref PubMed Scopus (50) Google Scholar) was expressed in bacteria, and the protein was partially purified as described above for UBCH7. cDNAs coding for UBCH5a (20Scheffner M. Huibregtse J.M. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8797-8801Crossref PubMed Scopus (235) Google Scholar) and UBCH5b and UBCH5c (19Jensen J.P. Bates P.W. Yang M. Vierstra R.D. Weissman A.M. J. Biol. Chem. 1995; 270: 30408-30414Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) were obtained from Dr. Allan Weissman (National Institutes of Health). cDNAs coding for human CDC34/UBC3 (24Plon S.E. Leppig K.A. Do H.-N. Groundine M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10484-10488Crossref PubMed Scopus (82) Google Scholar), E2-25K (34Kalchman M.A. Graham R.K. Xia G. Koide H.B. Hodgson J.G. Graham K.C. Goldberg Y.P. Gietz R.D. Pickart C.M. Hayden M.R. J. Biol. Chem. 1996; 271: 19385-19394Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar), and E2-20K (35Kaiser 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) were cloned by reverse transcription-polymerase chain reaction from 293 cells. Active-site, dominant-negative mutants of the different E2 enzymes (C85A UBCH5a, C85A UBCH5b, C85A UBCH5c, C93A CDC34/UBC3, C92A E2-25K, and C87A E2-20K) were generated by a two-step polymerase chain reaction as described (36Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2102) Google Scholar). For bacterial expression, the cDNAs coding for wild-type and mutant UBCH5a, UBCH5b, and UBCH5c, for CDC34/UBC3, and for E2-25K and E2-20K were subcloned into the pT7-7 vector (37Tabor S. Ausubel F.A. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing/Wiley-Interscience, New York1990: 16.2.1-16.2.11Google Scholar) following their modification by polymerase chain reaction to contain an N-terminal His6 tag. For transient expression in mammalian cells, the cDNAs were subcloned into the pCAGGS vector (38Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4597) Google Scholar), whereas for tetracycline-inducible expression, they were subcloned into the pUHG10-3 vector (39Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2040) Google Scholar). Sequences of all the constructs were verified using the ABI 310 or ABI 377 autosequencers (Perkin-Elmer). Identification of the transfected cDNAs was carried out by reverse transcription-polymerase chain reaction of cellular RNA following induction by doxycycline and primers from noncoding regions in the vectors used for transfection. Amplified cDNAs were diagnosed by specific restriction analysis. For purification of the His-tagged E2 enzymes, BL21(DE3) cells, transformed with the appropriate expression constructs, were cultured in 2× YT medium, and isopropyl-β-d-thiogalactopyranoside (400 μm) was added when A600 nm attained 0.6. The induced proteins were purified over Ni2+-nitrilotriacetic acid-agarose according to the manufacturer's instructions. For transient expression of the various E2 enzymes, 293 cells were transfected with the various E2 constructs using the DEAE-dextran method (40Guan J.L. Rose J.K. Cell. 1984; 37: 779-787Abstract Full Text PDF PubMed Scopus (47) Google Scholar). Transfection efficiency was always >75% as determined by parallel transfection with a gene encoding green fluorescent protein. Experiments were carried out 40–48 h following transfection. Stable transformants of HeLa Tet-on cells (CLONTECH) for inducible expression of mutant E2 enzymes were established by calcium phosphate transfection as described (41Iwai K. Klausner R.D. Rouault T.A. EMBO J. 1995; 14: 5350-5357Crossref PubMed Scopus (193) Google Scholar). Protein expression was induced by the addition of 1 μg/ml doxycycline for 48 h. Induction of the proteins was monitored by Western blot analysis using anti-His tag antibody. [35S]Methionine-labeled IκBα complexed to HeLa p50/p65 was generated as described (9Alkalay I. Yaron A. Hatzubai A. Orian A. Ciechanover A. Ben Neriah Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10599-10603Crossref PubMed Scopus (391) Google Scholar, 10Yaron A. Gonen H. Alkalay I. Hatzubai A. Jung S. Beyth S. Mercurio F. Manning A.M. Ciechanover A. Ben Neriah Y. EMBO J. 1997; 16: 6486-6494Crossref PubMed Scopus (202) Google Scholar). Briefly, cDNAs coding for wild-type or S32A,S36A mutant IκBα were translated/transcribed in vitro in wheat germ extract in the presence of [35S]methionine. For phosphorylation and incorporation into endogenous cellular NF-κB complex, the labeled protein was incubated in HeLa cell extract in the presence of okadaic acid. Following incubation, anti-p65 antibody was added, and the immune complex was immobilized on protein A-Sepharose beads. The washed immobilized complex was used as a substrate in the different conjugation assays. Conjugation of IκBα to ubiquitin was monitored essentially as described (9Alkalay I. Yaron A. Hatzubai A. Orian A. Ciechanover A. Ben Neriah Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10599-10603Crossref PubMed Scopus (391) Google Scholar, 10Yaron A. Gonen H. Alkalay I. Hatzubai A. Jung S. Beyth S. Mercurio F. Manning A.M. Ciechanover A. Ben Neriah Y. EMBO J. 1997; 16: 6486-6494Crossref PubMed Scopus (202) Google Scholar, 30Blumenfeld N. Gonen H. Mayer A. Smith C.E. Siegel N.R. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1994; 269: 9574-9581Abstract Full Text PDF PubMed Google Scholar, 31Abu Hatoum O. Gross-Mesilaty S. Breitschopf K. Hoffman A. Gonen H. Ciechanover A. Bengal E. Mol. Cell. Biol. 1998; 18: 5670-5677Crossref PubMed Google Scholar). Briefly, the reaction mixture contained (in final volume of 25 μl) 4 μl of packed and washed protein A-Sepharose beads containing the labeled IκBα protein (∼20,000 cpm), 40 mm Tris-HCl (pH 7.6), 5 mmMgCl2, 2 mm dithiothreitol, 5 μg of ubiquitin, and 0.5 μg of the isopeptidase inhibitor ubiquitin aldehyde (42Hershko A. Rose I.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1829-1833Crossref PubMed Scopus (157) Google Scholar). Crude HeLa cell extract (60 μg), Fraction II (45 μg of protein), Fraction I (15 μg; as a source of E2), and Fraction IIA (25 μg; as a source of E3); E1 (0.75 μg); E2-14K (0.75 μg); E2-C (0.5 μg); E2-8A (0.75 μg); UBCH7 (0.75 μg); and E2-25K (1.0 μg) were added as indicated. UBCH5a, UBCH5b, and UBCH5c were added at 0.75 μg or as indicated, whereas CDC34/UBC3 was added at 1.25 μg or as indicated. The different Cys-to-Ala mutant species of UBCH5a, UBCH5b, and UBCH5c and of CDC34/UBC3 were added as indicated. E2-20K was added at 0.8 μg or as indicated. The biological activity of the different E2 enzymes was monitored using formation of E2-S∼ubiquitin thiol ester in the presence of E1 as described (29Hershko A. Heller H. Elias S. Ciechanover A. J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar). The IκBα phosphopeptide and S32A,S36A peptide were added at 40 μm. In mixtures containing the peptides, bestatin was added at 20 μg/ml and was incubated for 15 min at room temperature with all the components of the reaction except for the peptides and the labeled substrate. Following addition of the peptides, the reaction mixture was further incubated for 5 min at 30 °C prior to the addition of the labeled substrate. All mixtures containing the peptides contained also 2 μm okadaic acid. When complete HeLa cell extract (25 μg) was used as a source of endogenous substrates, endogenous E1 and E2 and E3 enzymes were inactivated by N-ethylmaleimide (10 mm; 10 min at room temperature) followed by neutralization with dithiothreitol (6 mm; 1 min at room temperature). E1, the different E2 enzymes, Fraction IIA (as a source of E3 enzymes), and125I-labeled ubiquitin (0.1 μg, ∼100,000 cpm) were added as described above and in the figure legends. The complete reaction mixtures were incubated for 30 min at 37 °C in the presence of ATP (0.5 mm ATP and ATP-regenerating system) (30Blumenfeld N. Gonen H. Mayer A. Smith C.E. Siegel N.R. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1994; 269: 9574-9581Abstract Full Text PDF PubMed Google Scholar, 31Abu Hatoum O. Gross-Mesilaty S. Breitschopf K. Hoffman A. Gonen H. Ciechanover A. Bengal E. Mol. Cell. Biol. 1998; 18: 5670-5677Crossref PubMed Google Scholar). Reactions were terminated by the addition of 12.5 μl of 3-fold concentrated sample buffer and, following boiling, were resolved via SDS-PAGE (10%). Gels were dried, and [35S]methionine-labeled proteins were visualized using a PhosphorImager (Fuji, Japan). 125I-Labeled proteins were visualized following exposure to Kodak XAR-5 film. The fate of IκBα was monitored in cells that were stably or transiently transfected with the different species of E2 enzymes. Following incubation in the presence of TNF-α (10 ng/ml), cells were harvested at the indicated time points, lysed in sample buffer, and resolved via SDS-PAGE (10%). The resolved proteins were blotted onto nitrocellulose paper, and the inhibitor was visualized by Western blot analysis using a specific antibody, a secondary horseradish peroxidase-conjugated antibody, and ECL reaction as described (31Abu Hatoum O. Gross-Mesilaty S. Breitschopf K. Hoffman A. Gonen H. Ciechanover A. Bengal E. Mol. Cell. Biol. 1998; 18: 5670-5677Crossref PubMed Google Scholar). Degradation of bacterially expressed MyoD was followed in crude HeLa cell Fraction II by Western blot analysis as described (31Abu Hatoum O. Gross-Mesilaty S. Breitschopf K. Hoffman A. Gonen H. Ciechanover A. Bengal E. Mol. Cell. Biol. 1998; 18: 5670-5677Crossref PubMed Google Scholar). HeLa Tet-on cells stably transfected with C85A UBCH5b and C85A UBCH5c were labeled with [35S]methionine (100 μCi/ml) for 5 min (pulse). Following removal of the labeling amino acid, the cells were incubated for the indicated time periods in the presence of excess unlabeled methionine (chase), and degradation was monitored by measuring release of trichloroacetic acid-soluble radioactivity into the medium as described (43Gropper R. Brandt R.A. Elias S. Bearer C.F. Mayer A. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1991; 266: 3602-3610Abstract Full Text PDF PubMed Google Scholar). Cell extracts derived from uninduced or doxycycline-induced HeLa cells stably transfected with Cys-to-Ala mutant UBCH5b and UBCH5c or from 293 cells transiently transfected with Cys-to-Ala mutant CDC34/UBC3 were resolved via SDS-PAGE (10%). Resolved proteins were blotted onto nitrocellulose paper and probed with anti-ubiquitin antibody as described (31Abu Hatoum O. Gross-Mesilaty S. Breitschopf K. Hoffman A. Gonen H. Ciechanover A. Bengal E. Mol. Cell. Biol. 1998; 18: 5670-5677Crossref PubMed Google Scholar). Protein concentration was determined according to Bradford (44Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar) using bovine serum albumin as a standard. Ubiquitin was iodinated, using the chloramine-T method, as described (29Hershko A. Heller H. Elias S. Ciechanover A. J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar). To identify the ubiquitin carrier proteins involved in conjugation of pIκBα in the context of the heterotrimeric pIκBα·p50·p65 complex, a cell-free system was reconstituted. The system contained E1, Fraction IIA (as a source of E3), and different species of purified E2 enzymes. As shown in Fig. 1A, only UBCH5b and UBCH5c conjugated the inhibitor in a specific manner. The adducts are of high molecular mass, but more important, they are specific to the phosphorylated species of the inhibitor: they are not generated when the S32A,S36A mutant species of IκBα is used as a substrate. UBCH5a, UBCH7, and E2-8A also conjugated the inhibitor; however, the conjugates are mostly of the low molecular mass type and do not appear to be specific: S32A,S36A IκBα is also targeted. E2-20K, E2-25K, E2-C, and E2-14K did not conjugate the inhibitor at all. To further establish the specificity of these two E2 enzymes, we tested the effect of a specific phosphopeptide that spans the phosphorylation domain of IκBα. This peptide blocks specifically the conjugation reaction by interfering with the recognition by E3 (10Yaron A. Gonen H. Alkalay I. Hatzubai A. Jung S. Beyth S. Mercurio F. Manning A.M. Ciechanover A. Ben Neriah Y. EMBO J. 1997; 16: 6486-6494Crossref PubMed Scopus (202) Goog" @default.
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• W2100041351 title "Identification of the Ubiquitin Carrier Proteins, E2s, Involved in Signal-induced Conjugation and Subsequent Degradation of IκBα" @default.
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• W2100041351 cites W1484625070 @default.
• W2100041351 cites W1492571211 @default.
• W2100041351 cites W1513849811 @default.
• W2100041351 cites W1521408518 @default.
• W2100041351 cites W1528476113 @default.
• W2100041351 cites W153290133 @default.
• W2100041351 cites W1592932769 @default.
• W2100041351 cites W1768681792 @default.
• W2100041351 cites W1958512987 @default.
• W2100041351 cites W1978186903 @default.
• W2100041351 cites W1978611748 @default.
• W2100041351 cites W1981765089 @default.
• W2100041351 cites W1985476964 @default.
• W2100041351 cites W1989844987 @default.
• W2100041351 cites W1990638976 @default.
• W2100041351 cites W1991930087 @default.
• W2100041351 cites W2005275397 @default.
• W2100041351 cites W2008744347 @default.
• W2100041351 cites W2011401872 @default.
• W2100041351 cites W2018560829 @default.
• W2100041351 cites W2020547495 @default.
• W2100041351 cites W2027179660 @default.
• W2100041351 cites W2029276835 @default.
• W2100041351 cites W2037221825 @default.
• W2100041351 cites W2041156762 @default.
• W2100041351 cites W2045350410 @default.
• W2100041351 cites W2048162805 @default.
• W2100041351 cites W2049748848 @default.
• W2100041351 cites W2057254962 @default.
• W2100041351 cites W2067680656 @default.
• W2100041351 cites W2070853191 @default.
• W2100041351 cites W2073148985 @default.
• W2100041351 cites W2075739751 @default.
• W2100041351 cites W2089865526 @default.
• W2100041351 cites W2098598252 @default.
• W2100041351 cites W2124398251 @default.
• W2100041351 cites W2132570074 @default.
• W2100041351 cites W2137691728 @default.
• W2100041351 cites W2155678413 @default.
• W2100041351 cites W2158708535 @default.
• W2100041351 cites W2159979956 @default.
• W2100041351 cites W2166987747 @default.
• W2100041351 cites W2728081960 @default.
• W2100041351 cites W2762075333 @default.
• W2100041351 cites W4233535695 @default.
• W2100041351 cites W4293247451 @default.
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