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• W2079196169 abstract "The inactivation of the prototype NF-κB inhibitor, IκBα, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. We identify valosin-containing protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, that co-precipitates with IκBα immune complexes. The ubiquitinated IκBα conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-κB. In ultracentrifugation analysis, physically associated VCP and ubiquitinated IκBα complexes sediment in the 19 S fractions, while the unmodified IκBα sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of IκBα are critical for VCP binding, which in turn is necessary but not sufficient for IκBα degradation; while the N-terminal domain of IκBα is required in all three reactions, both N- and C-terminal domains are required in degradation. Further, VCP co-purifies with the 26 S proteasome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. Our results suggest that VCP may provide a physical and functional link between IκBα and the 26 S proteasome and play an important role in the proteasome-mediated degradation of IκBα. The inactivation of the prototype NF-κB inhibitor, IκBα, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. We identify valosin-containing protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, that co-precipitates with IκBα immune complexes. The ubiquitinated IκBα conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-κB. In ultracentrifugation analysis, physically associated VCP and ubiquitinated IκBα complexes sediment in the 19 S fractions, while the unmodified IκBα sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of IκBα are critical for VCP binding, which in turn is necessary but not sufficient for IκBα degradation; while the N-terminal domain of IκBα is required in all three reactions, both N- and C-terminal domains are required in degradation. Further, VCP co-purifies with the 26 S proteasome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. Our results suggest that VCP may provide a physical and functional link between IκBα and the 26 S proteasome and play an important role in the proteasome-mediated degradation of IκBα. Transcription factor NF-κB is involved in a large variety of processes, such as cell growth, transcriptional regulation, immune, inflammatory, and stress responses (reviewed in Refs. 1Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1217) Google Scholar, 2Verma I.M. Stevenson J.K. Schwarz E.M. Antwerp D.V. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1665) Google Scholar, 3Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2935) Google Scholar, 4Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5592) Google Scholar). NF-κB is a homo- or heterodimer consisting of various combinations of the family members, including NFκB1 (p50 and precursor p105), c-Rel, RelA, NFκB2 (p52 and precursor p100), RelB, and Drosophilaproteins Dorsal and Dif. Unlike many other transcription factors that are localized in the nucleus, the NF-κB dimeric factor is sequestered in the cytoplasm of most cells through binding to a family of inhibitor proteins, IκB. In response to extracellular stimuli, the inhibitors are partially or entirely degraded, thus liberating the DNA-binding dimer for translocation to the nucleus. The I6B family contains IκBα, IκBβ, IκBγ, Bcl-3, the precursor proteins p105 and p100, and the Drosophila protein Cactus. All members of the IκB family contain 5–8 ankyrin motifs, thought to be involved in protein-protein interactions. It has been shown that when the precursor protein p105 is involved as the inhibitor, the processing from p105 to the active p50 occurs through the ubiquitin-dependent proteasome (Ub-Pr) 1The abbreviations used are: Ub-Pr, ubiquitin-dependent proteasome; Ub, ubiquitin; Ub-Iκβα, ubiquitinated IκBα; IP, immunoprecipitation; RIPA, radioimmune precipitation; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; ATPγS, adenosine 5′-O-(thiotriphosphate); VCP, valosin-containing protein. pathway, which degrades the C-terminal ankyrin-containing domain of p105 (5Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1922) Google Scholar,6Orian A. Whiteside S. Israel A. Stancovski I. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1995; 270: 21707-21714Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). 2C.-C. H. Li, unpublished observations. For the prototype complex that contains p50, p65, and IκBα, upon stimulation the entire NF-κB complex becomes hyperphosphorylated. The induced phosphorylation of IκBα does not lead to its immediate dissociation from the complex; rather, it signals for rapid IκBα degradation, thus liberating the active p50·p65 dimer for translocation to the nucleus (7Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1317) Google Scholar, 8Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1172) Google Scholar, 9Li C.-C.H. Dai R.-M. Longo D.L. Biochem. Biophys. Res. Commun. 1995; 215: 292-301Crossref PubMed Scopus (56) Google Scholar, 10Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar, 11Traenckner E.B.-M. Heike L.P. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (934) Google Scholar, 12Scherer D.C. Brockman J.A. Chen Z. Maniatis T. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11259-11263Crossref PubMed Scopus (502) Google Scholar, 13Baldi L. Brown K. Franzoso G. Siebenlist U. J. Biol. Chem. 1996; 271: 376-379Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 14Roff M. Thompson J. Rodriguez M.S. Jacque J.-M. Baleux F. Arenzana-Seisdedos F. Hay R.T. J. Biol. Chem. 1996; 271: 7844-7850Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 15DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar). We and others showed that the degradation of IκBα is sensitive to proteasome inhibitors and is ubiquitin-dependent. Recently, it was further shown that signal-induced phosphorylation precedes IκBα degradation and targets IκBα to the Ub-Pr pathway (7Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1317) Google Scholar, 8Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1172) Google Scholar, 9Li C.-C.H. Dai R.-M. Longo D.L. Biochem. Biophys. Res. Commun. 1995; 215: 292-301Crossref PubMed Scopus (56) Google Scholar, 10Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar, 11Traenckner E.B.-M. Heike L.P. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (934) Google Scholar, 12Scherer D.C. Brockman J.A. Chen Z. Maniatis T. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11259-11263Crossref PubMed Scopus (502) Google Scholar, 13Baldi L. Brown K. Franzoso G. Siebenlist U. J. Biol. Chem. 1996; 271: 376-379Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 14Roff M. Thompson J. Rodriguez M.S. Jacque J.-M. Baleux F. Arenzana-Seisdedos F. Hay R.T. J. Biol. Chem. 1996; 271: 7844-7850Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 15DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar). It was proposed that the inactivation of IκBα occurs through a series of ordered processes including phosphorylation, ATP-dependent multiple ubiquitin conjugation, and proteasome-mediated proteolysis. However, the link between ubiquitination and proteasome-mediated degradation remains unclear. The extralysosomal, energy-dependent Ub-Pr pathway is a major mechanism used to regulate many critical cellular proteins that must be rapidly destroyed for normal growth and metabolism (reviewed in Refs. 16Goldberg A.L. Science. 1995; 268: 522-523Crossref PubMed Scopus (294) Google Scholar, 17Jentsch S. Schlenker S. Cell. 1995; 82: 881-884Abstract Full Text PDF PubMed Scopus (236) Google Scholar, 18Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-223Crossref PubMed Scopus (785) Google Scholar, 19Hochstrasser M. Cell. 1996; 84: 813-815Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 20Hilt W. Wolf D.H. Trends Biol. Sci. 1996; 21: 96-102Abstract Full Text PDF PubMed Scopus (363) Google Scholar, 21Coux L. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2239) Google Scholar). The rapidly growing list of the substrates for the Ub-Pr pathway includes mitotic cyclins, G1 cyclins, cyclin-dependent kinase inhibitors p27, Sic1 protein, proto-oncogene products p53, c-Myc, c-Jun, and c-Mos, NF-κB inhibitors, yeast MATα2 transcription factor, major histocompatibility complex molecules, and others. The Ub-Pr proteolytic pathway is ATP-dependent and present in both cytoplasm and nucleus. The pathway consists of two distinct, sequential steps. The target protein is first conjugated with multiple ubiquitin (Ub) molecules that mark the substrate for destruction. The Ub-tagged target is then translocated to (probably with the help of molecular chaperones) and degraded by a large protease complex with an apparent sedimentation coefficient of 26 S. The 26 S proteasome is a multisubunit complex, consisting of a central cylinder-shaped 20 S multicatalytic proteinase core and a 19 S cap-like regulatory complex attached to each end of the cylinder. The terminal cap structure consists of at least 18 distinct subunits with molecular masses of 35–110 kDa and has ATPase and ubiquitin conjugate binding activities. It is presumed that ATP hydrolysis by the 19 S complex provides the energy needed for the chaperoning and unfolding of substrates degraded within the proteasome cylinder. VCP (22Koller K.J. Brownstein M.J. Nature. 1987; 325: 542-545Crossref PubMed Scopus (100) Google Scholar, 23Egerton M. Ashe O.R. Chen D. Druker B.J. Burgess W.H. Samelson L.E. EMBO J. 1992; 11: 3533-3540Crossref PubMed Scopus (122) Google Scholar, 24Egerton M. Samelson L.E. J. Biol. Chem. 1994; 269: 11435-11441Abstract Full Text PDF PubMed Google Scholar), the mammalian homolog of the cell division cycle protein Cdc48p in yeast and p97 in Xenopus, is a member of a recently identified AAA family (reviewed in Ref. 25Confalonieri F. Duguet M. BioEssays. 1995; 17: 639-650Crossref PubMed Scopus (314) Google Scholar). The family members are characterized by having ATPase domain(s) with striking sequence similarities and having ring structures consisting of homooligomers. Despite the high sequence and structural homology, these proteins unexpectedly are implicated in a large variety of seemingly unrelated biological functions. These functions reviewed in Ref. 25Confalonieri F. Duguet M. BioEssays. 1995; 17: 639-650Crossref PubMed Scopus (314) Google Scholarinclude control of cell division cycle, T cell activation (23Egerton M. Ashe O.R. Chen D. Druker B.J. Burgess W.H. Samelson L.E. EMBO J. 1992; 11: 3533-3540Crossref PubMed Scopus (122) Google Scholar, 24Egerton M. Samelson L.E. J. Biol. Chem. 1994; 269: 11435-11441Abstract Full Text PDF PubMed Google Scholar), membrane fusion (26Zhang L. Ashendel C.L. Becker G.W. Morre J. J. Cell Biol. 1994; 127: 1871-1883Crossref PubMed Scopus (73) Google Scholar, 27Latterich M. Frohlich K.-U. Schekman R. Cell. 1995; 82: 885-893Abstract Full Text PDF PubMed Scopus (336) Google Scholar, 28Acharya U. Jacobs J.-M. Peters R. Watson N. Farquhar M.G. Malhotra V. Cell. 1995; 82: 895-904Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 29Rabouille C. Levine T.P. Peters J.-M. Warren G. Cell. 1995; 82: 905-914Abstract Full Text PDF PubMed Scopus (312) Google Scholar), and vesicle-mediated transport, peroxisome assembly and biogenesis, gene expressions, and the Ub-Pr degradation pathways (30Ghislain M. Dohmen R.J. Levy F. Varshavsky A. EMBO J. 1996; 15: 4884-4899Crossref PubMed Scopus (236) Google Scholar). Furthermore, at least six family members have been identified as subunits of the 26 S proteasome (subunits 4, 6, 7, 8, 10, and TBP-1) (31Dubiel W. Ferrell K. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22699-22702Abstract Full Text PDF PubMed Google Scholar, 32Dubiel W. Ferrell K. Rechsteiner M. FEBS Lett. 1993; 323: 276-278Crossref PubMed Scopus (77) Google Scholar, 33Dubiel W. Ferrell K. Rechsteiner M. Biol. Chem. Hoppe-Seyler. 1994; 375: 237-240Crossref PubMed Scopus (37) Google Scholar, 34Akiyama K. Yokota K. Kagawa S. Shimbara N. DeMartino G.N. Slaughter C.A. Noda C. Tanaka K. FEBS Lett. 1995; 363: 151-156Crossref PubMed Scopus (59) Google Scholar, 35Rubin D.M. Coux O. Wefes I. Hengartner C. Young R.A. Goldberg A. Finley D. Nature. 1996; 379: 655-657Crossref PubMed Scopus (143) Google Scholar, 36DeMartino G.N. Proske R.J. Moomaw C.R. Strong A.A. Song X. Hisamatsu H. Tanaka K. Slaughter C.A. J. Biol. Chem. 1996; 271: 3112-3118Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), and it is likely that other family proteins will fill this function as well (31Dubiel W. Ferrell K. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22699-22702Abstract Full Text PDF PubMed Google Scholar, 37Rechsteiner M. Hoffman L. Dubiel W. J. Biol. Chem. 1993; 268: 6065-6068Abstract Full Text PDF PubMed Google Scholar). The apparent paradox between the striking sequence homology and the large diversity of functions in this family suggests that these proteins have a basic and critical role in cellular function, and the involvement in many other functions may be indirect. During the course of studying the molecular mechanisms involved in IκBα degradation, we detected VCP physically associated with IκBα complexes both in vivo and in vitro. In this report, we demonstrate that VCP is involved in the proteasome-mediated degradation of IκBα, and VCP is co-purified with the 26 S proteasome. Consistent with this hypothesis, VCP indeed has in vitro ATPase activity and an apparent sedimentation coefficient of 19 S (Ref. 24Egerton M. Samelson L.E. J. Biol. Chem. 1994; 269: 11435-11441Abstract Full Text PDF PubMed Google Scholar and this study), the same as that of the regulatory complex of the 26 S proteasome. We propose that physical association of VCP with Ub-tagged IκBα targets IκBα to the Ub-Pr pathway. Two interchangeably used human B cell lines, DB (38Beckwith M. Longo D.L. O'Connell C.D. Moratz C.M. Urba W.J. J. Natl. Cancer Inst. 1990; 82: 501-509Crossref PubMed Scopus (71) Google Scholar) and CA46 (39O'Connor P.M. Jackman J. Jondle D. Bhatia K. Magrath I. Kohn K.W. Cancer Res. 1993; 53: 4776-4780PubMed Google Scholar), were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. Both analyses were performed as described previously (9Li C.-C.H. Dai R.-M. Longo D.L. Biochem. Biophys. Res. Commun. 1995; 215: 292-301Crossref PubMed Scopus (56) Google Scholar,40Li C.-C.H. Korner M. Ferris D.K. Chen E. Dai R.-M. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar, 41Li C.-C.H. Dai R.-M. Chen E. Longo D.L. J. Biol. Chem. 1994; 269: 30089-30092Abstract Full Text PDF PubMed Google Scholar) with minor modifications. Cells were labeled with 0.1 mCi/ml (1000 Ci/mmol) [35S]methionine/cysteine (ICN) at a density of 5 × 106/ml for approximately 16 h, washed twice with phosphate-buffered saline, and lysed in RIPA buffer (20 mm Tris/HCl, pH 7.6, 2 mm EDTA, 150 mm NaCl, 1% deoxycholate, 1% Triton X-100) containing protease inhibitors (1% aprotinin, 70 μg/ml phenylmethanesulfonyl fluoride, 40 μg/ml Tos-Phe-CH2Cl, 5 μg/ml Tos-Lys-CH2Cl, 5 μg/ml leupeptin). The lysates were clarified by centrifugation at 12,000 × g for 30 min and incubated with antisera. The immune complexes were collected with Protein A-Sepharose beads, washed with RIPA buffer, boiled, resolved by SDS-polyacrylamide gel electrophoresis (PAGE), electrophoretically transferred onto polyvinylpyrrolidone membranes, and analyzed by autoradiography. IPs shown in Figs. 1 A and 2 Bwere conducted in Buffer I (1% Nonidet P-40, 1% bovine serum albumin in phosphate-buffered saline) containing protease inhibitors, 100 μm calpain inhibitor I, and 10 mmiodoacetamide, and the precipitates were washed with Buffer I without bovine serum albumin. For reimmunoprecipitation experiments, the immune complexes isolated from the first IP were boiled in RIPA containing 1% SDS, diluted, and subjected to the second IP. For immunoblot analysis, equal amounts of protein or immune complexes were resolved by SDS-PAGE and transferred onto membranes. The membrane was blocked, washed, and incubated with antiserum (typically at 1:3,000), followed by reaction with peroxidase-conjugated anti-rabbit immunoglobulin serum (Boehringer Mannheim), and developed by the enhanced chemiluminescence detection system (ECL) (Amersham Corp.). When serial blotting analyses were performed, previous reactivity was stripped off from the membrane.Figure 2Generation of VCP antisera and co-immunoprecipitation of high M r Ub-IκBα in VCP immune complexes. A, DB cells were metabolically labeled with [35S]methionine/cysteine and lysed in RIPA buffer containing 0.1% SDS. The cell lysates were immunoprecipitated in the same buffer with the specified preimmune sera (P) or the corresponding anti-VCP immune sera (I). Immune complexes were separated by SDS-PAGE, transferred to membrane, and visualized by autoradiography. B, 35S-labeled DB cell lysates were subjected to single IPs (lanes 1–4) or sequential double IPs (lanes 5–13) with the specified antisera. Three times as much lysates were used in each of lanes 5–13 as inlanes 1–4. After SDS-PAGE, Western transfer, and autoradiography (top; lanes 1–4 are from a 2-day exposure, and lanes 5–13 are from a 20-day exposure), the membrane was immunoblotted (IB) with anti-VCP-3 (bottom).View Large Image Figure ViewerDownload (PPT) All antisera used in this study are polyclonal rabbit sera. Anti-IκBα-1 and anti-IκBα-2 (40Li C.-C.H. Korner M. Ferris D.K. Chen E. Dai R.-M. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar, 9Li C.-C.H. Dai R.-M. Longo D.L. Biochem. Biophys. Res. Commun. 1995; 215: 292-301Crossref PubMed Scopus (56) Google Scholar) were independently generated against synthetic peptide corresponding to residues 300–317 of the human IκBα sequence (42Haskill S. Beg A.A. Tompkins S.M. Morris J.S. Yurochko A.D. Sampson-Johannes A. Mondal K. Ralph P. Baldwin Jr., A.S. Cell. 1991; 65: 1281-1289Abstract Full Text PDF PubMed Scopus (586) Google Scholar). Anti-IκBα-3 (SC-847) and -5 (SC-371) were purchased from Santa Cruz Biotechnology, Inc., and anti-IκBα-4 (06–494) was purchased from Upstate Biotechnology, Inc. Anti-VCP-1 and anti-VCP-2, raised against peptides of residues 792–806 and 20–40 of murine VCP (23Egerton M. Ashe O.R. Chen D. Druker B.J. Burgess W.H. Samelson L.E. EMBO J. 1992; 11: 3533-3540Crossref PubMed Scopus (122) Google Scholar), respectively, were kindly provided by L. Samelson (NIH). Anti-VCP-3, -4, -5, -6, -7, and -8 were generated against glutathione S-transferase (GST)-VCP (full-length) and residues 792–806, 721–734, 184–197, 240–253, and 167–180 of murine VCP (23Egerton M. Ashe O.R. Chen D. Druker B.J. Burgess W.H. Samelson L.E. EMBO J. 1992; 11: 3533-3540Crossref PubMed Scopus (122) Google Scholar), respectively. Anti-Ub polyclonal antisera were purchased from either Sigma or Biogenesis or obtained from M. Rechsteiner (University of Utah, Salt Lake City, Utah) (43Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar). Antisera to subunits 4 and 5 and to the 26 S proteasome were kindly provided by M. Rechsteiner (43Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar, 44Deveraux Q. Jensen C. Rechsteiner M. J. Biol. Chem. 1995; 270: 23726-23729Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Well resolved protein bands were sliced from Coomassie Blue-stained gel and subjected to an in-gel partial V8 digestion (45Cleveland D.W. Fischer S.G. Kirschner M.W. Laemmli U.K. J. Biol. Chem. 1977; 252: 1102-1106Abstract Full Text PDF PubMed Google Scholar). The proteolytic fragments were resolved by 15% SDS-PAGE, transferred onto a polyvinylpyrrolidone membrane, and stained with Coomassie Blue. The well separated bands were sliced and subjected to N-terminal peptide sequencing using an ABI 494A sequencer. IκBα (7Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1317) Google Scholar) or VCP (23Egerton M. Ashe O.R. Chen D. Druker B.J. Burgess W.H. Samelson L.E. EMBO J. 1992; 11: 3533-3540Crossref PubMed Scopus (122) Google Scholar) was synthesized in a coupled in vitro transcription and translation reaction from a rabbit reticulocyte lysate system (Promega) in the presence of [35S]cysteine (for IκBα) or [35S]methionine (for VCP). GST-IκBα expression plasmid was constructed by inserting the EcoRI fragment of IκBα (7Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1317) Google Scholar) into pGEX-4T-2 vector (Pharmacia Biotech Inc.) (9Li C.-C.H. Dai R.-M. Longo D.L. Biochem. Biophys. Res. Commun. 1995; 215: 292-301Crossref PubMed Scopus (56) Google Scholar). The GST-VCP (24Egerton M. Samelson L.E. J. Biol. Chem. 1994; 269: 11435-11441Abstract Full Text PDF PubMed Google Scholar) or GST-IκBα fusion proteins were prepared according to the manufacturer (Pharmacia). Glutathione-Sepharose beads containing GST or GST fusion proteins were mixed with 35S-labeledin vitro translated proteins or unlabeled B cell lysates in a total volume of 225 μl of binding buffer (35 mm Tris, pH 7.6, 50 mm NaCl, 0.1% Nonidet P-40, 0.5 mmdithiothreitol including protease inhibitors). After incubation for 2 h at 4 °C and three washes with binding buffer, the bound products were analyzed by SDS-PAGE followed by Western transfer, autoradiography, and immunoblotting. Velocity sedimentation centrifugation was carried out in 10–40% glycerol gradients in a total volume of 13 ml of 12 mm Tris-HCl, pH 7.5, 50 mm NaCl, 2 mm CaCl2, 1 mm MgCl2. Freshly prepared CA46 cell lysate in a volume of 200 μl was loaded onto the gradient and centrifuged at 4 °C, in an SW41 rotor at 36,000 rpm for 16 h. Fractions of 0.5 ml were collected and analyzed by immunoblotting or IP. Protein markers were centrifuged in separate tubes and included thyroglobulin (19 S), immunoglobulin G (7 S), and bovine serum globulin (4.5 S). Cytosolic fractions were prepared by lysing cells in a hypotonic buffer (5 mm Hepes, pH 8.0, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol) on ice for 10 min. The lysates were clarified by low speed centrifugation for 10 min. The supernatant was further centrifuged at 100,000 × g for 1–2 h, and the supernatant, S100, was stored in aliquots at −70 °C. Proteasome-enriched (Pr+), and proteasome-depleted (Pr−) fractions were extracted from CA46 cells as described by Palombella et al. (5Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1922) Google Scholar). The highly purified (26 S-1) and the partially purified (26 S-2) proteasomes were isolated from human red blood cells as described (46Hoffman L. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22362-22368Abstract Full Text PDF PubMed Google Scholar). Deletion mutants of IκBα encoding amino acids 37–317, and 1–242 were constructed as described by Brockman et al. (10Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar). The deletion mutant encoding amino acids 23–317 was constructed by polymerase chain reaction using specific oligonucleotide primers (5′-GGGAAACTTCTCGTCCGCGCCATGCGGCTACTGGACGACCGC-3′ and 5′-GGTCTAGATCATAACGTCAGACGCTGGCCT-3′) and the wild-type IκBα cDNA (7Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1317) Google Scholar) as a template. Amplified product was purified and cloned into pCRII vector (TA cloning kit, Invitrogen). The site-specific mutants, S32A/S36A and K21R/K22R, were kindly provided by D. Ballard (12Scherer D.C. Brockman J.A. Chen Z. Maniatis T. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11259-11263Crossref PubMed Scopus (502) Google Scholar). The wild-type and mutants of IκBα were synthesized in reticulocyte lysate-based in vitrotranscription/translation system (Promega) in the presence of [35S]cysteine and used as the substrates in the Ub-Pr degradation assay (9Li C.-C.H. Dai R.-M. Longo D.L. Biochem. Biophys. Res. Commun. 1995; 215: 292-301Crossref PubMed Scopus (56) Google Scholar). S100 was extracted from CA46 cells (8Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1172) Google Scholar) and used as enzyme source. Master reaction mixture containing 5 μl of substrate, 8 μg of dialyzed ubiquitin (Sigma), 12 mmTris-HCl, pH 7.5, 60 mm KCl, 3.5 mmMgCl2, 5 mm CaCl2, 1 mmdithiothreitol, and 1 mm ATP was prepared and aliquoted into four tubes on ice. At various time points, 50 μg of S100 was added to individual tubes to start the reaction at 37 °C. The final volume in each reaction was adjusted to 50 μl. All of the reactions were simultaneously terminated by boiling and analyzed by SDS-PAGE followed by Western transfer and autoradiography. The ubiquitinated IκBα (Ub-IκBα) conjugates were generated by slightly modified Ub-conjugation assays (6Orian A. Whiteside S. Israel A. Stancovski I. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1995; 270: 21707-21714Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 8Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1172) Google Scholar), which were essentially the same as the degradation assay except that 1 μg/ml okadaic acid, 1 mmATPγS, and 100 μm calpain inhibitor I were included in the reaction, and the reactions were carried out for 90 min. Two-dimensional gel electrophoresis analysis was carried out as described previously (44Deveraux Q. Jensen C. Rechsteiner M. J. Biol. Chem. 1995; 270: 23726-23729Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The highly purified 26 S proteasome isolated from human red blood cells was resolved by two-dimensional isoelectric focusing followed by SDS-gel electrophoresis. IP performed on cell lysates containing active NF-κB detected IκBα, p50, p65, and an unidentified 90-kDa cellular protein (p90) (Refs. 9Li C.-C.H. Dai R.-M. Longo D.L. Biochem. Biophys. Res. Commun. 1995; 215: 292-301Crossref PubMed Scopus (56) Google Scholar and 40Li C.-C.H. Korner M. Ferris D.K. Chen E. Dai R.-M. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar; Fig. 1 A, top, lanes 1–4). p90 was detected in complexes precipitated by IκBα antisera from various sources and was not detected by the preimmune serum (Ref. 9Li C.-C.H. Dai R.-M. Longo D.L. Biochem. Biophys. Res. Commun. 1995; 215: 292-301Crossref PubMed Scopus (56) Google Scholar; Fig. 1 C, top, lanes 1 and 6) or the anti-IκBα immune serum preincubated with the immunogenic peptide (40Li C.-C.H. Korner M. Ferris D.K. Chen E. Dai R.-M. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar). When the p90-containing IκBα complex was subjected to the in vitro Ub-Pr degradation assay, unlike IκBα molecules, p90 was resistant to degradation (9Li C.-C.H. Dai R.-M. Longo D.L. Biochem. Biophys. Res. Commun. 1995; 215: 292-301Crossref PubMed Scopus (56) Google Scholar). To identify p90, IκBα immune complexes were resolved on SDS-polyacrylamide gel, and the p90 band was visualized by Coomassie Blue staining, excised, and subjected to N-terminal peptide sequencing. Although the N terminus of p90 was blocked, peptide sequencing of two of the V8 proteolytic fragments of p90 revealed a total of 20 residues identical to those of the murine valosin-containing protein, VCP (23Egerton M. Ashe O.R. Chen D. Druker B.J. Burgess W.H. Samelson L.E. EMBO J. 1992; 11: 3533-3540Crossref PubMed Scopus (122) Google Scholar) (Fig. 1 B). IP followed by immunoblotting with either C terminus-specific anti-VCP-1 or N terminus-specific anti-VCP-2, kindly provided by L. Samelson (National Institutes of Health, Bethesda, MD), confirmed that the p90 was VCP (Fig. 1 A, bottom). VCP was co-precipitated with IκBα in nondenatured lysate (Fig. 1 C, lanes 2), but not in boiled lysate (lanes 7), indicating that VCP physically associates with an IκBα-containing complex. When the recipro" @default.
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