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• W2020547495 abstract "Nuclear factor κB1 (NF-κB) is a heterodimeric complex that regulates transcription of many genes involved in immune and inflammatory responses. Its 50-kDa subunit (p50) is generated by the ubiquitin-proteasome pathway from a 105-kDa precursor (p105). We have reconstituted this proteolytic process in HeLa cell extracts and purified the responsible enzymes. Ubiquitination of p105 requires E1, and either of two types of E2s, E2–25K (for which p105 is the first proven substrate) or a member of the UBCH5 (UBC4) family. It also requires a new E3 of 50 kDa, which we call E3κB. This set of enzymes differs from the E2s and E3 reported by others to catalyze p105 ubiquitination in reticulocytes. The ubiquitinating enzymes purified here, together with 26S proteasomes, allowed formation of p50. Thus, the 26S proteasome provides all the proteolytic activities necessary for p105 processing. Interestingly, in the reconstituted system, as observed in cells, the C-terminally truncated form of p105, p97, was processed into p50 more efficiently than normal p105, even when both species were ubiquitinated to a similar extent. Therefore, some additional mechanism involving the C-terminal region of p105 influences the proteolytic processing of the ubiquitinated precursor. Nuclear factor κB1 (NF-κB) is a heterodimeric complex that regulates transcription of many genes involved in immune and inflammatory responses. Its 50-kDa subunit (p50) is generated by the ubiquitin-proteasome pathway from a 105-kDa precursor (p105). We have reconstituted this proteolytic process in HeLa cell extracts and purified the responsible enzymes. Ubiquitination of p105 requires E1, and either of two types of E2s, E2–25K (for which p105 is the first proven substrate) or a member of the UBCH5 (UBC4) family. It also requires a new E3 of 50 kDa, which we call E3κB. This set of enzymes differs from the E2s and E3 reported by others to catalyze p105 ubiquitination in reticulocytes. The ubiquitinating enzymes purified here, together with 26S proteasomes, allowed formation of p50. Thus, the 26S proteasome provides all the proteolytic activities necessary for p105 processing. Interestingly, in the reconstituted system, as observed in cells, the C-terminally truncated form of p105, p97, was processed into p50 more efficiently than normal p105, even when both species were ubiquitinated to a similar extent. Therefore, some additional mechanism involving the C-terminal region of p105 influences the proteolytic processing of the ubiquitinated precursor. NF-κB 1The abbreviations used are: NF-κB, nuclear factor κB1; Ub, ubiquitin; UBC, Ub carrier protein; DTT, dithiothreitol; FI, fraction I; FII, fraction II; PAGE, polyacrylamide gel electrophoresis; WGE, wheat germ extract; GST, glutathioneS-transferase: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase. is a ubiquitous transcription factor that regulates the expression of multiple genes involved in immune and inflammatory responses (1Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2935) Google Scholar). Greater knowledge about the mechanisms of NF-κB activation is therefore of major importance for understanding human disease and may indicate new targets for pharmacological intervention. NF-κB is a member of the Rel family of dimeric transcription factors present in many organisms (2Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4602) Google Scholar). The prototype of this family is a heterodimer of a p50 (NF-κB1) and a p65 (RelA) subunit. Its activity is regulated primarily at the posttranslational level, by two separate processes (3Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1217) Google Scholar). The p50 subunit is generated from a relatively stable precursor, p105, which undergoes proteolytic processing in the cytoplasm (4Blank V. Kourilsky P. Israel A. EMBO J. 1991; 10: 4159-4167Crossref PubMed Scopus (127) Google Scholar, 5Fan C.M. Maniatis T. Nature. 1991; 354: 395-398Crossref PubMed Scopus (239) Google Scholar, 6Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1922) Google Scholar). In this process, the C-terminal part of p105 is degraded, and the remaining N-terminal half of the molecule serves as the p50 subunit of NF-κB. However, in uninduced cells, the p50/p65 (NF-κB) complex is maintained in an inactive form in the cytoplasm by the inhibitor IκBα, which associates with p50/p65 and prevents its migration to the nucleus (7Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Crossref PubMed Scopus (1689) Google Scholar, 8Beg A.A. Ruben S.M. Scheinman R.I. Haskill S. Rosen C.A. Baldwin Jr., A.S. Genes Dev. 1992; 6: 1899-1913Crossref PubMed Scopus (614) Google Scholar, 9Henkel T. Zabel U. van Zee K. Muller J.M. Fanning E. Baeuerle P.A. Cell. 1992; 68: 1121-1133Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 10Ganchi P.A. Sun S.C. Greene W.C. Ballard D.W. Mol. Biol. Cell. 1992; 3: 1339-1352Crossref PubMed Scopus (204) Google Scholar, 11Zabel U. Henkel T. Silva M.S. Baeuerle P.A. EMBO J. 1993; 12: 201-211Crossref PubMed Scopus (268) Google Scholar). The final activation of NF-κB involves the proteolytic destruction of IκBα (12Henkel T. Machleidt T. Alkalay I. Kronke M. Ben-Neriah Y. Baeuerle P.A. Nature. 1993; 365: 182-185Crossref PubMed Scopus (1039) Google Scholar), which is triggered by its phosphorylation following a variety of stimuli (13Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1317) Google Scholar, 14Chen Z.J. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1172) Google Scholar). The kinases p90rsk (15Ghoda L. Lin X. Greene W.C. J. Biol. Chem. 1997; 272: 21281-21288Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 16Schouten G.J. Vertegaal A.C.O. Whiteside S.T. Israel A. Toebes M. Dorsman J.C. Vandereb A.J. Zantema A. EMBO J. 1997; 16: 3133-3144Crossref PubMed Scopus (207) Google Scholar) and CHUK (17Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z.D. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 18DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1917) Google Scholar, 19Israel A. Nature. 1997; 388: 519-521Crossref PubMed Scopus (30) Google Scholar) have very recently been shown to be involved in this process. Previous data have shown that the processing of the p105 precursor and the degradation of IκBα both require ubiquitin (Ub) conjugation to these polypeptides, leading to their proteolytic digestion by the 26S proteasome (6Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1922) Google Scholar, 14Chen Z.J. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1172) Google Scholar, 20Traenckner E.B.M. Wilk S. Baeuerle P.A. EMBO J. 1994; 13: 5433-5441Crossref PubMed Scopus (656) Google Scholar,21Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar). The Ub-proteasome system is a major pathway for degradation of intracellular proteins in eukaryotic cells (22Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2239) Google Scholar, 23Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1461) Google Scholar). In this pathway, substrates are marked for degradation by covalent attachment of poly-Ub chain(s) (24Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1210) Google Scholar). In this process, the Ub-activating protein, E1, utilizes ATP to form a high energy Ub-thiol ester and then transfers the activated Ub to an E2 (Ub carrier protein (UBC)), forming an E2-Ub thiol ester. The Ub is then linked to the substrate in a reaction requiring E3, a Ub-protein ligase (24Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1210) Google Scholar, 25Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (756) Google Scholar). Cells contain a large number of E2s (23Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1461) Google Scholar), each of which acts on a limited spectrum of protein substrates (26King R.W. Peters J.M. Tugendreich S. Rolfe M. Hieter P. Kirschner M.W. Cell. 1995; 81: 279-288Abstract Full Text PDF PubMed Scopus (831) Google Scholar, 27Aristarkhov A. Eytan E. Moghe A. Admon A. Hershko A. Ruderman J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4294-4299Crossref PubMed Scopus (121) Google Scholar). The E3s seem to provide most of the substrate specificity of the ubiquitination process, although only a limited number have been identified. Once ubiquitinated, proteins are usually rapidly degraded to small peptides by the 26S proteasome (22Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2239) Google Scholar). The proteolytic core of this 2000-kDa complex is the 20S proteasome (28Löwe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1381) Google Scholar), which is sandwiched at each end by the 19S complex (PA700) (29Peters J.M. Franke W.W. Kleinschmidt J.A. J. Biol. Chem. 1994; 269: 7709-7718Abstract Full Text PDF PubMed Google Scholar, 30Chu-Ping M. Vu J.H. Proske R.J. Slaughter C.A. Demartino G.N. J. Biol. Chem. 1994; 269: 3539-3547Abstract Full Text PDF PubMed Google Scholar). The 19S complex contains multiple activities, including an isopeptidase that catalyzes the release of free Ub (31Eytan E. Armon T. Heller H. Beck S. Hershko A. J. Biol. Chem. 1993; 268: 4668-4674Abstract Full Text PDF PubMed Google Scholar, 32Kam Y.A. Xu W. Demartino G.N. Cohen R.E. Nature. 1997; 385: 737-740Crossref PubMed Scopus (370) Google Scholar) and several ATPases, the likely function of which is to facilitate the unfolding of substrates and their translocation into the 20S proteasome (33Rubin D.M. Finley D. Curr. Biol. 1995; 5: 854-858Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), where degradation proceeds in a processive fashion (34Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). The finding that the Ub-proteasome pathway is responsible for the limited processing of p105 was quite surprising, because its other known substrates undergo complete degradation. To dissect the mechanisms of this process and to define its components, we undertook to identify the enzymes necessary for p105 ubiquitination and p50 generation in HeLa cell extracts and to reconstitute this process with purified proteins. All chemicals were of analytical grade. DE52 was obtained from Whatman; the Bio-Scale CHT20-I column was from Bio-Rad; the MonoQ, Superose 6, Superose 12, and Sephacryl S100 HR HiPrep 16/60 columns and [35S]methionine (SJ1515, >37 TBq/mmol) were from Amersham Pharmacia Biotech, and iodine-125 (NEZ-033A, 629 GBq/mg) from NEN Life Science Products. Cytoplasmic HeLa cell extracts were kindly provided by Dr. V. J. Palombella (ProScript, Cambridge, MA), Dr. R. Reed (Harvard Medical School) or Dr. P. A. Sharp (Massachusetts Institute of Technology), and purified rabbit reticulocyte E1 and E2s were provided by Dr. Z. Chen (ProScript). Strains, plasmids, and recombinant proteins were generously provided by the following colleagues: E. coli strains expressing plant 6His-Ub and 6His-UbR48 by Dr. J. Callis (University of California, Davis) (35Beers E.P. Callis J. J. Biol. Chem. 1993; 268: 21645-21649Abstract Full Text PDF PubMed Google Scholar); strain expressing the GST-yeast Ub fusion by Dr. J. M. Huibregtse (Rutgers University) (36Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1993) Google Scholar); plasmids encoding human UBCH5C (37Jensen 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) and human E2F1 (UBCH7) (38Nuber U. Schwarz S. Kaiser P. Schneider R. Scheffner M. J. Biol. Chem. 1996; 271: 2795-2800Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) by Dr. A. M. Weissman (Bethesda) and Dr. M. Scheffner (Heidelberg), respectively. UBCH5C and E2F1 were purified by cation-exchange chromatography of the flow-through of a DE52 column using an HiTrap SP column (Amersham Pharmacia Biotech). Recombinant bovine E2–25K (39Chen Z.J. Niles E.G. Pickart C.M. J. Biol. Chem. 1991; 266: 15698-15704Abstract Full Text PDF PubMed Google Scholar) was provided by Dr. C. M. Pickart (Johns Hopkins University). The C170S form of E2–25K used here has a serine in place of cysteine 170 (nonactive site Cys). Purified recombinant human UBC2 and UBCH5B (also called UBC4) (40Rolfe M. Beer-Romero P. Glass S. Eckstein J. Berdo I. Theodoras A. Pagano M. Draetta G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3264-3268Crossref PubMed Scopus (104) Google Scholar) by Drs. R. W. King and J. M. Peters (Harvard Medical School). HeLa cell cytoplasmic extracts were centrifuged for 20 min at 10,000 × g (4 °C) to remove debris. The supernatant was dialyzed against 3 mmpotassium phosphate (pH 7.0), 1 mm DTT, 10% glycerol and loaded onto a DE52 column equilibrated with the same buffer. The flow-through (fraction I) was collected, the column was washed with the equilibration buffer supplemented with 20 mm KCl, and the bound proteins (fraction II) were eluted with a buffer containing 20 mm Tris-HCl (pH 7.5), 0.5 m KCl, 5 mm MgCl2, 0.5 mm ATP, 1 mm DTT, 10% glycerol. FII was dialyzed overnight against the same buffer without KCl. In some experiments, FI and FII were prepared from extracts depleted of proteasomes by ultracentrifugation at 100,000 × g for 5 h or 200,000 ×g for 3 h (4 °C). In those cases, after sedimentation of the proteasomes, MgCl2 and ATP were omitted in the buffers used to prepare the FII. Ubiquitin was covalently bound to a CH-activated Sepharose matrix (Amersham Pharmacia Biotech), as recommended by the manufacturer. The final concentration of ubiquitin was about 20 mg/ml of gel. Fraction II was supplemented with 5 mm MgCl2, 2 mm ATP and mixed with the Ub-Sepharose for 1 h at room temperature (with shaking). The ubiquitinating enzymes were sequentially eluted as follows (41Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar, 42Tamura T. Tanaka K. Tanahashi N. Ichihara A. FEBS Lett. 1991; 292: 154-158Crossref PubMed Scopus (30) Google Scholar): E1 with 20 mm Tris, pH 7.5, 2 mm AMP, and 2 mm NaPPi; E2s with 20 mm Tris, pH 7.5, 20 mm DTT, and 100 mm KCl; other ubiquitin-binding proteins with 50 mm Tris, pH 9.0, 1m KCl, 2 mm DTT. Pellets obtained by centrifugation at 200,000 × g for 3 h of the HeLa cytoplasmic extracts were resuspended in Buffer A (50 mmTris, pH 7.5, 5 mm MgCl2, 0.5 mmEDTA, 1 mm ATP, 1 mm DTT, 10% glycerol). After centrifugation at 10,000 × g for 20 min, the supernatant was centrifuged again at 200,000 × g for 30 min to remove polysomes. The supernatant was loaded onto a MonoQ 10/10 column equilibrated with Buffer A, and the proteins were eluted with a NaCl gradient (0–500 mm). The fractions were assayed for activity against the fluorogenic proteasome substrate Suc-LLVY-MCA (Bachem). The active fractions (≈320 mmNaCl) were pooled, diluted twice in Buffer A, and loaded onto a MonoQ 5/5 column. The proteins were eluted with a 200–500 mmNaCl gradient. Fractions active against Suc-LLVY-MCA were pooled and concentrated using a Centricon-50 (Amicon) concentrator and loaded onto a Superose 6 column equilibrated with Buffer A containing 100 mm NaCl. 20S and 26S proteasomes were identified by their activity against Suc-LLVY-MCA in the presence or absence of 0.02% SDS. At this concentration, SDS greatly activates the 20S proteasomes but inhibits the 26S proteasomes, allowing easy discrimination of the two forms (43Hough R. Pratt G. Rechsteiner M. J. Biol. Chem. 1987; 262: 8303-8313Abstract Full Text PDF PubMed Google Scholar). Fractions containing 26S proteasomes and 20S proteasomes were pooled separately and stored frozen at −70 °C. The p105T, p97T, and p60Tth constructs (5Fan C.M. Maniatis T. Nature. 1991; 354: 395-398Crossref PubMed Scopus (239) Google Scholar) were generously provided by Dr. Tom Maniatis (Harvard University). The proteins were translated in vitro in a wheat germ extract using the coupled transcription/translation TNT system of Promega, as recommended by the manufacturer. Unless specified otherwise, the reaction mixture was diluted 3-fold after translation in the assay buffer (20 mm Tris, pH 7.5, 50 mmKCl, 5 mm MgCl2, 1 mm DTT), and the labeled protein was separated from the free [35S]methionine using a Nick Spin column (Amersham Pharmacia Biotech) equilibrated in the same buffer. The wheat germ extract (WGE) containing the in vitro-translated p97 and p105 used in Figs. 7 and 8 was kindly provided by Dr. M. A. Read (ProScript).Figure 8Comparison of the ubiquitination and processing of the truncated version p97 and the full-length p105. A, the processing of p97 is more efficient than the processing of p105. The ubiquitination and conversion of p97 or p105 to p50 were monitored at 37 °C in an 80-μl reaction. At the times indicated, 20 μl were removed and analyzed by SDS-PAGE and autoradiography. The substrates were incubated in the presence of 2 mm ATP, 0.5 mg/ml Ub, 10 μg/ml human E1, 2 μm of each recombinant E2–25K and UBCH5C, and an ATP-regenerating system. The source of E3κB was the FII depleted of ubiquitinating enzymes by Ub-affinity chromatography and of proteasomes by ultracentrifugation (5 μl (about 20 μg of protein) per 20 μl of reaction mixture). The translation mixtures containing p97 or p105 were diluted 20-fold. As indicated at the bottom of the figure, either buffer (−26S) or 2 μg of purified HeLa cell 26S proteasome (+26S) was added. B, although ubiquitination of p105 and p97 was similar, processing was less efficient for ubiquitinated p105. The ubiquitination and processing of p97 (1 μl of the undiluted translation mixture) (left) and p105 (0.5 μl of the undiluted translation mixture) (right) were monitored as in Fig. 7 A. The following enzymes were added: E2s, 2 μm final concentration of E2–25K and UBCH5C; E3, 4 μl of purified E3κB; 26S, 2 μg of purified HeLa cell 26S proteasomes. The bottom two panels show quantification using a Fujix Bas 1000 (Fuji) of the radioactivity that accumulated in each lane as p50 or as ubiquitinated proteins (brackets). In each case, the values obtained in the absence of E3κB and of proteasomes were subtracted.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Unless specified otherwise, reactions were carried out in the assay buffer (20 mm Tris, pH 7.5, 50 mm KCl, 5 mmMgCl2, 1 mm DTT) supplemented with other components as indicated. When the processing of p105 was studied, an ATP-regenerating system (0.1 mg/ml creatine phosphokinase, 10 mm creatine phosphate) was added. The products were separated by SDS-PAGE (44Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), and the gels were dried and analyzed using a PhosphorImager (Molecular Dynamics) or a Fujix Bas 1000 (Fuji) and autoradiography. Reactions were carried out for 10 min at 37 °C in 10 mm Tris, pH 7.6, 10 mm MgCl2, 2 mm ATP, 0.1 mm DTT in the presence of 125I-labeled Ub (50–100 μg/ml) and 50 units/ml of inorganic pyrophosphatase. Reactions were stopped by addition of 1 volume of 2× sample buffer (120 mm Tris, pH 6.8, 4% SDS, 4 m urea, 20% glycerol) containing or not containing 100 mm DTT. The products were analyzed by SDS-PAGE as described above. Prior studies have shown that p105 and its C-terminally truncated forms p60 and p97 can be converted into the NF-κB subunit p50, when expressed in cells or added to crude cell extracts (5Fan C.M. Maniatis T. Nature. 1991; 354: 395-398Crossref PubMed Scopus (239) Google Scholar, 6Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1922) Google Scholar). To learn more about this process,35S-labeled p60, translated in WGE, was added to a cytoplasmic extract (S100) of HeLa cells, and its fate was analyzed at different times by SDS-PAGE and autoradiography (Fig. 1). Within minutes, very high molecular weight forms of p60 appeared, which entered the resolving gel only slightly. This heterogenous 35S-labeled material then disappeared concomitantly with an increase in the mature, processed p50 (although some p50 also appeared within 5 min of p60 addition). Thus the high molecular weight forms behave like intermediates in the proteolytic processing of p60 and probably correspond to ubiquitinated forms of p60. To verify this conclusion, the extract was supplemented with various recombinant species of Ub: wild-type Ub; UbR48, a mutated form of Ub that has a defect in Ub chain formation due to the replacement of lysine 48 by an arginine (45Finley D. Sadis S. Monia B.P. Boucher P. Ecker D.J. Crooke S.T. Chau V. Mol. Cell. Biol. 1994; 14: 5501-5509Crossref PubMed Scopus (303) Google Scholar) (both with a His6 tag); and GST-Ub, a hybrid molecule in which the enzyme glutathioneS-transferase has been fused to the N-terminal end of normal Ub (36Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1993) Google Scholar). Addition of the modified Ub species significantly altered the events from those seen upon addition of normal Ub. With UbR48, we found p60 conjugates of reduced size (data not shown), probably because of the premature termination of the Ub-chain due to the K48R mutation. Moreover, addition of UbR48 to the crude extract inhibited the production of p50, although this inhibition was not complete, probably due to the presence of endogenous normal Ub in this extract. These observations are consistent with previous work showing that UbR48 inhibits p105 processing (6Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1922) Google Scholar). It is noteworthy that the effect of UbR48 on p105 ubiquitination and processing rules out a possible explanation of how p105 may be converted to p50: that p105 is modified by addition of an atypical type of poly-Ub chain, which directs the protein toward limited processing instead of complete degradation. Indeed, lysine 48 of Ub is the residue commonly used for complete degradation of proteins by the proteasome (45Finley D. Sadis S. Monia B.P. Boucher P. Ecker D.J. Crooke S.T. Chau V. Mol. Cell. Biol. 1994; 14: 5501-5509Crossref PubMed Scopus (303) Google Scholar). On the other hand, in the presence of GST-Ub, bands of very high molecular weight accumulated (data not shown, see below), which presumably correspond to poly-GST-Ub adducts of p60 because GST-Ub is about four times larger than normal Ub. Because the type of Ub added determined the size of the high molecular weight derivatives of p60, these derivatives must represent Ub-conjugated forms of the molecule. Furthermore, when the same extract was incubated with ATPγS, an ATP analog that supports ubiquitination of proteins but not the proteolytic activity of the 26S complex (46Scheffner M. Munger K. Huibregtse J.M. Howley P.M. EMBO J. 1992; 11: 2425-2431Crossref PubMed Scopus (98) Google Scholar), high molecular weight ubiquitinated forms of p60 accumulated, but p50 was not formed (data not shown). A similar accumulation of ubiquitinated species was obtained when the extract was depleted of proteasomes by prolonged ultracentrifugation, before being incubated with ATP (Fig. 2). These results confirm that the ubiquitinated species are indeed intermediates in the production of the p50 subunit, as was proposed previously (6Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1922) Google Scholar, 47Orian 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). To identify the enzymes required for the formation of these Ub conjugates, the HeLa extract was loaded onto a DE52 column (Whatman). The proteins that did not bind (FI) were collected and, after washing the column, the bound proteins (FII) were eluted with 0.5 m KCl. After extensive dialysis, both fractions were assayed for their ability to support the processing of p60 into p50. In accord with previous findings (6Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1922) Google Scholar) with FII from rabbit reticulocytes, we found that HeLa FII, when supplemented with Ub, was fully competent at p50 formation (Fig. 2). Moreover, the addition of FI did not increase the yield of p50 (Fig. 2). Thus, it is most likely that the HeLa FII contains all of the enzymes necessary for NF-κB1 processing. It has been reported that FI (from reticulocytes) contains an E2 essential for p105 processing and that this E2 could also be provided in the reaction by the WGE in which p60 (or p105) is translated (47Orian 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). However, our subsequent experiments clearly showed that HeLa FII contains E2 and E3 ubiquitination enzymes supporting p60 (or p105) ubiquitination and processing. FII was then further fractionated to isolate the enzymes active in p60 ubiquitination. Ammonium sulfate precipitation resolved two fractions that independently had some activity in ubiquitinating p60 (data not shown): FIIA, which precipitated between 0 and 40% (NH4)2SO4, and FIIB, which precipitated between 40 and 90% (NH4)2SO4. However, mixing these two fractions resulted in more than an additive effect in promoting p60 ubiquitination (data not shown). Thus, each of these fractions appeared to be enriched in distinct component(s) of the ubiquitination pathway. These components were then further purified by anion-exchange chromatography of FIIA and FIIB, using a MonoQ column. To assay their ability to support ubiquitination of p60, each fraction derived from FIIA was combined with FIIB, and each from FIIB was combined with FIIA, in the presence of E1 and GST-Ub. We used GST-Ub rather than normal Ub because the larger conjugates formed with GST-Ub accumulate at the top of the acrylamide gel and are easily detected. After anion-exchange chromatography of FIIA and FIIB, two protein fractions were obtained: A (from FIIA), which eluted at about 250 mm NaCl, and B (from FIIB), which eluted at about 100 mm NaCl. These two fractions, when mixed, supported efficient formation of Ub conjugates when p60 or p105 was used as the substrate (Fig. 3 A). Interestingly, the decrease in the p60 and p105 bands (which accounted for 30% or less of the substrate added, as analyzed with a PhosphorImager) could not account for the amount of labeled protein accumulating as conjugates. Therefore, the bands of lower molecular weight (most likely degradation products or products of premature termination of translation) apparently can also be ubiquitinated by these enzymes. The poly-ubiquitination of a substrate involves the successive thiol ester linkage of Ub to E1 and then to E2 and, in some cases, to E3 (24Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1210) Google Scholar,25Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (756) Google Scholar). Using 125I-Ub, such a thiol ester adduct can be detected after SDS-PAGE, provided that the sample is not exposed to a reducing agent. We analyzed fractions A and B for their content of enzymes capable of forming a thiol ester linkage with125I-Ub. As shown in Fig. 3 B, one DTT-sensitive band of about 110 kDa was detected in fraction A after electrophoresis. This band comigrated with the band formed when Ub was incubated with E1 purified from rabbit reticulocytes, and thus it corresponded to the human E1. No E2 could be detected in fraction A. In fraction B, no Ub-thiol ester could be detected, unless reticulocyte E1 was added. With E1 present, two Ub-protein adducts of about 27.5 and 33 kDa were evident under nonreducing conditions, but they disappeared if the sample was boiled in the presence of DTT. These two bands therefore must correspond to distinct ubiquitin carrier proteins (E2s) linked to Ub by a thiol ester. Finally, mixing fractions A and B allowed formation of the same two Ub-thiol esters, without exogenous E1 addition, and did not reveal any additional E2 (Fig. 3 B). In addition, some high molecular weight radiolabeled bands (Fig. 3 B, vertical bar) were formed that were DTT-resistant and therefore corresponded to Ub conjugates of proteins in fractions A and B. Neither fraction A, which contains E1, nor fraction B, which contains two E2s, was able by itself to conjugate Ub to p60 or p105, even if reticulocyte E1 was added (Fig. 3 A). Significant conjugation to p60 or p105 occurred only when fractions A and B were mixed. Thus, in addition to E1, fr" @default.
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• W2020547495 title "Enzymes Catalyzing Ubiquitination and Proteolytic Processing of the p105 Precursor of Nuclear Factor κB1" @default.
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