Critical Role for Lysines 21 and 22 in Signal-induced, Ubiquitin-mediated Proteolysis of IkB-α
1996; Elsevier BV; Volume: 271; Issue: 1 Linguagem: Inglês
10.1074/jbc.271.1.376
ISSN1083-351X
AutoresLucia Baldi, Keith Brown, Guido Franzoso, Ulrich Siebenlist,
Tópico(s)T-cell and Retrovirus Studies
ResumoThe NF-κB transcription factor induces rapid transcription of many genes in response to a variety of extracellular signals. NF-κB is readily activated from normally inhibited cytoplasmic stores by induced proteolytic degradation of IκB-α, a principal inhibitor of this transcription factor. Following the inhibitor's degradation, NF-κB is free to translocate to the nucleus and induce gene transcription. The IκB-α inhibitor is targeted for degradation by signal-induced phosphorylation of two closely spaced serines in its NH2 terminus (Ser32 and Ser36). Proteolytic degradation appears to be carried out by proteasomes which can recognize ubiquitinated intermediates of the IκB-α inhibitor. We provide evidence which supports a ubiquitin-mediated mechanism. Amino acid substitutions of two adjacent potential ubiquitination sites in the NH2 terminus of IκB-α (Lys21 and Lys22) almost completely block the rapid, signal-induced degradation of the mutant protein, while they do not interfere with induced phosphorylation. The mutant IκB-α also does not permit signal-induced activation of NF-κB bound to it. The data suggest that ubiquitination at either of the two adjacent lysines (21 and 22) is required for degradation following induced phosphorylation at nearby serines 32 and 36. Such dependence on ubiquitination of specific sites for protein degradation is unusual. This mechanism of degradation may also apply to IκB-β, an inhibitor related to and functionally overlapping with IκB-α, as well as to cactus, an IκB homolog of Drosophila. The NF-κB transcription factor induces rapid transcription of many genes in response to a variety of extracellular signals. NF-κB is readily activated from normally inhibited cytoplasmic stores by induced proteolytic degradation of IκB-α, a principal inhibitor of this transcription factor. Following the inhibitor's degradation, NF-κB is free to translocate to the nucleus and induce gene transcription. The IκB-α inhibitor is targeted for degradation by signal-induced phosphorylation of two closely spaced serines in its NH2 terminus (Ser32 and Ser36). Proteolytic degradation appears to be carried out by proteasomes which can recognize ubiquitinated intermediates of the IκB-α inhibitor. We provide evidence which supports a ubiquitin-mediated mechanism. Amino acid substitutions of two adjacent potential ubiquitination sites in the NH2 terminus of IκB-α (Lys21 and Lys22) almost completely block the rapid, signal-induced degradation of the mutant protein, while they do not interfere with induced phosphorylation. The mutant IκB-α also does not permit signal-induced activation of NF-κB bound to it. The data suggest that ubiquitination at either of the two adjacent lysines (21 and 22) is required for degradation following induced phosphorylation at nearby serines 32 and 36. Such dependence on ubiquitination of specific sites for protein degradation is unusual. This mechanism of degradation may also apply to IκB-β, an inhibitor related to and functionally overlapping with IκB-α, as well as to cactus, an IκB homolog of Drosophila. INTRODUCTIONThe transcription factor complexes known collectively as NF-κB function primarily as mediators of inducible transcription in response to a variety of environmental signals (for recent reviews, see (1.Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2010) Google Scholar, 2.Siebenlist U. Brown K. Franzoso G. Baeuerle P.A. Inducible Gene Expression. I. Birkhaeuser, Boston1995: 93-141Google Scholar, 3.Baeuerle P.A. Henkel T. Annu. Rev. 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The various NF-κB dimers usually lie dormant in the cytoplasm of cells, kept there by inhibitory ankyrin-containing members of the IκB family of proteins, in particular IκB-α. IκB-α strongly associates with p50/p65 heterodimers and appears to shield the nuclear localization sequences contained in both subunits; this is presumed to be the mechanism by which this protein retains the heterodimers in the cytoplasm(6.Beg 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 (608) Google Scholar, 7.Ganchi P.A. Sun S.C. Greene W.C. Ballard D.W. Mol. Biol. Cell. 1992; 3: 1339-1352Crossref PubMed Scopus (203) Google Scholar, 8.Henkel T. Zabel U. van Zee K. Muller J.M. Fanning E. Baeuerle P. Cell. 1992; 68: 1121-1133Abstract Full Text PDF PubMed Scopus (302) Google Scholar, 9.Zabel U. Henkel T. dos Santos Silva M. Baeuerle P. EMBO J. 1993; 12: 201-211Crossref PubMed Scopus (265) Google Scholar). Activation of NF-κB proceeds via rapid, signal-induced proteolytic degradation of the inhibitor, liberating the transcription factor which is now free to translocate to the nucleus (10.Brown K. Park S. Kanno T. Franzoso G. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2532-2536Crossref PubMed Scopus (575) Google Scholar, 11.Beg A.A. Finco T.S. Nantermet P.V. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 3301-3310Crossref PubMed Google Scholar, 12.Cordle S.R. Donald R. Read M.A. Hawiger J. J. Biol. Chem. 1993; 268: 11803-11810Abstract Full Text PDF PubMed Google Scholar, 13.Henkel T. Machleidt T. Alkalay I. Kronke M. Ben-Neriah Y. Bauerle P.A. Nature. 1993; 365: 182-185Crossref PubMed Scopus (1034) Google Scholar, 14.Rice N.R. Ernst M.K. EMBO J. 1993; 12: 4685-4695Crossref PubMed Scopus (284) Google Scholar, 15.Sun S-C. Ganchi P.A. Ballard D.W. Greene W.C Science. 1993; 259: 1912-1915Crossref PubMed Scopus (951) Google Scholar, 16.Sun S-C. Ganchi P.A. Beraud C. Ballard D.W. Greene W.C Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1346-1350Crossref PubMed Scopus (162) Google Scholar, 17.Chiao P.J. Miyamoto S. Verma I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 28-32Crossref PubMed Scopus (390) Google Scholar). Degradation is carried out by proteasomes and is preceded by signal-induced phosphorylation of IκB-α itself(18.Finco T.S. Beg A.A. Baldwin Jr., A.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11884-11888Crossref PubMed Scopus (293) Google Scholar, 19.Miyamoto S. Maki M. Schmitt M.J. Hatanaka M. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12740-12744Crossref PubMed Scopus (218) Google Scholar, 20.Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1915) Google Scholar, 21.Traenckner E.B.-M. Wilk S. Baeuerle P.A. EMBO J. 1994; 13: 5433-5441Crossref PubMed Scopus (652) Google Scholar, 22.Alkalay I. Yaron A. Hatzubai A. Jung S. Avraham A. Gerlitz O. Passhut-Lavon I. Ben-Neriah Y. Mol. Cell. Biol. 1995; 15: 1294-1301Crossref PubMed Google Scholar, 23.DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar, 24.Lin Y.-C. Brown K. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 552-556Crossref PubMed Scopus (256) Google Scholar). Induced phosphorylation occurs on two closely spaced serines in the NH2 terminus of the protein (amino acids 32 and 36), mediated by an as yet unknown kinase(s)(25.Brown K. Gertsberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1310) Google Scholar, 26.Brockman 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, 27.Traenckner E.B.-M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (929) Google Scholar, 28.Whiteside S.T. Ernst M.K. LeBail O. Laurent-Winter C. Rice N. Israel A. Mol. Cell. Biol. 1995; 15: 5339-5345Crossref PubMed Google Scholar). It has recently been shown that signal-induced phosphorylation can lead to ubiquitination of IκB-α(29.Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes & Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1161) Google Scholar). Since ubiquitin-tagged proteins are generally subject to proteasome-mediated proteolysis(30.Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1588) Google Scholar), these observations suggest that degradation of IκB-α is triggered by ubiquitination. However, a ubiquitin-independent mechanism of degradation is not necessarily excluded, since only a fraction of the total pool of IκB-α could be shown to be ubiquitinated (under conditions of proteolysis inhibition). Furthermore, precedents exist for a ubiquitin-independent, but proteasome-dependent degradation mechanism(31.Murakami Y. Matsufuji S. Kameji T. Hayashi S.-I. Igarashi K. Tamura T. Tanaka K. Ichihara A. Nature. 1992; 360: 597-599Crossref PubMed Scopus (666) Google Scholar). Therefore, we sought to demonstrate ubiquitin-dependence by investigating the requirement for lysines in IκB-α degradation, because lysines are the sites at which ubiquitin is ligated(30.Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1588) Google Scholar). Here we provide evidence which strongly suggests that rapid, signal-regulated degradation of IκB-α proceeds primarily via a ubiquitin-dependent mechanism. An IκB-α mutant bearing conservative substitutions at two potential ubiquitination sites is remarkably resistant to signal-regulated degradation. The results imply that two adjacent NH2-terminal lysines (Lys21 and Lys22) are the primary targets of signal-induced ubiquitination. That specific lysines play such an important role in ubiquitin-mediated protein degradation is uncommon(30.Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1588) Google Scholar).MATERIALS AND METHODSSite-directed MutagenesisMutations in full-length, human IκB-α cDNA (32.Haskill 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 (582) Google Scholar) were generated essentially as described previously(25.Brown K. Gertsberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1310) Google Scholar, 33.Bressler P. Brown K. Timmer W. Bours V. Siebenlist U. Fauci A.S. J. Virol. 1993; 67: 288-293Crossref PubMed Google Scholar). In each case, lysine codons were substituted with arginine codons (for positions 21, 22, 47 and 67: AAG was changed to AGG; for 38: AAA was changed to CGA; for 87: AAG was changed to CGG). The mutated IκB-α cDNAs were excised from a Bluescript vector (Stratagene, La Jolla, CA) with EcoRI (1550 base pairs) and subcloned into the PMT2T mammalian expression vector(34.Israel D.I. Kaufmann R.J. Nucleic Acids Res. 1989; 17: 4589-4604Crossref PubMed Scopus (49) Google Scholar). Mutations were confirmed by subsequent DNA sequence analysis.Transient TransfectionsNtera-2, human embryonal carcinoma cells were transfected via calcium phosphate-mediated transfer as described elsewhere (35.Bours V. Burd P. Brown K. Villalobos J. Park S. Ryseck R.P. Bravo R. Kelly K. Siebenlist U. Mol. Cell. Biol. 1992; 12: 685-695Crossref PubMed Google Scholar) with the following vectors: a CAT 1The abbreviations used are: CATchloramphenicol acetyltransferasePMAphorbol 12-myristate 13-acetate. reporter plasmid containing the tandemly repeated κB sites of human immunodeficiency virus (5 μg)(35.Bours V. Burd P. Brown K. Villalobos J. Park S. Ryseck R.P. Bravo R. Kelly K. Siebenlist U. Mol. Cell. Biol. 1992; 12: 685-695Crossref PubMed Google Scholar); the p65-PMT2T expression vector (0.2 μg)(35.Bours V. Burd P. Brown K. Villalobos J. Park S. Ryseck R.P. Bravo R. Kelly K. Siebenlist U. Mol. Cell. Biol. 1992; 12: 685-695Crossref PubMed Google Scholar); PMT2T vectors carrying the wild-type or mutant IκB-αs (see above) (depending on the experiment, between 0.15 and 0.6 μg were found to be required for near-maximal inhibition, which was the point used in the experiments). Cells were stimulated with PMA (10 ng/ml) for 6 h prior to harvesting, stimulation starting at about 36 h post-transfection, and CAT activity was measured as previously described(25.Brown K. Gertsberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1310) Google Scholar, 35.Bours V. Burd P. Brown K. Villalobos J. Park S. Ryseck R.P. Bravo R. Kelly K. Siebenlist U. Mol. Cell. Biol. 1992; 12: 685-695Crossref PubMed Google Scholar, 36.Franzoso G. Bours V. Park S. Tomita-Yamaguchi M. Kelly K. Siebenlist U. Nature. 1992; 359: 339-342Crossref PubMed Scopus (264) Google Scholar, 37.Franzoso G. Bours V. Azarenko V. Park S. Tomita-Yamaguchi M. Kanno T. Brown K. Siebenlist U. EMBO J. 1993; 12: 3893-3901Crossref PubMed Scopus (145) Google Scholar, 38.Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (423) Google Scholar).Permanent TransfectionsEL-4 murine T lymphoma cells were maintained in RPMI medium in the presence of 10% FCS (Life Technologies, Inc.) and were stably transfected with the various PMT2T-IκB-α vectors, together with a plasmid conferring neomycin resistance, as described elsewhere(25.Brown K. Gertsberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1310) Google Scholar). Briefly, 20 μg of PMT2T-IκB-α DNA and 2 μg of neomycin-resistant plasmid DNA were electroporated into 107 EL-4 cells using the Bio-Rad Gene Pulser, set at 250 V, 960 microfarads. Stable neomycin-resistant transfectants were selected after 3-4 weeks of incubation with geneticin (G418, Life Technologies, Inc.), used at 400 μg/ml during the first week and at 200 μg/ml thereafter. The resulting cell lines were screened for expression of human IκB-α using a rabbit polyclonal antibody directed against full-length IκB-α (10.Brown K. Park S. Kanno T. Franzoso G. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2532-2536Crossref PubMed Scopus (575) Google Scholar, 25.Brown K. Gertsberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1310) Google Scholar) and ECL technology (Amersham Corp.). Cells were stimulated with PMA (40 ng/ml) and ionomycin (2 μM), and calpain inhibitor I was used at 100 μM, starting 30 min prior to stimulation.RESULTSSince ubiquitin is ligated to proteins through lysine residues, we substituted the lysines in IκB-α by site-directed mutagenesis and tested the resulting mutant proteins for defects in signal-dependent degradation. Human IκB-α (Mad-3) contains lysine at positions 21, 22, 38, 47, 67, 87, 98, 177, and 238 (32.Haskill 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 (582) Google Scholar). Lysines 22, 38, 87, and 238 are perfectly conserved in pig, rat, and chicken (pp40) IκB-α; lysines 21, 47, 67, and 98 are absent in chicken; and lysine 177 is not conserved at all(39.de Martin R. Vanhove B. Cheng Q. Hofer E. Csizmadia V. Winkler H. Bach F.H. EMBO J. 1993; 12: 2773-2779Crossref PubMed Scopus (290) Google Scholar). We substituted each of the NH2-terminal lysines (21, 22, 38, 47, 67, and 87) and the pair, 21 + 22, with arginine residues to block ubiquitination of these sites which all lie near the inducibly phosphorylated serines 32 and 36. Arginine was chosen so as not to change the charge of the protein. As an initial test of the mutant proteins, we transiently transfected expression constructs for the various IκB-α mutants into Ntera-2 embryonal carcinoma cells, together with an expression construct for NF-κB/p65. Undifferentiated Ntera-2 cells do not express significant levels of endogenous NF-κB or IκB proteins and are thus ideal for evaluating the activities of the transfected proteins without interference by endogenous counterparts(33.Bressler P. Brown K. Timmer W. Bours V. Siebenlist U. Fauci A.S. J. Virol. 1993; 67: 288-293Crossref PubMed Google Scholar, 35.Bours V. Burd P. Brown K. Villalobos J. Park S. Ryseck R.P. Bravo R. Kelly K. Siebenlist U. Mol. Cell. Biol. 1992; 12: 685-695Crossref PubMed Google Scholar, 36.Franzoso G. Bours V. Park S. Tomita-Yamaguchi M. Kelly K. Siebenlist U. Nature. 1992; 359: 339-342Crossref PubMed Scopus (264) Google Scholar, 37.Franzoso G. Bours V. Azarenko V. Park S. Tomita-Yamaguchi M. Kanno T. Brown K. Siebenlist U. EMBO J. 1993; 12: 3893-3901Crossref PubMed Scopus (145) Google Scholar, 38.Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (423) Google Scholar, 40.Segars J.H. Nagata T. Bours V. Medin I.A. Franzoso G. Blanco J.C.G. Drew P.D. Becker K.G. An J. Tang T. Stephany D.A. Neel B. Siebenlist U. Ozato K. Mol. Cell. Biol. 1993; 13: 6157-6169Crossref PubMed Google Scholar). Transfected p65 potently transactivated a cotransfected κB-dependent CAT reporter, while coexpression of wild-type IκB-α or any of the lysine-substituted mutants severely inhibited p65-mediated transactivation (25.Brown K. Gertsberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1310) Google Scholar) (data not shown). Therefore, these mutations did not interfere with the inhibitory activity of the protein, which was expected, since inhibition does not require the NH2-terminal domain of IκB-α(25.Brown K. Gertsberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1310) Google Scholar, 41.Inoue J.I. Kerr L.D. Rashid D. Davis N. Bose Jr., H.R. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4333-4337Crossref PubMed Scopus (107) Google Scholar, 42.Hatada E.N. Naumann M. Scheidereit C. EMBO J. 1993; 12: 2781-2788Crossref PubMed Scopus (78) Google Scholar, 43.Ernst M.K. Dunn L.L. Rice N.R. Mol. Cell. Biol. 1995; 15: 872-882Crossref PubMed Google Scholar). We then tested whether the lysine mutations interfered with the signal-induced degradation of the proteins bearing them, as measured by the ability of a PMA stimulus to relieve inhibition and thus allow p65-mediated transactivation of the CAT reporter (Fig. 1; inducibility of mutants is shown as percent of inducibility of wild-type IκB-α; inducibility is measured as the ratio of CAT activity of PMA-stimulated cells/unstimulated cells). None of the individual lysine mutations significantly interfered with signal-induced transactivation, suggesting that no single lysine is critical for the signal-induced degradation of IκB-α (Fig. 1; column 1, wild-type (wt); columns 2, 3, 5-8, mutants K21R, K22R, K38R, K47R, K67R, and K87R (R21, R22, R38, R47, R67, and R87, respectively). However, an IκB-α mutant bearing substitutions at both lysines 21 and 22 (K21R/K22R (R21R22 mutant) had a dramatic effect; this mutant did not allow significant activation of NF-κB in PMA-stimulated, transfected Ntera-2 cells (Fig. 1, column 4). The data suggest that at least one of the two lysines at positions 21 and 22 has to be present for rapid signal-induced degradation of IκB-α to occur, since elimination of both lysines effectively blocked NF-κB activation, while substitution of either lysine alone was of little consequence.To confirm these interpretations and to rule out a potential defect in phosphorylation of the K21R/K22R mutant, we directly evaluated mutants for phosphorylation and degradation. Murine EL-4 T cells were permanently transfected with the various IκB-α mutants, and then the cells were stimulated with PMA and ionomycin. We showed previously that exogenously derived human IκB-α is subject to the same signal-induced phosphorylation and degradation as the endogenous murine IκB-α(25.Brown K. Gertsberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1310) Google Scholar). Endogenous murine IκB-α serves as an internal positive control in these experiments. Since it migrates slightly faster than the transfected human protein, both proteins could be simultaneously visualized(25.Brown K. Gertsberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1310) Google Scholar). Among the mutants tested, only the K21R/K22R mutant was resistant to signal-induced degradation in the EL-4 cells (Fig. 2), while all other transfected human (h) mutant proteins bearing individual lysine substitutions appeared to be degraded as efficiently as the endogenous murine (m) IκB-α (Fig. 2, K21R, K22R, K38R, K47R, K67R, and K87R (R21, R22, R38, R47, R67, and R87). All mutant proteins, including the double mutant K21R/K22R, were rapidly phosphorylated in response to signals, as indicated by the shift in mobility of IκB-α in the presence of calpain inhibitor I, which inhibits proteasomes (20.Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1915) Google Scholar, 21.Traenckner E.B.-M. Wilk S. Baeuerle P.A. EMBO J. 1994; 13: 5433-5441Crossref PubMed Scopus (652) Google Scholar, 22.Alkalay I. Yaron A. Hatzubai A. Jung S. Avraham A. Gerlitz O. Passhut-Lavon I. Ben-Neriah Y. Mol. Cell. Biol. 1995; 15: 1294-1301Crossref PubMed Google Scholar, 23.DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar, 24.Lin Y.-C. Brown K. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 552-556Crossref PubMed Scopus (256) Google Scholar) (Fig. 2, data not shown for K38R, K47R, K67R, and K87R). This is as expected for the rapidly degraded IκB-α proteins. In the case of the double mutant (K21R/K22R), the proteasome inhibitors were not needed to see the phosphorylation, since this mutant was not efficiently degraded. The presence of the proteasome inhibitor did, however, increase the amount of the K21R/K22R IκB-α mutant observed, suggesting that these mutations may not completely block induced degradation. Nonetheless, the K21R/K22R mutation afforded this IκB-α significant protection from degradation, and the results are consistent with the Ntera-2 experiments shown in Fig. 1.Figure 2:Signal-induced degradation and phosphorylation of wild-type and mutant IκB-α expressed in stably transfected EL-4 cells. EL-4 murine T cells were permanently transfected with wild-type (wt) or mutated (mt), human (h) IκB-α expression vectors (see "Materials and Methods"), as indicated. Cells were stimulated with PMA and ionomycin (Iono) for 15 min in the presence (+) or absence of calpain inhibitor I (Calp. Inhib.), which inhibits proteasome activity. Both transfected human and endogenous murine (m) IκB-α, as well as the inducibly phosphorylated form of human IκB-α (IκB-αP) were visualized by Western analysis (see "Materials and Methods"). (The inducibly phosphorylated mutated IκB-α migrates to almost the same position as the uninduced form and is not readily distinguished here(25.Brown K. Gertsberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1310) Google Scholar).) Only the K21R/K22R (R21R22) mutation in human IκB-α blocked rapid degradation (while the murine wild-type protein was degraded in the same cells), which resulted in an accumulation of the inducibly phosphorylated form, even in the absence of proteasome inhibitors.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONWe have demonstrated a critical requirement for the presence of either of two lysines at positions 21 and 22 in signal-induced degradation of IκB-α and, as a consequence, in signal-induced transactivation by NF-κB/p65. The substitution of both lysines 21 and 22 with arginines in IκB-α (K21R/K22R) caused a severe block to rapid signal-induced degradation of that IκB-α in stably transfected EL-4 cells. The mutant protein was, however, still phosphorylated at nearby serine sites (S32 and S36), indicating that the defect lies downstream of the phosphorylation step; it also suggests that the protein was not grossly altered by these conservative substitutions, since the kinase(s) acitivity on this substrate appears unaffected. The K21R/K22R mutant prevented the signal-induced, p65-mediated transactivation of a κB-dependent reporter in transient transfection experiments using Ntera-2 cells. In contrast to the K21R/K22R mutant, mutants bearing substitutions of individual lysines, including those at residue 21 or 22, had no measurable effect and behaved like wild type. Taken together these data demonstrate that either of the two lysines at positions 21 and 22 is necessary for rapid degradation (but not for phosphorylation) and they provide a compelling argument for obligatory ubiquitination prior to degradation of IκB-α: at least one of the two potential ubiquitination sites must be present for rapid signal-induced degradation to proceed. Although degradation is dramatically inhibited, it appears not to be absolutely blocked (see Fig. 2); this may indicate that ubiquitination can also occur at other sites, albeit less efficiently, or that another mechanism allows for a slower degradation. Finally, the data do not tell us if Lys21 or Lys22 are sufficient for ubiquitin-mediated degradation. It is possible, for example, that some other, not necessarily specific, lysine is necessary also. To formally test this less likely possibility would require a mutant IκB-α bearing substitutions of all lysines other than 21 and 22.Individual ubiquitination sites do not usually play a dominant role in protein degradation, where often multiple functional ubiquitination sites exist and targeted mutations have little effect(30.Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1588) Google Scholar), although the degradation of Mos may be another exception to this rule(44.Nishizawa M. Furuno N. Okazaki K. Tanaka H. Ogawa Y. Sagata N. EMBO J. 1993; 12: 4021-4027Crossref PubMed Scopus (102) Google Scholar). It is possible that ubiquitination of IκB-α may be specifically directed to lysines 21 and 22, or, alternatively, that these lysines are the only ones accessible for ligation (no other lysines exist NH2-terminal to the phosphorylation sites). This latter possibility may be supported by the observation that ubiquitination occurs with IκB-α still bound to NF-κB(29.Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes & Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1161) Google Scholar), which should partially shield the inhibitor. The central part of IκB-α consists of 6 ankyrin repeats whose primary function is to interact with NF-κB; this part may be largely buried in the cleft between the two NF-κB subunits, as suggested by x-ray crystallographic data of p50 homodimers(45.Muller C.W. Rey F.A. Sodeoka M. Verdine G.L. Harrison S.C. Nature. 1995; 373: 311-317Crossref PubMed Scopus (465) Google Scholar, 46.Ghosh G. Van Duyne S. Sigler P.B. Nature. 1995; 373: 303-310Crossref PubMed Scopus (503) Google Scholar). The fairly short COOH-terminal region of IκB-α is required for inhibition of DNA binding by NF-κB, implying that it too may interact with NF-κB proteins(25.Brown K. Gertsberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1310) Google Scholar, 43.Ernst M.K. Dunn L.L. Rice N.R. Mol. Cell. Biol. 1995; 15: 872-882Crossref PubMed Google Scholar). By contrast, the NH2-terminal part of the protein is not required for these functions, rather, it must be accessible to a kinase(s) to allow inducible phosphorylation. An as yet undetermined protein may then recognize the phosphorylated protein, presumably by recognizing the phosphorylated serines or local changes induced in the protein as a consequence of phosphorylation (no major conformational changes are expected, since the phosphorylated species remains tightly bound to NF-κB and continues to inhibit)(18.Finco T.S. Beg A.A. Baldwin Jr., A.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11884-11888Crossref PubMed Scopus (293) Google Scholar, 19.Miyamoto S. Maki M. Schmitt M.J. Hatanaka M. Verma I.M. Proc. Natl. Acad. Sci. U. S. 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It remains to be shown whether highly ubiquitinated IκB-α is removed from the complex just prior to degradation, or if degradation is initiated while ubiquitinated IκB-α is still in the complex. In contrast to bound IκB-α, the free unbound form may present additional sites for ubiquitination.Chicken IκB-α (pp40) contains only one of the two lysine residues important for degradation (the Lys equivalent to that at position 22 in the human protein), suggesting that a single substitution of that lysine may be sufficient to block rapidly inducible degradation of pp40. Recently IκB-β was cloned and shown to be inducibly degraded in response to certain signals, such as interleukin-1 and lipopolysaccharide(47.Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (692) Google Scholar). While IκB-α and IκB-β share high overall similarity, their NH2-terminal regions are surprisingly different, save for a few conserved amino acids; however, these few amino acids appear to be highly significant in that they suggest shared regulatory features of these proteins (Fig. 3). Both inducibly phosphorylated serines and a few surrounding residues are conserved as is the lysine equivalent to that at position 22 in IκB-α. (This is the only lysine in the entire NH2-terminal part of the IκB-β protein, which may suggest that it is absolutely required for signaling in that protein). This limited but significant conservation of functional sites can also be found in cactus, the Drosophila homolog of IκB proteins (48.Geisler R. Bergmann A. Nuesslein-Volhard C. Cell. 1992; 71: 613-621Abstract Full Text PDF PubMed Scopus (194) Google Scholar, 49.Kidd S. Cell. 1992; 71: 623-635Abstract Full Text PDF PubMed Scopus (183) Google Scholar) (see Fig. 3), suggesting that all three proteins may be regulated in a similar fashion. Cactus, which contains a much larger NH2-terminal domain than either IκB-α or IκB-β, may offer additional sites for regulation.Figure 3:Sequence comparisons of IκB-α, IκB-β, and cactus. Functionally important residues of IκB-α are conserved in IκB-β and cactus. This includes both inducibly phosphorylated serines, 32 and 36, a few surrounding residues, and at least one of the two NH2-terminal lysines important for degradation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) INTRODUCTIONThe transcription factor complexes known collectively as NF-κB function primarily as mediators of inducible transcription in response to a variety of environmental signals (for recent reviews, see (1.Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2010) Google Scholar, 2.Siebenlist U. Brown K. Franzoso G. Baeuerle P.A. Inducible Gene Expression. I. Birkhaeuser, Boston1995: 93-141Google Scholar, 3.Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4582) Google Scholar, 4.Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1216) Google Scholar, 5.Miyamoto S. Verma I. Adv. Cancer Res. 1995; 66: 255-292Crossref PubMed Google Scholar)). Stress- and pathogen-related signals in particular are known to activate NF-κB, leading to induced expression of a large number of genes, including many genes which encode functions relevant to immune responses. In most cell types, p50-p65 heterodimers represent the vast majority of the rapidly inducible NF-κB complexes, although several other NF-κB dimers may coexist and may become activated also. All NF-κB dimers are composed of members of the Rel/NF-κB family of polypeptides, and in vertebrates this family is comprised of p50 (NF-κB1), p65 (RelA), c-Rel, p52 (NF-κB2), and RelB. The various NF-κB dimers usually lie dormant in the cytoplasm of cells, kept there by inhibitory ankyrin-containing members of the IκB family of proteins, in particular IκB-α. IκB-α strongly associates with p50/p65 heterodimers and appears to shield the nuclear localization sequences contained in both subunits; this is presumed to be the mechanism by which this protein retains the heterodimers in the cytoplasm(6.Beg 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 (608) Google Scholar, 7.Ganchi P.A. Sun S.C. Greene W.C. Ballard D.W. Mol. Biol. Cell. 1992; 3: 1339-1352Crossref PubMed Scopus (203) Google Scholar, 8.Henkel T. Zabel U. van Zee K. Muller J.M. Fanning E. Baeuerle P. 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EMBO J. 1993; 12: 4685-4695Crossref PubMed Scopus (284) Google Scholar, 15.Sun S-C. Ganchi P.A. Ballard D.W. Greene W.C Science. 1993; 259: 1912-1915Crossref PubMed Scopus (951) Google Scholar, 16.Sun S-C. Ganchi P.A. Beraud C. Ballard D.W. Greene W.C Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1346-1350Crossref PubMed Scopus (162) Google Scholar, 17.Chiao P.J. Miyamoto S. Verma I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 28-32Crossref PubMed Scopus (390) Google Scholar). Degradation is carried out by proteasomes and is preceded by signal-induced phosphorylation of IκB-α itself(18.Finco T.S. Beg A.A. Baldwin Jr., A.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11884-11888Crossref PubMed Scopus (293) Google Scholar, 19.Miyamoto S. Maki M. Schmitt M.J. Hatanaka M. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12740-12744Crossref PubMed Scopus (218) Google Scholar, 20.Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1915) Google Scholar, 21.Traenckner E.B.-M. Wilk S. Baeuerle P.A. EMBO J. 1994; 13: 5433-5441Crossref PubMed Scopus (652) Google Scholar, 22.Alkalay I. Yaron A. Hatzubai A. Jung S. Avraham A. Gerlitz O. Passhut-Lavon I. Ben-Neriah Y. Mol. Cell. Biol. 1995; 15: 1294-1301Crossref PubMed Google Scholar, 23.DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar, 24.Lin Y.-C. Brown K. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 552-556Crossref PubMed Scopus (256) Google Scholar). Induced phosphorylation occurs on two closely spaced serines in the NH2 terminus of the protein (amino acids 32 and 36), mediated by an as yet unknown kinase(s)(25.Brown K. Gertsberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1310) Google Scholar, 26.Brockman 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, 27.Traenckner E.B.-M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (929) Google Scholar, 28.Whiteside S.T. Ernst M.K. LeBail O. Laurent-Winter C. Rice N. Israel A. Mol. Cell. Biol. 1995; 15: 5339-5345Crossref PubMed Google Scholar). It has recently been shown that signal-induced phosphorylation can lead to ubiquitination of IκB-α(29.Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes & Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1161) Google Scholar). Since ubiquitin-tagged proteins are generally subject to proteasome-mediated proteolysis(30.Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1588) Google Scholar), these observations suggest that degradation of IκB-α is triggered by ubiquitination. However, a ubiquitin-independent mechanism of degradation is not necessarily excluded, since only a fraction of the total pool of IκB-α could be shown to be ubiquitinated (under conditions of proteolysis inhibition). Furthermore, precedents exist for a ubiquitin-independent, but proteasome-dependent degradation mechanism(31.Murakami Y. Matsufuji S. Kameji T. Hayashi S.-I. Igarashi K. Tamura T. Tanaka K. Ichihara A. Nature. 1992; 360: 597-599Crossref PubMed Scopus (666) Google Scholar). Therefore, we sought to demonstrate ubiquitin-dependence by investigating the requirement for lysines in IκB-α degradation, because lysines are the sites at which ubiquitin is ligated(30.Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1588) Google Scholar). Here we provide evidence which strongly suggests that rapid, signal-regulated degradation of IκB-α proceeds primarily via a ubiquitin-dependent mechanism. An IκB-α mutant bearing conservative substitutions at two potential ubiquitination sites is remarkably resistant to signal-regulated degradation. The results imply that two adjacent NH2-terminal lysines (Lys21 and Lys22) are the primary targets of signal-induced ubiquitination. That specific lysines play such an important role in ubiquitin-mediated protein degradation is uncommon(30.Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1588) Google Scholar).
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