The IκB Kinase (IKK) Complex Is Tripartite and Contains IKKγ but Not IKAP as a Regular Component
2000; Elsevier BV; Volume: 275; Issue: 38 Linguagem: Inglês
10.1074/jbc.m003902200
ISSN1083-351X
AutoresDaniel Krappmann, Eunice N. Hatada, Sebastian Tegethoff, Jun Li, Anke Klippel, Klaus Giese, Patrick A. Baeuerle, Claus Scheidereit,
Tópico(s)interferon and immune responses
ResumoA critical step in the activation of NF-κB is the phosphorylation of IκBs by the IκB kinase (IKK) complex. IKKα and IKKβ are the two catalytic subunits of the IKK complex and two additional molecules, IKKγ/NEMO and IKAP, have been described as further integral members. We have analyzed the function of both proteins for IKK complex composition and NF-κB signaling. IKAP and IKKγ belong to distinct cellular complexes. Quantitative association of IKKγ was observed with IKKα and IKKβ. In contrast IKAP was complexed with several distinct polypeptides. Overexpression of either IKKγ or IKAP blocked tumor necrosis factor α induction of an NF-κB-dependent reporter construct, but IKAP in addition affected several NF-κB-independent promoters. Whereas specific down-regulation of IKKγ protein levels by antisense oligonucleotides significantly reduced cytokine-mediated activation of the IKK complex and subsequent NF-κB activation, a similar reduction of IKAP protein levels had no effect on NF-κB signaling. Using solely IKKα, IKKβ, and IKKγ, we could reconstitute a complex whose apparent molecular weight is comparable to that of the endogenous IKK complex. We conclude that while IKKγ is a stoichiometric component of the IKK complex, obligatory for NF-κB signaling, IKAP is not associated with IKKs and plays no specific role in cytokine-induced NF-κB activation. A critical step in the activation of NF-κB is the phosphorylation of IκBs by the IκB kinase (IKK) complex. IKKα and IKKβ are the two catalytic subunits of the IKK complex and two additional molecules, IKKγ/NEMO and IKAP, have been described as further integral members. We have analyzed the function of both proteins for IKK complex composition and NF-κB signaling. IKAP and IKKγ belong to distinct cellular complexes. Quantitative association of IKKγ was observed with IKKα and IKKβ. In contrast IKAP was complexed with several distinct polypeptides. Overexpression of either IKKγ or IKAP blocked tumor necrosis factor α induction of an NF-κB-dependent reporter construct, but IKAP in addition affected several NF-κB-independent promoters. Whereas specific down-regulation of IKKγ protein levels by antisense oligonucleotides significantly reduced cytokine-mediated activation of the IKK complex and subsequent NF-κB activation, a similar reduction of IKAP protein levels had no effect on NF-κB signaling. Using solely IKKα, IKKβ, and IKKγ, we could reconstitute a complex whose apparent molecular weight is comparable to that of the endogenous IKK complex. We conclude that while IKKγ is a stoichiometric component of the IKK complex, obligatory for NF-κB signaling, IKAP is not associated with IKKs and plays no specific role in cytokine-induced NF-κB activation. tumor necrosis factor α interleukin 1β IκB kinase IKK-associated protein reverse transcriptase-polymerase chain reaction dithiothreitol polyacrylamide gel electrophoresis hemagglutinin polymerase II NF-κB transcription factors play a pivotal role in many cellular processes such as inflammation, immune response, cell proliferation, and apoptosis (1Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2935) Google Scholar, 2May M.J. Ghosh S. Semin. Cancer Biol. 1997; 8: 63-73Crossref PubMed Scopus (337) Google Scholar, 3Luque I. Gelinas C. Semin. Cancer Biol. 1997; 8: 103-111Crossref PubMed Scopus (140) Google Scholar, 4Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5592) Google Scholar, 5Wulczyn F.G. 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Phosphorylated IκBs are bound by a βTrCP containing ubiquitin ligase (E3) complex, polyubiquitinated and subsequently degraded by the 26 S proteasome (7Maniatis T. Genes Dev. 1999; 13: 505-510Crossref PubMed Scopus (369) Google Scholar). Most NF-κB-inducing stimuli trigger activation of an IκB kinase (IKK) complex with a high apparent molecular mass of 700–900 kDa (8Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1855) Google Scholar, 9Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (307) Google Scholar), which has specificity for the amino-terminal phosphoacceptor sites in IκBα or -β. The kinase complex contains two catalytic subunits termed IKKα (IKK1) and IKKβ (IKK2) (8Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. 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Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar). Both kinases are stimulated by proinflammatory cytokines and their activation kinetics match that of IκBα phosphorylation. Highly purified recombinant IKKα and IKKβ can phosphorylate IκBα and IκBβ directly at the correct sites, thus no further downstream kinases are required for IκB phosphorylation (15Li J. Peet G.W. Pullen S.S. Schembri King J. Warren T.C. Marcu K.B. Kehry M.R. Barton R. Jakes S. J. Biol. Chem. 1998; 273: 30736-30741Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16Zandi E. Chen Y. Karin M. Science. 1998; 281: 1360-1363Crossref PubMed Google Scholar). IKKα and IKKβ also inducibly phosphorylate the NF-κB precursor protein p105 at three carboxyl-terminal serines and thereby trigger proteolysis of the precursor (17Heissmeyer V. Krappmann D. Wulczyn F.G. Scheidereit C. EMBO J. 1999; 18: 4766-4778Crossref PubMed Scopus (173) Google Scholar). The IKK complex appears to contain a IKKα/β heterodimer (9Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (307) Google Scholar), although in some cell types IKKβ homodimers are found as well (18Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar). The recent generation of IKKα- or IKKβ-deficient mice has established the requirement of IKKβ for activation of NF-κB by proinflammatory stimuli (19Tanaka M. Fuentes M.E. Yamaguchi K. Durnin M.H. Dalrymple S.A. Hardy K.L. Goeddel D.V. Immunity. 1999; 10: 421-429Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 20Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (857) Google Scholar, 21Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Crossref PubMed Scopus (824) Google Scholar). In contrast, IKKα was found to be dispensable for these stimuli but was essential for morphogenic functions, including differentiation and proliferation of epidermal keratinocytes and skeletal development (22Hu Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Crossref PubMed Scopus (713) Google Scholar, 23Li Q. Lu Q. Hwang J.Y. Buscher D. Lee K.F. Izpisua Belmonte J.C. Verma I.M. Genes Dev. 1999; 13: 1322-1328Crossref PubMed Scopus (418) Google Scholar, 24Takeda K. Takeuchi O. Tsujimura T. Itami S. Adachi O. Kawai T. Sanjo H. Yoshikawa K. Terada N. Akira S. Science. 1999; 284: 313-316Crossref PubMed Scopus (538) Google Scholar). The IKKα knock-out model also demonstrated that the signal responsiveness and activity of the resulting IKKβ homodimer in these animals is fully functional. A predominant role of IKKβ for proinflammatory signaling is also evident from the observation that mutation of two amino acids in the activation loop of IKKβ, but not in IKKα, blocks IKK activation by NIK or cytokines (25Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (754) Google Scholar). In addition to the two kinases, the IKK complex has been reported to contain regulatory subunits. IKKγ (NEMO, IKKAP1) has been obtained by complementation cloning (26Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar) and by microsequencing of the purified protein (18Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar, 27Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar). Murine IKKγ (NEMO) restored the defect of mutant cell lines which had lost the ability to activate NF-κB (26Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar). IKKγ has an extended coiled-coil structure prediction, forms dimers and trimers in vitro (26Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar, 27Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar), and directly binds to IKKβ but not to IKKα (27Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar). IKKγ is required for activation of NF-κB by TNFα, IL-1β, lipopolysaccharide, phorbol 12-myristate 13-acetate, double stranded RNA, or the human T-cell lymphotrophic virus (HTLV-1) Tax, as shown with IKKγ-deficient cells or by overexpression of antisense cDNA (26Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar, 27Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar). Most, if not all of the cellular IKKα and -β seems to be bound to IKKγ, the absence of which results in a shift of the apparent molecular mass of the IKK complex from 800–900 to 300–400 kDa (26Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar). A carboxyl-terminal truncated IKKγ, which still binds to IKKα and -β, acts as an inhibitor of cytokine-induced, but not of basal IKK kinase activity, when overexpressed (27Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar). The COOH-terminal domains containing a leucine zipper and potential zinc finger could thus be interaction sites for signal transmitting molecules which activate IKKα or -β (18Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar, 27Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar). IKKγ was also isolated in a yeast two-hybrid screen using the adenovirus-encoded E3-14.7K protein as a bait and was shown to interact with RIP and NIK in transfected cells (28Li Y. Kang J. Friedman J. Tarassishin L. Ye J. Kovalenko A. Wallach D. Horwitz M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1042-1047Crossref PubMed Scopus (157) Google Scholar). The same adenovirus protein also interacts with Fip2, which shares 40% similarity with IKKγ and also contains coiled coils and a leucine zipper (29Li Y. Kang J. Horwitz M.S. Mol. Cell. Biol. 1998; 18: 1601-1610Crossref PubMed Scopus (177) Google Scholar). A further IKK-associated protein (IKAP) was isolated by affinity purification of the IKK complex using immobilized IκBα (30Cohen L. Henzel W.J. Baeuerle P.A. Nature. 1998; 395: 292-296Crossref PubMed Scopus (271) Google Scholar). IKAP is a 150-kDa protein with an amino-terminal WD40-like repeat domain. IKAP was found to co-elute with large molecular size range proteins (900 kDa) and co-eluted with IKK activity. IKAP co-purified with IKKα, IKKβ, NIK, RelA, IκBα, and further proteins of 105, 100, 82, 80, 65, and 58 kDa. IKAP was reported to directly and independently interact with recombinant or transfected IKKα, IKKβ, and NIK. The sequestration of IKKs and NIK suggested a function of IKAP as a scaffold protein (30Cohen L. Henzel W.J. Baeuerle P.A. Nature. 1998; 395: 292-296Crossref PubMed Scopus (271) Google Scholar). It is not clear whether all endogenous IKK complexes associate with IKAP or only a subset of IKKs. Furthermore, it is not known whether IKKγ and IKAP are present in the same IKK complexes and how many other components are commonly associated with heterodimers of IKKα and IKKβ. In this study we have analyzed the composition of cellular IKKα·IKKβ complexes. We come to the conclusion that with regard to composition the major IKK complex is only tripartite and consists exclusively of IKKα, -β, and -γ. With in vitro reconstitution experiments we show that an IKKα·β·γ complex displays a gel filtration profile which, like that of endogenous IKK complexes, corresponds to a large size of more than 800 kDa relative to standard proteins. Various protein-protein interaction and functional assays demonstrate that cellular IKK complexes do not contain IKAP as an intrinsic component. IKAP appears as part of a novel complex containing additional proteins of 100, 70, 45, and 39 kDa. Overexpression of IKAP interferes with the activity of a set of different NF-κB-dependent as well as independent reporter genes, suggesting a function in a more general gene expression mechanism. HeLa and 293 cells were grown in Dulbeco's modified Eagle's medium supplemented with 10% fetal calf serum, 1 mm sodium pyruvate, and 100 units/ml penicillin/streptomycin. For stimulation, cells were treated with 20 ng/ml TNFα (Biomol) or 10 ng/ml IL-1β (Promega). 293 cells were transiently transfected by the calcium phosphate precipitation method as described previously (17Heissmeyer V. Krappmann D. Wulczyn F.G. Scheidereit C. EMBO J. 1999; 18: 4766-4778Crossref PubMed Scopus (173) Google Scholar). DNA constructs and amounts are indicated in the figure legend. Stimulation using 50 ng/ml TNFα was carried out 6 h prior to lysis. Cells were lysed 24 h after transfection and luciferase measurement was done with the dual luciferase reporter kit (Promega) according to the manufacturer's protocol. For transfection of antisense oligonucleotides (GeneBlocs) HeLa cells were plated in 96-well dishes at 4000 cells/well the evening before transfection. Cells were transfected in triplicate with antisense or control oligonucleotides for up to 72 h in the presence of serum using a lipid-based delivery system (Atugen AG). Transfection efficiency was greater than 80% (data no shown). Cells were harvested and RNA or protein extracts were prepared 48 h after transfection. Relative amounts of mRNA were determined by Real Time TaqManTM PCR analysis using the ABI Prism 7700 system (PE Applied Biosystems). IKKγ was amplified by PCR from a BJAB cDNA and cloned into theHindIII/BamHI pcDNA3 (Invitrogen), which contains a sequence coding for a HA epitope cloned intoBamHI/XbaI. For bacterial expression IKKγ cDNA was cloned into pGEX-6-P1 (Amersham Pharmacia Biotech). pRL-TKluc was obtained from Promega. Flag-IKAPpRK5, ELAMlucpRK5, pRcHA-IKKα, and pRcHA-IKKβ were described elsewhere (13Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1595) Google Scholar, 30Cohen L. Henzel W.J. Baeuerle P.A. Nature. 1998; 395: 292-296Crossref PubMed Scopus (271) Google Scholar). Antisense oligonucleotides (GeneBlocs) complementary to IKKγ and IKAP mRNA were generated (Atugen AG): 17794:24058 GB3.3 (5′-GTTTGAGATCTTCCAGCTGCATT-3′); 17783:24047 GB3.3 (5′-AGCACTTGGACAATCACCACATT-3′); 17785:24049 GB3.3 (5′-GACACCTGTCAGTCAGACCAAGG-3′). A randomized control oligonucleotide GBC 5′-{N}23-3′ was used in parallel. Anti-IKKγ antibody was raised against a peptide comprising amino acids 57–72 of the human IKKγ molecule and anti-IKAP antibody was raised against a peptide comprising the very carboxyl terminus of human IKAP (amino acids 1313–1332). Peptide synthesis and immunization was done by Eurogentec. Furthermore, IKKγ antibody (FL-419) from Santa Cruz was used for Western blotting. Polyclonal antibodies against p65 (A), IκBα (C-21), IκBβ (N-20), HA (Y-11) were obtained from Santa Cruz. Monoclonal antibodies were as follows: IKKα antibody (B78-1) and mouse IgG1isotype control, Pharmingen; IKKβ antibody, 10AG2,BIOSOURCE; Flag antibody, M5, Sigma. Whole cell extracts were analyzed by electrophoretic mobility shift assay and Western blotting essentially as described previously (31Krappmann D. Wulczyn F.G. Scheidereit C. EMBO J. 1996; 15: 6716-6726Crossref PubMed Scopus (180) Google Scholar). For the preparation of cytoplasmic, nuclear and chromatin extracts HeLa cells were washed with phosphate-buffered saline and swollen in buffer A (1 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 1 mm dithiothreitol (DTT) plus protease inhibitors, 0.4 mm Pefabloc, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A) for 10 min on ice. 0.15% Nonidet P-40 was added, thoroughly mixed, and spun down in a microcentrifuge. The supernatant was centrifuged for 10 min at 14,000 rpm and after addition of 10% glycerol used as cytoplasmic extract. The nuclei were shaken for 15 min in buffer C (20 mm HEPES, pH 7.9, 20% glycerol, 0.42 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 1 mm DTT, plus protease inhibitors (see above)) and after centrifugation for 10 min at 14,000 rpm the supernatant was used as a nuclear extract. The pellet was washed once with ∼10 volumes of buffer C and directly taken up and boiled for 15 min in SDS loading buffer to elute proteins that are tightly bound to chromatin. Gel filtration analysis of endogenous proteins was either carried out from whole cell extracts of HeLa cells or cytoplasmic extracts of 70Z/3, 1.3E2 cells, or extracts of transiently transfected 293 cells. ∼2 × 107 HeLa cells were lysed in a 300-μl volume of 100 mm Tris, pH 7.5, 300 mm NaCl, 0.3% Nonidet P-40, 2 mm EDTA, 1 mm DTT, 10 mm NaF, 8 mm β-glycerophosphate, 0.1 mm orthovanadate, 10% glycerol plus protease inhibitors (0.4 mm Pefabloc, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A). 300 μl of low salt buffer (10 mm Tris, pH 7.5, 1 mm DTT plus protease inhibitors, see above) were added for dilution and 500 μl were applied to the gel filtration. Extracts from 70Z/3 cells and 1.3E2 cells were prepared as described (26Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar). 6 mg of protein were analyzed by gel filtration. 100-mm plates of 293 cells were transiently transfected using calcium phosphate precipitation. Cells were lysed 48 h after transfection in 50 mm Tris, pH 7.5, 100 mmNaCl, 0.1% Nonidet P-40, 1 mm EDTA, 5% glycerol, 10 mm NaF, 8 mm β-glycerophosphate, 0.1 mm orthovanadate and protease inhibitors (see above). Equal volumes for each transfection were used for gel filtration analysis. Bacterially expressed glutathione S-transferase-IKKγ was bound to glutathione-Sepharose 4B and glutathioneS-transferase was cleaved by PreScission protease (Amersham Pharmacia Biotech). Equal amounts (∼200 ng) of purified baculovirally expressed IKKα (15Li J. Peet G.W. Pullen S.S. Schembri King J. Warren T.C. Marcu K.B. Kehry M.R. Barton R. Jakes S. J. Biol. Chem. 1998; 273: 30736-30741Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and/or purified bacterially expressed IKKγ were mixed and incubated on ice for 45 min in 100 μl of BC-100 containing 50 mm Tris, pH 7.5, 100 mm NaCl, 1 mm EDTA, 1 mm DTT, and 5 μg of bovine serum albumin before gel filtration analysis. All gel filtration chromatography was carried out on a Superose 6 column (Amersham Pharmacia Biotech). 500-μl fractions were recovered and every other fraction was analyzed by Western blotting. The column was calibrated with the molecular mass marker proteins thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa) (Amersham Pharmacia Biotech). For in vitroco-immunoprecipitations proteins were translated in rabbit reticulocyte lysate in the presence of [35S]methionine using thein vitro transcription/translation kit from Promega. For immunoprecipitations, 3–10 μl of the translated products were mixed and preincubated for 1 h at 4 °C in HEPES, pH 7.9, 100 mm KCl, 0.5% Nonidet P-40, 0.5 mm EDTA, 0.5 mm DTT, 0.4 mm Pefabloc, and 1 μg/ml leupeptin, pepstatin, and aprotinin. After pre-clearance with protein A-Sepharose for 1 h at 4 °C, HA or Flag antibody and fresh protein A-Sepharose were added and incubated for another hour. The samples were extensively washed in immunoprecipitation buffer, resuspended in SDS loading buffer, and analyzed by SDS-PAGE and autoradiography. For co-immunoprecipitation of endogenous proteins whole cell extracts of HeLa cells were prepared using 100 mm Tris, pH 7.5, 300 mm NaCl, 0.3% Nonidet P-40, 2 mm EDTA, 1 mm DTT, 10 mm NaF, 8 mmβ-glycerophosphate, 0.1 mm orthovanadate, 10% glycerol plus protease inhibitors (see above). Extracts were diluted 1:1 with 10 mm Tris, pH 7.5, 1 mm DTT plus protease inhibitors (see above). After preclearing for 1 h with protein A-Sepharose the extracts were incubated for 4 h with the antibodies and protein A-Sepharose. Precipitates were washed four times with 50 mm Tris, pH 7.5, 150 mm NaCl, 0.15% Nonidet P-40, 1 mm EDTA, boiled in SDS loading buffer, and analyzed by Western blotting. HeLa cells were labeled for 5 h with 100 μCi/ml [35S]methionine essentially as described previously (32Krappmann D. Emmerich F. Kordes U. Scharschmidt E. Dorken B. Scheidereit C. Oncogene. 1999; 18: 943-953Crossref PubMed Scopus (238) Google Scholar). Cells were lysed in 20 mm Tris, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40, 1 mm EDTA, 1 mm DTT, 10 mmNaF, 8 mm β-glycerophosphate, 0.1 mmorthovanadate, 10% glycerol plus protease inhibitors (0.4 mm Pefabloc, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A). Extracts were precleared with protein A-Sepharose for 1 h and immunoprecipitation was carried out overnight at 4 °C. Precipitates were washed three times with lysis buffer and boiled for 5 min in SDS loading buffer. The supernatant was applied on a SDS-PAGE and analyzed by autoradiography. To assay for IκB kinase activity, HeLa cells were transfected with antisense oligonucleotides against IKAP (GB47), IKKγ (GB58), or control GeneBlocs (GBC) and 48 h later the cells were treated for 5 min with TNFα or IL-1β. Cellular lysis, immunoprecipitation using IKKα antibody, and the in vitro kinase assay were performed exactly as described previously (32Krappmann D. Emmerich F. Kordes U. Scharschmidt E. Dorken B. Scheidereit C. Oncogene. 1999; 18: 943-953Crossref PubMed Scopus (238) Google Scholar). IKKγ and IKAP have each been suggested to stably associate with IKKα and IKKβ (26Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar, 27Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar, 30Cohen L. Henzel W.J. Baeuerle P.A. Nature. 1998; 395: 292-296Crossref PubMed Scopus (271) Google Scholar). To analyze the contribution of IKKγ and IKAP we tested if both proteins are part of the same complex with IKKα and IKKβ in whole cell extracts from HeLa cells fractionated by gel filtration (Fig.1 A). IKKα, IKKβ, and IKKγ had an identical elution profile (fractions 12–16), with an apparent molecular mass between 700 and 900 kDa relative to molecular mass markers, as reported earlier (26Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar). In contrast, IKAP was predominantly found in fractions containing proteins with lower apparent molecular mass (fractions 16–20), while only small amounts of IKAP co-eluted with the IKK complex and trailed into still much larger apparent sizes. In fact, IKAP peak elution roughly coincided with NF-κB·IκB complexes at an apparent molecular mass of 440–670 kDa (fractions 18–22). However, no association of IKAP and NF-κB·IκB complexes was observed after affinity purification with a p65 antibody (data not shown). Next we asked, whether the IKK complex disruption seen in IKKγ-deficient cells (26Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar) would affect the gel filtration elution profile of IKAP. We analyzed 70Z/3 cells and the 70Z/3-derived mutant cell line, 1.3E2, that exhibits impaired NF-κB activation and lacks IKKγ (26Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar, 33Rooney J.W. Emery D.W. Sibley C.H. Immunogenetics. 1990; 31: 73-78Crossref PubMed Scopus (19) Google Scholar, 34Courtois G. Whiteside S.T. Sibley C.H. Israel A. Mol. Cell. Biol. 1997; 17: 1441-1449Crossref PubMed Google Scholar). In 1.3E2 cells, IKKα and -β elute at smaller apparent molecular masses, between 450 and 600 kDa (Fig.1 B). Thus, loss of IKKγ in 1.3E2 cells leads to a similar migration decrease of the IKKα·β complex compared with the parental 70Z/3 cells, as that observed between Tax-transformed Rat-1 fibroblasts and their IKKγ lacking variant (26Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar). Despite the shift of IKKα peak elution from fraction 14 to fraction 18 in the absence of IKKγ, the elution of IKAP complexes remained unchanged with maximal elution around fraction 18. This strongly suggests that IKAP is not part of a regular complex with IKKα and IKKγ. To compare associations between IKKs and IKAP or IKKγ directly, we performed co-immunoprecipitations of in vitro translated epitope-tagged proteins (Fig.2 A). HA-IKKγ was mixed with Flag-IKAP. Whereas HA antibody efficiently precipitated HA-IKKγ and Flag antibody pulled down Flag-IKAP, neither protein was co-precipitated with the other (lanes 1–4). We also added Myc-tagged IKKα and IKKβ to the IKKγ and IKAP mixture (lanes 5–8). IKKα and IKKβ could be efficiently co-precipitated with HA-IKKγ, but under these conditions no significant association was seen with IKAP after precipitation with either HA or Flag antibodies. Similar results were obtained in transiently transfected 293 cells, where we observed tight association between IKKα, IKKβ, and IKKγ, but only a very weak association between IKKα, IKKβ, and IKAP (data not shown). These observations raised the question whether association between cellular IKKs and IKAP could be detected in intact cells. Whole cell extracts of HeLa cells were used for immunoprecipitations and subsequent Western blotting (Fig. 2 B). IKAP was specifically precipitated with the IKAP antibody (lane 3), but only very small amounts of IKAP were precipitated with antibodies directed against either IKKα or IKKγ (lanes 5 and 6) which were also seen with the IgG1 control (lane 4). Furthermore, the IKAP antibody did not co-precipitate IKKα (lane 3). Conversely, co-immunoprecipitation of IKKα and IKKγ using identical conditions was observed with either antibody (lanes 5 and6). We therefore conclude that IKAP is not stably associated with the IKK complex. Since NF-κB activation by the IKK complex is a cytoplasmic process, components which are involved in this process are expected to reside in the cytoplasm. We analyzed the cytoplasmic and nuclear distribution of IKAP, IKKα, and IKKγ and p65 in HeLa cells (Fig.3 A). Whereas IKKα, IKKγ, IKAP, and p65 were all found predominantly in
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