Characterization of the IκB-kinase NEMO Binding Domain
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m206494200
ISSN1083-351X
AutoresMichael J. May, Ralf Marienfeld, Sankar Ghosh,
Tópico(s)Cell Adhesion Molecules Research
ResumoProinflammatory activation of NF-κB requires an upstream kinase complex (IκB-kinase; IKK) composed of two catalytic subunits (IKKα and IKKβ) and a noncatalytic regulatory component named NEMO (NF-κB essentialmodulator). NEMO interacts with a COOH-terminal sequence within both IKKs termed the NEMO-binding domain (NBD), and a cell-permeable NBD peptide blocks NEMO/IKKβ interactions and inhibits tumor necrosis factor-α-induced NF-κB. We report here that a peptide encompassing the NBD not only blocked association of both IKKs with NEMO but also disrupted preformed NEMO/IKK complexes in vitro. Furthermore, peptide blocking and alanine-scanning mutation studies revealed differences between the NBDs of IKKα and IKKβ, and mutational analysis of the IKKβ NBD identified the physical properties required at each position to maintain association with NEMO. Finally, we demonstrate that loss of NEMO-binding by IKKβ through deletion of the NBD renders it catalytically active and that potential phosphorylation within the IKKβ NBD may serve as a signal to down-regulate IKK activity. Our findings therefore provide critical insight into the physical properties of the NBD that will be valuable for the design of drugs aimed at disrupting the IKK complex and also reveal potential regulatory mechanisms controlling the function of the IKK complex. Proinflammatory activation of NF-κB requires an upstream kinase complex (IκB-kinase; IKK) composed of two catalytic subunits (IKKα and IKKβ) and a noncatalytic regulatory component named NEMO (NF-κB essentialmodulator). NEMO interacts with a COOH-terminal sequence within both IKKs termed the NEMO-binding domain (NBD), and a cell-permeable NBD peptide blocks NEMO/IKKβ interactions and inhibits tumor necrosis factor-α-induced NF-κB. We report here that a peptide encompassing the NBD not only blocked association of both IKKs with NEMO but also disrupted preformed NEMO/IKK complexes in vitro. Furthermore, peptide blocking and alanine-scanning mutation studies revealed differences between the NBDs of IKKα and IKKβ, and mutational analysis of the IKKβ NBD identified the physical properties required at each position to maintain association with NEMO. Finally, we demonstrate that loss of NEMO-binding by IKKβ through deletion of the NBD renders it catalytically active and that potential phosphorylation within the IKKβ NBD may serve as a signal to down-regulate IKK activity. Our findings therefore provide critical insight into the physical properties of the NBD that will be valuable for the design of drugs aimed at disrupting the IKK complex and also reveal potential regulatory mechanisms controlling the function of the IKK complex. Activation of the inducible transcription factor NF-κB is an essential signal transduction pathway that is rapidly and transiently elicited in response to proinflammatory cytokines. Many genes that are induced or up-regulated by proinflammatory stimuli contain specific NF-κB binding sites (κB sites) within their promoters (1Karin M. Delhase M. Semin. Immunol. 2000; 12: 85-98Crossref PubMed Scopus (860) Google Scholar, 2Ghosh S. Karin M. Cell. 2002; 109: 81-96Abstract Full Text Full Text PDF PubMed Scopus (3292) Google Scholar). Hence, cytokines generated during inflammatory responses such as interleukin-1 and tumor necrosis factor (TNF-α), 1The abbreviations used for: TNF-α, tumor necrosis factor-α; IκB, inhibitor of κB; IKK, IκB kinase; NBD, NEMO-binding domain; GST, glutathioneS-transferase; HLH, helix-loop-helix. induce altered patterns of gene expression that are dependent upon the activity of NF-κB. It is perhaps not surprising therefore that the NF-κB activation pathway has emerged as an extremely attractive target for the development of anti-inflammatory drugs (3D'Acquisto F. May M.J. Ghosh S. Mol. Intervent. 2002; 2: 22-35Crossref PubMed Scopus (202) Google Scholar). Immense effort from many workers has revealed crucial details of the molecular mechanisms underlying proinflammatory activation of NF-κB (2Ghosh S. Karin M. Cell. 2002; 109: 81-96Abstract Full Text Full Text PDF PubMed Scopus (3292) Google Scholar). In the majority of resting cells, NF-κB remains sequestered in the cytoplasm through association with members of an inhibitory family of proteins named inhibitors of κB (IκB) that are typified by IκBα. Stimuli that activate NF-κB induce phosphorylation of IκB proteins at two specific NH2-terminal serine residues that “tags” the proteins for ubiquitination and subsequent degradation by the proteasome. Loss of bound IκB reveals a nuclear localization sequence on NF-κB allowing it to translocate to the nucleus, where it binds to DNA and interacts with distinct combinations of co-factors and other transcription factors to initiate or up-regulate gene expression. Various additional regulatory mechanisms such as stimulus-induced phosphorylation of the NF-κB p65 subunit that renders it transcriptionally active (4Madrid L.V. Wang C.Y. Guttridge D.C. Schottelius A.J. Baldwin Jr., A.S. Mayo M.W. Mol. Cell. Biol. 2000; 20: 1626-1638Crossref PubMed Scopus (587) Google Scholar, 5Wang D. Westerheide S.D. Hanson J.L. Baldwin Jr., A.S. J. Biol. Chem. 2000; 275: 32592-32597Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 6Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar) and repression by DNA-bound NF-κB complexes associated with histone deacetylases (7Zhong H. May M.J. Jimi E. Ghosh S. Mol. Cell. 2002; 9: 625-636Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar, 8Ashburner B.P. Westerheide S.D. Baldwin Jr., A.S. Mol. Cell. Biol. 2001; 21: 7065-7077Crossref PubMed Scopus (627) Google Scholar) play critical roles in governing the NF-κB activation pathway. Arguably, however, the most important point of control of proinflammatory NF-κB activity is the activation and regulation of the kinase responsible for IκB phosphorylation. IκB-kinase (IKK) functional activity resides in a high molecular weight complex composed of a core of three separate protein subunits (1Karin M. Delhase M. Semin. Immunol. 2000; 12: 85-98Crossref PubMed Scopus (860) Google Scholar, 2Ghosh S. Karin M. Cell. 2002; 109: 81-96Abstract Full Text Full Text PDF PubMed Scopus (3292) Google Scholar). Catalytic function of the complex is provided by two of the subunits, named IKKα and IKKβ, that exhibit striking structural similarity. These kinases are of comparable size, with IKKβ being slightly longer (756 amino acids compared with 745 for IKKα), and they share ∼52% identity throughout their entire length. IKKα and IKKβ both contain a single NH2-terminal catalytic domain, a centrally positioned leucine zipper motif through which they heterodimerize, and COOH-terminal helix-loop-helix (HLH) domain that may function to regulate the catalytic activity of the IKK complex (9Kwak Y.T. Guo J. Shen J. Gaynor R.B. J. Biol. Chem. 2000; 275: 14752-14759Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar,10Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (752) Google Scholar). Despite their structural similarities, elegant genetic studies have demonstrated that IKKα and IKKβ play distinct roles with respect to NF-κB activation. Thus, IKKβ is responsible for proinflammatory cytokine-induced IκBα phosphorylation and subsequent activation of “classical” NF-κB complexes containing the p50 and p65 subunits (11Chu W.M. Ostertag D. Li Z.W. Chang L. Chen Y. Hu Y. Williams B. Perrault J. Karin M. Immunity. 1999; 11: 721-731Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 12Li 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 (822) Google Scholar, 13Senftleben U. Li Z.W. Baud V. Karin M. Immunity. 2001; 14: 217-230Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 14Tanaka 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), whereas IKKα plays a significant although poorly defined role in keratinocyte differentiation that is independent of its catalytic activity and NF-κB activation (15Li 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 (417) Google Scholar, 16Hu Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Crossref PubMed Scopus (714) Google Scholar, 17Takeda 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 (539) Google Scholar). Recently, however, it has been demonstrated that IKKα catalytic activity is required for RANKL (receptoractivator of NF-κBligand)-induced NF-κB activation in mammary epithelial cells (18Cao Y. Bonizzi G. Seagroves T.N. Greten F.R. Johnson R. Schmidt E.V. Karin M. Cell. 2001; 107: 763-775Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar) and plays a crucial role in B-lymphocyte development (19Kaisho T. Takeda K. Tsujimura T. Kawai T. Nomura F. Terada N. Akira S. J. Exp. Med. 2001; 193: 417-426Crossref PubMed Scopus (157) Google Scholar,20Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1136) Google Scholar). Intriguingly, however, the role of IKKα in B-cell development does not involve IκB phosphorylation, but it appears to function as an upstream kinase in a pathway leading to processing of the NF-κB2/p100 subunit to p52 (20Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1136) Google Scholar). The third protein within the IKK complex is named NEMO (NF-κB essential modulator) but is also referred to as IKKγ, although it does not possess a catalytic domain (21Yamaoka 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 (950) Google Scholar, 22Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar, 23Mercurio 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). Evidence obtained from genetically altered mice clearly demonstrates that NEMO is absolutely critical for proinflammatory activation of the IKK complex (24Rudolph D. Yeh W.C. Wakeham A. Rudolph B. Nallainathan D. Potter J. Elia A.J. Mak T.W. Genes Dev. 2000; 14: 854-862PubMed Google Scholar, 25Schmidt-Supprian M. Bloch W. Courtois G. Addicks K. Israel A. Rajewsky K. Pasparakis M. Mol. Cell. 2000; 5: 981-992Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 26Makris C. Godfrey V.L. Krahn-Senftleben G. Takahashi T. Roberts J.L. Schwarz T. Feng L. Johnson R.S. Karin M. Mol. Cell. 2000; 5: 969-979Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Furthermore, mutations within NEMO that render it disfunctional have been associated with two X-linked pathologies named incontinentia pigmenti and “ectodermal dysplasia associated with immunodeficiency” (25Schmidt-Supprian M. Bloch W. Courtois G. Addicks K. Israel A. Rajewsky K. Pasparakis M. Mol. Cell. 2000; 5: 981-992Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 27Smahi A. Courtois G. Vabres P. Yamaoka S. Heuertz S. Munnich A. Israel A. Heiss N.S. Klauck S.M. Kioschis P. Wiemann S. Poustka A. Esposito T. Bardaro T. Gianfrancesco F. Ciccodicola A. D'Urso M. Woffendin H. Jakins T. Donnai D. Stewart H. Kenwrick S.J. Aradhya S. Yamagata T. Levy M. Lewis R.A. Nelson D.L. Nature. 2000; 405: 466-472Crossref PubMed Scopus (607) Google Scholar,28Jain A. Ma C.A. Liu S. Brown M. Cohen J. Strober W. Nat. Immunol. 2001; 2: 223-228Crossref PubMed Scopus (326) Google Scholar). Thus, NEMO remains the only protein known to function in the NF-κB pathway that has been linked to human genetic diseases. Nevertheless, the precise function of NEMO in IKK activation is not yet known. It has been proposed that NEMO plays a regulatory role by recruiting the IKK complex to ligated cytokine receptors, where IKKs may be activated by as yet poorly defined mechanisms that possibly include oligomerization, conformational changes, or proximity-induced transphosphorylation events (29Ye J. Xie X. Tarassishin L. Horwitz M.S. J. Biol. Chem. 2000; 275: 9882-9889Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 30Li X.H. Fang X. Gaynor R.B. J. Biol. Chem. 2001; 276: 4494-4500Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 31Poyet J.L. Srinivasula S.M. Lin J.H. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 32Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 33Devin A. Lin Y. Yamaoka S. Li Z. Karin M. Liu Z. Mol. Cell. Biol. 2001; 21: 3986-3994Crossref PubMed Scopus (129) Google Scholar). Additionally or alternatively, NEMO may facilitate the recruitment of upstream IKK activators such as kinases that specifically target the activation loops within the catalytic domains of the IKK subunits or enable the interaction of the IKKs with IκB proteins (22Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar, 34Yamamoto Y. Kim D.W. Kwak Y.T. Prajapati S. Verma U. Gaynor R.B. J. Biol. Chem. 2001; 276: 36327-36336Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In a previous study, we identified the molecular mechanism through which NEMO interacts with both IKKs (35May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Crossref PubMed Scopus (618) Google Scholar). We found that an NH2-terminal α-helical region of NEMO associates with a hexapeptide sequence (Leu-Asp-Trp-Ser-Trp-Leu) within the extreme COOH terminus of both kinases and named this region the NEMO-binding domain (NBD). Furthermore, a short cell-permeable peptide spanning the IKKβ NBD disrupted the association of NEMO with IKKβ in vitro, blocked TNF-α-induced NF-κB activation in cells, and effectively ameliorated responses in distinct animal models of inflammation. Hence, we have proposed that the NBD is an attractive target for the development of anti-inflammatory drugs aimed at disrupting the IKK complex. For such drugs to be developed, it will be necessary to obtain a clear understanding of the relative contributions and importance of each amino acid within the NBD to the interaction with NEMO. Therefore, to understand the NBD in greater detail and to investigate the function of NEMO in the IKK complex, we report here the results of in vitro peptide blocking studies and an in depth mutational analysis of the IKKβ NBD. Conservative or nonconservative substitution mutations of critical NBD residues demonstrated the required nature of the amino acids at each position for binding function and revealed significant differences between the NBDs of IKKα and IKKβ. Furthermore, we found that loss of NEMO binding function through deletion of the NBD rendered IKKβ as catalytically active as the TNF-α-induced kinase and that a mutation mimicking phosphorylation within the IKKβ NBD reduced its ability to activate NF-κB. We therefore conclude that in addition to being critical for proinflammatory IKK activation, association with NEMO may also play a significant role in maintaining the basal activity of the IKK complex. HeLa and COS cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 2 mml-glutamine, penicillin (50 units/ml), and streptomycin (50 μg/ml). Mouse anti-FLAG (M2) and anti-FLAG-coupled agarose beads were purchased from Sigma. Mouse Anti-Xpress was purchased from Invitrogen, and rabbit anti-NEMO was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The horseradish peroxidase-conjugated secondary antibodies against either rabbit or mouse IgG were both from AmershamBiosciences. TNF-α was purchased from R & D Systems. Small scale Fmoc synthesis of peptides was carried out on a Rainin Symphony instrument at the HHMI Biopolymer-Keck Foundation Biotechnology Resource Laboratory at Yale University. Peptides were characterized by matrix-assisted laser desorption ionization mass spectrometry and analytical reverse phase high pressure liquid chromatography analysis. Peptides were dissolved in Me2SO to stocks of between 20 and 50 mm. Full-length cDNA clones of IKKα and IKKβ were generous gifts from Dr. Michael Karin (University of California, San Diego). All subcloning and mutagenesis of the IKKs was performed by PCR using cloned Pfu DNA polymerase (Stratagene). All PCRs used primers containing restriction sites that enabled cloning into appropriate vectors (sequences available upon request). The wild-type and mutated IKKβ cDNAs were inserted into the KpnI and NotI restriction sites of pcDNA-3 or pcDNA-3.1-Xpress (Invitrogen), and all IKKα cDNAs were inserted into the EcoRI andXhoI sites of the same vectors. FLAG-tagged IKKβ was constructed by subcloning into pFLAG-CMV-2 (Sigma). A cDNA encoding human NEMO was obtained as previously described (35May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Crossref PubMed Scopus (618) Google Scholar). GST-NEMO was constructed by subcloning the full-length cDNA into theEcoRI and XhoI sites of pGEX-4T1 (AmershamBiosciences). The GST-NEMO protein was then made in Escherichia coli (BL21) by treating transformed bacteria with 0.4 mm isopropyl-1-thio-β-d-galactopyranoside and following the protocol for protein recovery provided with the vector. For GST pull-down analysis, pcDNA-3 containing IKKα or IKKβ was subjected to anin vitro transcription and translation reaction in the presence of [35S]methionine using the TNT-T7 Quick system from Promega. Labeled proteins (1 μl of reticulocyte lysate) were incubated with GST-NEMO (1 μg) in 100 μl of TNT (50 mmTris, pH 7.5, 200 mm NaCl, 1% Triton X-100) at 4 °C for 30 min, and then 20 μl of a 50% (v/v) slurry of glutathione beads (Amersham Biosciences) was added and incubated a further 15 min. Proteins were then precipitated and washed extensively in TNT before the addition of sample buffer (20 μl). Samples were then separated by SDS-PAGE (10%), and resulting gels were fixed and examined autoradiographically. In some experiments, NBD peptides were added to GST-NEMO in TNT either 15 min before or after the addition of the IKK proteins. For transient transfection studies, 1 × 106 COS cells grown in six-well trays were transfected with vector alone (pcDNA3.1-Xpress) or the IKKs together with FLAG-tagged NEMO (1 μg of total DNA) using the Fugene6 transfection reagent (Roche Diagnostics). All DNA/Fugene6 incubations were performed at a ratio of 1 μg of DNA to 3 μl of Fugene6 according to the manufacturer's recommended protocol in OptiMEM medium (Invitrogen). After 48 h, cells were lysed in 500 μl of TNT, and then complexes were immunoprecipitated using anti-FLAG-coupled agarose beads. A portion of each lysate (5%) was retained for analysis (preimmunoprecipitation). Precipitated proteins were analyzed by immunoblotting using epitope-specific (anti-FLAG or anti-Xpress) antibodies that were visualized using enhanced chemiluminescence reagents from AmershamBiosciences. Luciferase reporter assays were performed as described previously (6Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar, 7Zhong H. May M.J. Jimi E. Ghosh S. Mol. Cell. 2002; 9: 625-636Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar, 35May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Crossref PubMed Scopus (618) Google Scholar). Briefly, 2.5 × 105 HeLa cells grown on 12-well plates were transiently transfected using Fugene6 with the NF-κB-dependent reporter construct pBIIx-luciferase (0.2 μg/well) together with either vector alone (control), wild-type IKKβ, or the NBD point or truncation mutants indicated (up to 1.0 μg/well each). For dose-response experiments, the total concentration of transfected DNA was maintained by transfecting appropriate amounts of vector alone (pcDNA-3). Cells were lysed in passive lysis buffer (Promega) 24–48 h after transfection and luciferase activity was measured using a Luciferase assay kit from Promega. For immune complex kinase assays, 1 × 106 HeLa cells grown on six-well plates were transiently transfected with 1 μg of FLAG-IKKβ constructs using the Fugene6 reagent as described above. Forty-eight hours after transfection, the cells were treated with TNF-α (10 ng/ml) for 10 min then lysed on ice in 500 μl of TNT for 15 min. Protein content in each lysate was determined using a Bio-Rad protein assay kit and then normalized between samples. Proteins in lysates were immunoprecipitated using anti-FLAG (M2)-coupled agarose beads for 1 h at 4 °C, and then precipitates were washed extensively in TNT and then kinase buffer (20 mm HEPES, pH 7.5, 20 mm MgCl2, 1 mm EDTA, 2 mm NaF, 2 mmβ-glycerophosphate, 1 mm dithiothreitol, 10 μm ATP). Precipitates were then incubated for 15 min at 30 °C in 20 μl of kinase buffer containing GST-IκBα-(1–90) and 10 μCi of γ-32P-labeled ATP (Amersham Biosciences). The substrate was then precipitated using glutathione-agarose (AmershamBiosciences) and washed extensively with TNT. Beads were then suspended in 20 μl of sample buffer, and samples were separated by SDS-PAGE (10%). Kinase activity was determined by autoradiography. An equal portion of each cell lysate (10%) was precipitated using anti-FLAG (M2)-agarose beads and then processed for immunoblotting using anti-FLAG to ensure equal loading of proteins. In an earlier study, we demonstrated that a cell-permeable NBD peptide blocked the interaction of NEMO with IKKβ (35May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Crossref PubMed Scopus (618) Google Scholar). To further assess the effects of the NBD peptide in the absence of a cell-permeabilizing sequence, we performed in vitrobinding assays using a peptide of only 11 amino acids surrounding the NBD of IKKβ (Fig. 1 A). We previously used a control peptide containing tryptophan (Trp) to arginine (Ala) substitutions that did not inhibit NEMO binding to IKKβ (35May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Crossref PubMed Scopus (618) Google Scholar); however, to maintain the amino acid content of the wild-type peptide, we employed a scrambled control (Fig. 1 A) for the present study. To test the effects of the peptides on the IKK/NEMO interaction, we performed in vitro glutathioneS-transferase (GST) pull-down analysis using a bacterially expressed version of full-length NEMO fused with GST (GST-NEMO) together with in vitro transcribed and translated and35S-labeled IKK proteins. Consistent with our previous findings, preincubation of GST-NEMO with the wild-type NBD peptide dose-dependently inhibited its ability to interact with IKKβ, whereas identical concentrations of the scrambled peptide had no effect (Fig. 1 B). Neither peptide blocked the association of GST-IκBα with 35S-labeled IKKα (data not shown); nor did they disrupt the association of GST-NEMO with the glutathione beads used for precipitation (Fig. 1 B), verifying that the blocking effects are specific for the NBD. Although previous workers suggested that NEMO only interacted with IKKβ within the IKK complex (22Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar, 23Mercurio 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), we found that IKKα also contains a sequence within its extreme COOH terminus identical to the IKKβ NBD (35May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Crossref PubMed Scopus (618) Google Scholar). Moreover, we showed by in vitro pull-down assays that recombinant IKKα could bind to NEMO and that the interaction was maintained by the NBD. We therefore wanted to determine whether the NBD peptide could inhibit the association of NEMO with IKKα, and, as shown in Fig. 1 C we found that the wild-type but not the scrambled peptide effectively blocked the interaction. Identical results from such in vitro analysis were obtained using the cell-permeable NBD peptides (data not shown). Taken together, the experiments depicted in Fig. 1, A–C, reinforce our conclusion that the NBD peptide specifically blocks the interaction of NEMO with the IKK complex and further demonstrate that it prevents NEMO binding to both IKK subunits in vitro. For the preceding experiments, we preincubated GST-NEMO with the NBD peptides for 15 min prior to adding the 35S-labeled IKK proteins. However, any in vivo effects of compounds designed to target the NBD would presumably depend upon disruption of preformed IKK complexes. We therefore sought to determine whether the NBD peptide could dissociate a preformed interaction between NEMO and the IKKsin vitro. To test this, we used pull-down assays to compare the effects of preincubating the peptides with adding them after incubation of GST-NEMO with IKKα or IKKβ. As expected, the scrambled peptide had no effect on the interaction with either kinase when tested using both incubation protocols (Fig. 1 D,lanes 2and 5). In contrast, preincubation with the wild-type peptide blocked binding of both kinases to GST-NEMO, and although less dramatic, the peptide also reduced binding when added after complex formation (Fig. 1 D, compare lanes 3 and 6). These results therefore lead us to conclude that the NBD peptide can disrupt preformed NEMO/IKK interactions. During these studies, we consistently observed that concentrations of NBD peptide that completely blocked association of IKKα with GST-NEMO (500–1000 μm) did not fully inhibit the interaction with IKKβ (Fig. 1 D, lane 3). To explore this further, we performed a series of dose-response experiments in which we directly compared the effects of the peptide on both kinases, and as shown in Fig. 2 A, a notable difference in effective concentrations was observed. Moreover, densitometric analysis of data obtained from a series of identical experiments (n = 11) clearly demonstrated that effective blockage of IKKα/NEMO interactions occurred at lower concentrations of peptide compared with disruption of IKKβ/NEMO (Fig. 2 B). To try to determine the molecular basis for these differences, we performed alanine-scanning mutagenesis to identify the IKKα NBD residues that contribute to its association with NEMO. Similar analysis of the IKKβ NBD revealed that the aspartate (Asp738) and two tryptophan residues (Trp739 and Trp741) were critical for NEMO association (TableI) (35May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Crossref PubMed Scopus (618) Google Scholar). Consistent with this, truncation of IKKα immediately before the NBD (1–737) along with alanine substitution of the first tryptophan residue (Trp740) prevented NEMO binding (Fig. 2 C, lane 6), whereas mutation of the two leucines (Leu738and Leu743) and the serine residue (Ser741) had no effect (lanes 4, 7, and9). In contrast to IKKβ, however, we observed marked differences when the aspartate (Asp739) and second tryptophan (Trp742) residues of the IKKα NBD were replaced with alanine (Table I). Thus, whereas mutations at these positions within IKKβ completely blocked NEMO association, alanine substitution of Asp739 did not affect binding (Fig.2 C, lane 5), and mutation of Trp742 reduced but did not completely block the interaction of IKKα with NEMO (lane 8). The similarities and differences in NBD residue requirements for NEMO binding between IKKα and IKKβ are summarized in Table I.Table IComparison of the effects of single alanine mutations within the NBD of IKKα and IKKβ on the ability of each kinase to interact with NEMONBD positionResidueEffect of alanine substitution (NEMO binding)IKKα-(738–743)IKKβ-(737–742)1Leu++2Asp+−3Trp−−4Ser++5Trp−/+−6Leu++NEMO binding ability was defined as strong (+), weak (−/+), or absent (−). Open table in a new tab NEMO binding ability was defined as strong (+), weak (−/+), or absent (−). It remains formally possible that in the transfection experiments described above, IKKα interacted indirectly with NEMO via association with endogenous NEMO-bound IKKβ and that the mutations within the IKKα NBD affected the formation of such complexes. We therefore tested the ability of each of the IKKα NBD mutants to associate with FLAG-tagged IKKβ and found that they all formed complexes (Fig.2 C, lanes 10–18). Identical results were obtained using GST pull-down analysis (data not shown), and, combined with our previous findings (35May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Crossref PubMed Scopus (618) Google Scholar), we conclude that the effects of the IKKα NBD mutations were through preventing its direct interaction with NEMO. Therefore, when taken together, the data presented in Figs. 1 and 2 strongly suggest that IKKα binds directly to NEMO, albeit w
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