{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T01:42:19Z","timestamp":1781660539893,"version":"3.54.5"},"reference-count":52,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5339","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1997,10,31]]},"abstract":"<jats:p>Activation of the transcription factor nuclear factor kappa B (NF-\u03baB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit I\u03baB. A large multiprotein complex, the I\u03baB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible I\u03baB kinase activity that phosphorylates I\u03baB-\u03b1 and I\u03baB-\u03b2. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-\u03baB\u2013dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-\u03baB signaling pathway.<\/jats:p>","DOI":"10.1126\/science.278.5339.860","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:37:56Z","timestamp":1027762676000},"page":"860-866","source":"Crossref","is-referenced-by-count":1730,"title":["IKK-1 and IKK-2: Cytokine-Activated I\u03baB Kinases Essential for NF-\u03baB Activation"],"prefix":"10.1126","volume":"278","author":[{"given":"Frank","family":"Mercurio","sequence":"first","affiliation":[{"name":"F. Mercurio, H. Zhu, B. W. Murray, B. Bennett, J. Li, D. Young, M. Barbosa, A. Manning, Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA."},{"name":"A. Shevchenko and M. Mann, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany."},{"name":"A. Rao, Center for Blood Research and the Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hengyi","family":"Zhu","sequence":"additional","affiliation":[{"name":"F. Mercurio, H. Zhu, B. W. Murray, B. Bennett, J. Li, D. Young, M. Barbosa, A. Manning, Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA."},{"name":"A. Shevchenko and M. Mann, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany."},{"name":"A. Rao, Center for Blood Research and the Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brion W.","family":"Murray","sequence":"additional","affiliation":[{"name":"F. Mercurio, H. Zhu, B. W. Murray, B. Bennett, J. Li, D. Young, M. Barbosa, A. Manning, Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA."},{"name":"A. Shevchenko and M. Mann, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany."},{"name":"A. Rao, Center for Blood Research and the Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrej","family":"Shevchenko","sequence":"additional","affiliation":[{"name":"F. Mercurio, H. Zhu, B. W. Murray, B. Bennett, J. Li, D. Young, M. Barbosa, A. Manning, Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA."},{"name":"A. Shevchenko and M. Mann, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany."},{"name":"A. Rao, Center for Blood Research and the Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brydon L.","family":"Bennett","sequence":"additional","affiliation":[{"name":"F. Mercurio, H. Zhu, B. W. Murray, B. Bennett, J. Li, D. Young, M. Barbosa, A. Manning, Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA."},{"name":"A. Shevchenko and M. Mann, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany."},{"name":"A. Rao, Center for Blood Research and the Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian wu","family":"Li","sequence":"additional","affiliation":[{"name":"F. Mercurio, H. Zhu, B. W. Murray, B. Bennett, J. Li, D. Young, M. Barbosa, A. Manning, Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA."},{"name":"A. Shevchenko and M. Mann, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany."},{"name":"A. Rao, Center for Blood Research and the Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David B.","family":"Young","sequence":"additional","affiliation":[{"name":"F. Mercurio, H. Zhu, B. W. Murray, B. Bennett, J. Li, D. Young, M. Barbosa, A. Manning, Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA."},{"name":"A. Shevchenko and M. Mann, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany."},{"name":"A. Rao, Center for Blood Research and the Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miguel","family":"Barbosa","sequence":"additional","affiliation":[{"name":"F. Mercurio, H. Zhu, B. W. Murray, B. Bennett, J. Li, D. Young, M. Barbosa, A. Manning, Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA."},{"name":"A. Shevchenko and M. Mann, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany."},{"name":"A. Rao, Center for Blood Research and the Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthias","family":"Mann","sequence":"additional","affiliation":[{"name":"F. Mercurio, H. Zhu, B. W. Murray, B. Bennett, J. Li, D. Young, M. Barbosa, A. Manning, Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA."},{"name":"A. Shevchenko and M. Mann, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany."},{"name":"A. Rao, Center for Blood Research and the Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony","family":"Manning","sequence":"additional","affiliation":[{"name":"F. Mercurio, H. Zhu, B. W. Murray, B. Bennett, J. Li, D. Young, M. Barbosa, A. Manning, Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA."},{"name":"A. Shevchenko and M. Mann, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany."},{"name":"A. Rao, Center for Blood Research and the Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anjana","family":"Rao","sequence":"additional","affiliation":[{"name":"F. Mercurio, H. Zhu, B. W. Murray, B. Bennett, J. Li, D. Young, M. Barbosa, A. Manning, Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA."},{"name":"A. Shevchenko and M. Mann, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany."},{"name":"A. Rao, Center for Blood Research and the Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1101\/gad.9.22.2723"},{"key":"e_1_3_1_2_3","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.iy.12.040194.001041"},{"key":"e_1_3_1_2_4","doi-asserted-by":"crossref","unstructured":"; A. Manning Drug Discovery Today 1 151 (1996);","DOI":"10.1016\/1359-6446(96)10015-5"},{"key":"e_1_3_1_2_5","doi-asserted-by":"publisher","DOI":"10.1056\/NEJM199704103361506"},{"key":"e_1_3_1_2_6","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)81318-5"},{"key":"e_1_3_1_2_7","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1016\/S0960-9822(06)00046-7","volume":"7","author":"Baichwal V. R.","year":"1997","unstructured":"Baichwal V. R., Baeuerle P. A., Curr. Biol. 7, 94 (1997).","journal-title":"Curr. Biol."},{"key":"e_1_3_1_3_2","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/0092-8674(88)90382-0","volume":"53","author":"Baeuerle P. A.","year":"1988","unstructured":"Baeuerle P. A., Baltimore D., Cell 53, 211 (1988);","journal-title":"Cell"},{"key":"e_1_3_1_3_3","doi-asserted-by":"crossref","first-page":"1689","DOI":"10.1101\/gad.3.11.1689","volume":"3","author":"___","year":"1989","unstructured":"___, Genes Dev. 3, 1689 (1989).","journal-title":"Genes Dev."},{"key":"e_1_3_1_4_2","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1016\/0092-8674(95)90511-1","volume":"80","author":"Thompson J. E.","year":"1995","unstructured":"Thompson J. E., Philips R. J., Erdjument-Bromage H., Tempst P., Ghosh S., Cell 80, 573 (1995);","journal-title":"Cell"},{"key":"e_1_3_1_4_3","doi-asserted-by":"publisher","DOI":"10.1126\/science.271.5252.1128"},{"key":"e_1_3_1_4_4","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1146\/annurev.cb.10.110194.002201","volume":"10","author":"Siebenlist U.","year":"1994","unstructured":"Siebenlist U., Franzoso G., Brown K., Annu. Rev. Cell Biol. 10, 405 (1994).","journal-title":"Annu. Rev. Cell Biol."},{"key":"e_1_3_1_5_2","doi-asserted-by":"crossref","first-page":"2064","DOI":"10.1101\/gad.7.11.2064","volume":"7","author":"Beg A. A.","year":"1993","unstructured":"Beg A. A., Baldwin A. S., Genes Dev. 7, 2064 (1993);","journal-title":"Genes Dev."},{"key":"e_1_3_1_5_3","doi-asserted-by":"crossref","first-page":"427","DOI":"10.1016\/0168-9525(93)90106-R","volume":"9","author":"Gilmore T. D.","year":"1993","unstructured":"Gilmore T. D., Morin P. J., Trends Genet. 9, 427 (1993);","journal-title":"Trends Genet."},{"key":"e_1_3_1_5_4","first-page":"4770","volume":"13","author":"Diaz-Meco M. T.","year":"1993","unstructured":"Diaz-Meco M. T., et al., Mol. Cell. Biol. 13, 4770 (1993);","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_5_5","doi-asserted-by":"crossref","first-page":"1281","DOI":"10.1016\/0092-8674(91)90022-Q","volume":"65","author":"Haskill S.","year":"1991","unstructured":"Haskill S., et al., Cell 65, 1281 (1991).","journal-title":"Cell"},{"key":"e_1_3_1_6_2","doi-asserted-by":"crossref","first-page":"1899","DOI":"10.1101\/gad.6.10.1899","volume":"6","author":"Beg A. A.","year":"1992","unstructured":"Beg A. A., et al., Genes Dev. 6, 1899 (1992);","journal-title":"Genes Dev."},{"key":"e_1_3_1_6_3","doi-asserted-by":"crossref","first-page":"1294","DOI":"10.1128\/MCB.15.3.1294","volume":"15","author":"Alkalay I.","year":"1995","unstructured":"Alkalay I., et al., Mol. Cell. Biol. 15, 1294 (1995);","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_6_4","doi-asserted-by":"crossref","unstructured":"; J. A. DiDonato F. Mercurio M. Karin ibid. p. 1302 (1995); A. Hershko and A. Ciechanover Annu. Rev. Biochem. 61 761 (1992);","DOI":"10.1146\/annurev.bi.61.070192.003553"},{"key":"e_1_3_1_6_5","doi-asserted-by":"crossref","first-page":"11259","DOI":"10.1073\/pnas.92.24.11259","volume":"92","author":"Scherer D. C.","year":"1995","unstructured":"Scherer D. C., Brockman J., Chen Z., Maniatis T., Ballard D., Proc. Natl. Acad. Sci. U.S.A. 92, 11259 (1995);","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_6_6","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.271.13.7844"},{"key":"e_1_3_1_7_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.7878466"},{"key":"e_1_3_1_7_3","doi-asserted-by":"crossref","first-page":"2876","DOI":"10.1002\/j.1460-2075.1995.tb07287.x","volume":"14","author":"Traenckner E. B. M.","year":"1995","unstructured":"Traenckner E. B. M., et al., EMBO J. 14, 2876 (1995);","journal-title":"EMBO J."},{"key":"e_1_3_1_7_4","doi-asserted-by":"crossref","first-page":"5339","DOI":"10.1128\/MCB.15.10.5339","volume":"15","author":"Whiteside S. T.","year":"1995","unstructured":"Whiteside S. T., et al., Mol. Cell. Biol. 15, 5339 (1995);","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_7_5","first-page":"1295","volume":"16","author":"DiDonato J. A.","year":"1996","unstructured":"DiDonato J. A., et al., ibid. 16, 1295 (1996).","journal-title":"ibid."},{"key":"e_1_3_1_8_2","unstructured":"HeLa S3 cells were harvested and resuspended in two packed cell pellet volumes of WCE lysis buffer [20 mM tris-HCl (pH 8.0) 0.5 M NaCl 0.25% Triton X-100 1 mM EDTA 1 mM EGTA 10 mM \u03b2-glycerophosphate 10 mM NaF 10 mM 4-nitrophenyl phosphate (PNPP) 300 \u03bcM Na 3 VO 4 1 mM benzamidine 2 \u03bcM phenylmethylsulfonyl fluoride (PMSF) aprotinin at 10 \u03bcg\/ml leupeptin at 1 \u03bcg\/ml pepstatin at 1 \u03bcg\/ml 1 mM dithiothreitol (DTT)] and gently rotated at 4\u00b0C for 45 min; the lysate was centrifuged at 60 000 rpm for 60 min in a Ti50.1 rotor at 4\u00b0C. The supernatant was dialyzed into 50 mM Q buffer [20 mM tris-HCl (pH 8.0) 50 mM NaCl 0.5 mM EDTA 0.5 mM EGTA 0.025% Brij 35 10 mM \u03b2-glycerophosphate 10 mM NaF 10 mM PNPP 300 \u03bcM Na 3 VO 4 aprotinin at 10 \u03bcg\/ml leupeptin at 1 \u03bcg\/ml pepstatin at 1 \u03bcg\/ml 1 mM DTT] and chromatographed on a Mono Q column (Pharmacia). Mono Q fractions containing I\u03baB-\u03b1 kinase activity were pooled concentrated and fractionated on a Superdex 200 gel-filtration column (Pharmacia). Fractions containing I\u03baB-\u03b1 kinase activity were pooled and directly fractionated on a Mono Q column. Fractions containing I\u03baB-\u03b1 kinase activity were pooled and fractionated on a phenyl Superose (Pharmacia) column equilibrated with PS buffer [20 mM tris-HCl (pH 8.0) 0.25 mM EDTA 0.25 mM EGTA 1.2 M ammonium sulfate 10 mM \u03b2-glycerophosphate 10 mM NaF 10 mM PNPP 300 \u03bcM Na 3 VO 4 1 mM benzamidine 2 \u03bcM PMSF aprotinin at 10 \u03bcg\/ml leupeptin at 1 \u03bcg\/ml pepstatin at 1 \u03bcg\/ml 1 mM DTT] and the kinase was eluted in a gradient to PS buffer with no ammonium sulfate. Fractions containing I\u03baB kinase activity were subjected to immunoblot analysis with anti-RelA anti\u2013I\u03baB-\u03b2 and anti\u2013MKP-1 (Santa Cruz Biotechnology Inc.) or anti\u2013MEKK-1 or anti\u2013P-Tyr (Upstate Biotechnology)."},{"key":"e_1_3_1_9_2","unstructured":"IKK signalsome kinase assay. Samples from column fractions or immunoprecipitates were subjected to an in vitro kinase assay. Kinase assays were done in kinase buffer [20 mM Hepes (pH 7.7) 2 mM MgCl 2 2 mM MnCl 2 10 \u03bcM adenosine triphosphate (ATP) 1 to 3 \u03bcCi of [\u03b3- 32 P]ATP 10 mM \u03b2-glycerophosphate 10 mM NaF 10 mM PNPP 300 \u03bcM Na 3 VO 4 1 mM benzamidine 2 \u03bcM PMSF aprotinin at 10 \u03bcg\/ml leupeptin at 1 \u03bcg\/ml pepstatin at 1 \u03bcg\/ml 1 mM DTT] at 30\u00b0C for 30 to 60 min in the presence of the indicated substrate. The kinase reaction was stopped by addition of 6\u00d7 SDS\u2013polyacrylamide gel electrophoresis (PAGE) sample buffer subjected to SDS-PAGE analysis and visualized by autoradiography. GST\u2013I\u03baB substrates were expressed and purified from Escherichia coli with glutathione Sepharose 4B beads. His-tagged RelA\u2013streptavidin-tagged I\u03baB-\u03b1 WT and His-tagged Rel A\u2013streptavidin-tagged I\u03baB-\u03b1 (S32A S36A) mutant complexes were purified from Sf9 cells coinfected with the indicated baculoviral expression constructs."},{"key":"e_1_3_1_10_2","unstructured":"F. Mercurio B. Bennett B. Murray L. Ransone H. Zhu A. Manning unpublished data."},{"key":"e_1_3_1_11_2","doi-asserted-by":"crossref","first-page":"853","DOI":"10.1016\/S0092-8674(00)81064-8","volume":"84","author":"Chen Z. J.","year":"1996","unstructured":"Chen Z. J., Parent L., Maniatis T., Cell 84, 853 (1996).","journal-title":"Cell"},{"key":"e_1_3_1_12_2","first-page":"213","volume":"88","author":"Lee F. S.","year":"1997","unstructured":"Lee F. S., Hagler J., Chen Z. J., Maniatis T., ibid. 88, 213 (1997);","journal-title":"ibid."},{"key":"e_1_3_1_12_3","doi-asserted-by":"crossref","first-page":"13234","DOI":"10.1074\/jbc.271.22.13234","volume":"271","author":"Hirano M.","year":"1996","unstructured":"Hirano M., et al., J. Biol. Chem. 271, 13234 (1996);","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_12_4","doi-asserted-by":"publisher","DOI":"10.1038\/385540a0"},{"key":"e_1_3_1_13_2","unstructured":"Two 150-mm plates of HeLa cells were either stimulated with TNF-\u03b1 or not; whole-cell lysates were prepared and diluted with 3 vol of PD buffer [40 mM tris-HCl (pH 8.0) 500 mM NaCl 0.1% Nonidet P-40 6 mM EDTA 6 mM EGTA 10 mM \u03b2-glycerophosphate 10 mM NaF 10 mM PNPP 300 \u03bcM Na 3 VO 4 1 mM benzamidine 2 \u03bcM PMSF aprotinin at 10 \u03bcg\/ml leupeptin at 1 \u03bcg\/ml pepstatin at 1 \u03bcg\/ml 1 mM DTT]; 2 to 4 \u03bcg of the indicated antibody was added and samples were incubated on ice for 1 to 2 hours. Protein A or G beads (10 \u03bcl) were added and samples were incubated for 1 hour at 4\u00b0C. The immunoprecipitate was then washed three times with PD buffer and once with kinase buffer without ATP and then subjected to a standard kinase assay. There was no loss in I\u03baB kinase activity when the immunoprecipitate was subjected to more rigorous washing\u2014RIPA buffer [50 mM tris-HCl (pH 8.0) 150 mM NaCl 1 mM EDTA 0.4% deoxycholate 1% Nonidet P-40 10 mM \u03b2-glycerophosphate 10 mM NaF 10 mM PNPP 300 \u03bcM Na 3 VO 4 1 mM benzamidine 2 \u03bcM PMSF aprotinin at 10 \u03bcg\/ml leupeptin at 1 \u03bcg\/ml pepstatin at 1 \u03bcg\/ml 1 mM DTT] or up to 3.5 M urea."},{"key":"e_1_3_1_14_2","unstructured":"Peptide phosphorylation was done as described (8) with synthetic peptides (100 \u03bcM) (Alpha Diagnostics International San Antonio TX). Reactions were for 1 hour at room temperature and were terminated by the addition of SDS-PAGE loading buffer. SDS-PAGE with a 16% tris Tricine gel (Novex) or a 4 to 20% tris glycine gel (Novex) was used to characterize the reaction products. Gels were washed dried in vacuo and exposed to autoradiographic film. Peptide sequences were as follows: I\u03baB-\u03b1 (21\u201341) CKKERLLDDRHDSGLDSMKDEE; I\u03baB-\u03b1 (21\u201341) S\u2192T mutant CKKERLLDDRHDTGLDTMKDEE; c-Fos (222\u2013241) DLTGGPEVAT(PO 3 )PESEEAFLP; MKP-1 CPTNSALNYLKSPITTSPS; c-Jun (56\u201370) CNSDLLTSPDVGLLK; c-Jun (65\u201379) CVGLLKLASPELERL. Abbreviations for amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; Y Tyr. Incorporation of 32 P into GST\u2013I\u03baB-\u03b1 (1\u201354) was measured in a discontinuous assay as described above (8). GST\u2013I\u03baB-\u03b1 (1\u201354) and ATP were 0.56 and 3 \u03bcM respectively. Enzymatic reactions (32 \u03bcl) were carried out for 1 hour at room temperature and terminated with the addition of trichloroacetic acid (TCA) (150 \u03bcl per well; 12.5% w\/v). After 20 min the TCA precipitate was collected on 96-well glass fiber plates (Packard) and washed 10 times with about 0.3 ml of Dulbecco's phosphate-buffered saline (pH 7.4) per well (Sigma). Scintillation fluid (0.50 ml; MicroScint Packard) was added to each well and 32 P was detected by scintillation counting. Less than 10% of the ATP was consumed in the reaction."},{"key":"e_1_3_1_15_2","unstructured":"For large-scale IKK signalsome purification HeLa S3 cells were stimulated for 7 min with TNF-\u03b1 at 20 ng\/ml (R&D Systems) and harvested; whole-cell lysate was prepared (1.2 g of total protein) and about 5 mg of anti\u2013MKP-1 (Santa Cruz Biotechnology Inc.) was added and incubated at 4\u00b0C for 2 hours with gentle rotation. Then 50 ml of protein A\u2013agarose (Calbiochem) was added and incubated for 2 hours. The immunoprecipitate was then sequentially washed four times with PD buffer two times with RIPA buffer two times with PD buffer once with 3.5 M urea-PD buffer and three times with PD buffer. The immunoprecipitate was then made into a thick slurry by adding 10 ml of PD buffer and 25 mg of the MKP-1 peptide to which the antibody was generated (Santa Cruz Biotechnology Inc.); then it was incubated overnight at 4\u00b0C with gentle rotation. Salt was removed from the eluted protein on PD10 columns (Pharmacia) equilibrated with 50 mM Q buffer and eluate was chromatographed on a Mono Q column (Pharmacia). Fractions containing I\u03baB kinase activity were pooled concentrated and subjected to preparative SDS-PAGE; protein bands were visualized with colloidal blue stain (Novex) and the bands were excised and sequenced."},{"key":"e_1_3_1_16_2","unstructured":"Coomassie blue\u2013stained bands were excised and digested with trypsin as described. A small portion of the supernatant was removed for analysis by MALDI peptide mapping as described (16). The program PeptideSearch (EMBL Heidelberg) was used to compare the peptide mass map from the IKK-1 band with a protein sequence database. Eight measured peptide masses matched those calculated for peptides from CHUK within 30 ppm (18). The peptide mass map of the IKK-2 band did not result in a clear identification and therefore the sample was subjected to nanoelectrospray mass spectrometry (25). The peptide mixture was micropurified on a capillary containing 50 nl of Poros R2 resin (Perseptive Biosystems Framingham MA). The peptides were washed and then step-eluted with 0.5 \u03bcl of 50% MeOH in 5% formic acid into a nanoelectrospray needle. This needle was transferred to an APIII mass spectrometer (Perkin-Elmer Sciex Toronto Canada) and the sample was sprayed for about 20 min. During this time peptide ions apparent from the mass spectrum were selected and isolated and then fragmented in the collision chamber of the mass spectrometer. From the tandem mass spectra short stretches of sequence were assembled into peptide sequence tags (18) and compared with a protein sequence database or an EST database by using PeptideSearch. Three peptides matched the IKK-1 sequence. A1 IIDLGYAK; A2 VEVALSNIK; A3 SIQLDLER. Three other peptides matched human EST sequences in the EST database: B1 ALELLPK; B2 VIYTQLSK; B6 LLLQAIQSFEK. These three sequences all match EST clone . The peptide B4 with the sequence LGTGGFGNVIR was found in clone . After the full-length IKK-2 sequence was obtained (19) two more peptides (B3 ALDDILNLK; B5 DLKPENIVLQQGEQR) were found in the sequence. Peptide A1 is present in both IKK-1 and IKK-2 sequences."},{"key":"e_1_3_1_17_2","doi-asserted-by":"crossref","first-page":"14440","DOI":"10.1073\/pnas.93.25.14440","volume":"93","author":"Shevchenko A.","year":"1996","unstructured":"Shevchenko A., et al., Proc. Natl. Acad. Sci. U.S.A. 93, 14440 (1996).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_18_2","first-page":"537","volume":"41","author":"Connely M. A.","year":"1995","unstructured":"Connely M. A., Marcu K. B., Cell. Mol. Biol. Res. 41, 537 (1995).","journal-title":"Cell. Mol. Biol. Res."},{"key":"e_1_3_1_19_2","doi-asserted-by":"crossref","first-page":"4390","DOI":"10.1021\/ac00096a002","volume":"66","author":"Mann M.","year":"1994","unstructured":"Mann M., Wilm M. S., Anal. Chem. 66, 4390 (1994);","journal-title":"Anal. Chem."},{"key":"e_1_3_1_19_3","doi-asserted-by":"crossref","first-page":"466","DOI":"10.1038\/379466a0","volume":"379","author":"Wilm M.","year":"1996","unstructured":"Wilm M., et al., Nature 379, 466 (1996);","journal-title":"Nature"},{"key":"e_1_3_1_19_4","doi-asserted-by":"crossref","unstructured":"; M. Mann Trends Biol. Sci. 21 494 (1996).","DOI":"10.1016\/S0968-0004(96)30042-X"},{"key":"e_1_3_1_20_2","unstructured":"Several peptides were identical matches to human EST clones. All the EST clones were similar to human and mouse CHUK-1 (IKK-1). These clones were obtained (Genome Systems Inc.) and the precise nucleotide sequence was determined and used to design primers to clone human IKK-2 by polymerase chain reaction (PCR) from a human HeLa cell cDNA library (Clontech Inc.). Several IKK-2 cDNA clones were isolated and sequenced. Full-length mouse IKK-1 and a partial human IKK-1 nucleotide sequence were available in the comprehensive database. Primers were designed to clone by PCR the human and mouse IKK-1 cDNAs. The partial human IKK-1 coding region was used to probe a HeLa cDNA phage library (Stratagene) to obtain the full-length human IKK-1 cDNA clone by standard procedures."},{"key":"e_1_3_1_21_2","unstructured":"For in vitro translation studies HA-tagged IKK-1 and Flag-tagged IKK-2 were in vitro translated in RRLs either separately or alone exactly as described in the manufacturer's protocol (Promega)."},{"key":"e_1_3_1_22_2","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1994.tb06361.x"},{"key":"e_1_3_1_23_2","unstructured":"For immunocytochemistry HeLa cells were transiently transfected with either HA-tagged IKK-1 or Flag-tagged IKK-2 as indicated. Cells were washed with PBS fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 30 min and permeabilized with wash buffer (0.5% Triton X-100 0.01% azide in PBS). Cells were blocked with wash buffer containing donkey serum (5%) (Jackson Immunoresearch Laboratories) and probed with primary antibody\u2014anti-RelA (polyclonal antibody) 1:2000 (Santa Cruz); anti-HA (monoclonal antibody) 1:1000 (Eastman Kodak); or anti-Flag (monoclonal antibody) 1:1000 (Babco)\u2014followed by secondary antibody\u2014donkey antibody to rabbit (fluorescein isothiocyanate) conjugate 1:100; donkey antibody to mouse (Texas Red conjugate) 1:100 (Jackson Immunoresearch Laboratories). Cover slips were mounted with polyvinyl alcohol and 1 4-diazabicyclo[2.2.2]octane (Sigma) mounting medium and the slides were viewed under fluorescence with a Nikon Microphot-FXA microscope; the images were then scored and photographed."},{"key":"e_1_3_1_24_2","doi-asserted-by":"crossref","first-page":"6288","DOI":"10.1073\/pnas.91.14.6288","volume":"91","author":"Kumar A.","year":"1994","unstructured":"Kumar A., Haque J., Lacoste J., Hiscott J., Williams B. R. G., Proc. Natl. Acad. Sci. U.S.A. 91, 6288 (1994);","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_24_3","first-page":"4770","volume":"13","author":"Diaz-Meco M. T.","year":"1993","unstructured":"Diaz-Meco M. T., et al., Mol. Cell. Biol. 13, 4770 (1993);","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_24_4","first-page":"1842","volume":"13","author":"Diaz-Meco M. T.","year":"1994","unstructured":"Diaz-Meco M. T., et al., EMBO J. 13, 1842 (1994);","journal-title":"EMBO J."},{"key":"e_1_3_1_24_5","first-page":"3133","volume":"16","author":"Schouten G. J.","year":"1997","unstructured":"Schouten G. J., et al., ibid. 16, 3133 (1997).","journal-title":"ibid."},{"key":"e_1_3_1_25_2","doi-asserted-by":"publisher","DOI":"10.1038\/41493"},{"key":"e_1_3_1_25_3","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1016\/S0092-8674(00)80344-X","volume":"90","author":"Regnier C. H.","year":"1997","unstructured":"Regnier C. H., et al., Cell 90, 373 (1997).","journal-title":"Cell"},{"key":"e_1_3_1_26_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1021\/ac9509519","volume":"68","author":"Wilm M.","year":"1996","unstructured":"Wilm M., Mann M., Anal. Chem. 68, 1 (1996).","journal-title":"Anal. Chem."},{"key":"e_1_3_1_27_2","unstructured":"We thank I. Verma and R. Mueller for the gift of the I\u03baB-\u03b1 COOH-terminus construct and helpful discussions; J. DiDonato for the I\u03baB-\u03b1 vectors; H. Raymon and N. Richard for assistance with immunocytochemistry; S. Kim A. LaPointe and B. Lee for technical assistance; K. Davis and E. Carlson for help with compiling the manuscript and figures; and D. Anderson and A. Lewis for helpful comments and support. A.R. contributed to this work during a sabbatical at Signal Pharmaceuticals."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.278.5339.860","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T05:36:37Z","timestamp":1705124197000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.278.5339.860"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1997,10,31]]},"references-count":52,"journal-issue":{"issue":"5339","published-print":{"date-parts":[[1997,10,31]]}},"alternative-id":["10.1126\/science.278.5339.860"],"URL":"https:\/\/doi.org\/10.1126\/science.278.5339.860","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1997,10,31]]}}}