Role of IKKγ/NEMO in Assembly of the IκB Kinase Complex
2001; Elsevier BV; Volume: 276; Issue: 6 Linguagem: Inglês
10.1074/jbc.m008353200
ISSN1083-351X
AutoresXiaohua Li, Xiaoqun Fang, Richard B. Gaynor,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoIKKγ/NEMO is a protein that is critical for the assembly of the high molecular weight IκB kinase (IKK) complex. To investigate the role of IKKγ/NEMO in the assembly of the IKK complex, we conducted a series of experiments in which the chromatographic distribution of extracts prepared from cells transiently expressing epitope-tagged IKKγ/NEMO and the IKKs were examined. When expressed alone following transfection, IKKα and IKKβ were present in low molecular weight complexes migrating between 200 and 400 kDa. However, when coexpressed with IKKγ/NEMO, both IKKα and IKKβ migrated at ∼600 kDa which was similar to the previously described IKK complex that is activated by cytokines such as tumor necrosis factor-α. When either IKKα or IKKβ was expressed alone with IKKγ/NEMO, IKKβ but not IKKα migrated in the higher molecular weight IKK complex. Constitutively active or inactive forms of IKKβ were both incorporated into the high molecular weight IKK complex in the presence of IKKγ/NEMO. The amino-terminal region of IKKγ/NEMO, which interacts directly with IKKβ, was required for formation of the high molecular weight IKK complex and for stimulation of IKKβ kinase activity. These results suggest that recruitment of the IKKs into a high molecular complex by IKKγ/NEMO is a crucial step involved in IKK function. IKKγ/NEMO is a protein that is critical for the assembly of the high molecular weight IκB kinase (IKK) complex. To investigate the role of IKKγ/NEMO in the assembly of the IKK complex, we conducted a series of experiments in which the chromatographic distribution of extracts prepared from cells transiently expressing epitope-tagged IKKγ/NEMO and the IKKs were examined. When expressed alone following transfection, IKKα and IKKβ were present in low molecular weight complexes migrating between 200 and 400 kDa. However, when coexpressed with IKKγ/NEMO, both IKKα and IKKβ migrated at ∼600 kDa which was similar to the previously described IKK complex that is activated by cytokines such as tumor necrosis factor-α. When either IKKα or IKKβ was expressed alone with IKKγ/NEMO, IKKβ but not IKKα migrated in the higher molecular weight IKK complex. Constitutively active or inactive forms of IKKβ were both incorporated into the high molecular weight IKK complex in the presence of IKKγ/NEMO. The amino-terminal region of IKKγ/NEMO, which interacts directly with IKKβ, was required for formation of the high molecular weight IKK complex and for stimulation of IKKβ kinase activity. 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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. Nature. 2000; 405: 466-472Crossref PubMed Scopus (607) Google Scholar). Gene disruption studies of the IKKγ/NEMO gene demonstrate that although male mice die in utero, heterozygous female mice develop granulocytic infiltration and both hyperproliferation and increased apoptosis of keratinocytes similar to that seen in incontinentia pigmenti (48Makris C. Godfrey V.L. Krahn-Senftleben G. Takahashi T. Roberts J.L. Schwarz A.T. Feng L. Johnson R.S. Karin M. Mol. Cell. 2000; 5: 969-979Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 50Schmidt-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). Homozygous deletion of IKKγ/NEMO leads to embryonic lethality due to massive hepatic apoptosis (48Makris C. Godfrey V.L. Krahn-Senftleben G. Takahashi T. Roberts J.L. Schwarz A.T. Feng L. Johnson R.S. Karin M. Mol. Cell. 2000; 5: 969-979Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 49Rudolph 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). Mouse embryo fibroblasts isolated from these mice exhibit extreme defects in stimulating the NF-κB pathway in response to a variety of well characterized activators of this pathway (48Makris C. Godfrey V.L. Krahn-Senftleben G. Takahashi T. Roberts J.L. Schwarz A.T. Feng L. Johnson R.S. Karin M. Mol. Cell. 2000; 5: 969-979Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 49Rudolph 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, 50Schmidt-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). However, the mechanism by which IKKγ/NEMO activates the NF-κB pathway remains to be determined. In the current study, we utilized a biochemical approach to address the function of IKKγ/NEMO in the recruitment of IKKα and IKKβ into the IKK complex. We demonstrated that the amino terminus of IKKγ/NEMO that interacts with IKKβ is crucial for formation of the high molecular weight IKK complex. Moreover, we found that IKKγ/NEMO stimulates the ability of IKKβ but not IKKα to phosphorylate IκBα. These results further establish that IKKγ/NEMO association with the IKK complex is critical for stimulation of IKK kinase activity. The murine IKKγ/NEMO coding sequence (28Yamaoka 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) (GenBankTM accession number AF069542-1) was obtained by PCR using a mouse spleen cDNA library. Two oligonucleotide primers complementary to the 5′- and 3′-coding regions of mouse IKKγ/NEMO were used in PCR assays. The PCR product was cloned into the expression vector pCMV5/Myc1 fusing the Myc tag to the 5′ of IKKγ/NEMO sequence. An amino-terminal deletion that contains amino acid residues 101–412 of IKKγ/NEMO was constructed by PCR followed by cloning of the product into pCMV5/Myc1. The carboxyl-terminal deletions of IKKγ/NEMO containing either amino acids 1–270 or 1–312 were constructed utilizing PCR to generate these fragments followed by cloning into pCMV5/Myc1. The leucine zipper mutations were constructed by substitution of leucine residues at positions 315, 322, and 329 with methionine in the IKKγ/NEMO gene cloned into pCMV5/Myc1. Wild-type and mutant IKKβ were cloned into plasmid pCMVFl such that the FLAG tag was fused to the 5′ end of IKKβ (12Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar,35Yin M.-J. Christerson L.B. Yamamoto Y. Kwak Y.-T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). IKKα was cloned into plasmid pRcBactHA and the HA tag was fused to the 5′ end of IKKα (12Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar, 35Yin M.-J. Christerson L.B. Yamamoto Y. Kwak Y.-T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). The cDNA clone containing Myc-tagged CREB was previously described (52Yin M. Paulssen E. Seeler J. Gaynor R. J. Virol. 1995; 69: 6209-6218Crossref PubMed Google Scholar). COS cells grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum were transfected using Fugene-6 (Roche Molecular Biochemicals) as described by the manufacturer. For a typical cell fractionation experiment, cells cultured overnight in 100-mm plates were transfected with 0.6 μg of each DNA construct. Cells from five transfected plates were pooled, and S100 extracts were prepared as detailed (53Li X.-H. Murphy K.M. Palka K.T. Surabhi R.M. Gaynor R.B. J. Biol. Chem. 1999; 274: 34417-34424Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) except that the extracts were directly loaded onto a Superdex 200 column without dialysis. The S100 extract containing a total of 2 mg of protein was chromatographed through a Superdex-200 column (Amersham Pharmacia Biotech) in buffer D (20 mm HEPES (pH 7.9), 0.1 m KCl, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 20% glycerol, and 0.2 mm EDTA) (54Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar), and fractions of 1 ml each were collected. Western blotting was done with 30 μg of protein obtained from each of the column fractions as previously described (53Li X.-H. Murphy K.M. Palka K.T. Surabhi R.M. Gaynor R.B. J. Biol. Chem. 1999; 274: 34417-34424Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The antibodies used in this analysis are specified in the figure legends. For kinase assays, 50 μl of each column fraction was incubated overnight at 4 °C with 1–2 μg of the indicated antibodies in 150 μl 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 β-glycerophosphate, 10 mm NaF, 300 μmNa3VO4, and protease inhibitors (Roche Molecular Biochemicals)) (12Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar). Immune complexes were precipitated with protein A-agarose (Bio-Rad) for 1–3 h at 4 °C and analyzed byin vitro kinase assays in the presence of 5 μg of bacterially expressed GST fusion protein consisting of IκBα (aa 1–54) or with serine residues 32 and 36 changed to alanine (53Li X.-H. Murphy K.M. Palka K.T. Surabhi R.M. Gaynor R.B. J. Biol. Chem. 1999; 274: 34417-34424Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). After incubation at 30 °C for 30 min, the reactions were mixed with protein sample buffer (50 mm Tris (pH 8.0), 2% SDS, 0.1% bromphenol blue, 10% glycerol, and β-mecaptoethanol), heated at 95 °C for 3 min, and loaded on a 12% SDS gel. The phosphoprotein products were visualized by autoradiography. COS cells (1.3 × 105) were transfected with either 0.5 μg of IKKβ alone or in the presence of 0.5 μg of wild-type or mutant IKKγ/NEMO mutants. After 24 h post-transfection, cells were grown overnight in 2 ml of Dulbecco's modified Eagle's medium lacking phosphate (Life Technologies, Inc.) followed by incubating 3 h in 1 ml of Dulbecco's modified Eagle's medium in the presence of 50 μCi of [32P]orthophosphate (PerkinElmer Life Sciences). Cells were then collected in 300 μl of PD buffer. The32P-labeled IKKβ protein was immunoprecipitated from 200 μl of cell extract using 4 μg of anti-FLAG monoclonal antibody (Sigma, M2) or 4 μg of anti-HA monoclonal antibody (Roche Molecular Biochemicals, 12CA5). The immunoprecipitates were washed with PD buffer, and the phosphoproteins were resolved on a 10% SDS-polyacrylamide gel and visualized by autoradiography. It has been shown previously that cytokines such as TNFα stimulate IKK activity present in a high molecular complex migrating between 600 and 900 kDa on gel filtration columns (12Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar, 14Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1589) Google Scholar,23Chen Z.J. Parent L. Maniatis T. 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