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• W2000989306 abstract "IKKγ/NEMO is an essential regulatory component of the IκB kinase complex that is required for NF-κB activation in response to various stimuli including tumor necrosis factor-α and interleukin-1β. To investigate the mechanism by which IKKγ/NEMO regulates the IKK complex, we examined the ability of IKKγ/NEMO to recruit the IκB proteins into this complex. IKKγ/NEMO binding to wild-type, but not to a kinase-deficient IKKβ protein, facilitated the association of IκBα and IκBβ with the high molecular weight IKK complex. Following tumor necrosis factor-α treatment of HeLa cells, the majority of the phosphorylated form of endogenous IκBα was associated with the high molecular weight IKK complex in HeLa cells and parental mouse embryo fibroblasts but not in IKKγ/NEMO-deficient cells. Finally, we demonstrate that IKKγ/NEMO facilitates the association of the IκB proteins and IKKβ and leads to increases in IKKβ kinase activity. These results suggest that an important function of IKKγ/NEMO is to facilitate the association of both IKKβ and IκB in the high molecular weight IKK complex to increase IκB phosphorylation. IKKγ/NEMO is an essential regulatory component of the IκB kinase complex that is required for NF-κB activation in response to various stimuli including tumor necrosis factor-α and interleukin-1β. To investigate the mechanism by which IKKγ/NEMO regulates the IKK complex, we examined the ability of IKKγ/NEMO to recruit the IκB proteins into this complex. IKKγ/NEMO binding to wild-type, but not to a kinase-deficient IKKβ protein, facilitated the association of IκBα and IκBβ with the high molecular weight IKK complex. Following tumor necrosis factor-α treatment of HeLa cells, the majority of the phosphorylated form of endogenous IκBα was associated with the high molecular weight IKK complex in HeLa cells and parental mouse embryo fibroblasts but not in IKKγ/NEMO-deficient cells. Finally, we demonstrate that IKKγ/NEMO facilitates the association of the IκB proteins and IKKβ and leads to increases in IKKβ kinase activity. These results suggest that an important function of IKKγ/NEMO is to facilitate the association of both IKKβ and IκB in the high molecular weight IKK complex to increase IκB phosphorylation. tumor necrosis factor hemagglutinin glutathione S-transferase amino acids mouse embryo fibroblast cytomegalovirus The NF-κB proteins are critical for activating the expression of cellular genes that are involved in the control of the immune and inflammatory response and in protecting cells from apoptosis in response to a variety of stress stimuli (1Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2919) Google Scholar, 2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5552) Google Scholar, 3Barnes P.J. Int. J. Biochem. 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Mercurio F. Ben-Neriah Y. Nature. 1998; 396: 590-594Crossref PubMed Scopus (568) Google Scholar, 31Winston J.T. Strack P. Beer-Romero P. Chu C. Elledge S.J. Harper J.W. Genes Dev. 1999; 13: 270-283Crossref PubMed Scopus (808) Google Scholar, 32Spencer E. Jiang J. Chen Z.J. Genes Dev. 1999; 13: 284-294Crossref PubMed Scopus (372) Google Scholar). IκB mutants in which these serine residues are changed to alanine are resistant to proteasome-mediated degradation and thus prevent the nuclear translocation of the NF-κB proteins (33Wang C.Y. Mayo M.W. Baldwin A.S.J. Science. 1996; 274: 784-787Crossref PubMed Scopus (2505) Google Scholar). IKKγ/NEMO was initially identified in a genetic complementation assay as a factor that could restore NF-κβ activation in cells that were resistant to a variety of stimuli that normally induce the NF-κB pathway (34Yamaoka 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 (945) Google Scholar). IKKγ/NEMO was also identified independently in biochemical studies as an essential component of the high molecular weight IKK complex (35Mercurio 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, 36Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (845) Google Scholar). Finally, this factor was characterized as a factor known as FIP-3 that bound to the adenovirus E3 protein and could inhibit TNFα-induced apoptosis (37Li 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 (155) Google Scholar). IKKγ/NEMO in conjunction with IKKα and IKKβ is a component of the high molecular weight IKK complex, which migrates between 600 and 900 kDa following gel filtration chromatography (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 (1841) 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 (1575) Google Scholar, 29Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (867) Google Scholar, 34Yamaoka 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 (945) Google Scholar, 35Mercurio 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, 36Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (845) Google Scholar, 37Li 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 (155) Google Scholar, 38Courtois G. Whiteside S.T. Sibley C.H. Israel A. Mol. Cell. Biol. 1997; 17: 1441-1449Crossref PubMed Google Scholar, 39Harhaj E.W. Good L. Xiao G. Uhlik M. Cvijic M.E. Rivera-Walsh I. Sun S.C. Oncogene. 2000; 19: 1448-1456Crossref PubMed Scopus (92) Google Scholar, 40Krappmann D. Hatada E.N. Tegethoff S. Li J. Klippel A. Giese K. Baeuerle P.A. Scheidereit C. J. Biol. Chem. 2000; 275: 29779-29787Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 41Li X.-H. Fang X. Gaynor R.B. J. Biol. Chem. 2001; 276: 4494-4500Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Biochemical fractionation and coimmunoprecipitation studies demonstrate that IKKα, IKKβ, and IKKγ/NEMO interact in this IKK complex (35Mercurio 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, 36Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (845) Google Scholar,40Krappmann D. Hatada E.N. Tegethoff S. Li J. Klippel A. Giese K. Baeuerle P.A. Scheidereit C. J. Biol. Chem. 2000; 275: 29779-29787Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Cells that do not express IKKγ/NEMO are unable to assemble the high molecular weight IKK complex and increase IKK activity in response to agents that stimulate the NF-κB pathway (35Mercurio 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, 36Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (845) Google Scholar). Although IKKγ/NEMO itself does not have kinase activity, it is essential for NF-κB activation (34Yamaoka 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 (945) Google Scholar, 35Mercurio 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, 36Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (845) Google Scholar). The mechanism by which IKKγ/NEMO activates the NF-κB pathway has been the subject of intense investigation. Mutagenesis of IKKγ/NEMO has been performed in an attempt to define important functional domains (35Mercurio 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, 36Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (845) Google Scholar, 42Chu Z.-L. Shin Y.-A. Yang J.-M. DiDonato J.A. Ballard D.W. J. Biol. Chem. 1999; 274: 15297-15300Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 43May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Crossref PubMed Scopus (614) Google Scholar). IKKγ/NEMO has a molecular mass of 48 kDa and contains a leucine zipper and two coiled-coil motifs. Residues in the amino-terminal 100 amino acids of this protein are critical for interactions with IKKβ (43May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Crossref PubMed Scopus (614) Google Scholar). IKKβ preferentially associates with IKKγ/NEMO (34Yamaoka 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 (945) Google Scholar, 36Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (845) Google Scholar), although IKKα has also been shown to directly associate with IKKγ/NEMO (22Tanaka 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 (491) Google Scholar, 40Krappmann D. Hatada E.N. Tegethoff S. Li J. Klippel A. Giese K. Baeuerle P.A. Scheidereit C. J. Biol. Chem. 2000; 275: 29779-29787Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The coiled-coil domains in IKKγ/NEMO mediate its oligomerization, which is critical for activating IKK kinase activity (44Poyet J.-L. Srinivasula S.M. Lin J.-H. Fernandes-Alnemri T.F. Yamaoka S. Tasichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), while its carboxyl terminus is involved in the recruitment of upstream kinases, which are critical for activating IKK (45Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). For example, a kinase known as RIP, which is recruited to the TNF receptor following TNFα treatment of cells, binds to IKKγ/NEMO and leads to the subsequent association of IKKα and IKKβ (37Li 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 (155) Google Scholar, 45Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). In contrast, the association of the A20 protein with IKKγ/NEMO decreases TNFα-mediated activation of the NF-κB pathway (45Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). The viral transactivator Tax has also been shown to bind to IKKγ/NEMO and stimulate IKK kinase activity (42Chu Z.-L. Shin Y.-A. Yang J.-M. DiDonato J.A. Ballard D.W. J. Biol. Chem. 1999; 274: 15297-15300Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 46Harhaj E.W. Sun S.C. J. Biol. Chem. 1999; 274: 22911-22914Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 47Jin D.Y. Giordano V. Kibler K.V. Nakano H. Jeang K.T. J. Biol. Chem. 1999; 274: 17402-17405Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) as has the cellular protein CIKS (48Leonardi A. Chariot A. Claudio E. Cunningham K. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10494-10499Crossref PubMed Scopus (131) Google Scholar). These results indicate that IKKγ/NEMO can interact with a variety of different regulatory proteins that are important in the activation of the NF-κB pathway in response to various stimuli. Genetic studies have also been utilized to study the role of IKKγ/NEMO in regulating the NF-κB pathway (49Makris 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 (334) Google Scholar, 50Rudolph 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, 51Schmidt-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 (371) Google Scholar). Disruption of a single copy of the IKKγ/NEMO gene, which is located on the X chromosome, results in the death of male mice in utero, while female mice develop granulocytic infiltration and both hyperproliferation and increased apoptosis of keratinocytes (49Makris 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 (334) Google Scholar,51Schmidt-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 (371) Google Scholar). The homozygous deletion of IKKγ/NEMO results in embryonic lethality in both male and female mice due to TNFα-induced hepatic apoptosis (49Makris 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 (334) Google Scholar, 50Rudolph 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). Fibroblasts isolated from these mice are defective in activating the NF-κB pathway in response to a variety of stimulators of this pathway. In humans, mutation of a single copy of the IKKγ/NEMO gene is associated with a syndrome known as incontinentia pigmenti, an X-linked defect that results in lethality in males and a granulocytic infiltration of the skin in females (52Smahi 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 (603) Google Scholar). Recently, another syndrome due to mutations in the putative zinc finger domain in the C terminus of IKKγ/NEMO has been described (53Jain A. Ma C.A. Liu S. Brown M. Cohen J. Strober W. Nat. Immunol. 2001; 2: 223-228Crossref PubMed Scopus (321) Google Scholar, 54Zonana J. Elder M.E. Schneider L.C. Orlow S.J. Moss C. Golabi M. Shapira S.K. Farndon P.A. Wara D.W. Emmal S.A. Ferguson B.M. Am. J. Hum. Genet. 2000; 67: 1555-1562Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 55Doffinger R. Smahi A. Bessia C. Geissmann F. Feinberg J. Durandy A. Bodemer C. Kenwrick S. Dupuis-Girod S. Blanche S. Wood P. Rabia S.H. Headon D.J. Overbeek P.A. Le Deist F. Holland S.M. Belani K. Kumararatne D.S. Fischer A. Shapiro R. Conley M.E. Reimund E. Kalhoff H. Abinun M. Munnich A. Israel A. Courtois G. Casanova J.L. Nat. Genet. 2001; 27: 277-285Crossref PubMed Scopus (668) Google Scholar). These mutations, which impair but do not eliminate NF-κB function, result in an X-linked immunodeficiency syndrome characterized by hyper-IgM production and hypohydrotic ectodermal dysplasia. Thus, both biochemical and genetic studies indicate a critical role for IKKγ/NEMO in regulating NF-κB activation. Although IKKγ/NEMO is critical for activation of the NF-κB pathway, the exact mechanisms involved in its regulation remain to be elucidated. Previously, we demonstrated that interactions between IKKγ/NEMO and IKKβ are critical for the formation of the high molecular weight IKK complex (41Li X.-H. Fang X. Gaynor R.B. J. Biol. Chem. 2001; 276: 4494-4500Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In this study, we addressed the role of IKKγ/NEMO in facilitating interactions between IκB and IKKβ. IKKγ/NEMO was critical for the association of IκB and IKKβ with the high molecular weight IKK complex. Furthermore, we found that IκB in the high molecular weight complex was preferentially phosphorylated. Finally, we demonstrated that IKKγ/NEMO enhanced the association of the IκBα and IKKβ and increased IKKβ kinase activity. Thus, the ability of IKKγ/NEMO to both stimulate the association of the IκB proteins with IKKβ and increase IKKβ kinase activity is likely important in activating the NF-κB pathway. The wild-type murine IKKγ/NEMO cDNA was cloned into the CMV expression vector pCMV5 fusing the Myc tag to the amino terminus of IKKγ/NEMO sequence (41Li X.-H. Fang X. Gaynor R.B. J. Biol. Chem. 2001; 276: 4494-4500Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). An N-terminal IKKγ/NEMO deletion containing amino acid residues 101–412 and a C-terminal IKKγ/NEMO deletion containing amino acids 1–312 were also cloned downstream of the Myc epitope in pCMV5 (41Li X.-H. Fang X. Gaynor R.B. J. Biol. Chem. 2001; 276: 4494-4500Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Wild-type human IKKβ was cloned into pCMV5 constructs containing either amino-terminal Myc, FLAG, or influenza hemagglutinin (HA) epitopes (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 (1841) Google Scholar, 56Yin 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 for the kinase-defective (K/M) mutant at residue 44 in IKKβ was described previously (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 (1841) Google Scholar, 56Yin 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 IκBα, IκBα mutant (SS/AA), and IκBβ proteins were each cloned into pCMV vectors with FLAG epitopes at their amino terminus (57Liu L. Kwak Y.-T. Bex F. Garcia-Martinez L.F. Li X.-H. Meek K. Lane W.S. Gaynor R.B. Mol. Cell Biol. 1998; 18: 4221-4234Crossref PubMed Google Scholar). The constructs to express the GST-IκBα and GST-IκBβ proteins were previously described (57Liu L. Kwak Y.-T. Bex F. Garcia-Martinez L.F. Li X.-H. Meek K. Lane W.S. Gaynor R.B. Mol. Cell Biol. 1998; 18: 4221-4234Crossref PubMed Google Scholar) as were the GST-IKKγ/NEMO constructs encoding either wild type, ΔN (aa 101–412), or ΔC (aa 1–312) (41Li X.-H. Fang X. Gaynor R.B. J. Biol. Chem. 2001; 276: 4494-4500Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). COS, mouse embryo fibroblasts (MEFs) (a gift of Xiaodong Wong), and IKKγ/NEMO knock-out cells (a gift of Michael Karin) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (49Makris 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 (334) Google Scholar). HeLa cells were maintained in Iscove's modified Dulbecco's medium and supplemented with the same components as above. Transfections were carried out using Fugene-6 (Roche Molecular Biochemicals) as described by the manufacturer. For a typical cell fractionation experiment, COS cells cultured overnight in 100-mm plates were transfected with 2.0 μg of each DNA construct. Cytoplasmic extracts were prepared from either 107 transfected COS cells or nontransfected cells including 108 HeLa, 2.5 × 108 MEFs, and 2.5 × 108 IKKγ knock-out as detailed previously (41Li X.-H. Fang X. Gaynor R.B. J. Biol. Chem. 2001; 276: 4494-4500Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). TNFα (20 ng/ml) and MG-132 (25 μm) were purchased from Roche Molecular Biochemicals and Calbiochem, respectively. For protein fractionation, the cytoplasmic extracts of cells were prepared according to Dignam (58Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9150) Google Scholar) with slight modifications. Cells, washed twice with cold phosphate-buffered saline, were harvested by scraping from the culture dishes and precipitated by centrifugation at 1500 rpm for 10 min. Cell pellets were resuspended in buffer A (10 mmHepes (pH 7.9), 1 mm EDTA, 10 mm KCl, 1 mm dithiothreitol) supplemented with phosphatase inhibitors (50 mm NaF, 50 mm glycerol phosphate, 1 mm sodium orthovanadate, 0.1 μm okadaic acid) and proteinase inhibitors (Roche Molecular Biochemicals). After incubation on ice for 15 min, the cells were disrupted with 20 strokes through a 25-gauge needle and centrifuged at 14,000 rpm for 15 min. The supernatants were mixed with 0.11 volume of buffer B (0.3m Hepes (pH 7.9), 30 mm MgCl2, 1.4m KCl) and centrifuged again at 100,000 ×g for 60 min. The supernatants were assayed for protein concentration according to the Bradford method, and a total of 2 mg of protein was subjected to Superdex-200 column (Amersham Pharmacia Biotech) chromatography 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 EGTA), and 1-ml fractions were collected. Protein markers (Sigma) used for the Superdex-200 column were bovine thyroglobulin (669 kDa), horse spleen apoferritin (443 kDa), β-amylase (200 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa). Western blotting was done with 30 μg of protein obtained from each of the column fractions including monoclonal antibodies directed against the HA epitope (12CA5), the FLAG epitope (M2; Sigma), and the Myc epitope (Roche Molecular Biochemicals) and polyclonal antibodies directed against IκBα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; Sc-371), phospho-IκBα (Ser32) antibody (Cell Signaling Technology), IKKβ (Santa Cruz Biotechnology; Sc-7607), and IKKγ (Santa Cruz Biotechnology; Sc-8330). The antibodies used are specified in the figure legends. Cytoplasmic lysates of COS cells containing 300 μg of protein were incubated with either 2.5 μg of the 12CA5 monoclonal antibody directed against the HA epitope, the M2 monoclonal antibody directed against the FLAG epitope, or the Myc monoclonal antibody. Following this, 20 μl of protein A-agarose beads were added and mixed for 1 h at 4 °C, and the immunoprecipitates were washed three times with modified buffer D (20 mm Hepes (pH 7.9), 100 mm KCl, 200 mm NaCl, 0.2 mm EDTA, 20% glycerol, 0.5 mm dithiotheitol, 0.06% Nonidet P-40) containing proteinase inhibitors (Roche Molecular Biochemicals). Electrophoresis on a 6% SDS-polyacrylamide gel was performed, and the gel was subjected to immunoblotting with specific antibodies and chemiluminescence reagents (Amersham Pharmacia Biotech). For kinase assays, 50 μl of each column fraction was incubated overnight at 4 °C with 1–2 μg of anti-IKKα (Sc-7606) obtained from Santa Cruz Biotechnology 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)). 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. 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. To better understand the mechanism by which IKKγ/NEMO stimulates IKKβ kinase activity, we transfected expression vectors encoding wild-type epitope-tagged IKKβ and a variety of epitope-tagged IKKγ/NEMO constructs into COS cells (Fig.1A). Following immunoprecipitation of IKKβ, we assayed its ability to phosphorylate GST fusion" @default.
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• W2000989306 title "IKKγ/NEMO Facilitates the Recruitment of the IκB Proteins into the IκB Kinase Complex" @default.
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