Title: Nuclear Role of IκB Kinase-γ/NF-κB Essential Modulator (IKKγ/NEMO) in NF-κB-dependent Gene Expression
Abstract: The IκB kinase (IKK) complex, which is composed of the two kinases IKKα and IKKβ and the regulatory subunit IKKγ/nuclear factor-κB (NF-κB) essential modulator (NEMO), is important in the cytokine-induced activation of the NF-κB pathway. In addition to modulation of IKK activity, the NF-κB pathway is also regulated by other processes, including the nucleocytoplasmic shuttling of various components of this pathway and the post-translational modification of factors bound to NF-κB-dependent promoters. In this study, we explored the role of the nucleocytoplasmic shuttling of components of the IKK complex in the regulation of the NF-κB pathway. IKKγ/NEMO was demonstrated to shuttle between the cytoplasm and the nucleus and to interact with the nuclear coactivator cAMP-responsive element-binding protein-binding protein (CBP). Using both in vitro and in vivo analysis, we demonstrated that IKKγ/NEMO competed with p65 and IKKα for binding to the N terminus of CBP, inhibiting CBP-dependent transcriptional activation. These results indicate that, in addition to the key role of IKKγ/NEMO in regulating cytokine-induced IKK activity, its ability to shuttle between the cytoplasm and the nucleus and to bind to CBP can lead to transcriptional repression of the NF-κB pathway. The IκB kinase (IKK) complex, which is composed of the two kinases IKKα and IKKβ and the regulatory subunit IKKγ/nuclear factor-κB (NF-κB) essential modulator (NEMO), is important in the cytokine-induced activation of the NF-κB pathway. In addition to modulation of IKK activity, the NF-κB pathway is also regulated by other processes, including the nucleocytoplasmic shuttling of various components of this pathway and the post-translational modification of factors bound to NF-κB-dependent promoters. In this study, we explored the role of the nucleocytoplasmic shuttling of components of the IKK complex in the regulation of the NF-κB pathway. IKKγ/NEMO was demonstrated to shuttle between the cytoplasm and the nucleus and to interact with the nuclear coactivator cAMP-responsive element-binding protein-binding protein (CBP). Using both in vitro and in vivo analysis, we demonstrated that IKKγ/NEMO competed with p65 and IKKα for binding to the N terminus of CBP, inhibiting CBP-dependent transcriptional activation. These results indicate that, in addition to the key role of IKKγ/NEMO in regulating cytokine-induced IKK activity, its ability to shuttle between the cytoplasm and the nucleus and to bind to CBP can lead to transcriptional repression of the NF-κB pathway. The nuclear factor-κB (NF-κB) 1The abbreviations used are: NF-κBnuclear factor-κBTNFαtumor necrosis factor-αIKKIκB kinaseNEMONF-κB essential modulatorCBPcAMP-responsive element-binding protein-binding proteinMEFsmouse embryo fibroblastsHAhemagglutininFITCfluorescein isothiocyanateGSTglutathione S-transferasePBSphosphate-buffered saline.1The abbreviations used are: NF-κBnuclear factor-κBTNFαtumor necrosis factor-αIKKIκB kinaseNEMONF-κB essential modulatorCBPcAMP-responsive element-binding protein-binding proteinMEFsmouse embryo fibroblastsHAhemagglutininFITCfluorescein isothiocyanateGSTglutathione S-transferasePBSphosphate-buffered saline. 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: 13-20Google Scholar, 2Baldwin Jr., A.S. Annu. Rev. 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A. 1999; 96: 1042-1047Google Scholar). nuclear factor-κB tumor necrosis factor-α IκB kinase NF-κB essential modulator cAMP-responsive element-binding protein-binding protein mouse embryo fibroblasts hemagglutinin fluorescein isothiocyanate glutathione S-transferase phosphate-buffered saline. nuclear factor-κB tumor necrosis factor-α IκB kinase NF-κB essential modulator cAMP-responsive element-binding protein-binding protein mouse embryo fibroblasts hemagglutinin fluorescein isothiocyanate glutathione S-transferase phosphate-buffered saline. IKKγ/NEMO interacts with IKKα and IKKβ and is a component of the high molecular mass IKK complex, which migrates between 600 and 900 kDa following gel filtration chromatography (12Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Google Scholar, 13Mercurio 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. 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Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Google Scholar, 13Mercurio 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: 1526-1538Google Scholar, 14Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Google Scholar). IKKγ/NEMO has a molecular mass of 48 kDa and contains several domains (21Chu Z.L. Shin Y.A. Yang J.M. DiDonato J.A. Ballard D.W. J. Biol. Chem. 1999; 274: 15297-15300Google Scholar, 22May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Google Scholar), including an N-terminal domain, which is involved in its interactions with IKKβ (22May M.J. D'Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Science. 2000; 289: 1550-1554Google Scholar); a coiled-coil domain, which mediates its oligomerization, which is critical in stimulating IKK activity (23Poyet J.L. Srinivasula S.M. Lin J.H. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Google Scholar); and a C-terminal domain, which is involved in the recruitment of upstream factors such as receptor-interacting protein that are involved in IKK activation (15Li 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: 1042-1047Google Scholar, 24Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Google Scholar). Thus, the structure of IKKγ/NEMO is consistent with its role as a scaffold that is critical for function. Genetic studies have also demonstrated an important role of IKKγ/NEMO in regulating the NF-κB pathway (25Makris 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: 969-979Google Scholar, 26Rudolph D. Yeh W.C. Wakeham A. Rudolph B. Nallainathan D. Potter J. Elia A.J. Mak T.W. Genes Dev. 2000; 14: 854-862Google Scholar, 27Schmidt-Supprian M. Bloch W. Courtois G. Addicks K. Israël A. Rajewsky K. Pasparakis M. Mol. Cell. 2000; 5: 981-992Google Scholar). Disruption of the IKKγ/NEMO gene (which is located on the X chromosome) in male mice and homozygous deletion in female mice result in embryonic lethality due to TNFα-induced hepatocyte apoptosis (25Makris 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: 969-979Google Scholar, 27Schmidt-Supprian M. Bloch W. Courtois G. Addicks K. Israël A. Rajewsky K. Pasparakis M. Mol. Cell. 2000; 5: 981-992Google Scholar). Female mice with a deletion of a single copy of IKKγ/NEMO develop granulocytic infiltration and both hyperproliferation and increased apoptosis of keratinocytes (25Makris 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: 969-979Google Scholar, 27Schmidt-Supprian M. Bloch W. Courtois G. Addicks K. Israël A. Rajewsky K. Pasparakis M. Mol. Cell. 2000; 5: 981-992Google Scholar). Fibroblasts isolated from IKKγ/NEMO-null 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 granulocytic infiltration of the skin in females (28Smahi A. Courtois G. Vabres P. Yamaoka S. Heuertz S. Munnich A. Israël 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: 466-472Google Scholar). Another syndrome has been described in humans that is due to mutations in the putative zinc finger domain in the C terminus of IKKγ/NEMO that impair, but do not eliminate, NF-κB function, resulting in an X-linked immunodeficiency syndrome characterized by hyper-IgM production and hypohydrotic ectodermal dysplasia (29Jain A. Ma C.A. Liu S. Brown M. Cohen J. Strober W. Nat. Immunol. 2001; 2: 223-228Google Scholar, 30Doffinger 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. Israël A. Courtois G. Casanova J.L. Nat. Genet. 2001; 27: 277-285Google Scholar, 31Zonana 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: 1555-1562Google Scholar). Thus, both biochemical and genetic studies indicate a critical role for IKKγ/NEMO in regulating NF-κB activation. Recently, we (32Yamamoto Y. Verma U.N. Prajapati S. Kwak Y.T. Gaynor R.B. Nature. 2003; 423: 655-659Google Scholar) and others (33Anest V. Hanson J.L. Cogswell P.C. Steinbrecher K.A. Strahl B.D. Baldwin Jr., A.S. Nature. 2003; 423: 659-663Google Scholar) demonstrated that, in addition to the cytoplasmic role of the IKK complex, one of its components (IKKα) can also function in the nucleus to stimulate cytokine-induced expression of NF-κB-responsive genes. IKKα was found to interact with CBP and, in conjunction with p65, is recruited in a cytokine-dependent manner to NF-κB-responsive promoters, where it is critical for the phosphorylation and subsequent acetylation of specific residues in histone H3 to activate gene expression (32Yamamoto Y. Verma U.N. Prajapati S. Kwak Y.T. Gaynor R.B. Nature. 2003; 423: 655-659Google Scholar, 33Anest V. Hanson J.L. Cogswell P.C. Steinbrecher K.A. Strahl B.D. Baldwin Jr., A.S. Nature. 2003; 423: 659-663Google Scholar). The nuclear levels of IKKα result from its ability to shuttle between the cytoplasm and the nucleus in a CRM1-dependent fashion (34Birbach A. Gold P. Binder B.R. Hofer E. de Martin R. Schmid J.A. J. Biol. Chem. 2002; 277: 10842-10851Google Scholar). In this study, we demonstrate that IKKγ/NEMO is present in both the nucleus and cytoplasm of HeLa cells and that leptomycin B treatment increases its nuclear localization. This observation suggests that IKKγ/NEMO constitutively shuttles between cytoplasmic and nuclear compartments in a CRM1-dependent manner, as has been demonstrated for other proteins involved in the regulation of the NF-κB pathway (35Huang T.T. Kudo N. Yoshida M. Miyamoto S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1014-1019Google Scholar, 36Johnson C. Van Antwerp D. Hope T.J. EMBO J. 1999; 18: 6682-6693Google Scholar, 37Rodriguez M.S. Thompson J. Hay R.T. Dargemont C. J. Biol. Chem. 1999; 274: 9108-9115Google Scholar, 38Tam W.F. Lee L.H. Davis L. Sen R. Mol. Cell. Biol. 2000; 20: 2269-2284Google Scholar, 39Harhaj E.W. Sun S.C. Mol. Cell. Biol. 1999; 19: 7088-7095Google Scholar). Mammalian two-hybrid and in vitro binding assays demonstrated that nuclear IKKγ/NEMO bound to the N terminus of CBP, repressing NF-κB-regulated genes. These studies indicate that IKKγ/NEMO, like IKKα, can regulate NF-κB-dependent gene expression via its interactions with factors in both the cytoplasm and the nucleus. Cell Lines and Reagents—HeLa and 293 cells were purchased from American Type Culture Collection (Manassas, VA). Mouse embryo fibroblasts (MEFs) and IKKγ/NEMO-/- cells were gifts from Drs. Xiaodong Wong and Michael Karin, respectively. These cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone Laboratories), 2 mml-glutamine, and penicillin/streptomycin. Polyclonal antibodies directed against IKKα (sc-7182), IKKβ (sc-7607), IKKγ/NEMO (sc-8330), p65 (sc-372), and CBP (sc-583) were obtained from Santa Cruz Biotechnology. Monoclonal antibodies directed against IKKγ/NEMO and transcription factor IIB (BD Biosciences), the Myc epitope (Pharmingen), the hemagglutinin (HA) epitope (12CA5, Roche Applied Science), and the FLAG epitope (M2, Sigma) were used in immunoprecipitation and Western blot analysis. Donkey anti-rabbit, anti-mouse, and anti-goat antibodies conjugated to either fluorescein isothiocyanate (FITC) or rhodamine Red-X were obtained from Jackson ImmunoResearch Laboratories, Inc. Plasmid Constructs—The pCMV5 expression vectors encoding FLAG- or Myc-tagged IKKα, IKKβ, IKKγ, or p65 or HA-tagged CBP were described previously (32Yamamoto Y. Verma U.N. Prajapati S. Kwak Y.T. Gaynor R.B. Nature. 2003; 423: 655-659Google Scholar, 40Yamamoto Y. Kim D.W. Kwak Y.T. Prajapati S. Verma U. Gaynor R.B. J. Biol. Chem. 2001; 276: 36327-36336Google Scholar, 41Yin 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: 875-884Google Scholar). Gal4-CBP constructs were kindly provided by Dr. Tucker Collins, and fusions of the VP16 activation domain with IKKα, IKKβ, or IKKγ/NEMO were constructed in pCMV5 as described previously (32Yamamoto Y. Verma U.N. Prajapati S. Kwak Y.T. Gaynor R.B. Nature. 2003; 423: 655-659Google Scholar). Glutathione S-transferase (GST)-CBP fusions were constructed by generating the desired fragments from full-length CBP using PCR and subsequently cloning these fragments with GST into the pGEX vector (42Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Google Scholar). All PCR products were verified by DNA sequencing. Expression and Purification of GST-CBP Fusion Proteins—Recombinant GST-CBP fusion proteins were expressed in bacterial strain BL21 and lysed in HMK buffer (50 mm Tris-HCl (pH 7.5), 100 mm NaCl, and 1 mm phenylmethylsulfonyl fluoride), and the bacterial lysates were incubated with 0.5 ml of packed glutathione-conjugated agarose beads (Sigma) overnight at 4 °C. After three washes, the GST fusion proteins bound to the glutathione beads were stored at 4 °C in HMK buffer. Protein expression was assessed by SDS-PAGE and Coomassie Blue staining. Luciferase Reporter Assays—293T or HeLa cells were plated at 50% confluence in 6-well tissue culture plates. After 24 h, the cells were transfected using Genejuice transfection reagent (Novagen) with the indicated DNA constructs and a Gal4-luciferase reporter. A Rous sarcoma virus-β-galactosidase expression vector was included in the transfection assays to control for differences in transfection efficiency, and a pCMV5 plasmid was added to standardize DNA quantities. Between 30 and 36 h post-transfection, the cells were washed twice with cold phosphate-buffered saline (PBS), and the reporter activity was measured using the luciferase assay system (Promega). All transfections were performed in duplicate and repeated at least three times. Protein Association and Western Blot Analysis—For GST pull-down analysis with endogenous proteins, whole cell lysates were prepared from HeLa cells in lysis buffer A (40 mm Tris-HCl (pH 8.0), 500 mm NaCl, 6 mm EDTA, 6 mm EGTA, 10 mm β-glycerophosphate, 10 mm NaF, 1 mm Na3VO4, 1 mm dithiothreitol, 0.1% Nonidet P-40, and protease inhibitors (Roche Applied Science)), and equal protein amount of cell lysate were mixed with beads containing equal protein amount of GST-CBP or -GST and incubated overnight at 4 °C on a rotatory shaker. Following incubation, the beads were extensively washed with HMK buffer, with HMK buffer with 500 mm NaCl, and with HMK buffer with 0.1% Triton X-100, respectively, before a final wash with HMK buffer. Proteins bound to the beads were eluted by adding protein loading buffer and heating to 100 °C for 5 min and resolved on a 10% SDS-polyacrylamide gel; transferred to nitrocellulose membranes (Amersham Biosciences); and probed with antibodies to IKKα, IKKβ, IKKγ/NEMO, and p65. For GST pull-down analysis with transiently expressed proteins, expression plasmids encoding FLAG-tagged IKKα, IKKβ, IKKγ/NEMO, and p65 were transfected into 293T cells. Thirtysix hours post-transfection, whole cell lysates were prepared in PD buffer and incubated with GST-CBP or GST beads, and assays were performed as described above, with the Western blots being probed with anti-FLAG antibody. For immunoprecipitation and Western blot analysis, 293T cells were transfected with expression vectors encoding HA-tagged CBP in combination with FLAG-tagged IKKα, IKKβ, IKKγ/NEMO, or p65 or Myctagged IKKγ/NEMO as indicated. Cell lysates were prepared in lysis buffer B (50 mm HEPES (pH 7.5), 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EGTA, 10 mm sodium pyrophosphate, 10 mm NaF, and 10 mm Na3VO4). Immunoprecipitation was performed with anti-FLAG antibody or mouse IgG as a control, and the precipitates were captured on protein A-agarose beads and extensively washed. The bound proteins were resolved by SDS-PAGE; transferred to nitrocellulose membrane; and probed with anti-HA antibody 12CA5, anti-FLAG antibody M2, or anti-Myc antibody as indicated. Western blots were analyzed by enhanced chemiluminescence (Amersham Biosciences) after labeling with horseradish peroxidase-conjugated antimouse or anti-rabbit secondary antibody. Immunofluorescence and Confocal Microscopy—The cellular localization of IKKα, IKKβ, IKKγ/NEMO, and p65 was analyzed using both endogenous as well as transiently expressed proteins. For these experiments, HeLa cells and MEFs alone or transfected with expression vectors encoding the indicated Myc epitope-tagged constructs were cultured on coverslips and either untreated or treated with leptomycin B (Sigma) at final concentration of 10 ng/ml for 2 h. Coverslips were washed two times with PBS, and the cells were fixed with 3.7% formaldehyde for 10 min, followed by a brief permeabilization with 0.5% Triton X-100 in PBS. The cells were blocked for 30 min with 3% normal donkey serum in PBS and then incubated for 1 h with primary antibodies as indicated in the figures (diluted 1:50 to 1:200 in 1% normal donkey serum in PBS). The coverslips were washed three times with PBS and then incubated for 1 h with secondary antibodies conjugated to FITC or rhodamine Red-X (diluted 1:400 in 1% normal donkey serum in PBS). Nuclei were visualized by staining for lamin B. Samples were washed three times and then treated with Aquamount (Polyscience), and the results were analyzed using an MRC 1000 laser scanning confocal microscope (Bio-Rad). Cellular Localization of IKKγ/NEMO—Immunofluorescence studies were performed with untreated HeLa cells to analyze the localization of IKKα, IKKβ, IKKγ/NEMO, and p65. As demonstrated previously (32Yamamoto Y. Verma U.N. Prajapati S. Kwak Y.T. Gaynor R.B. Nature. 2003; 423: 655-659Google Scholar), IKKα was localized predominantly in the nucleus, whereas both IKKβ and p65 were localized predominantly in the cytoplasm (Fig. 1A). IKKγ/NEMO was found to be localized in both the cytoplasm and the nucleus (Fig. 1A). As a positive control, TNFα stimulation was found to induce the nuclear translocation of p65, but did not significantly change the cytoplasmic or nuclear distribution of IKKγ/NEMO or IKKα (data not shown). Next, we addressed whether IKKγ/NEMO can constitutively shuttle between the cytoplasm and the nucleus, as do other components of the NF-κB pathway, including IKKα (34Birbach A. Gold P. Binder B.R. Hofer E. de Martin R. Schmid J.A. J. Biol. Chem. 2002; 277: 10842-10851Google Scholar), NF-κB-inducing kinase (34Birbach A. Gold P. Binder B.R. Hofer E. de Martin R. Schmid J.A. J. Biol. Chem. 2002; 277: 10842-10851Google Scholar), and IκBα and p65 (35Huang T.T. Kudo N. Yoshida M. Miyamoto S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1014-1019Google Scholar, 36Johnson C. Van Antwerp D. Hope T.J. EMBO J. 1999; 18: 6682-6693Google Scholar, 37Rodriguez M.S. Thompson J. Hay R.T. Dargemont C. J. Biol. Chem. 1999; 274: 9108-9115Google Scholar, 38Tam W.F. Lee L.H. Davis L. Sen R. Mol. Cell. Biol. 2000; 20: 2269-2284Google Scholar, 39Harhaj E.W. Sun S.C. Mol. Cell. Biol. 1999; 19: 7088-7095Google Scholar). HeLa cells were treated with leptomycin B, a selective inhibitor of CRM1-dependent nuclear export (43Ossareh-Nazari B. Bachelerie F. Dargemont C. Science. 1997; 278: 141-144Google Scholar, 44Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Google Scholar), and the localization of IKKγ/NEMO, IKKα, IKKβ, and p65 was then determined. In the presence of leptomycin B, both IKKγ/NEMO and p65 became predominantly localized in the nucleus, without marked changes in the distribution of IKKα and IKKβ (Fig. 1B). To confirm these results, we next evaluated the distribution of Myc-tagged IKKα, IKKβ, and IKKγ/NEMO expressed in HeLa cells in both the presence and absence of leptomycin B. Similar to the results seen with endogenous IKKγ/NEMO, transfected Myc-tagged IKKγ/NEMO became predominantly localized to the nucleus following leptomycin B treatment (Fig. 2B). No significant change in the distribution of IKKα or IKKβ was noted following leptomycin B treatment (Fig. 2B). Next, we characterized the cellular localization of several Myc-tagged IKKγ/NEMO constructs in the presence and absence of leptomycin B treatment. Expression vectors encoding Myc-tagged wild-type IKKγ/NEMO, an N-terminal truncation mutant (IKKγ/NEMO-(101-412)), and two C-terminal truncation mutants (IKKγ/NEMO-(1-358) and IKKγ/NEMO-(1-306)) were analyzed following their transfection into HeLa cells. All of these IKKγ/NEMO constructs, except IKKγ/NEMO-(1-306), were predominantly localized in the cytoplasm and become localized to the nucleus following leptomycin B treatment (Fig. 3). In contrast, IKKγ/NEMO-(1-306) was present predominantly in the nucleus in both the presence and absence of leptomycin B (Fig. 3). Classical nuclear export sequences contain leucine-rich domains with variations of the motif LXXXLXXLX(L/I) (39Harhaj E.W. Sun S.C. Mol. Cell. Biol. 1999; 19: 7088-7095Google Scholar, 43Ossareh-Nazari B. Bachelerie F. Dargemont C. Science. 1997; 278: 141-144Google Scholar, 44Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Google Scholar). IKKγ/NEMO contains the sequence LLXXXLXXL between residues 328 and 336, which is similar, although not an exact match, to a consensus nuclear export sequence. These experiments suggest that a region in the C terminus of IKKγ/NEMO is involved in regulating its nuclear export. IKKγ/NEMO Interacts with CBP—The results of the immunofluorescence studies indicate that IKKγ/NEMO translocated to the nucleus either constitutively or in response to a yet unidentified stimulus. However, no previous studies have indicated a nuclear role of IKKγ/NEMO. Recently, we (32Yamamoto Y. Verma U.N. Prajapati S. Kwak Y.T. Gaynor R.B. Nature. 2003; 423: 655-659Google Scholar) and others (33Anest V. Hanson J.L. Cogswell P.C. Steinbrecher K.A. Strahl B.D. Baldwin Jr., A.S. Nature. 2003; 423: 659-663Google Scholar) demonstrated that IKKα can interact with the N terminus of the coactivator CBP, resulting in the phosphorylation and subsequent acetylation of histone H3, leading to increases in NF-κB-dependent gene expression. Because the N terminus of CBP is also the site of p65 interaction, we hypothesized that IKKγ/NEMO might also interact with CBP to alter NF-κB-regulated gene expression in a manner similar to IKKα and p65. First, the interaction of IKKγ/NEMO and CBP was characterized using the mammalian two-hybrid system. HeLa cells were transfected with a Gal4-luciferase reporter and Gal4-CBP constructs in conjunction with VP16 fusions with IKKγ/NEMO, IKKα, IKKβ, or p65. We have demonstrated previously that the fusion of VP16 with either IKKα or p65 can interact with Gal4-CBP to stimulate luciferase reporter activity (32Yamamoto Y. Verma U.N. Prajapati S. Kwak Y.T. Gaynor R.B. Nature. 2003; 423: 655-659Google Scholar). Luciferase reporter activity assayed at 30-36 h post-transfection demonstrated strong interactions of IKKα, IKKγ/NEMO, and p65 with the N terminus of CBP and only a minimal association of IKKα and IKKγ/NEMO with the C terminus of CBP (Fig. 4A). IKKβ did not interact with CBP (Fig. 4A). It is interesting to note that p65, IKKα, and IKKγ/NEMO could all interact with the N terminus of CBP. Next, the interaction of IKKγ/NEMO with CBP was characterized using in vitro binding assays of HeLa cell lysate with either GST or GST-CBP. HeLa cell lysate was incubated with either GST-CBP or GST immobilized on glutathione beads, and the bound proteins were subjected to Western blot analysis with antibody directed against IKKα, IKKβ, IKKγ/NEMO, or p65. There was strong interaction between IKKγ/NEMO and CBP, which was comparable with those seen with p65 and IKKα (Fig. 4B). Consistent with our previous results (32Yamamoto Y. Verma U.N. Prajapati S. Kwak Y.T. Gaynor R.B. Nature. 2003; 423: 655-659Google Scholar), there was no interaction between IKKβ and CBP (Fig. 4B). Similar experiments were also performed with 293T cell extracts prepared from cells transfected with