Regulation of IκB Kinase (IKK)γ/NEMO Function by IKKβ-mediated Phosphorylation
2002; Elsevier BV; Volume: 277; Issue: 27 Linguagem: Inglês
10.1074/jbc.m201393200
ISSN1083-351X
AutoresShashi Prajapati, Richard B. Gaynor,
Tópico(s)Immune Cell Function and Interaction
ResumoThe IκB kinase (IKK) complex includes the catalytic components IKKα and IKKβ in addition to the scaffold protein IKKγ/NEMO. Increases in the activity of the IKK complex result in the phosphorylation and subsequent degradation of IκB and the activation of the NF-κB pathway. Recent data indicate that the constitutive activation of the NF-κB pathway by the human T-cell lymphotrophic virus, type I, Tax protein leads to enhanced phosphorylation of IKKγ/NEMO by IKKβ. To address further the significance of IKKβ-mediated phosphorylation of IKKγ/NEMO, we determined the sites in IKKγ/NEMO that were phosphorylated by IKKβ, and we assayed whether IKKγ/NEMO phosphorylation was involved in modulating IKKβ activity. IKKγ/NEMO is rapidly phosphorylated following treatment of cells with stimuli such as tumor necrosis factor-α and interleukin-1 that activate the NF-κB pathway. By using both in vitro and in vivo assays, IKKβ was found to phosphorylate IKKγ/NEMO predominantly in its carboxyl terminus on serine residue 369 in addition to sites in the central region of this protein. Surprisingly, mutation of these carboxyl-terminal serine residues increased the ability of IKKγ/NEMO to stimulate IKKβ kinase activity. These results indicate that the differential phosphorylation of IKKγ/NEMO by IKKβ and perhaps other kinases may be important in regulating IKK activity. The IκB kinase (IKK) complex includes the catalytic components IKKα and IKKβ in addition to the scaffold protein IKKγ/NEMO. Increases in the activity of the IKK complex result in the phosphorylation and subsequent degradation of IκB and the activation of the NF-κB pathway. Recent data indicate that the constitutive activation of the NF-κB pathway by the human T-cell lymphotrophic virus, type I, Tax protein leads to enhanced phosphorylation of IKKγ/NEMO by IKKβ. To address further the significance of IKKβ-mediated phosphorylation of IKKγ/NEMO, we determined the sites in IKKγ/NEMO that were phosphorylated by IKKβ, and we assayed whether IKKγ/NEMO phosphorylation was involved in modulating IKKβ activity. IKKγ/NEMO is rapidly phosphorylated following treatment of cells with stimuli such as tumor necrosis factor-α and interleukin-1 that activate the NF-κB pathway. By using both in vitro and in vivo assays, IKKβ was found to phosphorylate IKKγ/NEMO predominantly in its carboxyl terminus on serine residue 369 in addition to sites in the central region of this protein. Surprisingly, mutation of these carboxyl-terminal serine residues increased the ability of IKKγ/NEMO to stimulate IKKβ kinase activity. These results indicate that the differential phosphorylation of IKKγ/NEMO by IKKβ and perhaps other kinases may be important in regulating IKK activity. tumor necrosis factor-α interleukin 1 IκB kinase glutathione S-transferase Rous sarcoma virus cytomegalovirus The NF-κB pathway is a critical regulator of the cellular response to a variety of stimuli including the cytokines, TNFα1 and IL-1, bacterial and viral infection, double-stranded RNA, and the human T-cell leukemia virus transactivator protein Tax (1Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2951) Google Scholar, 2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5646) Google Scholar, 3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4657) Google Scholar, 4Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (307) Google Scholar). The ability to activate rapidly and subsequently silence the NF-κB pathway in response to a variety of extracellular stimuli suggests that both positive and negative regulation is involved in its control. The further characterization of the mechanisms that regulate this pathway will be important for better understanding how NF-κB is involved in the control of the host immune and inflammatory responses. The members of the NF-κB family of transcription factors, which include p105/50, p100/52, p65, c-Rel, and RelB, contain a Rel homology domain that mediates their heterodimerization and homodimerization properties and DNA-binding properties (2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5646) Google Scholar). These proteins are sequestered in the cytoplasm of most cells where they are bound to a family of inhibitory proteins known as IκB (1Baeuerle P.A. Baltimore D. 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This process leads to the nuclear translocation of the NF-κB proteins, which then bind to consensus DNA sequences located upstream of a variety of cellular genes that are involved in the control of the immune and the inflammatory response and prevent apoptosis (1Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2951) Google Scholar, 2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5646) Google Scholar, 3Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4657) Google Scholar, 5Karin M. Oncogene. 1999; 18: 6867-6874Crossref PubMed Scopus (1016) Google Scholar, 6Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4155) Google Scholar, 7Pahl H.L. Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3495) Google Scholar). Activation of the IκB kinases, which phosphorylate the IκB proteins on the amino-terminal serine residues to result in their degradation, is a critical process that regulates the NF-κB pathway (8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1930) Google Scholar, 9Mercurio 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 (1861) Google Scholar, 10Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 11Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1073) Google Scholar, 12Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1610) Google Scholar). The IκB kinases, IKKα and IKKβ, are components of a 600–900-kDa complex that is composed of these two catalytic subunits (8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1930) Google Scholar, 9Mercurio 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 (1861) Google Scholar, 10Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 11Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1073) Google Scholar, 12Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1610) Google Scholar) in addition to a scaffold protein IKKγ/NEMO (13Li 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 (157) Google Scholar, 14Mercurio 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, 15Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (857) Google Scholar, 16Yamaoka 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 (955) Google Scholar). Stimulation of IKK activity by cytokine treatment has been demonstrated to involve both activation of mitogen-activated protein 3-kinases and/or IKK autophosphorylation (17Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (755) Google Scholar, 18Yin 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 and IKKβ have 52% amino acid identity, and their domain structure is similar with an amino-terminal kinase, leucine zipper, and helix-loop-helix motifs (8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1930) Google Scholar, 9Mercurio 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 (1861) Google Scholar, 10Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 11Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1073) Google Scholar, 12Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1610) Google Scholar). Although these kinases have a similar structure and are able to both homodimerize and heterodimerize, IKKβ is at least 20-fold more active in phosphorylation of the IκB proteins as compared with IKKα (9Mercurio 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 (1861) Google Scholar, 14Mercurio 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, 18Yin 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, 19Kwak Y.T. Guo J. Shen J. Gaynor R.B. J. Biol. Chem. 2000; 275: 14752-14759Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Studies using fibroblasts isolated from IKKα (20Li Q., Lu, Q. Hwang J.Y. Buscher D. Lee K.F. 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Immunity. 1999; 10: 421-429Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar) knock-out mice also demonstrate that IKKβ is the dominant kinase in regulating NF-κB activity. IKKγ/NEMO is critical for increasing IKK activity in response to all known stimuli. This protein contains several distinct domains including an amino-terminal domain that mediates its interaction with IKKβ, a coiled-coil domain that is important in its oligomerization, a leucine zipper of as yet uncharacterized function, and the carboxyl terminus that mediates the recruitment of upstream kinases that are involved in modulating IKK activity (13Li 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 (157) Google Scholar, 14Mercurio 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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Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). The interaction of IKKγ/NEMO with IKKα and IKKβ is critical for the assembly of this high molecular weight IKK complex that leads to the recruitment of IκB proteins and the stimulation of IKKβ activity (8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1930) Google Scholar, 12Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1610) Google Scholar, 14Mercurio 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, 15Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (857) Google Scholar, 16Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. 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Cells lacking IKKγ/NEMO are unable to assemble the high molecular weight IKK complex and exhibit severe defects in IKK activation in response to agents that stimulate the NF-κB pathway (14Mercurio 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, 15Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (857) Google Scholar, 16Yamaoka 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 (955) Google Scholar). IKKγ/NEMO also binds to a variety of proteins other than IKKs including the adaptor protein RIP, which is involved in TNFα-mediated activation of IKK (13Li 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 (157) Google Scholar, 30Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar), A20 which decreases TNFα-mediated activation of IKK (30Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar), the human T-cell lymphotrophic virus, type I, Tax protein which stimulates IKK activity (26Chu 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 (162) Google Scholar, 34Harhaj E.W. Sun S.C. J. Biol. Chem. 1999; 274: 22911-22914Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 35Jin D.Y. Giordano V. Kibler K.V. Nakano H. Jeang K.T. J. Biol. 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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 (633) Google Scholar). Thus, IKKγ/NEMO plays a central role in the activation of the NF-κB pathway in response to a variety of different stimuli. Recently it was demonstrated (40Carter R.S. Geyer B.C. Xie M. Acevedo-Suarez C.A. Ballard D.W. J. Biol. Chem. 2001; 276: 24445-24448Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) that the human T-cell lymphotrophic virus, type I, Tax protein, which results in the constitutive activation of the NF-κB pathway, leads to constitutive phosphorylation of both IKKβ and IKKγ/NEMO. Furthermore, IKKβ was shown to phosphorylate IKKγ/NEMO using in vitro kinase assays. These results suggested that IKKβ and IKKγ/NEMO could potentially regulate the function of each other. In this study, bothin vivo and in vitro assays were utilized to characterize IKKγ/NEMO phosphorylation by IKKβ. Our results indicate that IKKβ phosphorylation of IKKγ/NEMO appears to be important for regulating its functional properties. The murine IKKγ/NEMO cDNA was cloned into the CMV expression vector pCMV5 in which the Myc epitope tag was fused to the amino terminus of IKKγ/NEMO (27Li X.H. Fang X. Gaynor R.B. J. Biol. Chem. 2001; 276: 4494-4500Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). An amino-terminal IKKγ/NEMO deletion containing amino acid residues 101–412 was constructed using PCR, whereas carboxyl-terminal truncations of IKKγ/NEMO containing amino acids 1–394, 1–358, 1–312, 1–270, 1–180, 1–137, and 1–105, respectively, were constructed by placing a stop codon (TAG) into the IKKγ/NEMO cDNA using oligonucleotide-directed mutagenesis with a QuickChange Kit (Stratagene). Alanine residues were substituted for serine residues at positions 369 and 375, threonine residue 147, and serine residues 148, 156, and 158. The presence of these mutations was confirmed by DNA sequencing. The IKKγ/NEMO cDNAs were then cloned downstream of the Myc epitope in pCMV5 and GST in pGEX-KG (27Li X.H. Fang X. Gaynor R.B. J. Biol. Chem. 2001; 276: 4494-4500Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Wild-type human IKKα and IKKβ as well as the cDNAs for the IKK kinase-defective K44M and either the constitutively active or inactive IKK mutants for IKKβ (S177E/S181E and S177A/S181A) and for IKKα (S176E/S180E and S176A/S180A), respectively, were cloned into a pCMV5 construct containing a FLAG epitope (9Mercurio 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 (1861) Google Scholar, 18Yin 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 GST fusion proteins containing the wild-type IκB extending from amino acids 1–54 and mutant IκBα (S32A/S36A) were described previously (41Liu 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). 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (37Makris 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 (340) Google Scholar). HeLa cells were maintained in Iscove's modified Dulbecco's media and supplemented with the same components as above. Transfections were carried out using FuGENE-6 (Roche Molecular Biochemicals) as described by the manufacturer. Cytoplasmic extracts from 293T cells and HeLa cells were prepared as described previously (27Li X.H. Fang X. Gaynor R.B. J. Biol. Chem. 2001; 276: 4494-4500Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). IKKγ/NEMO−/− mouse embryo fibroblasts (37Makris 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 (340) Google Scholar) were plated in 35-mm tissue culture wells with Dulbecco's modified Eagle's media. After 24 h, the cells were transfected with CMV expression vectors encoding either wild-type or mutant Myc-tagged IKKγ/NEMO (0.3 μg), an NF-κB luciferace reporter (0.1 μg), and an RSV-β-galactosidase expression vector (0.1 μg). After 18 h of transfection, the cells were treated with TNFα (10 ng/ml) for 6 h. The cells were then treated with reporter lysis buffer (Promega), and luciferase activity was determined according to the manufacturer's protocol (Promega). The transfection efficiency was monitored by assaying β-galactosidase activity. All transfections were performed in triplicate and repeated in three independent experiments. To determine the interactions between wild-type and mutant IKKγ/NEMO and IKKβ, wild-type or mutant CMV-IKKβ (0.1 μg) and either wild-type or mutant CMV-IKKγ/NEMO vectors (1.0 μg) were transfected into 293T cells. Extracts (400 μg) were then prepared in PD buffer (40 mm Tris-HCl, pH 8.0, 500 mm NaCl, 6.0 mm EGTA, 6.0 mm EDTA, 10 mmβ-glycerophosphate, 0.5 mm dithiothreitol, 10 mm NaF, 300 μm sodium vanadate, and protease inhibitors (Roche Molecular Biochemicals)), incubated for 2–4 h at 4 °C with the Myc monoclonal antibody (2.0 μg), directed against the Myc epitope, followed by the addition of 20 μl of protein A-agarose beads for 1 h at 4 °C. The immunoprecipitates were washed three times with PD buffer. Electrophoresis on a 10% SDS-polyacrylamide gel was performed, and the gel was subjected to immunoblotting with specific antibodies and developed using chemiluminescence reagents (Amersham Biosciences). Western blotting of different extracts was performed with monoclonal antibodies directed against the FLAG epitope (M2, Sigma) and the Myc epitope (Roche Molecular Biochemicals) or polyclonal antibodies directed against IκBα (Santa Cruz Biotechnology, sc-371), IKKβ (Santa Cruz Biotechnology, sc-7607), and IKKγ (Santa Cruz Biotechnology, sc-8330) as indicated. To assay IKKβ phosphorylation of IKKγ/NEMO, 293T cells were transfected with wild-type or mutant Myc-tagged CMV-IKKγ/NEMO (4.0 μg) or FLAG-tagged CMV-IKKβ (2.0 μg) and harvested 30 h post-transfection. Cytoplasmic extracts (400 μg) were incubated overnight at 4 °C with 1–2 μg of anti-Myc or anti-FLAG monoclonal antibodies, followed by the addition of protein A-agarose (Bio-Rad) for 1–3 h at 4 °C and washed three times with ELB buffer (50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 5.0 mm NaF, 5.0 mmβ-glycerophosphate, and 1.0 mm sodium vanadate).In vitro kinase assays were performed for 30 min at 30 °C in the presence of kinase buffer containing 1.0 mm dithiothreitol, 10 μm ATP, and 10 μCi of [γ-32P]ATP, and the reactions were stopped with protein sample buffer and heated at 95 °C for 3 min as described previously (27Li X.H. Fang X. Gaynor R.B. J. Biol. Chem. 2001; 276: 4494-4500Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The samples were then subjected to SDS-PAGE on a 12% polyacrylamide gel and visualized by autoradiography. To assay IKKβ phosphorylation of wild-type and mutant GST-IKKγ/NEMO proteins, wild-type or mutant CMV-IKKβ (2.0 μg) was transfected into 293T cells, and at 30 h post-transfection, the cells were harvested and lysed in PD buffer. The extracts were immunoprecipitated with the M2 FLAG monoclonal antibody, and following the addition of protein A-agarose, in vitro kinase assays with the GST-IKKγ/NEMO substrate (10.0 μg) were performed as described above. Finally, to assay increases in IKKβ activity by wild-type and mutant IKKγ/NEMO, 293T cells were cotransfected with CMV expression vectors encoding IKKγ/NEMO (0.4 μg) and IKKβ (0.01 μg), respectively. At 30 h post-transfection, cellular lysates (200 μg) were prepared in PD buffer, and in vitro kinase assays with a GST-IκBα substrate (10.0 μg) were performed as described above. For in vivo labeling, HeLa cells at 60% confluence were grown in Dulbecco's modified Eagle's medium lacking either phosphate or methionine (Invitrogen) in the absence of serum for 4 h. At that time, either 50 μCi of [32P]orthophosphate (50 μCi/ml) or [35S]methionine (50 μCi/ml) (PerkinElmer Life Sciences) was added for 4 h. The cells were then treated with either TNFα (20 ng/ml) (Roche Molecular Biochemicals) or IL-1 (20 ng/ml) (Roche Molecular Biochemicals) for the indicated times and then harvested in PD buffer. The cellular lysates (200 μl) were incubated overnight at 4 °C with a monoclonal antibody directed against IKKγ/NEMO (BD PharMingen); the immunoprecipitates were isolated following the addition of 20 μl of protein A-agarose and washed with PD and then RIPA buffer, and the labeled IKKγ/NEMO proteins were resolved on a 10% SDS-polyacrylamide gel and visualized by autoradiography. In vitro kinase assays were performed with immunopurified IKKβ and GST-IKKγ/NEMO in the presence of [γ-32P]ATP. The 32P-labeled GST-IKKγ/NEMO was subjected to SDS-PAGE, and the32P-labeled GST-IKKγ/NEMO species was excised from the gel and subjected to trypsin digestion overnight. The trypsin-digested IKKγ/NEMO protein was applied to a reverse-phase high pressure liquid chromatography (Applied Biosystems, 130 A Separation system), and the fractions were collected. The majority of the counts were found in fractions 20 and 21, and the phosphopeptides isolated from these high pressure liquid chromatography fractions were analyzed by a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Voyager-DETM, Biospectrometry Workstation, Perspective Biosystems), and the amino acid sequence was analyzed by protein microsequencing using an Applied Biosystems 494 protein sequencer. Cytoplasmic extract (200 μg) was prepared from 293T cells transfected with Myc-tagged CMV-IKKγ/NEMO and FLAG-tagged CMV-IKKβ and immunoprecipitated with Myc antibody to isolate the IKKγ/NEMO and IKKβ. In vitrokinase assays were then performed with [γ-32P]ATP, and the reactions were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane, and the 32P-labeled IKKγ/NEMO species was isolated and subjected to hydrolysis in 6.0 n HCl at 110 °C for 1 h (42Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (996) Google Scholar). The 32P-labeled IKKγ/NEMO residues and the unlabeled phosphoserine, phosphothreonine, and phosphotyrosine standards were analyzed by one-dimensional electrophoresis using thin layer cellulose chromatography. The cellulose TLC plate was then stained with 2% ninhydrin (Sigma) followed by autoradiography. Previous data demonstrated that IKKγ/NEMO is a phosphoprotein (17Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (755) Google Scholar, 40Carter R.S. Geyer B.C. Xie M. Acevedo-Suarez C.A. Ballard D.W. J. Biol. Chem. 2001; 276: 24445-24448Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 43Tarassishin L. Horwitz M.S. Biochem. Biophys. Res. Commun. 2001; 285: 555-560Crossref PubMed Scopus (16) Google Scholar) and t
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