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• W2016410864 abstract "The IκB-Kinase (IKK) complex is a multisubunit protein complex crucial for signal-induced phosphorylation of the IκB proteins and thus controls the activity of the transcription factor NF-κB. Besides the two kinases IKKα and IKKβ, the IKK complex contains NEMO/IKKγ, an additional subunit with regulatory and adaptor functions. NEMO not only confers structural stability to the IKK complex but also participates in the activation process of the IKK complex by linking the IKK subunits to upstream activators. In this study we analyze the IKKβ-mediated phosphorylation of the IKK-binding domain of NEMO. In vitro, IKKβ phosphorylates three serine residues in the domain of NEMO at positions 43, 68, and 85. However, mutational analysis revealed that only the phosphorylation of serine 68 in the center of the IKK-binding domain plays an essential role for the formation and the function of the IKK complex. Thus, Ser68 phosphorylation attenuates the amino-terminal dimerization of NEMO as well as the IKKβ-NEMO interaction. In contrast, the NEMO-IKKα interaction was only mildly affected by the phosphorylation of Ser68. However, functional analysis revealed that Ser68 phosphorylation primarily affects the activity of IKKα. Furthermore, in complementation experiments of NEMO-deficient murine embryonic fibroblasts, a S68A-NEMO mutant enhanced, whereas a S68E mutant decreased, TNF-α-induced NF-κB activity, thus emphasizing the inhibitory role of the Ser68 phosphorylation on the signal-induced NF-κB activity. Finally, we provide evidence that the protein phosphatase PP2A is involved in the regulation of the Ser68-based mechanism. In summary, we provide evidence for a signal-induced phosphorylation-dependent alteration of the IKK complex emphasizing the dynamic nature of this multisubunit kinase complex. The IκB-Kinase (IKK) complex is a multisubunit protein complex crucial for signal-induced phosphorylation of the IκB proteins and thus controls the activity of the transcription factor NF-κB. Besides the two kinases IKKα and IKKβ, the IKK complex contains NEMO/IKKγ, an additional subunit with regulatory and adaptor functions. NEMO not only confers structural stability to the IKK complex but also participates in the activation process of the IKK complex by linking the IKK subunits to upstream activators. In this study we analyze the IKKβ-mediated phosphorylation of the IKK-binding domain of NEMO. In vitro, IKKβ phosphorylates three serine residues in the domain of NEMO at positions 43, 68, and 85. However, mutational analysis revealed that only the phosphorylation of serine 68 in the center of the IKK-binding domain plays an essential role for the formation and the function of the IKK complex. Thus, Ser68 phosphorylation attenuates the amino-terminal dimerization of NEMO as well as the IKKβ-NEMO interaction. In contrast, the NEMO-IKKα interaction was only mildly affected by the phosphorylation of Ser68. However, functional analysis revealed that Ser68 phosphorylation primarily affects the activity of IKKα. Furthermore, in complementation experiments of NEMO-deficient murine embryonic fibroblasts, a S68A-NEMO mutant enhanced, whereas a S68E mutant decreased, TNF-α-induced NF-κB activity, thus emphasizing the inhibitory role of the Ser68 phosphorylation on the signal-induced NF-κB activity. Finally, we provide evidence that the protein phosphatase PP2A is involved in the regulation of the Ser68-based mechanism. In summary, we provide evidence for a signal-induced phosphorylation-dependent alteration of the IKK complex emphasizing the dynamic nature of this multisubunit kinase complex. The transcription factor NF-κB plays a crucial role in the initiation of innate and adaptive immune responses, in inflammation and tumorigenesis (1Schulze-Luehrmann J. Ghosh S. Immunity. 2006; 25: 701-715Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 2Hayden M.S. West A.P. Ghosh S. Oncogene. 2006; 25: 6758-6780Crossref PubMed Scopus (949) Google Scholar, 3Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3380) Google Scholar). In its inactive state NF-κBis bound to small cytoplasmic proteins, the IκB proteins. Stimulation with a wide variety of agonists, for example pro-inflammatory cytokines like TNF-α, 2The abbreviations used are:TNF-αtumor necrosis factor αIKKIκB kinaseEGSethylene glycol bis (succinimidylsuccinate)NEMONF-κB essential modulatorIPimmunoprecipitationMODminimal oligomerization domainMEFmurine embryonic fibroblastWCEwhole cell extractERK2extracellular signal-regulated kinase 2GSTglutathione S-transferaseIBDIKK-binding domain bacterial components like lipopolysaccharide or by antigen receptors, funnels in the activation of a multisubunit IκB-kinase complex, which phosphorylates the IκB proteins at two specific serine residues. This phosphorylation marks the IκB proteins for proteasomal degradation setting NF-κB free, which then translocates into the nucleus and supports the expression of various pro-inflammatory or anti-apoptotic gene products. The IκB kinase (IKK) complex is composed of two catalytically active subunits, termed IKKα (IKK1) and IKKβ (IKK2), as well as NEMO/IKKγ, a subunit with regulatory and adaptor functions (4Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1595) Google Scholar, 5DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1917) Google Scholar). Analysis of mice deficient for either IKKα or IKKβ suggested that the IKKβ subunit is the major IKK regulating the canonical NF-κB pathway, and the IKKα subunit is crucial for a second, alternative NF-κB pathway leading to the activation of RelB-p52 heterodimers (6Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1144) Google Scholar, 7Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Crossref PubMed Scopus (824) Google Scholar). In contrast to the canonical NF-κB pathway, which depends on the presence of NEMO, the alternative pathway is NEMO-independent but requires the protein kinase NIK. However, recent data suggest that, instead of a clear assignment of IKKα and IKKβ to the alternative and the canonical NF-κB pathway, respectively, both kinases contribute to the activation of NF-κB by the canonical pathway with only gradual differences (8Solt L.A. Madge L.A. Orange J.S. May M.J. J. Biol. Chem. 2007; 282: 8724-8733Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 9Schmidt-Supprian M. Courtois G. Tian J. Coyle A.J. Israel A. Rajewsky K. Pasparakis M. Immunity. 2003; 19: 377-389Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). tumor necrosis factor α IκB kinase ethylene glycol bis (succinimidylsuccinate) NF-κB essential modulator immunoprecipitation minimal oligomerization domain murine embryonic fibroblast whole cell extract extracellular signal-regulated kinase 2 glutathione S-transferase IKK-binding domain Although a precise model regarding the molecular mechanism underlying IKK activation is still missing, it became clear that various post-translational modifications at the different IKK subunits are involved in this process. Besides the phosphorylation of IKKα and IKKβ at two serine residues in their T-loop, the ubiquitination and occasionally the phosphorylation at a specific serine residue of NEMO seem to be required (10Zhou H. Wertz I. O'Rourke K. Ultsch M. Seshagiri S. Eby M. Xiao W. Dixit V.M. Nature. 2003; 427: 167-171Crossref PubMed Scopus (453) Google Scholar, 11Lamsoul I. Lodewick J. Lebrun S. Brasseur R. Burny A. Gaynor R.B. Bex F. Mol. Cell. Biol. 2005; 25: 10391-10406Crossref PubMed Scopus (120) Google Scholar, 12Wu Z.H. Shi Y. Tibbetts R.S. Miyamoto S. Science. 2006; 311: 1141-1146Crossref PubMed Scopus (423) Google Scholar). This serine residue at position 85 of NEMO has been identified as a protein kinase Cα, and more recently as an ATM target-site crucial for the IKK activation induced by genotoxic stress (12Wu Z.H. Shi Y. Tibbetts R.S. Miyamoto S. Science. 2006; 311: 1141-1146Crossref PubMed Scopus (423) Google Scholar, 13Tarassishin L. Horwitz M.S. Biochem. Biophys. Res. Commun. 2001; 285: 555-560Crossref PubMed Scopus (16) Google Scholar). In addition, overexpression of the TNF-α-receptor I or the human T-cell lymphotrophic virus I Tax protein induces the IKKβ-mediated NEMO phosphorylation at several additional serine residues, primarily at the serine residues 31 and 43 in the amino terminus and 376 and 377 in the carboxyl terminus of NEMO (14Carter R.S. Pennington K.N. Ungurait B.J. Ballard D.W. J. Biol. Chem. 2003; 278: 19642-19648Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). However, no physiological functions for these IKKβ-mediated NEMO phosphorylation steps have been described. Ubiquitination of the carboxyl terminus of NEMO is another crucial step in the TNF-α or TCR-induced NF-κB activation process. One hypothesis is that the ubiquitination either leads to a structural alteration of the IKK complex, probably inducing a proximity-induced IKK activation (10Zhou H. Wertz I. O'Rourke K. Ultsch M. Seshagiri S. Eby M. Xiao W. Dixit V.M. Nature. 2003; 427: 167-171Crossref PubMed Scopus (453) Google Scholar, 16Huang T.T. Feinberg S.L. Suryanarayanan S. Miyamoto S. Mol. Cell. Biol. 2002; 22: 5813-5825Crossref PubMed Scopus (99) Google Scholar, 17Huang T.T. Wuerzberger-Davis S.M. Wu Z.H. Miyamoto S. Cell. 2003; 115: 565-576Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar), or might be important for the interaction of the IKK complex with upstream regulators for example the TNF-α-induced NEMO-RIP interaction (18Wu C.J. Conze D.B. Li T. Srinivasula S.M. Ashwell J.D. Nat Cell Biol. 2006; 8: 398-406Crossref PubMed Scopus (508) Google Scholar, 19Ea C.K. Deng L. Xia Z.P. Pineda G. Chen Z.J. Mol Cell. 2006; 22: 245-257Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar). However, post-translational modification of NEMO, like its phosphorylation or ubiquitination, might also positively influence its oligomerization status, which was for instance observed upon TNF-α stimulation or Tax overexpression (20Poyet J.L. Srinivasula S.M. Lin J.H. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 21Huang G.J. Zhang Z.Q. Jin D.Y. FEBS Lett. 2002; 531: 494-498Crossref PubMed Scopus (30) Google Scholar, 22Fontan E. Traincard F. Levy S.G. Yamaoka S. Veron M. Agou F. FEBS J. 2007; 274: 2540-2551Crossref PubMed Scopus (32) Google Scholar). Two different domains mediate the oligomerization of NEMO, one domain, the minimal oligomerization domain (MOD), is located in the carboxyl terminus, spanning amino acids 246–365, and mediates a NEMO trimerization; the second domain is located in the amino terminus overlapping the IKK-binding domain of NEMO (23Tegethoff S. Behlke J. Scheidereit C. Mol. Cell. Biol. 2003; 23: 2029-2041Crossref PubMed Scopus (111) Google Scholar, 24Marienfeld R.B. Palkowitsch L. Ghosh S. Mol. Cell. Biol. 2006; 26: 9209-9219Crossref PubMed Scopus (42) Google Scholar). Importantly, the formation of both homotypic NEMO interactions is necessary for a functional IKK complex. Thus, peptides interfering with the carboxyl-terminal oligomerization like the BA-CC2 and BA-LZ peptides have been used to inhibit the signal-induced NF-κB activation (25Agou F. Courtois G. Chiaravalli J. Baleux F. Coic Y.M. Traincard F. Israel A. Veron M. J. Biol. Chem. 2004; 279: 54248-54257Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In contrast, peptides corresponding to the IKK-binding domain of NEMO have either no or at best a mild effect, probably due to the additional binding of these peptides to IKKα or IKKβ (24Marienfeld R.B. Palkowitsch L. Ghosh S. Mol. Cell. Biol. 2006; 26: 9209-9219Crossref PubMed Scopus (42) Google Scholar). Here, we analyzed the impact of the phosphorylation of the amino-terminal IKK-binding domain of NEMO. Three serine residues are located in the α-helical part of the IKK-binding domain of NEMO at positions 43, 68, and 85. We show here that all three serine residues are IKKβ target sites in vitro. However, by using NEMO mutants with either phospho-inhibiting serine to alanine or phospho-mimetic serine to glutamic acid substitutions at the positions Ser43, Ser68, or Ser85 of NEMO, we observed only in the case of the Ser68 phosphorylation a negative effect on the amino-terminal NEMO dimerization as well as the IKKβ-NEMO interaction. As a consequence we observed a significant reduction in the TNF-α-induced NF-κB activity in NEMO-deficient cells reconstituted with the phospho-mimetic S68E-NEMO mutant. Collectively, our data suggest that the activity of the IKK complex is negatively regulated by the NEMO phosphorylation at position Ser68. Cell Culture, Stable and Transient Transfection, and Reagents—Human 293 cells (obtained from ATCC), NEMO-deficient murine embryonic fibroblasts (MEFs), and normal wild-type MEFs were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 μg/ml). For transient transfections of 2 × 105 293 cells, the CaPO4-transfection method was used according to standard protocols. Stable transfection of MEFs was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Human recombinant TNFα was purchased by R&D Systems (Minneapolis, MN), and okadaic acid was from Calbiochem. Plasmids and Antibodies—Expression vectors encoding FLAG-tagged human full-length NEMO, NEMO1–197, NEMO1–100, wild type and dominant negative IKKβ, Xpress-IKKα, and Xpress-IKKβ have been described elsewhere (26May M.J. Marienfeld R.B. Ghosh S. J. Biol. Chem. 2002; 277: 45992-46000Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The 3xκB firefly luciferase reporter plasmid and the Renilla luciferase reporter plasmid were described previously (27Marienfeld R. May M.J. Berberich I. Serfling E. Ghosh S. Neumann M. J. Biol. Chem. 2003; 278: 19852-19860Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The GST-NEMO expression vectors were generated by inserting the wild type or the mutated NEMO cDNA in-frame into the EcoRI and XhoI sites of the pGex 4T1 vector. Specific antibodies recognizing the FLAG-epitope or the Xpress-epitope were purchased from Sigma and Invitrogen, respectively. Antibodies specific for IKKα, IKKβ, IκBα, ERK2, or NEMO were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). The site-directed mutagenesis was performed using the Quick-Change site directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. Immunoprecipitation, GST Pulldown, and Immunoblotting—The immunoprecipitation and immunoblotting procedures were performed as described previously (27Marienfeld R. May M.J. Berberich I. Serfling E. Ghosh S. Neumann M. J. Biol. Chem. 2003; 278: 19852-19860Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In brief, 0.5–1 mg of protein extracts were mixed with 25 μl of bovine serum albumin-blocked FLAG-antibody (M2, Sigma) linked to agarose beads. The samples were incubated for 1–12 h at 4 °C with agitation. After incubation, the precipitates were washed extensively in TnT buffer (20 mm Tris, pH 8.0, 200 mm NaCl, 1% Triton X-100, 1 mm PMSF, 2 μm leupeptin, 1 mm dithiothreitol). The resulting immunopurified proteins were used for immunoblotting experiments. In case of the GST pulldown analysis 1μg of the GST fusion protein was mixed with 1μl of the 35S-labeled in vitro translated protein sample and incubated under agitation for 1 h prior to a pulldown assay with 25 μl of glutathione-conjugated beads (Sigma). The samples were washed extensively with TnT buffer and separated on a SDS-gel. The gel was stained with Coomassie, fixed, dried, and subjected to autoradiography. For the immunoblotting analysis, either the immunopurified protein complexes, or, as indicated, 50–100 μg of a protein extract were loaded on a standard SDS-polyacrylamide gel (PAA; polyacrylamide). SDS-PAGE and the transfer to nitrocellulose (Schleicher & Schuell) or nylon membranes (Immobilon polyvinylidene difluoride-membrane, Millipore) were performed using standard protocols. The membrane was blocked with 5% milk powder in TBS+Tween 20 prior to the incubation with the primary antibody (1:1000 in TBS+Tween 20), subsequently washed three times for 5 min each, and incubated in a TBS-Tween 20 solution containing either horseradish peroxidase-conjugated or IRDye700/800-conjugated secondary antibody (1:5000). The detection was performed using either ECL substrates from Amersham Biosciences or the Odyssey infrared scanning system (LICOR). In Vitro Kinase Assay and in Vivo Phosphorylation Studies—For the in vitro kinase assay the indicated proteins were individually expressed in 293 HEK cells prior to an anti-FLAG immunoprecipitation. The resulting immunocomplexes were washed extensively with TnT and finally with kinase-assay buffer to equilibrate the samples. Beads carrying either the immunopurified IKK or the immunopurified NEMO proteins were mixed, and the kinase reaction was perform at 30 °C for 30 min after adding 10 μCi of [γ-32P]ATP in kinase reaction buffer. The samples were subsequently washed extensively with TnT buffer and phosphate-buffered saline prior to a separation by SDS-PAGE. The separated proteins were transferred to nitrocellulose membrane, and the phosphorylation was monitored by autoradiography. For the in vivo phosphorylation studies 293 HEK cells were transiently transfected with the indicated expression vectors. After 24 h the cells were incubated for further 18 h in phosphate-free Dulbecco's modified Eagle's medium with 5% dialyzed calf-serum prior to incubation with 100 μCi/ml [32P]orthophosphate. The cells were treated as indicated, harvested, and lysed in TnT, and the resulting extracts were subjected to an anti-FLAG IP. The precipitated proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane, and the resulting membrane was used for autoradiography to monitor the phosphorylation and subsequently subjected to immunoblot analysis. Luciferase Reporter Assay—For the reporter gene assays, 293 cells were transiently transfected as described above. In general, we used 200 ng of the NF-κB-dependent reporter construct along with 15 ng of a Renilla luciferase reporter construct under the control of the human β-actin promoter, which leads to constitutive expression of the Renilla luciferase. 1 × 105 293 cells were transfected in one well of a 24-well plate. Equal DNA concentrations in each experiment (1.2 μg/well) were maintained by adding the appropriate empty vector to the DNA mixture. The cells were lysed after 24 h with TnT, and the firefly and Renilla luciferase activities were measured according to the protocol for the dual luciferase system (Promega). The resulting firefly luciferase values were normalized by the values of the Renilla luciferase. The experiments were done in parallel and were repeated at least three times. Gel Shift Analysis—For the gel shift analysis (electrophoretic mobility shift assay) 10 μg of nuclear proteins from untreated or stimulated cells was incubated on ice for 20 min in a reaction containing 0.3 ng of 32P-labeled κB-specific oligonucleotide, 1 μg of pdI:dC, and 3 μl of a binding buffer. The samples were separated on a native 5% polyacrylamide (PAA) gel, and the gel was dried and subjected to autoradiography. For supershift or competition analysis, the indicated sample was preincubated with either a 100-fold molar excess of the cold κB-oligonucleotide or with 0.2 μg of the anti-RelA antibody. IKKβ Phosphorylates the IKK-binding Domain of NEMO at Multiple Sites in Vitro—Oligomerization of the adaptor protein NEMO is crucial for the function of the IKK complex (23Tegethoff S. Behlke J. Scheidereit C. Mol. Cell. Biol. 2003; 23: 2029-2041Crossref PubMed Scopus (111) Google Scholar, 25Agou F. Courtois G. Chiaravalli J. Baleux F. Coic Y.M. Traincard F. Israel A. Veron M. J. Biol. Chem. 2004; 279: 54248-54257Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 28Agou F. Ye F. Goffinont S. Courtois G. Yamaoka S. Israel A. Veron M. J. Biol. Chem. 2002; 277: 17464-17475Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Yet, whether the activity of the IKK complex is regulated by an alteration of the oligomerization of NEMO described by Poyet et al. remains unclear (20Poyet J.L. Srinivasula S.M. Lin J.H. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Interestingly, NEMO is a phospho protein targeted by different serine/threonine kinases in vivo and in vitro, and the identified serine target sites are located in both oligomerization domains (14Carter R.S. Pennington K.N. Ungurait B.J. Ballard D.W. J. Biol. Chem. 2003; 278: 19642-19648Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 29Prajapati S. Gaynor R.B. J. Biol. Chem. 2002; 277: 24331-24339Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In this study we analyzed the role of the IKKβ-dependent phosphorylation at different serine residues at positions 43, 68, and 85 within in the amino-terminal IKK-binding domain (IBD) of NEMO for the activity of the IKK complex. To determine which of the three serine residues are phosphorylated by IKKβ in vitro, we performed in vitro kinase assays using ectopically expressed wild-type FLAG-NEMO or NEMO mutants with serine to alanine or cysteine substitutions at the positions 43 (S43A), 68 (S68A), or 85 (S85C) either separately or combined as a triple mutant (SIIIA/C-NEMO) alone or in conjunction with separately expressed FLAG-tagged IKKβ (for a schematic representation of the NEMO mutants used and the location of the serine residues see Fig. 1A). As depicted in Fig. 1B, we observed a high IKKβ-mediated phosphorylation of all FLAG-NEMO variants, which remained unchanged by the substitution of the single serine residues or by the combined substitution of all three serine residues (Fig. 1B, lanes 6–11). In contrast, none of the NEMO proteins were significantly phosphorylated in the absence of FLAG-tagged IKKβ (Fig. 1B, lanes 1–5) or in the presence of a dominant negative mutant of IKKβ (Fig. 1C, lanes 7–12). However, previous studies suggested that the phosphorylation of a specific IKK target site in NEMO might be masked by other phosphorylation events in NEMO due to the variety of IKK target sites in this protein (14Carter R.S. Pennington K.N. Ungurait B.J. Ballard D.W. J. Biol. Chem. 2003; 278: 19642-19648Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 29Prajapati S. Gaynor R.B. J. Biol. Chem. 2002; 277: 24331-24339Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Consistently, we observed a significant reduction of the IKKβ-mediated NEMO phosphorylation with all three single mutants (Fig. 1D) when we compared the different serine mutants with a wild-type version in the background of an amino-terminal FLAG-NEMO fragment, spanning the first hundred amino acids of NEMO (NEMO1–100), in a similar in vitro kinase assay. However, the reduction in the in vitro phosphorylation of S85C-NEMO1–100 is less pronounced (for a quantification see Fig. 1D, middle panel). Moreover, the effect on the IKKβ-mediated NEMO phosphorylation was even more dramatic in case of the SIIIA/C-NEMO1–100 triple mutant (Fig. 1D, lane 5). Based on these data, we concluded that all three serine residues located in the IBD, Ser43, Ser68, and Ser85, are IKKβ target sites in vitro. Phosphorylation of Serine 68 in the IKK-binding Domain of NEMO Attenuates the Amino-terminal NEMO Dimerization—Because the IKK-binding domain of NEMO is also crucial for the amino-terminal NEMO dimerization, we next compared the dimerization potential of the three single NEMO1–100 point mutants and the wild-type NEMO1–100. As shown in Fig. 2A, all mutants tested displayed an increased dimer formation, however, the dimerization of S68A-NEMO1–100 was significantly pronounced (Fig. 2A, lane 3), arguing for a critical role of Ser68 for the NEMO dimerization. The critical role of Ser68 was further supported by experiments with ectopic expressed full-length NEMO-proteins. Again, we observed an enhanced NEMO dimerization in case of S68A-NEMO and the SIIIA/C-triple mutant (Fig. 2B, lanes 4 and 6; right side: quantification of the dimer/monomer ratio in %). More strikingly, imitating the Ser68 phosphorylation by the substitution of Ser68 with a phospho-mimetic glutamic acid led to a significant decrease in NEMO dimerization (Fig. 2C, lane 4), further emphasizing the negative effect of the Ser68 phosphorylation on the amino-terminal NEMO dimerization. In order to analyze whether the Ser68 phosphorylation alters the global oligomerization status of NEMO, we treated whole cell extracts (WCEs) from 293 cells transfected with wild-type, S68A-, or S68E-NEMO with the cross-linker EGS. Without cross-linking S68A-NEMO slightly enhanced, and S68E-NEMO reduced, the formation of NEMO-dimers and higher molecular weight complexes (NEMOX) compared with wild-type NEMO (lanes 2–4). In addition, the NEMO-dimers mostly disappeared upon heating the samples for 10 min at 95 °C (lanes 6–8) with the exception of a remaining S68A-NEMO dimerization, thus once more demonstrating the positive effect of this mutation on the amino-terminal NEMO dimerization. However, cross-linking the proteins with EGS converted the different NEMO variants equally in NEMO-dimers and NEMO-based high molecular weight protein complexes (Fig. 2D, lanes 10–12), suggesting that only the amino-terminal dimerization, but not the global oligomerization status of NEMO is affected by the phosphorylation of Ser68, most likely due to an unaffected carboxyl-terminal oligomerization of NEMO. Phosphorylation of Ser68 Has a Negative Effect on the IKKβ-NEMO Interaction—Having established the negative effect of the Ser68 phosphorylation on the NEMO dimerization, we next analyzed whether the interaction of NEMO with IKKα or IKKβ is also altered by the substitution of Ser43, Ser68, or Ser85. For this, we performed an in vitro interaction study with ectopically expressed FLAG-tagged NEMO proteins in combination with in vitro transcribed/translated IKKα or IKKβ. Similar to the NEMO dimerization, only the IKKβ interaction with S68E-NEMO was significantly reduced (Fig. 3A, lane 5, middle panel), whereas the IKKα interaction of S68E-NEMO was, if all, only slightly affected (Fig. 3A, lane 5, upper panel). The specific negative effect of the S68E substitution on the IKKβ-NEMO interaction was also evident in in vivo interaction studies of IKKβ with the different NEMO variants (Fig. 3C, lane 4) in co-immunoprecipitation assays, whereas the S68E-NEMO-IKKα interaction was only slightly reduced (Fig. 3B, lane 4). Interestingly, a similar negative effect on the IKKα interaction was also observed with the S85C-NEMO mutant (Fig. 3B, lane 8). Moreover, the S68A-NEMO mutant displayed an enhanced IKKβ interaction as shown in a head-to-head comparison of wild-type NEMO, S68E-, and S68A-NEMO (Fig. 3D, a quantification of three independent experiments is given on the right part). However, because under these experimental conditions additional post-translational modifications of the ectopic expressed NEMO proteins could influence the interaction with the IKKs, we performed another in vitro interaction study where we compared the interaction of in vitro translated IKKα or IKKβ with bacterial expressed GST-NEMO wild-type, S68A, or S68E (Fig. 3E). Again, we observed a negative effect on the IKKβ interaction only in case of S68E-NEMO (Fig. 3E, lane 8). In summary, we concluded from these interaction studies that both protein-protein interactions mediated by the IBD, the NEMO dimerization, and the interaction with IKKβ, are negatively affected by the phosphorylation of Ser68 or by substitution of Ser68 with a glutamate residue, whereas binding of NEMO to IKKα is only modestly affected. Ser68 Is a Phospho-acceptor Site in Vivo—In a previous study serine 43, but not serine 68, has been identified as major in vivo IKKβ target site in NEMO (14Carter R.S. Pennington K.N. Ungurait B.J. Ballard D.W. J. Biol. Chem. 2003; 278: 19642-19648Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Given the specific importance of the Ser68 phosphorylation for the NEMO dimerization and the NEMO-IKKβ interaction, we next asked whether Ser68 is also a phospho-acceptor site in vivo. Although the increased NEMO dimerization (Fig. 2) and IKKβ-NEMO interaction (Fig. 3E) observed with S68A-NEMO already suggested a basal in vivo phosphorylation of Ser68, we also wanted to prove the in vivo phosphorylation of NEMO at Ser68 more formally. For this, 293 HEK cells, transiently transfected with expression vectors for either wild-type FLAG-NEMO, FLAG-S68A-NEMO, or, as a control, FLAG-S43A-NEMO, were metabolically labeled with [32P]orthophosphate and stimulated with TNF-α for the indicated time points prior to an anti-FLAG IP. As shown in Fig. 4A, all NEMO variants used display a high basal phosphorylation status. Interestingly, the basal phosphorylation of S68A-NEMO was even slightly enhanced. Because we observed in a similar in vivo phosphorylation experiment a strong reduction of the basal NEMO phosphorylation by co-transfection of a dominant negative mutant of IKKβ (Fig. 4B, compare lanes 2 and 3), we concluded that the basal" @default.
• W2016410864 created "2016-06-24" @default.
• W2016410864 creator A5004620989 @default.
• W2016410864 creator A5044844412 @default.
• W2016410864 creator A5071658023 @default.
• W2016410864 creator A5073950283 @default.
• W2016410864 date "2008-01-01" @default.
• W2016410864 modified "2023-09-27" @default.
• W2016410864 title "Phosphorylation of Serine 68 in the IκB Kinase (IKK)-binding Domain of NEMO Interferes with the Structure of the IKK Complex and Tumor Necrosis Factor-α-induced NF-κB Activity" @default.
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