Regulation of I B Kinase (IKK) /NEMO Function by IKK -mediated Phosphorylation*

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1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 27, Issue of July 5, pp , by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Regulation of I B Kinase (IKK) /NEMO Function by IKK -mediated Phosphorylation* Received for publication, February 11, 2002, and in revised form, April 16, 2002 Published, JBC Papers in Press, April 23, 2002, DOI /jbc.M Shashi Prajapati and Richard B. Gaynor From the Division of Hematology-Oncology, Department of Medicine, Harold Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas The I B kinase (IKK) complex includes the catalytic components IKK and IKK in addition to the scaffold protein IKK /NEMO. Increases in the activity of the IKK complex result in the phosphorylation and subsequent degradation of I B and the activation of the NF- B pathway. Recent data indicate that the constitutive activation of the NF- B pathway by the human T-cell lymphotrophic virus, type I, Tax protein leads to enhanced phosphorylation of IKK /NEMO by IKK. To address further the significance of IKK -mediated phosphorylation of IKK /NEMO, we determined the sites in IKK / NEMO that were phosphorylated by IKK, and we assayed whether IKK /NEMO phosphorylation was involved in modulating IKK activity. IKK /NEMO is rapidly phosphorylated following treatment of cells with stimuli such as tumor necrosis factor- and interleukin-1 that activate the NF- B pathway. By using both in vitro and in vivo assays, IKK was found to phosphorylate IKK /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 IKK /NEMO to stimulate IKK kinase activity. These results indicate that the differential phosphorylation of IKK /NEMO by IKK and perhaps other kinases may be important in regulating IKK activity. The NF- B pathway is a critical regulator of the cellular response to a variety of stimuli including the cytokines, TNF 1 and IL-1, bacterial and viral infection, double-stranded RNA, and the human T-cell leukemia virus transactivator protein Tax (1 4). The ability to activate rapidly and subsequently silence the NF- B 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- B is involved in the control of the host immune and inflammatory responses. The members of the NF- B family of transcription factors, which include p105/50, p100/52, p65, c-rel, and RelB, contain a Rel homology domain that mediates their heterodimerization * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed: Division of Hematology-Oncology, Dept. of Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX Tel.: ; Fax: ; gaynor@utsw.swmed.edu. 1 The abbreviations used are: TNF, tumor necrosis factor- ; IL-1, interleukin 1; IKK, I B kinase; GST, glutathione S-transferase; RSV, Rous sarcoma virus; CMV, cytomegalovirus. This paper is available on line at and homodimerization properties and DNA-binding properties (2). These proteins are sequestered in the cytoplasm of most cells where they are bound to a family of inhibitory proteins known as I B (1, 3). Treatment of cells with a variety of stimuli including the cytokines TNF and IL-1 stimulate the activity of kinases that phosphorylate I B on amino-terminal serine residues resulting in its ubiquitination and degradation by the proteasome (3 6). This process leads to the nuclear translocation of the NF- B 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 (1 3, 5 7). Activation of the I B kinases, which phosphorylate the I B proteins on the amino-terminal serine residues to result in their degradation, is a critical process that regulates the NF- B pathway (8 12). The I B kinases, IKK and IKK, are components of a kDa complex that is composed of these two catalytic subunits (8 12) in addition to a scaffold protein IKK / NEMO (13 16). Stimulation of IKK activity by cytokine treatment has been demonstrated to involve both activation of mitogen-activated protein 3-kinases and/or IKK autophosphorylation (17, 18). IKK and IKK have 52% amino acid identity, and their domain structure is similar with an amino-terminal kinase, leucine zipper, and helix-loop-helix motifs (8 12). Although these kinases have a similar structure and are able to both homodimerize and heterodimerize, IKK is at least 20- fold more active in phosphorylation of the I B proteins as compared with IKK (9, 14, 18, 19). Studies using fibroblasts isolated from IKK (20 22) and IKK (23 25) knock-out mice also demonstrate that IKK is the dominant kinase in regulating NF- B activity. IKK /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 IKK, 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 (13 16, 26 30). The interaction of IKK /NEMO with IKK and IKK is critical for the assembly of this high molecular weight IKK complex that leads to the recruitment of I B proteins and the stimulation of IKK activity (8, 12, 14 16, 25, 27, 31 33). Cells lacking IKK /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- B pathway (14 16). IKK / NEMO also binds to a variety of proteins other than IKKs including the adaptor protein RIP, which is involved in TNF mediated activation of IKK (13, 30), A20 which decreases TNF -mediated activation of IKK (30), the human T-cell lymphotrophic virus, type I, Tax protein which stimulates IKK activity (26, 34, 35), and the CIKS protein which also increases

2 24332 IKK Phosphorylation of IKK /NEMO IKK activity (36). Disruption of a single copy of the IKK / NEMO gene, which is located on the X chromosome, in mice or in humans results in male lethality due to hepatic apoptosis, whereas females heterozygous for this defect develop a severe skin disease known as incontentia pigmentia (37 39). Thus, IKK /NEMO plays a central role in the activation of the NF- B pathway in response to a variety of different stimuli. Recently it was demonstrated (40) that the human T-cell lymphotrophic virus, type I, Tax protein, which results in the constitutive activation of the NF- B pathway, leads to constitutive phosphorylation of both IKK and IKK /NEMO. Furthermore, IKK was shown to phosphorylate IKK /NEMO using in vitro kinase assays. These results suggested that IKK and IKK /NEMO could potentially regulate the function of each other. In this study, both in vivo and in vitro assays were utilized to characterize IKK /NEMO phosphorylation by IKK. Our results indicate that IKK phosphorylation of IKK / NEMO appears to be important for regulating its functional properties. MATERIALS AND METHODS DNA Constructs The murine IKK /NEMO cdna was cloned into the CMV expression vector pcmv5 in which the Myc epitope tag was fused to the amino terminus of IKK /NEMO (27). An amino-terminal IKK /NEMO deletion containing amino acid residues was constructed using PCR, whereas carboxyl-terminal truncations of IKK /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 IKK /NEMO cdna using oligonucleotidedirected 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 IKK /NEMO cdnas were then cloned downstream of the Myc epitope in pcmv5 and GST in pgex-kg (27). Wild-type human IKK and IKK as well as the cdnas for the IKK kinase-defective K44M and either the constitutively active or inactive IKK mutants for IKK (S177E/S181E and S177A/S181A) and for IKK (S176E/S180E and S176A/S180A), respectively, were cloned into a pcmv5 construct containing a FLAG epitope (9, 18). The GST fusion proteins containing the wild-type I B extending from amino acids 1 54 and mutant I B (S32A/S36A) were described previously (41). Transfections and Cellular Fractionation 293T cells were maintained in Dulbecco s modified Eagle s medium supplemented with 10% fetal bovine serum (37). 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 (27). IKK /NEMO / mouse embryo fibroblasts (37) 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 IKK /NEMO (0.3 g), an NF- B luciferace reporter (0.1 g), and an RSV- -galactosidase expression vector (0.1 g). After 18 h of transfection, the cells were treated with TNF (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 -galactosidase activity. All transfections were performed in triplicate and repeated in three independent experiments. Immunoprecipitation and Immunoblotting To determine the interactions between wild-type and mutant IKK /NEMO and IKK, wildtype or mutant CMV-IKK (0.1 g) and either wild-type or mutant CMV-IKK /NEMO vectors (1.0 g) were transfected into 293T cells. Extracts (400 g) 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 mm -glycerophosphate, 0.5 mm dithiothreitol, 10 mm NaF, 300 M sodium vanadate, and protease inhibitors (Roche Molecular Biochemicals)), incubated for 2 4 hat4 C with the Myc monoclonal antibody (2.0 g), directed against the Myc epitope, followed by the addition of 20 l of protein A-agarose beads for 1hat4 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 I B (Santa Cruz Biotechnology, sc-371), IKK (Santa Cruz Biotechnology, sc-7607), and IKK (Santa Cruz Biotechnology, sc-8330) as indicated. Kinase Assays To assay IKK phosphorylation of IKK /NEMO, 293T cells were transfected with wild-type or mutant Myc-tagged CMV- IKK /NEMO (4.0 g) or FLAG-tagged CMV-IKK (2.0 g) and harvested 30 h post-transfection. Cytoplasmic extracts (400 g) were incubated overnight at 4 C with 1 2 g of anti-myc or anti-flag monoclonal antibodies, followed by the addition of protein A-agarose (Bio-Rad) for 1 3 hat4 C and washed three times with ELB buffer (50 mm Tris-HCl, ph 8.0, 100 mm NaCl, 5.0 mm NaF, 5.0 mm -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 M ATP, and 10 Ci of [ - 32 P]ATP, and the reactions were stopped with protein sample buffer and heated at 95 C for 3 min as described previously (27). The samples were then subjected to SDS-PAGE on a 12% polyacrylamide gel and visualized by autoradiography. To assay IKK phosphorylation of wild-type and mutant GST-IKK / NEMO proteins, wild-type or mutant CMV-IKK (2.0 g) 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-IKK / NEMO substrate (10.0 g) were performed as described above. Finally, to assay increases in IKK activity by wild-type and mutant IKK / NEMO, 293T cells were cotransfected with CMV expression vectors encoding IKK /NEMO (0.4 g) and IKK (0.01 g), respectively. At 30 h post-transfection, cellular lysates (200 g) were prepared in PD buffer, and in vitro kinase assays with a GST-I B substrate (10.0 g) were performed as described above. In Vivo Phosphorylation 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 Ci of [ 32 P]orthophosphate (50 Ci/ml) or [ 35 S]methionine (50 Ci/ml) (PerkinElmer Life Sciences) was added for 4 h. The cells were then treated with either TNF (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 l) were incubated overnight at 4 C with a monoclonal antibody directed against IKK /NEMO (BD PharMingen); the immunoprecipitates were isolated following the addition of 20 l of protein A-agarose and washed with PD and then RIPA buffer, and the labeled IKK /NEMO proteins were resolved on a 10% SDS-polyacrylamide gel and visualized by autoradiography. Mass Spectrometry In vitro kinase assays were performed with immunopurified IKK and GST-IKK /NEMO in the presence of [ - 32 P]ATP. The 32 P-labeled GST-IKK /NEMO was subjected to SDS- PAGE, and the 32 P-labeled GST-IKK /NEMO species was excised from the gel and subjected to trypsin digestion overnight. The trypsin-digested IKK /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-DE TM, Biospectrometry Workstation, Perspective Biosystems), and the amino acid sequence was analyzed by protein microsequencing using an Applied Biosystems 494 protein sequencer. Phosphoamino Acid Analysis Cytoplasmic extract (200 g) was prepared from 293T cells transfected with Myc-tagged CMV-IKK /NEMO and FLAG-tagged CMV-IKK and immunoprecipitated with Myc antibody to isolate the IKK /NEMO and IKK. In vitro kinase assays were then performed with [ - 32 P]ATP, and the reactions were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane, and the 32 P-labeled IKK /NEMO species was isolated and subjected to hydrolysis in 6.0 N HCl at 110 C for 1 h (42). The 32 P-labeled IKK /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.

3 IKK Phosphorylation of IKK /NEMO FIG. 1.IKK /NEMO is a phosphoprotein. A, HeLa cells were incubated with serum-free Dulbecco s modified Eagle s media either lacking methionine or phosphate followed by the addition of [ 35 S]methionine or [ 32 P]orthophosphate for 4 h. Cells were either untreated (lane 1) or treated with either TNF (20 ng/ml) (lanes 2 and 3) or IL-1 (20 ng/ml) (lanes 4 and 5) for 10 and 30 min. IKK /NEMO was immunoprecipitated with a rabbit polyclonal antibody (sc-8330) and subjected to SDS-PAGE and autoradiography (1st and 2nd panels). In vitro kinase assays were performed with a GST-I B substrate and IKK (sc-7607) immunoprecipitated from TNF - and IL-1-treated cells (3rd panel). Western blot analysis with a rabbit polyclonal directed against IKK /NEMO (4th panel), IKK (5th panel), or antibody directed against I B (sc-371) (6th panel) was also performed with a portion of these unlabeled extracts. B, 293T cells were either transfected with 0.2 g of Myc-tagged CMV expression vectors either alone (lane 1), encoding IKK /NEMO (lane 2), or transfected with IKK /NEMO and 0.5 g of FLAG-tagged wild-type IKK (lane 3), IKK (S177E/S181E) (lane 5), IKK (S177A/S181A) (lane 7), IKK (lane 9), IKK (S176E/S180E) (lane 11), IKK (S177A/S180A) (lane 13), or each of these kinases alone (lanes 4, 6, 8, 10 12, and 14). Following in vivo orthophosphate labeling of the transfected 293T cells, extracts were prepared, and the epitope-tagged IKK /NEMO was immunoprecipitated (IP) with Myc monoclonal antibody followed by SDS-PAGE and autoradiography (top panel). A portion of each of these samples was also analyzed by Western blot analysis with the Myc and FLAG monoclonal antibodies (bottom panels). C, 293T cells were transfected with CMV expression vectors encoding Myc-tagged IKK /NEMO (4.0 g) and IKK (2.0 g), and extracts were immunoprecipitated with Myc monoclonal antibody, and in vitro kinase assays were performed in the presence of [ - 32 P]ATP. The radiolabeled IKK /NEMO was subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and subjected to autoradiography. Following acid hydrolysis, the sample (lane 1) and unlabeled phosphotyrosine (P-Y, lane 2), phosphothreonine (P-T, lane 3), and phosphoserine (P-S, lane 4) standards were subjected to one-dimensional electrophoresis on thin larger chromatography, stained with 2% ninhydrin, and autoradiography was performed. RESULTS IKK /NEMO Is Phosphorylated following Treatment of Cells with Cytokines That Activate the NF- B Pathway Previous data demonstrated that IKK /NEMO is a phosphoprotein (17, 40, 43) and that IKK was able to phosphorylate IKK /NEMO directly (40). To characterize further IKK /NEMO phosphorylation, we first performed in vivo [ 32 P]orthophosphate and [ 35 S]methionine labeling of HeLa cells in either the absence or presence of TNF and IL-1 which activate the NF- B pathway (Fig. 1A). Following immunoprecipitation of endogenous IKK / NEMO, SDS-PAGE and autoradiography were performed. There was little phosphorylation of endogenous IKK /NEMO in untreated cells (Fig. 1A, lane 1, top panel). However, treatment of HeLa cells with TNF resulted in a rapid, but transient, increase in IKK /NEMO phosphorylation (Fig. 1A, lanes 2 and 3, top panel). Similar patterns of IKK /NEMO phospho-

4 24334 IKK Phosphorylation of IKK /NEMO rylation were seen following treatment of cells with IL-1 (Fig. 1A, lanes 4 and 5). There was little difference in [ 35 S]methionine labeling of IKK /NEMO in response to these different stimuli (Fig. 1A, 2nd panel, lanes 1 5). TNF and IL-1 induction of IKK activity (Fig. 1A, 3rd panel, lanes 1 5) mirrored the increase in IKK /NEMO phosphorylation. Western blot analysis of a portion of the unlabeled cellular extracts demonstrated similar levels of the IKK /NEMO and IKK proteins, and both TNF and IL-1 induced the degradation of I B (Fig. 1A, 4th to 6th panels). These results suggest that IKK /NEMO undergoes rapid phosphorylation in response to TNF and IL-1 with similar kinetics to activation of IKK. Because the kinetics of IKK /NEMO phosphorylation appeared to correlate with increases in IKK activity, we asked whether either wild-type, constitutively active, or kinase-defective constructs of IKK or IKK could result in changes in IKK /NEMO phosphorylation. In vivo [ 32 P]orthophosphate labeling was performed on 293T cells that were transfected with either Myc-tagged IKK /NEMO, wild-type, or mutant IKK constructs or both IKK /NEMO and IKK. The samples were then analyzed following immunoprecipitation with a Myc monoclonal antibody and SDS-PAGE and autoradiography. There was little phosphorylation of IKK /NEMO alone (Fig. 1B, lane 2, top panel) but increased phosphorylation in the presence of the wild-type and constitutively active (S177E/S181E) IKK (Fig. 1B, lanes 3 and 5, top panel). In contrast, there was no detectable phosphorylation of IKK /NEMO with IKK (S177A/ S181A) (Fig. 1B, lane 7, top panel). The epitope-tagged wildtype and constitutively active IKK proteins that coimmunoprecipitated with IKK /NEMO were also highly phosphorylated (Fig. 1B, lanes 3 and 5, top panel). Neither wild-type, constitutively active (S176E/S180E), or kinase-defective (S176A/S180A) IKK mutants were able to stimulate IKK / NEMO phosphorylation (Fig. 1B, lanes 9 14, top panel). Western blot analysis indicated that IKK, IKK, and IKK /NEMO were expressed at similar levels in each of these transfections (Fig. 1B, lower panels). Thus, there was a marked enhancement of IKK /NEMO phosphorylation in the presence of wildtype and constitutively active IKK but not IKK. Finally, we addressed the amino acid residues in IKK / NEMO that were phosphorylated by IKK. Expression vectors encoding FLAG-tagged IKK and Myc-tagged IKK /NEMO were cotransfected into 293T cells, and following immunoprecipitation of the epitope-tagged IKK /NEMO and the associated IKK in vitro kinase assays were performed. The reactions were then subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane followed by autoradiography. Analysis of the 32 P-labeled IKK /NEMO species indicated that IKK /NEMO was phosphorylated predominantly on serine residues (Fig. 1C). In summary, phosphorylation of IKK / NEMO was enhanced in response to treatment of cells with TNF and IL-1. Furthermore, IKK expression results in enhanced IKK /NEMO phosphorylation. Domains in IKK /NEMO That Are Phosphorylated by IKK Because IKK /NEMO phosphorylation was increased by cytokines that stimulate IKK activity and also by the transfection of wild-type or constitutively active IKK, weattempted to identify the regions in IKK /NEMO that were phosphorylated by IKK. The amino acid sequence of IKK /NEMO is indicated as are the positions of the carboxyl-terminal deletion mutants and potential sites of IKK phosphorylation (Fig. 2A). In addition, the structural domains in IKK /NEMO and a schematic of the various deletion mutants that were assayed in in vitro kinase assays are shown (Fig. 2B). Immunopurified epitope-tagged IKK produced following transfection of 293T cells was utilized in in vitro kinase assays FIG. 2. Amino acid sequence and schematic of IKK /NEMO mutants. A, the amino acid sequence of IKK /NEMO is shown with the position of serine and threonine residues that were mutated to define sites of IKK phosphorylation, and the positions where stop codons were inserted to construct carboxyl-terminal deletion mutants are indicated in boldface. B, the domains in IKK /NEMO including the amino-terminal domain that is critical for IKK interaction, the coiled-coil domain that facilitates IKK /NEMO oligomerization, the leucine zipper (LZ), and the carboxyl-terminal interaction domain are indicated. A schematic of the IKK /NEMO mutants that were constructed by insertion of stop codons into the coding sequence of IKK /NEMO using oligonucleotide-directed mutagenesis is shown. with GST fusion proteins containing either wild-type or carboxyl-terminal deletions of IKK /NEMO. Following SDS- PAGE and autoradiography, wild-type IKK /NEMO was found to be phosphorylated by IKK (Fig. 3A, lane 1, top panel). There was a similar level of IKK -mediated phosphorylation of the IKK /NEMO mutant C-(1 394), whereas further deletions, C-(1 358) and C-(1 312), exhibited markedly reduced phosphorylation by IKK (Fig. 3A, lanes 2 4, top panel). Surprisingly, further deletions of IKK /NEMO such as C-(1 270) and C-(1 180) exhibited increased phosphorylation by IKK (Fig. 3A, lanes 5 and 6, top panel). However, the IKK /NEMO deletion mutants, C-(1 137) and C-(1 105), again exhibited severely reduced phosphorylation by IKK (Fig. 3A, lanes 7 and 8, top panel). Similar results were seen with an amino-terminal deletion of IKK /NEMO N-( ), which is unable to bind IKK (Fig. 3A, lane 9, top panel). There was no phosphorylation of GST-IKK /NEMO by the kinase-defective mutant IKK (K/M) (Fig. 3A, lane 10, top panel). The activity of wild-type IKK was demonstrated by its ability to phosphorylate GST- I B (Fig. 3A, lane 12, top panel). These results suggest that IKK is potentially capable of phosphorylating two domains of IKK /NEMO that extend between residues 358 and 394 and 137 and 180. To address further the domains in IKK /NEMO that were phosphorylated by IKK, CMV expression vectors encoding Myc-tagged wild-type and mutant IKK /NEMO constructs and FLAG-tagged IKK were cotransfected into 293T cells. Following immunoprecipitation of either epitope-tagged IKK (Fig. 3B, top panel) orikk /NEMO (Fig. 3B, middle panel) with monoclonal antibodies directed against these epitopes, in vitro kinase assays were performed, and the samples were analyzed by SDS-PAGE and autoradiography. Wild-type IKK, but not

5 IKK Phosphorylation of IKK /NEMO FIG. 3. Domains in IKK /NEMO that are phosphorylated by IKK. A, immunopurified IKK was isolated from 293T cells that were transfected with 2.0 g of a CMV expression vector encoding epitope-tagged IKK and used in in vitro kinase assays (lanes 1 9) with the indicated GST-IKK /NEMO proteins (10.0 g) followed by SDS-PAGE and autoradiography. IKK (K44M) was also assayed with GST-IKK /NEMO (lane 10). A Coomassie-stained gel of each of these GST fusion proteins is indicated (bottom panel). IKK was also analyzed in in vitro kinase assays with GST (10.0 g) and GST-I B (10.0 g) substrates (lanes 11 and 12). B, CMV expression vectors alone (lane 1) or encoding Myc-tagged wild-type (WT) or mutant IKK /NEMO constructs (4.0 g) were transfected into 293T cells with 2.0 g of either wild-type IKK (lanes 2 10) or wild-type IKK alone (lane 11). In addition, IKK (K44M) was transfected either with wild-type IKK /NEMO (lane 12) or alone (lane 13). Extracts were prepared and immunoprecipitated (IP) with FLAG monoclonal antibody (top panel) or Myc monoclonal antibody (middle panel) followed by in vitro kinase assays, SDS-PAGE, and autoradiography. Western blot analysis was performed on a portion of these extracts with Myc and FLAG monoclonal antibodies to detect the epitope-tagged IKK /NEMO and IKK proteins, respectively (bottom panels). the IKK (K/M) mutant, resulted in phosphorylation of IKK / NEMO and was itself autophosphorylated (Fig. 3B, lanes 2 and 12, top and middle panels). Similar to the results seen with IKK and the GST-IKK /NEMO mutants, there was a marked reduction in the ability of IKK to phosphorylate IKK /NEMO when the regions between amino acid residues 358 and 394 and 137 and 180 were deleted (Fig. 3B, lanes 4 and 8, top and middle panels). Western blot analysis revealed similar levels of epitope-tagged IKK /NEMO and IKK proteins in these transfections (Fig. 3B, bottom panels). These results indicate that at least two regions of IKK /NEMO are phosphorylated by IKK. Sites in IKK /NEMO That Are Targets of IKK Phosphorylation Next mass spectrometry was utilized to identify domains in GST-IKK /NEMO that were phosphorylated in vitro by IKK. Following in vitro kinase assays with IKK and GST-IKK /NEMO in the presence of [ - 32 P]ATP, SDS-PAGE was performed, and the 32 P-labeled GST-IKK /NEMO species was excised from the polyacrylamide gel and subjected to digestion with trypsin. The trypsin-digested IKK /NEMO protein was then applied to a reverse-phase high pressure liquid chromatograph, and the fractions were collected. Two fractions (fractions 20 and 21), which contained the majority of the 32 P-labeled peptides, were analyzed by matrix-assisted laser desorption ionization time-of-flight and then subjected to peptide microsequencing. This analysis indicated that fraction 20 was composed entirely of a peptide corresponding to amino acids of IKK /NEMO, whereas fraction 21 was composed of a peptide corresponding to amino acids of IKK /NEMO (data not shown). An examination of the sequences in these two domains indicated that between residues 353 and 378 there was a threonine residue at position 356 and serine residues at positions 369 and 375, whereas between residues 144 and 159 there was a threonine residue at position 147 and serine residues at positions 148, 156, and 158 (Fig. 2A). These data and our mutagenesis studies, which indicated that a region of IKK /NEMO between 358 and 394 was a target for IKK phosphorylation, suggested that serine residues 369 and 375 may be targets for IKK mediated phosphorylation. These serine residues were mutated to alanine. Mutagenesis of IKK /NEMO also indicated that another region between residues 137 and 180 was a target for IKK phosphorylation. Thus, the serine and threonine residues contained within the phosphopeptides identified by mass spectrometry were also mutated to alanine. Fi-

6 24336 IKK Phosphorylation of IKK /NEMO FIG. 4.Identification of residues in IKK /NEMO that are phosphorylated by IKK. A, serine and threonine residues were changed to alanine in GST-IKK /NEMO at amino acid residues 369 and 375 (lane 2), amino acid residues 147, 148, 156, and 158 (lane 3), or all of these residues (lane 4) and assayed using in vitro kinase assays with IKK followed by SDS-PAGE and autoradiography. The IKK was also assayed in in vitro kinase assays with GST and GST-I B substrates (lanes 5 and 6). WT, wild type. B, GST-IKK /NEMO fusion proteins (10.0 g) containing IKK /NEMO amino acid residues (lane 1) or this region of IKK /NEMO with mutation of residues 147 and 148 (lane 2), 156 and 158 (lane 3), or all of these residues (lane 4) were assayed in in vitro kinase assays with IKK followed by SDS-PAGE and autoradiography. An in vitro kinase assay with IKK and GST and GST-I B is shown in lanes 5 and 6. nally, residues 147, 148, 156 and 158, and 369 and 375 were all mutated to alanine in the context of a GST-IKK /NEMO fusion protein (Fig. 4A). In vitro kinase assays using these GST-IKK / NEMO fusion proteins indicated that substitution of serine residues 369 and 375 with alanine markedly reduced its phosphorylation by IKK (Fig. 4A, lanes 1 and 2, top panel). In contrast, there was little change in phosphorylation of GST- IKK /NEMO by IKK when threonine residue 147 and serine residues 148, 156, and 158 were substituted with alanine (Fig. 4A, lane 3, top panel). A GST-IKK /NEMO protein containing a combination of these two sets of mutations exhibited a marked reduction in phosphorylation by IKK similar to that seen with the mutation of serine residues 369 and 375 (Fig. 4A, lane 4, top panel). These results suggested that either residues 369 and 375 alone or in combination were the predominant sites in IKK /NEMO for phosphorylation by IKK. It was important to address whether mutation of either residues 147, 148, 156, and 158 altered IKK phosphorylation of GST-IKK /NEMO. For these studies, we utilized a GST- IKK /NEMO fusion protein extending between residues 1 and 180 to assay the role of these residues in the absence of the carboxyl-terminal phosphorylation sites. Site-directed mutants in which alanine was substituted for threonine residue 147 and serine residue 148 (Fig. 4B, lane 2, top panel), serine residues 156 and 158 (Fig. 4B, lane 3, top panel), or all of these residues (Fig. 4B, lane 4, top panel) resulted in decreased phosphorylation by IKK. These results suggested that there were likely multiple residues that were phosphorylated by IKK in this region of IKK /NEMO. Amino Acid Residues in IKK /NEMO Phosphorylated by IKK To confirm the previous GST-IKK /NEMO mutagenesis results and also address the specific residues in the carboxyl terminus of IKK /NEMO that were phosphorylated by IKK, expression vectors encoding Myc-tagged wild-type and mutant IKK /NEMO and FLAG-tagged IKK were transfected into 293T cells. Following immunoprecipitation with epitope-specific antibodies directed against either IKK (top panel) or IKK /NEMO (middle panel), in vitro kinase assays were performed and analyzed following SDS-PAGE and autoradiography. IKK phosphorylation of the IKK /NEMO mutant in which serine residues 369 and 375 were changed to alanine was reduced as compared with that seen with wild-type IKK / NEMO (Fig. 5A, lanes 2 and 3, top and middle panels). IKK phosphorylated the IKK /NEMO mutant containing substitutions of residues 147, 148, 156, and 158 with alanine to a similar level as seen with wild-type IKK /NEMO (Fig. 5A, lane 4, top and middle panels). Combining these mutations resulted in decreased IKK /NEMO phosphorylation by IKK similar to the results seen with the mutation of residues 369 and 375 (Fig. 5A, lane 5, top and middle panels). Thus, IKK phosphorylation of IKK /NEMO residues between 144 and 159 was only detected in the absence of the carboxyl terminus of IKK /NEMO. Because serine residues 369 and 375 appeared to be critical sites in IKK /NEMO for IKK phosphorylation, alanine substitutions were introduced into each of these two serine residues individually. Each of these epitope-tagged IKK /NEMO constructs was transfected with IKK into 293T cells and immunoprecipitated with either Myc or FLAG monoclonal antibodies, and in vitro kinase assays were performed. This analysis revealed that mutation of serine residue 369 resulted in a 60% reduction of phosphorylation by IKK (Fig. 5B, lanes 1 3, top and middle panels). In contrast, mutation of serine residue 375 resulted in only a 20% decrease in IKK /NEMO phosphorylation by IKK (Fig. 5B, lane 4, top panel). When both serine residues 369 and 375 were changed to alanine, there was a 50 70% decrease in IKK /NEMO phosphorylation (Fig. 5B, lane 5, top and middle panels). Thus, the carboxyl terminus of IKK /NEMO is the major site of phosphorylation by IKK. Finally, we addressed whether alterations in IKK /NEMO phosphorylation affected its interaction with IKK. CMV expression vectors encoding epitope-tagged wild-type and mutant IKK /NEMO and either wild-type IKK (Fig. 5C, top panel) or the IKK (S177A/S181A) mutant (Fig. 5C, lower panel) were transfected into 293T cells. Following immunoprecipitation of Myc-tagged IKK /NEMO, Western blot analysis was performed with FLAG antibody to detect the epitope-tagged IKK. These results indicated that there were similar levels of interaction between wild-type and mutant IKK /NEMO with either wild-type or kinase-defective IKK (Fig. 5C). These results suggest that changes in IKK /NEMO phosphorylation did not dramatically alter its interaction with IKK. IKK /NEMO Phosphorylation Regulates Both IKK and NF- B Activation Previously, cotransfection assays were utilized to demonstrate that IKK /NEMO resulted in the recruitment of IKK into the high molecular weight IKK complex and stimulated the ability of IKK to phosphorylate I B (27, 33). It was important to address whether alterations in the phosphorylation of IKK /NEMO led to changes in its ability to enhance IKK phosphorylation of I B and/or stimulate NF- B luciferase activity. For these studies, expression vectors encoding IKK alone or together with either wild-type or mutant IKK /NEMO were transfected into 293T cells. The FLAGtagged IKK was immunoprecipitated and assayed in in vitro kinase assays with a GST-I B substrate. Wild-type IKK / NEMO markedly enhanced IKK phosphorylation of I B (Fig. 6A, lanes 2 and 9, top panel). Next we addressed whether mutation of threonine residue 356 in IKK /NEMO, which did

7 IKK Phosphorylation of IKK /NEMO FIG. 5. In vitro kinase assays of cotransfected IKK /NEMO and IKK. A, CMV expression vectors (4.0 g) encoding Myc-tagged IKK /NEMO including wild-type (WT, lane 2), alanine substitution of residues 369 and 375 (lane 3), residues 147, 148, 156, and 158 (lane 4), or all of these residues (lane 5) were cotransfected with FLAG-tagged IKK (2.0 g) into 293T cells and immunoprecipitated (IP) with either FLAG antibody (top panel) or Myc antibody (middle panel), and in vitro kinase assays were performed followed by SDS-PAGE and autoradiography. Western blot analysis of these extracts with Myc and FLAG monoclonal antibodies was also performed (bottom panels). B, expression vectors encoding Myc-tagged IKK /NEMO corresponding to wild type (lane 2), alanine substitution of residue 369 (lane 3), 375 (lane 4), both of these residues (lane 5), or IKK alone (lane 6) were transfected into 293T cells with FLAG-tagged IKK, and following immunoprecipitation with either FLAG (top panel) or Myc (middle panel) antibodies, in vitro kinase assays were performed as described. PhosphorImager quantitation of the 32 P-labeled IKK /NEMO proteins revealed relative values in the top panel of0(lane 1), 1.0 (lane 2), 0.39 (lane 3), 0.79 (lane 4), and 0.51 (lane 5) and relative values in the middle panel of 0 (lane 1), 1.0 (lane 2), 0.50 (lane 3), 1.10 (lane 4), and 0.32 (lane 5). Western blot analysis of these extracts with Myc and FLAG antibody was also performed (bottom panel). C, expression vectors encoding wild-type or mutant CMV-IKK /NEMO (1.0 g) were transfected with wild-type (top panel) or the S177A/S181A mutant (lower panel) FLAG-tagged IKK (0.1 g), and extracts were immunoprecipitated with Myc antibody, and Western blot analysis with FLAG antibody was performed (top part of each panel). Western blot analysis of these extracts with Myc and FLAG antibody was also performed (bottom part of each panel). not appear to be a site of IKK phosphorylation, altered the ability of IKK /NEMO to stimulate IKK. This IKK /NEMO mutant resulted in similar levels of IKK activity to that seen with wild-type IKK /NEMO (Fig. 6A, lane 3, top panel). Surprisingly, mutation of serine residue 369 resulted in a 3-fold increase in IKK /NEMO stimulation of IKK activity as compared with wild-type IKK /NEMO (Fig. 6A, lane 4, top panel), whereas mutation of residue 375 resulted in a 2.5-fold increase in IKK /NEMO stimulation of IKK activity (Fig. 6, lane 5, top panel). Mutation of both serine residues 369 and 375 resulted in approximately a 6-fold increase in the ability of IKK /NEMO to stimulate IKK activity as compared with wild-type IKK / NEMO (Fig. 6A, lane 6, top panel). Finally, mutation of residues 147, 148, 156, and 158 resulted in a 2 3-fold increase in the ability of IKK /NEMO to stimulate IKK activity as compared with wild-type IKK /NEMO (Fig. 6A, lane 7, top panel), whereas a combination of these mutations and the carboxylterminal mutations resulted in more than a 6-fold increase in IKK /NEMO stimulation of IKK activity (Fig. 6A, lane 8, top panel). These results were seen in three independent experiments. Western blot analysis indicated similar levels of expression of both IKK /NEMO and IKK in these assays (Fig. 6A, lanes 1 9, middle and lower panels). Similar results with these mutants were seen when IKK /NEMO and IKK were immunoprecipitated with Myc antibody directed against IKK / NEMO, and IKK activity was assayed in in vitro kinase assays with a GST-I B substrate (data not shown). Thus, these data suggest that phosphorylation of IKK /NEMO likely reduces its ability to stimulate IKK activity. Next we assayed whether IKK /NEMO mutants exhibited changes in their ability to activate an NF- B luciferase reporter construct. Transfection of an NF- B reporter alone or in the presence of CMV expression vectors encoding wild-type or mutant IKK /NEMO was performed using IKK /NEMO / mouse embryo fibroblasts (37). An RSV- -galactosidase expression vector was included in these assays to control for changes in transfection efficiency. Following transfection, the cells were treated with TNF for 6 h prior to harvesting and assays of luciferase activity. These studies demonstrated that wild-type IKK /NEMO stimulated NF- B reporter activity 2.5-fold (Fig. 6B, lanes 1 and 2), whereas an IKK /NEMO construct that contained a mutation of serine residue 369 to alanine resulted in a 4.5-fold increase in NF- B reporter activity (Fig. 6B, lanes 1 and 4). Mutation of serine residues 369 and 375 in IKK /NEMO to alanine resulted in a 6-fold increase in NF- B luciferase activity (Fig. 6B, lane 6). Mutation of residues 147, 148, 156, and 158 either alone or in combination with mutation of residues 369 and 375 also resulted in the increased ability of IKK /NEMO to stimulate NF- B luciferase activity as compared with wild-type IKK /NEMO (Fig. 6B, lanes 7 and 8). It is interesting to note that mutation of all the potential IKK phosphorylation sites in IKK /NEMO did not result in further enhancement of NF- B luciferase activity as compared with mutation of either residues 369 and 375 or 147, 148, 156, and 158 alone. Western blot analysis of these lysates revealed similar levels of IKK /NEMO expression (Fig. 6B, lower panel). These results, which were repeated in three independent experiments, indicated that mutation of serine residues 369 and 375 increased the ability of IKK /NEMO to stimulate NF- B activity. DISCUSSION In this study, the role of IKK -mediated phosphorylation of IKK /NEMO on regulating its function was explored. IKK was found to phosphorylate IKK /NEMO predominantly on serine residue 369 in the carboxyl terminus of IKK /NEMO using both in vitro and in vivo assays. Mutation of serine residues 369 and 375 increased the ability of IKK /NEMO to stimulate IKK phosphorylation of I B and activate an NF- B reporter construct. Mutation of the putative phosphorylation sites at residues 147, 148, 156, and 158 did not markedly alter IKK /NEMO phosphorylation unless the carboxyl terminus was eliminated. These results suggest that phosphorylation of IKK /NEMO by IKK and perhaps other kinases may be important in modulating its function.

8 24338 IKK Phosphorylation of IKK /NEMO FIG. 6. IKK /NEMO phosphorylation is important for activation of IKK activity and NF- B activity. A, CMV expression vectors alone (lane 1) encoding Myc-tagged IKK /NEMO (0.4 g) corresponding to wild-type (WT, lane 2), an alanine substitution of threonine residue 356 (lane 3), alanine substitution of serine residues 369 (lane 4), and 375 (lane 5) or both residues 369 and 375 (lane 6), alanine substitution of threonine residue 147 and serine residues 148, 156, and 158 (lane 7), both sets of these mutations (lane 8), or IKK alone (lane 9) were transfected into 293T cells. Extracts of 200 g were prepared and immunoprecipitated with the FLAG monoclonal antibody followed by in vitro kinase assays with GST-I B (10.0 g) and SDS-PAGE and autoradiography (top panel). PhosphorImager analysis of this gel revealed relative values of 0 (lane 1), 1.0 (lane 2), 0.9 (lane 3), 3.0 (lane 4), 2.6 (lane 5), 5.8 (lane 6), 2.8 (lane 7), 6.2 (lane 8), and 0.16 (lane 9). The extracts were also analyzed for IKK /NEMO and IKK expression using Myc and FLAG monoclonal antibodies, respectively (bottom panels). B, IKK /NEMO / mouse embryo fibroblasts were cotransfected with CMV expression vectors encoding either wild-type or mutant Myctagged IKK /NEMO (0.3 g), an NF- B luciferase reporter vector (0.1 g), and an RSV- -galactosidase expression vector (0.1 g). At 18 h post-transfection, the cells were treated with TNF (10 ng/ml) and harvested 6 h later. Luciferase and -galactosidase activity was then determined. The luciferase activity of the NF- B reporter alone (lane 1) and the activity of this reporter in the presence of the different IKK / NEMO constructs (lanes 2 8) are shown. These experiments were repeated three times, and the average of triplicate samples from one experiment is shown with the error bars denoting the S.E. of the mean. The expression levels of the Myc-tagged IKK /NEMO constructs transfected into the IKK /NEMO / cells are shown in the lower panel of this figure. IKK /NEMO is required for NF- B activation in response to a variety of different signals (13 16). Thus, the mechanisms by which IKK /NEMO leads to activation of the NF- B pathway has been the subject of intense investigation. The ability of IKK /NEMO to regulate the NF- B pathway is likely mediated via its differential interactions with factors that regulate the NF- B pathway. Transient expression assays of IKK /NEMO indicate that IKK /NEMO results in the recruitment of IKK into the high molecular weight IKK complex and stimulate its kinase activity (27, 33). Furthermore, IKK /NEMO can enhance the association of the I B proteins with the IKK complex to facilitate its phosphorylation and subsequent degradation (33). IKK /NEMO has also been demonstrated to be able to increase IKK activity in a reconstituted yeast system to result in the assembly of the high molecular weight IKK complex (44). IKK /NEMO can bind to other proteins including the adaptor protein RIP at early times following TNF binding to the TNF receptor (13, 30) and the NF- B inhibitory protein A20 at later times post-tnf treatment to reduce TNF -mediated effects on the NF- B pathway (30). These findings suggest that the differential association of IKK /NEMO with cellular regulatory proteins is important for modulating TNF -induced activation of the NF- B pathway. Thus IKK /NEMO interacts with a variety of cellular proteins that are likely critical in modulating IKK activity. Post-translational modifications such as phosphorylation are important in altering protein function (45). In this study, we addressed the ability of IKK to phosphorylate IKK /NEMO and then determined whether IKK -mediated phosphorylation of IKK /NEMO altered its ability to stimulate IKK activity. First, we demonstrated that endogenous IKK /NEMO was rapidly phosphorylated following treatment of cells with activators of the NF- B pathway including TNF and IL-1. It is interesting to note that the kinetics of IKK /NEMO phosphorylation correlate with the increases in IKK activity in response to these agents. These results in conjunction with data obtained from transient expression assays and in vitro kinase assays with IKK and IKK /NEMO suggest that IKK can directly phosphorylate IKK /NEMO. The level of phosphorylated IKK /NEMO increased rapidly following cytokine stimulation. The rapid decrease in both IKK /NEMO phosphorylation and IKK activity following cytokine treatment suggests that both of these proteins could potentially be targets of phosphatases such as protein phosphatase 2A which reduces IKK activity following cytokine treatment (8). The transient phosphorylation of IKK /NEMO following cytokine stimulation may result in its differential interactions with a variety of cellular proteins including IKK, I B, A20, and RIP (9, 13 16, 30, 33, 36). Our studies suggest that at least two regions of IKK /NEMO are phosphorylated by IKK. However, additional sites of IKK phosphorylation of IKK /NEMO are indeed likely. Other kinases such as protein kinase C have also been demonstrated to phosphorylate serine residues in IKK /NEMO at positions that differ from those identified using IKK (43). In contrast to the results seen with mutation of residues in IKK /NEMO that are targets for IKK -mediated phosphorylation, mutation of serine residues 85 and 141 that are phosphorylated by protein kinase C result in the reduced ability of IKK /NEMO to stimulate IKK phosphorylation of I B (43). Given the specific association of IKK /NEMO and IKK and the temporal relationship between the increased IKK activity and IKK / NEMO phosphorylation, our data and that of a previous study (40) suggest that IKK is an important, although not likely the only, kinase involved in phosphorylating IKK /NEMO. The major site of IKK phosphorylation in the carboxyl terminus of IKK /NEMO is serine residue 369, although serine residue 375 may also potentially be phosphorylated by IKK. IKK was also found to phosphorylate IKK /NEMO in a region between amino acids 137 and 180 at potential sites including residues 147, 148, 156, and 158. It is likely that IKK predominantly phosphorylates the carboxyl terminus of IKK /NEMO when not bound to other proteins. However, the carboxyl terminus of IKK /NEMO is capable of binding to a variety of proteins such as A20 and RIP (30), and it is possible that this binding may lead to conformational changes that make additional domains in IKK /NEMO more accessible to phosphorylation by IKK. It is also possible that phosphorylation of IKK / NEMO may be temporally regulated with the carboxylterminal residues initially phosphorylated by IKK and then either IKK or other kinases phosphorylating additional residues in IKK /NEMO.

9 IKK Phosphorylation of IKK /NEMO The results of this study suggest that IKK -mediated phosphorylation of IKK /NEMO leads to its decreased ability to stimulate IKK and activate an NF- B reporter construct. Initially, the nonphosphorylated form of IKK /NEMO may preferentially bind to IKK to stimulate its ability to phosphorylate I B. Subsequent phosphorylation of IKK /NEMO by IKK may perhaps alter its conformation, its ability to oligomerize (46), or its interaction with other cellular proteins. This can potentially decrease IKK activity and/or lead to IKK / NEMO association with other regulators of the NF- B pathway. Changes in IKK /NEMO phosphorylation may in part be responsible for its ability to both activate and inhibit NF- Bdependent gene expression under different conditions (30). For example, mutagenesis of IKK indicated that in certain instances reduction in IKK binding to IKK /NEMO can result in increased IKK -mediated activation of NF- B reporter constructs (28). One possible explanation for this finding is that the decreased interaction of IKK and IKK /NEMO can lead to reduced IKK /NEMO phosphorylation and thus decreased activation of IKK. Finally, we noted that although the majority of IKK /NEMO is located in the cytoplasm, a small portion of IKK /NEMO is also present in the nucleus. Thus phosphorylation of IKK /NEMO may also serve to target the nuclear import of IKK /NEMO. Further studies will be needed to address the kinetics of phosphorylation of IKK /NEMO and identify additional kinases and/or phosphatases that are able to modulate its phosphorylation state. The slightly different effects of the IKK /NEMO phosphorylation site mutants on activating IKK and stimulating NF- B luciferase activity suggest that these different IKK /NEMO phosphorylation sites may in fact play multiple roles in IKK /NEMO regulation of the NF- B pathway. 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