Role of IKK /NEMO in Assembly of the I B Kinase Complex*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 6, Issue of February 9, pp , by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Role of IKK /NEMO in Assembly of the I B Kinase Complex* Received for publication, September 12, 2000, and in revised form, October 20, 2000 Published, JBC Papers in Press, November 15, 2000, DOI /jbc.M Xiao-Hua Li, Xiaoqun Fang, 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 IKK /NEMO is a protein that is critical for the assembly of the high molecular weight I B kinase (IKK) complex. To investigate the role of IKK /NEMO in the assembly of the IKK complex, we conducted a series of experiments in which the chromatographic distribution of extracts prepared from cells transiently expressing epitope-tagged IKK /NEMO and the IKKs were examined. When expressed alone following transfection, IKK and IKK were present in low molecular weight complexes migrating between 200 and 400 kda. However, when coexpressed with IKK /NEMO, both IKK and IKK migrated at 600 kda which was similar to the previously described IKK complex that is activated by cytokines such as tumor necrosis factor-. When either IKK or IKK was expressed alone with IKK /NEMO, IKK but not IKK migrated in the higher molecular weight IKK complex. Constitutively active or inactive forms of IKK were both incorporated into the high molecular weight IKK complex in the presence of IKK / NEMO. The amino-terminal region of IKK /NEMO, which interacts directly with IKK, was required for formation of the high molecular weight IKK complex and for stimulation of IKK kinase activity. These results suggest that recruitment of the IKKs into a high molecular complex by IKK /NEMO is a crucial step involved in IKK function. The NF- B proteins are a family of transcription factors that regulate the expression of a variety of cellular genes involved in the control of the immune and the inflammatory response (1 4). NF- B is sequestered in the cytoplasm of most cells where it is bound to a family of inhibitory proteins known as I B (2, 5, 6). A variety of agents including the cytokines interleukin-1 and TNF, 1 endotoxin, double-stranded RNA, and the viral transactivator Tax activate the NF- B pathway (4, 7 11). These agents stimulate upstream kinases that result in the activation of two related I B kinases, IKK and IKK (9, 12 16). IKK phosphorylation of amino-terminal serine residues in both I B and I B results in their ubiquitination via interaction with -TrCP and subsequent degradation by the proteasome (10, 17 26). Following I B degradation, the NF- B * This work was supported by National Institutes of Health Grant CA74128 and a grant from the Welch Foundation. 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- ; IKK, I B kinase; PCR, polymerase chain reaction; GST, glutathione S-transferase; CMV, cytomegalovirus; HA, hemagglutinin; aa, amino acid; CREB, camp-responsive element-binding protein proteins translocate from the cytoplasm to the nucleus where they activate the expression of specific cellular genes (8). Both IKK and IKK are components of a high molecular weight complex migrating between 600 and 900 kda that phosphorylates the I B proteins (12, 14, 23, 27 30). These kinases have 52% amino acid identity and a similar domain structure that includes amino-terminal kinase, leucine zipper, and helixloop-helix motifs (9, 12 16). IKK and IKK can both homodimerize and heterodimerize, and this process is critical for their kinase activity. Although these kinases have a number of similarities, IKK has at least a 20-fold higher level of kinase activity for I B than does IKK (12, 29, 31 35). The mitogen-activated protein kinase kinase family members NIK (36, 37) and MEKK1 (27, 31, 32, 35) can stimulate IKK activity via phosphorylation of serine residues in their activation loop. Mutation of serine residues to alanine in the activation loop at positions 176 and 180 in IKK and positions 177 and 181 in IKK inactivates IKK kinase activity, whereas replacement of these serine residues with glutamates results in the generation of constitutively active kinases (12, 29, 38, 39). Whether phosphorylation of IKK by either NIK or MEKK1 is the critical event that leads to stimulation of IKK kinase activity or whether other mechanisms such as IKK phosphorylation of IKK (39, 58) or IKK autophosphorylation (38) regulate this process remains to be determined. In addition to IKK and IKK, there are additional components of the IKK complex. A protein known as IKK /NEMO has also been shown to be a critical component of the IKK complex (28 30). This 48-kDa glutamine-rich protein contains a leucine zipper domain and two coiled-coil motifs but has no known enzymatic activity. IKK /NEMO was first identified in a genetic complementation assay as a cellular factor that was able to restore NF- B activation to cells that did not respond to a variety of activators of this pathway (28). IKK /NEMO was also isolated independently as a component of the high molecular weight IKK complex (29, 30) and as a factor, designated FIP-3, that binds to the adenovirus E3 protein and inhibits the cytolytic effects of TNF (40). Cells lacking IKK /NEMO are unable to form the high molecular weight IKK complex or respond to cytokines that activate this pathway (28 30, 41, 42). Mutagenesis of the IKK /NEMO indicates that several distinct domains are critical for its function (29, 30, 43). These include the amino-terminal 100 amino acids that mediate the direct interactions of IKK /NEMO with IKK, the carboxyl terminus which likely functions in the recruitment of upstream kinases to the IKK complex, and a coiled-coil domain that mediates oligomerization of IKK /NEMO. Although IKK preferentially binds to IKK /NEMO as compared with IKK (28, 30), IKK has also been found to bind to IKK /NEMO using extracts prepared from IKK knock-out cells (44). The kinase RIP which is recruited to the p55 TNF receptor following TNF treatment binds to IKK /NEMO (45). The recruitment of IKK / NEMO by RIP leads to the subsequent association of IKK and This paper is available on line at

2 Role of IKK /NEMO in Assembly of the IKK Complex 4495 IKK. Another protein, A20 which functions to inhibit NF- B activation, also binds to IKK /NEMO and may serve to downregulate TNF signaling pathway (45). Finally, the human T-cell lymphotrophic virus, type I, Tax protein can bind to IKK /NEMO to facilitate the activation of the IKKs (43, 46, 47). These results indicate that IKK /NEMO may serve to link various activators of the NF- B pathway to the IKK complex. Recent murine gene disruption studies (48 50) and genetic analysis of families lacking IKK /NEMO (51) demonstrate its essential role in regulating the anti-apoptotic and inflammatory properties of the NF- B pathway. Mutations in the IKK / NEMO gene on the X chromosome are the cause of incontinentia pigmenti, an X-linked dominant genetic disorder of the skin that is lethal in males (51). Gene disruption studies of the IKK /NEMO gene demonstrate that although male mice die in utero, heterozygous female mice develop granulocytic infiltration and both hyperproliferation and increased apoptosis of keratinocytes similar to that seen in incontinentia pigmenti (48, 50). Homozygous deletion of IKK /NEMO leads to embryonic lethality due to massive hepatic apoptosis (48, 49). Mouse embryo fibroblasts isolated from these mice exhibit extreme defects in stimulating the NF- B pathway in response to a variety of well characterized activators of this pathway (48 50). However, the mechanism by which IKK /NEMO activates the NF- B pathway remains to be determined. In the current study, we utilized a biochemical approach to address the function of IKK /NEMO in the recruitment of IKK and IKK into the IKK complex. We demonstrated that the amino terminus of IKK /NEMO that interacts with IKK is crucial for formation of the high molecular weight IKK complex. Moreover, we found that IKK /NEMO stimulates the ability of IKK but not IKK to phosphorylate I B. These results further establish that IKK /NEMO association with the IKK complex is critical for stimulation of IKK kinase activity. EXPERIMENTAL PROCEDURES DNA Constructs The murine IKK /NEMO coding sequence (28) (GenBank TM accession number AF ) was obtained by PCR using a mouse spleen cdna library. Two oligonucleotide primers complementary to the 5 - and 3 -coding regions of mouse IKK /NEMO were used in PCR assays. The PCR product was cloned into the expression vector pcmv5/myc1 fusing the Myc tag to the 5 of IKK /NEMO sequence. An amino-terminal deletion that contains amino acid residues of IKK /NEMO was constructed by PCR followed by cloning of the product into pcmv5/myc1. The carboxyl-terminal deletions of IKK /NEMO containing either amino acids or were constructed utilizing PCR to generate these fragments followed by cloning into pcmv5/myc1. The leucine zipper mutations were constructed by substitution of leucine residues at positions 315, 322, and 329 with methionine in the IKK /NEMO gene cloned into pcmv5/myc1. Wildtype and mutant IKK were cloned into plasmid pcmvfl such that the FLAG tag was fused to the 5 end of IKK (12, 35). IKK was cloned into plasmid prcbactha and the HA tag was fused to the 5 end of IKK (12, 35). The cdna clone containing Myc-tagged CREB was previously described (52). Transfection and Cellular Fractionation COS cells grown in Dulbecco s modified Eagle s medium with 10% fetal bovine serum were transfected using Fugene-6 (Roche Molecular Biochemicals) as described by the manufacturer. For a typical cell fractionation experiment, cells cultured overnight in 100-mm plates were transfected with 0.6 g of each DNA construct. Cells from five transfected plates were pooled, and S100 extracts were prepared as detailed (53) except that the extracts were directly loaded onto a Superdex 200 column without dialysis. The S100 extract containing a total of 2 mg of protein was chromatographed through a Superdex-200 column (Amersham Pharmacia Biotech) in buffer D (20 mm HEPES (ph 7.9), 0.1 M KCl, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 20% glycerol, and 0.2 mm EDTA) (54), and fractions of 1 ml each were collected. Western Blotting and Immunoprecipitation/Kinase Assays Western blotting was done with 30 g of protein obtained from each of the column fractions as previously described (53). The antibodies used in this analysis are specified in the figure legends. For kinase assays, 50 l of each column fraction was incubated overnight at 4 C with 1 2 g of the indicated antibodies in 150 l of PD buffer (40 mm Tris-HCl (ph 8.0), 500 mm NaCl, 0.1% Nonidet P mm EDTA, 6 mm EGTA, 10 mm -glycerophosphate, 10 mm NaF, 300 M Na 3 VO 4, and protease inhibitors (Roche Molecular Biochemicals)) (12). Immune complexes were precipitated with protein A-agarose (Bio-Rad) for 1 3 h at 4 C and analyzed by in vitro kinase assays in the presence of 5 g of bacterially expressed GST fusion protein consisting of I B (aa 1 54) or with serine residues 32 and 36 changed to alanine (53). After incubation at 30 C for 30 min, the reactions were mixed with protein sample buffer (50 mm Tris (ph 8.0), 2% SDS, 0.1% bromphenol blue, 10% glycerol, and -mecaptoethanol), heated at 95 C for 3 min, and loaded on a 12% SDS gel. The phosphoprotein products were visualized by autoradiography. In Vivo Phosphorylation Assay COS cells ( ) were transfected with either 0.5 g ofikk alone or in the presence of 0.5 g of wild-type or mutant IKK /NEMO mutants. After 24 h post-transfection, cells were grown overnight in 2 ml of Dulbecco s modified Eagle s medium lacking phosphate (Life Technologies, Inc.) followed by incubating 3 h in 1 ml of Dulbecco s modified Eagle s medium in the presence of 50 Ci of [ 32 P]orthophosphate (PerkinElmer Life Sciences). Cells were then collected in 300 l of PD buffer. The 32 P-labeled IKK protein was immunoprecipitated from 200 l of cell extract using 4 g of anti-flag monoclonal antibody (Sigma, M2) or 4 g of anti-ha monoclonal antibody (Roche Molecular Biochemicals, 12CA5). The immunoprecipitates were washed with PD buffer, and the phosphoproteins were resolved on a 10% SDS-polyacrylamide gel and visualized by autoradiography. RESULTS IKK /NEMO Recruits the IKKs into a High Molecular Weight Complex It has been shown previously that cytokines such as TNF stimulate IKK activity present in a high molecular complex migrating between 600 and 900 kda on gel filtration columns (12, 14, 23, 29, 30). Examination of the chromatographic distribution of the endogenous IKK, IKK, and IKK / NEMO proteins isolated from cytoplasmic fractions of COS cells indicated that these proteins were present in a similarly sized high molecular weight complex in both untreated and TNF -treated cells (Fig. 1). In extracts prepared from TNF treated cells, as compared with untreated cells, there was increased kinase activity for the I B substrate in immunoprecipitates isolated using either IKK or IKK antibody. There was no phosphorylation of an I B mutant in which serine residues 32 and 36 were changed to alanine (data not shown). The level of these proteins was not increased by TNF treatment (Fig. 1). In an attempt to assay the effects of IKK /NEMO on the assembly of the IKK complex, COS cells were transfected with expression vectors encoding epitope-tagged IKK and IKK in the presence or absence of epitope-tagged IKK /NEMO. Cytoplasmic extracts were subjected to gel filtration, and the column fractions were analyzed by Western blot analysis and by IKK kinase activity assay (Fig. 2). In extracts prepared from cells transfected with IKK and IKK in the absence of IKK / NEMO, the majority of the epitope-tagged IKK and IKK proteins was detected in fractions corresponding to a molecular mass of kda (Fig. 2A). The majority of the respective IKK kinase activity for the I B substrate was also detected in these same column fractions (Fig. 2A). The overlapping, yet slightly different chromatographic positions between IKK and IKK may reflect heterodimeric and homodimeric pools of these kinases. In contrast to these results, coexpression of IKK and IKK with IKK /NEMO resulted in the migration of these epitopetagged IKK and IKK proteins to a position corresponding to a molecular mass of 600 kda (Fig. 2B). The kinase activity of the IKK proteins correlated with the presence of these proteins in the 600-kDa fractions. The epitope-tagged IKK /NEMO protein was detected only in extracts prepared from cells that were transfected with the IKK /NEMO cdna and were mainly present in the kDa fractions, regardless of whether it was

3 4496 Role of IKK /NEMO in Assembly of the IKK Complex FIG. 1.Chromatographic distribution of IKK in untreated and TNF -treated cells. COS cells were either untreated (untr) or treated with TNF (20 ng/ml) for 10 min. Cytoplasmic (S100) extracts were prepared and subjected to chromatography on a Superdex-200 column (Amersham Pharmacia Biotech). The fractions derived from the indicated cells (left of figures) were analyzed by Western blotting using antibodies (Ab) directed against either IKK (Santa Cruz Biotechnology, sc-7606), IKK (Santa Cruz Biotechnology, sc-7607), or IKK (Santa Cruz Biotechnology, sc-8330). In vitro kinase activity was analyzed utilizing a GST/I B (aa 1 54) substrate following immunoprecipitation of the column fractions with either IKK (sc-7606) or IKK (sc- 7607) polyclonal antibodies. Molecular weight markers and column fraction numbers are indicated at the top and bottom of the figure, respectively. The positions of the IKKs and GST/I B substrate are indicated at the right of these figures. coexpressed with the IKKs (Fig. 2B) or was expressed alone (Fig. 2C). There was no IKK activity using an I B mutant in which serine residues 32 and 36 were changed to alanine (data not shown). These results suggest that IKK /NEMO has the ability to recruit IKK and IKK into a large protein complex, although the majority of IKK /NEMO does not necessarily comigrate with this complex. Assembly of the IKK Complex by IKK /NEMO Is Mediated through Interaction with IKK To address the mechanism by which IKK /NEMO leads to changes in the chromatographic mobility of the IKKs, we performed experiments in which either epitope-tagged IKK or IKK alone was expressed in the presence or absence of epitope-tagged IKK /NEMO. In extracts prepared from COS cells transfected with IKK in the absence of IKK /NEMO, the majority of the epitope-tagged IKK protein was detected migrating at 400 kda (Fig. 3A). The majority of the IKK kinase activity was also detected in these fractions (Fig. 3A). When IKK was coexpressed with IKK / NEMO, the chromatographic position of both the IKK protein and kinase activity was virtually unchanged when compared with that seen when the IKK was expressed alone (Fig. 3A). The epitope-tagged IKK /NEMO protein was detected only in the cells that were transfected with this cdna, and its chromatographic position was at kda (Fig. 3A). Similar experiments were performed to determine whether IKK /NEMO altered the chromatographic distribution of IKK. In the absence of IKK /NEMO, the majority of the epitope-tagged IKK protein and kinase activity was found in fractions migrating at kda. A small fraction of IKK kinase activity was also seen migrating in higher molecular FIG. 2.Effect of IKK /NEMO on the chromatographic distribution of cotransfected IKK and IKK. COS cells were transfected with CMV expression vectors containing cdnas encoding IKK and IKK (A), IKK, IKK, and IKK /NEMO (B), or IKK /NEMO only (C). Fractions isolated from a Superdex-200 column were subjected to either Western blotting or immunoprecipitation and in vitro kinase assays utilizing the GST/I B (aa 1 54) substrate. Monoclonal antibodies (Ab) used against the epitope-tagged proteins included anti-ha (Roche Molecular Biochemicals, 12CA5) for IKK, anti-flag (Sigma, M2) for IKK, and anti-myc (9E10) for IKK /NEMO. Molecular weight markers and fraction numbers are indicated at the top and bottom of the figures, respectively. The position of the IKKs and GST/I B are indicated at the right of the figure. fractions likely due to its binding to endogenous IKK /NEMO (Fig. 3B). In the presence of transfected IKK /NEMO, the majority of the epitope-tagged IKK protein and kinase activity was found in fractions migrating at 600 kda (Fig. 3B). The epitope-tagged IKK /NEMO was detected mainly in fractions migrating at 500 kda in cells coexpressing IKK and IKK / NEMO (Fig. 3B). These results suggest that IKK /NEMO alters the chromatographic behavior of IKK but not IKK. Next we addressed the specificity of IKK /NEMO to alter the chromatographic mobility of IKK. An unrelated epitopetagged protein, the camp-responsive element-binding protein (CREB) which has no known role in regulating the IKK complex, was coexpressed with IKK, and their chromatographic mobility was determined. Both the epitope-tagged IKK protein and IKK kinase activity were detected in column fractions migrating at 200 kda, indicating that CREB unlike IKK /NEMO is unable to alter IKK mobility (Fig. 3C). Taken together, these results suggest that IKK /NEMO is a factor directly involved in changing the chromatographic position of the IKKs. Furthermore, IKK but not IKK, serves as a primary target for IKK /NEMO in the process of formation of the high molecular weight IKK complex. IKK Recruitment by IKK /NEMO Is Independent of Its Kinase Activity To demonstrate whether IKK /NEMO requires an active IKK kinase to result in its incorporation into the high molecular weight IKK complex, epitope-tagged IKK

4 Role of IKK /NEMO in Assembly of the IKK Complex 4497 FIG. 3.Effect of IKK /NEMO on the chromatographic distribution of transfected IKK or IKK. COS cells were transfected with IKK alone (top three panels) orikk and IKK /NEMO (bottom three panels) (A), IKK alone (top three panels) orikk and IKK /NEMO (bottom three panels) (B), or IKK and CREB (C). Western blotting and in vitro kinase assays performed on fractions isolated from the Superdex-200 column were performed using the same antibodies (Ab) as described in Fig. 2. C, the Myc-tagged CREB was detected by anti-myc antibody. Molecular weight markers and fraction numbers are indicated at the top and the bottom of the figure, respectively. The positions of the IKKs and GST/I B are indicated at the right of the figure. mutants were transfected into COS cells in either the presence or the absence of IKK /NEMO. Either the constitutively active IKK mutant, IKK (SS/EE), which contains substitutions of serine residues 177 and 181 with glutamic acid residues or a noninducible IKK kinase mutant, IKK (SS/AA), in which these same serine residues in the activation loop were substituted with alanine were assayed (12, 29). Similar to the results seen with the wild-type IKK protein when expressed alone, the epitope-tagged IKK (SS/EE) protein was detected in fractions corresponding to 200 kda (Fig. 4A). When the IKK (SS/ EE) protein was coexpressed with the epitope-tagged IKK / NEMO, the IKK (SS/EE) protein and kinase activity was detected in the higher molecular weight IKK complex (Fig. 4A). The chromatographic distribution of the kinase defective IKK (SS/AA) protein was then assayed. In the absence of IKK /NEMO, the majority of epitope-tagged IKK(SS/AA) protein was present in lower molecular weight fractions migrating between 200 and 400 kda (Fig. 4B). In the presence of the epitope-tagged IKK /NEMO, the IKK (SS/AA) protein was detected in the high molecular weight IKK complex (Fig. 4B). No FIG. 4.Effect of IKK /NEMO on the chromatographic distribution of IKK mutants. COS cells were transfected with IKK (SS/EE) either alone (top three panels) orikk (SS/EE) and IKK /NEMO (bottom three panels) (A) or IKK (SS/AA) alone (top three panels) or IKK (SS/AA) and IKK /NEMO (bottom three panels) (B). Western blotting and in vitro kinase assays were performed on the column fractions using the same antibodies (Ab) as described in Fig. 2. Molecular weight markers and column fraction numbers are indicated at the top and bottom of the figure, respectively. The positions of IKK and the GST/ I B substrate are indicated at the right of the figure. detectable kinase activity was detected in any of the fractions containing the kinase-defective IKK (SS/AA) protein regardless of whether or not IKK /NEMO was coexpressed (Fig. 4B). The chromatographic position of IKK /NEMO was unchanged in cells expressing IKK (SS/EE) and IKK (SS/AA) (Fig. 4, A and B). These results suggest that incorporation of IKK into high molecular weight IKK complex by IKK /NEMO is not dependent on the kinase activity of IKK. The Amino-terminal Domain of IKK /NEMO Is Necessary for Assembly of the IKK Complex Studies were next performed to determine which domains in IKK /NEMO are responsible for recruiting IKK into the high molecular weight complex. Epitope-tagged IKK /NEMO deletion mutants lacking either the carboxyl-terminal 100 amino acid residues (aa 1 312) or the amino-terminal 100 residues (aa ) were transfected into COS cells along with both IKK and IKK. Similar to the results obtained with wild-type IKK /NEMO shown in Fig. 2A, the mutant lacking the carboxyl-terminal portion of IKK /NEMO was able to recruit the majority of the epitopetagged IKK protein and kinase activity into the high molecular weight IKK complex (Fig. 5A). However, this IKK /NEMO mutant consistently failed to shift efficiently the majority of the epitope-tagged IKK into the higher molecular complex (Fig. 5A). The epitope-tagged IKK /NEMO mutant lacking the amino-terminal portion of this protein was unable to shift either IKK or IKK into the high molecular weight IKK complex (Fig. 5B). Finally, mutations that changed leucine residues

5 4498 Role of IKK /NEMO in Assembly of the IKK Complex FIG. 5.Effect of IKK /NEMO mutants on the chromatographic distribution of IKK and IKK. COS cells were transfected with either IKK, IKK, and a carboxyl-terminal deletion mutant of IKK / NEMO (aa 1 312) (A), IKK, IKK, and an amino-terminal deletion mutant of IKK /NEMO (aa ) (B), or IKK and a IKK /NEMO mutant that contains substitutions of leucine residues with methionine at 315, 322, and 329 positions in the leucine zipper motif (C). Molecular weight markers and column fraction numbers are indicated at the top and bottom of the figure, respectively. The position of the IKKs and the GST/I B substrate are indicated at the right of the figure. Ab, antibody. 315, 322, and 329 in the leucine zipper motif of IKK /NEMO to methionine did not have major effects on the ability of IKK / NEMO to shift the chromatographic distribution of IKK (Fig. 5C). These results suggest that the amino-terminal portion of IKK /NEMO is critical for the chromatographic shift of the IKKs. IKK /NEMO Is Critical for Stimulating IKK Kinase Activity To demonstrate whether IKK /NEMO is involved in activation of the IKKs, COS cells were transfected with either epitope-tagged IKK or IKK cdnas in the presence of increasing amounts of IKK /NEMO. IKK kinase activity was then assayed following immunoprecipitation of either the epitope-tagged IKK or IKK proteins. In the absence of the transfected epitope-tagged IKK /NEMO, there was little IKK activity detected (Fig. 6A, upper panel, lane 2). However, IKK kinase activity was dramatically increased by transfecting increasing amounts of IKK /NEMO (Fig. 6A, lanes 3 6). Immunoprecipitation of the epitope-tagged IKK /NEMO from these extracts followed by in vitro kinase assays of the associated IKK activity gave similar results (data not shown). In contrast, IKK /NEMO resulted in very little stimulation of IKK activity (Fig. 6B). This result was consistently seen regardless of the amount of IKK that was transfected (data not shown). To confirm the specificity of IKK /NEMO induction of IKK kinase activity, epitope-tagged IKK mutants in either the activation loop, IKK (SS/AA) and IKK (SS/EE), or the kinasedefective mutant IKK (K/M) were expressed in either the presence or the absence of IKK /NEMO. Whereas IKK /NEMO stimulated wild-type IKK activity, it failed to stimulate the kinase activity of any of the IKK mutants (Fig. 6C). These results would be consistent with a potential role of IKK / NEMO in stimulating the phosphorylation of serine residues in the IKK activation loop with resultant increases in kinase activity. Domains in IKK /NEMO That Regulate the Kinase Activity and Phosphorylation of IKK Next we determined which domains in IKK /NEMO were important for stimulation of the IKK kinase activity. COS cells were transfected with epitopetagged IKK alone or with either the wild-type or mutated IKK /NEMO cdnas. Mutations in either the carboxyl terminus, the amino terminus, or the leucine zipper of IKK /NEMO reduced its ability to stimulate IKK kinase activity (Fig. 7A, upper panel). The amounts of the epitope-tagged IKK and IKK /NEMO proteins were comparable with these transfections (Fig. 7A, bottom two panels). Finally, we addressed whether the ability of IKK /NEMO to stimulate IKK kinase activity correlated with the phosphorylation of IKK. Epitope-tagged wild-type or mutant IKK / NEMO cdnas were transfected into COS cells in the presence of IKK. Cells were then incubated with [ 32 P]orthophosphate, and the epitope-tagged IKK proteins were immunoprecipitated. Analysis of these samples following SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated that wild-type IKK /NEMO and both the carboxyl-terminal deletion and leucine-zipper mutants stimulated IKK phosphorylation. However, the amino-terminal deletion of IKK /NEMO prevented IKK phosphorylation (Fig. 7B). The increased phosphorylation of IKK in the presence of IKK /NEMO appears to require an intact IKK activation loop as phosphorylation of the mutants IKK (SS/EE) and IKK (SS/AA) was not induced by IKK /NEMO (data not shown). Taken together, these results suggest that IKK /NEMO is involved in activation of IKK kinase activity possibly through stimulation of IKK phosphorylation. DISCUSSION IKK /NEMO is an essential factor required for NF- B activation (28, 41, 42, 48 50). The ability of IKK /NEMO to stimulate the NF- B pathway is likely due to its effects on the assembly of the IKK complex (28 30) and the recruitment of upstream kinases that increase IKK activity (45). However, the mechanisms involved in the assembly of the IKK complex by IKK /NEMO has not been elucidated. The results presented here using transient expression assays and column chromatography indicate that the expression of IKK /NEMO leads to the recruitment of the IKKs into a high molecular weight complex. Moreover, IKK /NEMO can stimulate IKK phosphorylation of the I B protein. The ability to assay IKK /NEMO recruitment of IKK into the high molecular weight IKK complex allowed us to identify domains in these proteins that are involved in this process. We found that the amino terminus of IKK /NEMO is crucial for recruitment of IKKs into the high molecular weight IKK complex. These results are consistent with previous data indicating that the amino terminus of IKK /NEMO is critical for its association with IKK (29, 30, 43). Neither the carboxyl terminus nor the leucine zipper domains of IKK /NEMO were required for recruitment of IKK into the IKK complex. However, both domains are important for the ability of IKK /NEMO to stimulate maximally IKK kinase activity. This suggests that the car-

6 Role of IKK /NEMO in Assembly of the IKK Complex 4499 FIG. 6. IKK /NEMO is critical for stimulation of IKK kinase activity. A, COS cells ( ) were transfected with either a CMV expression vector alone (lane 1), 0.05 gofikk alone (lane 2), 0.05 g of IKK and increasing amounts of the CMV vector containing IKK /NEMO of g (lane 3), 0.05 g (lane 4), 0.25 g (lane 5), or 0.5 g (lane 6). The total DNA amounts were adjusted to the same level with the vector DNA for the different transfections. In vitro IKK kinase activity was assayed using the FLAG monoclonal antibody (Sigma, M2), and Western blotting was performed with the FLAG antibody to assay the expression of IKK and the Myc monoclonal antibody (Roche Molecular Biochemicals, 9E10) to assay IKK /NEMO. B, COS cells were transfected with an IKK expression vector (0.5 g) rather than an IKK vector as in A. The HA monoclonal antibody (Roche Molecular Biochemicals, 12CA5) was used to detect IKK. C, COS cells were transfected with vector alone (lane 1), IKK (0.05 g), or the mutants indicated in the presence or absence of IKK /NEMO (lanes 2 9). In vitro kinase activity and protein expression were determined. Ab, antibody. FIG. 7.Domains in IKK /NEMO that regulate IKK kinase activity and phosphorylation. A, COS cells were transfected with vector alone (lane 1), IKK (0.05 g) alone (lane 2), or IKK (0.05 g) with either 0.1 g of the wild-type or mutant IKK /NEMO cdnas as indicated (lanes 3 7). In vitro kinase assays were performed to determine IKK kinase activity (top panel), and Western blotting was performed to detect the expression of IKK and IKK /NEMO (bottom two panels). PhosphorImager analysis of seven sets of transfections were quantitated as follows: lane 1, 3.04; lane 2, 24.74; lane 3, 100; lane 4, 36.01; lane 5, 38.79; lane 6, 23.43; and lane 7, B, COS cells were transfected for 36 h with vector alone (lane 1), 0.5 g ofikk alone (lane 2), or 0.5 g ofikk and the indicated IKK /NEMO mutants (0.5 g) (lanes 3 7). The cells were either incubated with [ 32 P]orthophosphate for 3hat37 Corharvested for Western analysis (bottom two panels). The radiolabeled proteins were immunoprecipitated using anti-flag monoclonal antibody (Sigma, M2) and subjected to electrophoresis and autoradiography (top panel). boxyl terminus and possibly the leucine zipper of IKK /NEMO are involved in additional functions such as the recruitment of upstream kinases including RIP into the IKK complex (45). These results are consistent with a role for different domains in IKK /NEMO in regulating distinct functions that lead to activation of the NF- B pathway. We also determined whether IKK kinase activity was necessary for its recruitment into the high molecular weight IKK complex. We found that both constitutively active and kinasedefective IKK proteins were recruited into the IKK complex by IKK /NEMO. These results suggest that activation of IKK kinase activity is not a requirement to facilitate IKK association with the IKK complex. In the absence of IKK /NEMO, both IKK and IKK migrate in lower molecular weight complexes. Although IKK /NEMO does not appear to efficiently recruit IKK alone into the high molecular weight IKK complex, it is able to recruit IKK through its interactions with IKK. A carboxyl-terminal deletion mutant of IKK /NEMO consistently exhibited defects in recruiting IKK into the high molecular weight complex. Previous data suggest that IKK can associate with IKK /NEMO in IKK -deficient fibroblasts (44) and in cotransfection assays performed in 293 cells (46). These results raise the possibility that the carboxyl terminus and potentially other domains of IKK /NEMO can facilitate IKK binding via either a direct association or by indirect association with upstream kinases that interact with IKK / NEMO. Thus, IKK /NEMO leads to the recruitment of both IKK and IKK into the IKK complex. The role of IKK /NEMO in regulating IKK kinase activity and stimulating the NF- B pathway has previously been stud-

7 4500 Role of IKK /NEMO in Assembly of the IKK Complex ied using transient expression assays (29, 30, 42, 43, 55). Antisense IKK reduces IKK activation in response to upstream activators (30, 43); whereas the expression of carboxyl-terminal IKK /NEMO mutants functions as dominant negative inhibitors of IKK activity (29, 30). These results are consistent with a positive role of IKK /NEMO on activating the NF- B pathway. However, overexpression of wild-type IKK /NEMO in transient expression assays can in some cases reduce IKK activation likely by a squelching mechanism (40). We were able to demonstrate that IKK /NEMO markedly stimulates IKK kinase activity. This stimulation is dependent on intact serine residues in the IKK activation loop. The amino terminus of IKK /NEMO is crucial for this process although the carboxyl terminus and leucine zipper region of IKK /NEMO are also involved. In contrast, IKK /NEMO does not significantly stimulate IKK kinase activity. The ability of IKK /NEMO to stimulate IKK activity is likely mediated in part by the ability of IKK /NEMO to increase phosphorylation of serine residues in the IKK activation loop. The amino terminus of IKK /NEMO is important for both increasing IKK phosphorylation and stimulating IKK kinase activity suggesting that IKK /NEMO association with IKK is critical for both of these events. Whether this process is due to IKK / NEMO-mediated stimulation of IKK autophosphorylation or the ability of IKK /NEMO to recruit other kinases remains to be determined. Thus, IKK /NEMO appears to be important for regulating both IKK phosphorylation and kinase activity. A model to address IKK /NEMO function would suggest that its interaction with IKK /IKK is critical for formation of the high molecular weight IKK complex that is responsive to various activators of the NF- B pathway (28 30). The amino terminus of IKK /NEMO is critical for IKK association with the high molecular weight complex, whereas the carboxyl terminus of IKK /NEMO likely mediates interactions with upstream kinases such as RIP (45, 57) or MEKK1 2 or potentially regulates IKK activation of IKK kinase activity (39, 58). Thus, IKK /NEMO likely serves as a unique scaffold protein to facilitate the assembly and activity of the IKK complex. Further studies on the regulation of IKK /NEMO will be important in defining its ability to regulate NF- B and potentially other signal transduction pathways. Acknowledgments We thank Sharon Johnson and Alex Herrera for preparation of the manuscript and the figures, respectively. We also thank Dean Ballard for providing us with IKK /NEMO deletion mutants. REFERENCES 1. Baeuerle, P. A., and Baltimore, D. (1996) Cell 87, Baldwin, A. S. (1996) Annu. Rev. Immunol. 14, Barnes, P. J. (1997) Int. J. Biochem. Cell Biol. 29, Ghosh, S., May, M. J., and Kopp, E. B. (1998) Annu. Rev. Immunol. 16, Beg, A. A., Ruben, S. M., Scheinman, R. I., Haskill, S., Rosen, C. A., and Baldwin, A. S., Jr. (1992) Genes Dev. 6, Beg, A. A., Finco, T. S., Nantermet, P. V., and Baldwin, A. S. J. (1993) Mol. Cell. Biol. 13, Li, X.-H., and Gaynor, R. B. (1999) Gene Expr. 7, Pahl, H. L. (1999) Oncogene 18, Karin, M. (1999) Oncogene 18, Karin, M., and Ben-Neriah, Y. (2000) Annu. Rev. Immunol. 18, Li, X.-H., and Gaynor, R. B. (2000) AIDS Res. Human Retroviruses, 16, Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J., Young, D. B., Barbosa, M., and Mann, M. (1997) Science 278, Woronicz, J. D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D. V. (1997) Science 278, Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91, X.-H. Li and R. B. Gaynor, unpublished observations. 15. Regnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M. (1997) Cell 90, DiDonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M. (1997) Nature 388, Brockman, J. A., Scherer, D. C., MsKinsey, T. A., Hall, S. M., Qi, X., Lee, W. Y., and Ballard, D. W. (1995) Mol. Cell. Biol. 15, Brown, K., Gerstberger, S., Carlson, L., Fransozo, G., and Siebenlist, U. (1995) Science 267, Traenckner, E. B. M., Pahl, H. L., Henkel, T., Schmidt, K. N., Wilk, S., and Baeuerle, P. A. (1995) EMBO J. 14, Whiteside, S. T., Ernst, M. K., LeBail, O., Laurent-Winter, C., Rice, N., and Israel, A. (1995) Mol. Cell. Biol. 15, DiDonato, J., Mercurio, F., Rosette, C., Wu-Li, J., Suyang, H., Ghosh, S., and Karin, M. (1996) Mol. Cell. Biol. 16, Chen, Z., Hagler, J., Palombella, V. J., Melandri, F., Scherer, D., Ballard, D., and Maniatis, T. (1995) Genes Dev. 9, Chen, Z. J., Parent, L., and Maniatis, T. (1996) Cell 84, Yaron, A., Hatzubai, A., Davis, M., Lavon, I., Amit, S., Manning, A. M., Andersen, J. S., Mann, M., Mercurio, F., and Ben-Neriah, Y. (1998) Nature 396, Winston, J. T., Strack, P., Beer-Romero, P., Chu, C., Elledge, S. J., and Harper, J. W. (1999) Genes Dev. 13, Spencer, E., Jiang, J., and Chen, Z. J. (1999) Genes Dev. 13, Lee, F. S., Hagler, J., Chen, Z. J., and Maniatis, T. (1997) Cell 88, Yamaoka, S., Courtois, G., Bessia, C., Whiteside, S. T., Weil, R., Agou, F., Kirk, H. E., Kay, R. J., and Israel, A. (1998) Cell 93, Mercurio, F., Murray, B. W., Shevchenko, A., Bennett, B. L., Young, D. B., Li, J. W., Pascual, G., Motiwala, A., Zhu, H., Mann, M., and Manning, A. M. (1999) Mol. Cell. Biol. 19, Rothwarf, D. M., Zandi, E., Natoli, G., and Karin, M. (1998) Nature 395, Lee, F. S., Peters, R. T., Dang, L. C., and Maniatis, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, Nakano, H., Shindo, M., Sakon, S., Nishinaka, S., Mihara, M., Yagita, H., and Okumura, K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, Zandi, E., Chen, Y., and Karin, M. (1998) Science 281, Kwak, Y. T., Guo, J., Shen, J., and Gaynor, R. B. (2000) J. Biol. Chem. 275, Yin, M.-J., Christerson, L. B., Yamamoto, Y., Kwak, Y.-T., Xu, S., Mercurio, F., Barbosa, M., Cobb, M. H., and Gaynor, R. B. (1998) Cell 93, Malinin, N. L., Boldin, M. P., Kovalenko, A. V., and Wallach, D. (1997) Nature 385, Ling, L., Cao, Z., and Goeddel, D. V. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, Delhase, M., Hayakawa, M., Chen, Y., and Karin, M. (1999) Science 284, Yamamoto, Y., Yin, M. J., and Gaynor, R. B. (2000) Mol. Cell. Biol. 20, Li, Y., Kang, J., Friedman, J., Tarassishin, L., Ye, J., Kovalenko, A., Wallach, D., and Horwitz, M. S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, Courtois, G., Whiteside, S. T., Sibley, C. H., and Israel, A. (1997) Mol. Cell. Biol. 17, Harhaj, E. W., Good, L., Xiao, G., Uhlik, M., Cvijic, M. E., Rivera-Walsh, I., and Sun, S. C. (2000) Oncogene 19, Chu, Z.-L., Shin, Y.-A., Yang, J.-M., DiDonato, J. A., and Ballard, D. W. (1999) J. Biol. Chem. 274, Tanaka, M., Fuentes, M. E., Yamaguchi, K., Durnin, M. H., Dalrymple, S. A., Hardy, K. L., and Goeddel, D. V. (1999) Immunity 10, Zhang, S. Q., Kovalenko, A., Cantarella, G., and Wallach, D. (2000) Immunity 12, Harhaj, E. W., and Sun, S. C. (1999) J. Biol. Chem. 274, Jin, D. Y., Giordano, V., Kibler, K. V., Nakano, H., and Jeang, K. T. (1999) J. Biol. Chem. 274, Makris, C., Godfrey, V. L., Krahn-Senftleben, G., Takahashi, T., Roberts, J. L., Schwarz, A. T., Feng, L., Johnson, R. S., and Karin, M. (2000) Mol. Cell 5, Rudolph, D., Yeh, W. C., Wakeham, A., Rudolph, B., Nallainathan, D., Potter, J., Elia, A. J., and Mak, T. W. (2000) Genes Dev. 14, Schmidt-Supprian, M., Bloch, W., Courtois, G., Addicks, K., Israel, A., Rajewsky, K., and Pasparakis, M. (2000) Mol. Cell 5, Smahi, A., Courtois, G., Vabres, P., Yamaoka, S., Heuertz, S., Munnich, A., Israel, A., Heiss, N. S., Klauck, S. M., Kioschis, P., Wiemann, S., Poustka, A., Esposito, T., Bardaro, T., Gianfrancesco, F., Ciccodicola, A., D Urso, M., Woffendin, H., Jakins, T., Donnai, D., Stewart, H., Kenwrick, S. J., Aradhya, S., Yamagata, T., Levy, M., and Lewis, R. A. (2000) Nature 405, Yin, M., Paulssen, E., Seeler, J., and Gaynor, R. (1995) J. Virol. 69, Li, X.-H., Murphy, K. M., Palka, K. T., Surabhi, R. M., and Gaynor, R. B. (1999) J. Biol. Chem. 274, Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, Li, Q., Van Antwerp, D., Mercurio, F., Lee, K. F., and Verma, I. M. (1999) Science 284, Li, Q., Lu, Q., Hwang, J. Y., Buscher, D., Lee, K. F., Izpisua-Belmonte, J. C., and Verma, I. M. (1999) Genes Dev. 13, Inohara, N., Koseki, T., Lin, J., del Peso, L., Lucas, P. C., Chen, F. F., Ogura, Y., and Nunez, G. (2000) J. Biol. Chem. 275, O Mahony, A., Lin, X., Geleziunas, R., and Greene, W. C. (2000) Mol. Cell. Biol. 20,