Role of IKKgamma /NEMO in Assembly of the Ikappa B Kinase Complex*

Xiao-Hua Li, Xiaoqun Fang, and Richard B. GaynorDagger

From the Division of Hematology-Oncology, Department of Medicine, Harold Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8594

Received for publication, September 12, 2000, and in revised form, October 20, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IKKgamma /NEMO is a protein that is critical for the assembly of the high molecular weight Ikappa B kinase (IKK) complex. To investigate the role of IKKgamma /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 IKKgamma /NEMO and the IKKs were examined. When expressed alone following transfection, IKKalpha and IKKbeta were present in low molecular weight complexes migrating between 200 and 400 kDa. However, when coexpressed with IKKgamma /NEMO, both IKKalpha and IKKbeta migrated at ~600 kDa which was similar to the previously described IKK complex that is activated by cytokines such as tumor necrosis factor-alpha . When either IKKalpha or IKKbeta was expressed alone with IKKgamma /NEMO, IKKbeta but not IKKalpha migrated in the higher molecular weight IKK complex. Constitutively active or inactive forms of IKKbeta were both incorporated into the high molecular weight IKK complex in the presence of IKKgamma /NEMO. The amino-terminal region of IKKgamma /NEMO, which interacts directly with IKKbeta , was required for formation of the high molecular weight IKK complex and for stimulation of IKKbeta kinase activity. These results suggest that recruitment of the IKKs into a high molecular complex by IKKgamma /NEMO is a crucial step involved in IKK function.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The NF-kappa 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-kappa B is sequestered in the cytoplasm of most cells where it is bound to a family of inhibitory proteins known as Ikappa B (2, 5, 6). A variety of agents including the cytokines interleukin-1 and TNFalpha ,1 endotoxin, double-stranded RNA, and the viral transactivator Tax activate the NF-kappa B pathway (4, 7-11). These agents stimulate upstream kinases that result in the activation of two related Ikappa B kinases, IKKalpha and IKKbeta (9, 12-16). IKK phosphorylation of amino-terminal serine residues in both Ikappa Balpha and Ikappa Bbeta results in their ubiquitination via interaction with beta -TrCP and subsequent degradation by the proteasome (10, 17-26). Following Ikappa B degradation, the NF-kappa B proteins translocate from the cytoplasm to the nucleus where they activate the expression of specific cellular genes (8).

Both IKKalpha and IKKbeta are components of a high molecular weight complex migrating between 600 and 900 kDa that phosphorylates the Ikappa 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 helix-loop-helix motifs (9, 12-16). IKKalpha and IKKbeta can both homodimerize and heterodimerize, and this process is critical for their kinase activity. Although these kinases have a number of similarities, IKKbeta has at least a 20-fold higher level of kinase activity for Ikappa B than does IKKalpha (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 IKKalpha and positions 177 and 181 in IKKbeta 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 IKKbeta by either NIK or MEKK1 is the critical event that leads to stimulation of IKKbeta kinase activity or whether other mechanisms such as IKKalpha phosphorylation of IKKbeta (39, 58) or IKKbeta autophosphorylation (38) regulate this process remains to be determined.

In addition to IKKalpha and IKKbeta , there are additional components of the IKK complex. A protein known as IKKgamma /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. IKKgamma /NEMO was first identified in a genetic complementation assay as a cellular factor that was able to restore NF-kappa B activation to cells that did not respond to a variety of activators of this pathway (28). IKKgamma /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 TNFalpha (40). Cells lacking IKKgamma /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 IKKgamma /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 IKKgamma /NEMO with IKKbeta , 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 IKKgamma /NEMO. Although IKKbeta preferentially binds to IKKgamma /NEMO as compared with IKKalpha (28, 30), IKKalpha has also been found to bind to IKKgamma /NEMO using extracts prepared from IKKbeta knock-out cells (44). The kinase RIP which is recruited to the p55 TNF receptor following TNFalpha treatment binds to IKKgamma /NEMO (45). The recruitment of IKKgamma /NEMO by RIP leads to the subsequent association of IKKalpha and IKKbeta . Another protein, A20 which functions to inhibit NF-kappa B activation, also binds to IKKgamma /NEMO and may serve to down-regulate TNFalpha signaling pathway (45). Finally, the human T-cell lymphotrophic virus, type I, Tax protein can bind to IKKgamma /NEMO to facilitate the activation of the IKKs (43, 46, 47). These results indicate that IKKgamma /NEMO may serve to link various activators of the NF-kappa B pathway to the IKK complex.

Recent murine gene disruption studies (48-50) and genetic analysis of families lacking IKKgamma /NEMO (51) demonstrate its essential role in regulating the anti-apoptotic and inflammatory properties of the NF-kappa B pathway. Mutations in the IKKgamma /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 IKKgamma /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 IKKgamma /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-kappa B pathway in response to a variety of well characterized activators of this pathway (48-50). However, the mechanism by which IKKgamma /NEMO activates the NF-kappa B pathway remains to be determined.

In the current study, we utilized a biochemical approach to address the function of IKKgamma /NEMO in the recruitment of IKKalpha and IKKbeta into the IKK complex. We demonstrated that the amino terminus of IKKgamma /NEMO that interacts with IKKbeta is crucial for formation of the high molecular weight IKK complex. Moreover, we found that IKKgamma /NEMO stimulates the ability of IKKbeta but not IKKalpha to phosphorylate Ikappa Balpha . These results further establish that IKKgamma /NEMO association with the IKK complex is critical for stimulation of IKK kinase activity.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA Constructs-- The murine IKKgamma /NEMO coding sequence (28) (GenBankTM accession number AF069542-1) was obtained by PCR using a mouse spleen cDNA library. Two oligonucleotide primers complementary to the 5'- and 3'-coding regions of mouse IKKgamma /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 IKKgamma /NEMO sequence. An amino-terminal deletion that contains amino acid residues 101-412 of IKKgamma /NEMO was constructed by PCR followed by cloning of the product into pCMV5/Myc1. The carboxyl-terminal deletions of IKKgamma /NEMO containing either amino acids 1-270 or 1-312 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 IKKgamma /NEMO gene cloned into pCMV5/Myc1. Wild-type and mutant IKKbeta were cloned into plasmid pCMVFl such that the FLAG tag was fused to the 5' end of IKKbeta (12, 35). IKKalpha was cloned into plasmid pRcBactHA and the HA tag was fused to the 5' end of IKKalpha (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 µg 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 µg 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 µl of each column fraction was incubated overnight at 4 °C with 1-2 µg of the indicated antibodies in 150 µl of PD buffer (40 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.1% Nonidet P-40. 6 mM EDTA, 6 mM EGTA, 10 mM beta -glycerophosphate, 10 mM NaF, 300 µM Na3VO4, 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 µg of bacterially expressed GST fusion protein consisting of Ikappa Balpha (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 beta -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 (1.3 × 105) were transfected with either 0.5 µg of IKKbeta alone or in the presence of 0.5 µg of wild-type or mutant IKKgamma /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 µCi of [32P]orthophosphate (PerkinElmer Life Sciences). Cells were then collected in 300 µl of PD buffer. The 32P-labeled IKKbeta protein was immunoprecipitated from 200 µl of cell extract using 4 µg of anti-FLAG monoclonal antibody (Sigma, M2) or 4 µg 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IKKgamma /NEMO Recruits the IKKs into a High Molecular Weight Complex-- It has been shown previously that cytokines such as TNFalpha 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 IKKalpha , IKKbeta , and IKKgamma /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 TNFalpha -treated cells (Fig. 1). In extracts prepared from TNFalpha -treated cells, as compared with untreated cells, there was increased kinase activity for the Ikappa Balpha substrate in immunoprecipitates isolated using either IKKalpha or IKKbeta antibody. There was no phosphorylation of an Ikappa Balpha mutant in which serine residues 32 and 36 were changed to alanine (data not shown). The level of these proteins was not increased by TNFalpha treatment (Fig. 1).



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Fig. 1.   Chromatographic distribution of IKK in untreated and TNFalpha -treated cells. COS cells were either untreated (untr) or treated with TNFalpha (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 IKKalpha (Santa Cruz Biotechnology, sc-7606), IKKbeta (Santa Cruz Biotechnology, sc-7607), or IKKgamma (Santa Cruz Biotechnology, sc-8330). In vitro kinase activity was analyzed utilizing a GST/Ikappa Balpha (aa 1-54) substrate following immunoprecipitation of the column fractions with either IKKalpha (sc-7606) or IKKbeta (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/Ikappa Balpha substrate are indicated at the right of these figures.

In an attempt to assay the effects of IKKgamma /NEMO on the assembly of the IKK complex, COS cells were transfected with expression vectors encoding epitope-tagged IKKalpha and IKKbeta in the presence or absence of epitope-tagged IKKgamma /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 IKKalpha and IKKbeta in the absence of IKKgamma /NEMO, the majority of the epitope-tagged IKKalpha and IKKbeta proteins was detected in fractions corresponding to a molecular mass of 200-400 kDa (Fig. 2A). The majority of the respective IKK kinase activity for the Ikappa Balpha substrate was also detected in these same column fractions (Fig. 2A). The overlapping, yet slightly different chromatographic positions between IKKalpha and IKKbeta may reflect heterodimeric and homodimeric pools of these kinases.



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Fig. 2.   Effect of IKKgamma /NEMO on the chromatographic distribution of cotransfected IKKalpha and IKKbeta . COS cells were transfected with CMV expression vectors containing cDNAs encoding IKKalpha and IKKbeta (A), IKKalpha , IKKbeta , and IKKgamma /NEMO (B), or IKKgamma /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/Ikappa Balpha (aa 1-54) substrate. Monoclonal antibodies (Ab) used against the epitope-tagged proteins included anti-HA (Roche Molecular Biochemicals, 12CA5) for IKKalpha , anti-FLAG (Sigma, M2) for IKKbeta , and anti-Myc (9E10) for IKKgamma /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/Ikappa Balpha are indicated at the right of the figure.

In contrast to these results, coexpression of IKKalpha and IKKbeta with IKKgamma /NEMO resulted in the migration of these epitope-tagged IKKalpha and IKKbeta 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 IKKgamma /NEMO protein was detected only in extracts prepared from cells that were transfected with the IKKgamma /NEMO cDNA and were mainly present in the 400-500-kDa fractions, regardless of whether it was coexpressed with the IKKs (Fig. 2B) or was expressed alone (Fig. 2C). There was no IKK activity using an Ikappa Balpha mutant in which serine residues 32 and 36 were changed to alanine (data not shown). These results suggest that IKKgamma /NEMO has the ability to recruit IKKalpha and IKKbeta into a large protein complex, although the majority of IKKgamma /NEMO does not necessarily comigrate with this complex.

Assembly of the IKK Complex by IKKgamma /NEMO Is Mediated through Interaction with IKKbeta -- To address the mechanism by which IKKgamma /NEMO leads to changes in the chromatographic mobility of the IKKs, we performed experiments in which either epitope-tagged IKKalpha or IKKbeta alone was expressed in the presence or absence of epitope-tagged IKKgamma /NEMO. In extracts prepared from COS cells transfected with IKKalpha in the absence of IKKgamma /NEMO, the majority of the epitope-tagged IKKalpha protein was detected migrating at ~400 kDa (Fig. 3A). The majority of the IKKalpha kinase activity was also detected in these fractions (Fig. 3A). When IKKalpha was coexpressed with IKKgamma /NEMO, the chromatographic position of both the IKKalpha protein and kinase activity was virtually unchanged when compared with that seen when the IKKalpha was expressed alone (Fig. 3A). The epitope-tagged IKKgamma /NEMO protein was detected only in the cells that were transfected with this cDNA, and its chromatographic position was at ~400-500 kDa (Fig. 3A).



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Fig. 3.   Effect of IKKgamma /NEMO on the chromatographic distribution of transfected IKKalpha or IKKbeta . COS cells were transfected with IKKalpha alone (top three panels) or IKKalpha and IKKgamma /NEMO (bottom three panels) (A), IKKbeta alone (top three panels) or IKKbeta and IKKgamma /NEMO (bottom three panels) (B), or IKKbeta 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/Ikappa Balpha are indicated at the right of the figure.

Similar experiments were performed to determine whether IKKgamma /NEMO altered the chromatographic distribution of IKKbeta . In the absence of IKKgamma /NEMO, the majority of the epitope-tagged IKKbeta protein and kinase activity was found in fractions migrating at 200-300 kDa. A small fraction of IKKbeta kinase activity was also seen migrating in higher molecular fractions likely due to its binding to endogenous IKKgamma /NEMO (Fig. 3B). In the presence of transfected IKKgamma /NEMO, the majority of the epitope-tagged IKKbeta protein and kinase activity was found in fractions migrating at ~600 kDa (Fig. 3B). The epitope-tagged IKKgamma /NEMO was detected mainly in fractions migrating at 500 kDa in cells coexpressing IKKbeta and IKKgamma /NEMO (Fig. 3B). These results suggest that IKKgamma /NEMO alters the chromatographic behavior of IKKbeta but not IKKalpha .

Next we addressed the specificity of IKKgamma /NEMO to alter the chromatographic mobility of IKKbeta . An unrelated epitope-tagged protein, the cAMP-responsive element-binding protein (CREB) which has no known role in regulating the IKK complex, was coexpressed with IKKbeta , and their chromatographic mobility was determined. Both the epitope-tagged IKKbeta protein and IKKbeta kinase activity were detected in column fractions migrating at ~200 kDa, indicating that CREB unlike IKKgamma /NEMO is unable to alter IKKbeta mobility (Fig. 3C). Taken together, these results suggest that IKKgamma /NEMO is a factor directly involved in changing the chromatographic position of the IKKs. Furthermore, IKKbeta but not IKKalpha , serves as a primary target for IKKgamma /NEMO in the process of formation of the high molecular weight IKK complex.

IKKbeta Recruitment by IKKgamma /NEMO Is Independent of Its Kinase Activity-- To demonstrate whether IKKgamma /NEMO requires an active IKKbeta kinase to result in its incorporation into the high molecular weight IKK complex, epitope-tagged IKKbeta mutants were transfected into COS cells in either the presence or the absence of IKKgamma /NEMO. Either the constitutively active IKKbeta mutant, IKKbeta (SS/EE), which contains substitutions of serine residues 177 and 181 with glutamic acid residues or a noninducible IKKbeta kinase mutant, IKKbeta (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 IKKbeta protein when expressed alone, the epitope-tagged IKKbeta (SS/EE) protein was detected in fractions corresponding to ~200 kDa (Fig. 4A). When the IKKbeta (SS/EE) protein was coexpressed with the epitope-tagged IKKgamma /NEMO, the IKKbeta (SS/EE) protein and kinase activity was detected in the higher molecular weight IKK complex (Fig. 4A).



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Fig. 4.   Effect of IKKgamma /NEMO on the chromatographic distribution of IKKbeta mutants. COS cells were transfected with IKKbeta (SS/EE) either alone (top three panels) or IKKbeta (SS/EE) and IKKgamma /NEMO (bottom three panels) (A) or IKKbeta (SS/AA) alone (top three panels) or IKKbeta (SS/AA) and IKKgamma /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 IKKbeta and the GST/Ikappa Balpha substrate are indicated at the right of the figure.

The chromatographic distribution of the kinase defective IKKbeta (SS/AA) protein was then assayed. In the absence of IKKgamma /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 IKKgamma /NEMO, the IKKbeta (SS/AA) protein was detected in the high molecular weight IKK complex (Fig. 4B). No detectable kinase activity was detected in any of the fractions containing the kinase-defective IKKbeta (SS/AA) protein regardless of whether or not IKKgamma /NEMO was coexpressed (Fig. 4B). The chromatographic position of IKKgamma /NEMO was unchanged in cells expressing IKKbeta (SS/EE) and IKKbeta (SS/AA) (Fig. 4, A and B). These results suggest that incorporation of IKKbeta into high molecular weight IKK complex by IKKgamma /NEMO is not dependent on the kinase activity of IKKbeta .

The Amino-terminal Domain of IKKgamma /NEMO Is Necessary for Assembly of the IKK Complex-- Studies were next performed to determine which domains in IKKgamma /NEMO are responsible for recruiting IKKbeta into the high molecular weight complex. Epitope-tagged IKKgamma /NEMO deletion mutants lacking either the carboxyl-terminal 100 amino acid residues (aa 1-312) or the amino-terminal 100 residues (aa 101-412) were transfected into COS cells along with both IKKalpha and IKKbeta . Similar to the results obtained with wild-type IKKgamma /NEMO shown in Fig. 2A, the mutant lacking the carboxyl-terminal portion of IKKgamma /NEMO was able to recruit the majority of the epitope-tagged IKKbeta protein and kinase activity into the high molecular weight IKK complex (Fig. 5A). However, this IKKgamma /NEMO mutant consistently failed to shift efficiently the majority of the epitope-tagged IKKalpha into the higher molecular complex (Fig. 5A). The epitope-tagged IKKgamma /NEMO mutant lacking the amino-terminal portion of this protein was unable to shift either IKKalpha or IKKbeta into the high molecular weight IKK complex (Fig. 5B). Finally, mutations that changed leucine residues 315, 322, and 329 in the leucine zipper motif of IKKgamma /NEMO to methionine did not have major effects on the ability of IKKgamma /NEMO to shift the chromatographic distribution of IKKbeta (Fig. 5C). These results suggest that the amino-terminal portion of IKKgamma /NEMO is critical for the chromatographic shift of the IKKs.



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Fig. 5.   Effect of IKKgamma /NEMO mutants on the chromatographic distribution of IKKalpha and IKKbeta . COS cells were transfected with either IKKalpha , IKKbeta , and a carboxyl-terminal deletion mutant of IKKgamma /NEMO (aa 1-312) (A), IKKalpha , IKKbeta , and an amino-terminal deletion mutant of IKKgamma /NEMO (aa 101-412) (B), or IKKbeta and a IKKgamma /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/Ikappa Balpha substrate are indicated at the right of the figure. Ab, antibody.

IKKgamma /NEMO Is Critical for Stimulating IKKbeta Kinase Activity-- To demonstrate whether IKKgamma /NEMO is involved in activation of the IKKs, COS cells were transfected with either epitope-tagged IKKalpha or IKKbeta cDNAs in the presence of increasing amounts of IKKgamma /NEMO. IKK kinase activity was then assayed following immunoprecipitation of either the epitope-tagged IKKalpha or IKKbeta proteins. In the absence of the transfected epitope-tagged IKKgamma /NEMO, there was little IKKbeta activity detected (Fig. 6A, upper panel, lane 2). However, IKKbeta kinase activity was dramatically increased by transfecting increasing amounts of IKKgamma /NEMO (Fig. 6A, lanes 3-6). Immunoprecipitation of the epitope-tagged IKKgamma /NEMO from these extracts followed by in vitro kinase assays of the associated IKKbeta activity gave similar results (data not shown). In contrast, IKKgamma /NEMO resulted in very little stimulation of IKKalpha activity (Fig. 6B). This result was consistently seen regardless of the amount of IKKalpha that was transfected (data not shown).



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Fig. 6.   IKKgamma /NEMO is critical for stimulation of IKKbeta kinase activity. A, COS cells (2 × 105) were transfected with either a CMV expression vector alone (lane 1), 0.05 µg of IKKbeta alone (lane 2), 0.05 µg of IKKbeta and increasing amounts of the CMV vector containing IKKgamma /NEMO of 0.005 µg (lane 3), 0.05 µg (lane 4), 0.25 µg (lane 5), or 0.5 µg (lane 6). The total DNA amounts were adjusted to the same level with the vector DNA for the different transfections. In vitro IKKbeta 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 IKKbeta and the Myc monoclonal antibody (Roche Molecular Biochemicals, 9E10) to assay IKKgamma /NEMO. B, COS cells were transfected with an IKKalpha expression vector (0.5 µg) rather than an IKKbeta vector as in A. The HA monoclonal antibody (Roche Molecular Biochemicals, 12CA5) was used to detect IKKalpha . C, COS cells were transfected with vector alone (lane 1), IKKbeta (0.05 µg), or the mutants indicated in the presence or absence of IKKgamma /NEMO (lanes 2-9). In vitro kinase activity and protein expression were determined. Ab, antibody.

To confirm the specificity of IKKgamma /NEMO induction of IKKbeta kinase activity, epitope-tagged IKKbeta mutants in either the activation loop, IKKbeta (SS/AA) and IKKbeta (SS/EE), or the kinase-defective mutant IKKbeta (K/M) were expressed in either the presence or the absence of IKKgamma /NEMO. Whereas IKKgamma /NEMO stimulated wild-type IKKbeta activity, it failed to stimulate the kinase activity of any of the IKKbeta mutants (Fig. 6C). These results would be consistent with a potential role of IKKgamma /NEMO in stimulating the phosphorylation of serine residues in the IKKbeta activation loop with resultant increases in kinase activity.

Domains in IKKgamma /NEMO That Regulate the Kinase Activity and Phosphorylation of IKKbeta -- Next we determined which domains in IKKgamma /NEMO were important for stimulation of the IKKbeta kinase activity. COS cells were transfected with epitope-tagged IKKbeta alone or with either the wild-type or mutated IKKgamma /NEMO cDNAs. Mutations in either the carboxyl terminus, the amino terminus, or the leucine zipper of IKKgamma /NEMO reduced its ability to stimulate IKKbeta kinase activity (Fig. 7A, upper panel). The amounts of the epitope-tagged IKKbeta and IKKgamma /NEMO proteins were comparable with these transfections (Fig. 7A, bottom two panels).



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Fig. 7.   Domains in IKKgamma /NEMO that regulate IKKbeta kinase activity and phosphorylation. A, COS cells were transfected with vector alone (lane 1), IKKbeta (0.05 µg) alone (lane 2), or IKKbeta (0.05 µg) with either 0.1 µg of the wild-type or mutant IKKgamma /NEMO cDNAs as indicated (lanes 3-7). In vitro kinase assays were performed to determine IKKbeta kinase activity (top panel), and Western blotting was performed to detect the expression of IKKbeta and IKKgamma /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, 69.94. B, COS cells were transfected for 36 h with vector alone (lane 1), 0.5 µg of IKKbeta alone (lane 2), or 0.5 µg of IKKbeta and the indicated IKKgamma /NEMO mutants (0.5 µg) (lanes 3-7). The cells were either incubated with [32P]orthophosphate for 3 h at 37 °C or harvested 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).

Finally, we addressed whether the ability of IKKgamma /NEMO to stimulate IKKbeta kinase activity correlated with the phosphorylation of IKKbeta . Epitope-tagged wild-type or mutant IKKgamma /NEMO cDNAs were transfected into COS cells in the presence of IKKbeta . Cells were then incubated with [32P]orthophosphate, and the epitope-tagged IKKbeta proteins were immunoprecipitated. Analysis of these samples following SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated that wild-type IKKgamma /NEMO and both the carboxyl-terminal deletion and leucine-zipper mutants stimulated IKKbeta phosphorylation. However, the amino-terminal deletion of IKKgamma /NEMO prevented IKKbeta phosphorylation (Fig. 7B). The increased phosphorylation of IKKbeta in the presence of IKKgamma /NEMO appears to require an intact IKKbeta activation loop as phosphorylation of the mutants IKKbeta (SS/EE) and IKKbeta (SS/AA) was not induced by IKKgamma /NEMO (data not shown). Taken together, these results suggest that IKKgamma /NEMO is involved in activation of IKKbeta kinase activity possibly through stimulation of IKKbeta phosphorylation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IKKgamma /NEMO is an essential factor required for NF-kappa B activation (28, 41, 42, 48-50). The ability of IKKgamma /NEMO to stimulate the NF-kappa 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 IKKgamma /NEMO has not been elucidated.

The results presented here using transient expression assays and column chromatography indicate that the expression of IKKgamma /NEMO leads to the recruitment of the IKKs into a high molecular weight complex. Moreover, IKKgamma /NEMO can stimulate IKKbeta phosphorylation of the Ikappa Balpha protein. The ability to assay IKKgamma /NEMO recruitment of IKKbeta 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 IKKgamma /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 IKKgamma /NEMO is critical for its association with IKKbeta (29, 30, 43). Neither the carboxyl terminus nor the leucine zipper domains of IKKgamma /NEMO were required for recruitment of IKKbeta into the IKK complex. However, both domains are important for the ability of IKKgamma /NEMO to stimulate maximally IKKbeta kinase activity. This suggests that the carboxyl terminus and possibly the leucine zipper of IKKgamma /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 IKKgamma /NEMO in regulating distinct functions that lead to activation of the NF-kappa B pathway.

We also determined whether IKKbeta kinase activity was necessary for its recruitment into the high molecular weight IKK complex. We found that both constitutively active and kinase-defective IKKbeta proteins were recruited into the IKK complex by IKKgamma /NEMO. These results suggest that activation of IKKbeta kinase activity is not a requirement to facilitate IKKbeta association with the IKK complex. In the absence of IKKgamma /NEMO, both IKKalpha and IKKbeta migrate in lower molecular weight complexes. Although IKKgamma /NEMO does not appear to efficiently recruit IKKalpha alone into the high molecular weight IKK complex, it is able to recruit IKKalpha through its interactions with IKKbeta . A carboxyl-terminal deletion mutant of IKKgamma /NEMO consistently exhibited defects in recruiting IKKalpha into the high molecular weight complex. Previous data suggest that IKKalpha can associate with IKKgamma /NEMO in IKKbeta -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 IKKgamma /NEMO can facilitate IKKalpha binding via either a direct association or by indirect association with upstream kinases that interact with IKKgamma /NEMO. Thus, IKKgamma /NEMO leads to the recruitment of both IKKalpha and IKKbeta into the IKK complex.

The role of IKKgamma /NEMO in regulating IKK kinase activity and stimulating the NF-kappa B pathway has previously been studied using transient expression assays (29, 30, 42, 43, 55). Antisense IKKgamma reduces IKK activation in response to upstream activators (30, 43); whereas the expression of carboxyl-terminal IKKgamma /NEMO mutants functions as dominant negative inhibitors of IKKbeta activity (29, 30). These results are consistent with a positive role of IKKgamma /NEMO on activating the NF-kappa B pathway. However, overexpression of wild-type IKKgamma /NEMO in transient expression assays can in some cases reduce IKK activation likely by a squelching mechanism (40).

We were able to demonstrate that IKKgamma /NEMO markedly stimulates IKKbeta kinase activity. This stimulation is dependent on intact serine residues in the IKKbeta activation loop. The amino terminus of IKKgamma /NEMO is crucial for this process although the carboxyl terminus and leucine zipper region of IKKgamma /NEMO are also involved. In contrast, IKKgamma /NEMO does not significantly stimulate IKKalpha kinase activity. The ability of IKKgamma /NEMO to stimulate IKKbeta activity is likely mediated in part by the ability of IKKgamma /NEMO to increase phosphorylation of serine residues in the IKKbeta activation loop. The amino terminus of IKKgamma /NEMO is important for both increasing IKKbeta phosphorylation and stimulating IKKbeta kinase activity suggesting that IKKgamma /NEMO association with IKKbeta is critical for both of these events. Whether this process is due to IKKgamma /NEMO-mediated stimulation of IKKbeta autophosphorylation or the ability of IKKgamma /NEMO to recruit other kinases remains to be determined. Thus, IKKgamma /NEMO appears to be important for regulating both IKKbeta phosphorylation and kinase activity.

A model to address IKKgamma /NEMO function would suggest that its interaction with IKKalpha /IKKbeta is critical for formation of the high molecular weight IKK complex that is responsive to various activators of the NF-kappa B pathway (28-30). The amino terminus of IKKgamma /NEMO is critical for IKKbeta association with the high molecular weight complex, whereas the carboxyl terminus of IKKgamma /NEMO likely mediates interactions with upstream kinases such as RIP (45, 57) or MEKK12 or potentially regulates IKKalpha activation of IKKbeta kinase activity (39, 58). Thus, IKKgamma /NEMO likely serves as a unique scaffold protein to facilitate the assembly and activity of the IKK complex. Further studies on the regulation of IKKgamma /NEMO will be important in defining its ability to regulate NF-kappa B and potentially other signal transduction pathways.


    ACKNOWLEDGEMENTS

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 IKKgamma /NEMO deletion mutants.


    FOOTNOTES

* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger 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 75390-8594. Tel.: 214-648-4996; Fax: 214-648-4152; E-mail: gaynor@utsw.swmed.edu.

Published, JBC Papers in Press, November 15, 2000, DOI 10.1074/jbc.M008353200

2 X.-H. Li and R. B. Gaynor, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: TNFalpha , tumor necrosis factor-alpha ; IKK, Ikappa B kinase; PCR, polymerase chain reaction; GST, glutathione S-transferase; CMV, cytomegalovirus; HA, hemagglutinin; aa, amino acid; CREB, cAMP-responsive element-binding protein.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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