Role of IKKγ/NEMO in Assembly of the IKK Complex

Transcription

1 JBC Papers in Press. Published on November 15, 2000 as Manuscript M Role of /NEMO in Assembly of the IKK Complex Xiao Hua Li, Xiaoqun Fang and Richard B. Gaynor Division of Hematology-Oncology Department of Medicine Harold Simmons Cancer Center University of Texas Southwestern Medical Center, Dallas, Texas Correspondence: Richard B. Gaynor Division of Hematology-Oncology Department of Medicine U.T. Southwestern Medical Center 5323 Harry Hines Boulevard Dallas, Texas Phone: (214) FAX: (214) Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

2 Summary /NEMO is a protein that is critical for the assembly of the high molecular weight IKK complex. In order to investigate the role of /NEMO in the assembly of the IKK complex, we conducted series of experiments in which the chromatographic distribution of extracts prepared from cells transiently expressing epitope-tagged /NEMO and the IKKs were examined. When expressed alone following transfection, IKKα and IKKβ were present in low molecular weight complexes migrating between kda. However, when coexpressed with /NEMO, both IKKα and IKKβ migrated at ~600 kda which was similar to the previously described IKK complex that is activated by cytokines such as TNFα. When either IKKα or IKKβ was expressed alone with /NEMO, IKKβ but not IKKα migrated in the higher molecular weight IKK complex. Constitutively active or inactive forms of IKKβ were both incorporated into the high molecular weight IKK complex in the presence of /NEMO. The N-terminal region of /NEMO, which interacts directly with IKKβ, was required for formation of the high molecular weight IKK complex and for stimulation of IKKβ kinase activity. These results suggest that recruitment of the IKKs into a high molecular complex by /NEMO is a crucial step involved in IKK function. 2

3 Introduction The NF-κB 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-κB is sequestered in the cytoplasm of most cells where it is bound to a family of inhibitory proteins known as IκB (2,5,6). A variety of agents including the cytokines IL-1 and TNFα, endotoxin, double-stranded RNA and the viral transactivator Tax activate the NF-κΒ pathway (4,7-11). These agents stimulate upstream kinases which result in the activation of two related IκB kinases, IKKα and IKKβ (9,12-16). IKK phosphorylation of amino-terminal serine residues in both and IκBβ results in their ubiquitination via interaction with β- TrCP and subsequent degradation by the proteasome (10,17-26). Following IκB degradation, the NF-κΒ proteins translocate from the cytoplasm to the nucleus where they activate the expression of specific cellular genes (8). Both IKKα and IKKβ are components of a high molecular weight complex migrating between kda that phosphorylates the IκB proteins (12,14,23,27-30). These kinases have 52% amino acid identity and a similar domain structure which includes N-terminal kinase, leucine zipper and helix-loop-helix motifs (9,12-16). IKKα and IKKβ can both 3

4 homodimerize and heterodimerize and this process is critical for their kinase activity. Although these kinases have a number of similarities, IKKβ has at least a 20-fold higher level of kinase activity for IκB than does IKKα (12,29,31-35). The MAP 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 IKKα and positions 177 and 181 in IKKβ inactivates IKK kinase activity while replacement of these serine residues with glutamates results in the generation of constitutively active kinases (12,29,38,39). Whether phosphorylation of IKKβ by either NIK or MEKK1 is the critical event that leads to stimulation of IKKβ kinase activity or whether other mechanisms such as IKKα phosphorylation of IKKβ (38, 51) or IKKβ autophosphorylation (38) regulate this process remains to be determined. In addition to IKKα and IKKβ, there are additional components of the IKK complex. A protein known as /NEMO has also been shown to be a critical component of the IKK complex (28-30). This 48 kda glutaminerich protein contains a leucine zipper domain and two coiled-coil motifs but has no known enzymatic activity. /NEMO was first identified in a 4

5 genetic complementation assay as a cellular factor that was able to restore NF-κB activation to cells that did not respond to a variety of activators of this pathway (28). /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 TNFα (40). Cells lacking /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 /NEMO indicates that several distinct domains are critical for its function (29,30,43). These include the aminoterminal 100 amino acids that mediate the direct interactions of /NEMO with IKKβ, the carboxy-terminus which likely functions in the recruitment of upstream kinases to the IKK complex, and a coiled-coil domain which mediates oligomerization of /NEMO. Although IKKβ preferentially binds to /NEMO as compared to IKKα (28,30), IKKα has also been found to bind to /NEMO using extracts prepared from IKKβ knock-out cells (44). The kinase RIP which is recruited to the p55 TNF receptor following TNFα treatment binds to /NEMO (45). The recruitment of /NEMO by RIP leads to the subsequent association of IKKα and IKKβ. Another protein, A20 which functions to inhibit NF-κB 5

6 activation, also binds to /NEMO and may serve to downregulate TNFα signaling pathway (45). Finally, the HTLV-I Tax protein can bind to /NEMO to facilitate the activation of the IKKs (43,46,47). These results indicate that /NEMO may serve to link various activators of the NF-κB pathway to the IKK complex. Recent murine gene disruption studies (48-50) and genetic analysis of families lacking /NEMO (51) demonstrate its essential role in regulating the anti-apoptotic and inflammatory properties of the NF-κB pathway. Mutations in the /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 /NEMO gene demonstrate that while 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 /NEMO leads to embryonic lethality due to massive hepatic apoptosis (48,49). MEFs isolated from these mice exhibit extreme defects in stimulating the NF-κB pathway in response to a variety of wellcharacterized activators of this pathway (48-50). However, the mechanism 6

7 by which /NEMO activates the NF-κB pathway remains to be determined. In the current study, we utilized a biochemical approach to address the function of /NEMO in the recruitment of IKKα and IKKβ into the IKK complex. We demonstrated that the N-terminus of /NEMO which interacts with IKKβ is crucial for formation of the high molecular weight IKK complex. Moreover, we found that /NEMO stimulates the ability of IKKβ but not IKKα to phosphorylate. These results further establish that /NEMO association with the IKK complex is critical for stimulation of IKK kinase activity. Experimental Procedures DNA constructs. The murine /NEMO coding sequence (28) (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 /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 /NEMO sequence. An N-terminal 7

8 deletion which contains amino acid residues of /NEMO was constructed by PCR followed by cloning of the product into pcmv5/myc1. The C-terminal deletions of /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 /NEMO gene cloned into pcmv5/myc1. Wildtype and mutant IKKβ were cloned into plasmid pcmvfl such that the Flag tag was fused to the 5 end of IKKβ (12,35). IKKα was cloned into plasmid prcbactha and the HA tag was fused to the 5 end of IKKα (12,35). The cdna clone containing Myc-tagged CREB was previously described (52). Transfection and cellular fractionation. COS cells grown in DMEM with 10% fetal bovine serum were transfected using Fugene-6 (Roche) 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 8

9 Superdex-200 column (Pharmacia) in buffer D [20 mm HEPES (ph 7.9), 0.1 M KCl, 0.5 mm dithiothreitol, 0.5 mm PMSF, 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 mm EDTA, 6 mm EGTA, 10 mm β- glycerophosphate, 10 mm NaF, 300 um Na3V04, and protease inhibitors (Roche) (12). Immune complexes were precipitated with protein A-agarose (Bio-Rad) for 1-3 hours at 4 C and analyzed by in vitro kinase assays in the presence of 5 µg of bacterially expressed GST fusion protein consisting of (aa1-54) or with serine residues 32 and 36 changed to alanine (53). After incubation at 30 C for 30 minutes, the reactions were mixed with protein sample buffer (50 mm Tris ph 8.0, 2% SDS, 0.1% bromophenol blue, 10% glycerol and β-mecaptoethanol), heated at 95 C for 3 minutes and 9

10 loaded on a 12% SDS gel. The phosphoprotein products were visualized by autoradiography. In vivo phosphorylation assay. COS cells (1.3 x 10 5 ) were transfected with either 0.5 µg of IKKβ alone or in the presence of 0.5 µg of wild-type or mutant /NEMO mutants. After 24 hours post-transfection, cells were grown overnight in 2 ml of Dulbecco's Modified Eagle medium lacking phosphate (GibcoBRL) followed by incubating 3 hours in 1 ml of Dulbecco's Modified Eagle medium in the presence of 50 µci of 32 P- orthophosphate (NEN). Cells were then collected in 300 µl of PD buffer. The 32 P-labeled IKKβ 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, 12CA5). The immunoprecipitates were washed with PD buffer and the phosphoproteins were resolved on a 10 % SDS polyacrylamide gel and visualized by autoradiography. 10

11 Results /NEMO recruits the IKKs into a high molecular weight complex It has been previously shown that cytokines such as TNFα stimulate IKK activity present in a high molecular complex migrating between 600 to 900 kda on gel-filtration columns (12,14,23,29,30). Examination of the chromatographic distribution of the endogenous IKKα, IKKβ and /NEMO proteins isolated from cytoplasmic fractions of COS cells indicated that these proteins were present in a similar-sized high molecular weight complex in both untreated and TNFα-treated cells (Figure 1). In extracts prepared from TNFα-treated cells, as compared to untreated cells, there was increased kinase activity for the substrate in immunoprecipitates isolated using either IKKα or IKKβ antibody. There was no phosphorylation of an mutant in which serine residues 32 and 36 were changed to alanine (data not shown). The level of these proteins was not increased by TNFα treatment (Figure 1). In an attempt to assay the effects of /NEMO on the assembly of the IKK complex, COS cells were transfected with expression vectors encoding epitope-tagged IKKα and IKKβ in the presence or absence of epitope-tagged /NEMO. Cytoplasmic extracts were subjected to gel- 11

12 filtration and the column fractions were analyzed by Western blot analysis and by IKK kinase activity assay (Figure 2). In extracts prepared from cells transfected with IKKα and IKKβ in the absence of /NEMO, the majority of the epitope-tagged IKKα and IKKβ proteins were detected in fractions corresponding to a molecular weight of kda (Figure 2A). The majority of the respective IKK kinase activity for the substrate was also detected in these same column fractions (Figure 2A). The overlapping, yet slightly different chromatographic positions between IKKα and IKKβ, may reflect heterodimeric and homodimeric pools of these kinases. In contrast to these results, coexpression of IKKα and IKKβ with /NEMO resulted in the migration of these epitope-tagged IKKα and IKKβ proteins to a position corresponding to a molecular weight of ~600 kda (Figure 2B). The kinase activity of the IKK proteins correlated with the presence of these proteins in the 600 kda fractions. The epitope-tagged /NEMO protein was detected only in extracts prepared from cells that were transfected with the /NEMO cdna and was mainly present in the kda fractions, regardless of whether it was coexpressed with the IKKs (Figure 2B) or was expressed alone (Figure 2C). There was no IKK activity using an mutant in which serine residues 32 and 36 were 12

13 changed to alanine (data not shown). These results suggest that /NEMO has the ability to recruit IKKα and IKKβ into a large protein complex, although the majority of /NEMO does not necessarily comigrate with this complex. Assembly of the IKK complex by /NEMO is mediated through interaction with IKKβ To address the mechanism by which /NEMO leads to changes in the chromatographic mobility of the IKKs, we performed experiments in which either epitope-tagged IKKα or IKKβ alone was expressed in the presence or absence of epitope-tagged /NEMO. In extracts prepared from COS cells transfected with IKKα in the absence of /NEMO, the majority of the epitope-tagged IKKα protein was detected migrating at ~400 kda (Figure 3A). The majority of the IKKα kinase activity was also detected in these fractions (Figure 3A). When IKKα was coexpressed with /NEMO, the chromatographic position of both the IKKα protein and kinase activity was virtually unchanged when compared with that seen when the IKKα was expressed alone (Figure 3A). The epitope-tagged /NEMO protein was detected only in the cells that were transfected 13

14 with this cdna and its chromatographic position was at ~ kda (Figure 3A). Similar experiments were performed to determine whether /NEMO altered the chromatographic distribution of IKKβ. In the absence of /NEMO, the majority of the epitope-tagged IKKβ protein and kinase activity was found in fractions migrating at kda. A small fraction of IKKβ kinase activity was also seen migrating in higher molecular fractions likely due to its binding to endogenous /NEMO (Figure 3B). In the presence of transfected /NEMO, the majority of the epitope-tagged IKKβ protein and kinase activity was found in fractions migrating at ~600 kda (Figure 3B). The epitope-tagged /NEMO was detected mainly in fractions migrating at 500 kda in cells coexpressing IKKβ and /NEMO (Figure 3B). These results suggest that /NEMO alters the chromatographic behavior of IKKβ but not IKKα. Next we addressed the specificity of /NEMO to alter the chromatographic mobility of IKKβ. An unrelated elpitope-tagged protein, the camp-responsive element binding protein (CREB) which has no known role in regulating the IKK complex, was coexpressed with IKKβ and their chromatographic mobility was determined. Both the epitope-tagged IKKβ protein and IKKβ kinase activity were detected in column fractions 14

15 migrating at ~200 kda, indicating that CREB unlike /NEMO is unable to alter IKKβ mobility (Figure 3C). Taken together, these results suggest that /NEMO is a factor directly involved in changing the chromatographic position of the IKKs. Furthermore, IKKβ but not IKKα, serves as a primary target for /NEMO in the process of formation of the high molecular weight IKK complex. IKKβ recruitment by /NEMO is independent of its kinase activity To demonstrate whether /NEMO requires an active IKKβ kinase to result in its incorporation into the high molecular weight IKK complex, epitope-tagged IKKβ mutants were transfected into COS cells in either the presence or the absence of /NEMO. Either the constitutively active IKKβ mutant, IKKβ(SS/EE), which contains substitutions of serine residues 177 and 181 with glutamic acid residues or a non-inducible IKKβ kinase mutant, IKKβ (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 IKKβ protein when expressed alone, the epitope-tagged IKKβ(SS/EE) protein was detected in fractions corresponding to ~200 kda (Figure 4A). When the IKKβ (SS/EE) protein was coexpressed with the epitope-tagged /NEMO, the IKKβ(SS/EE) 15

16 protein and kinase activity was detected in the higher molecular weight IKK complex (Figure 4A). The chromatographic distribution of the kinase defective IKKβ (SS/AA) protein was then assayed. In the absence of /NEMO, the majority of epitope-tagged IKK(SS/AA) protein was present in lower molecular weight fractions migrating between kda (Figure 4B). In the presence of the epitope-tagged /NEMO, the IKKβ(SS/AA) protein was detected in the high molecular weight IKK complex (Figure 4B). No detectable kinase activity was detected in any of the fractions containing the kinase defective IKKβ(SS/AA) protein regardless of whether or not /NEMO was coexpressed (Figure 4B). The chromatographic position of /NEMO was unchanged in cells expressing IKKβ(SS/EE) and IKKβ(SS/AA) (Figure 4A and 4B). These results suggest that incorporation of IKKβ into high molecular weight IKK complex by /NEMO is not dependent on the kinase activity of IKKβ. The N-terminal domain of /NEMO is necessary for assembly of the IKK complex Studies were next performed to determine which domains in /NEMO are responsible for recruiting IKKβ into the high molecular 16

17 weight complex. Epitope-tagged /NEMO deletion mutants lacking either the C-terminal 100 amino acid residues (1-312) or the N-terminal 100 residues ( ) were transfected into COS cells along with both IKKα and IKKβ. Similar to the results obtained with wild-type /NEMO shown in Figure 2A, the mutant lacking the C-terminal portion of /NEMO was able to recruit the majority of the epitope-tagged IKKβ protein and kinase activity into the high molecular weight IKK complex (Figure 5A). However, this /NEMO mutant consistently failed to efficiently shift the majority of the epitope-tagged IKKα into the higher molecular complex (Figure 5A). The epitope-tagged /NEMO mutant lacking the N-terminal portion of this protein was unable to shift either IKKα or IKKβ into the high molecular weight IKK complex (Figure 5B). Finally, mutations that changed leucine residues 315, 322, and 329 in the leucine-zipper motif of /NEMO to methionine did not have major effects on the ability of /NEMO to shift the chromatographic distribution of IKKβ (Figure 5C). These results suggest that the N-terminal portion of /NEMO is critical for the chromatographic shift of the IKKs. 17

18 The N-terminus of /NEMO is critical for stimulating IKKβ kinase activity To demonstrate whether /NEMO is involved in activation of the IKKs, COS cells were transfected with either epitope-tagged IKKα or IKKβ cdnas in the presence of increasing amounts of /NEMO. IKK kinase activity was then assayed following immunoprecipitation of either the epitope-tagged IKKα or IKKβ proteins. In the absence of the transfected epitope-tagged /NEMO, there was little IKKβ activity detected (Figure 6A, upper panel, lane 2). However, IKKβ kinase activity was dramatically increased by transfecting increasing amounts of /NEMO (Figure 6A, lanes 3-6). Immunoprecipitation of the epitope-tagged /NEMO from these extracts followed by in vitro kinase assays of the associated IKKβ activity gave similar results (data not shown). In contrast, /NEMO resulted in very little stimulation of IKKα activity (Figure 6B). This result was consistently seen regardless of the amount of IKKα that was transfected (data not shown). To confirm the specificity of /NEMO induction of IKKβ kinase activity, epitope-tagged IKKβ mutants in either the activation loop, IKKβ(SS/AA) and IKKβ(SS/EE), or the kinase-defective mutant IKKβ(K/M) were expressed in either the presence or the absence of 18

19 /NEMO. Whereas /NEMO stimulated wild-type IKKβ activity, it failed to stimulate the kinase activity of any of the IKKβ mutants (Figure 6C). These results would be consistent with a potential role of /NEMO in stimulating the phosphorylation of serine residues in the IKKβ activation loop with resultant increases in kinase activity. Domains in /NEMO that regulate the kinase activity and phosphorylation of IKKβ Next we determined which domains in /NEMO were important for stimulation of the IKKβ kinase activity. COS cells were transfected with epitope-tagged IKKβ alone or with either the wild-type or mutated /NEMO cdnas. Mutations in either the C-terminus, the N-terminus or the leucine-zipper of /NEMO reduced its ability to stimulate IKKβ kinase activity (Figure 7A, upper panel). The amount of the epitope-tagged IKKβ and /NEMO proteins were comparable in these transfections (Figure 7A, bottom two panels). Finally we addressed whether the ability of /NEMO to stimulate IKKβ kinase activity correlated with the phosphorylation of IKKβ. Epitopetagged wild-type or mutant /NEMO cdnas were transfected into COS cells in the presence of IKKβ. Cells were then incubated with 32 P- 19

20 orthophosphate and the epitope-tagged IKKβ proteins were immunoprecipitated. Analysis of these samples following SDS-PAGE and autoradiography demonstrated that wild-type /NEMO and both the C- terminal deletion and leucine-zipper mutants stimulated IKKβ phosphorylation. However, the N-terminal deletion of /NEMO prevented IKKβ phosphorylation (Figure 7B). The increased phosphorylation of IKKβ in the presence of /NEMO appears to require an intact IKKβ activation loop as phosphorylation of the mutants IKKβ(SS/EE) and IKKβ(SS/AA) was not induced by /NEMO (data not shown). Taken together, these results suggest that /NEMO is involved in activation of IKKβ kinase activity possibly through stimulation of IKKβ phosphorylation. Discussion /NEMO is an essential factor required for NF-κB activation (28,41,42,48-50). The ability of /NEMO to stimulate the NF-κB pathway is likely due to its effects on the assembly of the IKK complex (29,30) and the recruitment of upstream kinases that increase IKK activity 20

21 (45). However, the mechanisms involved in the assembly of the IKK complex by /NEMO has not been elucidated. The results presented here using transient expression assays and column chromatography indicate that the expression of /NEMO leads to the recruitment of the IKKs into a high molecular weight complex. Moreover, /NEMO can stimulate IKKβ phosphorylation of the protein. The ability to assay /NEMO recruitment of IKKβ 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 /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 /NEMO is critical for its association with IKKβ (29,30,43). Neither the carboxyl-terminus nor the leucine-zipper domains of /NEMO were required for recruitment of IKKβ into the IKK complex. However, both domains are important for the ability of /NEMO to maximally stimulate IKKβ kinase activity. This suggests that the C-terminus and possibly the leucine zipper of /NEMO are involved in additional functions such as the recruitment of upstream kinases including RIP into the IKK complex (45). These results are 21

22 consistent with a role for different domains in /NEMO in regulating distinct functions that lead to activation of the NF-κB pathway. We also determined whether IKKβ kinase activity was necessary for its recruitment into the high molecular weight IKK complex. We found that both constitutively active and kinase-defective IKKβ proteins were recruited into the IKK complex by /NEMO. These results suggest that activation of IKKβ kinase activity is not a requirement to facilitate IKKβ association with the IKK complex. In the absence of /NEMO, both IKKα and IKKβ migrate in lower molecular weight complexes. Although /NEMO does not appear to efficiently recruit IKKα alone into the high molecular weight IKK complex, it is able to recruit IKKα through its interactions with IKKβ. A C-terminal deletion mutant of /NEMO consistently exhibited defects in recruiting IKKα into the high molecular weight complex. Previous data suggest that IKKα can associate with /NEMO in IKKβ deficient fibroblasts (44) and in cotransfection assays performed in 293 cells (46). These results raise the possibility that the C- terminus and potentially other domains of /NEMO can facilitate IKKα binding via either a direct association or by indirect association with upstream kinases that interact with /NEMO. Thus, /NEMO leads to the recruitment of both IKKα and IKKβ into the IKK complex. 22

23 The role of /NEMO in regulating IKK kinase activity and stimulating the NF-κB pathway has previously been studied using transient expression assays (29,30,42,43,55). Antisense reduces IKK activation in response to upstream activators while the expression of C- terminal /NEMO mutants function as dominant negative inhibitors of IKKβ activity (49). These results are consistent with a positive role of /NEMO on activating the NF-κB pathway. However, overexpression of wild-type /NEMO in transient expression assays can in some cases reduce IKK activation likely by a squelching mechanism (56). We were able to demonstrate that /NEMO markedly stimulates IKKβ kinase activity. This stimulation is dependent on intact serine residues in the IKKβ activation loop. The N-terminus of /NEMO is crucial for this process although the C-terminus and leucine zipper region of /NEMO are also involved. In contrast, /NEMO does not significantly stimulate IKKα kinase activity. The ability of /NEMO to stimulate IKKβ activity is likely in part mediated by the ability of /NEMO to increase phosphorylation of serine residues in the IKKβ activation loop. The amino-terminus of /NEMO is important for both increasing IKKβ phosphorylation and stimulating IKKβ kinase activity suggesting that /NEMO association with IKKβ is critical for both of 23

24 these events. Whether this process is due to /NEMO-mediated stimulation of IKKβ autophosphorylation or the ability of /NEMO to recruit other kinases remains to be determined. Thus, /NEMO appears to be important for regulating both IKKβ phosphorylation and kinase activity. A model to address /NEMO function would suggest that its interaction with IKKα/IKKβ is critical for formation of the high molecular weight IKK complex that is responsive to various activators of the NF-κB pathway (28-30). The N-terminus of /NEMO is critical for IKKβ association with the high molecular weight complex while the C-terminus of /NEMO likely mediates interactions with upstream kinases such as RIP (45,57) or MEKK1 (XHL and RBG, unpublished observations) or potentially regulates IKKα activation of IKKβ kinase activity (39,58). Thus, /NEMO likely serves as a unique scaffold protein to facilitate the assembly and activity of the IKK complex. Further studies on the regulation of /NEMO will be important in defining its ability to regulate NF-κB and potentially other signal transduction pathways. 24

25 Acknowledgements We thank Sharon Johnson and Alex Herrera for preparation of the manuscript and the figures, respectively. Also we thank Dean Ballard for providing us with /NEMO deletion mutants. This work was supported by NIH grant (CA74128) and a grant from the Welch Foundation. 25

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33 Figure Legends Figure 1. Chromatographic distribution of IKK in untreated and TNFα-treated cells. COS cells were either untreated or treated with TNFα (20 ng/ml) for 10 min. Cytoplasmic (S100) extracts were prepared and subjected to chromatography on a Superdex-200 column (Pharmacia). The fractions derived from the indicated cells (left of figures) were analyzed by Western blotting using antibodies directed against either IKKα (Santa Cruz, sc-7606), IKKβ (Santa Cruz, sc-7607) or (Santa Cruz, sc-8330). In vitro kinase activity was analyzed utilizing a GST/(aa1-54) substrate following immunoprecipitation of the column fractions with either IKKα (sc-7606) or IKKβ (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/ substrate are indicated at the right of these figures. Figure 2. Effect of /NEMO on the chromatographic distribution of cotransfected IKKα and IKKβ. COS cells were transfected with CMV expression vectors containing cdnas encoding (A) IKKα and IKKβ (B) IKKα, IKKβ and /NEMO or (C) /NEMO only. Fractions 33

34 isolated from a Superdex-200 column were subjected to either Western blotting or immunoprecipitation and in vitro kinase assays utilizing the GST/(aa1-54) substrate. Monoclonal antibodies used against the epitope-tagged proteins included anti-ha (Roche, 12CA5) for IKKα; anti- Flag (Sigma, M2) for IKKβ; and anti-myc (9E10) for /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/ are indicated at the right of the figure. Figure 3. Effect of /NEMO on the chromatographic distribution of transfected IKKα or IKKβ. COS cells were transfected with (A) IKKα alone (top three panels) or IKKα and /NEMO (bottom three panels); (B) IKKβ alone (top three panels) or IKKβ and /NEMO (bottom three panels) or (C) IKKβ and CREB. Western blotting and in vitro kinase assays were performed on fractions isolated from the Superdex- 200 column were performed using the same antibodies as described in Figure 2. In part 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 position of the IKKs and GST/ are indicated at the right of the figure. 34

35 Figure 4. Effect of /NEMO on the chromatographic distribution of IKKβ mutants. COS cells were transfected with (A) IKKβ(SS/EE) either alone (top three panels) or IKKβ(SS/EE) and /NEMO (bottom three panels) or (B) IKKβ(SS/AA) alone (top three panels) or IKKβ(SS/AA) and /NEMO (bottom three panels). Western blotting and in vitro kinase assays were performed on the column fractions using the same antibodies as described in Figure 2. Molecular weight markers and column fraction numbers are indicated at the top and bottom of the figure, respectively. The positions of IKKβ and the GST/ substrate are indicated at the right of the figure. Figure 5. Effect of /NEMO mutants on the chromatographic distribution of IKKα and IKKβ. COS cells were transfected with either (A) IKKα, IKKβ and a C-terminal deletion mutant of /NEMO (aa 1-312) (B) IKKα, IKKβ and a N-terminal deletion mutant of /NEMO (aa ) or (C) IKKβ and a /NEMO mutant that contains substitutions of leucine residues with methionine at 315, 322, and 329 positions in the leucine-zipper motif. Molecular weight markers and column fraction numbers are indicated at the top and bottom of the figure, 35

36 respectively. The position of the IKKs and the GST/ substrate are indicated at the right of the figure. Figure 6. The N-terminal portion of /NEMO is critical for stimulation of IKKβ kinase activity and phosphorylation. (A) COS cells (2 x 10 5 ) were transfected with either a CMV expression vector alone (lane 1), 0.05 µg of IKKβ alone (lane 2), 0.05 µg of IKKβ and increasing amounts of the CMV vector containing /NEMO of 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 IKKβ 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 IKKβ and the Myc monoclonal antibody (Roche, 9E10) to assay /NEMO. (B) COS cells were transfected with an IKKα expression vector (0.5 µg) rather than an IKKβ vector as in A. The HA monoclonal antibody (Roche, 12CA5) was used to detect IKKα. (C) COS cells were transfected with vector alone (lane 1), IKKβ (0.05 µg) or the mutants indicated in the presence or absence of /NEMO (lanes 2-9). In vitro kinase activity and protein expression were determined. 36

37 Figure 7. Domains in /NEMO that regulate IKKβ kinase activity and phosphorylation. (A) COS cells were transfected with vector alone (lane 1), IKKβ (0.05 µg) alone (Lane 2), or IKKβ (0.05 µg) with either 0.1 µg of the wild-type or mutant /NEMO cdnas as indicated (lanes 3-7). In vitro kinase assays were performed to determine IKKβ kinase activity (top panel) and Western blotting was performed to detect the expression of IKKβ and /NEMO (bottom two panels). Phosphorimager analysis of seven sets of transfections were quantitated: lane 1, 3.04 ; lane 2, 24.74; lane 3, 100; lane 4, 36.01; lane 5, 38.79; lane 6, and lane 7, (B) COS cells were transfected for 36 hours with vector alone (lane 1), 0.5 µg of IKKβ alone (lane 2) or 0.5 µg of IKKβ and the indicated /NEMO mutants (0.5 µg) (lanes 3-7). The cells were either incubated with 32 P-orthophosphate for 3 hour at 37 o 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). 37

38 Figure untr IKKα TNF IKKα untr TNF IKKβ IKKβ Western untr TNF untr TNF untr TNF IKKα Ab KA IKKβ Ab KA

39 Figure 2 A. C IKKα IKKβ Western IKKα/β IKKα Ab KA IKKβ Ab KA B IKKα IKKβ Western IKKα/β/γ IKKα Ab KA IKKβ Ab KA Western

40 Figure 3 A. B IKKβ IKKβ Western IKKβ Ab KA IKKβ/γ IKKβ Western IKKβ Ab KA IKKα IKKα/γ IKKα IKKα Western IKKα Ab KA Western IKKα Ab KA C. IKKβ/CREB IKKβ CREB Western IKKβ Ab KA

41 Figure 4 A IKKβ(SS/EE) IKKβ(SS/EE)/γ IKKβ IKKβ Western IKKβ Ab KA Western IKKβ Ab KA B IKKβ(SS/AA) IKKβ Western IKKβ Ab KA IKKβ(SS/AA)/γ IKKβ Western IKKβ Ab KA

42 Figure 5 A IKKα IKKβ Western IKKα/β/ γ(1-312) IKKα Ab KA IKKβ Ab KA B IKKα IKKβ Western IKKα/β/ γ( ) IKKα Ab KA IKKβ Ab KA C IKKβ/ γ(lz) IKKβ Western IKKβ Ab KA

43 Figure 6 A. B. Vector IKKβ Vector IKKα IKKβ Ab KA IKKα Ab KA IKKβ IKKα Western Western C. IKKβ: : WT AA EE K/M IKKβ Ab KA IKKβ Western

44 32 P-IKKβ IKKβ Western Figure 7 A. Vector IKKβ IKKβ/γ IKKβ/γ (1-312) IKKβ/γ (1-270) IKKβ/γ ( ) IKKβ/γ (LZ) GTS- IKKβ Ab KA B. vector IKKβ IKKβ/γ IKKβ/γ (1-312) IKKβ/γ (1-270) IKKβ/γ ( ) IKKβ/γ (LZ) IKKβ Western