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Molecular and Cellular Biology, May 2000, p. 3655-3666, Vol. 20, No. 10
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Ikappa B Kinase alpha  (IKKalpha ) Regulation of IKKbeta Kinase Activity

Yumi Yamamoto, Min-Jean Yin, and Richard B. Gaynor*

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

Received 4 October 1999/Returned for modification 12 November 1999/Accepted 23 February 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Two related kinases, Ikappa B kinase alpha  (IKKalpha ) and IKKbeta , phosphorylate the Ikappa B proteins, leading to their degradation and the subsequent activation of gene expression by NF-kappa B. IKKbeta has a much higher level of kinase activity for the Ikappa B proteins than does IKKalpha and is more critical than IKKalpha in modulating tumor necrosis factor alpha activation of the NF-kappa B pathway. These results indicate an important role for IKKbeta in activating the NF-kappa B pathway but leave open the question of the role of IKKalpha in regulating this pathway. In the current study, we demonstrate that IKKalpha directly phosphorylates IKKbeta . Moreover, IKKalpha either directly or indirectly enhances IKKbeta kinase activity for Ikappa Balpha . Finally, transfection studies to analyze NF-kappa B-directed gene expression suggest that IKKalpha is upstream of IKKbeta in activating the NF-kappa B pathway. These results indicate that IKKalpha , in addition to its previously described ability to phosphorylate Ikappa Balpha , can increase the ability of IKKbeta to phosphorylate Ikappa Balpha .


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The NF-kappa B proteins are a family of transcription factors that activate a variety of cellular genes involved in control of the inflammatory response and in regulating cellular growth (2, 3). 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 (2-4). A variety of extracellular stimuli, including tumor necrosis factor alpha (TNF-alpha ), lipopolysaccharide, and interleukin-1 (IL-1), lead to the activation of signal transduction pathways that result in the phosphorylation of two amino-terminal serine residues in the Ikappa B proteins (1, 5-7, 12, 32, 33). The Ikappa B proteins are then ubiquitinated on amino-terminal lysine residues via interaction with beta -TrCP (29, 31, 34, 37). After the formation of the ubiquitin-ligase complex, Ikappa B is degraded by the 26S proteasome (8, 9).

Two related kinases that phosphorylate amino-terminal serine residues 32 and 36 in Ikappa Balpha and 19 and 23 in Ikappa Bbeta have been described (13, 22, 27, 35, 40). These Ikappa B kinases are components of a 700-kDa kinase complex whose activity is markedly increased by treatment of cells with activators of the NF-kappa B pathway, such as TNF-alpha and IL-1 (8, 9, 15, 28). Other components of this complex include NEMO or Ikappa B kinase gamma  (IKKgamma ), which is required for in vivo activation of IKK kinase activity (23, 28, 36), and IKAP, which may function as a scaffold protein (10). IKKalpha and IKKbeta have a high degree of sequence homology and similar structural domains, including a conserved kinase domain in addition to leucine zipper and helix-loop-helix domains (13, 22, 27, 35, 40). The leucine zipper domain of these kinases facilitates their ability to homodimerize and heterodimerize (13, 22, 27, 35, 40). Although these kinases have a number of similarities, IKKbeta has a 20- to 50-fold-higher level of kinase activity for Ikappa B than does IKKalpha (16, 22, 24, 38, 39, 41).

TNF-alpha activation of the NF-kappa B pathway is mediated by multiple adapter proteins which lead to activation of NF-kappa B-inducing kinase (NIK) (21), which is capable of directly phosphorylating IKKalpha in its activation loop at serine residue 176 (20). However, other upstream kinases have also been demonstrated to activate the NF-kappa B pathway. For example, mitogen-activated protein/extracellular signal-regulated kinase 1 (MEKK1) can activate both IKKalpha and IKKbeta kinase activity (15, 16, 24, 38, 39). Other upstream kinases, such as TAK1, MEKK2, and MEKK3, can also directly or indirectly lead to activation of the Ikappa B kinases (26, 42). These results suggest that multiple signal transduction pathways can likely modulate IKK function.

Recent data suggest that TNF-alpha - and IL-1-mediated increases in the phosphorylation of IKKbeta and potentially IKKalpha may be important in the regulation of their kinase activity (11). Both IKKalpha and IKKbeta contain a canonical MAP kinase kinase activation loop motif with the sequence Ser-X-X-X-Ser that has similarities to domains found in other MAP kinases (13, 22, 27, 35, 40). Phosphorylation of two closely spaced serine residues in this domain, at positions 176 and 180 in IKKalpha and positions 177 and 181 in IKKbeta , has been shown to be important for IKK kinase activity (22). For example, mutation of these serine residues to alanine in both IKKalpha and IKKbeta can inactivate their ability to phosphorylate Ikappa B. Moreover, replacement of these serine residues with glutamates results in the generation of proteins that have constitutively active IKK kinase activity (22). However, a recent study indicates that the serine residues in the activation loop of IKKbeta but not IKKalpha are critical for modulating IKK kinase activity (11). The reason for the discrepancy between these studies remains unclear. NIK (20) and MEKK1 (16) can phosphorylate serine residues in the activation loop of the IKK proteins, although it is possible that autophosphorylation of these residues by the IKK proteins themselves may also provide a mechanism for activating IKK kinase activity.

Recent gene disruption studies of the murine IKK genes indicate their importance in mammalian development (14, 18, 19, 30). For example, disruption of the murine IKKalpha genes results in animals that die shortly after they are born (14, 18, 30). These mice have a number of developmental abnormalities, including those of the axial skeleton, limbs, and skin. In two studies, mice lacking IKKalpha are not impaired for activation of the NF-kappa B pathway or Ikappa B degradation following treatment with inflammatory cytokines (14, 30). However, another study indicates that mice lacking IKKalpha are somewhat defective in activating the NF-kappa B pathway (18). In contrast, mice lacking IKKbeta die as embryos due to extensive liver damage from uncontrolled apoptosis (19). Moreover, in these mice there are marked defects in activation of the NF-kappa B pathway by proinflammatory cytokines such as TNF-alpha (19). These results indicate that IKKbeta appears to be more critical than IKKalpha in activating the NF-kappa B pathway.

In the current study, we address the role of IKKalpha in activating the NF-kappa B pathway. We demonstrate that IKKalpha directly phosphorylates IKKbeta . Furthermore, we demonstrate that IKKalpha either directly or indirectly increases the ability of IKKbeta to phosphorylate Ikappa Balpha . Finally, transfection studies suggest that IKKalpha is upstream of IKKbeta in mediating activation of NF-kappa B-directed gene expression. These results suggest that IKKalpha may modulate IKKbeta kinase activity to regulate the NF-kappa B pathway.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

DNA constructs. The cDNAs for wild-type IKKalpha and the IKKalpha mutants K44M (K/M), S176A/S180A (SS/AA), and K44M Delta HLH, in which amino acids 560 to 744 in the carboxy terminus of IKKalpha have been deleted, contain amino-terminal influenza virus hemagglutinin sequences and were cloned downstream of the cytomegalovirus (CMV) promoter in pCMV5. The cDNAs for wild-type IKKbeta and the IKKbeta mutants K44M (K/M) and S177A/S181A (SS/AA) contain amino-terminal Flag sequences and were cloned downstream of the CMV promoter in pCMV5 (22). The cDNAs for wild-type NIK and the dominant negative NIK mutant K429A/K430A (KK/AA) were cloned downstream of the CMV promoter in pCMV5 and contained an amino-terminal Myc tag (38). The cDNAs for wild-type MEKK1 and dominant negative MEKK1 mutant D1369A (D/A) contain a carboxy-terminal influenza virus hemagglutinin epitope (38) and were also cloned downstream of the CMV promoter in pCMV5.

Wild-type and mutant IKKalpha and IKKbeta cDNAs tagged with six histidines or with influenza virus hemagglutinin were each cloned into the baculovirus expression vector pAcHLT, and recombinant baculoviruses were generated by cotransfection with the Baculo Gold DNA and transfer vectors (PharMingen). The recombinant baculoviruses were used to infect Sf9 cells at a multiplicity of infection of 5 to express the different IKK proteins. The baculovirus-produced IKK proteins were purified by nickel-agarose chromatography and then immunoprecipitated with the 12CA5 monoclonal antibody. These recombinant IKK proteins were assayed in in vitro kinase assays as described below.

Transfections. COS cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, and transfections were performed with Fugene 6 (Boehringer). COS cells were transfected with DNA concentrations ranging from 0.10 to 1.0 µg of either wild-type or kinase-defective Flag epitope-tagged IKKbeta constructs or wild-type or kinase-defective influenza virus hemagglutinin-tagged IKKalpha cDNAs (38). The wild-type or dominant negative NIK and MEKK1 mutant constructs have been described previously (38). For assays of NF-kappa B-directed gene expression, a human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-luciferase construct was transfected into COS cells in the presence of the indicated wild-type or mutant IKKalpha and IKKbeta constructs, and luciferase activity was assayed 30 h posttransfection (38). A CMV-beta -galactosidase plasmid was also incorporated into each transfection.

[32P]orthophosphate labeling of IKK proteins. COS cells were maintained in DMEM with 10% fetal bovine serum and transfected with either the indicated IKKalpha or IKKbeta cDNAs or either wild-type or mutant NIK or MEKK1 constructs. Before labeling the cells, the culture medium was changed to serum-free and either phosphate-free or methionine-free DMEM. Either [32P]orthophosphate (50 µCi/ml) or [35S]methionine (50 µCi/ml) was then added to the cells and incubated for 3 h. TNF-alpha (20 ng/µl) was added for 5 to 7 min before harvesting the cells. The cells were washed three times with cold phosphate-buffered saline, and the cell pellets were lysed on ice for 15 min in PD buffer (500 mM NaCl, 50 mM Tris-HCl [pH 8.0], 0.5% NP-40, 1 mM sodium orthovanadate, 1 mM NaF, 0.5 mM beta -glycerophosphate, and protease inhibitors).

Immunoprecipitation and kinase assays. Cell lysates from either [32P]orthophosphate- and [35S]methionine-labeled cells or nonlabeled cells were incubated with 50 µl of 12CA5 supernatant or 500 ng of anti-Flag M2 antibody for 2 h on ice. To assay endogenous IKK labeling with either [32P]orthophosphate or [35S]methionine, 50 µg of the cellular lysate was immunoprecipitated with rabbit polyclonal antibody directed against IKKalpha (Santa Cruz). Then 20 µl of protein A-agarose was added to each of the immunoprecipitates and incubated for 1 h at 4°C. The immunoprecipitates were washed twice with 10 volumes of 50 mM Tris-HCl (pH 8.0)-100 mM NaCl-protease inhibitor, and protein loading buffer was added prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography.

For kinase assays, immunoprecipitates from cellular lysates (50 µg) were incubated in kinase reaction buffer containing 10 µCi of [gamma -32P]ATP, 1 mM ATP, 5 mM MgCl2, 1 mM dithiothreitol, 100 mM NaCl, and 50 mM Tris-HCl (pH 8.0) at 30°C for 15 min (38). The substrates in these kinase reactions were either glutathione-S-transferase (GST)-Ikappa Balpha (2 µg), wild type (amino acids 1 to 54) or mutant (S32/S36right-arrowA32/A36), or baculovirus-produced polyhistidine- and Flag-tagged IKKalpha or IKKbeta proteins (500 ng). These proteins were produced by baculovirus expression, purified by nickel-agarose chromatography, and then subjected to chromatography on a Q-Sepharose column. Proteins were quantitated and analyzed following SDS-PAGE, silver staining, and Western blot analysis with anti-Flag M2 monoclonal antibody (38).

Chromatography of IKK proteins. COS cells (108) were cotransfected with epitope-tagged expression vectors containing either IKKalpha K/M and IKKbeta or IKKalpha and IKKbeta . Cells were harvested by centrifugation for 10 min at 2,000 rpm (Beckman bench-top centrifuge, CH3.7 rotor). Pelleted cells were washed twice in cold phosphate-buffered saline and resuspended in 5 volumes of buffer A (10 mM HEPES [pH 7.9], 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol) supplemented with phosphatase inhibitors (50 mm NaF, 50 mm glycerophosphate, 0.125 µM okadaic acid, and 1 mM sodium orthovanadate) and proteinase inhibitors (Roche Molecular Biochemicals). After incubation for 15 min on ice, the cells were lysed with 40 strokes of a Kontes all-glass Dounce homogenizer (B-type pestle). The nuclei were pelleted by centrifugation at 2,000 rpm. The supernatant was mixed with 0.11 volume of buffer B (0.3 M HEPES [pH 7.9], 0.03 M MgCl2) and then centrifuged for 60 min at 100,000 × g. The supernatant was dialyzed for 5 to 8 h against 20 volumes of buffer D (20 mM HEPES [pH 7.9], 0.1 M KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20% glycerol, 0.2 mM EDTA).

Equal amounts of proteins (2.5 mg) were fractionated on a Superdex 200 column (Amersham Pharmacia Biotech). Protein markers (Sigma) used for the column include bovine thyroglobulin (669 kDa), horse spleen apoferritin (443 kDa), beta -amylase (200 kDA), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.5 kDa). The column fractions were immunoprecipitated with either the M2 or 12CA5 monoclonal antibody, and in vitro kinase assays were performed as indicated. Western blot analysis of the column fractions with these monoclonal antibodies was also performed.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

TNF-alpha induces endogenous IKK phosphorylation. Stimulation of IKKbeta kinase activity correlates with increases in its phosphorylation (11). To further analyze the role of phosphorylation in IKK function, we first tested the ability of TNF-alpha treatment or transfection of MEKK1 and NIK constructs to induce phosphorylation of endogenous IKK. COS cells were labeled with either [32P]orthophosphate (Fig. 1A, top panel) or [35S]methionine (Fig. 1A, middle panel) for 3 h prior to harvest. The IKK proteins were then immunoprecipitated with a polyclonal antibody directed against IKKalpha that immunoprecipitates the IKKalpha -IKKbeta heterodimer (38).


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  		FIG. 1.   Activators of the NF-kappa B pathway increase IKK phosphorylation. (A) COS cells were either untreated (lane 1), treated with TNF-alpha (20 ng/ml) for 5 to 7 min prior to harvest (lane 2), or transfected with 2 µg of expression vectors containing wild-type NIK or MEKK1 (lanes 3 and 4) or dominant negative mutants of NIK and MEKK1 (lanes 5 and 6). Cells were labeled with either [32P]orthophosphate (top panel) or [35S]methionine (middle panel) for 3 h prior to harvesting the cells. Immunoprecipitation was performed with IKKalpha polyclonal antibody (Santa Cruz) followed by SDS-PAGE and autoradiography. Extracts were also analyzed for IKK expression in Western blot analysis with IKKalpha antibody (lower panel). (B) COS cells were not transfected (lane 1) or transfected with 1 µg of the influenza virus hemagglutinin-tagged IKKalpha (lanes 2 and 3) or 0.2 µg of the Flag-tagged IKKbeta (lanes 4 and 5) cDNAs in either the absence (lanes 2 and 4) or presence (lanes 3 and 5) of TNF-alpha . Cells were labeled with [32P]orthophosphate for 3 h prior to harvest, and TNF-alpha treatment was performed for 7 min prior to harvest. Immunoprecipitation was performed with either 12CA5 (lanes 1, 2, and 3, top panel) or the M2 (Flag) (lanes 4 and 5, top panel) monoclonal antibody using 50 µg of the cell lysate, followed by SDS-PAGE and autoradiography. Western blot analysis of the transfected IKK cDNAs was performed with 12CA5 (lanes 2 and 3, lower panel) or the M2 (lanes 4 and 5, lower panel) monoclonal antibody.

TNF-alpha treatment of COS cells stimulated the phosphorylation of the IKK proteins (Fig. 1A, lanes 1 and 2, top panel). Transfection of either of two kinases, NIK and MEKK1, that have been demonstrated to increase the IKK kinase activity (15, 16, 20, 24, 25) also increased IKK phosphorylation (Fig. 1A, lanes 3 and 4, top panel). In contrast, transfection of dominant negative mutants of MEKK1 and NIK did not significantly alter endogenous IKK phosphorylation (Fig. 1A, lanes 5 and 6, top panel). Similar quantities of [35S]methionine-labeled extracts prepared from TNF-alpha -treated or wild-type or mutant NIK- or MEKK1-transfected COS cells did not demonstrate differences in the levels of the [35S]methionine-labeled IKK proteins (Fig. 1A, lanes 1 to 6, middle panel). Western blot analysis confirmed the presence of similar amounts of IKKalpha in each of these extracts (Fig. 1A, lanes 1 to 6, lower panel).

We also determined whether TNF-alpha increased the phosphorylation of influenza virus hemagglutinin-tagged IKKalpha or Flag-tagged IKKbeta cDNAs following transfection of each of these constructs into COS cells. The 32P-labeled IKKalpha and IKKbeta proteins were immunoprecipitated with the 12CA5 and Flag monoclonal antibodies, respectively. The phosphorylation of both IKKalpha and IKKbeta was increased following TNF-alpha treatment (Fig. 1B, lanes 2 to 5, top panel). There were similar amounts of the IKKalpha proteins in both untreated and TNF-alpha -treated extracts (Fig. 1B, lanes 2 to 5, lower panel). These results indicate that activators of the NF-kappa B pathway such as TNF-alpha increase the phosphorylation of both the IKKalpha and IKKbeta proteins.

TNF-alpha induces phosphorylation of IKKalpha and IKKbeta . To address the mechanism by which activators of the NF-kappa B pathway increase the phosphorylation of IKKalpha and IKKbeta , epitope-tagged cDNAs encoding each of these proteins were transfected into COS cells (Fig. 2). [32P]orthophosphate labeling of the transfected COS cells was performed in either the presence or absence of TNF-alpha . Dominant negative mutants of either IKKbeta , IKKalpha , MEKK1, or NIK were included in these transfection assays as indicated.


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  		FIG. 2.   IKKbeta phosphorylation is inhibited by dominant negative IKKalpha mutants. (A and C) COS cells were transfected with HA-tagged IKKalpha or Flag-tagged IKKbeta cDNAs alone (lanes 1 and 2) or in the presence of similar amounts of IKKalpha , IKKbeta , NIK, or MEKK1 dominant negative mutants as indicated (lanes 3 to 6). The transfected cells were labeled with [32P]orthophosphate (top panel) or [35S]methionine (lower panel) for 3 h and either untreated (lane 1) or treated with TNF-alpha (20 ng/ml) for 5 to 7 min (lanes 2 to 6). Cell lysates were prepared, and 50 µg of this lysate was incubated with the 12CA5 antibody to immunoprecipitate the IKKalpha protein or with the Flag antibody M2 to immunoprecipitate the IKKbeta protein. The immunoprecipitates were subjected to SDS-PAGE, and autoradiography was performed. (B and D) COS cells were transfected with the kinase-defective IKKalpha or IKKbeta mutant SS/AA (lanes 1 and 2) or K/M (lanes 3 and 4). The cells were labeled with either [32P]orthophosphate (top panel) or [35S]methionine (lower panel) for 3 h prior to harvest in the absence (lanes 1 and 3) or presence of TNF-alpha for 5 to 7 min (lanes 2 and 4). The cell lysates were immunoprecipitated and subjected to SDS-PAGE.

TNF-alpha treatment of COS cells strongly induced the phosphorylation of IKKalpha (Fig. 2A, lanes 1 and 2). TNF-alpha induction of IKKalpha phosphorylation was not inhibited by cotransfection of either of two IKKbeta dominant negative mutants (Fig. 2A, lanes 3 and 4) or a dominant negative MEKK1 mutant (Fig. 2A, lane 5). In contrast, TNF-alpha -induced IKKalpha phosphorylation was blocked by a dominant negative NIK mutant (Fig. 2B, lane 6). TNF-alpha treatment did not increase the phosphorylation of an IKKalpha mutant in the activation loop motif (Fig. 2B, lanes 1 and 2) or a catalytically inactive IKKalpha mutant (Fig. 2B, lanes 3 and 4).

COS cells were next transfected with an epitope-tagged IKKbeta cDNA in either the presence or absence of TNF-alpha and labeled with either [32P]orthophosphate or [35S]methionine. TNF-alpha induced the phosphorylation of IKKbeta (Fig. 2C, lanes 1 and 2). Cotransfection of dominant negative IKKalpha mutants decreased TNF-alpha -induced phosphorylation of IKKbeta (Fig. 2C, lanes 3 and 4). A dominant negative NIK mutant also decreased IKKbeta phosphorylation, while a dominant negative MEKK1 mutant did not decrease and in fact slightly increased IKKbeta phosphorylation (Fig. 2C, lanes 5 and 6). There was no significant change in the level of [35S]methionine-labeled IKKbeta proteins (Fig. 2C, lower panel). TNF-alpha treatment did not induce phosphorylation of an IKKbeta mutant in the two serine residues in its activation loop (Fig. 2D, lanes 1 and 2) or of a catalytically inactive IKKbeta mutant (Fig. 2D, lanes 3 and 4). These results are consistent with a role for IKKalpha in potentially modulating the phosphorylation state of IKKbeta .

IKKalpha induces phosphorylation of IKKbeta . To determine whether IKKalpha may potentially be involved in either directly or indirectly stimulating the phosphorylation of IKKbeta , we assayed the ability of IKKalpha to modulate the phosphorylation of IKKbeta . COS cells were transfected with an epitope-tagged IKKbeta cDNA either alone or in the presence of wild-type IKKalpha , a constitutively active IKKalpha construct, or two dominant negative IKKalpha mutants. The COS cells were labeled with either [32P]orthophosphate or [35S]methionine, and the Flag epitope-tagged IKKbeta protein was immunoprecipitated with the M2 monoclonal antibody.

Both wild-type and constitutively active IKKalpha constructs increased the phosphorylation of IKKbeta (Fig. 3A, lanes 1 to 3). In contrast, there was little or no increase in IKKbeta phosphorylation with either of two IKKalpha mutants, IKKalpha SS/AA or IKKalpha K/M (Fig. 3A, lanes 4 and 5). In vivo labeling of the IKKbeta proteins with [35S]methionine demonstrated that IKKalpha expression did not alter the level of the [35S]methionine-labeled IKKbeta proteins (Fig. 3A, lower panel). Similar results from three independent experiments indicate that IKKalpha can either directly or indirectly modulate the level of IKKbeta phosphorylation.


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  		FIG. 3.   IKKalpha increases IKKbeta phosphorylation. (A) COS cells were transfected with a Flag-tagged wild-type (WT) IKKbeta cDNA construct (0.5 µg) alone (lane 1) or in the presence of 0.5 µg of influenza virus hemagglutinin-tagged wild-type IKKalpha (lane 2), a constitutively active IKKalpha construct (lane 3), or the mutant IKKalpha construct SS/AA or K/M (lanes 4 and 5), as indicated above the top panel. (B) COS cells were transfected with an influenza virus hemagglutinin-tagged wild-type (WT) IKKalpha cDNA construct (0.5 µg) alone (lane 1) or in the presence of 0.5 µg of Flag-tagged wild-type IKKbeta (lane 2), a constitutively active IKKalpha construct (lane 3), or the mutant IKKalpha construct SS/AA or K/M (lanes 4 and 5), as indicated above the top panel. In both panels A and B, the cells were labeled with either [32P]orthophosphate (top panel) or [35S]methionine (lower panel) for 3 h prior to cell harvest. The cell lysates (50 µg) were immunoprecipitated with the (A) anti-Flag M2 monoclonal antibody to immunoprecipitate the epitope-tagged IKKbeta proteins or (B) the 12CA5 monoclonal antibody to immunoprecipitate the epitope-tagged IKKalpha proteins. The immunoprecipitates were then subjected to SDS-PAGE, and autoradiography was performed.

To address whether IKKbeta could increase IKKalpha phosphorylation, COS cells were transfected with an influenza virus hemagglutinin-tagged IKKalpha construct either alone or in the presence of different IKKbeta constructs. COS cells were again labeled with either [32P]orthophosphate or [35S]methionine, and the influenza virus hemagglutinin-tagged IKKalpha protein was immunoprecipitated with the 12CA5 monoclonal antibody. Neither the wild-type nor the constitutively active IKKbeta constructs altered the amount of IKKalpha phosphorylation (Fig. 3B, lanes 1 to 3). The dominant negative IKKbeta mutants IKKbeta SS/AA and IKKbeta K/M also did not alter the phosphorylation of IKKalpha (Fig. 3B, lanes 4 and 5). In vivo labeling of the IKKalpha proteins with [35S]methionine demonstrated similar amounts of the IKKalpha proteins (Fig. 3B, lanes 1 to 5, lower panel). These results suggest that IKKbeta does not markedly alter IKKalpha phosphorylation.

IKKalpha increases IKKbeta phosphorylation in a high-molecular-weight IKK complex. It was important to address whether IKKalpha could stimulate IKKbeta kinase activity when these kinases were part of a high-molecular-weight IKK complex (8, 9, 15, 28). To address this point, COS cells were cotransfected with expression vectors containing wild-type IKKbeta and either wild-type IKKalpha or a catalytically defective IKKalpha K/M mutant. The IKKalpha constructs were tagged with the influenza virus hemagglutinin epitope, while IKKbeta was tagged with the Flag epitope. Cytoplasmic extracts were prepared at 30 h posttransfection and subjected to chromatography on a Superdex 200 column to isolate the high-molecular-weight IKK complex.

Fractions from the Superdex 200 column were immunoprecipitated with the anti-Flag M2 monoclonal antibody, and in vitro kinase assays were performed. IKKbeta phosphorylation was present at low levels in column fractions migrating between 400 and 600 kDa in the presence of the IKKalpha K/M protein (Fig. 4A, top panel). There was no detectable IKKalpha K/M autophosphorylation in these column fractions.


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  		FIG. 4.   IKKalpha phosphorylation of IKKbeta in the IKK complex. COS cells were transfected with expression vectors encoding (A) hemagglutinin-tagged IKKalpha K/M and Flag-tagged IKKbeta or (B) hemagglutinin-tagged wild-type IKKalpha and Flag-tagged IKKbeta . Cytoplasmic extracts were prepared at 30 h posttransfection and fractionated on a Superdex 200 column. Column fractions 7 to 14 were immunoprecipitated with the M2 monoclonal antibody, and in vitro kinase assays of these fractions were performed and analyzed by SDS-PAGE and autoradiography (top panel). The positions of the epitope-tagged IKKalpha and IKKbeta proteins transfected individually into COS cells are indicated in the last two lanes of panel B. Western blot analysis was performed with the 12CA5 monoclonal antibody to detect IKKalpha K/M and wild-type IKKalpha or with the M2 monoclonal antibody to detect IKKbeta (lower two panels in A and B). The column fractions and the molecular mass markers, which indicate the positions of the fractions eluted from the Superdex 200 column, are indicated at the bottoms and tops of the figures, respectively. (C) Column fraction 9 from the Superdex 200 column analyzed in panels A and B was immunoprecipitated with the 12CA5 antibody to isolate either IKKalpha K/M or wild-type IKKalpha followed by in vitro kinase assays, SDS-PAGE, and autoradiography.

In contrast, the column fractions containing both wild-type IKKalpha and IKKbeta showed markedly enhanced phosphorylation of both IKKbeta and IKKalpha (Fig. 4B, top panel). The positions of the phosphorylated wild-type IKKalpha and IKKbeta proteins which were transfected alone and immunoprecipitated followed by in vitro kinase assays are also shown (Fig. 4B, top panel). Western blot analysis indicated that there was similar expression of the IKKalpha and IKKbeta proteins in these Superdex 200 fractions (Fig. 4A and B, lower panels). Finally, we determined whether immunoprecipitation of either IKKalpha K/M or IKKalpha present in column fraction 9 with the 12CA5 monoclonal antibody also demonstrated differences in IKKbeta phosphorylation (Fig. 4C). This analysis demonstrated that the presence of wild-type IKKalpha but not IKKalpha K/M was associated with enhanced IKKbeta phosphorylation. Immunoprecipitation of these column fractions followed by Western blot analysis indicated that the epitope-tagged IKKalpha and IKKalpha K/M proteins both strongly associated with the epitope-tagged IKKbeta protein (data not shown). No IKKbeta phosphorylation was noted when the catalytically defective IKK mutants IKKalpha K/M and IKKbeta K/M were analyzed following cotransfection and Superdex 200 fractionation (data not shown). These results indicate that IKKalpha is associated with enhanced IKKbeta phosphorylation when these kinases are present as heterodimers in a high-molecular-weight IKK complex.

IKKalpha stimulates IKKbeta kinase activity. Next we investigated whether IKKalpha -mediated increases in IKKbeta phosphorylation correlate with its ability to stimulate IKKbeta kinase activity. First, an epitope-tagged IKKbeta cDNA was transfected into COS cells, the cells were either untreated or treated with TNF-alpha , and IKKbeta kinase activity was assayed. Next, dominant negative mutants of either IKKalpha , NIK, or MEKK1 were cotransfected with IKKbeta in the presence of TNF-alpha to determine their role in regulating IKKbeta kinase activity. Finally, we assayed the ability of wild-type and constitutively active IKKalpha proteins to stimulate IKKbeta kinase activity. The Flag-tagged IKKbeta protein in each of these transfections was immunoprecipitated with the M2 monoclonal antibody and assayed for its ability to phosphorylate the amino terminus of Ikappa Balpha (amino acids 1 to 54).

TNF-alpha treatment markedly increased IKKbeta kinase activity for the GST-Ikappa Balpha substrate (Fig. 5A, lanes 1 and 2). The TNF-alpha -mediated increase in IKKbeta kinase activity was blocked by two dominant negative IKKalpha mutants (Fig. 5A, lanes 3 and 4) and a dominant negative NIK mutant (Fig. 5A, lane 5) but not a dominant negative MEKK1 mutant (Fig. 5A, lane 6). Next we assayed the ability of IKKalpha to stimulate IKKbeta kinase activity. Transfection of wild-type IKKalpha markedly stimulated the ability of IKKbeta to phosphorylate GST-Ikappa Balpha (Fig. 5A, lanes 1 and 7). A constitutively active IKKalpha construct also markedly stimulated IKKbeta kinase activity for the GST-Ikappa Balpha substrate (Fig. 5A, lanes 1 and 8). IKKalpha mutants K/M and SS/AA did not stimulate IKKbeta kinase activity, and the immunoprecipitated IKKbeta did not phosphorylate a GST-Ikappa B construct mutant at serine residues 32 and 36 (data not shown). When the wild-type and the constitutively active IKKalpha constructs were transfected alone and immunoprecipitated with the 12CA5 antibody, they had very low kinase activity with the GST-Ikappa Balpha substrate (Fig. 5A, lanes 9 and 10). Western blot analysis demonstrated that there was little change in the level of the epitope-tagged IKKbeta proteins in either the presence or absence of IKKalpha (Fig. 5A, lower panel). These results suggested that IKKalpha can either directly or indirectly modulate IKKbeta kinase activity and that TNF-alpha induction of IKKbeta kinase activity may be mediated in part through effects on IKKalpha .


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  		FIG. 5.   IKKalpha stimulates IKKbeta kinase activity. (A) COS cells were transfected with a Flag-tagged wild-type (WT) IKKbeta cDNA construct (0.1 µg) (lanes 1 to 8) in the absence (lane 1) or presence of TNF-alpha (lanes 2 to 6). Either 0.3 µg of the dominant negative mutants IKKalpha SS/AA and IKKalpha K/M (lanes 3 and 4), NIK KK/AA (lane 5), or MEKK1 D/A (lane 6) or 0.3 µg of the wild-type or constitutively active IKKalpha constructs (lanes 7 and 8) was cotransfected with the wild-type IKKbeta construct. Either wild-type IKKalpha or the constitutively active IKKalpha construct was also transfected alone (lanes 9 and 10). (B) COS cells were transfected with an influenza virus hemagglutinin-tagged wild-type (WT) IKKalpha construct (1 µg) (lanes 1 to 8) in the absence (lane 1) or presence of TNF-alpha (lanes 2 to 6). Dominant negative mutants (1 µg), including IKKbeta SS/AA and K/M (lanes 3 and 4), NIK KK/AA (lane 5), and MEKK1 D/A (lane 6), or 1 µg of either wild-type IKKbeta (lane 7) or a constitutively active IKKbeta construct (lane 8) were cotransfected with the wild-type IKKalpha as indicated. Wild-type IKKbeta (lane 9) and a constitutively active IKKbeta construct (lane 10) were also transfected alone. Cell lysates (50 µg) were immunoprecipitated with (A) anti-Flag M2 antibody to immunoprecipitate IKKbeta protein (lanes 1 to 8) or (B) 12CA5 antibody to immunoprecipitate the IKKalpha protein (lanes 1 to 8). In lanes 9 and 10, the 12CA5 antibody was used to immunoprecipitate IKKalpha and the M2 antibody was used to immunoprecipitate IKKbeta . Kinase assays were performed with a GST-Ikappa Balpha (amino acids 1 to 54) substrate, and the reaction mixtures were subjected to SDS-PAGE and autoradiography (top panel). Cell lysates from these immunoprecipitates were also analyzed by Western blot analysis with the M2 or 12CA5 antibody to quantitate the epitope-tagged IKKbeta and IKKalpha proteins (lanes 1 to 8) (lower panel).

We next performed a similar analysis to address whether IKKbeta could increase the ability of IKKalpha to phosphorylate the GST-Ikappa Balpha substrate. First, we demonstrated that TNF-alpha treatment of COS cells transfected with IKKalpha resulted in increased IKKalpha kinase activity for the GST-Ikappa Balpha substrate (Fig. 5B, lanes 1 and 2). TNF-alpha induction of IKKalpha kinase activity was not decreased by cotransfection of either of two dominant negative IKKbeta mutants (Fig. 5B, lanes 3 and 4). However, a dominant negative NIK mutant, but not a dominant negative MEKK1 mutant, inhibited TNF-alpha stimulation of IKKalpha kinase activity (Fig. 5B, lanes 5 and 6). These results suggested that dominant negative IKKbeta mutants did not block TNF-alpha -mediated increases in IKKalpha kinase activity.

To determine the role of IKKbeta in modulating IKKalpha kinase activity, either wild-type IKKbeta or the constitutively active IKKbeta construct was cotransfected with IKKalpha . Immunoprecipitation of the epitope-tagged IKKalpha proteins resulted in increased IKK kinase activity for the GST-Ikappa Balpha substrate (Fig. 5B, lanes 7 and 8). However, transfection of either the wild-type or the constitutively active IKKbeta constructs alone, followed by immunoprecipitation with the M2 monoclonal antibody, demonstrated a level of kinase activity similar to that seen when both IKKalpha and IKKbeta were cotransfected (Fig. 5B, lanes 9 and 10). The immunoprecipitated IKKalpha and IKKbeta proteins did not phosphorylate a GST-Ikappa Balpha protein mutant at serine residues 32 and 36 (data not shown). Immunoprecipitation followed by Western blot analysis indicated that IKKbeta , which has a much higher level of kinase activity than does IKKalpha , coimmunoprecipitated with IKKalpha , resulting in enhanced phosphorylation of Ikappa Balpha (data not shown). These results are consistent with the inability of IKKbeta to directly stimulate IKKalpha kinase activity.

In vitro phosphorylation of IKKbeta by IKKalpha . To address whether IKKalpha could directly phosphorylate IKKbeta , we used an in vitro kinase assay in which the ability of wild-type or mutant IKKalpha proteins to phosphorylate IKKbeta was analyzed. Epitope-tagged wild-type and mutant IKKalpha proteins, produced following transfection of COS cells, were immunoprecipitated. These epitope-tagged IKKalpha proteins were used because they exhibit little autophosphorylation in the in vitro kinase assays. In contrast, the baculovirus-produced IKKalpha proteins are autophosphorylated and thus make analysis of the effects of IKKalpha on IKKbeta phosphorylation more difficult to interpret (data not shown). The immunoprecipitated IKKalpha proteins were assayed for their ability to phosphorylate a catalytically defective Flag-tagged IKKbeta K/M protein which was purified following baculovirus expression. This substrate was used because baculovirus-produced wild-type IKKbeta exhibited high levels of autophosphorylation which obscured IKKalpha -mediated effects on this substrate.

The immunoprecipitated IKKalpha proteins had little kinase activity when assayed in in vitro kinase assays without the addition of substrate (Fig. 6A, lanes 1 to 5, top panel). Western blot analysis demonstrated that equivalent amounts of these proteins were used in the kinase assay (Fig. 6A, lower panel). The baculovirus-produced IKKbeta K/M substrate itself exhibited a low level of kinase activity (Fig. 6A, lane 6). Kinase assays were then performed with the IKKbeta K/M substrate and each of the different immunoprecipitated IKKalpha proteins. The 32P-labeled IKKbeta K/M substrate that was generated in the kinase assays was immunoprecipitated with the M2 monoclonal antibody and analyzed following SDS-PAGE and autoradiography. Both the wild-type and the constitutively active IKKalpha proteins phosphorylated the IKKbeta K/M substrate (Fig. 6A, lanes 7 and 8). In contrast, there was no significant phosphorylation of IKKbeta (K/M) by the kinase-deficient IKKalpha mutants, including IKKalpha K/M Delta HLH, IKKalpha K/M, and IKKalpha SS/AA (Fig. 6A, lanes 9 to 11). Equal amounts of the IKKbeta K/M substrate were present in each of these kinase reactions as determined by Western blot analysis of portions of each kinase assay (Fig. 6A, lanes 6 to 11, lower panel).


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  		FIG. 6.   In vitro phosphorylation of IKKbeta by IKKalpha . (A) COS cells were transfected with the indicated influenza virus hemagglutinin-tagged IKKalpha constructs. Cellular extracts (50 µg) were immunoprecipitated with 12CA5 antibody for wild-type IKKalpha (lanes 1 and 7), a constitutively active IKKalpha construct (lanes 2 and 8), or the kinase-defective IKKalpha mutants K/M Delta HLH (lanes 3 and 9), SS/AA (lanes 4 and 10), and K/M (lanes 5 and 11). Kinase assays were performed in either the absence of substrate (lanes 1 to 5), with only the baculovirus-produced purified IKKbeta K/M substrate (500 ng) (lane 6), or in the presence of the different IKKalpha proteins and the IKKbeta K/M substrate (lanes 7 to 11) (top panel). Following kinase assays, the supernatant was isolated by centrifugation, and the 32P-labeled IKKbeta K/M substrate was immunoprecipitated with the anti-Flag M2 monoclonal antibody and analyzed by SDS-PAGE and autoradiography. Western blot analysis of the influenza virus hemagglutinin-tagged IKKalpha immunoprecipitates (lanes 1 to 5) used in these assays or a portion of the immunoprecipitated Flag-tagged IKKbeta K/M substrate from each of the kinase assays was analyzed (lanes 6 to 11) (lower panel). (B) The different IKKalpha proteins used in panel A were used in kinase assays in the absence of substrate (lanes 1 to 5) or in the presence of 500 ng of baculovirus-produced IKKbeta SS/AA (lanes 7 to 11) or IKKbeta K/M (lanes 13 to 15) (top panel). Western blot analysis of the different IKKalpha proteins from these assays was done with 12CA5 antibody (lanes 1 to 5) or the baculovirus-produced IKKbeta SS/AA (lanes 6 to 11) or IKKbeta K/M proteins was done with the M2 monoclonal antibody (lower panel).

We also determined whether the IKKalpha proteins (Fig. 6B, lanes 1 to 5) could phosphorylate a baculovirus-produced IKKbeta SS/AA protein in which alanines were substituted for the serine residues at positions 177 and 181 in the IKKbeta activation loop (Fig. 6B, lanes 6 to 10). There was no IKKalpha -mediated phosphorylation of this protein, although both the wild-type and constitutively active IKKalpha proteins used in this experiment could phosphorylate the baculovirus-produced IKKbeta K/M protein (Fig. 6B, lanes 13 and 14). There were equal quantities of the different IKKalpha proteins used in these assays (Fig. 6B, lanes 1 to 5, lower panel) and equal quantities of the baculovirus-produced IKKbeta SS/AA and IKKbeta K/M substrates in these assays (Fig. 6B, lanes 6 to 15, lower panel). These results suggest that IKKalpha likely phosphorylates the activation loop of IKKbeta .

IKKbeta does not phosphorylate IKKalpha in vitro. It was important to determine whether IKKbeta could phosphorylate an IKKalpha substrate in in vitro kinase assays. Wild-type or mutant IKKbeta proteins produced following transfection of COS cells were assayed for their ability to phosphorylate baculovirus-produced wild-type IKKalpha or the IKKalpha mutants SS/AA and K/M (Fig. 7A). The immunoprecipitated IKKbeta proteins did not result in background phosphorylation (Fig. 7A, lanes 1 to 5), while the baculovirus-produced IKKalpha protein exhibited a low level of autophosphorylation (Fig. 7A, lane 6). IKKalpha phosphorylation was not stimulated by the addition of wild-type, constitutively active, or mutant IKKbeta constructs (Fig. 7A, lanes 7 to 11). The IKKbeta proteins also did not increase the phosphorylation of the baculovirus-produced IKKalpha SS/AA (Fig. 7A, lanes 12 to 17) or IKKalpha K/M (Fig. 7A, lanes 18 and 19) substrates. Western blot analysis indicated that there were equivalent amounts of IKKbeta (Fig. 7B, lanes 1 to 5) and wild-type and mutant IKKalpha (Fig. 7B, lanes 6 to 19) substrates used in these kinase assays.


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  		FIG. 7.   IKKbeta does not phosphorylate IKKalpha in vitro. (A) COS cells were transfected with the indicated Flag-tagged IKKbeta constructs. The extracts (50 µg) were immunoprecipitated with M2 monoclonal antibody for wild-type IKKbeta (lanes 1 and 7), a constitutively active IKKbeta construct (lanes 2 and 8), or the kinase-defective IKKbeta mutants K/M Delta HLH (lanes 3 and 9), SS/AA (lanes 4 and 10), and K/M (lanes 5 and 11). Kinase assays were performed in either the absence of substrate (lanes 1 to 5), with 500 ng of the baculovirus-produced purified IKKalpha substrate alone (lane 6), the IKKalpha SS/AA substrate alone (lane 12), or the different IKKbeta proteins and either the IKKalpha (lanes 7 to 11), the IKKalpha SS/AA (lanes 13 to 17), or the IKKalpha K/M (lanes 18 and 19) substrate. Following kinase assays, the supernatant was isolated by centrifugation, and the 32P-labeled IKKalpha and IKKalpha SS/AA substrates were immunoprecipitated with the 12CA5 monoclonal antibody and analyzed by SDS-PAGE and autoradiography. (B) Western blot analysis was performed on a portion of the Flag-tagged IKKbeta immunoprecipitates (lanes 1 to 5) or a portion of the immunoprecipitated influenza virus hemagglutinin-tagged IKKalpha (lanes 6 to 11), IKKalpha SS/AA (lanes 12 to 17), or IKKalpha K/M (lanes 18 and 19) substrate from each of the kinase assays using the epitope-specific monoclonal antibodies. (C) The different immunoprecipitated IKKbeta proteins used in panel A were used in kinase assays with GST-Ikappa Balpha (amino acids 1 to 54) or mutant GST-Ikappa Balpha , in which serine residues 32 and 36 were changed to alanine. Following SDS-PAGE, autoradiography was performed.

Since the IKKbeta proteins did not enhance the in vitro phosphorylation of IKKalpha , it was important to address whether these IKKbeta proteins exhibited kinase activity with an Ikappa Balpha substrate. Each of the IKKbeta proteins used in part A were tested for their ability to phosphorylate GST fusion proteins containing the amino-terminal 54 amino acids of Ikappa Balpha or a mutant Ikappa Balpha protein in which serine residues 32 and 36 were changed to alanine. Wild-type and constitutively active IKKbeta proteins strongly phosphorylated wild-type GST-Ikappa Balpha (Fig. 7C, lanes 1 and 2), while the mutant IKKbeta proteins did not significantly phosphorylate this substrate (Fig. 7C, lanes 3 to 5). The IKKbeta proteins did not phosphorylate the GST-Ikappa Balpha protein mutant at serine residues 32 and 36 (Fig. 7C, lanes 6 to 10). These results indicate that although IKKbeta did not phosphorylate IKKalpha , it strongly phosphorylated the Ikappa Balpha substrate.

In vivo analysis of constitutively active IKK proteins. Finally, we addressed whether our results suggesting a role for IKKalpha in modulating IKKbeta phosphorylation and kinase activity could be correlated with in vivo studies regarding IKK activation of an NF-kappa B reporter construct. In these studies, TNF-alpha was not used to stimulate the activity of the transfected IKKalpha and IKKbeta cDNAs because this cytokine itself strongly activates NF-kappa B reporter constructs (24, 38). Instead, we tested the ability of dominant negative IKKalpha and IKKbeta mutants to alter the ability of constitutively active IKKalpha and IKKbeta constructs to activate gene expression of an NF-kappa B reporter construct.

An HIV-1 LTR-luciferase reporter construct which contains two NF-kappa B binding sites was transfected into COS cells with either a constitutively active IKKalpha or IKKbeta construct (Fig. 8). In addition, either of two dominant negative IKKalpha or IKKbeta mutants was also cotransfected. Thus, the ability of the dominant negative IKKalpha and IKKbeta mutants to prevent IKK activation of an NF-kappa B reporter construct could be assayed. Both of the constitutively active IKK constructs, IKKalpha SS/EE and IKKbeta SS/EE, activated gene expression from the HIV-1 LTR-luciferase reporter (Fig. 8). Neither of these constitutively active IKK constructs stimulated gene expression from an HIV-1 LTR-luciferase reporter construct with mutated NF-kappa B binding sites (data not shown). Cotransfection of either of the two dominant negative IKKbeta constructs prevented IKKalpha SS/EE activation of the NF-kappa B reporter construct (Fig. 8). This result may be explained by the fact that the IKKalpha SS/EE protein formed heterodimers with the IKKbeta dominant negative mutants and thus was not able to phosphorylate endogenous IKKbeta or endogenous Ikappa Balpha .


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  		FIG. 8.   IKKbeta dominant negative mutants inhibit NF-kappa B activation by a constitutively active IKKalpha construct. An HIV-1 LTR-luciferase construct (10 ng) was transfected into COS cells either alone (---), with a constitutively active IKKalpha SS/EE construct (0.5 µg) (lane 2), or with 0.25 µg of either IKKbeta SS/AA or IKKbeta K/M. The HIV-1 LTR-luciferase construct was also transfected with IKKbeta SS/EE alone (0.3 µg) or together with 0.5 µg of the IKKalpha K/M or SS/AA dominant negative mutant. Cells were harvested at 30 h posttransfection, and luciferase activity was quantitated and normalized by using a CMV-beta -galactosidase control plasmid. The results are the means of three independent experiments.

In contrast, neither of the dominant negative IKKalpha mutants was able to significantly inhibit IKKbeta SS/EE activation of the NF-kappa B reporter construct (Fig. 8). Since IKKbeta SS/EE does not require phosphorylation by IKKalpha for stimulation of its kinase activation, the dominant negative IKKalpha constructs would not be expected to alter IKKbeta SS/EE activation of the NF-kappa B reporter. These transfection studies provide indirect evidence that IKKalpha may modulate IKKbeta activation of the NF-kappa B pathway.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In this study, we present several lines of evidence that IKKalpha can modulate IKKbeta function. First, we demonstrate that dominant negative IKKalpha mutants prevent TNF-alpha -induced phosphorylation of IKKbeta . Second, we show that wild-type and constitutively active IKKalpha proteins stimulate IKKbeta phosphorylation both in transfection assays and following isolation of high-molecular-weight IKK complexes. Third, our data indicate that IKKalpha stimulates IKKbeta kinase activity for the Ikappa Balpha substrate. Finally, we demonstrate that IKKalpha can phosphorylate IKKbeta in in vitro kinase assays. These results suggest that IKKalpha likely modulates IKKbeta function.

Our studies utilized transient-expression assays to analyze IKKalpha function. Thus, we cannot rule out that these results might not entirely reflect those obtained with IKKalpha and IKKbeta are present in the high-molecular-weight IKK complex. However, we did demonstrate that the presence of IKKalpha and IKKbeta in a complex migrating between 400 and 700 kDa correlates with increases in IKKbeta phosphorylation. Although the size of this IKK complex is less than the 700 to 900 kDa of an IKK complex that has been described before (8, 9, 15, 28), it is likely that the IKK complex generated from transfection of IKKalpha and IKKbeta expression vectors lacks sufficient quantities of proteins like NEMO (23, 28, 36) or IKAP (10) that are components of the endogenous IKK complex. Overexpression of IKK proteins in transfection assays likely also accounts for the fact that wild-type IKKalpha and the constitutively active IKKalpha mutant have similar abilities to stimulate IKKbeta phosphorylation and kinase activity. When low concentrations of these plasmids are transfected into COS cells, the constitutively active IKKalpha mutant has a greater ability to stimulate IKKbeta phosphorylation and kinase activity for Ikappa Balpha than does wild-type IKKalpha (unpublished observations). However, when larger quantities of IKKalpha and the constitutively active IKKalpha mutant are transfected, these constructs have a similar ability to stimulate IKKbeta phosphorylation and kinase activity. Thus, it is important to note that several of the conclusions reached in this study are based on the results of transfection assays with IKKalpha and IKKbeta .

A recent study examined the patterns of phosphorylation of the IKKalpha and IKKbeta proteins in response to different activators of the NF-kappa B pathway, including TNF-alpha , IL-1, and NIK (11). In agreement with this study, we find that TNF-alpha treatment of cells markedly stimulates both IKKalpha and IKKbeta phosphorylation. However, catalytically inactive and activation loop mutants of IKKalpha and IKKbeta exhibit decreased in vivo phosphorylation in response to TNF-alpha . These data suggest that at least a portion of IKKalpha and IKKbeta phosphorylation in response to TNF-alpha treatment likely results from autophosphorylation of these kinases. In contrast to the results of this latter study, which indicate that mutations in the IKKalpha activation loop do not alter IKKalpha phosphorylation of Ikappa Balpha , our data and several previous studies indicate that such mutants exhibit defective kinase activity (20, 22). Thus, we suggest that phosphorylation of IKKalpha is critical for enhancing its ability to phosphorylate both Ikappa Balpha and IKKbeta .

IKKbeta appears to be the dominant kinase required for activating NF-kappa B, based on its higher level of activity for Ikappa Balpha compared with IKKalpha (17, 22, 24, 35, 38, 39) and the failure to activate the NF-kappa B pathway when this gene is disrupted in mice (18). IKKalpha , in addition to NIK (20) and MEKK1 (16), may also be involved in activating IKKbeta kinase activity. The fact that multiple kinases can activate IKKbeta kinase activity may explain the somewhat different results seen in IKKalpha knock-out mice. For example, two such studies found TNF-alpha induction of the NF-kappa B pathway to be intact (14, 19, 30), while another study found defects in activating the NF-kappa B pathway (18). Additional studies will be required to further define the specific roles of NIK, MEKK1, NEMO, and IKKalpha in regulating IKKbeta kinase activity.

In summary, IKKalpha may potentially have multiple effects on regulating the NF-kappa B pathway. First, it can phosphorylate Ikappa Balpha and Ikappa Bbeta to result in their ubiquitination and subsequent degradation by the proteasome. In addition, our data suggest that IKKalpha can phosphorylate IKKbeta . The physiologic relevance of IKKalpha in each of these processes will need to be better elucidated by both in vivo studies and reconstituted in vitro assay systems to more clearly determine the role of this kinase in regulating the NF-kappa B pathway.


    ACKNOWLEDGMENTS

We thank Sharon Johnson and Stephanie Guyer for preparation of the manuscript and figures, respectively.

This work was supported by grants from the NIH and the Veterans Administration.


    FOOTNOTES

* Corresponding author. Mailing address: Division of Hematology-Oncology, Department of Medicine, U.T. Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-8594. Phone: (214) 648-7570. Fax: (214) 648-8862. E-mail: gaynor{at}utsw.swmed.edu.


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Molecular and Cellular Biology, May 2000, p. 3655-3666, Vol. 20, No. 10
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