I B Kinase (IKK ) Regulation of IKK Kinase Activity

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. I B Kinase (IKK ) Regulation of IKK 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 Two related kinases, I B kinase (IKK ) and IKK, phosphorylate the I B proteins, leading to their degradation and the subsequent activation of gene expression by NF- B. IKK has a much higher level of kinase activity for the I B proteins than does IKK and is more critical than IKK in modulating tumor necrosis factor alpha activation of the NF- B pathway. These results indicate an important role for IKK in activating the NF- B pathway but leave open the question of the role of IKK in regulating this pathway. In the current study, we demonstrate that IKK directly phosphorylates IKK. Moreover, IKK either directly or indirectly enhances IKK kinase activity for I B. Finally, transfection studies to analyze NF- B-directed gene expression suggest that IKK is upstream of IKK in activating the NF- B pathway. These results indicate that IKK, in addition to its previously described ability to phosphorylate I B, can increase the ability of IKK to phosphorylate I B. * 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@utsw.swmed.edu. The NF- 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- B is sequestered in the cytoplasm of most cells, where it is bound to a family of inhibitory proteins known as I B (2 4). A variety of extracellular stimuli, including tumor necrosis factor alpha (TNF- ), 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 I B proteins (1, 5 7, 12, 32, 33). The I B proteins are then ubiquitinated on amino-terminal lysine residues via interaction with -TrCP (29, 31, 34, 37). After the formation of the ubiquitin-ligase complex, I B is degraded by the 26S proteasome (8, 9). Two related kinases that phosphorylate amino-terminal serine residues 32 and 36 in I B and 19 and 23 in I B have been described (13, 22, 27, 35, 40). These I B kinases are components of a 700-kDa kinase complex whose activity is markedly increased by treatment of cells with activators of the NF- B pathway, such as TNF- and IL-1 (8, 9, 15, 28). Other components of this complex include NEMO or I B kinase (IKK ), which is required for in vivo activation of IKK kinase activity (23, 28, 36), and IKAP, which may function as a scaffold protein (10). IKK and IKK 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, IKK has a 20- to 50-fold-higher level of kinase activity for I B than does IKK (16, 22, 24, 38, 39, 41). TNF- activation of the NF- B pathway is mediated by multiple adapter proteins which lead to activation of NF- Binducing kinase (NIK) (21), which is capable of directly phosphorylating IKK in its activation loop at serine residue 176 (20). However, other upstream kinases have also been demonstrated to activate the NF- B pathway. For example, mitogen-activated protein/extracellular signal-regulated kinase 1 (MEKK1) can activate both IKK and IKK 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 I B kinases (26, 42). These results suggest that multiple signal transduction pathways can likely modulate IKK function. Recent data suggest that TNF- - and IL-1-mediated increases in the phosphorylation of IKK and potentially IKK may be important in the regulation of their kinase activity (11). Both IKK and IKK 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 IKK and positions 177 and 181 in IKK, has been shown to be important for IKK kinase activity (22). For example, mutation of these serine residues to alanine in both IKK and IKK can inactivate their ability to phosphorylate I 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 IKK but not IKK 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 IKK 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 IKK are not impaired for activation of the NF- B pathway or I B degradation following treatment with inflammatory cytokines (14, 30). However, another study indicates that mice lacking IKK are somewhat defective in activating the NF- B pathway (18). In contrast, mice lacking IKK die as embryos due to extensive liver damage from 3655

3656 YAMAMOTO ET AL. MOL. CELL. BIOL. uncontrolled apoptosis (19). Moreover, in these mice there are marked defects in activation of the NF- B pathway by proinflammatory cytokines such as TNF- (19). These results indicate that IKK appears to be more critical than IKK in activating the NF- B pathway. In the current study, we address the role of IKK in activating the NF- B pathway. We demonstrate that IKK directly phosphorylates IKK. Furthermore, we demonstrate that IKK either directly or indirectly increases the ability of IKK to phosphorylate I B. Finally, transfection studies suggest that IKK is upstream of IKK in mediating activation of NF- Bdirected gene expression. These results suggest that IKK may modulate IKK kinase activity to regulate the NF- B pathway. MATERIALS AND METHODS DNA constructs. The cdnas for wild-type IKK and the IKK mutants K44M (K/M), S176A/S180A (SS/AA), and K44M HLH, in which amino acids 560 to 744 in the carboxy terminus of IKK have been deleted, contain aminoterminal influenza virus hemagglutinin sequences and were cloned downstream of the cytomegalovirus (CMV) promoter in pcmv5. The cdnas for wild-type IKK and the IKK 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 IKK and IKK 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 g of either wild-type or kinase-defective Flag epitope-tagged IKK constructs or wild-type or kinase-defective influenza virus hemagglutinin-tagged IKK cdnas (38). The wild-type or dominant negative NIK and MEKK1 mutant constructs have been described previously (38). For assays of NF- 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 IKK and IKK constructs, and luciferase activity was assayed 30 h posttransfection (38). A CMV -galactosidase plasmid was also incorporated into each transfection. [ 32 P]orthophosphate labeling of IKK proteins. COS cells were maintained in DMEM with 10% fetal bovine serum and transfected with either the indicated IKK or IKK 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 [ 32 P]orthophosphate (50 Ci/ml) or [ 35 S]methionine (50 Ci/ml) was then added to the cells and incubated for 3 h. TNF- (20 ng/ l) 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 -glycerophosphate, and protease inhibitors). Immunoprecipitation and kinase assays. Cell lysates from either [ 32 P]orthophosphate- and [ 35 S]methionine-labeled cells or nonlabeled cells were incubated with 50 l of 12CA5 supernatant or 500 ng of anti-flag M2 antibody for 2hon ice. To assay endogenous IKK labeling with either [ 32 P]orthophosphate or [ 35 S]methionine, 50 g of the cellular lysate was immunoprecipitated with rabbit polyclonal antibody directed against IKK (Santa Cruz). Then 20 l of protein A-agarose was added to each of the immunoprecipitates and incubated for1hat 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 g) were incubated in kinase reaction buffer containing 10 Ci of [ - 32 P]ATP, 1 mm ATP, 5 mm MgCl 2, 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)-I B (2 g), wild type (amino acids 1 to 54) or mutant (S32/S363A32/A36), or baculovirus-produced polyhistidine- and Flagtagged IKK or IKK 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 (10 8 ) were cotransfected with epitope-tagged expression vectors containing either IKK K/M and IKK or IKK and IKK. 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 MgCl 2, 10 mm KCl, 0.5 mm dithiothreitol) supplemented with phosphatase inhibitors (50 mm NaF, 50 mm glycerophosphate, 0.125 M 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 MgCl 2 ) 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), -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 TNF- induces endogenous IKK phosphorylation. Stimulation of IKK 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- treatment or transfection of MEKK1 and NIK constructs to induce phosphorylation of endogenous IKK. COS cells were labeled with either [ 32 P]orthophosphate (Fig. 1A, top panel) or [ 35 S]methionine (Fig. 1A, middle panel) for 3 h prior to harvest. The IKK proteins were then immunoprecipitated with a polyclonal antibody directed against IKK that immunoprecipitates the IKK -IKK heterodimer (38). TNF- 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 [ 35 S]methionine-labeled extracts prepared from TNF- -treated or wildtype or mutant NIK- or MEKK1-transfected COS cells did not demonstrate differences in the levels of the [ 35 S]methioninelabeled IKK proteins (Fig. 1A, lanes 1 to 6, middle panel). Western blot analysis confirmed the presence of similar amounts of IKK in each of these extracts (Fig. 1A, lanes 1 to 6, lower panel). We also determined whether TNF- increased the phosphorylation of influenza virus hemagglutinin-tagged IKK or Flagtagged IKK cdnas following transfection of each of these constructs into COS cells. The 32 P-labeled IKK and IKK proteins were immunoprecipitated with the 12CA5 and Flag monoclonal antibodies, respectively. The phosphorylation of both IKK and IKK was increased following TNF- treatment (Fig. 1B, lanes 2 to 5, top panel). There were similar amounts of the IKK proteins in both untreated and TNF- treated extracts (Fig. 1B, lanes 2 to 5, lower panel). These results indicate that activators of the NF- B pathway such as TNF- increase the phosphorylation of both the IKK and IKK proteins.

VOL. 20, 2000 IKK REGULATION OF IKK 3657 FIG. 1. Activators of the NF- B pathway increase IKK phosphorylation. (A) COS cells were either untreated (lane 1), treated with TNF- (20 ng/ml) for 5 to 7 min prior to harvest (lane 2), or transfected with 2 g 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 [ 32 P]orthophosphate (top panel) or [ 35 S]methionine (middle panel) for 3 h prior to harvesting the cells. Immunoprecipitation was performed with IKK polyclonal antibody (Santa Cruz) followed by SDS-PAGE and autoradiography. Extracts were also analyzed for IKK expression in Western blot analysis with IKK antibody (lower panel). (B) COS cells were not transfected (lane 1) or transfected with 1 g of the influenza virus hemagglutinin-tagged IKK (lanes 2 and 3) or 0.2 g of the Flag-tagged IKK (lanes 4 and 5) cdnas in either the absence (lanes 2 and 4) or presence (lanes 3 and 5) of TNF-. Cells were labeled with [ 32 P]orthophosphate for 3 h prior to harvest, and TNF- 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 g 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- induces phosphorylation of IKK and IKK. To address the mechanism by which activators of the NF- B pathway increase the phosphorylation of IKK and IKK, epitopetagged cdnas encoding each of these proteins were transfected into COS cells (Fig. 2). [ 32 P]orthophosphate labeling of the transfected COS cells was performed in either the presence or absence of TNF-. Dominant negative mutants of either IKK, IKK, MEKK1, or NIK were included in these transfection assays as indicated. TNF- treatment of COS cells strongly induced the phosphorylation of IKK (Fig. 2A, lanes 1 and 2). TNF- induction of IKK phosphorylation was not inhibited by cotransfection of either of two IKK dominant negative mutants (Fig. 2A, lanes 3 and 4) or a dominant negative MEKK1 mutant (Fig. 2A, lane 5). In contrast, TNF- -induced IKK phosphorylation was blocked by a dominant negative NIK mutant (Fig. 2B, lane 6). TNF- treatment did not increase the phosphorylation of an IKK mutant in the activation loop motif (Fig. 2B, lanes 1 and 2) or a catalytically inactive IKK mutant (Fig. 2B, lanes 3 and 4). COS cells were next transfected with an epitope-tagged IKK cdna in either the presence or absence of TNF- and labeled with either [ 32 P]orthophosphate or [ 35 S]methionine. TNF- induced the phosphorylation of IKK (Fig. 2C, lanes 1 and 2). Cotransfection of dominant negative IKK mutants decreased TNF- -induced phosphorylation of IKK (Fig. 2C, lanes 3 and 4). A dominant negative NIK mutant also decreased IKK phosphorylation, while a dominant negative MEKK1 mutant did not decrease and in fact slightly increased IKK phosphorylation (Fig. 2C, lanes 5 and 6). There was no significant change in the level of [ 35 S]methionine-labeled IKK proteins (Fig. 2C, lower panel). TNF- treatment did not induce phosphorylation of an IKK mutant in the two serine residues in its activation loop (Fig. 2D, lanes 1 and 2) or of a catalytically inactive IKK mutant (Fig. 2D, lanes 3 and 4). These results are consistent with a role for IKK in potentially modulating the phosphorylation state of IKK. IKK induces phosphorylation of IKK. To determine whether IKK may potentially be involved in either directly or indirectly stimulating the phosphorylation of IKK, weassayed the ability of IKK to modulate the phosphorylation of IKK. COS cells were transfected with an epitope-tagged IKK cdna either alone or in the presence of wild-type IKK, a constitutively active IKK construct, or two dominant negative IKK mutants. The COS cells were labeled with either [ 32 P]orthophosphate or [ 35 S]methionine, and the Flag epitope-tagged IKK protein was immunoprecipitated with the M2 monoclonal antibody. Both wild-type and constitutively active IKK constructs increased the phosphorylation of IKK (Fig. 3A, lanes 1 to 3). In contrast, there was little or no increase in IKK phosphorylation with either of two IKK mutants, IKK SS/AA or IKK K/M (Fig. 3A, lanes 4 and 5). In vivo labeling of the IKK proteins with [ 35 S]methionine demonstrated that IKK expression did not alter the level of the [ 35 S]methionine-labeled IKK proteins (Fig. 3A, lower panel). Similar results from three independent experiments indicate that IKK can either directly or indirectly modulate the level of IKK phosphorylation. To address whether IKK could increase IKK phosphorylation, COS cells were transfected with an influenza virus hemagglutinin-tagged IKK construct either alone or in the presence of different IKK constructs. COS cells were again labeled with either [ 32 P]orthophosphate or [ 35 S]methionine, and the influenza virus hemagglutinin-tagged IKK protein was immunoprecipitated with the 12CA5 monoclonal antibody. Neither the wild-type nor the constitutively active IKK constructs altered the amount of IKK phosphorylation (Fig. 3B, lanes 1 to 3). The dominant negative IKK mutants IKK SS/AA and IKK K/M also did not alter the phosphorylation of IKK (Fig. 3B, lanes 4 and 5). In vivo labeling of the IKK

3658 YAMAMOTO ET AL. MOL. CELL. BIOL. FIG. 2. IKK phosphorylation is inhibited by dominant negative IKK mutants. (A and C) COS cells were transfected with HA-tagged IKK or Flag-tagged IKK cdnas alone (lanes 1 and 2) or in the presence of similar amounts of IKK, IKK, NIK, or MEKK1 dominant negative mutants as indicated (lanes 3 to 6). The transfected cells were labeled with [ 32 P]orthophosphate (top panel) or [ 35 S]methionine (lower panel) for 3 h and either untreated (lane 1) or treated with TNF- (20 ng/ml) for 5 to 7 min (lanes 2 to 6). Cell lysates were prepared, and 50 g of this lysate was incubated with the 12CA5 antibody to immunoprecipitate the IKK protein or with the Flag antibody M2 to immunoprecipitate the IKK protein. The immunoprecipitates were subjected to SDS-PAGE, and autoradiography was performed. (B and D) COS cells were transfected with the kinase-defective IKK or IKK mutant SS/AA (lanes 1 and 2) or K/M (lanes 3 and 4). The cells were labeled with either [ 32 P]orthophosphate (top panel) or [ 35 S]methionine (lower panel) for 3 h prior to harvest in the absence (lanes 1 and 3) or presence of TNF- for 5 to 7 min (lanes 2 and 4). The cell lysates were immunoprecipitated and subjected to SDS-PAGE. proteins with [ 35 S]methionine demonstrated similar amounts of the IKK proteins (Fig. 3B, lanes 1 to 5, lower panel). These results suggest that IKK does not markedly alter IKK phosphorylation. IKK increases IKK phosphorylation in a high-molecular-weight IKK complex. It was important to address whether IKK could stimulate IKK 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 IKK and either wildtype IKK or a catalytically defective IKK K/M mutant. The IKK constructs were tagged with the influenza virus hemagglutinin epitope, while IKK 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.

VOL. 20, 2000 IKK REGULATION OF IKK 3659 FIG. 3. IKK increases IKK phosphorylation. (A) COS cells were transfected with a Flag-tagged wild-type (WT) IKK cdna construct (0.5 g) alone (lane 1) or in the presence of 0.5 g of influenza virus hemagglutinin-tagged wild-type IKK (lane 2), a constitutively active IKK construct (lane 3), or the mutant IKK 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) IKK cdna construct (0.5 g) alone (lane 1) or in the presence of 0.5 g of Flag-tagged wild-type IKK (lane 2), a constitutively active IKK construct (lane 3), or the mutant IKK 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 [ 32 P]orthophosphate (top panel) or [ 35 S]methionine (lower panel) for 3 h prior to cell harvest. The cell lysates (50 g) were immunoprecipitated with the (A) anti-flag M2 monoclonal antibody to immunoprecipitate the epitope-tagged IKK proteins or (B) the 12CA5 monoclonal antibody to immunoprecipitate the epitope-tagged IKK proteins. The immunoprecipitates were then subjected to SDS-PAGE, and autoradiography was performed. Fractions from the Superdex 200 column were immunoprecipitated with the anti-flag M2 monoclonal antibody, and in vitro kinase assays were performed. IKK phosphorylation was present at low levels in column fractions migrating between 400 and 600 kda in the presence of the IKK K/M protein (Fig. 4A, top panel). There was no detectable IKK K/M autophosphorylation in these column fractions. In contrast, the column fractions containing both wild-type IKK and IKK showed markedly enhanced phosphorylation of both IKK and IKK (Fig. 4B, top panel). The positions of the phosphorylated wild-type IKK and IKK 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 IKK and IKK proteins in these Superdex 200 fractions (Fig. 4A and B, lower panels). Finally, we determined whether immunoprecipitation of either IKK K/M or IKK present in column fraction 9 with the 12CA5 monoclonal antibody also demonstrated differences in IKK phosphorylation (Fig. 4C). This analysis demonstrated that the presence of wild-type IKK but not IKK K/M was associated with enhanced IKK phosphorylation. Immunoprecipitation of these column fractions followed by Western blot analysis indicated that the epitope-tagged IKK and IKK K/M proteins both strongly associated with the epitope-tagged IKK protein (data not shown). No IKK phosphorylation was noted when the catalytically defective IKK mutants IKK K/M and IKK K/M were analyzed following cotransfection and Superdex 200 fractionation (data not shown). These results indicate that IKK is associated with enhanced IKK phosphorylation when these kinases are present as heterodimers in a high-molecular-weight IKK complex. IKK stimulates IKK kinase activity. Next we investigated whether IKK -mediated increases in IKK phosphorylation correlate with its ability to stimulate IKK kinase activity. First, an epitope-tagged IKK cdna was transfected into COS cells, the cells were either untreated or treated with TNF-, and IKK kinase activity was assayed. Next, dominant negative mutants of either IKK, NIK, or MEKK1 were cotransfected with IKK in the presence of TNF- to determine their role in regulating IKK kinase activity. Finally, we assayed the ability of wild-type and constitutively active IKK proteins to stimulate IKK kinase activity. The Flag-tagged IKK protein in each of these transfections was immunoprecipitated with the M2 monoclonal antibody and assayed for its ability to phosphorylate the amino terminus of I B (amino acids 1 to 54). TNF- treatment markedly increased IKK kinase activity for the GST-I B substrate (Fig. 5A, lanes 1 and 2). The TNF- -mediated increase in IKK kinase activity was blocked by two dominant negative IKK 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 IKK to stimulate IKK kinase activity. Transfection of wild-type IKK markedly stimulated the ability of IKK to phosphorylate GST-I B (Fig. 5A, lanes 1 and 7). A constitutively active IKK construct also markedly stimulated IKK kinase activity for the GST-I B substrate (Fig. 5A, lanes 1 and 8). IKK mutants K/M and SS/AA did not stimulate IKK kinase activity, and the immunoprecipitated IKK did not phosphorylate a GST-I B construct mutant at serine residues 32 and 36 (data not shown). When the wild-type and the constitutively active IKK constructs were transfected alone and immunoprecipitated with the 12CA5 antibody, they had very low kinase activity with the GST-I B substrate (Fig. 5A, lanes 9 and 10). Western blot analysis demonstrated that there was little change in the level of the epitope-tagged IKK proteins in either the presence or absence of IKK (Fig. 5A, lower panel). These results suggested that IKK can either directly or indirectly modulate IKK

3660 YAMAMOTO ET AL. MOL. CELL. BIOL. FIG. 4. IKK phosphorylation of IKK in the IKK complex. COS cells were transfected with expression vectors encoding (A) hemagglutinin-tagged IKK K/M and Flag-tagged IKK or (B) hemagglutinin-tagged wild-type IKK and Flag-tagged IKK. 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 IKK and IKK 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 IKK K/M and wild-type IKK or with the M2 monoclonal antibody to detect IKK (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 IKK K/M or wild-type IKK followed by in vitro kinase assays, SDS-PAGE, and autoradiography. kinase activity and that TNF- induction of IKK kinase activity may be mediated in part through effects on IKK. We next performed a similar analysis to address whether IKK could increase the ability of IKK to phosphorylate the GST-I B substrate. First, we demonstrated that TNF- treatment of COS cells transfected with IKK resulted in increased IKK kinase activity for the GST-I B substrate (Fig. 5B, lanes 1 and 2). TNF- induction of IKK kinase activity was not decreased by cotransfection of either of two dominant negative IKK mutants (Fig. 5B, lanes 3 and 4). However, a dominant negative NIK mutant, but not a dominant negative MEKK1 mutant, inhibited TNF- stimulation of IKK kinase activity (Fig. 5B, lanes 5 and 6). These results suggested that dominant negative IKK mutants did not block TNF- -mediated increases in IKK kinase activity. To determine the role of IKK in modulating IKK kinase activity, either wild-type IKK or the constitutively active IKK construct was cotransfected with IKK. Immunoprecipitation of the epitope-tagged IKK proteins resulted in increased IKK kinase activity for the GST-I B substrate (Fig. 5B, lanes 7 and 8). However, transfection of either the wildtype or the constitutively active IKK constructs alone, followed by immunoprecipitation with the M2 monoclonal antibody, demonstrated a level of kinase activity similar to that seen when both IKK and IKK were cotransfected (Fig. 5B, lanes 9 and 10). The immunoprecipitated IKK and IKK proteins did not phosphorylate a GST-I B protein mutant at serine residues 32 and 36 (data not shown). Immunoprecipitation followed by Western blot analysis indicated that IKK, which has a much higher level of kinase activity than does IKK, coimmunoprecipitated with IKK, resulting in enhanced phosphorylation of I B (data not shown). These results are consistent with the inability of IKK to directly stimulate IKK kinase activity. In vitro phosphorylation of IKK by IKK. To address whether IKK could directly phosphorylate IKK, we used an in vitro kinase assay in which the ability of wild-type or mutant IKK proteins to phosphorylate IKK was analyzed. Epitopetagged wild-type and mutant IKK proteins, produced following transfection of COS cells, were immunoprecipitated. These epitope-tagged IKK proteins were used because they exhibit little autophosphorylation in the in vitro kinase assays. In contrast, the baculovirus-produced IKK proteins are autophosphorylated and thus make analysis of the effects of IKK on IKK phosphorylation more difficult to interpret (data not shown). The immunoprecipitated IKK proteins were assayed for their ability to phosphorylate a catalytically defective Flag-tagged IKK K/M protein which was purified following baculovirus expression. This substrate was used because baculovirus-produced wild-type IKK exhibited high levels of autophosphorylation which obscured IKK -mediated effects on this substrate. The immunoprecipitated IKK 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 IKK K/M substrate itself exhibited a low level of kinase activity (Fig. 6A, lane 6). Kinase assays were then performed with the IKK K/M substrate and each of the different immunoprecipitated IKK proteins. The 32 P-labeled IKK K/M substrate that was generated in the kinase

VOL. 20, 2000 IKK REGULATION OF IKK 3661 FIG. 5. IKK stimulates IKK kinase activity. (A) COS cells were transfected with a Flag-tagged wild-type (WT) IKK cdna construct (0.1 g) (lanes 1 to 8) in the absence (lane 1) or presence of TNF- (lanes 2 to 6). Either 0.3 g of the dominant negative mutants IKK SS/AA and IKK K/M (lanes 3 and 4), NIK KK/AA (lane 5), or MEKK1 D/A (lane 6) or 0.3 g of the wild-type or constitutively active IKK constructs (lanes 7 and 8) was cotransfected with the wild-type IKK construct. Either wild-type IKK or the constitutively active IKK construct was also transfected alone (lanes 9 and 10). (B) COS cells were transfected with an influenza virus hemagglutinin-tagged wild-type (WT) IKK construct (1 g) (lanes 1 to 8) in the absence (lane 1) or presence of TNF- (lanes 2 to 6). Dominant negative mutants (1 g), including IKK SS/AA and K/M (lanes 3 and 4), NIK KK/AA (lane 5), and MEKK1 D/A (lane 6), or 1 g of either wild-type IKK (lane 7) or a constitutively active IKK construct (lane 8) were cotransfected with the wild-type IKK as indicated. Wild-type IKK (lane 9) and a constitutively active IKK construct (lane 10) were also transfected alone. Cell lysates (50 g) were immunoprecipitated with (A) anti-flag M2 antibody to immunoprecipitate IKK protein (lanes 1 to 8) or (B) 12CA5 antibody to immunoprecipitate the IKK protein (lanes 1 to 8). In lanes 9 and 10, the 12CA5 antibody was used to immunoprecipitate IKK and the M2 antibody was used to immunoprecipitate IKK. Kinase assays were performed with a GST-I B (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 IKK and IKK proteins (lanes 1 to 8) (lower panel).

3662 YAMAMOTO ET AL. MOL. CELL. BIOL. FIG. 6. In vitro phosphorylation of IKK by IKK. (A) COS cells were transfected with the indicated influenza virus hemagglutinin-tagged IKK constructs. Cellular extracts (50 g) were immunoprecipitated with 12CA5 antibody for wild-type IKK (lanes 1 and 7), a constitutively active IKK construct (lanes 2 and 8), or the kinase-defective IKK mutants K/M 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 IKK K/M substrate (500 ng) (lane 6), or in the presence of the different IKK proteins and the IKK K/M substrate (lanes 7 to 11) (top panel). Following kinase assays, the supernatant was isolated by centrifugation, and the 32 P-labeled IKK 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 IKK immunoprecipitates (lanes 1 to 5) used in these assays or a portion of the immunoprecipitated Flag-tagged IKK K/M substrate from each of the kinase assays was analyzed (lanes 6 to 11) (lower panel). (B) The different IKK 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 IKK SS/AA (lanes 7 to 11) or IKK K/M (lanes 13 to 15) (top panel). Western blot analysis of the different IKK proteins from these assays was done with 12CA5 antibody (lanes 1 to 5) or the baculovirus-produced IKK SS/AA (lanes 6 to 11) or IKK K/M proteins was done with the M2 monoclonal antibody (lower panel). assays was immunoprecipitated with the M2 monoclonal antibody and analyzed following SDS-PAGE and autoradiography. Both the wild-type and the constitutively active IKK proteins phosphorylated the IKK K/M substrate (Fig. 6A, lanes 7 and 8). In contrast, there was no significant phosphorylation of IKK (K/M) by the kinase-deficient IKK mutants, including IKK K/M HLH, IKK K/M, and IKK SS/AA (Fig. 6A, lanes 9 to 11). Equal amounts of the IKK K/M substrate were

VOL. 20, 2000 IKK REGULATION OF IKK 3663 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). We also determined whether the IKK proteins (Fig. 6B, lanes 1 to 5) could phosphorylate a baculovirus-produced IKK SS/AA protein in which alanines were substituted for the serine residues at positions 177 and 181 in the IKK activation loop (Fig. 6B, lanes 6 to 10). There was no IKK -mediated phosphorylation of this protein, although both the wild-type and constitutively active IKK proteins used in this experiment could phosphorylate the baculovirus-produced IKK K/M protein (Fig. 6B, lanes 13 and 14). There were equal quantities of the different IKK proteins used in these assays (Fig. 6B, lanes 1 to 5, lower panel) and equal quantities of the baculovirusproduced IKK SS/AA and IKK K/M substrates in these assays (Fig. 6B, lanes 6 to 15, lower panel). These results suggest that IKK likely phosphorylates the activation loop of IKK. IKK does not phosphorylate IKK in vitro. It was important to determine whether IKK could phosphorylate an IKK substrate in in vitro kinase assays. Wild-type or mutant IKK proteins produced following transfection of COS cells were assayed for their ability to phosphorylate baculovirus-produced wild-type IKK or the IKK mutants SS/AA and K/M (Fig. 7A). The immunoprecipitated IKK proteins did not result in background phosphorylation (Fig. 7A, lanes 1 to 5), while the baculovirus-produced IKK protein exhibited a low level of autophosphorylation (Fig. 7A, lane 6). IKK phosphorylation was not stimulated by the addition of wild-type, constitutively active, or mutant IKK constructs (Fig. 7A, lanes 7 to 11). The IKK proteins also did not increase the phosphorylation of the baculovirus-produced IKK SS/AA (Fig. 7A, lanes 12 to 17) or IKK K/M (Fig. 7A, lanes 18 and 19) substrates. Western blot analysis indicated that there were equivalent amounts of IKK (Fig. 7B, lanes 1 to 5) and wild-type and mutant IKK (Fig. 7B, lanes 6 to 19) substrates used in these kinase assays. Since the IKK proteins did not enhance the in vitro phosphorylation of IKK, it was important to address whether these IKK proteins exhibited kinase activity with an I B substrate. Each of the IKK proteins used in part A were tested for their ability to phosphorylate GST fusion proteins containing the amino-terminal 54 amino acids of I B or a mutant I B protein in which serine residues 32 and 36 were changed to alanine. Wild-type and constitutively active IKK proteins strongly phosphorylated wild-type GST-I B (Fig. 7C, lanes 1 and 2), while the mutant IKK proteins did not significantly phosphorylate this substrate (Fig. 7C, lanes 3 to 5). The IKK proteins did not phosphorylate the GST-I B protein mutant at serine residues 32 and 36 (Fig. 7C, lanes 6 to 10). These results indicate that although IKK did not phosphorylate IKK, it strongly phosphorylated the I B substrate. In vivo analysis of constitutively active IKK proteins. Finally, we addressed whether our results suggesting a role for IKK in modulating IKK phosphorylation and kinase activity could be correlated with in vivo studies regarding IKK activation of an NF- B reporter construct. In these studies, TNF- was not used to stimulate the activity of the transfected IKK and IKK cdnas because this cytokine itself strongly activates NF- B reporter constructs (24, 38). Instead, we tested the ability of dominant negative IKK and IKK mutants to alter the ability of constitutively active IKK and IKK constructs to activate gene expression of an NF- B reporter construct. An HIV-1 LTR-luciferase reporter construct which contains two NF- B binding sites was transfected into COS cells with either a constitutively active IKK or IKK construct (Fig. 8). In addition, either of two dominant negative IKK or IKK mutants was also cotransfected. Thus, the ability of the dominant negative IKK and IKK mutants to prevent IKK activation of an NF- B reporter construct could be assayed. Both of the constitutively active IKK constructs, IKK SS/EE and IKK SS/EE, activated gene expression from the HIV-1 LTRluciferase reporter (Fig. 8). Neither of these constitutively active IKK constructs stimulated gene expression from an HIV-1 LTR-luciferase reporter construct with mutated NF- B binding sites (data not shown). Cotransfection of either of the two dominant negative IKK constructs prevented IKK SS/EE activation of the NF- B reporter construct (Fig. 8). This result may be explained by the fact that the IKK SS/EE protein formed heterodimers with the IKK dominant negative mutants and thus was not able to phosphorylate endogenous IKK or endogenous I B. In contrast, neither of the dominant negative IKK mutants was able to significantly inhibit IKK SS/EE activation of the NF- B reporter construct (Fig. 8). Since IKK SS/EE does not require phosphorylation by IKK for stimulation of its kinase activation, the dominant negative IKK constructs would not be expected to alter IKK SS/EE activation of the NF- B reporter. These transfection studies provide indirect evidence that IKK may modulate IKK activation of the NF- B pathway. DISCUSSION In this study, we present several lines of evidence that IKK can modulate IKK function. First, we demonstrate that dominant negative IKK mutants prevent TNF- -induced phosphorylation of IKK. Second, we show that wild-type and constitutively active IKK proteins stimulate IKK phosphorylation both in transfection assays and following isolation of high-molecular-weight IKK complexes. Third, our data indicate that IKK stimulates IKK kinase activity for the I B substrate. Finally, we demonstrate that IKK can phosphorylate IKK in in vitro kinase assays. These results suggest that IKK likely modulates IKK function. Our studies utilized transient-expression assays to analyze IKK function. Thus, we cannot rule out that these results might not entirely reflect those obtained with IKK and IKK are present in the high-molecular-weight IKK complex. However, we did demonstrate that the presence of IKK and IKK in a complex migrating between 400 and 700 kda correlates with increases in IKK 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 IKK and IKK 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 IKK and the constitutively active IKK mutant have similar abilities to stimulate IKK phosphorylation and kinase activity. When low concentrations of these plasmids are transfected into COS cells, the constitutively active IKK mutant has a greater ability to stimulate IKK phosphorylation and kinase activity for I B than does wild-type IKK (unpublished observations). However, when larger quantities of IKK and the constitutively active IKK mutant are transfected, these constructs have a similar ability to stimulate IKK 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 IKK and IKK. A recent study examined the patterns of phosphorylation of the IKK and IKK proteins in response to different activators

3664 YAMAMOTO ET AL. MOL. CELL. BIOL. FIG. 7. IKK does not phosphorylate IKK in vitro. (A) COS cells were transfected with the indicated Flag-tagged IKK constructs. The extracts (50 g) were immunoprecipitated with M2 monoclonal antibody for wild-type IKK (lanes 1 and 7), a constitutively active IKK construct (lanes 2 and 8), or the kinase-defective IKK mutants K/M 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 IKK substrate alone (lane 6), the IKK SS/AA substrate alone (lane 12), or the different IKK proteins and either the IKK (lanes 7 to 11), the IKK SS/AA (lanes 13 to 17), or the IKK K/M (lanes 18 and 19) substrate. Following kinase assays, the supernatant was isolated by centrifugation, and the 32 P-labeled IKK and IKK 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 IKK immunoprecipitates (lanes 1 to 5) or a portion of the immunoprecipitated influenza virus hemagglutinin-tagged IKK (lanes 6 to 11), IKK SS/AA (lanes 12 to 17), or IKK K/M (lanes 18 and 19) substrate from each of the kinase assays using the epitope-specific monoclonal antibodies. (C) The different immunoprecipitated IKK proteins used in panel A were used in kinase assays with GST-I B (amino acids 1 to 54) or mutant GST-I B, in which serine residues 32 and 36 were changed to alanine. Following SDS-PAGE, autoradiography was performed.

VOL. 20, 2000 IKK REGULATION OF IKK 3665 regulating the NF- B pathway. First, it can phosphorylate I B and I B to result in their ubiquitination and subsequent degradation by the proteasome. In addition, our data suggest that IKK can phosphorylate IKK. The physiologic relevance of IKK 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- 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. FIG. 8. IKK dominant negative mutants inhibit NF- B activation by a constitutively active IKK construct. An HIV-1 LTR-luciferase construct (10 ng) was transfected into COS cells either alone ( ), with a constitutively active IKK SS/EE construct (0.5 g) (lane 2), or with 0.25 g of either IKK SS/AA or IKK K/M. The HIV-1 LTR-luciferase construct was also transfected with IKK SS/EE alone (0.3 g) or together with 0.5 g of the IKK 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 -galactosidase control plasmid. The results are the means of three independent experiments. of the NF- B pathway, including TNF-, IL-1, and NIK (11). In agreement with this study, we find that TNF- treatment of cells markedly stimulates both IKK and IKK phosphorylation. However, catalytically inactive and activation loop mutants of IKK and IKK exhibit decreased in vivo phosphorylation in response to TNF-. These data suggest that at least a portion of IKK and IKK phosphorylation in response to TNF- treatment likely results from autophosphorylation of these kinases. In contrast to the results of this latter study, which indicate that mutations in the IKK activation loop do not alter IKK phosphorylation of I B, our data and several previous studies indicate that such mutants exhibit defective kinase activity (20, 22). Thus, we suggest that phosphorylation of IKK is critical for enhancing its ability to phosphorylate both I B and IKK. IKK appears to be the dominant kinase required for activating NF- B, based on its higher level of activity for I B compared with IKK (17, 22, 24, 35, 38, 39) and the failure to activate the NF- B pathway when this gene is disrupted in mice (18). IKK, in addition to NIK (20) and MEKK1 (16), may also be involved in activating IKK kinase activity. 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