NF-κB transcription factors play an important role in the activation of the IL-2 gene in response to TCR ligation. The release of NF-κB factors to the nucleus requires phosphorylation and degradation of the inhibitory κ-B proteins (IκBs). IκBα and IκBβ phosphorylation is dependent on dual signaling by the TCR and the CD28 accessory receptor. This pathway involves a multisubunit IκB kinase (IKK) complex, which includes the IKKα (IKK-1) and IKKβ (IKK-2) kinases. We demonstrate that stimulation of primary human CD4+ T cells by CD3/CD28 activates two distinct endogenous IKK complexes, a heterodimeric IKKα/β and a homodimeric IKKβ complex. IKKβ overexpression in a Jurkat cell line resulted in the formation of a constitutively active IKK complex, which was CD3/CD28 inducible. In contrast, ectopic expression of IKKα assembled into a complex with negligible IκB kinase activity. Moreover, IKKβ, but not IKKα, overexpression enhanced transcriptional activation of the CD28 response element in the IL-2 promoter. Conversely, only kinase-inactive IKKβ interfered in the activation of the IL-2 promoter. Sodium salicylate, an inhibitor of IKKβ, but not IKKα, activity, inhibited IL-2 promoter activation as well as IL-2 secretion and interfered in activation of both the heterodimeric as well as the homodimeric IKK complexes in primary CD4+ T cells. Taken together, these data demonstrate the presence of an IKKβ-mediated signaling pathway that is activated by TCR and CD28 coligation and regulates IL-2 promoter activity.

The outcome of TCR stimulation is regulated by the simultaneous engagement of costimulatory molecules, including the CD28 receptor (1, 2). Engagement of CD28 by its ligands, CD80 or CD86, which are expressed on APCs, promotes T cell proliferation and enhances the production of various cytokines and chemokines (3, 4, 5). Stimulation of naive T cells in the absence of CD28 coligation may lead to a state of nonresponsiveness, known as anergy (6). One of the prominent features of anergic T cells is their inability to produce IL-2; CD28 plays an important role in preventing anergy induction through transcriptional activation of the IL-2 gene (6). Mutational analysis of the minimal IL-2 promoter has revealed the presence of a CD28 response element (CD28RE)3 located at −160 to −150 relative to the start site (7, 8). CD28 cooperates with TCR/CD3 in the activation of AP-1 and NF-κB transcription factors, which have cognate binding sites in the CD28RE domain (9, 10, 11).

The NF-κB pathway, in addition to its role in TCR activation, also plays a role in T cell responsiveness to TNF-α, IL-1, and phorbol esters (12, 13). In resting cells, NF-κB transcription factors are sequestered in the cytoplasm by a group of inhibitory proteins known as the IκBs (12, 13, 14). Upon stimulation, IκBα and/or IκBβ are phosphorylated on specific serine residues and are subsequently degraded through the 26S proteosome (12, 13, 14, 15, 16). This allows the release and nuclear localization of NF-κB transcription factors. The phosphorylation of IκBs is mediated by a recently identified multicomponent signalsome, known as the IκB kinase (IKK) complex (16, 17, 18, 19, 20, 21, 22). This complex contains two serine kinase subunits, IKKα (IKK-1) and IKKβ (IKK-2), with relative molecular masses of 85 and 87 kDa, respectively (17, 18, 19, 20, 21, 22). IKKα and IKKβ genes have been cloned, and their expressed proteins have been purified (23, 24, 25). IKKα and IKKβ, which are nearly 50% homologous, are able to homo- and heterodimerize in vitro and in vivo through their C-terminal leucine zipper motifs (23, 24, 25). Although the active kinase complex purified from TNF-α- and IL-1-stimulated cells contains IKKα and IKKβ heterodimers, homodimerized versions of each IKK are capable of phosphorylating IκB proteins in vitro (23, 24, 25). Interestingly, purified IKKβ has substantially higher basal IκBα kinase activity than IKKα (23, 25). Furthermore, in transient transfection studies, expression of a kinase-inactive IKKα mutant has minimal effects on the kinase activity compared with the potent inhibitory effect of kinase-inactive IKKβ (19). These findings raise the possibility that, in addition to IKK heterodimers, homodimers may contribute to NF-κB signaling in vivo. Mercurio et al. have recently demonstrated the existence of heterodimeric IKKα/IKKβ as well as homodimeric IKKβ complexes that can be activated by TNF-α stimulation in HeLa cells (23). In contrast, SLB cells contained IKKα/IKKβ heterodimers but no homodimers (23). This suggests the existence of heterogeneous IKK complexes that may play differential roles in NF-κB activation in different cell types. The possible existence of homo- or heterodimeric IKK complexes in T cells has not been addressed.

Activation of the IKK complex is dependent on the action of upstream kinases (9, 18, 19, 20, 21, 22, 25, 26, 27, 28). NF-κB-inducing kinase (NIK) and MEKK1, members of the mitogen-activating protein kinase kinase kinase (MAP3K) family, have been shown to activate the IKK complex (9, 20, 21, 25, 26, 27, 28, 29). NIK copurifies with the IKK complex and has been shown to be involved in IKK activation by IL-1 and TNF-α (20, 21, 26, 27). Although overexpression of NIK leads to stimulation of IKKα and IKKβ activities in vivo, NIK preferentially phosphorylates IKKα and has little activity on IKKβ (26). At least one study has suggested that NIK is involved in CD28 costimulation (27). While MEKK1 also copurifies with IKK complexes (18), its overexpression leads to differential activation of IKKβ in Jurkat and other cell types (9, 29). HTLV-I tax protein also activates the NF-κB complex by interacting with MEKK1 and preferentially activating IKKβ kinase activity (30). However, in some cell types, MEKK1 has been linked to both IKKα and IKKβ activation (25, 31). Taken together, it is possible that several MAP3Ks may be involved in the activation of IKK complexes in T cells.

We were interested in determining whether signaling by TCR/CD3 and CD28 activates different IKK complexes in primary human T lymphocytes. In addition, we were interested in whether there is a functional relationship between IKKs and in the activation of the CD28RE in the IL-2 promoter. Here we describe that CD3 and CD28 costimulation results in the activation of IKKα/IKKβ heterodimeric and IKKβ homodimeric complexes in primary human CD4+ T cells. We also provide evidence that IKKβ, rather than IKKα, is critical for activation of the CD28RE in the IL-2 promoter.

OKT3 (anti-CD3) was obtained from Ortho Pharmaceuticals (Raritan, NJ), and the 9.3 mAb (anti-CD28) were provided by Bristol-Meyer Squibb (Princeton, NJ). The primary stimulating Abs were cross-linked with mAb 187.1 (Bristol-Meyer Squibb). For Western blotting and immunoprecipitation experiments, polyclonal anti-IKKα, anti-IKKβ, anti-IκBα, and anti-IκBβ Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Flag (M2) Abs were obtained from Sigma (St. Louis, MO). Anti-IKKγ (IKAP1) Abs and GST-IκBα1–54 have been described by us (23). HRP conjugated to protein A was obtained from Amersham (Arlington Heights, IL). Tosyl-activated magnetic beads and M-450 anti-CD4 beads were purchased from Dynal (Great Neck, NY). PMA and ionomycin were purchased from Sigma.

Jurkat T cells (clones BMS2, Jurkat-hIL-2-Luc) were grown in RPMI medium, supplemented with 10 mM HEPES (pH 7.4), 10% FCS, 2 mM glutamine, 100 U of penicillin, and 100 μg of streptomycin/ml. The Jurkat-hIL-2-luciferase cell line was provided by Dr. A. Weiss (Howard Hughes Medical Institute, University of California, San Francisco, CA), while Jurkat BMS2 was a gift from Dr. B. Mittler (Bristol Myers Squibb) (32).

Mononuclear cells were isolated from human peripheral blood by density centrifugation and depletion of adherent cells on plastic culture dishes. CD4+ T cells were positively selected with anti-CD4 Dynabeads according to the manufacturer’s instructions (Dynal). This yielded a T cell subset that was >98% positive for the CD4 marker as determined by dual-color CD4/CD8 flow cytometry. Isolated CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAb coupled to tosyl-activated magnetic beads as previously described (21). Cells were replenished with 40 U/ml rIL-2 (Chiron, Emeryville, CA) on day 3 and allowed to complete their growth cycle over the course of 12–13 days. At this point, IL-2 and beads were removed, and cells were allowed to return to their resting state over a 48-h period. Resting vs activated state was assessed by cell size analysis in a Coulter Counter (Hialeah, FL). Cells were subjected to flow cytometry for a second time to confirm that >95% of the cells remained CD4+.

IκB kinase constructs were previously described and characterized (21). Flag-tagged wild-type IKKα and IKKβ and kinase-inactive mutants (K44A) cloned in pRK5 vector were provided by Dr. M. Rothe (Tularik, San Francisco, CA) (21). Full-length kinase-inactive MEKK1 (K1253 to M), a gift from Dr. M. Karin (University of California, San Diego, CA) (33), was cloned in pcDNA3 (Invitrogen, Carlsbad, CA). The JNK-interacting protein 1 (JIP-1), cloned in CMV5 vector (34), was a gift from Dr. R. Davis (Howard Hughes Medical Institute, Worcester, MA) (33). The CD28RE/AP-1 luciferase reporter and related mutants, provided by Dr. A. Weiss (Howard Hughes Medical Institute) were cloned in pAEODLO vectors (8). CD28RE/AP-1 Luc contains four tandem copies of the sequence 5′-tttaaagaaattccaaagagtcatca-3′, which is situated 150–160 bp upstream from the start site of the IL-2 gene (8). For cellular transfection, we used the stated amounts of an individual or combination of plasmids for electroporation (240 V, 950 μF) into 1 × 107 Jurkat cells as previously described (9, 35).

CD4+ T cells (2 × 107) were lysed in kinase lysis buffer and cleared by centrifugation as previously described (23). For immunoprecipitation, 200 μg of precleared lysate was treated with 2 μg of anti-IKKα Abs, bound to protein A-Sepharose, and rocked for 2 h at 4°C (9, 17, 23). To completely remove all IKKα, this step was repeated once. The remaining supernatants were subjected to further immunoprecipitation using 2 μg of the antisera to either IKKβ or IKKγ. Immunoprecipitated complexes were washed extensively and subjected to immunoblotting as previously described (9, 23). IKKα, IKKβ, and IKKγ Abs were used at a 1/1000 dilution for primary staining. Protein A-conjugated HRP was used at a 1/2000 dilution, and blots were developed by enhanced chemiluminescence. Western blotting of cellular lysates was performed as described previously (9).

Jurkat cells or CD4+ resting T cells (1 × 107) in 1 ml of RPMI were left unstimulated or were stimulated with 2 μg/ml anti-CD3 or a combination of 2 μg/ml anti-CD3 and 2 μg/ml anti-CD28 mAb, secondarily cross-linked with 10 μg/ml mAb 187.1. In another group of experiments, primary human CD4+ T cells were treated with 20 mM sodium salicylate (36) for 2 h before stimulation as described above. All kinase assays were performed as described previously (23). Briefly, after cell lysates were precleared with protein A-Sepharose beads, 200 μg of lysate was treated with 2 μg of the specific anti-IKK protein A-Sepharose for 2 h. In a third variation, the immunoprecipitation was performed sequentially, first by adding anti-IKKα in two rounds, and then adding either anti-IKKβ or anti-IKKγ to the remaining supernatants (23). Immune complexes were washed and equilibrated in kinase buffer as previously described (23). Kinase reactions were initiated by the addition of 10 μCi of [γ-32P]ATP and 3 μg GST-IκB1–54 substrate. The reaction was conducted for 30 min at 30°C. Products were analyzed on SDS-PAGE and detected by autoradiography.

Twelve micrograms of the indicated reporter gene constructs were transiently transfected into 107 Jurkat cells (9). The cells were rested for 24 h and then stimulated for 6 h with 2 μg/ml anti-CD3, a combination of 2 μg/ml anti-CD3 and 2 μg/ml anti-CD28 mAb, or a combination of 100 nM PMA and 1 μg/ml ionomycin (PMA+I). The cells were washed and lysed in luciferase buffer (Analytical Luminescence, Ann Arbor, MI), and luciferase activity was measured in 50 μg of lysate in a Monolight 2010 luminometer (Analytical Luminescence). Transfection efficiency was monitored by cotransfection of a β-galactosidase plasmid (CMV-β-gal); β-galactosidase activity was used for adjusting luciferase values among cell populations transfected with different vector combinations (9).

Jurkat T cells (1 × 106) in 2 ml of RPMI were treated with either anti-CD3 or anti-CD3 plus anti-CD28 mAb in the presence or the absence of 20 mM sodium salicylate for 24 h. Cells were removed by centrifugation, and the supernatants were collected. Triplicate aliquots were analyzed by ELISA for the presence of IL-2 (UMAB Cytokine Core Laboratory, Baltimore, MD).

Nuclear protein was extracted from 107 Jurkat T cells, harvested at the indicated time to evaluate DNA binding of NF-κB as previously described (37). Briefly, after washing, the cell pellet was suspended in 1 ml of buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, and 1 mM DTT) containing 0.1% Triton X-100. After incubating for 10 min on ice, the lysates were centrifuged, and the nuclei were resuspended in 20–40 μl of buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 1 mM DTT). This suspension was incubated for 30 min on ice followed by centrifugation at 10,000 × g for 20 min. Double-stranded oligonucleotides containing a consensus NF-κB sequence (Promega, Madison, WI) were end labeled with T4 polynucleotide kinase in the presence of [γ-32P]dATP (Amersham). The DNA binding reaction was performed at room temperature for 30 min in a final volume of 15 μl, which contained 3–5 μg of nuclear extract, oligonucleotide probe (40 fmol), and binding buffer containing 100 μg/ml poly(dI-dC) as nonspecific competitor. Reactions were subjected to electrophoresis on nondenaturing 5% polyacrylamide gels in 0.5× TBE at 125 mA for 4 h at 4°C.

CD28 is an important costimulatory receptor that regulates cellular activation by the TCR/CD3 complex (1, 2, 3, 4, 5). While CD28 can independently signal T cells (38), this receptor also participates in signaling events that require TCR/CD3 coligation. This has been best demonstrated in the activation of the N-terminal c-Jun kinase (JNK) cascade, which regulates transcriptional activation of AP-1 response elements in the IL-2 promoter (9, 35, 39). More recently, CD28 has been shown to contribute to the activation of a composite CD28RE in the IL-2 promoter (8). Since NF-κB signaling is mediated through a multicomponent IκB kinase (IKK) complex, we wanted to determine whether immunoprecipitation of IKKα or IKKβ shows dual receptor requirement for the activation of IKK complexes. In vitro kinase assays using GST-IκBα as a substrate showed CD3- and CD28-inducible kinase activity in IKKα and IKKβ immunoprecipitates obtained from Jurkat T cells (Fig. 1,A). In contrast, ligation of CD3 alone had no effect on IKK activation, indicating that CD28 cooperates with CD3 in IKK activation. P+I treatment had the same effect as dual receptor ligation (Fig. 1 A). Similar cooperation between PMA and anti-CD28 mAb in IKK activation in Jurkat T cells has been demonstrated by Harhaj and Sun (11).

FIGURE 1.

CD3/CD28 costimulation of Jurkat T cells induces IKKα and IKKβ activities. A, Immune complex kinase assay using anti-IKKα and -IKKβ Abs to precipitate Jurkat cell lysates. Cells were left untreated or were treated with anti-CD3 or a combination of anti-CD3 and anti-CD28 mAb, secondarily cross-linked by 187.1 mAb, for 15 min. PMA (100 nM) and ionomycin (1 μM) treatment was used as a positive control. IKK complexes were immunoprecipitated from cleared lysates with Abs to IKKα or IKKβ, bound to protein A-Sepharose. Kinase assays were conducted using GST-IκBα1–54 as substrate. The fold increase in kinase activity was determined by phosphorimaging scanning. This experiment was repeated three times with similar results. B, Kinetics of IκBα and IκBβ phosphorylation and their degradation during stimulation of Jurkat T cells with anti-CD3 and anti-CD28 mAb for the indicated time periods. The top two panels show phosphorylation of GST-IκBα1–54 and GST-IκBβ1–44, respectively, by anti-IKKβ immunoprecipitates. The next two panels represent Western blotting of IκBα and IκBβ with specific Abs to show degradation of these proteins in crude cell lysates. IκBα and IκBβ migrated as 37- and 45-kDa proteins, respectively. In the bottom panel, EMSA was used to examine nuclear localization of NF-κB. Nuclear proteins were extracted from stimulated cells, and shift assays were conducted as described in Materials and Methods.

FIGURE 1.

CD3/CD28 costimulation of Jurkat T cells induces IKKα and IKKβ activities. A, Immune complex kinase assay using anti-IKKα and -IKKβ Abs to precipitate Jurkat cell lysates. Cells were left untreated or were treated with anti-CD3 or a combination of anti-CD3 and anti-CD28 mAb, secondarily cross-linked by 187.1 mAb, for 15 min. PMA (100 nM) and ionomycin (1 μM) treatment was used as a positive control. IKK complexes were immunoprecipitated from cleared lysates with Abs to IKKα or IKKβ, bound to protein A-Sepharose. Kinase assays were conducted using GST-IκBα1–54 as substrate. The fold increase in kinase activity was determined by phosphorimaging scanning. This experiment was repeated three times with similar results. B, Kinetics of IκBα and IκBβ phosphorylation and their degradation during stimulation of Jurkat T cells with anti-CD3 and anti-CD28 mAb for the indicated time periods. The top two panels show phosphorylation of GST-IκBα1–54 and GST-IκBβ1–44, respectively, by anti-IKKβ immunoprecipitates. The next two panels represent Western blotting of IκBα and IκBβ with specific Abs to show degradation of these proteins in crude cell lysates. IκBα and IκBβ migrated as 37- and 45-kDa proteins, respectively. In the bottom panel, EMSA was used to examine nuclear localization of NF-κB. Nuclear proteins were extracted from stimulated cells, and shift assays were conducted as described in Materials and Methods.

Close modal

IKK activation results in phosphorylation of IκBα and IκBβ (12, 13, 14, 15, 16), which, in turn, leads to ubiquitination and proteolytic degradation of these proteins and release of NF-κB transcription factors into the nucleus (12, 13, 14, 15, 16). While recombinant IκBα as well as IκBβ were substrates for CD3- and CD28-inducible IKK complexes, the former substrate was more robustly phosphorylated than the latter in vitro kinase assays (Fig. 1,B, top two panels). This finding agrees with previous studies that showed that IκBα is a better substrate for the IKKs than IκBβ (17). The kinetics of IκBα and IκBβ phosphorylation were coincident with the degradation of endogenous IκBα as well as the appearance of DNA-binding NF-κB complexes in the nucleus at approximately 15–30 min (Fig. 1,B, bottom three panels). Although de novo IκBα expression was detected at 60 min (due to the transcriptional activation of its gene by NF-κB), the intranuclear translocation of NF-κB factors still continues at this time point (Fig. 1,B, bottom panel). Since IκBβ degradation was still seen at 60 min, IκBβ probably plays a role in prolonged NF-κB activation in T cells. Taken together with the findings in Fig. 1 A, these data show cooperation between CD3 and CD28 in activating IKK kinases and NF-κB transcriptional activity in T cells.

To investigate IKK activation in primary human T lymphocytes, we generated CD4+ T cell blasts, which were rested for 48 h before restimulation. This was accomplished by using magnetic bead separation of a CD4+ subset from human peripheral blood, followed by expansion in tissue culture with anti-CD3- and anti-CD28 (9.3 mAb)-conjugated beads (32). After allowing these cells to return to the resting state, CD3 and CD28 coligation or P+I stimulation induced IKK activity (Fig. 2), which could be precipitated by either anti-IKKα or -IKKβ Abs. These data confirm that endogenous IKK complexes in primary T cells are activated by dual CD3 and CD28 coligation. While IKK activation was detected with both anti-IKKα and anti-IKKβ immune complexes, it was not clear from these assays whether CD3/CD28 coligation activated heterodimeric or homodimeric IKK complexes.

FIGURE 2.

Activation of IKKα and IKKβ in primary CD4+ T cells. CD4+ T cells were purified from human peripheral blood and expanded by stimulating with anti-CD3 and anti-CD28 immobilized on polystyrene magnetic beads (32 ). After entering the resting stage, cells were stimulated, and in vitro kinase assays performed as described in Fig. 1 A. The IKK activity presented for primary CD4+ was reproduced several times. Similar results were obtained with CD4+ T cells from three different donors.

FIGURE 2.

Activation of IKKα and IKKβ in primary CD4+ T cells. CD4+ T cells were purified from human peripheral blood and expanded by stimulating with anti-CD3 and anti-CD28 immobilized on polystyrene magnetic beads (32 ). After entering the resting stage, cells were stimulated, and in vitro kinase assays performed as described in Fig. 1 A. The IKK activity presented for primary CD4+ was reproduced several times. Similar results were obtained with CD4+ T cells from three different donors.

Close modal

Recombinant IKKα or IKKβ form homo- and heterodimeric kinase complexes in vitro, suggesting that heterogeneous IKK com- plexes may exist in vivo (23, 24). In this regard, Mercurio et al. have recently shown that HeLa cells contain two distinct IKK complexes, one consisting of IKKα/IKKβ heterodimers, and the other containing IKKβ homodimers (23). Moreover, both complex types could be activated by TNF-α, with heterodimeric complexes showing more abundant IKK activity than homodimeric complexes (23). However, not all cell types contain heterogeneous complexes (23). We were interested in determining the composition of IKK complexes in primary human T cells. First, we examined primary human CD4+ as well as Jurkat T cell lysates for expression of different IKK subunits. Western blot data indicated that IKKβ and IKKα were abundantly expressed in Jurkat and primary T cells (Fig. 3,A, lanes 1–4). The relative amounts of IKKα or IKKβ messages were not affected by cellular stimulation (data not shown). Secondly, we used a sequential immunoprecipitation approach, previously used in HeLa cells (23), to examine primary T cells for assembly of IKKα/IKKβ heterodimers and homodimers (23). This required preclearing of IKKα-containing complexes from CD4+ cell lysates with excess anti-IKKα Abs before precipitating any possible remaining IKKβ from the supernatant with IKKβ-specific antiserum (23). These immunoprecipitates were transferred to immunoblotting membranes, which were sequentially overlaid with IKKα and IKKβ antisera. As demonstrated in Fig. 3 B (lanes 1 and 4), anti-IKKα Abs precipitated both IKKα and IKKβ from the cell lysates, indicating the presence of a heterodimeric complex. The remaining supernatant was devoid of IKKα (lane 2), but still contained IKKβ protein, which could be precipitated by the anti-IKKβ antiserum (lane 6). These complexes were devoid of IKKα (lane 3). The residual amount of IKKβ protein after IKKα clearance (lane 6) was more abundant than the amount of IKKβ that associates with IKKα (lane 4). These results demonstrate the presence of a homodimeric IKKβ complex in primary human CD4+ T cells. No evidence was obtained, however, for the existence of IKKα homodimers in experiments in which we attempted to immune precipitate IKKα from lysates that were precleared with anti-IKKβ Abs (data not shown).

FIGURE 3.

Evidence for the existence of two distinct IKK complexes in primary CD4+ T cells. A, Western blot analysis of IKKα, IKKβ, and IKKγ expression in Jurkat and primary human CD4+ T cells. Sixty micrograms of lysate protein from Jurkat or CD4+ T cells were subjected to immunoblotting, using a 1/1000 dilution of the primary Abs, followed by a 1/2000 dilution of the protein A-conjugated HRP. IKKβ Ab reacted with two other unknown faster migrating proteins. Note that IKKγ stains as a 50/52-kDa doublet (40 ). Similar results were obtained upon blotting of CD4+ T cell lysates from three different donors. B, Immunoblotting for IKKs after sequential immunoprecipitation of IKKα and IKKβ from primary CD4+ T cells. Two hundred micrograms of lysate was treated initially with 2 μg of anti-IKKα bound to protein A-Sepharose. The immune complexes were removed, and the supernatant was reprecipitated with immobilized anti-IKKα. The IKKα-depleted supernatant was then precipitated with 2 μg of anti-IKKβ bound to protein A-Sepharose beads. The immunoprecipitates were washed extensively, resolved in a 10% SDS-PAGE, and blotted onto nylon membranes. Blots were developed as described in A. IKKα (1) presents the heterodimeric complex precipitated by the first round of anti-IKKα treatment. IKKα (2) lanes represent the second round of immunoprecipitation and demonstrate that the first round effectively removed IKKα. IKKβ homodimeric complexes were immunoprecipitated with anti-IKKβ from the IKKα-depleted lysate. Similar results were obtained with CD4+ T cells isolated from two other donors. C, Western blot showing the same experiment as that in B, except that the lysates, after the second round of IKKα precipitation, were subjected to either anti-IKKβ or anti-IKKγ immunoprecipitation. IKKα indicates the heterodimeric complex precipitated by first round of anti- IKKα treatment. IKKα (2 ) lanes shows that heterodimeric complexes are completely removed. IKKβ homodimeric complexes were immunoprecipitated with anti-IKKβ or anti-IKKγ from the IKKα depleted lysate. As expected, IKKγ Abs immunoprecipitated IKKβ from CD4+ T cell lysates of two other donors. D, CD3/CD28 coligation activates both heterodimeric and homodimeric IKK complexes. IKK complexes were immunoprecipitated in a sequential fashion as described in B and C above. The immunoprecipitates were examined for IκB kinase activity as described in Fig. 1 A. The indicated IKKα activity was detected with the first round of immunoprecipitation using anti-IKKα Abs. Subsequent immunoprecipitation with IKKα Ab did not reveal any residual IKK complexes containing this isoform. The activities associated with IKKβ homodimeric complexes were precipitated with anti-IKKβ or anti-IKKγ from lysates in which the heterodimeric complexes were completely removed. These experiments were reproduced three times.

FIGURE 3.

Evidence for the existence of two distinct IKK complexes in primary CD4+ T cells. A, Western blot analysis of IKKα, IKKβ, and IKKγ expression in Jurkat and primary human CD4+ T cells. Sixty micrograms of lysate protein from Jurkat or CD4+ T cells were subjected to immunoblotting, using a 1/1000 dilution of the primary Abs, followed by a 1/2000 dilution of the protein A-conjugated HRP. IKKβ Ab reacted with two other unknown faster migrating proteins. Note that IKKγ stains as a 50/52-kDa doublet (40 ). Similar results were obtained upon blotting of CD4+ T cell lysates from three different donors. B, Immunoblotting for IKKs after sequential immunoprecipitation of IKKα and IKKβ from primary CD4+ T cells. Two hundred micrograms of lysate was treated initially with 2 μg of anti-IKKα bound to protein A-Sepharose. The immune complexes were removed, and the supernatant was reprecipitated with immobilized anti-IKKα. The IKKα-depleted supernatant was then precipitated with 2 μg of anti-IKKβ bound to protein A-Sepharose beads. The immunoprecipitates were washed extensively, resolved in a 10% SDS-PAGE, and blotted onto nylon membranes. Blots were developed as described in A. IKKα (1) presents the heterodimeric complex precipitated by the first round of anti-IKKα treatment. IKKα (2) lanes represent the second round of immunoprecipitation and demonstrate that the first round effectively removed IKKα. IKKβ homodimeric complexes were immunoprecipitated with anti-IKKβ from the IKKα-depleted lysate. Similar results were obtained with CD4+ T cells isolated from two other donors. C, Western blot showing the same experiment as that in B, except that the lysates, after the second round of IKKα precipitation, were subjected to either anti-IKKβ or anti-IKKγ immunoprecipitation. IKKα indicates the heterodimeric complex precipitated by first round of anti- IKKα treatment. IKKα (2 ) lanes shows that heterodimeric complexes are completely removed. IKKβ homodimeric complexes were immunoprecipitated with anti-IKKβ or anti-IKKγ from the IKKα depleted lysate. As expected, IKKγ Abs immunoprecipitated IKKβ from CD4+ T cell lysates of two other donors. D, CD3/CD28 coligation activates both heterodimeric and homodimeric IKK complexes. IKK complexes were immunoprecipitated in a sequential fashion as described in B and C above. The immunoprecipitates were examined for IκB kinase activity as described in Fig. 1 A. The indicated IKKα activity was detected with the first round of immunoprecipitation using anti-IKKα Abs. Subsequent immunoprecipitation with IKKα Ab did not reveal any residual IKK complexes containing this isoform. The activities associated with IKKβ homodimeric complexes were precipitated with anti-IKKβ or anti-IKKγ from lysates in which the heterodimeric complexes were completely removed. These experiments were reproduced three times.

Close modal

Nonlymphoid cells have recently been shown to include a third noncatalytic IKK subunit, which has been designated IKKγ or IKK-associated protein (IKKAP1) (23, 40). Using an antiserum to IKKγ, we could demonstrate that Jurkat as well as primary human CD4+ T cells, in addition to IKKα and IKKβ, express IKKγ (Fig. 3,A, lanes 5 and 6). Since IKKγ has selective affinity for IKKβ (40), we asked whether IKKγ is present in the homodimeric IKKβ complexes shown in Fig. 3,B. In a further sequential immunoprecipitation experiment, anti-IKKγ Abs were used to immunoprecipitate this subunit from an IKKα-depleted CD4+ cell supernatant. Subsequent immunoblotting showed that IKKγ interacts with IKKβ complex in vivo in the absence of IKKα (Fig. 3,C, lanes 4 and 8). However, heterodimeric complexes also contained IKKγ (40) (not shown). Therefore, IKKγ is a common component of heterodimeric and homodimeric IKK complexes in T cells. Also notice that immunoprecipitation of IKKβ after IKKα depletion (Fig. 3,C, lanes 3 and 7), yielded positive blotting for IKKβ alone. This confirms the existence of IKKβ homodimers, as noted in Fig. 3 B.

Since the data in Fig. 3, B and C, indicate the existence of heterogeneous IKK complexes in primary human CD4+ T cells, we asked whether one or both complexes could be activated by CD3 and CD28 or P+I stimulation. This experiment was conducted in exactly the same way as shown in Fig. 3,C, except that the washed immune complexes were incubated with GST-IκBα and [γ-32P]ATP. Our data show that heterodimeric complexes, precipitated with anti-IKKα (Fig. 3,D, lanes 1–4), and homodimeric complexes, precipitated with either anti-IKKβ (lanes 5–8) or anti-IKKγ (lanes 9–12) after IKKα/IKKβ heterodimer depletion, harbored CD3- and CD28-inducible as well as PMA- and ionomycin-inducible IKK activities. Neither type of complex could be activated by anti-CD3 alone (Fig. 3 D, lanes 2, 6, and 10). These results demonstrate that two distinct IKK complexes are activated upon CD3/CD28 and P+I costimulation in primary human CD4+ T cells.

To study the biological significance of IKK activation by TCR/CD28, we concentrated on the IL-2 gene, since its promoter contains a composite c-Rel/AP-1 element, previously characterized as the CD28RE (7, 8, 9). CD28RE plays a critical role in CD3- and CD28-induced activation of the IL-2 gene (7, 8, 9). Moreover, CD28RE is a key integrator of TCR/CD3 and CD28 signaling pathways (7, 8, 9). In a Jurkat cell line transfected with a CD28RE/AP-1 luciferase gene construct, IKKα overexpression had no independent stimulatory effect (Fig. 4,A). In contrast, IKKβ overexpression dramatically enhanced CD3- and CD28-stimulated reporter activity. Similar trends were seen in PMA- and ionomycin-activated cells (Fig. 4,A). It is interesting to note that IKKα and IKKβ overexpression in Jurkat cells generate anti-Flag-precipitable complexes with different levels of IKK activity (Fig. 4,B). While immunoprecipitation of exogenously expressed IKKβ yielded constitutively active complexes, immunoprecipitation of Flag-tagged IKKα yielded a complex with negligible levels of kinase activity (Fig. 4,B). These differences were not due to different amounts of these kinases being expressed, since anti-Flag immunoblotting showed roughly equal amounts of the kinase proteins in the cellular lysate (Fig. 4,C). Since overexpressed IKKβ does not coprecipitate significant amounts of endogenous IKKα (not shown), the data in Fig. 4,B probably reflect the formation of active IKKβ homodimers. The activity of these complexes could be further increased by CD3 and CD28 or P+I stimulation (Fig. 4 B, lanes 5 and 6). While overexpressed IKKα was not biochemically active in Jurkat cells, we have previously shown that overexpression of Flag-tagged IKKα in a macrophage cell line can result in the formation of an LPS-inducible IKK complex (41).

FIGURE 4.

Effects of exogenously expressed IKKα and IKKβ on induction of IKK activity and activation of the CD28RE/AP-1 element. A, Reporter gene assay showing that IKKβ, but not IKKα, overexpression enhances CD28RE/AP-1 Luc activation. Cells were transfected with 6 μg of empty pCDNA1.1 vector, Flag-IKKα, or Flag-IKKβ plus 3 μg of 4XCD28RE/AP-1 Luc and 3 μg of pCMV-βGal. Cells were rested for 24 h. Duplicate aliquots were preincubated for 2 h with 20 mM sodium salicylate, then treated with anti-CD3 and anti-CD28 mAb or PMA (100 nM) and ionomycin (1 μM) for 6 h. Luciferase activity was measured in 50 μg of lysate in a Monolight 2010 luminometer (Analytical Luminescence). The fold increase in luciferase activity was calculated with reference to unstimulated values for cells transfected with 4XCD28RE/AP-1 Luc plus pCDNA1.1. Similar results were obtained in two other independent experiments. Cotransfected β-galactosidase was used to correct for the efficiency of transfection between different populations (9 ). B, Immune complex kinase assay showing that epitope-tagged IKKβ, but not tagged IKKα, is activated by CD3 and CD28 in Jurkat cells. Cells were transfected with either 20 μg of Flag-IKKα or Flag-IKKβ, rested for 24 h, and then stimulated with anti-CD3 and CD28 mAb or P+I for 15 min. Cell lysates were immunoprecipitated with anti-Flag (M2). These immunoprecipitates were examined for their abilities to phosphorylate GST-IκBα as described in Fig. 1 A. Note that exposure of the gel for a longer time period showed low levels of IKKα activity (not shown). IKKβ activity could be detected with as little as 2 μg of IKKβ/transfection. However, transfection with 40 μg of IKKα did not result in a substantial increase in the kinase activity. C, Anti-Flag Western blot showing the levels of exogenously expressed IKKs from A.

FIGURE 4.

Effects of exogenously expressed IKKα and IKKβ on induction of IKK activity and activation of the CD28RE/AP-1 element. A, Reporter gene assay showing that IKKβ, but not IKKα, overexpression enhances CD28RE/AP-1 Luc activation. Cells were transfected with 6 μg of empty pCDNA1.1 vector, Flag-IKKα, or Flag-IKKβ plus 3 μg of 4XCD28RE/AP-1 Luc and 3 μg of pCMV-βGal. Cells were rested for 24 h. Duplicate aliquots were preincubated for 2 h with 20 mM sodium salicylate, then treated with anti-CD3 and anti-CD28 mAb or PMA (100 nM) and ionomycin (1 μM) for 6 h. Luciferase activity was measured in 50 μg of lysate in a Monolight 2010 luminometer (Analytical Luminescence). The fold increase in luciferase activity was calculated with reference to unstimulated values for cells transfected with 4XCD28RE/AP-1 Luc plus pCDNA1.1. Similar results were obtained in two other independent experiments. Cotransfected β-galactosidase was used to correct for the efficiency of transfection between different populations (9 ). B, Immune complex kinase assay showing that epitope-tagged IKKβ, but not tagged IKKα, is activated by CD3 and CD28 in Jurkat cells. Cells were transfected with either 20 μg of Flag-IKKα or Flag-IKKβ, rested for 24 h, and then stimulated with anti-CD3 and CD28 mAb or P+I for 15 min. Cell lysates were immunoprecipitated with anti-Flag (M2). These immunoprecipitates were examined for their abilities to phosphorylate GST-IκBα as described in Fig. 1 A. Note that exposure of the gel for a longer time period showed low levels of IKKα activity (not shown). IKKβ activity could be detected with as little as 2 μg of IKKβ/transfection. However, transfection with 40 μg of IKKα did not result in a substantial increase in the kinase activity. C, Anti-Flag Western blot showing the levels of exogenously expressed IKKs from A.

Close modal

To determine whether overexpressed IKKα and IKKβ also yield differential effects on the full-length IL-2 promoter, we compared the effects of wild-type and kinase-inactive IKKs on a cotransfected IL-2 Luc promoter (Fig. 5). While kinase-inactive IKKα had a minor effect, kinase-inactive IKKβ was a potent (76% decrease) inhibitor of IL-2 Luc activity (Fig. 5). Wild-type IKKα and IKKβ had minimal effects (Fig. 5). The effect of kinase-inactive IKKβ was comparable to the effect of kinase-inactive MEKK1, which acts as a MAP3K in the JNK cascade (Fig. 5). We have recently shown that the JNK cascade plays a critical role in regulating the CD28RE/AP-1 element in the IL-2 (9). The role of JNK in this activation of this promoter is also demonstrated by the inhibitory effect of the JIP-1 (33), which sequesters endogenous JNK in the cell (Fig. 5). Taken together, these results indicate that MEKK1 and IKKβ play critical roles in the activation of the IL-2 promoter.

FIGURE 5.

Effect of exogenously expressed IKKs and kinase-inactive MEKK1 on the activation of a full-length IL-2 Luc reporter. A luciferase assay showed that wild-type and kinase-inactive mutants of MEKK1, IKKβ, as well as JIP-1 interfere in the activation of the IL-2 Luc reporter. Jurkat T cells were transfected with 12 μg of DNA comprised of 3 μg of IL-2 Luc reporter, 3 μg of pCMV β-gal, and 6 μg of each specific kinase. Cells were incubated for 24 h and stimulated with anti-CD3 mAb, anti-CD28 mAb, and 10 nM PMA for 6 h. Luciferase activity was measured as described above. β-Galactosidase values were used to correct for the efficiency of transfections between different populations. Similar results were obtained in two additional experiments.

FIGURE 5.

Effect of exogenously expressed IKKs and kinase-inactive MEKK1 on the activation of a full-length IL-2 Luc reporter. A luciferase assay showed that wild-type and kinase-inactive mutants of MEKK1, IKKβ, as well as JIP-1 interfere in the activation of the IL-2 Luc reporter. Jurkat T cells were transfected with 12 μg of DNA comprised of 3 μg of IL-2 Luc reporter, 3 μg of pCMV β-gal, and 6 μg of each specific kinase. Cells were incubated for 24 h and stimulated with anti-CD3 mAb, anti-CD28 mAb, and 10 nM PMA for 6 h. Luciferase activity was measured as described above. β-Galactosidase values were used to correct for the efficiency of transfections between different populations. Similar results were obtained in two additional experiments.

Close modal

To determine whether endogenous, rather than exogenous, IKKβ exerts similar effects on the IL-2 promoter, we made use of a recent demonstration that sodium salicylate inhibits IKKβ, but not IKKα, activity in vivo and in vitro (36). At a dose (20 mM) that was previously shown to inhibit IKKβ in vivo (36), sodium salicylate completely inhibited the activation of a full-length IL-2 promoter luciferase construct stably transfected into a Jurkat cell line (Fig. 6,A). Furthermore, sodium salicylate suppressed induction of IL-2 secretion in Jurkat cells stimulated by CD3 and CD28 or P+I (Table I). This drug was also effective in interfering in CD28RE/AP-1 activation during CD3 and CD28 or P+I stimulation, even in the presence of overexpressed IKKβ (Fig. 4 A).

FIGURE 6.

Effect of sodium salicylate on a stably transfected IL-2 Luc reporter and activation of heterogeneous IKK complexes. A, Luciferase assay showing the effect of sodium salicylate on IL-2 Luc activation in Jurkat cells. A Jurkat cell line, stably transfected with the −327 to +46 domain of the IL-2 promoter linked to a luciferase reporter was treated with 20 mM sodium salicylate for 2 h before stimulation with anti-CD3 mAb; anti-CD3 and anti-CD28 mAb; anti-CD3, anti-CD28, and PMA (10 nM); or PMA (100 nM) and ionomycin (1 μM) for 6 h. Luciferase activity was examined as described in Fig. 5. Similar results were obtained with two additional experiments. B, Immune complex kinase assays showed the effect of salicylate on the activation of heterogeneous IKK complexes in primary CD4+ T cells. CD4+ T cells were stimulated as indicated and fractionated by sequential immunoprecipitation as described in Fig. 3,D. Duplicate samples were treated with 20 mM sodium salicylate for 2 h before stimulation. Immune complex kinase assays were conducted as described in Fig. 1 A. Sodium salicylate also interfered with IKK kinase activity during in vitro addition to the immunoprecipitates. Results were reproduced three times.

FIGURE 6.

Effect of sodium salicylate on a stably transfected IL-2 Luc reporter and activation of heterogeneous IKK complexes. A, Luciferase assay showing the effect of sodium salicylate on IL-2 Luc activation in Jurkat cells. A Jurkat cell line, stably transfected with the −327 to +46 domain of the IL-2 promoter linked to a luciferase reporter was treated with 20 mM sodium salicylate for 2 h before stimulation with anti-CD3 mAb; anti-CD3 and anti-CD28 mAb; anti-CD3, anti-CD28, and PMA (10 nM); or PMA (100 nM) and ionomycin (1 μM) for 6 h. Luciferase activity was examined as described in Fig. 5. Similar results were obtained with two additional experiments. B, Immune complex kinase assays showed the effect of salicylate on the activation of heterogeneous IKK complexes in primary CD4+ T cells. CD4+ T cells were stimulated as indicated and fractionated by sequential immunoprecipitation as described in Fig. 3,D. Duplicate samples were treated with 20 mM sodium salicylate for 2 h before stimulation. Immune complex kinase assays were conducted as described in Fig. 1 A. Sodium salicylate also interfered with IKK kinase activity during in vitro addition to the immunoprecipitates. Results were reproduced three times.

Close modal
Table I.

Inhibition of IL-2 synthesis by sodium salicylatea

StimulationsIL-2 (pg/ml)
UntreatedSodium salicylate (20 mM)
Control 23.4 ± 2.8 17.4 ± 2.3 
αCD3 40.4 ± 2.9 1.5 ± 0.3 
αCD3+ αCD28 1346 ± 24.2 50.5 ± 3.1 
P+ I 1710 ± 1.0 32.2 ± 3.3 
StimulationsIL-2 (pg/ml)
UntreatedSodium salicylate (20 mM)
Control 23.4 ± 2.8 17.4 ± 2.3 
αCD3 40.4 ± 2.9 1.5 ± 0.3 
αCD3+ αCD28 1346 ± 24.2 50.5 ± 3.1 
P+ I 1710 ± 1.0 32.2 ± 3.3 
a

Jurkat T cells were left untreated or preincubated with 20 mM sodium salicylate for 2 h. Stimulations were carried out for 24 h as described. Supernatant from each sample was used to test for IL-2 levels by ELISA performed by University of Maryland at Baltimore Cytokine Core Laboratories (Baltimore, MD). Results represent the average of triplicate measurements.

In primary human CD4+ lymphocytes, we asked whether sodium salicylate had an effect on the activation of the homo- or heterodimeric IKK complexes demonstrated in Fig. 3,D. Prior salicylate treatment of intact lymphocytes almost completely inhibited activation of the homodimeric complex (Fig. 6,B, lanes 9–12), but also had a significant inhibitory effect on IKKα/β heterodimers (Fig. 6 B, lanes 1–4), although not to the same extent as the homodimeric complex. Similar to Jurkat cells, sodium salicylate interfered in the induction of IL-2 secretion during CD4+ cell stimulation by CD3 and CD28 (not shown). These data confirm that IKKβ plays a critical role in regulating NF-κB transcription factors that leads to activation of the IL-2 promoter.

In a previous study, we demonstrated that the c-Jun kinase and the NF-κB pathways synergize in the activation of the IL-2 gene in Jurkat T cells (9). In the present study we extend those findings by showing that IKK complexes play a critical role in primary T cells stimulated via the CD3 and CD28 receptors (Figs. 1 and 2). Moreover, biochemical analysis revealed the presence of at least two types of IKK complexes in CD4+ T lymphocytes, i.e., a heterodimeric complex containing equivalent amounts of IKKα and IKKβ, and a homodimeric complex containing IKKβ alone (Fig. 3). Both complexes included IKKγ, a recently discovered non kinase component of the IKK signalsome (Fig. 3,A) (23, 40). These findings agree with previous demonstration of heterogeneous IKK complexes in HeLa cells (23) and suggest that different IKK complexes may contribute to differential NF-κB signaling in human primary CD4+ T cells. Although the roles of different IKK complexes still need to be clarified, it is interesting that kinase-inactive IKKβ exerts a stronger effect on the IL-2 promoter than IKKα (Fig. 5 A). While there may be a number of explanations for this finding, our results suggest one of two possibilities. The first is that IKKβ homodimers rather than IKKα/β heterodimers play the dominant role in regulating the IL-2 promoter in response to CD3/CD28 costimulation. The second possibility is that homo- and heterodimers both contribute to the activation of the IL-2 promoter, but the regulation of these complexes involves a CD3/CD28-inducible component that primarily signals IKKβ. IKKγ, which binds IKKβ selectively (40), may play a role in this process.

Several lines of evidence support the idea that IKKβ is the predominant kinase regulated by CD3/CD28 coligation in primary human CD4+ T cells. First, IKKα depleted lysates from CD4+ cells contained an active kinase complex that could be precipitated by anti-IKKβ alone (Fig. 3). This finding is consistent with observations in other cell types that ectopically expressed, kinase-inactive IKKα minimally interfered in IKK activation, while overexpressed, kinase-inactive IKKβ was a potent inhibitor of IKK activation (19, 42). The contribution of IKKβ to CD3/CD28 costimulation was further demonstrated by pharmacological interference in IKK activation by a specific IKKβ inhibitor, sodium salicylate (36). Not only did this drug inhibit IKK activation, but it also abrogated transcriptional activation of the CD28RE as well as the full-length IL-2 promoter (Fig. 6). Although heterodimeric IKK complexes were also activated by CD3/CD28 costimulation, it is possible that initially this event is triggered by IKKβ. This will explain why sodium salicylate also interfered in the activation of the heterodimeric complex (Fig. 6,B). What role, if any, IKKα plays in the phosphorylation of the IκB proteins remains to be determined. In fact, IKKα has been shown to phosphorylate IκBα at carboxyl-terminal residues that are distinct from the serines (S32/S36) that play a role in proteolytic degradation (24, 25); the significance of that phosphorylation event still needs to be determined. Recently, several additional reports have appeared that indicate that IKKβ has a dominant effect in regulating IκB kinase activity (43, 44, 45, 46). Mutational alteration of the regulatory and catalytic domains of IKKα and IKKβ showed that IKKβ is the main target for TNF-α and IL-1 stimulation (43). Moreover, embryonic fibroblasts obtained from IKKα-deficient mice exhibit reasonably good IKK activation, while embryonic cells from IKKβ-deficient mice do not express significant IKK activity (44, 45, 46). These findings together with our observations that kinase-inactive IKKβ, but not kinase-inactive IKKα, interfered in the activation of the IL-2 promoter (Fig. 5) support our conclusions that IKKβ is the principal kinase involved in CD3/CD28-mediated NF-κB activation.

New information on the regulation of IKK complexes is emerging with the discovery of new IKK subunits. One example is IKKγ (IKKAP-1), a nonkinase subunit that was recently identified as an essential component of the heterodimeric IKK complex (23, 40). We show that IKKγ is also a component of the homodimeric IKKβ complexes in T cells (Fig. 3). This is probably due to the specific binding affinity of IKKγ for IKKβ (23, 40). Moreover, an anti-IKKγ antiserum immunoprecipitated active IKK complexes from IKKα-depleted lysates in primary human T cells (Fig. 3). While the role of IKKγ still needs to be clarified, one suggestion has been that it relays signals to IKKβ from afferent components in the IKK complex (40). A second novel protein that influences IKKβ activity is IKAP (note that IKAP is distinct from IKKAP-1/IKKγ), which appears to function as a scaffold protein regulating the assembly of IKK complexes (42). While in unstimulated cells most endogenous IKKβ appears to be IKAP associated (42), IL-1 stimulation results in the dissociation of IKKβ from IKAP (42). Similar to the role of JIP-1 in the JNK cascade, overexpression of IKAP abrogates IKK activity (42). While the role of IKAP in T cells still needs to be clarified, one possibility is that IKAP may play a role in the assembly of specific IKK complexes.

The apparent selectivity of IKKβ in the activation of the IL-2 promoter is compatible with our recent finding that a second, but interconnected, signaling cascade is activated in a synergistic fashion by CD3/CD28 coligation. Activation of the Jun kinase cascade is medicated by a MAP3K, MEKK1, that additionally activates IKKβ (9). This selectivity for IKKβ, but not IKKα, has also been demonstrated by other investigators (29, 30). The inhibitory effect of dominant-negative MEKK1 on the IL-2 promoter (Fig. 5) may therefore reflect a role of MEKK1 in both the JNK and NF-κB signaling pathways. That idea is compatible with the stimulatory effects of dominant-active MEKK1 on both the AP-1 and c-Rel binding sites in the CD28RE/AP-1 (9). While we do not know at this stage whether MEKK1 selectively activates homo- or heterodimeric IKK complexes, further studies to address that question are ongoing. The idea that heterogeneous IKK complexes may be selectively activated by different upstream kinases is further strengthened by the recent demonstration that the proto-oncogene, Cot/Tp1, a MAP3K-related serine-threonine kinase, also participates in IKK activation by CD3/CD28 (27). Interestingly, Cot interacts directly with IKKα and NIK, and it has been proposed that Cot acts upstream of NIK (27). Although ectopic expression of a kinase-inactive version of NIK inhibits IKKα and IKKβ activities, NIK only phosphorylates IKKα in vitro and in vivo (26). Since this implies that Cot may selectively signal IKKα, it is possible that this MAP3K may be regulating the activities of those complexes that contain IKKα, while MEKK1 may regulate the homodimeric complex. We are in the process of testing that hypothesis.

Finally, the data presented in this paper indicate that the composition of IKK signalsome may not be rigid, and its assembly is subject to regulation by the afferent signal as well as the cell type. Such regulation may be important for selective gene expression by the NF-κB pathway. We are currently pursuing the role of heterogeneous IKK complexes in selective gene activation during CD3/CD28 costimulation of primary human CD4+ T cells.

We thank Ivonne Castaneda and Peggy Shih for secretarial assistance and Martin Kaszubowski for technical support.

1

This work was supported by grants from the U.S. Public Health Service AG14992 and the Southern California Chapter of the Arthritis Foundation (to A.E.N.) and a National Institute of Allergy and Infectious Diseases Immunology training grant.

3

Abbreviations used in this paper: CD28RE, CD28 response element; IKK, IκB kinase; MAP3K, mitogen-activated protein kinase kinase kinase; MEKK1, mitogen-activated protein kinase kinase kinase 1; JNK, N-terminal Jun kinase; NIK, NF-κB- inducing kinase; IKAP, IKK complex-associated protein; IKKAP-1, IKK-associated protein 1; JIP-1, JNK-interacting protein; DA, dominant active; DN, dominant negative; P+I, PMA plus ionomycin; Tpl, tumor progression locus; Cot, cancer Osaka thyroid; Luc, luciferase.

1
Schwartz, R. H..
1992
. Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy.
Cell
71
:
1065
2
Turka, L. A., J. A. Ledbetter, A. Lee, C. H. June, C. B. Thompson.
1990
. CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes.
J. Immunol.
144
:
1646
3
Harding, F. A., J. G. McArthur, J. A. Gross, D. H. Raulet, J. P. Allison.
1992
. CD28-mediated signaling co-stimulates murine T cells and prevents induction of anergy in T-cell clones.
Nature
356
:
607
4
Herold, K. C., J. Lu, I. Rulifson, V. Vezys, D. Taub, M. J. Grusby, J. A. Bluestone.
1997
. Regulation of C-C chemokine production by murine T cells by CD28/B7 co-stimulation.
J. Immunol.
159
:
4150
5
Fraser, J. D., B. A. Irving, G. R. Crabtree, A. Weiss.
1991
. Regulation of interleukin-2 gene enhancer activity by the T-cell accessory molecule CD28.
Science
251
:
313
6
Schwartz, R. H..
1997
. T cell clonal anergy.
Curr. Opin. Immunol.
9
:
351
7
Fraser, J. D., A. Weiss.
1992
. Regulation of T-cell lymphokine gene transcription by the accessory molecule CD28.
Mol. Cell. Biol.
12
:
4357
8
Shapiro, V. S., K.E. Truitt, J. B. Imboden, A. Weiss.
1997
. CD28 mediates transcriptional upregulation of the interleukin-2 (IL-2) promoter through a composite element containing the CD28RE and NF-IL-2B AP-1 sites.
Mol. Cell. Biol.
17
:
4051
9
Kempiak, S. J., T. S. Hiura, A. E. Nel.
1999
. The Jun kinase cascade is responsible for activating the CD28 response element of the IL-2 promoter: proof of cross-talk with the IκB kinase cascade.
J. Immunol.
162
:
3176
10
Kang, S. M., B. Beverly, A. C. Tran, K. Brorson, R. H. Schwartz, M. J. Lenardo.
1992
. Transactivation by AP-1 is a molecular target of T cell clonal anergy.
Science
257
:
1134
11
Haraj, E. W., S-C. Sun.
1998
. IκB kinases serve as a target of CD28 signaling.
J. Biol. Chem.
273
:
25185
12
Bauerle, P. A., D. Baltimore.
1996
. NF-κB: ten years after.
Cell
87
:
13
13
Beg, A. A., T. S. Finco, P. V. Nantermet, A. S. Baldwin, Jr.
1993
. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of IκBα: a mechanism for NF-κB activation.
Mol. Cell. Biol.
13
:
3301
14
Baldwin, A. J..
1996
. The NF-κB and IκB proteins: new discoveries and insights.
Annu. Rev. Immunol.
14
:
649
15
Traenckner, E. B., H. L. Pahl, T. Henkel, K. N. Schmidt, S. Wilk, P. A. Baeuerle.
1995
. Phosphorylation of human IκB-α on serines 32 and 36 controls IκB-α proteolysis and NF-κB activation in response to diverse stimuli.
EMBO J.
14
:
2876
16
Stancovski, I., D. Baltimore.
1997
. NF-κB activation: the IκB kinase revealed?.
Cell
91
:
299
17
Mercurio, F., H. Zhu, B. W. Murray, A. Shevchenko, B. L. Bennett, J. Li, D. B. Young, M. Barbosa, M. Mann, A. Manning, et al
1997
. IKK-1 and IKK-2: cytokine-activated IκB kinases essential for NF-κB activation.
Science
278
:
860
18
Lee, F. S., J. Hagler, Z. J. Chen, T. Maniatis.
1997
. Activation of the IκBα kinase complex by MEKK1, a kinase of the JNK pathway.
Cell
88
:
213
19
Zandi, E., D. M. Rothwarf, M. Delhase, M. Hayakawa, M. Karin.
1997
. The IκB kinase complex (IKK) contains two kinase subunits, IKKα and IKKβ, necessary for IκB phosphorylation and NF-κB activation.
Cell
91
:
243
20
Regnier, C. H., H. Y. Song, X. Gao, D. V. Goeddel, Z. Cao, M. Rothe.
1997
. Identification and characterization of an IκB kinase.
Cell
90
:
373
21
Woronicz, J. D., X. Gao, Z. Cao, M. Rothe, D. Goeddel.
1997
. IκB kinase-B: NF-κB activation and complex formation with IκB kinase-α and NIK.
Science
278
:
866
22
DiDonato, J. A., M. Hayakawa, D. M. Rothwarf, E. Zandi, M. Karin.
1997
. A cytokine-responsive IκB kinase that activates the transcription factor NF-κB.
Nature
388
:
548
23
Mercurio, F., B. W. Murray., A. Shevchenko, B. L. Bennet, D. B. Young, G. J. W. Li., A. Pascual, H. Motiwala., M. Zue, M. Mann, et al
1999
. IκB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.
Mol. Cell. Biol.
19
:
1526
24
Zandi, E., Y. Chen, M. Karin.
1998
. Direct phosphorylation of IκB by IKKα and IKKβ: discrimination between free and NF-κB-bound substrate.
Science
281
:
1360
25
Lee, F. S., R. T. Peters, L. C. Dang, T. Maniatis.
1998
. MEKK1 activates both IκB kinaseα and IκB kinaseβ.
Proc. Natl. Acad. Sci. USA
95
:
9319
26
Ling, L., Z. Cao, D. Goeddel.
1998
. NF-κB-inducing kinase activates IKK-α by phosphorylation of Ser-176.
Proc. Natl. Acad. Sci. USA
95
:
3792
27
Lin, X., E. T. Cunningham, Jr, Y. Mu, R. Geleziunas, W. C. Greene.
1999
. The proto-oncogene Cot kinase participates in CD3/CD28 induction of NF-κB acting through the NF-κB-inducing kinase and IκB kinases.
Immunity
10
:
271
28
Malanin, N. L., M. P. Boldin, A. V. Kovalenko, D. Wallach.
1997
. MAP-3K related kinase involved in NF-κB induction by TNF, CD95 and IL-1.
Nature
385
:
540
29
Nakano, H., M. Shindo, S. Sakon, S. Nishinaka, M. Mihara, H. Yagita, K. Okumura.
1998
. Differential regulation of IκB kinase α and β by two upstream kinases, NF-κB-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1.
Proc. Natl. Acad. Sci USA
95
:
3537
30
Yin, M.-J., L. B. Christerson, Y. Yamamoto, Y.-T. Kwak, S. Xu, F. Mercurio, M. Barbosa, M. H. Cobb, R. B. Gaynor.
1998
. HTLV-I Tax protein binds to MEKK1 to stimulate IκB kinase activity and NF-κB activation.
Cell
93
:
875
31
Nemoto, S., J. A. Didonato, A. Lin.
1998
. Coordinate regulation of IKB kinases by mitogen-activated protein kinase kinase kinase 1 and NF-κB inducing kinase.
Mol. Cell. Biol.
18
:
7336
32
Levine, B. L., W. B. Bernstein, N. M Connors, T. Craighead, C. B. Thompson Lindsten, C. H. June.
1997
. Effects CD28 costimulation on long-term proliferation of CD4+ T cells in the absence of exogenous feeder cells.
J. Immunol.
159
:
5921
33
Minden, A., A. Lin, M. McMahon, C. Lange-Carter, B. Derijard, R. J. Davis, G. L. Johnson, M. Karin.
1994
. Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK.
Science
266
:
1719
34
Dickens, M., J. S. Rogers, J. Cavanagh, A. Raitano, Z. Xia, J. R. Halpern, M. E. Greenberg, C. L. Sawyers, R. J. Davis.
1997
. A cytoplasmic inhibitor of the JNK signal transduction pathway.
Science
277
:
693
35
Faris, M., N. Kokot, L. Lee, A. E. Nel.
1996
. Regulation of interleukin-2 transcription by inducible stable expression of dominant negative and dominant active mitogen-activated protein kinase kinase kinase in Jurkat T cells: evidence for the importance of Ras in a pathway that is controlled by dual receptor stimulation.
J. Biol. Chem.
271
:
27366
36
Yin, M.-J., Y. Yamamoto, R. B. Gaynor.
1998
. The anti-inflammatory agents aspirin and salicylate inhibit the activity of IκB kinase-B.
Nature
396
:
77
37
Marui, N., M. K. Offermann, R. Swerlick, C. Kunsch, C. A. Rosen, M. Ahmad, R. W. Alexander, R. M. Medford.
1993
. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells.
J. Clin. Invest.
92
:
1866
38
Rudd, C. E..
1996
. Upstream-downstream: CD28 cosignaling pathways and T cell function.
Immunity
4
:
527
39
Su, B., E. Jacinto, M. Hibi, T. Kallunki, M. Karin, Y. Ben-Neriah.
1994
. JNK is involved in signal integration during costimulation of T lymphocytes.
Cell
77
:
727
40
Rothwarf, D. M., E. Zandi, G. Natoli, M. Karin.
1998
. IKK-γ is an essential regulatory subunit of the IκB kinase complex.
Nature
395
:
297
41
Hiura, T. S., Kempiak S. J., A. E. Nel.
1999
. Activation of the human RANTES gene promoter in a macrophage cell line by lipopolysaccharide is depended on stress-activated protein kinases and the IκB kinase cascade: implications for exacerbation of allergic inflammation by environmental pollutants.
Clin. Immunol.
90
:
287
42
Cohen, L., W. J. Henzel, P. A. Baeuerle.
1998
. IKAP is a scaffold protein of the IκB kinase complex.
Nature
395
:
292
43
Delhase, M., M. Hayakawa, Y. Chen, M. Karin.
1999
. Positive and negative regulation of IκB kinase activity through IKKβ subunit phosphorylation.
Science
284
:
309
44
Hu, Y., V. Boud, M. Delhase, P. Zhang, T. Deerinck, M. Ellisman, R. Tohnson, M. Karin.
1999
. Abnormal morphogenesis but intact IKK activation in mice lacking the IKKα subunit of IκB kinase.
Science
284
:
316
45
Li, Q., D. V. Antwerp, F. Mercurio, K-F Lee, and Inder. M. Verma. 1999. Severe liver degeneration in mice lacking the IκB kinase 2 gene. Science 284:321.
46
Takeda, K., O. Takeuchi, T. Sujimura, S. Itami, K. Takeda, O. Dachi, T. Kawai, H. Sanjo, K. Yoshikawa, N. Terada, et al
1999
. Limb and skin abnormalities in mice lacking IKKα.
Science
284
:
313