When first identified, IKKα and IKKβ were viewed as functionally interchangeable IκB kinases that coexist within a macromolecular IKK signaling complex termed the signalsome. In the wake of targeted gene disruption studies, it is clear that these kinases play significantly different roles within the heterodimeric signalsome, IKKβ being the principal IκB kinase while the function of IKKα is less clear. We now demonstrate that activation of signalsomes containing heterodimeric IKKα-IKKβ complexes proceeds in a directional manner. Specifically, we show that a wide variety of NF-κB inducing MAP3Ks act through IKKα to induce phosphorylation of the activation loop residues of IKKβ in various cell lines. In contrast, kinase-deficient IKKβ exerts no inhibitory effects on NIK-induced phosphorylation of IKKα, underscoring the directional nature of this activation process. Our studies further indicate that phosphorylation of IKKβ induced by the physiological agonist TNF-α or the pathological stimulant HTLV-1 Tax similarly proceeds through IKKα to IKKβ. Interestingly, not all agonists require IKKα for induction of IKKβ phosphorylation. For example, we found that PKCθ is able to induce phosphorylation of IKKβ in the absence of IKKα. The addition of wild-type IKKα inhibits this PKCθ response, suggesting that expression of IKKα may disrupt IKKβ homodimeric complexes that may be selectively activated by PKCθ. These findings raise the intriguing possibility that different upstream activators couple preferentially to heterodimeric or homodimeric complexes, increasing signalling specificity.
Functional asymmetry within the heterodimeric signalsome was first suggested by the observation that IKKβ is a significantly more potent IκB kinase than IKKα. While both kinases are capable of phosphorylating IκBα in vitro, they do so with dramatically different efficiencies, with IKKβ exhibiting 50- to 60-fold greater activity than IKKα (28
). Additional support for disparate roles in NF-κB activation has come from the targeted inactivation of the IKKα and IKKβ genes in mice. Disruption of the Ikkβ
locus results in embryonic lethality at ~14 days of gestation due to massive hepatic cell apoptosis leading to liver degeneration, a phenotype remarkably similar to that seen in mice deficient in the RelA/p65 subunit of NF-κB (4
). This enhanced hepatocyte death is likely due to the loss of the antiapoptotic effects of NF-κB since IKKβ-deficient embryonic fibroblasts have severely depressed IκB kinase activity and diminished NF-κB activation in response to either TNF-α or IL-1 (31
). Indeed, IKKβ-deficient cells were 30-fold more sensitive to TNF-α-induced apoptosis than their wild-type counterparts (52
). The amount of IKKα protein was greater in homozygous IKKβ-deficient embryos than in wild-type embryos, suggesting that there is a selective pressure to enhance IKKα expression in IKKβ-deficient cells, although this up-regulation of IKKα does not fully compensate for the loss of IKKβ activity and therefore is unable to counteract the extensive cell death (52
). Of interest is the observation that IKKα continued to assemble into a minimally responsive ~900-kDa signalsome in these IKKβ-deficient cells (31
IKKα-defective animals survive to birth but die within 1 to 4 h of birth and exhibit a range of morphogenic abnormalities including a thickened, undifferentiated epidermis that appears to restrict extension of the limbs and a number of skeletal malformations (21
). Intriguingly, skin abnormalities, although not identical, have also been reported for mice deficient for IκBα, a negative regulator of NF-κB (26
). In this study we, like others, have shown that IKKα can similarly function as a negative regulator of basal IKKβ activity (29
). It is interesting to speculate whether these skin abnormalities may emerge as a consequence of disrupting the normal negative regulators of IKKβ activity and NF-κB activation.
Disruption of the Ikkα
locus surprisingly does not impair TNF-α induction of NF-κB, a finding confirmed in three independent studies. Of note, there is a quantitative decrease in the total level of NF-κB binding in these IKKα-deficient animals (21
). This result seems at odds with the abundance of IKKα expression in the wild-type animals, its tight association with IKKβ expression, and the high degree of sequence similarity shared by these genes. Indeed, the widespread assembly of IKKα with IKKβ in signalsomes in many tissues argues that IKKα plays a broader function than regulating epidermal development (63
). Moreover, previous studies with kinase-inactive or activation loop mutants of IKKα (15
) as well as transfection of IKKα-as constructs (14
) have all reported a negative impact on IKK activity underlying the conditional importance of IKKα expression. In view of our described findings, we propose that the IKKα-deficient animals have likely compensated for the loss of the IKKα regulator by assembling functional homodimeric IKKβ signalsomes (21
). These homodimeric IKKβ signalsomes (38
) may be positively selected for during embryogenesis in the IKKα-deficient animals to prevent the extensive apoptosis that would result from a loss of IKK activity. In view of the dramatic difference in the IκB-phosphorylating activities of these two kinases, we would argue that IKKα has mainly evolved to negatively regulate the high constitutive activity of IKKβ under basal conditions and to couple its activation in stimulated conditions to many upstream agonists. Likewise, a proportion of complexes consisting of IKKβ homodimers have evolved with an alternative regulatory mechanism, perhaps IKKγ, which also plays a role in coupling of the signalsome to different upstream activators. Therefore, loss of a regulating kinase like IKKα may be compensated for, but loss of the functional kinase, IKKβ, cannot be tolerated. The generation of IKKα and IKKβ conditional knockout and knock-in animals will no doubt clarify the nature of the physiological interplay between these two kinases in the regulation of NF-κB induction.
We have demonstrated directional activation of the heterodimeric IKK complex by a number of MAP3Ks known to play a role in NF-κB activation (19
). This activation occurs through phosphorylation of the serine residues within the activation loops of the IKKs. One recent report suggests that the activation loop serines of IKKβ are essential for NIK-induced IKK activation (12
). We find that these activation loop serines are phosphorylated in the presence of NIK but in an indirect manner dependent on the kinase activity of IKKα. In the same study, Delhase and colleagues report that homologous activation loop mutations in IKKα do not affect IκB phosphorylation (12
). This result is at odds with our observations that the activation loop mutant IKKαS176A blocks both IKKβ and IκBα phosphorylation induced by NIK. In support of our data, NIK was previously shown to phosphorylate IKKα on Ser-176 of its activation loop, but it did not phosphorylate IKKβ (35
). These data support a dual regulatory role for IKKα leading to the appropriate activation of IKKβ phosphorylation. As such, IKKα could be functionally viewed as a surrogate MAP2-like kinase connecting the upstream MAP3Ks to the downstream MAPK represented by IKKβ.
The precise nature of the interplay of MEKK1 with IKKα or IKKβ remains unclear. Some studies indicate MEKK1 interacts with, and activates, both IKKα and IKKβ (28
). However, other reports show that MEKK1 overexpression in 293 or Jurkat cells preferentially stimulates IKKβ kinase activity over IKKα (24
). In addition, Tax has been shown to bind and activate MEKK1, which then directly activates IKKβ but not IKKα (61
). However, more recent reports indicate that Tax binds to the signalsome by assembling with NEMO/IKKγ rather than by binding to IKKβ directly (9
). This interaction may be impaired in the presence of overexpressed upstream kinase-inactive MAP3Ks, which may also interact with IKKγ. We too find that within the heterodimeric signalsome, both MEKK1 and Tax induce IKKβ phosphorylation in a manner dependent on the kinase activity of IKKα. In agreement with our findings, kinase-inactive forms of both IKKα and IKKβ have been shown to block Tax and MEKK1 induction of IKK activity, clearly implicating both kinases in the pathway (10
Of interest is our finding that not all signals proceed through IKKα. We show that PKCθ appears to selectively target IKKβ for activation. Of note, this reaction may involve IKKβ homodimers since assembly of IKKβ into the heterodimeric complex inhibits its ability to serve as a target for PKCθ-mediated activation. These inconsistencies in activation of IKKα versus IKKβ by various upstream kinases may, in part, be reconciled by the existence of a number of distinct IKK complexes (38
). The larger ~700-kDa TNF-α-responsive complex was found to contain IKKα, IKKβ, and IKKAP1 (NEMO/IKKγ), while a ~300-kDa complex consisting of only IKKβ and IKKAP1 proved significantly less responsive to TNF-α-coupled induction (38
). It is possible, however, that the higher-molecular-weight complex also contains functional IKKβ homodimeric complexes. Moreover, the smaller IKKβ complexes may not respond to TNF-α but may couple to different activators. Different cell lines may contain varying amounts of these IKKα-β heterodimeric versus IKKβ homodimeric complexes, and these complexes may couple differentially to upstream activating signals. Our studies clearly show that, in the 293 and HeLa cell lines studied, transmission of the NF-κB-inducing signal is directional within the heterodimeric IKK signalsome.
In summary, we propose that, when present in the heterodimeric signalsome, IKKα exerts a dominant regulating effect on the phosphorylation and activation of IKKβ kinase activity. This regulatory role of IKKα is further underscored by the finding that mutations in the leucine zipper region of IKKα disrupts dimerization with IKKβ, resulting in a strong diminution of IκB phosphorylation (38
). Interestingly, mutations in the helix-loop-helix motifs of either kinase do not abolish their dimerization but do result in the loss of kinase activity (62
), likely reflecting a failure of the IKKs to bind NEMO/IKKγ/IKKAP1, an essential component of functional signalsomes (38
). IKKα is thus an essential regulatory component of the IKK heterodimeric signalsome that serves to couple the upstream activating signal to the IKKβ catalytic component of the complex.