In this study, we demonstrated that IKK1 and IKK2 represent bona fide IκB kinases and that distinct IKK complexes composed of different proteins exist in vivo. In addition, we have purified and cloned a novel component of these complexes which specifically interacts with IKK2 and participates in NF-κB activation.
Although IKK1 and IKK2 were identified as kinase components of the IKK signalsome, formal confirmation of their identities as bona fide IκB kinases is complicated by the fact that functional analysis was performed by transfection experiments in mammalian cells. It is possible that overexpressed proteins associate with other cellular proteins which themselves represent the authentic IκB kinase. For this reason, we expressed and purified IKK1 and IKK2 by using a baculovirus expression system and analyzed in detail the protein species obtained and the kinase activities associated with them. We also expressed a mutant form of IKK2 (IKK2EE) in which two serine residues contained within the MEKK-related activation loop were mutated to glutamic acid. In our previous studies, we reported that this mutant displayed constitutive kinase activity and was capable of inducing NF-κB translocation to the nucleus of transfected HeLa cells in the absence of any other stimuli (30
). We sought to determine if this mutation truly resulted in elevated levels of IκB kinase activity. Purified recombinant IKK1 and IKK2, expressed alone or together, associated as dimers in the absence of other proteins and exhibited IκB kinase activity with similar selectivity and kinetic parameters to those found from analysis of the endogenous IKK signalsome (30
). Detailed kinetic analysis revealed that both IKK1 and IKK2 display a preference for IκBα over IκBβ as a substrate. In addition, IKK2 showed a marked preference for phosphorylation of full-length IκBα compared to the truncated form, IκBα 1–54. Further support for this finding was provided by Burke et al., who demonstrated that a peptide corresponding to the C-terminal region of IκBα enhanced IKK signalsome phosphorylation of a peptide containing Ser32 and Ser36 (10
). In this study, the Km
for IκBα 1–317 was similar to that determined for recombinant IKK2EE. The Km
s of IKK2EE for free IκBα compared to that for IκBα in the context of a RelA-IκBα complex were also similar. We did not observe any significant difference in substrate selectivity for each of the IKK dimers formed, either IKK1-IKK2 heterodimers or IKK1 or IKK2 homodimers. Complexes containing the IKK2EE mutant consistently displayed greater levels of kinase activity, confirming a key role for the activation-loop serines in regulation of IKK activity. Based upon these characteristics, we conclude that IKK1 and IKK2 are bona fide IκB kinases and that full kinase activity can be reconstituted in vitro without the requirement for additional proteins.
In addition, recombinant IKK1 and IKK2 exhibited strong RelA-phosphorylating activity, again consistent with previous results demonstrating stimulus-dependent phosphorylation of IκBα and RelA by the endogenous IKK signalsome (30
). The residues of RelA targeted for phosphorylation by the IKKs are unknown, as is the potential physiologic role of this event. The level of RelA kinase activity associated with IKK1 and IKK2 is comparable to that observed for IκBα as determined by detailed kinetic analysis. These findings suggest that IKK-mediated RelA phosphorylation may play a physiologic role. Moreover, the IKKs do not appear to be general kinases for all Rel-related proteins in that they do not phosphorylate cRel or NF-κB p52. We are currently identifying the sites on RelA which are phosphorylated by the IKKs. Recently, inducible phosphorylation of RelA was demonstrated to be mediated by the catalytic subunit of protein kinase A, and this phosphorylation enhanced the transactivating potential of RelA-containing complexes (44
). In addition, RelA was found to undergo TNF-α-induced phosphorylation on Ser529 (36
). The relationship of these events, if any, to that mediated by IKK1 and IKK2 is under investigation.
IKK1 and IKK2 can form homo- and heterodimers in vitro (16
), and our finding of similar complexes in vivo is consistent with these kinases being able to variably associate. Whereas the HeLa cell line used in these studies contained both the IKK1-IKK2 heterodimer and the IKK2 homodimer, SLB cells contained only the IKK1-IKK2 heterodimer. Therefore, mechanisms must exist for the regulated assembly of the IKK complexes in different cells. The mechanism which regulates complex assembly remains unclear. Perhaps the relative levels of IKK1 and IKK2 expression dictate complex formation. Alternatively, IKK-associated proteins could influence the nature of complex formation, whereby selective protein-protein interactions facilitate the assembly of specific complexes. Interestingly, IKK1-IKK2 and IKK2-only complexes are subject to distinct modes of activation in that they display markedly different levels of activation in response to TNF-α treatment. The IKK1-IKK2 heterodimeric complex was potently activated by TNF-α, in contrast to the IKK2 homodimeric complex, which exhibited only a modest increase in activation. There may be physiologic conditions that preferentially activate the IKK2 homodimer. We did not observe any change in the composition or relative amounts of IKK1-IKK2 heterodimer in stimulated cells, suggesting that a dramatic reorganization of these complexes does not occur upon cellular activation. However, we cannot discount the possibility that other components of these complexes are dynamically regulated and affect IKK function upon cellular activation.
In an effort to better understand IKK regulation, studies were initiated to further elucidate the subunits comprising the respective IKK complexes. The IKK signalsome was originally purified with an anti-MKP-1 antibody; however, we were unable to identify MKP-1 as a component of the IKK complex, either by direct sequence determination or by using a panel of antibodies recognizing distinct epitopes on MKP-1. The identity of the MKP-1 epitope remains elusive, although we have been able to exclude IKK1, IKK2, and IKKAP1 as candidates. We determined that this antibody specifically immunoprecipitates the IKK1-IKK2 heterodimer complex but not the IKK2 homodimer complex. This finding suggests the presence of a protein in the IKK1-IKK2 complex that is not present in the IKK2-homodimeric complex. Studies to identify the IKK signalsome component that is recognized by the MKP-1 antibody are under way. In contrast, by virtue of its ability to bind IKK2, IKKAP1 was identified as a component of both the IKK1-IKK2 heterodimeric and IKK2 homodimeric complex in cells. IKKAP1 associates with IKK2 in vitro and in vivo via sequences contained within the N-terminal coiled-coil repeat region of IKKAP1. IKK2 binding studies established that the IKK2 binding domain of IKKAP1 resides within amino acids 68 through 235. In HeLa cells, transient overexpression of either the IKKAP1 N-terminal (ΔC IKKAP1) or C-terminal (ΔN IKKAP1) domain potently inhibited both IKK2 activation and RelA nuclear localization. These studies suggest that the C- and N-terminal domains of IKKAP1 play distinct and essential roles in IKK activation.
Yamaoka et al. recently described the identification of NEMO (NF-κB essential modulator) via genetic complementation studies of cells unresponsive to NF-κB activating stimuli (41
). NEMO is essential for activation of the NF-κB activation pathway. We report independent data showing the biochemical purification and cloning of a novel component of the IKK signalsome, IKKAP1, which is the human homolog of murine NEMO. Blast search analysis of the available gene databases identified two additional proteins related to IKKAP1: FIP-2, which displays significant sequence similarity to IKKAP1, and FIP-3, which is identical to IKKAP1 (24
). FIP-2 and FIP-3 were identified as E3 14.7-kDa interacting proteins, which are adenovirus proteins encoded by the early transcription region 3 (E3) and function to inhibit the cytolytic effects of TNF-α (25
). Interestingly, FIP-3 (IKKAP1/NEMO) associates with components of the TNF-α receptor complex including RIP (25
). Our immunocytochemical studies provide an intriguing observation where ΔN IKKAP1 displays stimulus-dependent subcellular localization to the cell membrane, perhaps mediated by direct association with the TNF-α receptor complex. We postulate that IKKAP1 provides a scaffold upon which IKK2-containing complexes could be localized to the upstream components of the NF-κB activation cascade. Indeed, JIP-1 (JNK-interacting protein 1) was recently demonstrated to function as a mammalian scaffold protein for the JNK signaling pathway. JIP-1 binds specific upstream components of the JNK pathway and facilitates signal transduction mediated by the bound proteins (38
). JIP-1 is highly selective for a given MAP kinase module, namely, MLK, MKK7, and JNK. This suggests that different scaffold proteins facilitate activation of JNK mediated by other MAP kinase modules. IKKAP1/FIP-3 and FIP-2 may play a similar role in the activation of NF-κB by diverse upstream signaling cascades.
The studies described herein begin to address issues regarding the functional divergence of IKK1 and IKK2. A preference for TNF-α-induced activation of IKK1-IKK2 heterodimers relative to IKK2 homodimers suggests that either IKK1 or IKK1-specific associated proteins are required for full activation of the IKK complex. Conversely, IKKAP1-mediated interaction with upstream activators can be achieved only if IKK2 is present. Thus, the IKK signalsome, by virtue of the functional diversity of IKK1 and IKK2 and their respective associated proteins, provides the potential to integrate the diverse array of signaling pathways known to activate NF-κB in different cell types.