The IκB Kinase (IKK) Complex Is Tripartite and Contains IKKγ but Not IKAP as a Regular Component

Abstract

A critical step in the activation of NF-B is the phosphorylation of IBs by the IB kinase (IKK) complex. IKK␣ and IKK␤ are the two catalytic subunits of the IKK complex and two additional molecules, IKK␥/NEMO and IKAP, have been described as further integral members. We have analyzed the function of both proteins for IKK complex composition and NF-B signaling. IKAP and IKK␥ belong to distinct cellular complexes. Quantitative association of IKK␥ was observed with IKK␣ and IKK␤. In contrast IKAP was complexed with several distinct polypeptides. Overexpression of either IKK␥ or IKAP blocked tumor necrosis factor ␣ induction of an NF-Bdependent reporter construct, but IKAP in addition affected several NF-B-independent promoters. Whereas specific down-regulation of IKK␥ protein levels by antisense oligonucleotides significantly reduced cytokinemediated activation of the IKK complex and subsequent NF-B activation, a similar reduction of IKAP protein levels had no effect on NF-B signaling. Using solely IKK␣, IKK␤, and IKK␥, we could reconstitute a complex whose apparent molecular weight is comparable to that of the endogenous IKK complex. We conclude that while IKK␥ is a stoichiometric component of the IKK complex, obligatory for NF-B signaling, IKAP is not associated with IKKs and plays no specific role in cytokine-induced NF-B activation.NF-B transcription factors play a pivotal role in many cellular processes such as inflammation, immune response, cell proliferation, and apoptosis (1-5). The prototype of the NF-B family is a heterodimer of the p50 and p65 (RelA) subunits. IB proteins (IB␣, IB␤, IB⑀, p105, and p100) retain NF-B in an inactive form in the cytoplasm. A conserved ankyrin repeat domain in these inhibitors masks nuclear translocation signals contained in the Rel homology domain of NF-B.In response to multiple stimuli, including TNF␣, 1 IL-1␤, phorbol ester, and lipopolysaccharides, NF-B is liberated from IB molecules and translocates to the nucleus (6). This critical step of NF-B activation is initiated by phosphorylation of IB proteins at conserved amino-terminal serine residues, e.g. at serines 32 and 36 of IB␣ or serines 19 and 23 of IB␤. Phosphorylated IBs are bound by a ␤TrCP containing ubiquitin ligase (E3) complex, polyubiquitinated and subsequently degraded by the 26 S proteasome (7).Most NF-B-inducing stimuli trigger activation of an IB kinase (IKK) complex with a high apparent molecular mass of 700 -900 kDa (8, 9), which has specificity for the amino-terminal phosphoacceptor sites in IB␣ or -␤. The kinase complex contains two catalytic subunits termed IKK␣ (IKK1) and IKK␤ (IKK2) (8, 10 -13). IKK␣ and IKK␤ are related molecules of 85 and 87 kDa, respectively, with an overall identity of about 44%. Both contain an NH 2 -terminal kinase domain, a leucine zipper and a COOH-terminal helix-loop-helix motif. IKK␣ and IKK␤ form homo-or heterodimers via their leucine zipper (for review, see Ref. 14). Both kinases are stimulated by proinflammatory cytokines and their activation kinetics match that of IB␣ p...

A Novel Specific Role for IκB Kinase Complex-associated Protein in Cytosolic Stress Signaling

“…2F). These observations are supported by an earlier study in which IKAP was also shown to localize mainly in the cytosol using cell fractionation and Western blot analysis, utilizing a different IKAP antibody from what we use in this study (2). We conclude that the vast majority of IKAP is cytoplasmic.…”

“…A later report by Krappmann et al (2) cast doubt on the role of IKAP in the NF-B pathway. Using a gel filtration approach, these authors were unable to detect IKAP in the fractions containing IKK complexes.…”

IKKγ/NEMO Facilitates the Recruitment of the IκB Proteins into the IκB Kinase Complex

“…This may lead to increased IKK␤ phosphorylation of IB␣ and IB␤ and thus result in enhanced IB degradation and NF-B nuclear translocation. Previous studies indicate that IKK␣, IKK␤, and IKK␥/ NEMO interact in the high molecular weight IKK complex (35,36,40). Our previous studies indicate that the high molecular weight IKK complex can be formed in COS cell extracts prepared following transfection of expression vectors encoding epitope-tagged IKK␣, IKK␤, and IKK␥/NEMO (41).…”

“…IKK␥/NEMO in conjunction with IKK␣ and IKK␤ is a component of the high molecular weight IKK complex, which migrates between 600 and 900 kDa following gel filtration chromatography (12, 14, 29, 34 -41). Biochemical fractionation and coimmunoprecipitation studies demonstrate that IKK␣, IKK␤, and IKK␥/NEMO interact in this IKK complex (35,36,40). Cells that do not express IKK␥/ NEMO are unable to assemble the high molecular weight IKK complex and increase IKK activity in response to agents that stimulate the NF-B pathway (35,36).…”

“…Residues in the aminoterminal 100 amino acids of this protein are critical for interactions with IKK␤ (43). IKK␤ preferentially associates with IKK␥/NEMO (34,36), although IKK␣ has also been shown to directly associate with IKK␥/NEMO (22,40). The coiled-coil domains in IKK␥/NEMO mediate its oligomerization, which is critical for activating IKK kinase activity (44), while its carboxyl terminus is involved in the recruitment of upstream kinases, which are critical for activating IKK (45).…”