The IB kinase (IKK) complex mediates activation of transcription factor NF-B by phosphorylation of IB proteins. Its catalytic subunits, IKK␣ and IKK␤, require association with the regulatory IKK␥ (NEMO) component to gain full basal and inducible kinase activity. However, the oligomeric composition of the IKK complex and its regulation by IKK␥ are poorly understood. We show here that IKK␥ predominantly forms tetramers and interacts with IKK␣ or IKK␤ in this state. We propose that tetramerization is accomplished by a prerequisite dimerization through a C-terminal coiled-coil minimal oligomerization domain (MOD). This is followed by dimerization of the dimers with their N-terminal sequences. Tetrameric IKK␥ sequesters four kinase molecules, yielding a ␥ 4 (␣/␤) 4 stoichiometry. Deletion of the MOD leads to loss of tetramerization and of phosphorylation of IKK␤ and IKK␥, although the kinase can still interact with the resultant IKK␥ monomers and dimers. Likewise, MOD-mediated IKK␥ tetramerization is required to enhance IKK␤ kinase activity when overexpressed in 293 cells and to reconstitute a lipopolysaccharide-responsive IKK complex in pre-B cells. These data thus suggest that IKK␥ tetramerization enforces a spatial positioning of two kinase dimers to facilitate transautophosphorylation and activation.A large number of physiological stimuli activate NF-B transcription factors to regulate gene expression in innate or adaptive immune responses in development and cellular growth control. In unstimulated cells, NF-B proteins are sequestered by the small cytosolic IB molecules ␣, ␤, and ε or by the precursor proteins NF-B1/p105 and NF-B2/p100 (24, 36). Upon cellular stimulation with proinflammatory agents, including tumor necrosis factor alpha (TNF-␣), interleukin 1 (IL-1), and bacterial lipopolysaccharide (LPS), or with antigens, viral pathogens, growth factors, or morphogens, these IBs are phosphorylated by an IB kinase (IKK) complex. Phosphorylated IB proteins are ubiquitinated by the SCF ␤TRCP ubiquitin ligase complex and subject to proteolysis by the proteasome, resulting in liberation and nuclear translocation of 32).The prevalent cellular IKK complex contains three components, two kinases, IKK␣ (also called IKK1) and IKK␤ (also called IKK2) and the noncatalytic IKK␥ (also called NEMO) protein. The kinases share 52% identity and contain an Nterminal catalytic domain, a central leucine zipper, and a C-terminal helix-loop-helix motif. The kinases form homo-or heterodimers via their leucine zippers, and their activity depends on their ability to dimerize (16,27,35,45). Direct interaction with IKK␥ is mediated by a short motif at the extreme C terminus of IKK␣ and IKK␤ (23). Deletion of this motif or introduction of a short peptide containing this sequence into cells impairs binding of both kinases to IKK␥ and activation of IKK kinase activity and of NF-B (23).Murine IKK␥ was identified by complementation cloning using NF-B activation-deficient rodent cells (39). The human homologue was obtained by biochemical purific...

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...