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

A critical step in the activation of NF-κB is the phosphorylation of IκBs by the IκB kinase (IKK) complex. IKKα and IKKβ are the two catalytic subunits of the IKK complex and two additional molecules, IKKγ/NEMO and IKAP, have been described as further integral members. We have analyzed the function of both proteins for IKK complex composition and NF-κB signaling. IKAP and IKKγ belong to distinct cellular complexes. Quantitative association of IKKγ was observed with IKKα and IKKβ. In contrast IKAP was complexed with several distinct polypeptides. Overexpression of either IKKγ or IKAP blocked tumor necrosis factor α induction of an NF-κB-dependent reporter construct, but IKAP in addition affected several NF-κB-independent promoters. Whereas specific down-regulation of IKKγ protein levels by antisense oligonucleotides significantly reduced cytokine-mediated activation of the IKK complex and subsequent NF-κB activation, a similar reduction of IKAP protein levels had no effect on NF-κB signaling. Using solely IKKα, IKKβ, and IKKγ, we could reconstitute a complex whose apparent molecular weight is comparable to that of the endogenous IKK complex. We conclude that while IKKγ is a stoichiometric component of the IKK complex, obligatory for NF-κB signaling, IKAP is not associated with IKKs and plays no specific role in cytokine-induced NF-κB activation.