The I kappa B kinase (IKK) complex is tripartite and contains IKK gamma but not IKAP as a regular component

A critical step in the activation of NF-kappa B is the phosphorylation of I kappa Bs by the I kappa B kinase (IKK) complex. IKK alpha and IKK beta are the two catalytic subunits of the IRK complex and two additional molecules , IKK gamma/NEMO and IKAP, have been described as further integral members. We have analyzed the function of both proteins for IKK complex composition and NF-kappa B signaling. IKAP and IKK gamma belong to distinct cellular c omplexes. Quantitative association of IKK gamma was observed with IKK alpha and IKK beta. In contrast IKAP was complexed with several distinct polypep tides. Overexpression of either IKK gamma or IKAP blocked tumor necrosis fa ctor alpha induction of an NF-kappa B-dependent reporter construct, but IKA P in addition affected several NF-kappa B-independent promoters. Whereas sp ecific down-regulation of IKK gamma protein levels by antisense oligonucleo tides significantly reduced cytokine-mediated activation of the IKK complex and subsequent NF-kappa B activation, a similar reduction of IKAP protein levels had no effect on NF-kappa B signaling. Using solely IKK alpha, IKK b eta, and IKK gamma, we could reconstitute a complex whose apparent molecula r weight is comparable to that of the endogenous IKK complex. We conclude t hat while IKK gamma is a stoichiometric component of the IKK complex, oblig atory for NF-kappa B signaling, IKAP is not associated with IKKs and plays no specific role in cytokine-induced NF-kappa B activation.