Regulation of the NF-κB activation pathway by isolated domains of FIP3/IKKγ, a component of the IκB-α kinase complex

FIP3, isolated as a type 2 adenovirus E3-14.7-kDa interacting protein, is an essential component of the multimeric I kappa B-alpha kinase (IKK) complex and has been shown to interact with various components (Fas receptor-interacting protein, NF-kappa B-inducing kinase, IKK beta) of the NF-kappa B activation pathway. FIP3 has also been shown to repress basal and tumor necrosis factor (TNF) alpha-induced NF-kappa B activity as well as to induce cell death when overexpressed, The adenovirus E3-14.7-kDa protein (E3-14.7K) is an inhibitor of TNF alpha-induced cell death. In the current study, we generated deletion mutants to map the domains of FIP3, which are responsible for its various functions, The NF-kappa B inhibitory activity and the E3-14.7K binding domains were mapped at the carboxyl half of the FIP3 protein. We also found that the carboxyl-terminal half of FIP3 blocked TNF alpha-induced I kappa B-alpha phosphorylation and subsequent degradation, which suggests that the stabilization of the cytoplasmic inhibitor of NF-kappa B underlies the FIP3 inhibition of NF-kappa B activity. The amino-terminal 119 amino acids were responsible for the FIP3-IKK beta and FIP3-IKK alpha interaction, and the middle of the protein (amino acids 201-300) appeared to be both the FIP3 self-association domain as well as the FIP3-Fas receptor-interacting protein interaction domain. Thus, FIP3 might serve as a scaffold protein to organize the various components of the I kappa B-alpha kinase complex. Whereas the full-length protein is required for efficient cell death, the amino-terminal 200 amino acids are sufficient to cause rounding and detachment of the cells from the monolayer.