Regulation of the NF-kappa B activation pathway by isolated domains of FIP3/IKK gamma, a component of the I kappa B-alpha kinase complex

FIP3, isolated as a type 2 adenovirus E3-14.7-kDa interacting protein, is a n essential component of the multimeric I kappa B-alpha kinase (IKK) comple x and has been shown to interact with various components (Fas receptor-inte racting protein, NF-kappa B-inducing kinase, IKK beta) of the NF-kappa B ac tivation pathway. FIP3 has also been shown to repress basal and tumor necro sis factor (TNF) alpha-induced NF-kappa B activity as well as to induce cel l death when overexpressed, The adenovirus E3-14.7-kDa protein (E3-14.7K) i s an inhibitor of TNF alpha-induced cell death. In the current study, we ge nerated 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. W e also found that the carboxyl-terminal half of FIP3 blocked TNF alpha-indu ced I kappa B-alpha phosphorylation and subsequent degradation, which sugge sts that the stabilization of the cytoplasmic inhibitor of NF-kappa B under lies the FIP3 inhibition of NF-kappa B activity. The amino-terminal 119 ami no acids were responsible for the FIP3-IKK beta and FIP3-IKK alpha interact ion, and the middle of the protein (amino acids 201-300) appeared to be bot h the FIP3 self-association domain as well as the FIP3-Fas receptor-interac ting protein interaction domain. Thus, FIP3 might serve as a scaffold prote in 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 detach ment of the cells from the monolayer.