Sites on FIP-3 (NEMO/IKK gamma) essential for its phosphorylation and NF-kappa B modulating activity

FIP-3 (NEMO/IKK gamma) is an essential modulator of the! activity of NF-kap paB by mechanisms that include alterations in the phosphorylation, ubiquina tion, and degradation of I kappaB alpha. The multiple protein-protein inter actions of FIP-3 (NEMO/IKK gamma) in a high molecular weight IKK complex in dicated that this protein may be a link between some of the receptor-proxim al upstream signal transduction molecules such as RIP and the down stream e ffects on IKBcu, Although FIP-3 (NEMO/IKK gamma) has no intrinsic kinase ac tivity, it has been shown to increase the kinase activity of IKK beta. In t his manuscript, the results of serine to alanine mutations at five sites on FIP-3 (NEMO/IKK gamma) are described, and functional assays demonstrated t hat two of these mutants affect both the phosphorylation and kinase activit y of IKK beta, Protein kinase C alpha appeared to be the kinase that was re quired for the posttranslational modification of FIP-3 (NEMO/IKK gamma). On e of the serine targets of the protein kinase Ca enzyme at amino acid 141 w as within a leucine zipper-like sequence of FIP-3 (NEMO/IKK gamma), which m ight affect its interactions with other proteins on the signal transduction pathway. The second serine, which augmented the inhibition, was at amino a cid 85 within the FIP-3 (NEMO/IKK gamma) interaction site with IKK beta, Wh en both serines were mutated simultaneously, the effect on IKK beta and I k appaB alpha phosphorylation was more profoundly affected. (C) 2001 Academic Press.