FIP-3 (NEMO/IKK gamma) is an essential modulator of the! activity of NF-kappaB by mechanisms that include alterations in the phosphorylation, ubiquination, and degradation of I kappaB alpha. The multiple protein-protein interactions of FIP-3 (NEMO/IKK gamma) in a high molecular weight IKK complex indicated that this protein may be a link between some of the receptor-proximal upstream signal transduction molecules such as RIP and the down stream effects on IKBcu, Although FIP-3 (NEMO/IKK gamma) has no intrinsic kinase activity, it has been shown to increase the kinase activity of IKK beta. In this manuscript, the results of serine to alanine mutations at five sites on FIP-3 (NEMO/IKK gamma) are described, and functional assays demonstrated that two of these mutants affect both the phosphorylation and kinase activity of IKK beta, Protein kinase C alpha appeared to be the kinase that was required for the posttranslational modification of FIP-3 (NEMO/IKK gamma). One of the serine targets of the protein kinase Ca enzyme at amino acid 141 was within a leucine zipper-like sequence of FIP-3 (NEMO/IKK gamma), which might affect its interactions with other proteins on the signal transduction pathway. The second serine, which augmented the inhibition, was at amino acid 85 within the FIP-3 (NEMO/IKK gamma) interaction site with IKK beta, When both serines were mutated simultaneously, the effect on IKK beta and I kappaB alpha phosphorylation was more profoundly affected.