Characterization of NF&kappaB activation by detection of green fluorescent protein-tagged I&kappaB degradation in living cells

Activation of the transcription factor NF&kappaB requires rapid degradation of its inhibitor, I&kappaBα. To facilitate the study of I&kappaBα degradation, we fused I&kappaBα protein to enhanced green fluorescent protein to construct I&kappaBa-enhanced green fluorescent protein (IG). We demonstrated by both flow cytometry and Western blot analysis that the half-life of IG in the presence of human tumor necrosis factor (TNF) α is approximately 5 min, which is similar to the half-life of native I&kappaBα. The degradation coincided with NF&kappaB translocation from the cytoplasm to the nucleus and NF&kappaB-mediated induction of transcription. Phorbol 12-myristate 18-acetate (PMA), but not forskolin, also induces degradation of IG fusion protein. The half-life of IG in the presence of PMA is approximately 15 min, longer than when induced with TNFα. Co-treatment with TNFα and PMA did not result in a synergistic effect on IG degradation, although they stimulate different kinases in two different signalling pathways. Degradation of IG was inhibited by mutations at serine residues 32 and 36, which are the target sites of the phosphorylation modification that initiates degradation of IG. We also demonstrated that basal degradation of IG in the presence of cycloheximide is inhibited by such mutations, suggesting that basal degradation of I&kappaBα also requires phosphorylation as the signal for degradation. Finally, we showed that the rate of TNFα-induced degradation of IG remains almost constant throughout the cell cycle, except at the mitotic phase, in which IG decades more slowly.