Characterization of NFkappaB activation by detection of green fluorescent protein-tagged IkappaB degradation in living cells.

Activation of the transcription factor NFkappaB requires rapid degradation of its inhibitor, IkappaBalpha. To facilitate the study of IkappaBalpha degradation, we fused IkappaBalpha protein to enhanced green fluorescent protein to construct IkappaBalpha-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) alpha is approximately 5 min, which is similar to the half-life of native IkappaBalpha. The degradation coincided with NFkappaB translocation from the cytoplasm to the nucleus and NFkappaB-mediated induction of transcription. Phorbol 12-myristate 13-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 TNFalpha. Co-treatment with TNFalpha and PMA did not result in a synergistic effect on IG degradation, although they stimulate different kinases in two different signaling 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 IkappaBalpha. We also demonstrated that basal degradation of IG in the presence of cycloheximide is inhibited by such mutations, suggesting that basal degradation of IkappaBalpha also requires phosphorylation as the signal for degradation. Finally, we showed that the rate of TNFalpha-induced degradation of IG remains almost constant throughout the cell cycle, except at the mitotic phase, in which IG degrades more slowly.