부산대학교 도서관

Activation of the transcription factor NFκB requires rapid degradation of its inhibitor, IκBα. To facilitate the study of IκBα degradation, we fused IκBα protein to enhanced green fluorescent protein to construct IκBα-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 IκBα. The degradation coincided with NFκB translocation from the cytoplasm to the nucleus and NFκB-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 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 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 IκBα. We also demonstrated that basal degradation of IG in the presence of cycloheximide is inhibited by such mutations, suggesting that basal degradation of IκBα 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 degrades more slowly.