Degradation of I kappa B alpha is limited by a postphosphorylation/ubiquitination event

Regulation of I kappaB alpha during activation was examined using EGFP. Sin gle cell analysis showed that both localisation- and cytokine-induced degra dation of I kappaB alpha are dependent on expression levels. Cells expressi ng higher levels of the inhibitor demonstrated an increase in nuclear I kap paB alpha EGFP with a pronounced enhancement in the nuclear/cytoplasmic rat io. Enhancing the levels of the endogenous I kappaB alpha by relA transfect ion caused significant reduction in IL-1-mediated degradation of the fusion protein. Similarly, I kappaB alpha EGFP-transfected cells showed an invers e correlation between the level of the fusion protein and IL-1-mediated deg radation. Comparing absolute levels demonstrated a biphasic response, with reduction in cells expressing over 15-fold that of endogenous levels. Furth er experiments using Western analysis showed a positive correlation between both phosphorylation and ubiquitination of I kappaB alpha EGFP, and the le vel the inhibitor. In contrast, and in agreement with the single cell analy sis, while IL-1 stimulation caused the expected degradation at lower levels of the fusion protein, breakdown of I kappaB alpha EGFP was totally inhibi ted at the higher transfection levels. The data show that turnover of I kap paB alpha is saturable and suggest that limitation of the pathway by enhanc ed inhibitor expression is regulated through a post phosphorylation/ubiquit ination event, at the level of degradation. (C) 2001 Academic Press.