This project is aimed at delineating the mechanisms by which stimulation of immune cells results in inactivation of the IkappaB-alpha inhibitor and subsequent activation of the NF-kappaB transcription factor. NF-kappaB is critical for the inducible expression of many genes whose encoded functions are required to counteract pathogens or stress. In addition, NF-kappaB regulates the expression of several viruses, including the human immunodeficiency virus (HIV). Therefore, blocking the activation of NF-kappaB could aid in the treatment of many inflammatory diseases and may also prevent the spread of HIV. An understanding of the mechanism(s) of activation of this transcription factor and the identification of the molecular components involved will provide potential targets for anti-inflammatory and anti-viral therapies. We have previously demonstrated that signal-induced activation of the NF-kappaB transcription factor involves rapid site-specific phosphorylation followed by proteolytic degradation of its cytoplasmic inhibitor IkappaB-alpha. Degradation is carried out by proteasomes in a ubiquitin-dependent fashion. We have determined that the inhibitor becomes ubiquitinated on specific lysines as a consequence of induced phosphorylation; the ubiquitinated species is then degraded by proteasomes. The phosphorylation and ubiquitination sites are located near the N-termius of IkappaB-alpha. Efficient degradation also requires PEST sequences located near the C-terminus. We demonstrate that the short N- and C-terminal domains of IkappaB-alpha are not only necessary but also sufficient to confer an inducible degradation phenotype. Attachment of these two domains to a completely unrelated protein caused signal-induced degradation of the chimeric protein. This research will aid in the identification of the molecular components which target the phosphorylated inhibitor for proteasomal degradation and the kinases which phosphorylate it in response to appropriate stimuli.