This project is aimed at delineating specific molecular mechanisms by which signals received by immune cells result in activation of genes critical to fighting diseases. In particular, we have focused on the activation of the family of transcription factors collectively referred to as NF-kB, because they are essential for inducing the expression of many important immune effectors. In addition, NF-kB factors are essential to expression of the HIV as well as several other viruses of clinical relevance. This project focuses on the transcription factor-proximal events which occur during signal-induced activation. The activation process proceeds via controlled degradation of inhibitors (IkBs) of the transcription factors, which allows the thus liberated NF-kB complexes to travel from the cytoplasm into the nucleus, where they exert their effects. An understanding of the molecular details of the degradation of these inhibitors is fundamental to understanding immune activation in healthy and diseased states. In addition, such knowledge will identify potential new targets for therapeutic intervention in inflammatory diseases as well as viral diseases in which NF-kB plays a critical role, such as AIDS. We have previously demonstrated that signal-induced activation of the NF-kB transcription factors involves rapid site-specific phosphorylation of IkB proteins by the IkB kinases (IKK-alpha and IKK-beta), followed by site-specific ubiquitination and finally, proteasome-dependent proteolytic degradation. The phosphorylation and ubiquitination sites are located near the N-terminus of IkB-alpha. We have now shown that efficient degradation of IkB-alpha also requires PEST sequences located near its C-terminus. We demonstrate that the short N- and C-terminal domains of IkB-alpha are not only necessary but also sufficient to confer an inducible degradation phenotype; attachment of these two domains to a completely unrelated protein causes signal-induced degradation of the chimeric protein. TNF and IL-1 mediated activation of the IkB kinases (IKKs) has been shown to depend on the NIK kinase; in addition to NIK, MEKK1, a distantly related kinase, has been shown to activate IKKs as well. We have set up an in vitro system to study the activation of IKKs. In particular, we have partially purified IKK complexes and we are investigating their composition and their regulation by NIK and MEKK1. We have also set up in vitro systems to dissect the molecular requirements for ubiquitination of phosphorylated IkB-alpha. Finally, we have used degradation-resistant mutants of IkBs, which are no longer phosphorylated by IKKs, to generate transgenic animals. Cells within these transgenic animals which express the dominant-negative mutant inhibitors are significantly repressed in their ability to inducibly activate NF-kB complexes. These transgenic mice are now being investigated for physiologic defects.
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