This project is focused on the elucidation of molecular signal transduction mechanisms involved in activation of NF-kappaB transcription factors, which are central to regulating immune responses in many cells. The work will not only lead to a better basic understanding of signaling processes, but, in addition, it will also provide a basis for new approaches to treating diseases: Once new proteins have been identified that help regulate NF-kappaB-mediated gene transcription and once their roles in various signaling paths have been identified, these proteins may also provide new targets for therapeutic intervention in NF-kappaB-associated diseases. Inflammatory and autoimmune diseases are often driven by undesirable activation of NF-kappaB. Expression of the human immunodeficiency virus (HIV) and other clinically relevant viruses depends on activation of NF-kappaB as does the survival of many types of cancer cells, in which NF-kappaB induces anti-apoptotic proteins. Thus NF-kappaB and the network of proteins that regulates these factors become potential targets for controlling inflammatory and autoimmune diseases, the spread of viruses and cancer growth. Most inflammatory signals stimulate the classical NF-kappaB activation pathway, which is mediated by the IKK kinase complex, the target of many signaling cascades. The IKK complex then phosphorylates the small inhibitory IkappaB proteins, which causes their proteolytic degradation, thereby freeing the NF-kappaB transcription factors to translocate to the nucleus and to regulate target genes. We have previously identified proximal components of IKK-mediated activation, including the adaptor CIKS and we are investigating this proteins in vivo function and the pathways this adaptor is part of. In addition to the classical pathway of activation, NF-kappaB can also be activated via a non-classical pathway, independent of the IKK complex. This second pathway involves processing of the p100 precursor form of NF kappaB2 to the p52 form, which leads to activation of p52/RelB heterodimers. Based on analyses of B cell defects in NF-kappaB2 knockout mice, we previously identified the TNF family member BAFF as the physiologic inducer of this second activation pathway in B cells. In addition, this pathway is activated by the lymphotoxin beta receptor (LTbetaR) in stromal cells, where it contributes importantly to stromal cell-dependent immune functions. We are investigating by what mechanisms these receptors signal the processing of p100 to p52. We have determined that the TNF-receptor-associated protein TRAF3 has an inhibitory role to play in processing, although the mechanism by which the BAFFR and LTbetaR signal processing remains to be determined. We are using FRET technology to assay possible interactions of various signaling proteins that are likely to be involved in the processing pathway. NF-kappaB2 (and thus the non-classical pathway) and the IkappaB-related regulator Bcl-3 appear to have redundant functions in immunologic tolerance, as lack of both proteins, but not either one alone, causes severe multi-organ inflammation. We are investigating the signaling paths and targets of these proteins responsible for immunologic tolerance. As part of our efforts to identify the regulation and function of both NF-kappaB2 and Bcl-3 we have now shown that Bcl-3?s activities are modulated by the GSK-3 kinase. NF-kappaB2 is also involved in obesity-induced insulin resistance and we are investigating the functions and regulation of this protein in insulin-responsive tissues.