Lymphotoxin-Beta Receptor Signaling

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MECHANISMS OF LTf'.R SIGNAL TRANSDUCTION The Lymphotoxin-p receptor (LTPR) signaling pathway orchestrates the development and organization of secondary lymphoid tissues through the activation of NFicB family of transcription factors, including NFicB1(relA/p50) and NFicB2(relB/p52). Biochemical and genetic evidence supports a model in which LTPR ligation activates NFxB via a stepwise event involving the serine kinases, NIK and IKKa, which control the proteolytic processing of p100->52. Members of the TRAF family of RING finger proteins, TRAF2, 3 and 5, are thought to act as signal adaptors for LTPR, however mice deficient in these TRAFs do not phenocopy the LTpR-/- mice, thus the role of TRAFs in LTf5R signaling in vivo is enigmatic. This project specifically seeks to define how TRAF3 is involved in LTpR signaling pathways that activate NFxfi. We propose that TRAFs function regulators restraining the activity of key enzymes and consider a model in which TRAFS and NIK are preassociated in an inactive complex, and upon ligation, LTpR competitively displaces NIK from TRAFS, liberating NIK to phosphorylate protein substrates. A variety of reagents, including crystals of TRAFS and genetically defined cell lines, have been assembled to define the roles of TRAFs in LTPR signaling pathway in relevant physiologic responses. Three aims are proposed to test this model: In the first aim the competitive displacement model predicts that the NIK-TRAF3 interaction will be affected by the point mutations that alter LTPR-TRAF3 interactions. Targeted mutations in LTPR, TRAFS and NIK will be examined for their effect on binding interactions, subcellular location and trafficking, enzymatic activity, NFxB processing and signaling in genetically defined cell lines to address this model. In aim 2, the structural basis of NIK-TRAF3 interaction will be determined by X-ray diffraction using NIK peptide-TRAFS crystals. The third aim will determine the physiologic consequence of the LTPR-TRAF interaction by introducing TRAF binding mutants of LTPR into mouse bone marrow cells creating chimeras to examine their effect on NFtcB dependent gene expression in vivo. These approaches will provide a direct assessment of the role of TRAFs in LTPR signaling pathway.