Identification and functional analysis of TNFR2-induced signaling complexes

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Tumor necrosis factor (TNF) is a pleiotropic cytokine acting through stimulation of two functional distinct receptors, namely TNF receptor-1 (TNFR1) and TNFR2. Studies on TNFR1 resulted in a detailed understanding of many aspects of TNF signaling and lead to discoveries of general relevance for research on cell death and inflammation. However, the mechanisms of TNFR2 signal transduction are still poorly understood. Therefore, it was the major aim of the project to improve the understanding of TNFR2 signaling to gain deeper insights into TNF biology in general. Against this background, we made the following major findings in the expired funding period of the project. First, we identified TNFR2 as an activator of the MAP3 kinase NIK and the alternative NFkappaB pathway. Further, we identified the deubiquitinase Cyld and Sharpin, a subunit of the linear ubiquitin chain assembly complex, as new components of the TNFR2 signaling complex. We also demonstrated for the first time that TNFR2 modulates non-apoptotic TNFR1 signaling and revealed considerable functional similarities between TNFR2 and the TWEAK receptor Fn14, another member of the TNF receptor family.Based on these results the following issues shall be addressed in the second funding period:1. Activation of NIK and IKK1 are pivotal steps in the activation of the alternative NFkappaB pathway but for both molecules NFkappaB-independent functions, particularly in the Notch and Wnt signaling pathways, have also been described. Based on our finding that TNFR2 activates NIK as well as the alternative NFkappaB pathway, we will evaluate whether there is a crosstalk between TNFR2 and Notch and/or Wnt signaling.2. We identified Cyld and Sharpin as parts of the TNFR2 signaling complex but have yet no information about their role in TNFR2 signal transduction. We will therefore analyze the effect of Cyld and Sharpin knockdown in our established cellular systems of TNFR2 signaling. If we should be able to identify TNFR2-induced NIK- and IKK1-mediated NFkappaB-independent cellular effects in the course of the project, these findings will also be analyzed with respect to a possible role of Cyld and Sharpin. 3. With very few exceptions, all components of the TNFR2 signaling complex are indirectly recruited via TRAF2, a multifunctional adaptor protein with E3 ligase activity. To decipher the complex functions of TRAF2 in TNFR2 signaling, we will generate TRAF2 knockdown/knockout cells and reconstitute them with known well characterized TRAF2 mutants that allow dissection of TRAF2-related functions.4. TNFR2 is primarily expressed in immune cells and endothelial cells. The results and ideas derived from our work on TNFR2 signaling mainly base so far on the use of tumor cell lines. We will therefore reevaluate these findings in corresponding primary cells of human and murine origin. The use of primary murine cells will also allow an analysis of cells derived from mice with mutated Sharpin or NIK.