FLIP proteins and TRAF2 control the quality of death receptor signaling

• Applicant: Dr. Daniela Siegmund
• Subject Area: Cell Biology ; Biochemistry
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 431867410

Death receptors (DRs) of the CD95-type (CD95, TRAILR1/DR4, TRAILR2/DR5) recruit caspase-8 directly by help of FADD. TNFR1-type DRs (TNFR1, DR3) stimulate the FADD-caspase-8 dyad, too, but secondarily in a cytoplasmic complex. Both DR types also trigger necroptosis via RIPK1 but FADD is here of differential relevance. FADD is required for CD95- but not for TNFR1-type DR-induced necroptosis and in the latter case even inhibits this response. CD95- and TNFR1-type DRs activate furthermore the classical NFkB pathway. Again there are differences: CD95-type DRs require FADD and caspase-8 for NFkB activation while both molecules are dispensable for this in the case of TNFR1-type DRs. We recently showed that RIPK1 and TRADD are crucially involved in a redundant manner in TNFR1- and CD95-type DR-induced NFkB signaling. We also found that TRADD and RIPK1 are redundantly required for TNF- but not TRAIL-induced apoptosis. The redundant role of TRADD and RIPK1 in DR-induced classical NFkB signaling points to a role of TRAF2 which interacts with both of these proteins and which is known to mediate NFkB signaling by non-DRs of the TNFRSF. Noteworthy, TRAF2 preferentially limits apoptotic TNFR1-type DR signaling but inhibits TNFR1- and CD95-type DR-induced necroptosis.The isoforms of cFLIP are potent regulators of DR-induced cytotoxicity. All cFLIPs are recruited to CD95-type DRs and to cytosolic complexes induced by TNFR1- and CD95-type DRs. All cFLIP isoforms show antiapoptotic activity but their function(s) in DR-induced necroptosis and NFkB activation are rather unclear. Recent years revealed that there is not a single TRADD-RIPK1-dependent pathway by which DRs engage the classical NFkB pathway but three: i) RIPK1-independent via TRADD, ii) TRADD-independent via RIPK1 without the need for RIPK1 kinase activity and iii) via RIPK1 under crucial involvement of its kinase activity. It is currently fully unclear whether cFLIPs and TRAF2 act one or more of these pathways in a similar fashion. In sum, there is currently an inconsistent picture of the central question: how cFLIPs and TRAF2 regulate the balance of apoptotic, necroptotic and NFkB signaling in a DR-type specific manner. Thus, I will clarify how the various cFLIP isoforms and TRAF2 control the common and DR-specific functions of caspase-8 and RIPK1 described above. Concretely, I will address following issues: - the still controversial role of different cFLIP isoforms for necroptosis induction and NFkB activation by death receptors - the relevance of TRAF2 for the different modes of DR-induced NFkB activation - the control of caspase-8-mediated functions by TRAF2 The pathway- and DR type-specific functions of cFLIP proteins and TRAF2 will be analyzed i) in TRAF2 and cFLIP KO cells reconstituted with functionally defined TRAF2 and cFLIP mutants and ii) in cells double-deficient in TRAF2 and other factors involved in DR signaling (e.g. TRADD, RIPK1) using a panel of established assays and methods.

• DFG Programme: Research Grants