FLIP proteins and TRAF2 control the quality of death receptor signaling

CD95型死亡受体(CD95、TRAILR1/DR4、TRAILR2/DR5)通过FADD直接募集caspase-8。TNFR1型DRs(TNFR1，DR3)也刺激FADD-caspase-8二联体，但在细胞质复合体中起辅助作用。这两种类型的DR也通过RIPK1引发坏死性下垂，但FADD在这里具有不同的相关性。CD95需要FADD，但TNFR1型DR诱导的坏死性下垂不需要FADD，在后一种情况下甚至可以抑制这一反应。CD95和TNFR1型DRs进一步激活经典的NFkB通路。同样也有不同之处：CD95型DRS需要FADD和caspase-8来激活NFkB，而对于TNFR1型DRS来说，这两个分子都是必不可少的。我们最近发现RIPK1和Tradd以冗余的方式参与了TNFR1和CD95型DR诱导的NFkB信号转导。我们还发现Tradd和RIPK1是肿瘤坏死因子诱导的细胞凋亡所必需的，而不是TRAIL诱导的细胞凋亡。Tradd和RIPK1在DR诱导的经典NFkB信号转导中的冗余作用表明，TRAF2与这两种蛋白相互作用，并通过TNFRSF的非DRS介导NFkB信号转导。值得注意的是，TRAF2优先限制凋亡的TNFR1型DR信号，但抑制TNFR1和CD95型DR诱导的坏死链。cFLIP的异构体是DR诱导的细胞毒性的有效调节因子。所有的cFLIP都被招募到CD95型DRs以及由TNFR1和CD95型DRs诱导的胞浆复合体。所有cFlIP异构体都具有抗凋亡活性，但它们在DR诱导的坏死性下垂和NFkB激活中的作用(S)尚不清楚。近年来发现，DRS参与经典的NFkB途径不是单一的Tradd-RIPK1依赖途径，而是三个途径：i)通过Tradd不依赖于RIPK1，ii)通过RIPK1不依赖于Tradd，而不需要RIPK1激活，以及iii)通过RIPK1参与其激酶活性的关键参与。目前还完全不清楚cFLIP和TRAF2是否以类似的方式作用于这些通路中的一个或多个。总而言之，目前对核心问题有一个不一致的图景：cFlips和TRAF2如何以DR类型特异性的方式调节凋亡、坏死和NFkB信号的平衡。因此，我将阐明各种cFlIP亚型和TRAF2如何控制上述caspase-8和RIPK1的共同和DR特异性功能。具体地说，我将解决以下问题：-不同的cFLIP亚型在死亡受体诱导坏死症和激活NFkB方面的作用仍然存在争议-TRAF2与DR诱导的NFkB激活的不同模式的相关性-TRAF2对caspase-8介导的功能的控制TRAF2将使用一系列已建立的分析和方法来分析：i)在TRAF2和cFLIP KO细胞中，由功能定义的TRAF2和cFLIP突变体重组的cFLIP KO细胞中；以及ii)在TRAF2和其他参与DR信号传递的因子(例如Tradd、IPRK1)中双缺陷的细胞中。

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.