RIP3, a Novel Apoptosis-inducing Kinase

Abstract

RIP3 is a novel gene product containing a N-terminal kinase domain that shares extensive homology with the corresponding domain in RIP (receptor-interacting protein) and RIP2. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 has a unique C terminus. RIP3 binds RIP through its unique C-terminal segment and by virtue of this interaction is recruited to the tumor necrosis factor (TNF) receptor-1 signaling complex. Previous studies have shown that RIP mediates TNF-induced activation of the anti-apoptotic NF-B pathway. RIP3, however, attenuates both RIP and TNF receptor-1-induced NF-B activation. Overexpression studies revealed RIP3 to be a potent inducer of apoptosis, capable of selectively binding to large prodomain initiator caspases.Tumor necrosis factor receptor-1 mediates both the proinflammatory and pro-apoptotic effects of the pleiotropic cytokine TNF (1, 2). 1 The proinflammatory effects are mediated by activation of the transcription factor NF-B. Recently it has been shown that NF-B activation results in both the transcriptional activation of proinflammatory genes, including , and in activation of a cell survival pathway mediated at least in part by induction of anti-apoptotic IAP family members (6 -8). Therefore, quite paradoxically, two diametrically opposed pathways emanate from TNFR-1: a cell death pathway and a cell survival pathway mediated by activation of NF-B.The intracellular segment of TNFR-1 contains a 70-amino acid homophilic interaction domain, dubbed the "death domain," which is required for both signaling and NF-B activation. Upon activation of TNFR-1, a multi-component signaling complex is assembled by a series of homophilic interactions (1). Initially, the death domain-containing platform adapter molecule TRADD is recruited to TNFR-1 by virtue of a homophilic death domain interaction (9). TRADD in turn binds the death adapter molecule FADD (10, 11), which interacts with the zymogen form of the initiator death protease caspase-8 (12). Subsequent activation of caspase-8 by an induced proximity mechanism leads to amplification of the death signal through proteolytic activation of downstream caspase zymogens (13). Studies done with FADD-deficient embryonic fibroblasts suggest that this is the major but not the only pro-apoptotic pathway engaged by TNFR-1, because instead of being completely resistant to TNF-induced apoptosis, 30% of FADD-null cells are still sensitive (14,15). Taken together, these studies suggest that there exists a subsidiary FADD-independent TNFR-1-initiated death pathway. Earlier biochemical studies indicated that the TNFR-1-associated adapter molecule RAIDD might fulfill this function by recruiting caspase-2 to the receptor signaling complex (16). However, caspase-2 null cells do not show any loss of sensitivity to TNF-induced cytotoxicity (17); therefore, the physiological significance of this interaction remains unclear. Initial biochemical studies also suggested a role for the TRADD-bindin...

RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition

“…Our results on the kinase-independent role of RIPK3 in TNF-mediated apoptosis are in line with early work reporting apoptosis induction upon ectopic expression of KD RIPK3 mutants. 37,48 It was also previously reported that RIPK3 could, directly or indirectly, interact with the prodomain of caspase-8 upon ectopic expression. 37 It is therefore possible that the contribution of RIPK3 to TNF-mediated apoptosis involves another mechanism than ROS production.…”

“…RIPK3 has emerged as an essential kinase in necroptosis induction [25][26][27] and, as a consequence, RIPK3 repression has become a proof of principle to highlight the contribution of necroptosis in a defined phenotype; although initial reports on RIPK3 indicated that its ectopic expression could also induce apoptosis. 37,38 To investigate whether RIPK3 could contribute to TNF-induced RIPK1-dependent apoptosis, we stimulated immortalized Ripk3 þ / þ and Ripk3 À / À MEFs with TNF þ SM and TNF þ TAK1i. Remarkably, we observed that RIPK3 deficiency provided partial protection to both apoptotic triggers (Figures 5a-d, Supplementary Figure S7).…”

RIPK1- and RIPK3-induced cell death mode is determined by target availability

“…Thus, in the context of the apoptotic machinery (the ripoptosome) it appears that, without activating the kinase, dimerization of the RIPKs induces apoptosis, probably via the C terminal domain. 8,9,22 If dimerization of the two different RIPK domains has different outcomes, what determines the mode of cell death? This is most clearly answered by examining the death of Ripk1 À / À cells following dimerization of RIPK3.…”

“…21 It is also unclear whether RIPK3 can contribute to apoptosis. Despite some reports to this effect, 8,9,22 RIPK3 has been described as the necroptotic 'switch', implying its activity precipitates necroptosis to the exclusion of apoptosis. [23][24][25] Here, we have directly activated RIP kinases without the confounding effects of multiple signals emanating from the target cell's cytokine receptors, allowing us to define more precisely the functions of RIPK1 and RIPK3.…”

“…6,7 RIPK3, like RIPK1, bears a kinase domain and RIP homology interaction motif (RHIM), but unlike RIPK1 does not have a DD. [8][9][10][11] RIPK3 is required for necroptosis. 12,13 Furthermore, RIPK1 appears to activate RIPK3 in this pathway, as cell death could be blocked by nec-1.…”