Identification of RIP3, a RIP-like kinase that activates apoptosis and NFκB

Abstract

The tumor necrosis factor receptor 1 (TNFR1) and the Fas receptor recruit complexes formed by the interactions between RIP kinase, TRADD, FADD and RAIDD - adaptor proteins that contain death domains - which in turn recruit other proteins to initiate signaling [1][2][3][4][5]. To identify proteins associated with the TNF signaling pathway, we performed a yeast two-hybrid interaction screen using RIP as bait. We isolated a kinase, RIP3, which shares homology with the kinase domain of RIP and RIP2 (also known as Rick or CARDIAK). RIP3 could be co-immunoprecipitated with RIP, TRAF2 and TNFR1 in mammalian cells. The carboxy-terminal domain of RIP3, like that of RIP, could activate the transcription factor NFkappaB and induce apoptosis when expressed in mammalian cells. Interestingly, this region shares no significant sequence homology to the death domain of RIP, the caspase-recruiting domain (CARD) of RIP2 [6][7][8] or any other apoptosis-inducing domain. As with RIP and RIP2, the kinase domain of RIP3 was not required for either NFkappaB activation or apoptosis induction. Overexpression of a dominant-negative mutant of RIP3 strongly inhibited the caspase activation but not the NFkappaB activation induced by TNFalpha. Therefore, RIP3 appears to function as an intermediary in TNFalpha-induced apoptosis.

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

“…6,31,35 To test whether the kinase activity of RIPK3 was required for either form of RIPK3 gyrase-induced death, we initially generated a K51A kinase-dead mutant of the RIPK3 gyrase vector. 9 We expressed it in WT MEFs, immunoprecipitated the RIPK3 gyrase and confirmed that the kinase was inactive in an in vitro assay (Figure 7a). The K51A RIPK3 gyrase protein did not cause death of caspase 8 À / À cells (Figure 7b) that are susceptible to necroptosis (Figure 4b) but lack detectable expression of endogenous RIPK3 (Figure 2a).…”

“…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.…”

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

“…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).…”