Distinct roles of RIP1-RIP3 hetero- and RIP3-RIP3 homo-interaction in mediating necroptosis

Abstract

Necroptosis is mediated by a signaling complex called necrosome, containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL). It is known that RIP1 and RIP3 form heterodimeric filamentous scaffold in necrosomes through their RIP homotypic interaction motif (RHIM) domain-mediated oligomerization, but the signaling events based on this scaffold has not been fully addressed. By using inducible dimer systems we found that RIP1-RIP1 interaction is dispensable for necroptosis; RIP1-RIP3 interaction is required for necroptosis signaling, but there is no necroptosis if no additional RIP3 protein is recruited to the RIP1-RIP3 heterodimer, and the interaction with RIP1 promotes the RIP3 to recruit other RIP3; RIP3-RIP3 interaction is required for necroptosis and RIP3-RIP3 dimerization is sufficient to induce necroptosis; and RIP3 dimer-induced necroptosis requires MLKL. We further show that RIP3 oligomer is not more potent than RIP3 dimer in triggering necroptosis, suggesting that RIP3 homo-interaction in the complex, rather than whether RIP3 has formed homo polymer, is important for necroptosis. RIP3 dimerization leads to RIP3 intramolecule autophosphorylation, which is required for the recruitment of MLKL. Interestingly, phosphorylation of one of RIP3 in the dimer is sufficient to induce necroptosis. As RIP1-RIP3 heterodimer itself cannot induce necroptosis, the RIP1-RIP3 heterodimeric amyloid fibril is unlikely to directly propagate necroptosis. We propose that the signaling events after the RIP1-RIP3 amyloid complex assembly are the recruitment of free RIP3 by the RIP3 in the amyloid scaffold followed by autophosphorylation of RIP3 and subsequent recruitment of MLKL by RIP3 to execute necroptosis.

Figure 1

The RHIM and DD domains are both capable of mediating the interaction of RIP1 proteins. (a) Schematic representation of murine RIP1 and its truncations. K, I, and D represent the kinase domain, intermediate sequence, and DD of RIP1, respectively. RHIM mut represents QIG529–531AAA mutation on the RHIM in RIP1. The amino acid number is indicated. (b) Flag-RIP1 was co-transfected with HA-RIP1 mutants in 293T cells. Thirty-six hours post transfection, cell lysates were immunoprecipitated with anti-Flag antibody. The cell lysates and immunoprecipitates were analyzed by western blotting. (c and d) Total internal reflection fluorescence (TIRF) of Flag-RFP-tagged DD (c) or RHIM domain (d). (e–g) Photobleaching step distribution for Flag-RFP-tagged DD (e and f) and RHIM domain (g)

Figure 2

The RIP1–RIP3 heterodimer itself cannot initiate necroptosis, but recruitment of additional RIP3 protein(s) to this heterodimer causes necroptosis. (a) Rip1^−/−Rip3^d and Rip1^−/−Rip3* MEF cells were infected with lentivirus encoding the RIP1 variants as indicated, cultured with or without zVAD (20 μM). Thirty hours after infection, cell death assay was performed using propidium iodide (PI) exclusion. (b) Schematic representation of the heterodimer adaptors (FKBP and FRB*) fused RIP1 and RIP3 variants. RHIM mut in RIP3 represents QIG449–451AAA mutation in RHIM domain. (c, d and f) Rip1^−/−Rip3^d MEF cells were infected with lentivirus as indicated, followed by the heterodimerizer AP21967 treatment (250 nM), with or without zVAD (20 μM). Cell loss was determined by ATP levels. (e and g) 293T cells were transfected with the indicated constructs. After 36 h, these cells were treated with mock or AP21967 (250 nM) for 1 h, followed by immunoprecipitation with anti-Myc antibody and subsequent western blotting analysis. All data above were represented as mean±S.E.M. of three independent experiments. *P<0.05. **<0.01. n.s., nonsignificant

Figure 3

RIP3 homo-dimerization is sufficient to trigger MLKL-dependent necroptosis. (a) Rip1^−/−Rip3^d MEF cells were infected with lentivirus as indicated, followed by AP21967 treatment (250 nM) for 12 h, with or without zVAD (20 μM). Cell loss was determined by ATP levels. (b and c) Rip3 KO and Mlkl KO L929 cells were infected with lentivirus as indicated. Thirty-six hours post infection, these cells were treated with AP21967 (250 nM) for various time intervals (b), or were pretreated with DMSO, zVAD (20 uM), or necrostatin-1 (Nec-1, 30 μM) for 30 min, followed by AP21967 treatment (250 nM) for 6 h. (c) Cell death was determined by PI exclusion. (d) The cells as indicated were treated with mock or AP21967 (250 nM) for 30 min. The cell lysates were fractionated on SuperoseTM 6 size-exclusion column. These fractions were then subjected to immunoprecipitation with anti-Flag antibody, followed by western blot analysis. (e) The Rip3-Flag knock-in L929 cells were treated with mock or TNF+zVAD for 2 h. The cell lysates were fractionated as described in d, followed by anti-Flag immunoprecipitation and western blot analysis. (f) The cells as indicated were treated with mock or AP21967 (250 nM) for 30 min, followed by DSS cross-link reaction as described in Methods. The cells were then lysed and immunoprecipitated with anti-Flag antibody followed by western blotting with anti-HA antibody. Western blotting of immunoprecipitates with anti-Flag antibody shows the equal immunoprecipitation of different samples. Western blotting of total cell lysates shows equal samples used in the immunoprecipitation. All data above were represented as mean±S.E.M. of three independent experiments. **P<0.01

Figure 4

Oligomerization of RIP3 is not more potent than dimerization of RIP3 in generating necroptosis signaling. (a) Wild-type L929 cells were infected with lentivirus encoding the RIP3 variants as indicated for 24 h. After mock or AP21967+zVAD treatment for 30 min, the cell lysates were fractionated and immunoprecipitated as described in Figure 3d, followed by western blotting analysis. The relative levels of protein were calculated by Image J and indicated. (b and c) Wild-type and Rip3 KO L929 cells were infected with lentivirus encoding the RIP3 variants as indicated. After 24 h, these cells were re-plated and treated with AP21967 as indicated concentrations for 6 h. The protein expression was determined by western blot analysis and the cell death was determined by ATP levels. The data were represented as mean±95% confidence interval (CI) of three independent experiments. n.s., nonsignificant

Figure 5

RIP3 homo-dimerization triggers intramolecular autophosphorylation. (a) The Rip3 KO L929 cells, expressing the indicated RIP3 variants, were treated with AP21967 (250 nM) for 30 min. The cell lysates were analyzed by western blotting with antibodies as indicated. (b–d) Rip3 KO and Mlkl KO L929 cells were infected with lentivirus as indicated. Thirty-six hours post infection, these cells were treated with mock or AP21967 (250 nM) for 30 min (b), or 6 h (c), or as indicated (d). AA represents T231A/S232A mutation. The cell lysates were analyzed by western blotting. The cell death was determined by PI exclusion. All data above were represented as mean±S.E.M. of three independent experiments. **P<0.01

Figure 6

RIP3 homo-dimerization leads to MLKL recruitment. (a and b) Rip3 KO and Mlkl KO L929 cells were infected with lentivirus as indicated. Thirty-six hours post infection, these cells were treated with mock or AP21967 (250 nM) for 30 min. The cell lysates were immunoprecipitated with anti-Flag antibody and then analyzed by western blotting. (c) Proposed model of signaling events in necrosome