Research ArticleCell death

Kinase domain dimerization drives RIPK3-dependent necroptosis

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Science Signaling  21 Aug 2018:
Vol. 11, Issue 544, eaar2188
DOI: 10.1126/scisignal.aar2188
  • Fig. 1 RIPK3+/D161N fetuses on a RIPK1-deficient background exhibit MLKL-dependent lethality.

    (A) Number of embryonic day 18.5 (E18.5) embryos recovered from intercrossing Ripk1+/− Ripk3+/D161N mice. Data are pooled from the analysis of 10 independent litters. (B) Whole-mount embryo imaging of E12.5 littermates. Images are representative of at least four embryos analyzed of each genotype analyzed between E12.5 and E13.5. (C) Immunohistochemical (IHC) analysis of phosphorylated RIPK3 in tissue sections from E11.5 placenta and liver. Images are representative of greater than three independent controls and three Ripk1/Ripk3+/D161N embryos. (D) Number of E18.5 embryos from crossing of Ripk1+/ Ripk3+/D161N Mlkl/ and Ripk1+/ Ripk3+/+ Mlkl/ mice. Data are pooled from the analysis of six independent litters. (E) Images of E18.5 littermates recovered from intercrossing Ripk1+/ Ripk3+/D161N and Ripk1+/ Ripk3+/ mice. Images are representative of the analysis of at least two independent litters.

  • Fig. 2 The RIPK3 kinase domain dimerizes using an interface similar to RAF.

    (A) Superposition of RIPK3 (orange; amber; PDB ID: 4M66) and BRAF (4MNF; navy; sky blue) molecular dimers, illustrating conserved dimer surface. Inlay panel shows a magnified view of conserved arginine in molecular handshake across dimer interface. (B) Open-book representation of the RIPK3 dimer interface surface (amber). Residues selected for mutation to validate dimer interface are colored navy blue. Additional residues that contact the opposite kinase (<3 Å) are colored sky blue. Residue numbers are given in white. (C) RIPK3 dimer interface surface with one of the RIPK3 molecules depicted with space-filling model and the second RIPK3 molecule represented as orange ribbon diagram. (D) A close-up view of the RIPK3 dimer interface (PDB ID: 4M66). Residues mutated in this study are represented by sticks and labeled in black. Transparent sticks indicate additional points of contact, which were not mutated in this study. (E and F) Co-immunoprecipitation (IP) analysis of RIPK3 kinase domain (1 to 313 amino acids) interaction in lysates of HEK293T cells cotransfected with pcDNA3-Flag RIPK3 kinase domain and the indicated pcDNA3-HA-RIPK3 kinase domain that were immunoprecipitated for Flag. Blots are representative of at least three independent experiments. Normalized band intensities are means ± SD from all experiments. (G) Split luciferase complementation assay assessment of assessment of RIPK3 dimerization in HEK293T cells transfected with constructs expressing WT RIPK3 kinase domain fused to the C-terminal domain of click beetle luciferase (CBGC-RIPK3 KD) and the indicated RIPK3 kinase domain mutations fused to the N terminus of click beetle luciferase (CBGN-RIPK3 KD). Luciferase activity data are means ± SD combined from at least two independent experiments. **P < 0.01 and ***P < 0.005 by Mann-Whitney test. AU, arbitrary units.

  • Fig. 3 Necroptosis is impaired by kinase domain dimerization–defective RIPK3.

    (A and B) Flow cytometry analysis of enhanced GFP (EGFP) abundance in Ripk3/ MEFs infected with lentiviruses encoding EGFP-P2A-WT RIPK3 or the indicated mutants and treated with TNF-α + z-VAD-fmk for 24 hours. Dot plots (A) are representative of at least three independent experiments. The relative frequencies of GFP+ cells (B) are means ± SD from all experiments. SSC, side scatter. (C and D) Flow cytometry analysis of EGFP abundance in Ripk3/ bone marrow cells transduced with lentiviral constructs encoding EGFP-P2A-WT RIPK3 or mutants and cultured for 6 days in macrophage colony-stimulating factor (M-CSF) to generate macrophages before treatment with poly(I:C)/z-VAD-fmk for 24 hours. (C) Dot plots (left) are representative of at least three independent experiments. The relative frequencies of GFP+ cells (D) are means ± SD from all experiments. (E) Western blot analysis for p-MLKL, MLKL, RIPK3, and β-actin from lysates of Ripk3/ MEFs transduced with the indicated RIPK3 mutant construct and treated with TZ for the indicated times. Cell lysates were cross-linked with bismaleimidohexane (BMH) or directly used for immunoblotting. Blots are representative of at least three independent experiments. Normalized band intensities are means ± SD from all experiments. (F) Co-immunoprecipitation analysis of RIPK1 interactions in lysates of Ripk3/ MEFs reconstituted with WT RIPK3 or RIPK3 R69H and treated with TZ for 4 hours that were immunoprecipitated for RIPK1. Blots are representative of at least three independent experiments. Normalized band intensities are means ± SD from all experiments. **P < 0.01 and ***P < 0.005 by Student’s t test.

  • Fig. 4 RIPK3 functions as an allosteric activator dependent on kinase domain homodimerization.

    (A) Autoradiography and Western blot analysis of the in vitro kinase activity from lysates of HEK293T cells transfected with pcDNA3-Flag WT, pcDNA3-Flag K51A, or pcDNA3-Flag V36F RIPK3 constructs and immunoprecipitated for Flag 48 hours later. Blots are representative of at least three independent experiments. Normalized band intensities are means ± SD from all experiments. (B) Ripk3/ MEFs were cotransduced with the indicated lentiviral constructs of RIPK3 fused to FRBP and FRB (B), which heterodimerize in the presence of AP21967. (C and D) CellTiter-Glo analysis of cellular viability in cells that received the indicated constructs and were pretreated with z-VAD-fmk for 1 hour before treatment with AP21967 for 6 hours. Data are means ± SD from three independent experiments. (E) Western blot analysis for p-MLKL, MLKL, and Flag in lysates from cells that received the indicated constructs and were pretreated with z-VAD-fmk before the addition of 250 nM AP21967 for the indicated time periods. Blots are representative of at least three independent experiments. Normalized band intensities are means ± SD from all experiments. (F) Model for allosteric activation of RIPK3 through kinase domain dimerization. *P < 0.05, **P < 0.01, and ***P < 0.005 by Student’s t test.

  • Fig. 5 Kinase-inactive RIPK3 D161N allosterically activates WT RIPK3 and induces its cis-autophosphorylation.

    (A and B) Western blot analysis for p-MLKL (A) or pRIK3 (B) in lysates of Ripk3/ MEFs cotransduced with the indicated constructs treated for the indicated times with 250 nM AP21967 + z-VAD-fmk. Blots are representative of three independent experiments. Normalized band intensities are means ± SD from all experiments. (C) CellTiter-Glo analysis of cell viability in Ripk3/ MEFs cotransduced with the indicated constructs treated with 250 nM AP21967 + TZ for 6 hours. Data are means ± SD pooled from at least three independent experiments. (D) Co-immunoprecipitation analysis of RIPK3 interactions in lysates of Ripk3/ MEFs cotransduced with the indicated constructs that were immunoprecipitated for Flag. Blots are representative of three independent experiments. Normalized band intensities are means ± SD from all experiments. (E) Western blot analysis of pRIPK3 in cell lysates from Ripk3/ MEFs cotransduced with the indicated RIPK3-FKBP fusion constructs and WT RIPK3. Blots are representative of three independent experiments. Normalized band intensities are means ± SD from all experiments. (F) Flow cytometery analysis of EGFP and mCherry abundance in Ripk3/Ripk1KO MEFs cotransduced with the indicated mCherry-RIPK3 construct and EGFP-RIPK3 D161N in the presence or absence of GSK′872. Dot plots are representative of two independent experiments. The frequencies of cells expressing both constructs are means ± SD from all experiments. (G) Model for activation of WT RIPK3 by RIPK3 D161N in the absence of RIPK1. *P < 0.05, **P < 0.01, and ***P < 0.005 by Student’s t test.

  • Fig. 6 RIPK3 D161N and RIPK3 inhibitor–induced apoptosis requires Arg69 and a stable R-spine.

    (A) Flow cytometry analysis of EGFP abundance in Ripk3/ MEFs or Ripk3/Caspase-8KO MEFs transduced with the indicated EGFP-P2A-RIPK3 constructs and treated with z-VAD-fmk for 3 days. The frequencies of GFP+ cells are means ± SD pooled from at least three independent experiments. (B) Flow cytometry analysis of EGFP abundance in Ripk3/ MEFs transduced with the indicated EGFP-P2A-RIPK3 constructs and treated with the indicated RIPK3 inhibitors. The frequencies of GFP+ cells are means ± SD pooled from at least three independent experiments. (C) Co-immunoprecipitation analysis of RIPK1 interactions in lysates of Ripk3/ MEFs transduced with the indicated RIPK3 constructs and treated with the GSK′872 and z-VAD-fmk for 3 hours that were immunoprecipitated for RIPK1. Blots are representative of three independent experiments. Normalized band intensities are means ± SD from all experiments. (D) RIPK3 kinase domain in the active conformation (amber; PDB ID: 4M66). Features of the active conformation include the αC positioned “in” as well as a closed R-spine. R-spine residue side chains are represented by gray sticks and spheres. (E) Flow cytometry analysis of EGFP abundance in Ripk3/ MEFs transduced with the indicated RIPK3 constructs and treated with z-VAD-fmk for 3 days. The frequencies of GFP+ cells are means ± SD pooled from at least three independent experiments. (F) Flow cytometry analysis of EGFP abundance in Ripk3/ MEFs transduced with the RIPK3 construct and treated with the indicated inhibitors. The frequencies of GFP+ cells are means ± SD pooled from at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.005 by Student’s t test. n.s., not significant.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/544/eaar2188/DC1

    Fig. S1. Biochemical analysis of the RIPK3 kinase domain.

    Fig. S2. Characterization of Ripk3/ MEFs after reconstitution with WT RIPK3 and dimerization-defective RIPK3 mutations.

    Fig. S3. Expression of RIPK3 V36F does not cause apoptosis or TZ-dependent necroptosis.

    Fig. S4. Characterization and kinetics of FKBP-FRB RIPK3 fusion system for induced heterodimerization.

    Fig. S5. RIPK3-induced apoptosis by RIPK3 D161N and the RIPK3 inhibitor GSK′872.

    Fig. S6. Model of the mechanism of RIPK3 D161N function.

  • This PDF file includes:

    • Fig. S1. Biochemical analysis of the RIPK3 kinase domain.
    • Fig. S2. Characterization of Ripk3/ MEFs after reconstitution with WT RIPK3 and dimerization-defective RIPK3 mutations.
    • Fig. S3. Expression of RIPK3 V36F does not cause apoptosis or TZ-dependent necroptosis.
    • Fig. S4. Characterization and kinetics of FKBP-FRB RIPK3 fusion system for induced heterodimerization.
    • Fig. S5. RIPK3-induced apoptosis by RIPK3 D161N and the RIPK3 inhibitor GSK′872.
    • Fig. S6. Model of the mechanism of RIPK3 D161N function.

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