ADK licenses constitutive RIPK1 symmetric dimethylation at R606, which inhibits TNFα-induced cell death. (A) RIPK1 was immunoprecipitated from primary mouse hepatocytes and was analyzed with mass spectrometry. Left: Enrichment efficiency of RIPK1 immunoprecipitation was confirmed with immunoblotting. Right: The mass spectrum data revealed that endogenous mouse RIPK1 was dimethylated at R606. (B) Amino acid sequences at R606 in RIPK1 DD across various mammalian species were aligned. R606 was highlighted in red. (C)Ripk1−/− MEFs were reconstituted with Flag-RIPK1 by lentivirus. The cells were treated with or without adenosine (1 mM). RIPK1 was immunoprecipitated using anti-Flag antibody, and SDMA or ADMA levels were determined with immunoblotting. (D)Ripk1−/− MEFs were reconstituted with Flag-RIPK1 by lentivirus. The cells were transfected with siNC or siAdk. RIPK1 was immunoprecipitated using anti-Flag antibody, and SDMA or ADMA levels were determined with immunoblotting. (E)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were treated with TNFα (10 ng/ml) for 15 min. The level of SDMA on RIPK1 was determined by immunoprecipitation and immunoblotting. (F)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were treated with TNFα (10 ng/ml) for 15 min. The level of RIPK1 R606me2s was determined with immunoblotting. (G) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with CHX (1 μM)/TNFα (10 ng/ml) for the indicated time. RIPK1 R606me2s, RIPK1, and ADK levels were analyzed with immunoblotting. (H) MEFs transfected with or without siAdk were stimulated with Flag-TNFα (10 ng/ml) for 15 min after the pretreatment of DZNep (10 μM), 5-ITu (20 μM), or adenosine (1 mM) for 1 h. Complex I was immunoprecipitated using anti-Flag antibody, and RIPK1 R606me2s level in complex I or whole-cell lysates was determined with immunoblotting. (I and J) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with CHX (1 μM)/TNFα (10 ng/ml) for the indicated time. Adenosine levels in the cells were measured and compared (I). SAM, SAH levels, and their ratios in the cells were measured and compared (J). (K and L)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were transfected with siAdk and subsequently treated with TNFα (10 ng/ml) for the indicated time (K) or 12 h (L) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (K). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (L). (M and N)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were treated with adenosine (1 mM) and subsequently treated with TNFα (10 ng/ml) for the indicated time (M) or 12 h (N) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (M). The levels of p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (N). Data are represented as the mean ± SD (I–K and M). Data are representative of n = 3 independent experiments (A–N). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (I–K and M). ***P < 0.001. Source data are available for this figure: SourceData F3.
Figure 3.

ADK licenses constitutive RIPK1 symmetric dimethylation at R606, which inhibits TNFα-induced cell death. (A) RIPK1 was immunoprecipitated from primary mouse hepatocytes and was analyzed with mass spectrometry. Left: Enrichment efficiency of RIPK1 immunoprecipitation was confirmed with immunoblotting. Right: The mass spectrum data revealed that endogenous mouse RIPK1 was dimethylated at R606. (B) Amino acid sequences at R606 in RIPK1 DD across various mammalian species were aligned. R606 was highlighted in red. (C)Ripk1−/− MEFs were reconstituted with Flag-RIPK1 by lentivirus. The cells were treated with or without adenosine (1 mM). RIPK1 was immunoprecipitated using anti-Flag antibody, and SDMA or ADMA levels were determined with immunoblotting. (D)Ripk1−/− MEFs were reconstituted with Flag-RIPK1 by lentivirus. The cells were transfected with siNC or siAdk. RIPK1 was immunoprecipitated using anti-Flag antibody, and SDMA or ADMA levels were determined with immunoblotting. (E)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were treated with TNFα (10 ng/ml) for 15 min. The level of SDMA on RIPK1 was determined by immunoprecipitation and immunoblotting. (F)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were treated with TNFα (10 ng/ml) for 15 min. The level of RIPK1 R606me2s was determined with immunoblotting. (G) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with CHX (1 μM)/TNFα (10 ng/ml) for the indicated time. RIPK1 R606me2s, RIPK1, and ADK levels were analyzed with immunoblotting. (H) MEFs transfected with or without siAdk were stimulated with Flag-TNFα (10 ng/ml) for 15 min after the pretreatment of DZNep (10 μM), 5-ITu (20 μM), or adenosine (1 mM) for 1 h. Complex I was immunoprecipitated using anti-Flag antibody, and RIPK1 R606me2s level in complex I or whole-cell lysates was determined with immunoblotting. (I and J) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with CHX (1 μM)/TNFα (10 ng/ml) for the indicated time. Adenosine levels in the cells were measured and compared (I). SAM, SAH levels, and their ratios in the cells were measured and compared (J). (K and L)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were transfected with siAdk and subsequently treated with TNFα (10 ng/ml) for the indicated time (K) or 12 h (L) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (K). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (L). (M and N)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were treated with adenosine (1 mM) and subsequently treated with TNFα (10 ng/ml) for the indicated time (M) or 12 h (N) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (M). The levels of p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (N). Data are represented as the mean ± SD (I–K and M). Data are representative of n = 3 independent experiments (A–N). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (I–K and M). ***P < 0.001. Source data are available for this figure: SourceData F3.

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