RIPK1 R606me2s suppresses DD-mediated RIPK1 dimerization and the formation of complex I and complex II to inhibit subsequent cell death. (A and B) Protein folding of RIPK1-DD-WT (50 µM) and RIPK1-DD-R606K (50 µM) was determined by the thermal shift assay. Data were collected in the presence of RIPK1-DD-WT or RIPK1-DD-R606K, leading to a rightward shift in the unfolding transition. The apparent melting temperature (Tm) is the peak in the derivative of the unfolding curve (dF/dT), which is used as an indicator of thermal stability. (C) HEK293T cells were transfected with expression vectors for Flag-RIPK1 WT or Flag-RIPK1-R606K in the presence or absence of Nec-1s (10 μM), respectively. The autophosphorylation of RIPK1 was determined with immunoblotting. (D) HEK293T cells were cotransfected with HA-tagged RIPK1-DD and Flag-tagged RIPK1-DD expression plasmids as indicated for 24 h. The cells were treated with DZNep (10 μM), siADK, or adenosine (1 mM). The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. DD, death domain. (E) HEK293T cells were cotransfected with Flag-tagged RIPK1-DD, Flag-tagged RIPK1-DD-R606K, HA-tagged RIPK1-DD, and HA-tagged RIPK1-DD-R606K expression plasmids as indicated for 24 h. The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. DD, death domain. (F) HEK293T cells were cotransfected with HA-tagged RIPK1 and Flag-tagged RIPK1 expression plasmids as indicated for 24 h. The cells were treated with DZNep (10 μM), siADK, or adenosine (1 mM). The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (G) HEK293T cells were cotransfected with Flag-tagged RIPK1, Flag-tagged RIPK1-R606K, HA-tagged RIPK1, and HA-tagged RIPK1-R606K expression plasmids as indicated for 24 h. The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (H) HEK293T cells were cotransfected with HA-tagged RIPK1-4A and Flag-tagged RIPK1-4A expression plasmids as indicated for 24 h. The cells were treated with DZNep (10 μM), siADK, or adenosine (1 mM). The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. 4A, amino acid sequence of IQIG in RIPK1 RHIM was mutated to AAAA. (I) HEK293T cells were cotransfected with Flag-tagged RIPK1-4A, Flag-tagged RIPK1-4A-R606K, HA-tagged RIPK1-4A, and HA-tagged RIPK1-4A-R606K expression plasmids as indicated for 24 h. The cells were lysed with 0.5% Nonidet P-40 buffer and divided equally into two parts. The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. 4A, amino acid sequence of IQIG in RIPK1 RHIM was mutated to AAAA. (J) HEK293T cells were cotransfected with Flag-RIPK1 expression plasmids along with HA-tagged TNFR1-DD expression plasmids for 24 h. The cells were treated with DZNep (10 μM), siADK, or adenosine (1 mM). The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (K) HEK293T cells were cotransfected with Flag-RIPK1-WT or Flag-RIPK1-R606K expression plasmids, as well as HA-tagged TNFR1-DD expression plasmids for 24 h. The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (L) HEK293T cells were cotransfected with Flag-RIPK1 expression plasmids along with HA-tagged FADD expression plasmids for 24 h. The cells were treated with DZNep (10 μM), siADK, or adenosine (1 mM). The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (M) HEK293T cells were cotransfected with Flag-RIPK1-WT or Flag-RIPK1-R606K expression plasmids, as well as HA-tagged FADD expression plasmids for 24 h. The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (N) HEK293T cells were cotransfected with Flag-RIPK1 expression plasmids along with HA-tagged TRADD expression plasmids for 24 h. The cells were treated with DZNep (10 μM), siADK, or adenosine (1 mM). The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (O) HEK293T cells were cotransfected with Flag-RIPK1-WT or Flag-RIPK1-R606K expression plasmids, as well as HA-tagged TRADD expression plasmids for 24 h. The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. Data are representative of n = 3 independent experiments (A–O). Source data are available for this figure: SourceData F4.
Figure 4.

RIPK1 R606me2s suppresses DD-mediated RIPK1 dimerization and the formation of complex I and complex II to inhibit subsequent cell death. (A and B) Protein folding of RIPK1-DD-WT (50 µM) and RIPK1-DD-R606K (50 µM) was determined by the thermal shift assay. Data were collected in the presence of RIPK1-DD-WT or RIPK1-DD-R606K, leading to a rightward shift in the unfolding transition. The apparent melting temperature (Tm) is the peak in the derivative of the unfolding curve (dF/dT), which is used as an indicator of thermal stability. (C) HEK293T cells were transfected with expression vectors for Flag-RIPK1 WT or Flag-RIPK1-R606K in the presence or absence of Nec-1s (10 μM), respectively. The autophosphorylation of RIPK1 was determined with immunoblotting. (D) HEK293T cells were cotransfected with HA-tagged RIPK1-DD and Flag-tagged RIPK1-DD expression plasmids as indicated for 24 h. The cells were treated with DZNep (10 μM), siADK, or adenosine (1 mM). The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. DD, death domain. (E) HEK293T cells were cotransfected with Flag-tagged RIPK1-DD, Flag-tagged RIPK1-DD-R606K, HA-tagged RIPK1-DD, and HA-tagged RIPK1-DD-R606K expression plasmids as indicated for 24 h. The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. DD, death domain. (F) HEK293T cells were cotransfected with HA-tagged RIPK1 and Flag-tagged RIPK1 expression plasmids as indicated for 24 h. The cells were treated with DZNep (10 μM), siADK, or adenosine (1 mM). The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (G) HEK293T cells were cotransfected with Flag-tagged RIPK1, Flag-tagged RIPK1-R606K, HA-tagged RIPK1, and HA-tagged RIPK1-R606K expression plasmids as indicated for 24 h. The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (H) HEK293T cells were cotransfected with HA-tagged RIPK1-4A and Flag-tagged RIPK1-4A expression plasmids as indicated for 24 h. The cells were treated with DZNep (10 μM), siADK, or adenosine (1 mM). The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. 4A, amino acid sequence of IQIG in RIPK1 RHIM was mutated to AAAA. (I) HEK293T cells were cotransfected with Flag-tagged RIPK1-4A, Flag-tagged RIPK1-4A-R606K, HA-tagged RIPK1-4A, and HA-tagged RIPK1-4A-R606K expression plasmids as indicated for 24 h. The cells were lysed with 0.5% Nonidet P-40 buffer and divided equally into two parts. The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. 4A, amino acid sequence of IQIG in RIPK1 RHIM was mutated to AAAA. (J) HEK293T cells were cotransfected with Flag-RIPK1 expression plasmids along with HA-tagged TNFR1-DD expression plasmids for 24 h. The cells were treated with DZNep (10 μM), siADK, or adenosine (1 mM). The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (K) HEK293T cells were cotransfected with Flag-RIPK1-WT or Flag-RIPK1-R606K expression plasmids, as well as HA-tagged TNFR1-DD expression plasmids for 24 h. The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (L) HEK293T cells were cotransfected with Flag-RIPK1 expression plasmids along with HA-tagged FADD expression plasmids for 24 h. The cells were treated with DZNep (10 μM), siADK, or adenosine (1 mM). The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (M) HEK293T cells were cotransfected with Flag-RIPK1-WT or Flag-RIPK1-R606K expression plasmids, as well as HA-tagged FADD expression plasmids for 24 h. The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (N) HEK293T cells were cotransfected with Flag-RIPK1 expression plasmids along with HA-tagged TRADD expression plasmids for 24 h. The cells were treated with DZNep (10 μM), siADK, or adenosine (1 mM). The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. (O) HEK293T cells were cotransfected with Flag-RIPK1-WT or Flag-RIPK1-R606K expression plasmids, as well as HA-tagged TRADD expression plasmids for 24 h. The interaction between Flag-tagged and HA-tagged proteins was determined with immunoprecipitation and immunoblotting. Data are representative of n = 3 independent experiments (A–O). Source data are available for this figure: SourceData F4.

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