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. 2012 Apr 27;287(18):14863-72.
doi: 10.1074/jbc.M111.288670. Epub 2012 Feb 23.

Intermediate domain of receptor-interacting protein kinase 1 (RIPK1) determines switch between necroptosis and RIPK1 kinase-dependent apoptosis

Linde Duprez  1 , Mathieu J M Bertrand, Tom Vanden Berghe, Yves Dondelinger, Nele Festjens, Peter Vandenabeele
Affiliations

Intermediate domain of receptor-interacting protein kinase 1 (RIPK1) determines switch between necroptosis and RIPK1 kinase-dependent apoptosis

Linde Duprez et al. J Biol Chem. .

Abstract

Receptor-interacting protein kinase 1 (RIPK1) is an important component of the tumor necrosis factor receptor 1 (TNFR1) signaling pathway. Depending on the cell type and conditions, RIPK1 mediates MAPK and NF-κB activation as well as cell death. Using a mutant form of RIPK1 (RIPK1ΔID) lacking the intermediate domain (ID), we confirm the requirement of this domain for activation of these signaling events. Moreover, expression of RIPK1ΔID resulted in enhanced recruitment of caspase-8 to the TNFR1 complex II component Fas-associated death domain (FADD), which allowed a shift from TNF-induced necroptosis to apoptosis in L929 cells. Addition of the RIPK1 kinase inhibitor necrostatin-1 strongly reduced recruitment of RIPK1 and caspase-8 to FADD and subsequent apoptosis, indicating a role for RIPK1 kinase activity in apoptotic complex formation. Our study shows that RIPK1 has an anti-apoptotic function residing in its ID and demonstrates a cellular system as an elegant genetic model for RIPK1 kinase-dependent apoptosis that, in contrast to the Smac mimetic model, does not rely on depletion of cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2).

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Figures

FIGURE 1.
FIGURE 1.
Absence of the RIPK1 intermediate domain inhibits TNF-induced necroptosis and prosurvival signaling. A, overview of the results of the structure-function analysis of RIPK1 in TNF signaling. A series of RIPK1 mutants was overexpressed in HEK293T or L929sACrmA cells as indicated, and their effect on activation of NF-κB, induction of necroptosis, and TNF-induced necroptosis was analyzed. −, no induction of NF-κB or necroptosis; +, induction of NF-κB or necroptosis; ND, not done due to toxic effect of overexpression. B, V5-tagged mRIPK1ΔDD and mRIPK1 were overexpressed together with FLAG-tagged hRIPK1 in HEK293T cells, and their interaction was analyzed by FLAG-mediated immunoprecipitation. C, parental and RIPK1ΔID expressing L929sA cells were stimulated with hTNF (10,000 IU/ml) for the indicated durations. Lysates were made and analyzed for phospho-IκB and total IκB (upper panel), phospho-JNK, and total JNK (middle panel), and phospho-p38 and total p38 (lower panel). An asterisk indicates a nonspecific band.
FIGURE 2.
FIGURE 2.
Ectopic expression of RIPK1ΔID induces a shift from necroptosis to apoptosis in response to TNF. A, parental and RIPK1ΔID-expressing L929sA cells were stimulated with hTNF (10,000 IU/ml) and cell death (% PI-positive cells) was analyzed by flow cytometry. B, cells from A were freeze-thawed, and their content of hypoploid DNA was analyzed by flow cytometry. C and D, parental and RIPK1ΔID-expressing L929sA cells were stimulated with hTNF (10,000 IU/ml) for the indicated time points. Lysates were collected and analyzed for caspase-3 cleavage (Casp-3; C) and caspase activity (D). Error bars represent standard deviation of biological triplicates. E, parental and RIPK1ΔID-expressing L929sA cells were stained with Hoechst 33242 (blue) and PI (red) for live-cell imaging and monitored for 12 h after stimulation with hTNF (10,000 IU/ml). Representative overlay snapshots of the fluorescent and transmitted light images at the indicated time points after hTNF stimulation are shown. Scale bar, 20 μm.
FIGURE 3.
FIGURE 3.
Ectopic expression of RIPK1ΔID promotes the formation of a caspase-8 activating complex. A, parental and RIPK1ΔID-expressing L929sA cells were stimulated with hTNF (10,000 IU/ml) after pretreatment with Z-VAD-fmk (20 μm). Lysates were made at the indicated time points, and FADD was immunoprecipitated. Levels of FADD, caspase-8, RIPK1, RIPK1ΔID, and TRADD in the immunocomplex were measured by Western blot. An asterisk indicates nonspecific bands. B and C, L929sA cells expressing RIPK1ΔID were transfected with siRNA targeting caspase-8 before stimulation with hTNF (10,000 IU/ml). Caspase activity (B) and cell death (C) (% PI-positive cells measured using a BDPathwayTM 855 instrument) were analyzed at the indicated time points. D, Western blot showing efficiency of caspase-8 knockdown. E and F, L929sA cells expressing RIPK1ΔID were transfected with siRNA targeting TRADD before stimulation with hTNF (10,000 IU/ml). Caspase activity (E) and cell death (F) (% PI-positive cells measured using a BDPathwayTM 855 instrument) were analyzed at the indicated time points. G, Western blot showing efficiency of TRADD knockdown. Error bars represent S.D. of biological triplicates. Ctrl, control.
FIGURE 4.
FIGURE 4.
TNF-induced formation of the caspase-8 activating complex II and consequent apoptosis are dependent on RIPK1 kinase activity. A and B, L929sA cells expressing RIPK1ΔID were pretreated with Nec-1 (10 μm) for 1 h and stimulated with hTNF (10,000 IU/ml). Cell death (% PI-positive cells) was analyzed by flow cytometry (A) or cell survival was measured 20 h after hTNF treatment by 5-diphenyltetrazolium bromide (MTT) B, error bars represent S.D. of biological triplicates. C, parental and RIPK1ΔID-expressing L929sA cells were stimulated with hTNF (10,000 IU/ml) after treatment with Z-VAD-fmk (20 μm) and Nec-1 (10 μm). Lysates were made at indicated time points, and FADD was immunoprecipitated. Levels of FADD, caspase-8, RIPK1, RIPK1ΔID, and TRADD in the immunocomplex were measured by Western blot. An asterisk indicates nonspecific bands. D, parental and RIPK1ΔID-expressing L929sA cells were stimulated with hTNF (10,000 IU/ml) for the indicated durations. Lysates were made and analyzed for cIAP1 levels by Western blot.
FIGURE 5.
FIGURE 5.
Ectopic expression of RIPK1ΔID in RIPK1-depleted cells shifts the cell death response to apoptosis inhibited by Nec-1. L929sA cells were transduced with a miRNA directed against the 3′ UTR of the RIPK1 mRNA (RIPK1i) and reconstituted with an empty vector (EV) or RIPK1ΔID. A, expression levels of endogenous RIPK1 and RIPK1ΔID in parental L929sA cells and L929sA-RIPK1i + EV or RIPK1ΔID were analyzed by Western blot. B and C, RIPK1-depleted L929sA cells reconstituted with empty vector (B) or RIPK1ΔID (C) were stimulated with hTNF (10,000 IU/ml) after treatment with Nec-1 (10 μm), and cell death levels were analyzed via PI uptake. D, lysates were made from cells in B and C, and caspase activity was analyzed via DEVDase activity. E, RIPK1-depleted L929sA cells reconstituted with RIPK1ΔID were stimulated with hTNF (10,000 IU/ml) after treatment with Z-VAD-fmk (20 μm), and cell death levels were analyzed via PI uptake. Error bars represent S.D. of biological duplicates. Ctrl, control.
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