RIPK1 both positively and negatively regulates RIPK3 oligomerization and necroptosis - PubMed (original) (raw)

RIPK1 both positively and negatively regulates RIPK3 oligomerization and necroptosis

S Orozco et al. Cell Death Differ. 2014 Oct.

Abstract

Necroptosis is a form of programmed cell death that depends on the activation of receptor interacting protein kinase-1 (RIPK1) and RIPK3 by receptors such as tumor necrosis factor (TNF) receptor-1. Structural studies indicate that activation of RIPK3 by RIPK1 involves the formation of oligomers via interactions of the RIP homotypic interaction motif (RHIM) domains shared by both proteins; however, the molecular mechanisms by which this occurs are not fully understood. To gain insight into this process, we constructed versions of RIPK3 that could be induced to dimerize or oligomerize in response to a synthetic drug. Using this system, we find that although the formation of RIPK3 dimers is itself insufficient to trigger cell death, this dimerization seeds a RHIM-dependent complex, the propagation and stability of which is controlled by caspase-8 and RIPK1. Consistent with this idea, we find that chemically enforced oligomerization of RIPK3 is sufficient to induce necroptosis, independent of the presence of the RHIM domain, TNF stimulation or RIPK1 activity. Further, although RIPK1 contributes to TNF-mediated RIPK3 activation, we find that RIPK1 intrinsically suppresses spontaneous RIPK3 activation in the cytosol by controlling RIPK3 oligomerization. Cells lacking RIPK1 undergo increased spontaneous RIPK3-dependent death on accumulation of the RIPK3 protein, while cells containing a chemically inhibited or catalytically inactive form of RIPK1 are protected from this form of death. Together, these data indicate that RIPK1 can activate RIPK3 in response to receptor signaling, but also acts as a negative regulator of spontaneous RIPK3 activation in the cytosol.

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Figures

Figure 1

RIPK3 dimerization seeds a RHIM-dependent necrosome complex. (a) Schematic representation of the dimerizable and oligomerizable RIPK3 constructs used in this study. These constructs were cloned upstream of a T2A-GFP sequence, such that RIPK3 constructs contain both N-terminal FLAG and C-terminal 2A epitope tags. (b and c) NIH-3T3 cells stably expressing RIPK3-1xFV were treated with indicated concentrations of AP1 (b), or with 30 nM AP1 in the presence or absence of 200 ng/ml TNFR1-Fc (c), and cell death was assessed over time using an IncuCyte imaging system. (d) NIH-3T3 cells stably expressing RIPK3ΔRHIM-1xFV were treated with increasing doses of dimerizer. (e) NIH-3T3 cells stably expressing RIPK3-1xFV were transfected with indicated siRNAs. Seventy-two hours later cells were treated with 30 nM AP1 and cell death was assessed. *_P_=0.0001 (f) NIH-3T3 cells stably expressing indicated constructs were treated as indicated, lysed and necrosome complexes were covalently cross-linked using DSS. Resulting complexes were resolved by western blotting. Nec1 and zVAD were used at 30 and 50 _μ_M, respectively, throughout

Figure 2

Receptor-independent RIPK3 oligomerization is regulated by RIPK1 and caspase-8. (a–c) NIH-3T3 cells stably expressing RIPK1-1xFV were treated with 30 nM AP1 and the indicated inhibitors, and cell death was assayed by IncuCyte. In b, cells were transfected with indicated siRNAs, then treated with AP1 72 h later. a: *P<0.0001, b: *_P_=0.0157, c: *_P_=0.0006. (d) NIH-3T3 cells stably expressing RIPK3-1xFV were treated with 30 nM AP1, as well as Nec1 or zVAD as indicated, and resulting complexes were resolved by western blotting. (e) NIH-3T3 cells expressing the indicated constructs were treated as shown for 30 min, then lysed and subjected to immunoprecipitation using an antibody to the FLAG epitopes expressed on the RIPK3 constructs. Immune complexes were purified and resolved using TruBlot reagents to avoid aspecific signals from immunoglobulins, as described. Nec1 and zVAD were used at 30 and 50 _μ_M, respectively, throughout

Figure 3

Chemically enforced RIPK3 oligomerization activates RIPK3 in the absence of the RHIM domain. (a–d) NIH-3T3 cells expressing RIPK3-2xFV (a and c) or RIPK3ΔC-2xFV (b and d) were treated as indicated, and cell death was assessed using an IncuCyte as described. (e) NIH-3T3 cells stably expressing indicated constructs were treated as indicated, lysed and necrosome complexes were covalently cross-linked using DSS. Resulting complexes were resolved by western blotting. (f) NIH-3T3 cells expressing the indicated constructs were treated as shown for 30 min, then lysed and subjected to immunoprecipitation using an antibody to the FLAG epitopes expressed on the RIPK3 constructs. Immune complexes were purified and resolved using TruBlot reagents to avoid aspecific signals from immunoglobulins, as described. Nec1 and zVAD were used at 30 and 50 _μ_M, respectively, throughout

Figure 4

The presence of RIPK1 inhibits spontaneous RIPK3 oligomerization and necroptosis. (a) NIH-3T3 cells expressing RIPK3-1xFV were transfected with indicated siRNAs, then with 30 nM AP1 or 30 _μ_M Nec1 72 h later. For scramble versus RIPK1 siRNA treated with AP1, *_P_=0.0024. (b) NIH-3T3 cells stably expressing DD-RIPK3 or DD-RIPK3K51A were treated with 1 _μ_M Shield drug for indicated times, then lysed and resolved by western blotting. Jackson immortalized fibroblasts (Jax) expressing endogenous RIPK3 are included as a control. NIH-3T3 cells expressing DD-RIPK3 were transfected with indicated siRNAs, then treated 72 h later with 1 ng/ml recombinant TNF (c) or 1 _μ_M Shield drug (d and e) and 30 _μ_M Nec1 as indicated. c: *P<0.0001, d: *P<0.0001, e: *_P_=0.0001, **P<0.0001

Figure 5

Catalytically inactive RIPK1 mimics the suppressive effects of Nec-1 on spontaneous RIPK3 oligomerization and necroptosis. (a) RIPK1/RIPK3 DKO MEF cells were stably transduced with wild-type murine RIPK1 or RIPK1K45A, then stably transduced with DD-RIPK3. These cell lines were treated with Shield drug as indicated, then lysed and resolved by western blotting. Jax cells are included as a control for endogenous RIPK expression. (b) The cell lines depicted in a were treated with Shield drug in combination with Nec-1 as indicated. *_P_=0.0154, **_P_=0.0027. (c and d) A speculative model for the control of RIPK3 activation by RIPK1; RHIM domains are depicted in green, death domains in red and death effector domains in yellow. (c) When RIPK1 is activated by receptor signals, it binds to and phosphorylates RIPK3 via RHIM–RHIM interactions. This leads to RHIM-driven RIPK3 oligomerization, MLKL recruitment and necroptosis. (d) In the absence of receptor signals, spontaneous RIPK3 activation recruits RIPK1, which in turn recruits caspase-8/FLIP complexes. This leads to inhibition of RIPK3 oligomerization, possibly via cIAP-mediated degradation of the nascent RIPK3 complex

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RIPK1 both positively and negatively regulates RIPK3 oligomerization and necroptosis - PubMed (original) (raw)