Co-operative and Hierarchical Binding of c-FLIP and Caspase-8: A Unified Model Defines How c-FLIP Isoforms Differentially Control Cell Fate

Abstract

The death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIPL only) or anti-apoptotic (c-FLIPL/c-FLIPS) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIPL/S binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate.

Graphical abstract

Figure 1

Procaspase-8 Dimerization Is Required for Procaspase-8 Cleavage of c-FLIP_L at the DISC (A) Domain structure of procaspase-8b showing cleavage sites, active site (C360), and dimerization residue (T467) (Machα1 numbering). Reconstituted TRAIL DISC was assembled using GST-TRAIL-R1/R2 intracellular domain (TRAIL-R1/R2-IcD), recombinant FADD (r-FADD), and ³⁵S-labeled recombinant procaspase-8b (³⁵S r-Casp-8b, 100 μl) wild-type (WT), highly active (E201A/D210A/D216A), non-cleavable Quadruple (D210A/D216A/D374A/D384A), active site (C360A), or dimerization (T467D) mutants. TRAIL-R1/R2 DISCs were analyzed for r-Casp-8b cleavage fragments, IETDase activity, and proteolytic cleavage of procaspase-3 (C163A) or Bid. See also Figure S1. (B) TRAIL-R1 DISC (r-DISC) assembled using indicated ³⁵S r-Casp-8b variants (100 μl) alone (WT only) or in combination with c-FLIP_L (25 μl). Beads were assessed for r-Casp-8b cleavage, c-FLIP_L cleavage, and IETDase activity. Domain structure of c-FLIP_L showing caspase-8 cleavage site (D376). Scheme shows intra-dimer/inter-dimer cleavage of c-FLIP_L when combined with r-Casp-8b WT or dimerization mutant (T467D). ^∗non-specific band.

Figure 2

c-FLIP_L/Procaspase-8 Heterodimerization Is Critical for c-FLIP-Mediated Procaspase-8 Activation (A) r-DISCs assembled with ³⁵S r-Casp-8b (85 μl) alone (−) or with increasing amounts of c-FLIP_S (0–75 μl) or c-FLIP_L (0–225 μl). Beads were analyzed for r-Casp-8b cleavage, c-FLIP_L/S, and IETDase activity; control (−) r-DISC activity expressed as 100%. (B) r-DISC reconstituted (16 hr at 16°C) using sub-optimal levels of ³⁵S r-Casp-8b (50 μl) with increasing amounts of c-FLIP_L (0–75 μl). Beads were analyzed as in (A); graph shows time-dependent IETD.AFC hydrolysis. (C) r-DISCs assembled (16 hr at 16°C) with ³⁵S r-Casp-8b (50 μl) alone (−) or with c-FLIP_L mutants (50 μl) shown in (D). Beads were analyzed for r-Casp-8b cleavage, c-FLIP_L, and IETDase activity. (D) Domain organization of c-FLIP_L/S showing c-FLIP_L caspase-8 cleavage site (D376), dimerization residue (Q468), and p43 fragment (FLIP_L Δ). Scheme shows intra-dimer/inter-dimer activation of r-Casp-8b WT when combined with c-FLIP_L WT or dimerization mutant (Q468D). ^∗non-specific band.

Figure 3

DISC Reconstitution Reveals Co-operative and Hierarchical Recruitment of c-FLIP_L/S and Procaspase-8 (A) r-DISCs assembled using ³⁵S r-Casp-8b C360A (100 μl) with increasing amounts of c-FLIP_L/S (0–75 μl). Beads were analyzed for FADD, r-Casp-8b, and c-FLIP_L/S binding and quantified by densitometry (bar graphs). (B) r-DISCs reconstituted with c-FLIP_L/S (85 μl) and increasing amounts of ³⁵S r-Casp-8b C360A (0–75 μl) were analyzed as in (A). (C) r-DISC was pre-assembled using TRAIL-R1-IcD, r-FADD, and ³⁵S r-Casp-8b C360A (160 μl), and beads were washed to remove unbound protein before re-incubating with increasing amounts of c-FLIP_S (0–150 μl). Beads and supernatants, post first and second bind, were analyzed for r-Casp-8b and c-FLIP_S. (D) TRAIL-R1-IcD pull downs from caspase-8 null Jurkat lysates combined with exogenous r-Casp-8b C360A (0–60 μl) were analyzed for FADD, procaspase-8, and c-FLIP_L/S binding and quantified by densitometry. ^∗non-specific band. See also Figure S2.

Figure 4

DISC-Bound FADD Recruits Procaspase-8 via Procaspase-8 DED1 Pocket (A) DISC model in which multiple procaspase-8 molecules are recruited to FADD via DED-mediated interactions. (B) Modeled structure of procaspase-8 DEDs showing residues in the DED1 pocket that potentially interact with the FL motif of FADD or DED2 of another procaspase-8 molecule. (C) r-DISC reconstituted using the indicated variants of ³⁵S r-Casp-8b (100 μl) and beads assessed for FADD and r-Casp-8b binding (R5E/Y8D; DM). See also Figure S3. Scheme shows effect of mutating either procaspase-8 DED2 FL motif or key residues in the DED1 pocket.

Figure 5

c-FLIP and Procaspase-8 Are Recruited to the DISC via Molecularly Distinct Mechanisms (A) Modeled structure of c-FLIP and FADD showing DED pocket residues or FL motifs that could potentially interact with another DED molecule. (B) r-DISC assembled using wild-type (WT), DED2 FL (F114G/L115G), or DED1 pocket (H7D) mutants of c-FLIP_S (50 μl) minus/plus ³⁵S r-Casp-8b C360A (100 μl). Beads were assessed for FADD, r-Casp-8b, and c-FLIP_S binding. Schemes depict c-FLIP recruitment to the DISC in the absence or presence of procaspase-8. (C) r-DISC reconstituted using WT or DED pocket mutant (H9D) of r-FADD (5 μg) with ³⁵S r-Casp-8b C360A (100 μl) and WT c-FLIP_S (50 μl), singly or in combination, and analyzed as in (B). Scheme showing FADD DED pocket mutant can only bind c-FLIP_S via procaspase-8:c-FLIP_S DED interactions. See also Figure S4.

Figure 6

c-FLIP_S Blocks Cell Death by Inhibiting DED-Mediated Procaspase-8 Oligomerization (A) HeLa cells were transfected with empty vector (EGFP), GFP-tagged caspase-8 DEDs (C8 DED1-DED2-EGFP), or GFP-tagged c-FLIP_S (c-FLIP_S-EGFP) for 24 hr before fixing and staining with Hoechst. Cells were imaged and a representative field for each transfection is shown. Far right panels show enlargement of areas arrowed in GFP panels. Scale bar, 20 μm. (B) Native TRAIL DISC isolated from 5 × 10⁸ control (vector) and c-FLIP_S expressing BJAB cells stimulated with bTRAIL. Affinity-purified TRAIL DISCs and cleared lysate supernatants (Inputs) were analyzed by western blotting. DISCs were analyzed by label-free quantitative LC-MS/MS to determine the amount of FADD, caspase-8, and c-FLIP (lower panel; mean ± SEM; n = 3). (C) Native CD95 DISC isolated from 2 × 10⁸ control (vector) and c-FLIP_S expressing HaCaT cells stimulated with Fc-CD95L. DISCs were analyzed and quantified as in (B) (lower panel; mean ± SEM; n = 3). (D) HeLa cells transfected with GFP-tagged caspase-8 DEDs in combination with c-FLIP_S tagged with a non-fluorescent EGFP mutant (C8 DED1-DED2-EGFP + c-FLIP_S-R96S-EGFP) and visualized as in (A). See also Figures S5–S7.

Figure 7

A Unified Model Defines How c-FLIP Isoforms Differentially Regulate Procaspase-8 Activation to Direct Cell Fate In this model, the priming event is FADD recruitment to ligated DR. Initiation proceeds via procaspase-8 recruitment to FADD, with procaspase-8 DED1 pocket binding to FADD FL motif. Procaspase-8, in turn, recruits and heterodimerizes with c-FLIP_L/S via a co-operative and hierarchical binding mechanism. The composition of this procaspase-8:c-FLIP heterodimer then constitutes a key decision step, which determines procaspase-8 activation and subsequent cell fate. Heterodimer composition is critically regulated by the ratio of unbound c-FLIP_L/S to procaspase-8; thus, at physiological levels, procaspase-8:c-FLIP_L heterodimer forms the first active protease at the DISC, exhibits localized activity and is an activator, promoting procaspase-8 oligomer assembly and cell death. In contrast, high levels of c-FLIP_L preclude procaspase-8 oligomer assembly, restricting caspase-8:c-FLIP_L heterodimer activity and inhibiting cell death. c-FLIP_S does not readily form DED oligomers; thus, high levels of c-FLIP_S disrupt procaspase-8 oligomer assembly, resulting in a catalytically inactive procaspase-8:c-FLIP_S heterodimer and inhibition of cell death.