Volume 35, Issue 3, Pages 265-279 (August 2009) Reconstitution of the Death-Inducing Signaling Complex Reveals a Substrate Switch that Determines CD95-Mediated Death or Survival  Michelle A. Hughes, Nicholas Harper, Michael Butterworth, Kelvin Cain, Gerald M. Cohen, Marion MacFarlane  Molecular Cell  Volume 35, Issue 3, Pages 265-279 (August 2009) DOI: 10.1016/j.molcel.2009.06.012 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 CD95 Intracellular Domain and Recombinant FADD Restore DISC-Mediated Procaspase-8 Activation in FADD Null Lysates (A) CD95 intracellular domain (CD95-IcD) (10 μg) pull-downs (16 hr at 23°C) from Jurkat E6.1 cell lysates (10 mg) were immunoblotted for FADD, caspase-8, and caspase-3. Bound proteins (beads, left) and free/released proteins and caspase-3 activity (DEVDase) are shown (supernatant, right). Asterisks, nonspecific band. (B) Wild-type (WT) and DD mutant (lpr) of CD95-IcD were assessed for FADD, procaspase-8, and caspase-8 activity (IETDase). (C) CD95-IcD pull-downs from FADD null lysates plus or minus recombinant FADD (r-FADD) (5 μg) were assessed for FADD, caspase-8, and IETDase. In (B) and (C), an equal input of GST and GST-CD95-IcD was confirmed by immunoblotting for GST. (D) CD95-IcD pull-downs from FADD null lysates with WT or DD/DED mutants of r-FADD were analyzed for FADD, caspase-8, and IETDase. Molecular Cell 2009 35, 265-279DOI: (10.1016/j.molcel.2009.06.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Recombinant Procaspase-8a/8b Restores DISC Activity in Caspase-8 Null Lysates (A) Putative cleavage fragments derived from procaspase-8a/8b are shown with the death effector domain (DED) and starred active site Cysteine (C360). Machα1 numbering is used throughout. (B) Putative dimeric uncleaved (i) or various combinations of cleaved (ii) procaspase-8b are shown. (C) CD95-IcD pull-downs from caspase-8 null lysates plus or minus 35S-labeled recombinant procaspase-8a/8b (r-Casp-8a/8b) (50 μl) were analyzed by SDS-PAGE and autoradiography. 35S-labeled empty vector (pcDNA3) and GST beads were used as controls. Beads were analyzed for FADD and IETDase activity, and supernatants were assayed for DEVDase activity. (D) CD95-IcD pull-downs from caspase-8 null lysates using active site WT or C360A mutants of 35S-labeled r-Casp-8a/8b, either singly or in 1:1 combinations. Beads were analyzed for 35S-labeled r-Casp-8 and IETDase activity and lysates for active caspase-3 subunits (Asp175 Ab). The scheme shows intermolecular/interdimer cleavage with combinations of WT and active site C360A (X) mutants of r-Casp-8a/8b. Solid and open arrows indicate intermolecular and interdimer cleavage, respectively, whereas cleavage is blocked with a dimer of two C360A mutants. Molecular Cell 2009 35, 265-279DOI: (10.1016/j.molcel.2009.06.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Reconstitution of a Functional CD95 DISC Shows a Critical Requirement for Both Dimerization and Cleavage of Procaspase-8 (A) Reconstituted DISC (r-DISC) with CD95-IcD (10 μg), r-FADD (5 μg), and 35S-labeled r-Casp-8a/8b (or pcDNA3 control) added in the indicated volumes. Beads and supernatants (Supt) were assessed for 35S-labeled r-Casp-8a/8b, r-FADD, and IETDase. GST immunoblotting (see Figure 1) validated equal inputs. †, the increased release of caspase-8 p18 subunit into Supt. (B) Critical mutants of r-Casp-8b (100 μl) were used to reconstitute the r-DISC and cleavage fragments and IETDase detected, as in (A). Caspase-8 bioassay for r-DISC or Supt catalytic activity employed an active site mutant (C163A) of recombinant procaspase-3 (r-Casp-3). Molecular Cell 2009 35, 265-279DOI: (10.1016/j.molcel.2009.06.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Procaspase-8 Activation in the r-DISC Is Not Replicated by Enforced Dimerization of Soluble Procaspase-8 (A) 35S-labeled r-Casp-8a (C360A) or r-Casp-8b (WT, Quadruple, or T467D) mutants were incorporated singly or in combination in the r-DISC, plus or minus z-VAD.FMK (5 μM). Beads were assayed for IETDase, for 35S-labeled fragments, and for the p10 (Asp384 mAb) and p43/41 (C15 mAb) subunits (immunoblotting) of r-Casp-8. (B) Kosmotrope-induced dimerization of recombinant noncleavable ΔDED-Casp-8b (D374/384A) double mutant (DM) was induced by Na2Citrate. IETDase (RFU/min/μg) activity was measured plus or minus (solid/open bars) kosmotrope (upper-left); mean ± SEM; n = 3. Bio-VAD.fmk labeling of active caspase-8 was detected by immunoblotting (middle-left). Right panels show the effect of kosmotrope on IVT-produced ΔDED-Casp-8b or full-length caspase-8b variants. Bio-VAD labeling is shown (middle-right) and quantified by densitometry (fold-increase; upper-right). Nonspecific capture by beads alone is shown for ΔDED-Casp-8b minus kosmotrope/Bio-VAD. Bottom panels show equal input of bacterial- or IVT-Casp-8b. Molecular Cell 2009 35, 265-279DOI: (10.1016/j.molcel.2009.06.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Two-Step Activation Mechanism for Procaspase-8 Reveals a Critical Switch in Catalytic Activity and Substrate Repertoire of the CD95 DISC (A and B) In the r-DISC, 35S-labeled r-Casp-8b (WT, Quadruple, or C360A; 100 μl) was used alone (WT only) or combined with (A) c-FLIPL (25 μl) or (B) RIP (60 μl). Beads were assessed for 35S-labeled r-Casp-8b cleavage (top), r-DISC IETDase activity, and (A) the p12 fragment of c-FLIPL or (B) cleaved RIP (bottom). Asterisks, nonspecific band. (C) r-DISC containing 35S-labeled r-Casp-8b (WT, Quadruple, C360A, or the ALPS-associated caspase-8 mutant, R248W) was assessed for r-Casp-8b cleavage fragments and IETDase (top), and r-DISC activity was bioassayed for r-procaspase-3 (C163A) or Bid cleavage (bottom). (D) In the r-DISC, 35S-labeled r-procaspase-8b (WT, Quadruple, or C360A; 100 μl) was used alone (WT) or in combination with 35S-labeled ΔDED-Casp-8b (C360A)-His6 (50 μl). Beads and Supts were assessed for 35S-labeled r-Casp-8b/ΔDED-Casp-8b-His6 cleavage (top) and the His6-tagged p10 fragment of ΔDED-Casp-8b (C360A)/p10 fragment of r-Casp-8b (His6/Asp384 Abs) (bottom). Molecular Cell 2009 35, 265-279DOI: (10.1016/j.molcel.2009.06.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Dimerization and Proteolytic Cleavage of Procaspase-8 in the DISC Are Essential for Triggering CD95-Induced Apoptosis (A) Apoptosis in duplicate Jurkat cell clones stably expressing WT or mutant procaspase-8b after 16 hr exposure to anti-CD95 (50 ng/ml) (top; mean ± SEM; n = 3). Cells were immunoblotted for caspase-8, caspase-3, and PARP cleavage (bottom). A longer blot exposure shows that the most CD95-sensitive caspase-8 mutant clones (1-A5; D210/216A and 1-C12; D210/216A/E201A) generated only caspase-8 p41/p10 and no p18 fragment (right). (B) Native CD95 DISC analysis of parental (A3) and caspase-8-deficient Jurkat cells or clones expressing WT or mutant procaspase-8b (Quadruple or D210/216A/E201A). Apoptosis was assessed after 4 hr at 37°C. Beads plus unstimulated lysates (u/s) controlled for nonspecific interactions. DISCs were blotted for FADD and caspase-8. Asterisks, nonspecific band. Molecular Cell 2009 35, 265-279DOI: (10.1016/j.molcel.2009.06.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 A Critical Substrate Switch Mechanism Determines CD95-Mediated Death or Survival From our studies with the r-DISC, we propose that initial recruitment of procaspase-8 into the DISC generates a tethered procaspase-8 dimer with low intrinsic catalytic activity and narrow substrate range (step 1, DISC “priming”). This, in turn, triggers proteolysis to remove the D374-D384 linker, thus generating the highly active caspase-8 p41/p10 form within the DISC (step 2). This “fully activated” DISC exhibits a broad substrate repertoire and now cleaves procaspase-3 and Bid, the downstream drivers of apoptotic cell death. In the absence of intersubunit linker cleavage, the procaspase-8 dimer exhibits a restricted substrate repertoire limited only to itself or c-FLIP and, consequently, signals for cell survival. Molecular Cell 2009 35, 265-279DOI: (10.1016/j.molcel.2009.06.012) Copyright © 2009 Elsevier Inc. Terms and Conditions