Volume 48, Issue 6, Pages 888-899 (December 2012) TRAF2 Sets a Threshold for Extrinsic Apoptosis by Tagging Caspase-8 with a Ubiquitin Shutoff Timer  Francois Gonzalvez, David Lawrence, Becky Yang, Sharon Yee, Robert Pitti, Scot Marsters, Victoria C. Pham, Jean-Philippe Stephan, Jennie Lill, Avi Ashkenazi  Molecular Cell  Volume 48, Issue 6, Pages 888-899 (December 2012) DOI: 10.1016/j.molcel.2012.09.031 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 48, 888-899DOI: (10.1016/j.molcel.2012.09.031) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Proteasomal Degradation of Activated Caspase-8 Dampens Proapoptotic DR Signaling (A) HCT116Bax+/− cells were pretreated for 1 hr with Btz (Btz, 50 nM), incubated with Apo2L/TRAIL for 14 hr, and assayed for viability. (B and C) HCT116Bax−/− cells were pretreated as in (A) and stimulated with Apo2L/TRAIL (Apo2L, 200 ng/ml, 4 hr). Cells were then plated at low density to form colonies (B) or analyzed by apoptosis assay (C). (D) HCT116Bax−/− cells were pretreated as in (A) and incubated with Apo2L/TRAIL (200 ng/ml). (E–G) HCT116Bax−/− cells were pretreated as in (A), stimulated with Apo2L/TRAIL, and assayed for caspase-8 (C8) and caspase-3/caspase-7 (C3/7) activity at 3 hr (E and F) or viability at 14 hr (G). (H) HCT116Bax−/− cells were pretreated as in (A) and stimulated with Apo2L/TRAIL (200 ng/ml, 2 hr). Stimulation was then continued for the indicated time in the absence or presence of z-VAD (20 μM), and the cells were lysed and analyzed by IB. (I) HCT116Bax−/− cells were pretreated as in (A) and stimulated with Apo2L/TRAIL (200 ng/ml). Cell lysates were analyzed for C8 activity. Data in (A), (C), (E)–(G), and (I) depict mean ± SEM of triplicates from one representative experiment of three or more. Molecular Cell 2012 48, 888-899DOI: (10.1016/j.molcel.2012.09.031) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 The Proteasome Targets the Cytosolic Pool of p43 and p18 (A and B) HCT116Bax−/− cells were pretreated for 1 hr with Btz (50 nM) and incubated with Apo2L/TRAIL (200 ng/ml). Cell lysates were subjected to IP with DR4 and DR5 antibodies. IP samples were analyzed by IB (A) or assayed for C8 activity (B). Of note, Btz had no effect on its own (gray bar). (C and D) DR4/5-immunodepleted lysates from (A) and (B) were subjected to IP with C8 antibody and analyzed by IB (C) or assayed for C8 activity (D). (E) HCT116Bax−/− cells were preincubated as in (A) and treated for 2 hr with Apo2L/TRAIL (200 ng/ml). Cells were fixed, stained with anticleaved C8 antibody (green) and DAPI (blue), and analyzed by confocal microscopy. (F and G) NB7 cells, stably expressing GFP, WT C8, or the S1 or S2 mutant variant of C8, were preincubated for 1 hr with MG132 (1 μM), stimulated with Apo2L/TRAIL (500 ng/ml, 6 hr), lysed, and analyzed by IB (F) or C8 activity assay (G). NB7 cells stably expressing GFP, WT C8, or a DR4:C8 chimera were preincubated and stimulated as in (F) and assayed for C8 activity. Data in (B), (D), (G), and (H) depict mean ± SEM of triplicates from one representative experiment of three or more. Molecular Cell 2012 48, 888-899DOI: (10.1016/j.molcel.2012.09.031) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 K48-Linked Polyubiquitination Tags Caspase-8 for Proteasomal Degradation (A) HCT116Bax−/− cells were pretreated for 1 hr with Btz (50 nM) and incubated with Apo2L/TRAIL (200 ng/ml, 2.5 hr). Cell lysates were subjected to denaturing IP with C8 antibody. (B and C) HCT116Bax−/− cells were pretreated and stimulated as in (A) and subjected to denaturing IP with K63-pUb (B) or K48-pUb antibody (C). (D and E) HCT116Bax−/− cells were treated with Apo2L/TRAIL (200 ng/ml), subjected to C8 IP under denaturing conditions, and analyzed by MS-AQUA to quantify K48 (D) and K63 (E) linkages. Where indicated, cells were pretreated for 1 hr with Btz (50 nM). Besides K48 and K63 linkages, a low amount of K11-modification (∼2 fmol) was detected, which was unaltered by Btz (not shown). Data represent a single MS-AQUA analysis from one of two independent experiments that showed similar ubiquitination profiles. (F and G) NB7 cells expressing a GFP control or WT caspase-8 were treated with M2-crosslinked Flag-Apo2L/TRAIL (500 ng/ml). Cell lysates were subjected to denaturing IP with antibody to K63-Ub (F) or K48-Ub (G). The asterisks refer to the heavy chains of the IP antibody. Molecular Cell 2012 48, 888-899DOI: (10.1016/j.molcel.2012.09.031) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 The Principal Site of K48-Linked Polyubiquitination Resides on p18 (A) HCT116Bax−/− cells were treated with Apo2L/TRAIL (200 ng/ml, 0.5 hr), subjected to denaturing C8 IP, and analyzed by MS-AQUA. Single ion chromatographic traces are shown for the isotopically labeled AQUA peptide (z = +3; upper left panel) and the endogenous peptide (z = +3; lower left panel). The high resolution full MS spectrum under these peaks revealed the presence of both the isotopic and endogenous peptide at a mass accuracy of <4 ppm, confirming that K224, K229, and K231 are ubiquitination sites (right panel). (B) HEK293T cells were transfected with GFP or Flag-tagged caspase-8 variants for 24 hr and subjected to denaturing Flag IP followed by IB with K48-Ub antibody. (C–E) NB7 cells stably expressing GFP or C8 variants were stimulated with Apo2L/TRAIL (500 ng/ml) and analyzed by IB (C) or assayed for C8 (D) and C3/7 (E) activity. (F) NB7 cells stably expressing C8 variants were stimulated with Apo2L/TRAIL for 24 hr and assayed for cell death. Data in (D)–(F) depict mean ± SEM of triplicates from one representative experiment of three or more. Molecular Cell 2012 48, 888-899DOI: (10.1016/j.molcel.2012.09.031) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 TRAF2 Inhibits Caspase-8 Activation and Associates with the DISC (A and B) HCT116Bax−/− cells were transfected with an siRNA pool or four single siRNAs targeting TRAF2 and stimulated with Apo2L/TRAIL (200 ng/ml, 3 hr). Cell lysates were analyzed by C8-Glo assay (A) and Live Green C8 imaging (B). (C and D) WT MEF, TRAF2 KO MEF, or TRAF2 KO MEFs reconstituted with WT TRAF2 (TRAF2KO+TRAF2WT) or a ΔRING TRAF2 mutant (TRAF2KO+TRAF2ΔRING) were treated with crosslinked Flag-Apo2L/TRAIL (0.5 μg/ml) and assayed for C8 activity at 14 hr (C) or cell death at 24 hr (D). (E) WT and TRAF2 KO MEFs were treated for 14 hr with anti-Fas antibody (Jo2, 0.5 μg/ml) and assayed for C8 enzymatic activity. (F and G) HeLa cells were transfected with the indicated siRNA, treated with anti-Fas antibody (CH11, 0.5 μg/ml), and assayed for C8 activity at 4 hr (F) or cell death at 24 hr (G). (H) HCT116Bax−/− cells were treated with Apo2L/TRAIL (500 ng/ml) and subjected to IP with DR4 and DR5 antibodies, followed by IB. (I) HCT116Bax−/− cells were treated with Apo2L/TRAIL (200 ng/ml), subjected to size exclusion chromatography as previously described (Jin et al., 2009), and high-MW fractions were analyzed by C8 IP and IB. Data in (A) and (C)–(G) depict mean ± SEM of triplicates from one representative experiment of three or more. Molecular Cell 2012 48, 888-899DOI: (10.1016/j.molcel.2012.09.031) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 TRAF2 Mediates K48-Polyubiquitination on p18 (A) HCT116Bax−/− cells transfected with nontargeted control (NTC) or pooled TRAF2 siRNAs were treated with crosslinked Flag-Apo2L/TRAIL (1 μg/ml) and analyzed by IB. (B) HCT116Bax−/− cells were transfected as in (A), stimulated for 2 hr with Apo2L/TRAIL, and treated with z-VAD (20 μM). Lysates were analyzed by IB. (C) HEK293T cells were transfected with T7-tagged, catalytically inactive mutant C8, ubiquitin (Ub), K48R-Ub, and TRAF2 siRNA, and analyzed by IB. (D) HEK293T cells were transfected with WT or mutant T7-C8, Ub, and TRAF2 siRNA and analyzed by IB. (E and F) HCT116Bax−/− cells were transfected with pooled TRAF2 siRNA (E) or cIAP1 and cIAP2 siRNAs (F) and stimulated with Apo2L/TRAIL (200 ng/ml) at 4°C. Cell lysates were subjected to denaturing C8 IP followed by IB. (G) HEK293T cells were transfected with Myc-tagged p18, TRAF2, or TRAF2ΔRING for 48 hr, and subjected to denaturing C8 IP followed by IB. Where indicated, cells were treated with MG132 (1 μM, 24 hr). (H and I) Recombinant TRAF2 (rTRAF2) was incubated with recombinant p18 (rp18) for 2 hr in a reaction mix containing E1 and E2 (UbcH5a, UbcH5b, and UbcH5c) enzymes and K48-only ubiquitin. Samples were then analyzed by IB (H) or subjected to denaturing C8 IP followed by IB (I). (J) HEK293T cells were cotransfected with Myc-tagged p18 plus TRAF2 or CUL3 constructs for 48 hr and analyzed by IB. (K and L) HCT116Bax−/− cells were transfected with indicated siRNAs, stimulated with crosslinked Flag-Apo2L/TRAIL (1 μg/ml), and subjected to TRAF2 (K) or CUL3 (L) IP followed by IB. Molecular Cell 2012 48, 888-899DOI: (10.1016/j.molcel.2012.09.031) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 TRAF2 Sets a Threshold for Caspase-8 Activation (A–C) HT29 cells were treated with Apo2L/TRAIL (500 ng/ml) in the presence or absence of TWEAK (250 ng/ml) and analyzed by IB at 14 hr (A) or assayed for C8 activity at 14 hr (B) or cell viability at 24 hr (C). Data in (B) and (C) depict mean ± SE of triplicates from one representative experiment of three or more. (D) Survival of WT mice injected intravenously with NTC or TRAF2 siRNA followed by intraperitoneal injection of agonistic anti-Fas antibody Jo2. n indicates number of mice analyzed. (E) WT mice were injected with NTC or TRAF2 siRNA and treated for 90 min with Jo2. Liver extracts were analyzed by IB. NI, noninjected WT mice. (F) Survival of Bid KO mice injected intravenously with NTC or TRAF2 siRNA followed by intraperitoneal injection of agonistic anti-Fas antibody Jo2. n indicates number of mice analyzed. (G) Bid KO mice were injected with NTC or TRAF2 siRNA and treated for 3 hr with Jo2. Liver extracts were analyzed by IB. NI, noninjected WT mice. Molecular Cell 2012 48, 888-899DOI: (10.1016/j.molcel.2012.09.031) Copyright © 2012 Elsevier Inc. Terms and Conditions