Article Figures & Data
Figures
- FIG 1
RNF31 is cleaved during the process of apoptosis. (A and B) WB analysis of the indicated proteins in HeLa cells treated either with TNF-α (40 ng/ml) and CHX (10 μg/ml) (A) or with TRAIL (100 ng/ml) (B). Ex., exposure. (C) WB analysis of lysates of BxPC-1, Panc-1, A549, HCT116, HT29, and HeLa cells stimulated with TNF-α (20 ng/ml) and CHX (10 μg/ml) for 6 h. (D) WB analysis of HeLa cells exposed to Dox (3 μg/ml) or CPT (20 μM). (E) WB analysis of HeLa cells treated with a Smac mimetic (20 μM).
- FIG 2
RNF31 is cleaved by caspases during apoptosis but not during necroptosis. (A) Nonpretreated HeLa cells or HeLa cells pretreated with Z-VAD-FMK (20 μM) were treated with TNF-α (20 ng/ml) alone, CHX (10 μg/ml) alone, or both TNF-α and CHX and were then subjected to WB analysis. (B) WB analysis of WT, FADD-deficient, and caspase 8-deficient Jurkat cells stimulated with TNF-α (20 ng/ml) and CHX (10 μg/ml). Asterisks indicate pretreatment with the pancaspase inhibitor Z-VAD-FMK.
- FIG 3
RNF31 is cleaved by the effector caspases caspase 3 and caspase 6. (A) WB analysis of A431 cells treated with Dox (3 μg/ml) or CPT (20 μM). (B and C) WB analysis of caspase 8-deficient Jurkat cells stimulated with Dox (3 μg/ml), CPT (20 μM), or 5-FU (20 μg/ml). (D) Results of an in vitro cleavage assay in which Myc-tagged RNF31 proteins were incubated with or without the indicated recombinant caspases for 2 h. C, caspase; IB, immunoblot.
- FIG 4
RNF31 is cleaved at aspartates 348, 387, and 390. (A) WB analysis of 293T cells transfected with RNF31 tagged with Myc at the N terminus, followed by treatment with TNF-α (40 ng/ml) and CHX (10 μg/ml). (B) Estimated sites of RNF31 cleavage by caspases. (C) WB analysis of 293T cells transfected with a plasmid encoding Myc-conjugated WT, D390A, D348/390A, or D348/387/390A RNF31. (D) Results of an in vitro cleavage assay in which recombinant WT or D348/387/390A mutant RNF31 was incubated with or without caspase 8, caspase 3, or caspase 6 for 1 h.
- FIG 5
Activation of the NF-κB pathway is suppressed by RNF31 cleavage. (A) Schematic diagram of RNF31 domains. PUB, PNGase/UBA- or UBX-containing proteins; UBA, ubiquitin associated; IBR, in between Ring fingers; LDD, linear ubiquitin chain-determining domain. (B) Luciferase analysis of lysates from 293T cells transfected with NF-κB luciferase reporter genes and the indicated RNF31 mutants with or without HOIL-1 and Sharpin. FL, full length. (C) WB analysis of lysates from the cells analyzed in panel B. ubi, ubiquitination. (D) Luciferase analysis of lysates from 293T cells transfected with NF-κB luciferase reporter genes and the indicated RNF31 mutants with HOIL-1 and Sharpin. Con, control; n.s., not significant. Triple asterisks indicate significant differences (***, P < 0.001.).
- FIG 6
NEMO and RIP1 are conjugated with linear ubiquitination chains by the C-terminal RNF31 fragment. (A and C) WB analysis of immunoprecipitates (IP) from 293T cells transfected with FLAG-NEMO (A) or FLAG-RIP1 (C) and the indicated LUBAC components using anti-FLAG beads. (B and D) WB analysis of immunoprecipitates from lysates (prepared with 2% SDS lysis buffer and boiling) of 293T cells transiently transfected with the indicated plasmids using anti-FLAG beads. HS, HOIL-1 and Sharpin.
- FIG 7
A cleavage-resistant RNF31 mutant enhances resistance to apoptosis. (A) MTT analysis of lysates from control KD (shCon), RNF31 KD (shRNF31), WT RNF31-reconstituted RNF31 KD (SR WT), and cleavage-resistant RNF31 mutant-reconstituted RNF31 KD (SR MT134) HeLa cells treated with TNF-α (100 ng/ml). Single and double asterisks indicate significant differences at P values of <0.05 and <0.01, respectively. OD, optical density. (B) WB analysis of control, RNF31-deficient, RKO-WT, and RKO-MT134 Jurkat cells. (C) WB analysis of nuclear lysates from RKO-WT and RKO-MT134 Jurkat cells treated with TNF-α (20 ng/ml). (D) Flow cytometry analysis of Jurkat RKO-WT and Jurkat RKO-MT134 cells treated with TNF-α (200 ng/ml) for 24 h. The percentage of annexin V-stained cells is given in each panel. (E) Sensitivities to apoptosis of Jurkat RKO-WT and Jurkat RKO-MT134 cells treated with TNF-α (200 ng/ml) for the indicated periods. (F) Flow cytometry analysis of Jurkat RKO-WT and Jurkat RKO-MT134 cells treated with TNF-α (200 ng/ml) plus cycloheximide for the times shown. ns, not significant. Double and triple asterisks indicate significant differences at P values of <0.01 and <0.001, respectively. (G) WB analysis of RKO-WT and RKO-MT134 Jurkat cells stimulated with TNF-α (50 ng/ml). (H) WB analysis of control, RNF31-deficient, RKO-WT, and RKO-MT134 Jurkat cells treated with TNF-α (10 ng/ml) and CHX (10 μg/ml). (I) WB analysis of Jurkat RKO-Mock, Jurkat RKO-FL, Jurkat RKO-NT, and Jurkat RKO-CT cells stimulated with TNF-α (10 ng/ml). (J) Flow cytometry analysis of Jurkat RKO, Jurkat RKO-FL, Jurkat RKO-CT, and Jurkat RKO-NT cells treated with TNF-α (100 ng/ml) for 24 h.
- FIG 8
Proposed model of the current study. Upon TNF-α stimulation, activation of the caspase cascade leads to the initiation of apoptosis. Simultaneously, NF-κB signaling is activated through the activation of the LUBAC/IKKs, which promotes cell survival by regulating gene expression. Here we report the negative regulatory loop from apoptosis to NF-κB signaling. Activated effector caspases cleave RNF31, suppressing NF-κB activation and accelerating the apoptosis process.
