Figure 1. ER stress induces caspase-mediated cleavage of IRE1 in the cytoplasmic linker region.

(A) Schematic representation of the human IRE1 protein and comparison of the amino acid sequences surrounding the predicted caspase cleavage sites in the linker region of IRE1 from different species (zFish, zebrafish; Dros, Drosophila melanogaster). (B, C) KMS11 (B) or OPM2 (C) cells were treated with 100 nM Tg, 5 μg/ml Tm, or 0.2 μg/ml BfeA for 16 hr, or 0.3 μg/ml SubAB for 3 hr. Cell lysates were analyzed by western blot (WB) with anti-IRE1α LD or anti-IRE1α CD antibody to detect the lumenal or cytoplasmic domains. (D, E) KMS11 (D) or OPM2 (E) cells were treated with 100 nM Tg, 5 μg/ml Tm, 0.2 μg/ml BfeA, or 1 mM DTT for 16 hr in the absence or presence of 20 μM zVAD. Samples were analyzed as in B for the presence of cleavage products. (F) A cDNA plasmid expressing either WT or doubly mutated IRE1 (D507A, D512A) was transiently transfected into KMS11 cells harboring CRISPR/Cas9-based IRE1 knockout. Cells were treated with either DMSO or 100 nM Tg for 16 hr and analyzed by WB as indicated. (G) Two independent KMS11 clones harboring CRISPR/Cas9-based BAX knockout were generated and validated for BAX deletion as compared to the parental cell line. Cells were treated with DMSO, 100 nM Tg, 0.5 μg/ml BfeA, 5 μg/ml Tm, or 1 mM DTT for 24 hr and analyzed by WB. (H) BMDCs were obtained from C57/BL6 mice and treated with 100 nM Tg in the absence or presence of 20 μM zVAD for the indicated times. Equal amounts of protein from cell lysates were analyzed by WB. B–H show representative results from at least three similar experiments. DMSO vehicle was used as control.

Figure 1—figure supplement 1. ER stress induces caspase-mediated cleavage of IRE1 in the cytoplasmic linker region.

(A) KMS11 cells harboring IRE1 knockout were transiently transfected with WT IRE1 or mutant IRE1 in which the Asp at position 507 or 512 was replaced by Ala. After 48 hr, cells were treated with DMSO or 100 nM Tg for another 24 hr and analyzed by WB. (B) KMS11 cells were transfected with a plasmid expressing either WT IRE1 or a D507A, D512A double mutant and analyzed as in A, in the absence or presence of 20 μM zVAD. (C) Purified caspase-3 or caspase-7 proteins were incubated with a purified recombinant IRE1 protein comprising the linker, kinase and RNase domains (IRE1 LKR) and analyzed by western blot. Asterisk indicates a background band, while arrow indicates the cleavage fragment. (D) Summary of cell lines that displayed similar IRE1 processing in response to treatment with Tg. (E, F) Corresponding WB data for most of the cell lines listed in the table. Cells were incubated with 100 nM Tg in the absence or presence of 20 μM zVAD for the indicated time (E) or 24 hr (F). WB data show representative results from at least three similar experiments. DMSO vehicle was used as control.