AIP1 Is Critical in Transducing IRE1-mediated Endoplasmic Reticulum Stress Response

Luyang Yu,Haifeng Zhang,Hong Chen,Wang Min,Fumihiko Urano,Shibo Tang,Yun He,Dianhong Luo,Zhe Xu

doi:10.1074/jbc.m710557200

Abstract

We have previously shown that ASK1-interacting protein 1 (AIP1) transduces tumor necrosis factor-induced ASK1-JNK signaling. Because endoplasmic reticulum (ER) stress activates ASK1-JNK signaling cascade, we investigated the role of AIP1 in ER stress-induced signaling. We created AIP1-deficient mice (AIP1-KO) from which mouse embryonic fibroblasts and vascular endothelial cells were isolated. AIP1-KO cells show dramatic reductions in ER stress-induced, but not oxidative stress-induced, ASK1-JNK activation and cell apoptosis. The ER stress-induced IRE1-JNK/XBP-1 axis, but not the PERK-CHOP1 axis, is blunted in AIP1-KO cells. ER stress induced formation of an AIP1-IRE1 complex, and the PH domain of AIP1 is critical for the IRE1 interaction. Furthermore, reconstitution of AIP1-KO cells with AIP1 wild type, not an AIP1 mutant with a deletion of the PH domain (AIP1-DeltaPH), restores ER stress-induced IRE1-JNK/XBP-1 signaling. AIP1-IRE1 association facilitates IRE1 dimerization, a critical step for activation of IRE1 signaling. More importantly, AIP1-KO mice show impaired ER stress-induced IRE1-dependent signaling in vivo. We conclude that AIP1 is essential for transducing the IRE1-mediated ER stress response.

Highlights

Adaptive endoplasmic reticulum (ER) stress response termed the unfolded protein response [1, 2]
TNF-induced apoptosis signal-regulating kinase-1 (ASK1)-JNK activation was reduced in ASK1-interacting protein 1 (AIP1)-KO endothelial cells (EC), consistent with our previous reports that AIP1 is critical for TNFinduced ASK1-JNK signaling (Fig. 1a)
We demonstrated that ER stress-induced IRE1JNK/XBP-1 axis, but not the PERK-CHOP1 axis, is blunted in AIP1-KO cells

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Treatment—Human umbilical vein EC were purchased from the Yale Vascular Biology and Therapeutic Endothelial Cell Culture Core Facility (Yale University). 10 ␮l of beads (per T-75 of mouse lung cells) were washed with 1 ml of buffer A (phosphate-buffered saline ϩ 2% fetal bovine serum) three times and resuspended in 100 ␮l of buffer A. Confluent mouse lung cells cultured in a T-75 flask were placed at 4 °C for 5 min and incubated with the beads at 4 °C for 1 h. Immunoprecipitation and Immunoblotting—BAEC after various treatments were washed twice with cold phosphatebuffered saline and lysed in 1.5 ml of cold lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Triton X-100, 0.75% Brij 96, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM sodium pyrophosphate, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, 1 mM EDTA) for 20 min on ice. Immunoprecipitation and immunoblotting were performed as described previously [14, 15].

TNF Tsg Tm kDa

RESULTS

ER TM

WT KO WT KO

DISCUSSION