Abstract LB-336: Specific serine phosphorylation of IRE-1 controls enhanced splicing of XBP-1 and regulated IRE-1-dependent decay (RIDD) (2017)

Abstract The endoplasmic reticulum (ER) stress sensor IRE-1 is critical for cancer survival and anti-tumor immunity. IRE-1 splices the mRNA of XBP-1 to activate the functional XBP-1s transcription factor or engages regulated IRE-1-dependent decay (RIDD) to degrade tissue-specific mRNAs. It is unclear how IRE-1 RNase activity is regulated to perform the two functions. Upon XBP-1 deficiency, IRE-1 switches to perform RIDD. We examined IRE-1 in XBP-1-deficient B cells, and discovered that IRE-1 undergoes glycosylation at asparagine 178 (N178) and phosphorylation at serine 729 (S729). We generated an anti-phospho-S729 antibody and confirmed that S729 is indeed phosphorylated in XBP-1-deficient B cells. Different from conventional ER stress inducers or Toll-like receptor ligands, subtilase cytotoxin (SubAB), an AB5 toxin produced by Shiga-toxigenic E. coli, has an unusual capability in causing rapid and strong phosphorylation of S729. To further understand the function of N178 and S729 of IRE-1, we generated two knock-in mouse models (N178Q and S729A). Compared with wild-type B cells and those carrying the N178Q mutation, B cells carrying the S729A mutation similarly respond to cues of differentiation by expressing XBP-1s, but they completely fail to respond to pharmacological ER stress inducers or SubAB, suggesting that phosphorylation of S729 is critically important for secondary ER stress response. To evaluate the role of N178, S729 and the kinase domain of IRE-1 in regulating RIDD, we crossed N178Q, S729A and ΔIRE-1 (deletion of amino acids 652 to 751 in the kinase domain) mice with B cell-specific XBP-1KO (CD19Cre/XBP-1f/f) mice to induce RIDD. RIDD was evidenced by the decreased mRNA levels of secretory immunoglobulin (Ig) μ heavy chains in XBP-1-deficient B cells. RIDD was blocked in S729A/XBP-1KO and ΔIRE-1/XBP-1KO but not in N178Q/XBP-1KO B cells. While deleting the kinase function of IRE-1 blocks both XBP-1 splicing and RIDD, mutating S729 only blocks RIDD, highlighting an important role of S729 in regulating RIDD. Since SubAB can efficiently trigger phosphorylation of S729 of IRE-1, we further demonstrated that exposure to SubAB indeed causes RIDD of secretory Ig μ heavy chains in wild-type B cells but not in those carrying the S729A mutation, further supporting the role of S729 in regulating RIDD. In addition, we showed that a potent inhibitor of the IRE-1 RNase, B-I09, can inhibit RIDD. The identification of phosphorylated S729 as a control for RIDD may help scientists devise new ways to specifically target this phosphorylation, contributing to the control of human cancer. Citation Format: Chih-Hang Tang, Shiun Chang, Adrienne Paton, James Paton, Dmitry Gabrilovich, Hidde Ploegh, Juan Del Valle, Chih-Chi Andrew Hu. Specific serine phosphorylation of IRE-1 controls enhanced splicing of XBP-1 and regulated IRE-1-dependent decay (RIDD) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-336. doi:10.1158/1538-7445.AM2017-LB-336