TBL2 is a novel PERK-binding protein that modulates stress-signaling and cell survival during endoplasmic reticulum stress.

Yoshinori Tsukumo,Toru Natsume,Akihiro Tomida,Shun-Ichiro Iemura,Aki Furuno,Satomi Tsukahara,Dong-Yan Jin

doi:10.1371/journal.pone.0112761

Abstract

Under ER stress, PKR-like ER-resident kinase (PERK) phosphorylates translation initiation factor eIF2α, resulting in repression of global protein synthesis and concomitant upregulation of the translation of specific mRNAs such as activating transcription factor 4 (ATF4). This PERK function is important for cell survival under ER stress and poor nutrient conditions. However, mechanisms of the PERK signaling pathway are not thoroughly understood. Here we identify transducin (beta)-like 2 (TBL2) as a novel PERK-binding protein. We found that TBL2 is an ER-localized type-I transmembrane protein and preferentially binds to the phosphorylated form of PERK, but not another eIF2α kinase GCN2 or ER-resident kinase IRE1, under ER stress. Immunoprecipitation analysis using various deletion mutants revealed that TBL2 interacts with PERK via the N-terminus proximal region and also associates with eIF2α via the WD40 domain. In addition, TBL2 knockdown can lead to impaired ATF4 induction under ER stress or poor nutrient conditions such as glucose and oxygen deprivation. Consistently, TBL2 knockdown rendered cells vulnerable to stresses similarly to PERK knockdown. Thus, TBL2 serves as a potential regulator of the PERK pathway.

Highlights

The unfolded protein response (UPR) is a survival stress response enabling the cell to cope with the accumulation of unfolded proteins in the endoplasmic reticulum (ER) causing ER stress
We examined the subcellular localization of transducin (beta)-like 2 (TBL2) and found it in the ER, as shown by co-localization with PKR-like ER-resident kinase (PERK) and ER-GFP (GFP with an ER localization signal, see Materials and Methods)
To verify whether TBL2 preferentially interacted with phospho-PERK, we investigated the interaction with the PERK kinase-dead form K621A (PERK-KD) or another typeI ER transmembrane kinase, inositol-requiring enzyme 1 (IRE1), which is another important sensor of the UPR [1,2]

Summary

Introduction

The unfolded protein response (UPR) is a survival stress response enabling the cell to cope with the accumulation of unfolded proteins in the endoplasmic reticulum (ER) causing ER stress. Three ER-membrane sensor proteins, PERK, activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1), play important roles in the UPR signaling [1,2]. These sensor proteins are activated in response to ER stress and transmit the signals to activate both transcriptional and translational gene expression programs. ATF4 expression is detected in hypoxic- and nutrientdeprived regions where it plays an important role in maintaining metabolic homeostasis and promoting cancer cell survival by transcriptionally regulating amino acid uptake and biosynthesis, autophagy, redox balance and angiogenesis [7]. We show transducin (beta)-like 2 (TBL2) as a novel PERK-binding protein

Methods

Results

Conclusion