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. 2014 Nov 13;9(11):e112761.
doi: 10.1371/journal.pone.0112761. eCollection 2014.

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

Yoshinori Tsukumo  1 , Satomi Tsukahara  1 , Aki Furuno  1 , Shun-ichiro Iemura  2 , Toru Natsume  2 , Akihiro Tomida  1
Affiliations

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

Yoshinori Tsukumo et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. TBL2 is an ER-localized type-I transmembrane protein.
(A) The TBL2 domain structure was depicted by the SMART protein domain prediction program. TM: transmembrane region, WD40 domain, CC: coiled coil domain. bottom, topology of the TBL2 protein. Transmembrane domain prediction was done by using the TMHMM algorithm. (B) HT1080 cells were transiently transfected with each plasmid and then were fixed and analyzed by immunofluorescence using a confocal microscope. (C) Distribution of TBL2 in fractions of 293T cells. The cells were disrupted using a Dounce-type homogenizer and then each fraction (whole lysate [W], nuclei [N], mitochondria and ER [M], cytoplasm [C]) was separated by centrifugation and subjected to immunoblot analysis. (D) 293T cells were transfected with pTBL2 (V5-tag) and then the cells were disrupted using a Dounce-type homogenizer. The nucleus was removed by centrifugation and then the remaining crude cell extract, which contains microsome [M] and cytosol [C] fractions, was digested with trypsin for 5 min and subjected to immunoblot analysis.
Figure 2
Figure 2. TBL2 interacts with PERK in response to ER stress.
(A) 293T cells were transiently co-transfected with pTBL2 (V5-tag) and pFLAG-PERK, and then were treated with 300 nM thapsigargin for 1, or 3 h. The cell lysates were immunoprecipitated with anti-V5 antibody and immunoblotted with anti-FLAG or anti-V5 antibody. (B) 293T cells were transiently transfected with pFLAG-PERK or together with pTBL2 (non-tag). After immunoprecipitation with anti-FLAG conjugated beads, PERK-bound TBL2 protein was detected with anti-TBL2 antibody. (C) 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with 300 nM thapsigargin for 1 hour, 1 mM DTT for 30 min or with 5 mM histidinol (HisOH) for 4 hour. After immunoprecipitation with anti-FLAG antibody-conjugated beads, each protein was immunoblotted with the indicated antibody.
Figure 3
Figure 3. Preferential binding of TBL2 to phospho-PERK.
(A) 293T cells were transiently co-transfected with pTBL2 (V5-tag) and either pFLAG-PERK, pFLAG-PERK(K621A) or pFLAG-IRE1 and then were treated with 300 nM thapsigargin (Tg) for 2 h. The cell lysates were immunoprecipitated with anti-V5 antibody and immunoblotted with anti-FLAG or anti-V5 antibody. (B) 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with 300 nM thapsigargin (Tg), 4 µg/ml tunicamycin (Tu) or 10 mM 2-deoxyglucose (2DG) for 2 h. Endogenous PERK protein was detected with anti-PERK or anti–phospho-PERK antibody. (C) 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with the indicated doses of hydrogen peroxide (H2O2) for 4 hour. After immunoprecipitation with anti-FLAG antibody-conjugated beads, each protein was immunoblotted with the indicated antibody. (D) 786-O, 293 and 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with 300 nM thapsigargin (Tg) for 1 hour. After immunoprecipitation with anti-FLAG antibody-conjugated beads, each protein was immunoblotted with the indicated antibody.
Figure 4
Figure 4. Identification of PERK- and eIF2α-binding region.
(A) Schematic representations of each TBL2 mutant. (B) 293T cells were transiently transfected with each TBL2 mutant plasmid and then treated with 300 nM thapsigargin for 1 h. After immunoprecipitation, each sample was subjected to immunoblot analysis. (C) Schematic representations of each TBL2 mutant. (D) HT1080 cells were transiently co-transfected with pTBL2 mutant (FLAG, red) and pTBL2 WT (V5-tag, green) plasmids. After 24 hours, cells were fixed and then analyzed by immunofluorescence using confocal microscope. (E) 293T cells were transiently transfected with each TBL2 mutant plasmid and then treated with 300 nM thapsigargin for 0.5 or 1 h. After immunoprecipitation, each sample was subjected to immunoblot analysis.
Figure 5
Figure 5. Effects of TBL2 knockdown on the PERK pathway.
(A) 293 cells were transiently transfected with non-silencing siRNA, two TBL2 siRNAs (#1, #2) or PERK siRNAs (#1, #2). After 48 h, the cells were treated with 300 nM thapsigargin for 90 min and analyzed by immunoblot anaysis. (B) 293 cells were transiently transfected with non-silencing siRNA, two TBL2 siRNAs (#1, #2) or PERK siRNAs (#1, #2). The protein synthesis rate was measured by incorporating [35S]methionine/cysteine. The pulse labeling was carried out during the last 20 min of the 40-min thapsigargin (Tg) treatment (100 or 300 nM). Upper: autoradiography image of SDS-PAGE. Lower panel: TCA precipitation sample was measured using a scintillation counter.
Figure 6
Figure 6. TBL2 knockdown impairs ATF4 induction under glucose- and oxygen-deprived conditions.
(A) 293T cells were transiently transfected with pFLAG-TBL2. Then, the cells were incubated for 4 h under glc(−) and/or hypoxic conditions (Hy). After immunoprecipitation with anti-FLAG-conjugated beads, each sample was subjected to immunoblot analysis. (B) Control-, TBL2- or PERK-shRNA-expressing 786-O cells were incubated for 2 h under glc(−) and/or hypoxic conditions (Hy). Each sample was subjected to immunoblot analysis. (C) Control or TBL2 shRNA-expressing cells were exposed to glc(−) and/or hypoxia (Hy) for 2 h in the presence or absence of 10 µM MG132. (D) TBL2- or PERK-shRNA-expressing cells were incubated for the indicated times under glc(−) and hypoxic conditions (Hy). ATF4 mRNA induction under glc(−)/hypoxic conditions was measured by qRT-PCR. β-actin mRNA levels were used for normalization.
Figure 7
Figure 7. TBL2 plays an important role in cell growth after exposure to glocose and oxygen deprivation.
(A) TBL2 or PERK shRNA-expressing cells were exposed to glc(−) and/or hypoxia for 12 h and then the cells were reseeded. Relative cell numbers after 3 days were measured by MTT assay. Data is representative of three independent experiments (n = 3) *P<0.05; **P<0.01. (B) Control or TBL2 shRNA-expressing cells were treated with 2 µM thapsigargin for 24 h. Cell viability was calculated by measuring intracellular ATP level. Data is representative of three independent experiments (n = 3) *P<0.05; **P<0.01. (C) The shRNA-expressing cells were exposed to glc(−) and hypoxia for 12 h and were then immediately reseeded. Then the cells were incubated in normal medium for the indicated time (days). Cell numbers at the each time point were counted automatically using a Beckman Coulter Counter. Data is representative of three independent experiments (n = 3) *P<0.05; **P<0.01.
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Grants and funding

This work was supported in part by Grant-in-Aid for Young Scientists (B) (22700892 YT), a Grant-in-Aid for scientific research (B) (22300342, 25290061 AT), a Grant-in-Aid for challenging Exploratory Research (24650626 AT), from the Ministry of Education, Culture, Sports, Science and Technology of Japan, National Cancer Center Research and Development Fund (21-3-1) from the Ministry of Health, Labour and Welfare, a Grant from Kobayashi Foundation for Cancer Research, and from the Vehicle Racing Commemorative Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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