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

Tsukumo Y, Tsukahara S, Furuno A, Iemura S, Natsume T, Tomida A - PLoS ONE (2014)

Bottom Line: 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).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.Thus, TBL2 serves as a potential regulator of the PERK pathway.

View Article: PubMed Central - PubMed

Affiliation: Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231078&req=5

pone-0112761-g006: 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.

Mentions: We also examined the role of TBL2 under low glucose and hypoxic conditions, which arephysiological cell conditions observed in the tumor microenvironment or during ischemia and that cause the UPR [2], [3], [24], [25]. As expected, glucose withdrawal (glc(−)) induced a PERK-TBL2 interaction (Figure 6A). Although hypoxia alone did not trigger the interaction at this time point (4 h), hypoxia combined with glc(−) enhanced PERK-TBL2 interaction compared with glc(−) alone (Figure 6A). We examined the response to glc(−) and hypoxia using stably TBL2-shRNA-expressing cells. In control shRNA-expressing cells, the glc(−)/hypoxia combination stimulated a ATF4 protein expression more strongly than each stressor alone (Figure 6B). By contrast, but similarly to the results of siRNA experiments (Figure 5), ATF4 induction in TBL2-shRNA-expressing cells was impaired and the eIF2α phosphorylation was similar level to that in control cells (Figure 6B). In addition, impaired ATF4 induction in TBL2 knockdown cells was observed even in the presence of a proteasome inhibitor MG132 (Figure 6C), suggesting that TBL2 is unlikely involved in protein degradation of ATF4. In contrast to decrease in the expression at the protein level, ATF4 mRNA expression was largely unchanged in TBL2-shRNA-expressing cells compared to control cells (Figure 6D). Thus, TBL2 appears to mediate the post-transcriptional process of ATF4 expression under glc(−)/hypoxia.


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

Tsukumo Y, Tsukahara S, Furuno A, Iemura S, Natsume T, Tomida A - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231078&req=5

pone-0112761-g006: 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.
Mentions: We also examined the role of TBL2 under low glucose and hypoxic conditions, which arephysiological cell conditions observed in the tumor microenvironment or during ischemia and that cause the UPR [2], [3], [24], [25]. As expected, glucose withdrawal (glc(−)) induced a PERK-TBL2 interaction (Figure 6A). Although hypoxia alone did not trigger the interaction at this time point (4 h), hypoxia combined with glc(−) enhanced PERK-TBL2 interaction compared with glc(−) alone (Figure 6A). We examined the response to glc(−) and hypoxia using stably TBL2-shRNA-expressing cells. In control shRNA-expressing cells, the glc(−)/hypoxia combination stimulated a ATF4 protein expression more strongly than each stressor alone (Figure 6B). By contrast, but similarly to the results of siRNA experiments (Figure 5), ATF4 induction in TBL2-shRNA-expressing cells was impaired and the eIF2α phosphorylation was similar level to that in control cells (Figure 6B). In addition, impaired ATF4 induction in TBL2 knockdown cells was observed even in the presence of a proteasome inhibitor MG132 (Figure 6C), suggesting that TBL2 is unlikely involved in protein degradation of ATF4. In contrast to decrease in the expression at the protein level, ATF4 mRNA expression was largely unchanged in TBL2-shRNA-expressing cells compared to control cells (Figure 6D). Thus, TBL2 appears to mediate the post-transcriptional process of ATF4 expression under glc(−)/hypoxia.

Bottom Line: 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).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.Thus, TBL2 serves as a potential regulator of the PERK pathway.

View Article: PubMed Central - PubMed

Affiliation: Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan.

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.

Show MeSH
Related in: MedlinePlus