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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.

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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.
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pone-0112761-g004: 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.

Mentions: We constructed a number of TBL2 deletion mutants to determine which regions would be required for interaction with PERK (Figure 4A). In this analysis, we also examined whether TBL2 interacts with eIF2α because it is a well-characterized PERK substrate [4], [5]. Interestingly, TBL2 also associated with eIF2α under both normal and thapsigargin-treated conditions (Figure 4B right panel, lanes with “WT”). The mutants that lacked part of the WD40 domain, 131-447aa and 1-350aa, completely lost the ability to associate with eIF2α (Figure 4B), suggesting that a large region of the WD40 domain is required for interaction with eIF2α. Similar requirement of large region of WD40 domain for proper activity has been shown in previous reports on WD40 proteins, UAF1 and COP1 [19], [20]. Given that the WD40 domain forms a circularized, propeller structure consisting of each blade of WD40 repeats [21], all of the WD40 repeats may be required for correct folding of TBL2. Next, we found that the 32-447aa TBL2 mutant lacking the N-terminal TM region exhibited impaired interaction with phospho-PERK, probably due to its inability to be retained in the ER membrane (Figure 4B and 1B). The 75-447aa mutant completely lost the PERK interaction ability, suggesting that 32-74aa was crucial for phospho-PERK binding (Figure 4B). The C-terminal deletion of TBL2 (the 1-350aa mutant) also weakened the interaction with PERK but the mutant was still able to bind to PERK (Figure 4B). To determine which region binds to phospho-PERK, we constructed the del32-74aa mutant, which lacked the 32-74aa region only, and compared it with the 1-350aa (Figure 4C). Both mutants localized in the ER (Figure 4D). The del32-74aa mutant kept the association with eIF2α but completely lost the ability to interact with phospho-PERK (Figure 4E). In contrast, while the 1-350aa mutant could not associate with eIF2α (Figure 4E), it still had phospho-PERK binding ability (Figure 4E). Therefore, we concluded that TBL2 interacts with phospho-PERK via the 32-74aa region and also associates with eIF2α via the WD40 domain. Thus, TBL2 forms the complex via its distinct regions. In addition, a PERK mutant lacking its cytoplasmic region (PERK-DN) barely bound to TBL2 despite a greater expression levels than those of PERK-WT (Figure S1), suggesting that TBL2 interacts likely with the cytoplasmic region of PERK.


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)

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.
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Related In: Results  -  Collection

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pone-0112761-g004: 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.
Mentions: We constructed a number of TBL2 deletion mutants to determine which regions would be required for interaction with PERK (Figure 4A). In this analysis, we also examined whether TBL2 interacts with eIF2α because it is a well-characterized PERK substrate [4], [5]. Interestingly, TBL2 also associated with eIF2α under both normal and thapsigargin-treated conditions (Figure 4B right panel, lanes with “WT”). The mutants that lacked part of the WD40 domain, 131-447aa and 1-350aa, completely lost the ability to associate with eIF2α (Figure 4B), suggesting that a large region of the WD40 domain is required for interaction with eIF2α. Similar requirement of large region of WD40 domain for proper activity has been shown in previous reports on WD40 proteins, UAF1 and COP1 [19], [20]. Given that the WD40 domain forms a circularized, propeller structure consisting of each blade of WD40 repeats [21], all of the WD40 repeats may be required for correct folding of TBL2. Next, we found that the 32-447aa TBL2 mutant lacking the N-terminal TM region exhibited impaired interaction with phospho-PERK, probably due to its inability to be retained in the ER membrane (Figure 4B and 1B). The 75-447aa mutant completely lost the PERK interaction ability, suggesting that 32-74aa was crucial for phospho-PERK binding (Figure 4B). The C-terminal deletion of TBL2 (the 1-350aa mutant) also weakened the interaction with PERK but the mutant was still able to bind to PERK (Figure 4B). To determine which region binds to phospho-PERK, we constructed the del32-74aa mutant, which lacked the 32-74aa region only, and compared it with the 1-350aa (Figure 4C). Both mutants localized in the ER (Figure 4D). The del32-74aa mutant kept the association with eIF2α but completely lost the ability to interact with phospho-PERK (Figure 4E). In contrast, while the 1-350aa mutant could not associate with eIF2α (Figure 4E), it still had phospho-PERK binding ability (Figure 4E). Therefore, we concluded that TBL2 interacts with phospho-PERK via the 32-74aa region and also associates with eIF2α via the WD40 domain. Thus, TBL2 forms the complex via its distinct regions. In addition, a PERK mutant lacking its cytoplasmic region (PERK-DN) barely bound to TBL2 despite a greater expression levels than those of PERK-WT (Figure S1), suggesting that TBL2 interacts likely with the cytoplasmic region of PERK.

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