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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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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.
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pone-0112761-g001: 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.

Mentions: To address the molecular mechanisms of the PERK signaling pathway, we screened novel PERK-binding partners by analyzing PERK-coprecipitated proteins in transiently PERK-overexpressed 293T cells using direct nano-flow liquid chromatography/tandem mass spectrometry [16]. As a result, we identified TBL2 (also termed WS-beta-TRP, WBSCR13), the function of which is unknown [8]. The SMART protein domain prediction program (http://smart.embl.de/) suggested that TBL2 contained the N-terminal proximal transmembrane region (TM: 9-31aa), the WD40, and the C-terminal coiled coil domains (Figure 1A). In general, the WD40 and the coiled coil domains are known to engage in protein-protein interaction. In addition, hydropathy analysis of TBL2 with the TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM/) predicted one transmembrane domain corresponding to 9-31aa (Figure 1A, bottom). We examined the subcellular localization of TBL2 and found it in the ER, as shown by co-localization with PERK and ER-GFP (GFP with an ER localization signal, see Materials and Methods). In contrast, the del 1-31aa TBL2 mutant that lacked the putative TM region exhibited a broadly diffused staining pattern in the cell, suggesting that the TM region functions as ER-anchor region (Figure 1B). We further analyzed the intracellular distribution pattern of TBL2 in a cell fractionation experiment. Consistent with type I ER transmembrane proteins PERK and calnexin, TBL2 was enriched in the M fraction, which contains the ER and mitochondria, under both thapsigargin-treated or non-treated conditions (Figure 1C). The TBL2 protein, as well as PERK and calnexin, was also recovered in the N fraction probably because nuclear outer membrane is contiguous with ER membrane (Figure 1C).


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 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.
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Related In: Results  -  Collection

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Show All Figures
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pone-0112761-g001: 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.
Mentions: To address the molecular mechanisms of the PERK signaling pathway, we screened novel PERK-binding partners by analyzing PERK-coprecipitated proteins in transiently PERK-overexpressed 293T cells using direct nano-flow liquid chromatography/tandem mass spectrometry [16]. As a result, we identified TBL2 (also termed WS-beta-TRP, WBSCR13), the function of which is unknown [8]. The SMART protein domain prediction program (http://smart.embl.de/) suggested that TBL2 contained the N-terminal proximal transmembrane region (TM: 9-31aa), the WD40, and the C-terminal coiled coil domains (Figure 1A). In general, the WD40 and the coiled coil domains are known to engage in protein-protein interaction. In addition, hydropathy analysis of TBL2 with the TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM/) predicted one transmembrane domain corresponding to 9-31aa (Figure 1A, bottom). We examined the subcellular localization of TBL2 and found it in the ER, as shown by co-localization with PERK and ER-GFP (GFP with an ER localization signal, see Materials and Methods). In contrast, the del 1-31aa TBL2 mutant that lacked the putative TM region exhibited a broadly diffused staining pattern in the cell, suggesting that the TM region functions as ER-anchor region (Figure 1B). We further analyzed the intracellular distribution pattern of TBL2 in a cell fractionation experiment. Consistent with type I ER transmembrane proteins PERK and calnexin, TBL2 was enriched in the M fraction, which contains the ER and mitochondria, under both thapsigargin-treated or non-treated conditions (Figure 1C). The TBL2 protein, as well as PERK and calnexin, was also recovered in the N fraction probably because nuclear outer membrane is contiguous with ER membrane (Figure 1C).

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