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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 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.
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pone-0112761-g002: 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.

Mentions: To confirm binding of TBL2 to PERK, we performed immunoprecipitation and immunoblotting after cotransfection of PERK and TBL2 plasmids into 293T cells. As shown in Figure 2A, PERK coprecipitated with TBL2 when cells were treated with thapsigargin, a representative ER stress-inducing agent. Similarly, immunoprecipitation of PERK protein also showed thapsigargin-stimulated binding of endogenous or exogenous TBL2 protein (Figure 2B). The PERK-TBL2 interaction was also stimulated by treatment with other ER stress inducer DTT, but not amino acid starvation-mimicking agent histidinol [18] (Figure 2C). In contrast to PERK, TBL2 did not interact with GCN2, another eIF2α kinase (Figure 2C).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112761-g002: 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.
Mentions: To confirm binding of TBL2 to PERK, we performed immunoprecipitation and immunoblotting after cotransfection of PERK and TBL2 plasmids into 293T cells. As shown in Figure 2A, PERK coprecipitated with TBL2 when cells were treated with thapsigargin, a representative ER stress-inducing agent. Similarly, immunoprecipitation of PERK protein also showed thapsigargin-stimulated binding of endogenous or exogenous TBL2 protein (Figure 2B). The PERK-TBL2 interaction was also stimulated by treatment with other ER stress inducer DTT, but not amino acid starvation-mimicking agent histidinol [18] (Figure 2C). In contrast to PERK, TBL2 did not interact with GCN2, another eIF2α kinase (Figure 2C).

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