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KDEL receptor 1 regulates T-cell homeostasis via PP1 that is a key phosphatase for ISR.

Kamimura D, Katsunuma K, Arima Y, Atsumi T, Jiang JJ, Bando H, Meng J, Sabharwal L, Stofkova A, Nishikawa N, Suzuki H, Ogura H, Ueda N, Tsuruoka M, Harada M, Kobayashi J, Hasegawa T, Yoshida H, Koseki H, Miura I, Wakana S, Nishida K, Kitamura H, Fukada T, Hirano T, Murakami M - Nat Commun (2015)

Bottom Line: Mechanistic analysis shows that KDELR1 directly regulates protein phosphatase 1 (PP1), a key phosphatase for ISR in naïve T cells.T-Red KDELR1 does not associate with PP1, resulting in reduced phosphatase activity against eIF2α and subsequent expression of stress responsive genes including the proapoptotic factor Bim.These results demonstrate that KDELR1 regulates naïve T-cell homeostasis by controlling ISR.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan [2] Laboratory of Developmental Immunology, Graduate School of Frontier Biosciences, Graduate School of Medicine, and WPI Immunology Frontier Research Center, Osaka University, 2-2, Yamada-oka, Suita 565-0871, Japan.

ABSTRACT
KDEL receptors are responsible for retrotransporting endoplasmic reticulum (ER) chaperones from the Golgi complex to the ER. Here we describe a role for KDEL receptor 1 (KDELR1) that involves the regulation of integrated stress responses (ISR) in T cells. Designing and using an N-ethyl-N-nitrosourea (ENU)-mutant mouse line, T-Red (naïve T-cell reduced), we show that a point mutation in KDELR1 is responsible for the reduction in the number of naïve T cells in this model owing to an increase in ISR. Mechanistic analysis shows that KDELR1 directly regulates protein phosphatase 1 (PP1), a key phosphatase for ISR in naïve T cells. T-Red KDELR1 does not associate with PP1, resulting in reduced phosphatase activity against eIF2α and subsequent expression of stress responsive genes including the proapoptotic factor Bim. These results demonstrate that KDELR1 regulates naïve T-cell homeostasis by controlling ISR.

No MeSH data available.


ISR are enhanced in T-Red naïve T cells due to a functional defect of PP1.(a) ISR target genes in T-cell subsets and B cells were examined by qPCR. N, M and B indicate naïve, memory and B cells, respectively. Expression levels of WT populations were normalized as 1. Data represent the mean+s.d. (n=2). (b) Global translation in DP thymocytes (DP), naïve T cells from WT (W) or T-Red (T) mice was examined by in vivo puromycin labelling (right). Ponceau-S staining is shown on the left. Numbers below the blots represent the intensity ratio of puromycin/Ponceau-S. (c) Phosphorylated-eIF2α in T-cell subsets were examined by flow cytometry. Representative FACS histograms from three independent experiments (top) and mean fluorescence intensity (MFI; bottom) are shown. Data represent the mean+s.e.m. (n=3). (d) ISR target genes in naïve CD8 T cells from WT, T-Red or OT-I/T-Red mice were examined. Relative expressions to Hprt levels are shown. Data represent the mean+s.d. (n=2). One-way ANOVA with post hoc Dunnett's test was used. (e) Naïve T cells were cultured and examined for phosphorylated-eIF2α levels. In some cultures, PP1/GADD34 inhibitor salubrinal was added. (f) Radiolabelled, phosphorylated-eIF2α was incubated for the indicated times with cell lysates from WT or T-Red naïve T cells, followed by SDS–PAGE and phosphor imaging. The intensity at 10 min in each group was normalized as 1.0. (g) Flag-KDELR1 WT or T-Red mutants and Myc-WT PP1α were overexpressed in HEK293T cells and immunoprecipitated with anti-Flag beads, followed by blotting with anti-Flag or anti-Myc. (h) Association of KDELR1 mutants with WT PP1α. A schema of KDELR1 is shown to the right. Closed and open arrows indicate the location of RVEA and T-Red mutations, respectively. (i) Association of PP1α mutants with WT KDELR1. A schema of PP1α is shown to the right. Numbers below the blots in g–i represent the ratio of PP1α/KDELR1. (j) Association of KDELR1 and PP1α in naïve T cells examined by proximity ligation assay (PLA). Numbers of association signals per cell are shown. Data represent the mean+s.d. Representative images from more than three independent experiments are shown in b,d,f–h and i. Mice between 7 and 13 weeks old were used. *P<0.05; **P<0.01; ***P<0.001.
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f6: ISR are enhanced in T-Red naïve T cells due to a functional defect of PP1.(a) ISR target genes in T-cell subsets and B cells were examined by qPCR. N, M and B indicate naïve, memory and B cells, respectively. Expression levels of WT populations were normalized as 1. Data represent the mean+s.d. (n=2). (b) Global translation in DP thymocytes (DP), naïve T cells from WT (W) or T-Red (T) mice was examined by in vivo puromycin labelling (right). Ponceau-S staining is shown on the left. Numbers below the blots represent the intensity ratio of puromycin/Ponceau-S. (c) Phosphorylated-eIF2α in T-cell subsets were examined by flow cytometry. Representative FACS histograms from three independent experiments (top) and mean fluorescence intensity (MFI; bottom) are shown. Data represent the mean+s.e.m. (n=3). (d) ISR target genes in naïve CD8 T cells from WT, T-Red or OT-I/T-Red mice were examined. Relative expressions to Hprt levels are shown. Data represent the mean+s.d. (n=2). One-way ANOVA with post hoc Dunnett's test was used. (e) Naïve T cells were cultured and examined for phosphorylated-eIF2α levels. In some cultures, PP1/GADD34 inhibitor salubrinal was added. (f) Radiolabelled, phosphorylated-eIF2α was incubated for the indicated times with cell lysates from WT or T-Red naïve T cells, followed by SDS–PAGE and phosphor imaging. The intensity at 10 min in each group was normalized as 1.0. (g) Flag-KDELR1 WT or T-Red mutants and Myc-WT PP1α were overexpressed in HEK293T cells and immunoprecipitated with anti-Flag beads, followed by blotting with anti-Flag or anti-Myc. (h) Association of KDELR1 mutants with WT PP1α. A schema of KDELR1 is shown to the right. Closed and open arrows indicate the location of RVEA and T-Red mutations, respectively. (i) Association of PP1α mutants with WT KDELR1. A schema of PP1α is shown to the right. Numbers below the blots in g–i represent the ratio of PP1α/KDELR1. (j) Association of KDELR1 and PP1α in naïve T cells examined by proximity ligation assay (PLA). Numbers of association signals per cell are shown. Data represent the mean+s.d. Representative images from more than three independent experiments are shown in b,d,f–h and i. Mice between 7 and 13 weeks old were used. *P<0.05; **P<0.01; ***P<0.001.

Mentions: We next attempted to identify how the apoptotic pathway is activated in T-Red naïve T cells. We performed DNA microarray analysis using freshly isolated naïve CD4+ and CD8+ T cells from WT and T-Red mice. Because Bim is known to be a target of ISR11, we investigated this pathway. Several genes known to be involved in ISR were also upregulated in T-Red naïve T cells. Quantitative PCR analysis confirmed that ISR-related genes, including Asns, Chop, Trib3 and Vegfα, were significantly upregulated (Fig. 6a). Activated/memory T cells and B cells from T-Red mice also showed the upregulation of some stress genes, but the induction levels were much lower than in naïve T cells (Fig. 6a and Supplementary Fig. 5). In addition, activation of ISR is predicted to globally reduce translation in T-Red cells. Indeed, we found such a reduction in DP thymocytes and naïve T cells in T-Red mice (Fig. 6b).


KDEL receptor 1 regulates T-cell homeostasis via PP1 that is a key phosphatase for ISR.

Kamimura D, Katsunuma K, Arima Y, Atsumi T, Jiang JJ, Bando H, Meng J, Sabharwal L, Stofkova A, Nishikawa N, Suzuki H, Ogura H, Ueda N, Tsuruoka M, Harada M, Kobayashi J, Hasegawa T, Yoshida H, Koseki H, Miura I, Wakana S, Nishida K, Kitamura H, Fukada T, Hirano T, Murakami M - Nat Commun (2015)

ISR are enhanced in T-Red naïve T cells due to a functional defect of PP1.(a) ISR target genes in T-cell subsets and B cells were examined by qPCR. N, M and B indicate naïve, memory and B cells, respectively. Expression levels of WT populations were normalized as 1. Data represent the mean+s.d. (n=2). (b) Global translation in DP thymocytes (DP), naïve T cells from WT (W) or T-Red (T) mice was examined by in vivo puromycin labelling (right). Ponceau-S staining is shown on the left. Numbers below the blots represent the intensity ratio of puromycin/Ponceau-S. (c) Phosphorylated-eIF2α in T-cell subsets were examined by flow cytometry. Representative FACS histograms from three independent experiments (top) and mean fluorescence intensity (MFI; bottom) are shown. Data represent the mean+s.e.m. (n=3). (d) ISR target genes in naïve CD8 T cells from WT, T-Red or OT-I/T-Red mice were examined. Relative expressions to Hprt levels are shown. Data represent the mean+s.d. (n=2). One-way ANOVA with post hoc Dunnett's test was used. (e) Naïve T cells were cultured and examined for phosphorylated-eIF2α levels. In some cultures, PP1/GADD34 inhibitor salubrinal was added. (f) Radiolabelled, phosphorylated-eIF2α was incubated for the indicated times with cell lysates from WT or T-Red naïve T cells, followed by SDS–PAGE and phosphor imaging. The intensity at 10 min in each group was normalized as 1.0. (g) Flag-KDELR1 WT or T-Red mutants and Myc-WT PP1α were overexpressed in HEK293T cells and immunoprecipitated with anti-Flag beads, followed by blotting with anti-Flag or anti-Myc. (h) Association of KDELR1 mutants with WT PP1α. A schema of KDELR1 is shown to the right. Closed and open arrows indicate the location of RVEA and T-Red mutations, respectively. (i) Association of PP1α mutants with WT KDELR1. A schema of PP1α is shown to the right. Numbers below the blots in g–i represent the ratio of PP1α/KDELR1. (j) Association of KDELR1 and PP1α in naïve T cells examined by proximity ligation assay (PLA). Numbers of association signals per cell are shown. Data represent the mean+s.d. Representative images from more than three independent experiments are shown in b,d,f–h and i. Mice between 7 and 13 weeks old were used. *P<0.05; **P<0.01; ***P<0.001.
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Related In: Results  -  Collection

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f6: ISR are enhanced in T-Red naïve T cells due to a functional defect of PP1.(a) ISR target genes in T-cell subsets and B cells were examined by qPCR. N, M and B indicate naïve, memory and B cells, respectively. Expression levels of WT populations were normalized as 1. Data represent the mean+s.d. (n=2). (b) Global translation in DP thymocytes (DP), naïve T cells from WT (W) or T-Red (T) mice was examined by in vivo puromycin labelling (right). Ponceau-S staining is shown on the left. Numbers below the blots represent the intensity ratio of puromycin/Ponceau-S. (c) Phosphorylated-eIF2α in T-cell subsets were examined by flow cytometry. Representative FACS histograms from three independent experiments (top) and mean fluorescence intensity (MFI; bottom) are shown. Data represent the mean+s.e.m. (n=3). (d) ISR target genes in naïve CD8 T cells from WT, T-Red or OT-I/T-Red mice were examined. Relative expressions to Hprt levels are shown. Data represent the mean+s.d. (n=2). One-way ANOVA with post hoc Dunnett's test was used. (e) Naïve T cells were cultured and examined for phosphorylated-eIF2α levels. In some cultures, PP1/GADD34 inhibitor salubrinal was added. (f) Radiolabelled, phosphorylated-eIF2α was incubated for the indicated times with cell lysates from WT or T-Red naïve T cells, followed by SDS–PAGE and phosphor imaging. The intensity at 10 min in each group was normalized as 1.0. (g) Flag-KDELR1 WT or T-Red mutants and Myc-WT PP1α were overexpressed in HEK293T cells and immunoprecipitated with anti-Flag beads, followed by blotting with anti-Flag or anti-Myc. (h) Association of KDELR1 mutants with WT PP1α. A schema of KDELR1 is shown to the right. Closed and open arrows indicate the location of RVEA and T-Red mutations, respectively. (i) Association of PP1α mutants with WT KDELR1. A schema of PP1α is shown to the right. Numbers below the blots in g–i represent the ratio of PP1α/KDELR1. (j) Association of KDELR1 and PP1α in naïve T cells examined by proximity ligation assay (PLA). Numbers of association signals per cell are shown. Data represent the mean+s.d. Representative images from more than three independent experiments are shown in b,d,f–h and i. Mice between 7 and 13 weeks old were used. *P<0.05; **P<0.01; ***P<0.001.
Mentions: We next attempted to identify how the apoptotic pathway is activated in T-Red naïve T cells. We performed DNA microarray analysis using freshly isolated naïve CD4+ and CD8+ T cells from WT and T-Red mice. Because Bim is known to be a target of ISR11, we investigated this pathway. Several genes known to be involved in ISR were also upregulated in T-Red naïve T cells. Quantitative PCR analysis confirmed that ISR-related genes, including Asns, Chop, Trib3 and Vegfα, were significantly upregulated (Fig. 6a). Activated/memory T cells and B cells from T-Red mice also showed the upregulation of some stress genes, but the induction levels were much lower than in naïve T cells (Fig. 6a and Supplementary Fig. 5). In addition, activation of ISR is predicted to globally reduce translation in T-Red cells. Indeed, we found such a reduction in DP thymocytes and naïve T cells in T-Red mice (Fig. 6b).

Bottom Line: Mechanistic analysis shows that KDELR1 directly regulates protein phosphatase 1 (PP1), a key phosphatase for ISR in naïve T cells.T-Red KDELR1 does not associate with PP1, resulting in reduced phosphatase activity against eIF2α and subsequent expression of stress responsive genes including the proapoptotic factor Bim.These results demonstrate that KDELR1 regulates naïve T-cell homeostasis by controlling ISR.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan [2] Laboratory of Developmental Immunology, Graduate School of Frontier Biosciences, Graduate School of Medicine, and WPI Immunology Frontier Research Center, Osaka University, 2-2, Yamada-oka, Suita 565-0871, Japan.

ABSTRACT
KDEL receptors are responsible for retrotransporting endoplasmic reticulum (ER) chaperones from the Golgi complex to the ER. Here we describe a role for KDEL receptor 1 (KDELR1) that involves the regulation of integrated stress responses (ISR) in T cells. Designing and using an N-ethyl-N-nitrosourea (ENU)-mutant mouse line, T-Red (naïve T-cell reduced), we show that a point mutation in KDELR1 is responsible for the reduction in the number of naïve T cells in this model owing to an increase in ISR. Mechanistic analysis shows that KDELR1 directly regulates protein phosphatase 1 (PP1), a key phosphatase for ISR in naïve T cells. T-Red KDELR1 does not associate with PP1, resulting in reduced phosphatase activity against eIF2α and subsequent expression of stress responsive genes including the proapoptotic factor Bim. These results demonstrate that KDELR1 regulates naïve T-cell homeostasis by controlling ISR.

No MeSH data available.