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Cited by 159Endoplasmic Reticulum Stress and DiseasePI3K/AKT/mTOR signaling in cancerCellular transport and secretion

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Regulation of AKT Phosphorylation at Ser473 and Thr308 by Endoplasmic Reticulum Stress Modulates Substrate Specificity in a Severity Dependent Manner

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Yung, Hong Wa, Charnock-Jones, D. Stephen, Burton, Graham J.·2011·PLoS ONE
DOI 10.1371/journal.pone.0017894PMC PMC3061875
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Abstract

Endoplasmic reticulum (ER) stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473) confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308). The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.

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Figure 1 ER stress reduces AKT phosphorylation at Thr308, but increases it at Ser473, and alters target substrate specificity. In a dose-response study of tunicamycin, JEG-3 cells were treated with increasing concentrations of tunicamycin (0, 0.625, 1.25, 25 and 5 µg/ml) for 24 hours. Proteins were isolated for Western blotting analysis for GRP78, AKT, P-AKT(Thr308), P-AKT(Ser473), P-PDK1(Ser241), P-AKT substrate (RXRXXS/T), P-mTOR(Ser2448), P-HDM2(Ser166), and P-GSK-3β(Ser9). Ponceau S staining was used to show equivalent input of cell lysate. Densitometry of band intensity is expressed relative to untreated control (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are mean±SEM from 3 to 5 independent experiments. ** and * indicate P<0.01 and P<0.05. A) Increasing severity of ER stress gradually induces GRP78 expression and cell death. B) ER stress suppresses AKT phosphorylation and modulates downstream substrates specificity without affecting PDK1 phosphorylation. Arrowheads and arrows indicate AKT substrate phosphorylation levels going down and going up respectively with increasing ER stress. Because of the different abundances of the AKT substrates, the blots of phospho-AKT substrates necessitated different exposure times. Here, three exposures are merged into a single image in order to view all potential bands. The blot shown is a typical result from 3 independent experiments. C) An in vitro non-radioactive AKT kinase assay using GSK-3β fusion protein as the substrate showed increased overall AKT activity in tunicamycin-treated cells. A similar result was obtained from a repeat experiment. D) Normalisation between phosphorylated AKT and AKT indicates an increase of Ser473 phosphorylation but a decrease at Thr308.
Figure 1 ER stress reduces AKT phosphorylation at Thr308, but increases it at Ser473, and alters target substrate specificity. In a dose-response study of tunicamycin, JEG-3 cells were treated with increasing concentrations of tunicamycin (0, 0.625, 1.25, 25 and 5 µg/ml) for 24 hours. Proteins were isolated for Western blotting analysis for GRP78, AKT, P-AKT(Thr308), P-AKT(Ser473), P-PDK1(Ser241), P-AKT substrate (RXRXXS/T), P-mTOR(Ser2448), P-HDM2(Ser166), and P-GSK-3β(Ser9). Ponceau S staining was used to show equivalent input of cell lysate. Densitometry of band intensity is expressed relative to untreated control (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are mean±SEM from 3 to 5 independent experiments. ** and * indicate P<0.01 and P<0.05. A) Increasing severity of ER stress gradually induces GRP78 expression and cell death. B) ER stress suppresses AKT phosphorylation and modulates downstream substrates specificity without affecting PDK1 phosphorylation. Arrowheads and arrows indicate AKT substrate phosphorylation levels going down and going up respectively with increasing ER stress. Because of the different abundances of the AKT substrates, the blots of phospho-AKT substrates necessitated different exposure times. Here, three exposures are merged into a single image in order to view all potential bands. The blot shown is a typical result from 3 independent experiments. C) An in vitro non-radioactive AKT kinase assay using GSK-3β fusion protein as the substrate showed increased overall AKT activity in tunicamycin-treated cells. A similar result was obtained from a repeat experiment. D) Normalisation between phosphorylated AKT and AKT indicates an increase of Ser473 phosphorylation but a decrease at Thr308.CC BY · PubMed Central
Figure 2 Knock-down of ER stress-induced GRP78 expression by siGRP78 restored AKT phosphorylation at Ser473, but not at Thr308, and altered AKT substrates specificity. Cells were transfected with either siCon or siGRP78 RNA duplexes for 24 hour before treatment with tunicamycin for 24 hour. Proteins were extracted for immunoblot analysis with GRP78, P-PDK1(Ser241), PDK1, P-AKT(Thr308), P-AKT(Ser473), AKT, P-AKT substrate (RXRXXS/T), P-mTOR(Ser2448), P-HDM2(Ser166), P-FOXO1(Ser319) and P-GSK-3α/β(Ser21/9). Densitometry of band intensity is expressed relative to siCon untreated control (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are mean±SEM for 3 independent experiments. ** indicates P≤0.01; n.s indicates non-significant change. A & B) Down-regulation of GRP78 elevates Ser473 phosphorylation but not at Thr308. C) Knock-down of GRP78 alters AKT downstream substrates recognition. Arrows indicate the substrates changed their phosphorylation pattern in tunicamycin-treated siGRP78 cells.
Figure 2 Knock-down of ER stress-induced GRP78 expression by siGRP78 restored AKT phosphorylation at Ser473, but not at Thr308, and altered AKT substrates specificity. Cells were transfected with either siCon or siGRP78 RNA duplexes for 24 hour before treatment with tunicamycin for 24 hour. Proteins were extracted for immunoblot analysis with GRP78, P-PDK1(Ser241), PDK1, P-AKT(Thr308), P-AKT(Ser473), AKT, P-AKT substrate (RXRXXS/T), P-mTOR(Ser2448), P-HDM2(Ser166), P-FOXO1(Ser319) and P-GSK-3α/β(Ser21/9). Densitometry of band intensity is expressed relative to siCon untreated control (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are mean±SEM for 3 independent experiments. ** indicates P≤0.01; n.s indicates non-significant change. A & B) Down-regulation of GRP78 elevates Ser473 phosphorylation but not at Thr308. C) Knock-down of GRP78 alters AKT downstream substrates recognition. Arrows indicate the substrates changed their phosphorylation pattern in tunicamycin-treated siGRP78 cells.CC BY · PubMed Central
Figure 3 The interaction between AKT and GRP78 occurs in vivo. After tunicamycin treatment, JEG-3 cells were fixed in 100% methanol and subjected to a DuoLink Proximity Ligation Assay in situ and confocal microscopy. Arrows indicate cells staining positive with normal nuclear morphology, whereas stronger staining was seen in cells with condensed nuclei. All images were a single optical section taken with a 60X objective using the same PMT, gain, and offset setting. Scale bar = 31.75 µm for all panels.
Figure 3 The interaction between AKT and GRP78 occurs in vivo. After tunicamycin treatment, JEG-3 cells were fixed in 100% methanol and subjected to a DuoLink Proximity Ligation Assay in situ and confocal microscopy. Arrows indicate cells staining positive with normal nuclear morphology, whereas stronger staining was seen in cells with condensed nuclei. All images were a single optical section taken with a 60X objective using the same PMT, gain, and offset setting. Scale bar = 31.75 µm for all panels.CC BY · PubMed Central
Figure 4 Association of GRP78 with AKT prevents AKT phosphorylation at Ser473 but not at Thr308. A) Loss of AKT phosphorylation at Ser473 and Thr308 in the GRP78-IP complex. AKT was pulled down by anti-GRP78 (N-20) antibody and immunoblotted for P-AKT(Ser473), P-AKT (Thr308), AKT1 and GRP78 antibodies. B) No GRP78 was pulled down by P-AKT(Ser473) antibody.
Figure 4 Association of GRP78 with AKT prevents AKT phosphorylation at Ser473 but not at Thr308. A) Loss of AKT phosphorylation at Ser473 and Thr308 in the GRP78-IP complex. AKT was pulled down by anti-GRP78 (N-20) antibody and immunoblotted for P-AKT(Ser473), P-AKT (Thr308), AKT1 and GRP78 antibodies. B) No GRP78 was pulled down by P-AKT(Ser473) antibody.CC BY · PubMed Central
Figure 5 The interaction between AKT and GRP78 is likely a specific phenomenon upon ER stress in a variety of cell types. JEG-3 cells were treated with tunicamycin for 24 hour before protein isolation for immunoprecipitation followed by immunoblotting. Ponceau S staining and IgG heavy chain were used to show both equivalent input of cell lysates and antibodies respectively. A) AKT does not interact with many chaperones. AKT immunoprecipitated products were immunoblotting with antibodies against GRP94, HSP90, HSP70 and HSP40. Mouse IgG was used as a negative control and no interaction was observed. B) GRP78 does not bind to other AGC family member, p70 S6 kinase. P70 S6 kinase immunoprecipitated products were immunoblotting with GRP78 and p70 S6 kinase. C) The interaction between GRP78 and AKT is also found in HeLa, JAR and primary HUVECs. D) Binding of GRP78 with AKT is observed following another ER stress inducer, thapsigargin.
Figure 5 The interaction between AKT and GRP78 is likely a specific phenomenon upon ER stress in a variety of cell types. JEG-3 cells were treated with tunicamycin for 24 hour before protein isolation for immunoprecipitation followed by immunoblotting. Ponceau S staining and IgG heavy chain were used to show both equivalent input of cell lysates and antibodies respectively. A) AKT does not interact with many chaperones. AKT immunoprecipitated products were immunoblotting with antibodies against GRP94, HSP90, HSP70 and HSP40. Mouse IgG was used as a negative control and no interaction was observed. B) GRP78 does not bind to other AGC family member, p70 S6 kinase. P70 S6 kinase immunoprecipitated products were immunoblotting with GRP78 and p70 S6 kinase. C) The interaction between GRP78 and AKT is also found in HeLa, JAR and primary HUVECs. D) Binding of GRP78 with AKT is observed following another ER stress inducer, thapsigargin.CC BY · PubMed Central
Figure 6 A schematic model proposing how the severity of ER stress might modulate AKT target substrate specificity, and the consequent pathologies.
Figure 6 A schematic model proposing how the severity of ER stress might modulate AKT target substrate specificity, and the consequent pathologies.CC BY · PubMed Central

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Metadata

Authors
Yung, H. W., Charnock-Jones, D. S., & Burton, G. J.
Affiliations
University of Cambridge (GB)
Corresponding author
Burton, Graham J.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2011-03-21
Volume
6
Issue
3
Pages
e17894
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0017894
PMC
PMC3061875
OpenAlex
W2058236541
Cited by
158
References
41
Citations by year
2026: 6 · 2025: 5 · 2024: 9 · 2023: 7 · 2022: 12 · 2021: 10 · 2020: 15 · 2019: 14 · 2018: 8 · 2017: 16 · 2016: 12 · 2015: 10 · 2014: 15 · 2013: 7 · 2012: 9
FWCI
5.21
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Apollo (University of Cambridge) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
  • Apollo (cc-by)
Funders
Wellcome Trust (084804, 084804/2/08/Z, 084804/Z/08/Z)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Endoplasmic Reticulum Stress and Disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › PI3K/AKT/mTOR signaling in cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords
endoplasmic reticulum stress, Akt phosphorylation, GRP78, PDK1, plasma membrane, apoptosis, cell proliferation, p70 S6 kinase, molecular chaperones, proximity ligation assay
MeSH terms
Endoplasmic Reticulum Chaperone BiP, Endoplasmic Reticulum, Heat-Shock Proteins, Humans, Phosphorylation, Serine, Substrate Specificity, Threonine, Down-Regulation, Proto-Oncogene Proteins c-akt

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