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. 2011 Mar 21;6(3):e17894.
doi: 10.1371/journal.pone.0017894.

Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner

Hong Wa Yung  1 , D Stephen Charnock-Jones, Graham J Burton
Affiliations

Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner

Hong Wa Yung et al. PLoS One. .

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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 2
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 3
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 4
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 5
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 6
Figure 6. A schematic model proposing how the severity of ER stress might modulate AKT target substrate specificity, and the consequent pathologies.
See this image and copyright information in PMC

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