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

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.     

Aquatic birnavirus-induced ER stress-mediated death signaling contribute to downregulation of Bcl-2 family proteins in salmon embryo cells

Aquatic birnavirus induces mitochondria-mediated cell death, but whether connects to endoplasmic reticulum (ER) stress is still unknown. In this present, we characterized that IPNV infection triggers ER stress-mediated cell death via PKR/eIF2α phosphorylation signaling for regulating the Bcl-2 family protein expression in fish cells. The IPNV infection can induce ER stress as follows: (1) ER stress sensor ATF6 cleavaged; (2) ER stress marker GRP78 upregulation, and (3) PERK/eIF2α phosphorylation. Then, the IPNV-induced ER stress signals can induce the CHOP expression at early (6 h p.i.) and middle replication (12 h p.i.) stages. Moreover, IPNV-induced CHOP upregulation dramatically correlates to apparently downregulate the Bcl-2 family proteins, Bcl-2, Mcl-1 and Bcl-xL at middle replication stage (12 h p.i.) and produces mitochondria membrane potential (MMP) loss and cell death. Furthermore, with GRP78 synthesis inhibitor momitoxin (VT) and PKR inhibitor 2-aminopurine (2-AP) treatment for blocking GRP78 expression and eIF2α phosphorylation, PKR/PERK may involve in eIF2α phosphorylation/CHOP upregulation pathway that enhances the downstream regulators Bcl-2 family proteins expression and increased cell survival. Taken together, our results suggest that IPNV infection activates PKR/PERK/eIF2α ER stress signals for regulating downstream molecules CHOP upregulation and Bcl-2 family downregulation that led to induce mitochondria-mediated cell death in fish cells, which may provide new insight into RNA virus pathogenesis and disease.     

FAD-linked presenilin-1 mutants impede translation regulation under ER stress

FAD mutations in presenilin-1 (PS1) cause attenuation of the induction of the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP under ER stress, due to disturbed function of IRE1, the sensor for accumulation of unfolded protein in the ER lumen. PERK, an ER-resident transmembrane protein kinase, is also a sensor for the unfolded protein response (UPR), causing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation. Here, we report that the FAD mutant PS1 disturbs the UPR by attenuating both the activation of PERK and the phosphorylation of eIF2alpha. Consistent with the results of a disturbed UPR, inhibition of protein synthesis under ER stress was impaired in cells expressing PS1 mutants. These results suggest that mutant PS1 impedes general translational attenuation regulated by PERK and eIF2alpha, resulting in an increased load of newly synthesized proteins into the ER and subsequently increasing vulnerability to ER stress.     

Insulin induces chaperone and CHOP gene expressions in adipocytes

Adipocyte secretes bioactive proteins called adipocytokines, and biosynthesis of secretory proteins requires molecular chaperones and folding enzymes in endoplasmic reticulum (ER). ER chaperones are known to be induced by unfolded protein response (UPR) and growth factors, however, it has not been determined how ER chaperones expression is regulated in adipocytes. Here we show that insulin treatment induced GRP78 and ERO1L mRNA levels in 3T3-L1 adipocytes. Insulin also upregulated CHOP mRNA levels, but did not induce phosphorylation of eIF2α. Pretreatment with insulin protected 3T3-L1 adipocytes against thapsigargin-mediated phosphorylation of eIF2α but did not against DTT-mediated one. In vivo mice study showed that GRP78 and CHOP expressions were regulated by feeding conditions. These results suggest that insulin signaling is important to induce mRNA expressions of GRP78 and CHOP, and may have a protective role against UPR.     

Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

Disturbance of endoplasmic reticulum (ER) homeostasis causes ER stress and leads to activation of the unfolded protein response, which reduces the stress and promotes cell survival at the early stage of stress, or triggers cell death and apoptosis when homeostasis is not restored under prolonged ER stress. Here, we report that Cab45S, a member of the CREC family, inhibits ER stress-induced apoptosis. Depletion of Cab45S increases inositol-requiring kinase 1 (IRE1) activity, thus producing more spliced forms of X-box-binding protein 1 mRNA at the early stage of stress and leads to phosphorylation of c-Jun N-terminal kinase, which finally induces apoptosis. Furthermore, we find that Cab45S specifically interacts with 78-kDa glucose-regulated protein/immunoglobulin heavy chain binding protein (GRP78/BiP) on its nucleotide-binding domain. Cab45S enhances GRP78/BiP protein level and stabilizes the interaction of GRP78/BiP with IRE1 to inhibit ER stress-induced IRE1 activation and apoptosis. Together, Cab45S, a novel regulator of GRP78/BiP, suppresses ER stress-induced IRE1 activation and apoptosis by binding to and elevating GRP78/BiP, and has a role in the inhibition of ER stress-induced apoptosis.     

The role of endoplasmic reticulum‐related BiP/GRP78 in interferon gamma‐induced persistent Chlamydia pneumoniae infection

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN‐γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN‐γ‐induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN‐γ‐induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN‐γ‐induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor‐2α (eIF2α) and down‐regulation of the vesicle‐associated membrane protein‐associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN‐γ‐induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER‐related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN‐γ‐induced persistent infection.     

Down-regulation of glucose-regulated protein (GRP) 78 potentiates cytotoxic effect of celecoxib in human urothelial carcinoma cells

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has been reported to elicit anti-proliferative response in various tumors. In this study, we aim to investigate the antitumor effect of celecoxib on urothelial carcinoma (UC) cells and the role endoplasmic reticulum (ER) stress plays in celecoxib-induced cytotoxicity. The cytotoxic effects were measured by MTT assay and flow cytometry. The cell cycle progression and ER stress-associated molecules were examined by Western blot and flow cytometry. Moreover, the cytotoxic effects of celecoxib combined with glucose-regulated protein (GRP) 78 knockdown (siRNA), (-)-epigallocatechin gallate (EGCG) or MG132 were assessed. We demonstrated that celecoxib markedly reduces the cell viability and causes apoptosis in human UC cells through cell cycle G1 arrest. Celecoxib possessed the ability to activate ER stress-related chaperones (IRE-1α and GRP78), caspase-4, and CCAAT/enhancer binding protein homologous protein (CHOP), which were involved in UC cell apoptosis. Down-regulation of GRP78 by siRNA, co-treatment with EGCG (a GRP78 inhibitor) or with MG132 (a proteasome inhibitor) could enhance celecoxib-induced apoptosis. We concluded that celecoxib induces cell cycle G1 arrest, ER stress, and eventually apoptosis in human UC cells. The down-regulation of ER chaperone GRP78 by siRNA, EGCG, or proteosome inhibitor potentiated the cytotoxicity of celecoxib in UC cells. These findings provide a new treatment strategy against UC.     

The therapeutic mavericks: Potent immunomodulating chaperones capable of treating human diseases

Two major chaperones, calreticulin (CRT) and binding immunoglobulin protein (GRP78/BiP) dependent on their location, have immunoregulatory or anti-inflammatory functions respectively. CRT induces pro-inflammatory cytokines, dendritic cell (DC) maturation and activates cytotoxic T cells against tumours. By contrast, GRP78/BiP induces anti-inflammatory cytokines, inhibits DC maturation and heightens T-regulatory cell responses. These latter functions rebalance immune homeostasis in inflammatory diseases, such as rheumatoid arthritis. Both chaperones are therapeutically relevant agents acting primarily on monocytes/DCs. Endogenous exposure of CRT on cancer cell surfaces acts as an ‘eat-me’ signal and facilitates improved elimination of stressed and dying tumour cells by DCs. Therefore, therapeutics that promote endogenous CRT translocation to the cell surface can improve the removal of cancer cells. However, infused recombinant CRT dampens this cancer cell eradication by binding directly to the DCs. Low levels of endogenous BiP appear as a surface biomarker of endoplasmic reticulum (ER) stress in some types of tumour cells, a reflection of cells undergoing proliferation, in which resulting hypoxia and nutrient deprivation perturb ER homeostasis triggering the unfolded protein response, leading to increased expression of GRP78/BiP and altered cellular location. Conversely, infusion of an analogue of GRP78/BiP (IRL201805) can lead to long-term immune resetting and restoration of immune homeostasis. The therapeutic potential of both chaperones relies on them being relocated from their intracellular ER environment. Ongoing clinical trials are employing therapeutic interventions to either enhance endogenous cell surface CRT or infuse IRL201805, thereby triggering several disease-relevant immune responses leading to a beneficial clinical outcome.     

Endoplasmic reticulum chaperones inhibit the production of amyloid-beta peptides

Abeta (amyloid-beta peptides) generated by proteolysis of APP (beta-amyloid precursor protein), play an important role in the pathogenesis of AD (Alzheimer's disease). ER (endoplasmic reticulum) chaperones, such as GRP78 (glucose-regulated protein 78), make a major contribution to protein quality control in the ER. In the present study, we examined the effect of overexpression of various ER chaperones on the production of Abeta in cultured cells, which produce a mutant type of APP (APPsw). Overexpression of GRP78 or inhibition of its basal expression, decreased and increased respectively the level of Abeta40 and Abeta42 in conditioned medium. Co-expression of GRP78's co-chaperones ERdj3 or ERdj4 stimulated this inhibitory effect of GRP78. In the case of the other ER chaperones, overexpression of some (150 kDa oxygen-regulated protein and calnexin) but not others (GRP94 and calreticulin) suppressed the production of Abeta. These results indicate that certain ER chaperones are effective suppressors of Abeta production and that non-toxic inducers of ER chaperones may be therapeutically beneficial for AD treatment. GRP78 was co-immunoprecipitated with APP and overexpression of GRP78 inhibited the maturation of APP, suggesting that GRP78 binds directly to APP and inhibits its maturation, resulting in suppression of the proteolysis of APP. On the other hand, overproduction of APPsw or addition of synthetic Abeta42 caused up-regulation of the mRNA of various ER chaperones in cells. Furthermore, in the cortex and hippocampus of transgenic mice expressing APPsw, the mRNA of some ER chaperones was up-regulated in comparison with wild-type mice. We consider that this up-regulation is a cellular protective response against Abeta.     

Promastigote to amastigote differentiation of Leishmania is markedly delayed in the absence of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation

The parasitic protozoan Leishmania is the etiological agent of human leishmaniasis worldwide. It undergoes cellular differentiation from the sandfly promastigote form into amastigotes within mammalian macrophages, a process that is essential for its intracellular survival. Here, we characterized the Leishmania infantum PERK eIF2alpha kinase homologue and addressed its role in the parasite's cytodifferentiation. We show that Leishmania PERK is an endoplasmic reticulum (ER) transmembrane protein that largely colocalizes with the ER BiP chaperone. The Leishmania PERK catalytic kinase domain undergoes autohyperphosphorylation and phosphorylates the translation initiation factor 2-alpha subunit (eIF2alpha) in vitro at threonine 166. We also report that PERK is post-translationally regulated specifically in the intracellular stage of the parasite or under ER stress, most likely through extensive autohyperphosphorylation. We have generated a PERK dominant negative mutant overexpressing a truncated PERK protein lacking the N-terminal luminal domain and showed that this mutant is impaired in eIF2alpha phosphorylation in response to ER stress or during amastigote differentiation. Most importantly, we showed that lack of eIF2alpha phosphorylation markedly delays the Leishmania differentiation process towards amastigote forms both in parasites grown axenically or within macrophages. These data highlight the importance of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation in the intracellular development of Leishmania.