Mechanism-Based Screen for G1/S Checkpoint Activators Identifies a Selective Activator of EIF2AK3/PERK Signalling

Stockwell, S.; Platt, Georgina M.; Barrie, S. E.; Zoumpoulidou, Georgia; Poele, Robert H. te; Aherne, G. Wynne; Wilson, Stuart C.; Sheldrake, Peter; McDonald, Edward; Venet, Mathilde; Soudy, Christelle; Elustondo, Frédéric; Rigoreau, Laurent; Blagg, Julian; Workman, Paul; Garrett, Michelle D.; Mittnacht, Sibylle

doi:10.1371/journal.pone.0028568

Cited by 94 publications

(76 citation statements)

References 54 publications

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“…1A, marked with a diamond). At least one of the proteins that did not recover was the short-lived cyclin D1 protein, whose translation has been shown to be blocked by eIF2a-dependent translational repression (Brewer and Diehl, 2000;Stockwell et al, 2012). Indeed, in our experiments cyclin D1 protein levels began decreasing within 0.5 hours of thapsigargin treatment and were undetectable by 2 hours (Fig.…”

Section: Resultsmentioning

confidence: 60%

Examination of a second node of translational control in the unfolded protein response

Preston¹,

Hendershot²

2013

Journal of Cell Science

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SummaryThe unfolded protein response (UPR) is a largely cytoprotective signaling cascade that acts to re-establish homeostasis of the endoplasmic reticulum (ER) under conditions of stress by inducing an early and transient block in general protein synthesis and by increasing the folding and degradative capacity of the cell through an extensive transcriptional program. It is well established that the mechanism for the early translational attenuation during ER stress occurs through phosphorylation of eukaryotic initiation factor 2 a (eIF2a) by activated PERK. Our data demonstrate that when eIF2a is dephosphorylated translation is not fully restored to pre-stressed levels. We found that this correlates with reduced mTOR activity and as a result decreases phosphorylation of 4E-BP1, which negatively regulates assembly of the eIF4F complex and cap-dependent translation. The decrease in mTOR activity and 4E-BP1 phosphorylation is associated with activation of AMP kinase, a negative regulator of mTOR, and in the case of some stress conditions, downregulation of signaling through key components of the PI3K pathway. Furthermore, we show that there is a subset of mRNAs that does not recover from UPR-induced translational repression, including those whose translation is particularly sensitive to loss of eIF4F, such as cyclin D1, Bcl-2 and MMP-9. Together these data implicate reduced mTOR activity and 4E-BP1 hypophosphorylation as a second, more restricted mechanism of translational control occurring somewhat later in the UPR.

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Section: Resultsmentioning

confidence: 60%

Examination of a second node of translational control in the unfolded protein response

Preston¹,

Hendershot²

2013

Journal of Cell Science

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“…ER stress has been reported to induce apoptosis in various cell types via the upregulation of protein translation mediated through the PERK-eIF2α pathway (24)(25)(26)(27). For instance, upon ER stress, the ER chaperone GRP78 dissociates from the PERK and initiates transphosphorylation with subsequent activation of the kinase (28).…”

Section: Discussionmentioning

confidence: 99%

PCSK9 regulates apoptosis in human lung adenocarcinoma A549 cells via endoplasmic reticulum stress and mitochondrial signaling pathways

Cui

Cao

et al. 2017

Experimental and Therapeutic Medicine

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Abstract. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the subtilisin family of PCs that encodes a neural apoptosis-regulated convertase 1. However, the precise role of PCSK9 in lung cancer cell apoptosis has remained elusive. In the present study, A549 human lung adenocarcinoma cells were transfected with PCSK9 small interfering (si)RNA to investigate the underlying mechanisms of apoptosis. The results indicated that PCSK9 siRNA exhibited anti-tumor activity by inducing apoptosis as determined by a Cell Counting Kit-8 and Hoechst staining analysis. In addition, PCSK9 siRNA significantly increased apoptosis of A549 cells in part via activation of caspase-3 and downregulation of the anti-apoptotic proteins survivin and X-linked inhibitor of apoptosis protein. Moreover, the results demonstrated that perturbations in the mitochondrial membrane were associated with the deregulation of the Bax/Bcl-2 ratio, which led to the release of cytochrome c after PCSK9 siRNA transfection. In addition, PCSK9 siRNA also induced endoplasmic reticulum stress (ERS) by increasing the levels of 78 kDa glucose-regulated protein (GRP78), GRP94, phosphorylated protein kinase R-like ER kinase and phosphorylated eukaryotic initiation factor 2α. Therefore, these results demonstrated that PCSK9 siRNA may exert its anti-tumor activity through inducing mitochondrial dysfunction and ERS-associated cell death in A549 cells.

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“…In cancer cells, the prolonged inactivation of eIF2-α result in reduced viability because of the loss of anti-apoptotic proteins, which are required for the survival of cancer cells (Fritsch et al 2007). Theoretically, a small molecule that could induce the phosphorylation of eIF2-α could sensitize cancer cells to paclitaxel (Stockwell et al 2012). In contrast to a previous cancer study (Lee do et al 2010), activation of PERK, which is a kinase upstream of eIF2-α, was shown to be beneficial in beta-amyloid-induced murine models of neurotoxicity.…”

Section: Discussionmentioning

confidence: 87%

“…The densitometric analysis results from western blotting are presented at the right panel. The bar means ± SD (n = 3; *p < 0.05; **p < 0.01) eIF2-α activity (Stockwell et al 2012). When CSM14.1 cells were incubated with AMC compounds, Cyclin D1 protein expression was downregulated in a dose-dependent manner within 4 h of compound exposure.…”

Section: Cap-dependent Translation Can Be Suppressed By Amc Compoundsmentioning

confidence: 99%

The small molecule ‘1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate’ and its derivatives regulate global protein synthesis by inactivating eukaryotic translation initiation factor 2-alpha

Hong

Nam

Kim

et al. 2016

Cell Stress and Chaperones

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By environmental stresses, cells can initiate a signaling pathway in which eukaryotic translation initiation factor 2-alpha (eIF2-α) is involved to regulate the response. Phosphorylation of eIF2-α results in the reduction of overall protein neogenesis, which allows cells to conserve resources and to reprogram energy usage for effective stress control. To investigate the role of eIF2-α in cell stress responses, we conducted a viability-based compound screen under endoplasmic reticulum (ER) stress condition, and identified 1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate (AMC-01) and its derivatives as eIF2-α-inactivating chemical. Molecular characterization of this signaling pathway revealed that AMC-01 induced inactivation of eIF2-α by phosphorylating serine residue 51 in a dose-and time-dependent manner, while the negative control compounds did not affect eIF2-α phosphorylation. In contrast with ER stress induction by thapsigargin, phosphorylation of eIF2-α persisted for the duration of incubation with AMC-01. By pathway analysis, AMC-01 clearly induced the activation of protein kinase RNA-activated (PKR) kinase and nuclear factor-κB (NF-κB), whereas it did not modulate the activity of PERK or heme-regulated inhibitor (HRI). Finally, we could detect a lower protein translation rate in cells incubated with AMC-01, establishing AMC-01 as a potent chemical probe that can regulate eIF2-α activity. We suggest from these data that AMC-01 and its derivative compounds can be used as chemical probes in future studies of the role of eIF2-α in protein synthesis-related cell physiology.

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Mechanism-Based Screen for G1/S Checkpoint Activators Identifies a Selective Activator of EIF2AK3/PERK Signalling

Cited by 94 publications

References 54 publications

Examination of a second node of translational control in the unfolded protein response

Examination of a second node of translational control in the unfolded protein response

PCSK9 regulates apoptosis in human lung adenocarcinoma A549 cells via endoplasmic reticulum stress and mitochondrial signaling pathways

The small molecule ‘1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate’ and its derivatives regulate global protein synthesis by inactivating eukaryotic translation initiation factor 2-alpha

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