The eIF2α serine 51 phosphorylation-ATF4 arm promotes HIPPO signaling and cell death under oxidative stress.

The HIPPO pathway is an evolutionary conserved regulator of organ size that controls both cell proliferation and death. This pathway has an important role in mediating cell death in response to oxidative stress through the inactivation of Yes-associated protein (YAP) and inhibition of anti-oxidant gene expression. Cells exposed to oxidative stress induce the phosphorylation of the alpha (α) subunit of the translation initiation factor eIF2 at serine 51 (eIF2αP), a modification that leads to the general inhibition of mRNA translation initiation. Under these conditions, increased eIF2αP facilitates the mRNA translation of activating transcription factor 4 (ATF4), which mediates either cell survival and adaptation or cell death under conditions of severe stress. Herein, we demonstrate a functional connection between the HIPPO and eIF2αP-ATF4 pathways under oxidative stress. We demonstrate that ATF4 promotes the stabilization of the large tumor suppressor 1 (LATS1), which inactivates YAP by phosphorylation. ATF4 inhibits the expression of NEDD4.2 and WWP1 mRNAs under pro-oxidant conditions, which encode ubiquitin ligases mediating the proteasomal degradation of LATS1. Increased LATS1 stability is required for the induction of cell death under oxidative stress. Our data reveal a previously unidentified ATF4-dependent pathway in the induction of cell death under oxidative stress via the activation of LATS1 and HIPPO pathway.

Evidence for eIF2α phosphorylation-independent effects of GSK2656157, a novel catalytic inhibitor of PERK with clinical implications.

The endoplasmic reticulum (ER)-resident protein kinase PERK is a major component of the unfolded protein response (UPR), which promotes the adaptation of cells to various forms of stress. PERK phosphorylates the α subunit of the translation initiation factor eIF2 at serine 51, a modification that plays a key role in the regulation of mRNA translation in stressed cells. Several studies have demonstrated that the PERK-eIF2α phosphorylation pathway maintains insulin biosynthesis and glucose homeostasis, facilitates tumor formation and decreases the efficacy of tumor treatment with chemotherapeutic drugs. Recently, a selective catalytic PERK inhibitor termed GSK2656157 has been developed with anti-tumor properties in mice. Herein, we provide evidence that inhibition of PERK activity by GSK2656157 does not always correlate with inhibition of eIF2α phosphorylation. Also, GSK2656157 does not always mimic the biological effects of the genetic inactivation of PERK. Furthermore, cells treated with GSK2656157 increase eIF2α phosphorylation as a means to compensate for the loss of PERK. Using human tumor cells impaired in eIF2α phosphorylation, we demonstrate that GSK2656157 induces ER stress-mediated death suggesting that the drug acts independent of the inhibition of eIF2α phosphorylation. We conclude that GSK2656157 might be a useful compound to dissect pathways that compensate for the loss of PERK and/or identify PERK pathways that are independent of eIF2α phosphorylation.

eIF2α phosphorylation bypasses premature senescence caused by oxidative stress and pro-oxidant antitumor therapies.

Eukaryotic cells respond to various forms of stress by blocking mRNA translation initiation via the phosphorylation of the alpha (α) subunit of eIF2 at serine 51 (S51) (eIFαP). An important role of eIF2αP is the regulation of redox homeostasis and adaptation of cells to oxidative stress. Herein, we demonstrate that eIF2αP guards cells from intracellular reactive oxygen species (ROS) via the inhibition of senescence. Specifically, genetic inactivation of either eIF2αP or eIF2α kinase PERK in primary mouse or human fibroblasts leads to proliferative defects associated with increased DNA damage, G2/M accumulation and induction of premature senescence. Impaired proliferation of either PERK or eIF2αP-deficient primary cells is caused by increased ROS and restored by anti-oxidant treatment. Contrary to primary cells, impaired eIF2αP in immortalized mouse fibroblasts or human tumor cells provides tolerance to elevated intracellular ROS levels. However, eIF2αP-deficient human tumor cells are highly susceptible to extrinsic ROS generated by the pro-oxidant drug doxorubicin by undergoing premature senescence. Our work demonstrates that eIF2αP determines cell destiny through its capacity to control senescence in response to oxidative stress. Also, inhibition of eIF2αP may be a suitable means to increase the anti-tumor effects of pro-oxidant drugs through the induction of senescence.

HDAC pharmacological inhibition promotes cell death through the eIF2α kinases PKR and GCN2.

Histone deacetylase inhibitors (HDACi) comprise a family of chemotherapeutic agents used in the clinic to treat cutaneous T-cell lymphoma and tested for the therapy of other malignancies. Previous reports have shown that eIF2α phosphorylation is induced upon treatment with HDACi. However the kinase responsible for this phosphorylation or the biological significance of this finding is not yet established. Herein, we show that eIF2α phosphorylation is not attributed to a specific eIF2α kinase, but rather different eIF2α kinases contribute to its upregulation in response to the HDACi, vorinostat. More importantly our data indicate that eIF2α phosphorylation acts in a cytoprotective manner, whereas the eIF2α kinases PKR and GCN2 promote vorinostat-induced apoptosis. These results reveal a dual nature for eIF2α kinases with potential implications in the treatment with histone deacetylase inhibitors.

Phosphorylation of eIF2 alpha in Sf9 cells: a stress, survival and suicidal signal.

An analysis of the stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF2alpha) involved in translation regulation, in the ovarian cells of Spodoptera frugiperda (Sf9) for its role in cell survival and death reveals that it stimulates casapase activation and cell death in the absence of BiP, a chaperone and stress marker of the endoplasmic reticulum (ER). While Phospho-JNK and GADD-153 levels are elevated in non-ER stress-induced eIF2alpha phosphorylation-mediated cell death, ATF4 levels are elevated both in response to ER and non-ER stress-induced eIF2alpha phosphorylation. Infection of Sf9 cells by wt and a mutant Deltapk2 baculovirus that harbor the anti-apoptotic p35 gene induces BiP expression. However, UV-induced eIF2alpha phosphorylation and caspase activation are mitigated more efficiently by wt, but not by Deltapk2 baculovirus that lacks pk2, an inhibitor of eIF2alpha kinase. z-VAD-fmk, a caspase inhibitor reduces the late stages, but not the initial stages of non-ER stress-induced eIF2alpha phosphorylation, thereby suggesting that eIF2alpha phosphorylation is a cause and consequence of caspase activation. The importance of BiP affecting the delicate balance between eIF2alpha phosphorylation-mediated cell survival and death is further supported by the findings that tunicamycin-treated cells expressing BiP resist eIF2alpha phosphorylation-mediated cell death and addition of a purified recombinant mutant phosphomimetic form, but not wt eIF2alpha, stimulates caspase activation in cell extracts devoid of BiP. These findings therefore suggest that eIF2alpha phosphorylation is primarily a stress signal and evokes adaptive or apoptotic responses depending on its cellular location, changes in gene expression, coincident signaling activities, and inter-protein interactions.

Intersubunit and interprotein interactions of alpha- and beta-subunits of human eIF2: Effect of phosphorylation.

Purified recombinant human subunits of eukaryotic initiation factor 2 (eIF2) expressed in bacteria are found to interact with each other to form alphabeta, alphagamma, and betagamma complexes in a pull-down experiment. Recombinant phosphorylated human eIF2alpha that cannot interact with purified eIF2B, the GDP/GTP exchange factor of eIF2, however interacts efficiently with eIF2B along with the beta-subunit of eIF2 of the rabbit reticulocyte lysates and also with the purified recombinant beta-subunit. These findings therefore suggest that the beta-subunit of eIF2 mediates the productive and non-productive interactions between eIF2 and 2B. Recombinant alpha and beta-subunits serve as substrates for not only kinases but also for caspase 3 and interestingly phosphorylated subunits resist caspase action. Phosphorylation also modifies the beta-subunit's interaction with Nck1, a cofactor of eIF2alpha phosphatase, but not with eIF5, the GTPase activating protein. These findings suggest that subunits of mammalian eIF2 interact with each other and the beta-subunit plays a critical role both in the regulation and function of eIF2.