GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation

In cells undergoing apoptosis, mitochondrial outer-membrane permeabilization (MOMP) is followed by caspase activation promoted by released cytochrome c. Although caspases mediate the apoptotic phenotype, caspase inhibition is generally not sufficient for survival following MOMP; instead cells undergo a "caspase-independent cell death" (CICD). Thus, MOMP may represent a point of commitment to cell death. Here, we identify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a critical regulator of CICD. GAPDH-expressing cells preserved their clonogenic potential following MOMP, provided that caspase activation was blocked. GAPDH-mediated protection of cells from CICD involved an elevation in glycolysis and a nuclear function that correlated with and was replaced by an increase in Atg12 expression. Consistent with this, protection from CICD reflected an increase in and a dependence upon autophagy, associated with a transient decrease in mitochondrial mass. Therefore, GAPDH mediates an elevation in glycolysis and enhanced autophagy that cooperate to protect cells from CICD.     

Praf2 is a novel Bcl-xL/Bcl-2 interacting protein with the ability to modulate survival of cancer cells

Increased expression of Bcl-xL in cancer has been shown to confer resistance to a broad range of apoptotic stimuli and to modulate a number of other aspects of cellular physiology, including energy metabolism, cell cycle, autophagy, mitochondrial fission/fusion and cellular adhesion. However, only few of these activities have a mechanistic explanation. Here we used Tandem Affinity purification to identify novel Bcl-xL interacting proteins that could explain the pleiotropic effects of Bcl-xL overexpression. Among the several proteins co-purifying with Bcl-xL, we focused on Praf2, a protein with a predicted role in trafficking. The interaction of Praf2 with Bcl-xL was found to be dependent on the transmembrane domain of Bcl-xL. We found that Bcl-2 also interacts with Praf2 and that Bcl-xL and Bcl-2 can interact also with Arl6IP5, an homologue of Praf2. Interestingly, overexpression of Praf2 results in the translocation of Bax to mitochondria and the induction of apoptotic cell death. Praf2 dependent cell death is prevented by the co-transfection of Bcl-xL but not by its transmembrane domain deleted mutant. Accordingly, knock-down of Praf2 increases clonogenicity of U2OS cells following etoposide treatment by reducing cell death. In conclusion a screen for Bcl-xL-interacting membrane proteins let us identify a novel proapoptotic protein whose activity is strongly counteracted exclusively by membrane targeted Bcl-xL.     

Akt protein kinase inhibits non-apoptotic programmed cell death induced by ceramide.

A growing body of evidence now suggests that programmed cell death (PCD) occurs via non-apoptotic mechanisms as well as by apoptosis. In contrast to apoptosis, however, the molecular mechanisms involved in the regulation of non-apoptotic PCD remain only poorly understood. Here we show that ceramide induces a non-apoptotic PCD with a necrotic-like morphology in human glioma cells. Characteristically, the cell death was not accompanied by loss of the mitochondrial transmembrane potential, cytosolic release of cytochrome c from mitochondria, or the activation of the caspase cascade. Consistent with these characteristics, this ceramide-induced cell death was inhibited neither by the overexpression of Bcl-xL nor by the pan-caspase inhibitor zVAD-fmk. However, strikingly, the ceramide-induced non-apoptotic cell death was inhibited by the activation of the Akt/protein kinase B pathway through the expression of a constitutively active version of Akt. The results for the first time indicate that the Akt kinase, known to play an essential role in survival factor-mediated inhibition of apoptotic cell death, is also involved in the regulation of non-apoptotic PCD.     

The E3 ligase UBR2 regulates cell death under caspase deficiency via Erk/MAPK pathway

Escape from cell death is a key event in cancer establishment/progression. While apoptosis is often considered as the main cell death pathway, upon caspase inhibition, cell death is rather delayed than blocked leading to caspase-independent cell death (CICD). Although described for years, CICD’s underlying mechanism remains to be identified. Here, we performed a genome-wide siRNA lethality screening and identified the RING-Type E3 Ubiquitin Transferase (UBR2) as a specific regulator of CICD. Strikingly, UBR2 downregulation sensitized cells towards CICD while its overexpression was protective. We established that UBR2-dependent protection from CICD was mediated by the MAPK/Erk pathway. We then observed that UBR2 is overexpressed in several cancers, especially in breast cancers and contributes to CICD resistance. Therefore, our work defines UBR2 as a novel regulator of CICD, found overexpressed in cancer cells, suggesting that its targeting may represent an innovative way to kill tumor cells.Subject terms: Cancer, Cell death     

Bcl-xL mediates a survival mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells

Among various molecular strategies by which prostate cancer cells evade apoptosis, phosphoinositide 3-kinase (PI3K)/Akt signaling represents a dominant survival pathway. However, different prostate cancer cell lines such as LNCaP and PC-3 display differential sensitivity to the apoptotic effect of PI3K inhibition in serum-free media, reflecting the heterogeneous nature of prostate cancer in apoptosis regulation. Whereas both cell lines are equally susceptible to LY294002-mediated Akt dephosphorylation, only LNCaP cells default to apoptosis, as evidenced by DNA fragmentation and cytochrome c release. In PC-3 cells, Akt deactivation does not lead to cytochrome c release, suggesting that the intermediary signaling pathway is short-circuited by an antiapoptotic factor. This study presents evidence that Bcl-xL overexpression provides a distinct survival mechanism that protects PC-3 cells from apoptotic signals emanating from PI3K inhibition. First, the Bcl-xL/BAD ratio in PC-3 cells is at least an order of magnitude greater than that of LNCaP cells. Second, ectopic expression of Bcl-xL protects LNCaP cells against LY294002-induced apoptosis. Third, antisense down-regulation of Bcl-xL sensitizes PC-3 cells to the apoptotic effect of LY294002. The physiological relevance of this Bcl-xL-mediated survival mechanism is further underscored by the protective effect of serum on LY294002-induced cell death in LNCaP cells, which is correlated with a multifold increase in Bcl-xL expression. In contrast to Bcl-xL, Bcl-2 expression levels are similar in both cells lines, and do not respond to serum stimulation, suggesting that Bcl-2 may not play a physiological role in antagonizing apoptosis signals pertinent to BAD activation in prostate cancer cells.     

Activation of the phosphatidylinositol 3-kinase/Akt pathway protects against interleukin-3 starvation but not DNA damage-induced apoptosis

Baf-3 cells are dependent on interleukin-3 (IL-3) for their survival and proliferation in culture. To identify anti-apoptotic pathways, we performed a retroviral-insertion mutagenesis on Baf-3 cells and selected mutants that have acquired a long term survival capacity. The phenotype of one mutant, which does not overexpress bcl-x and proliferates in the absence of IL-3, is described. We show that, in this mutant, Akt is constitutively activated leading to FKHRL1 phosphorylation and constitutive glycolytic activity. This pathway is necessary for the mutant to survive following IL-3 starvation but is not sufficient or necessary to protect cells from DNA damage-induced cell death. Indeed, inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in Baf-3 cells does not prevent the ability of IL-3 to protect cells against gamma-irradiation-induced DNA damage. This protective effect of IL-3 rather correlates with the expression of the anti-apoptotic Bcl-x protein. Taken together, these data demonstrate that the PI3K/Akt pathway is sufficient to protect cells from growth factor starvation-induced apoptosis but is not required for IL-3 inhibition of DNA damage-induced cell death.     

C6-Ceramide Nanoliposomes Target the Warburg Effect in Chronic Lymphocytic Leukemia

Ceramide is a sphingolipid metabolite that induces cancer cell death. When C6-ceramide is encapsulated in a nanoliposome bilayer formulation, cell death is selectively induced in tumor models. However, the mechanism underlying this selectivity is unknown. As most tumors exhibit a preferential switch to glycolysis, as described in the “Warburg effect”, we hypothesize that ceramide nanoliposomes selectively target this glycolytic pathway in cancer. We utilize chronic lymphocytic leukemia (CLL) as a cancer model, which has an increased dependency on glycolysis. In CLL cells, we demonstrate that C6-ceramide nanoliposomes, but not control nanoliposomes, induce caspase 3/7-independent necrotic cell death. Nanoliposomal ceramide inhibits both the RNA and protein expression of GAPDH, an enzyme in the glycolytic pathway, which is overexpressed in CLL. To confirm that ceramide targets GAPDH, we demonstrate that downregulation of GAPDH potentiates the decrease in ATP after ceramide treatment and exogenous pyruvate treatment as well as GAPDH overexpression partially rescues ceramide-induced necrosis. Finally, an in vivo murine model of CLL shows that nanoliposomal C6-ceramide treatment elicits tumor regression, concomitant with GAPDH downregulation. We conclude that selective inhibition of the glycolytic pathway in CLL cells with nanoliposomal C6-ceramide could potentially be an effective therapy for leukemia by targeting the Warburg effect.     

Reactive oxygen species are involved in FasL-induced caspase-independent cell death and inflammatory responses

Fas-mediated caspase-dependent cell apoptosis has been well investigated. However, recent studies have shown that Fas can induce nonapoptotic caspase-independent cell death (CICD) when caspase activity is inhibited. Currently, the molecular mechanism of this alternative cell death mediated by Fas remains unclear. In this study, we investigated the signaling pathway of Fas-induced CICD in mouse embryonic fibroblasts (MEFs) whose caspase function was disrupted by the pan-caspase inhibitor Z-VAD-FMK and its coupling to inflammatory responses. Our results revealed that receptor-interacting protein 1 and tumor necrosis factor receptor-associated factor 2 play important roles in FasL-induced CICD. This death is associated with intracellular reactive oxygen species (ROS) production from mitochondria, as a ROS scavenger (BHA), antioxidants (trolox, NAC), and a mitochondrial respiratory chain uncoupler (rotenone) could prevent this event. Furthermore, delayed and sustained JNK activation, mitochondrial membrane potential breakdown, and loss of intracellular GSH were observed. In addition to CICD, FasL also induces cyclooxygenase-2 and MIP-2 gene upregulation, and both responses are attributed to ROS-dependent JNK activation. Taken together, these results demonstrate alternative signaling pathways of Fas upon caspase inhibition in MEFs that are unrelated to the classical apoptotic pathway, but steer cells toward necrosis and an inflammatory response through ROS production.     

Akt2 kinase suppresses glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-mediated apoptosis in ovarian cancer cells via phosphorylating GAPDH at threonine 237 and decreasing its nuclear translocation

Protein kinase B (Akt) plays important roles in regulation of cell growth and survival, but while many aspects of its mechanism of action are known, there are potentially additional regulatory events that remain to be discovered. Here we detected a 36-kDa protein that was co-immunoprecipitated with protein kinase Bβ (Akt2) in OVCAR-3 ovarian cancer cells. The protein was identified to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by MALDI-TOF/TOF MS, and the interaction of Akt2 and GAPDH was verified by reverse immunoprecipitation. Our further study showed that Akt2 may suppress GAPDH-mediated apoptosis in ovarian cancer cells. Overexpression of GAPDH increased ovarian cancer cell apoptosis induced by H(2)O(2), which was inhibited by Akt2 overexpression and restored by the PI3K/Akt inhibitor wortmannin or Akt2 siRNA. Akt2 phosphorylated Thr-237 of GAPDH and decreased its nuclear translocation, an essential step for GAPDH-mediated apoptosis. The interaction between Akt2 and GAPDH may be important in ovarian cancer as immunohistochemical analysis of 10 normal and 30 cancerous ovarian tissues revealed that decreased nuclear expression of GAPDH correlated with activation (phosphorylation) of Akt2. In conclusion, our study suggests that activated Akt2 may increase ovarian cancer cell survival via inhibition of GAPDH-induced apoptosis. This effect of Akt2 is partly mediated by its phosphorylation of GAPDH at Thr-237, which results in the inhibition of GAPDH nuclear translocation.     

Anchorless surface associated glycolytic enzymes from Lactobacillus plantarum 299v bind to epithelial cells and extracellular matrix proteins

An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated.     

13,15-N-cycloimide derivatives of chlorin p6 with isonicotinyl substituent are photosensitizers targeted to lysosomes.

Four monocationic cycloimide derivatives of chlorin p(6) (CICD) were studied as photosensitizers and compared to a structurally similar neutral derivative. Cationic CICD are highly photostable (quantum yield of photobleaching is about 1 x 10(-5), generate singlet oxygen under irradiation (quantum yields are 0.3-0.45), can be involved in a photo-induced substrate-dependent generation of superoxide radicals, but do not produce OH . 17,18-delta-lacton 13(2)-(N-methylisonicotinylamido)-13,15-cycloimide mesochlorin p(6) () and 13(2)-(N-methylisonicotinylamido)-13,15-cycloimide mesochlorin p(6) methyl ester () possess high cancer cell killing photodynamic activity, but they provide no photoinduced bactericidal effect. Substitution of an ethyl group with a hydroxyethyl or acetyl group at position 3 of the macrocycle results in a decrease in extinction and intracellular accumulation that finally leads to the reduced photocytotoxicity. Cationic CICD are targeted to lysosomes, and their intracellular penetration occurs most probably via caveolae-dependent endocytosis. Photodynamic treatment with cationic CICD results in the cell death via necrosis at both sub-phototoxic (40-70% of dead cells) and phototoxic (90-100% of dead cells) regimes of cell treatment. Irradiation induces lysosome damage, leakage of CICD from lysosomes and development of protease activity in cytoplasm, whereas mitochondria are not affected with irradiation. A positive charge of cationic CICD modified drastically an internalization pathway, sites of intracellular localization and mechanisms of photoinduced cytotoxicity as compared to previously studied neutral and anionic CICD. Our experiments with different CICD show that varying charge and structure of substituents it is possible to modulate many cellular properties of CICD in order to find the best molecular template of the advanced near-IR photosensitizer for photodynamic therapy.     

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Phosphorylation by Protein Kinase Cδ (PKCδ) Inhibits Mitochondria Elimination by Lysosomal-like Structures following Ischemia and Reoxygenation-induced Injury

After cardiac ischemia and reperfusion or reoxygenation (I/R), damaged mitochondria propagate tissue injury by promoting cell death. One possible mechanism to protect from I/R-induced injury is the elimination of damaged mitochondria by mitophagy. Here we identify new molecular events that lead to mitophagy using a cell culture model and whole hearts subjected to I/R. We found that I/R induces glyceraldehyde-3-phosphate dehydrogenase (GAPDH) association with mitochondria and promotes direct uptake of damaged mitochondria into multiorganellar lysosomal-like (LL) structures for elimination independently of the macroautophagy pathway. We also found that protein kinase C δ (PKCδ) inhibits GAPDH-driven mitophagy by phosphorylating the mitochondrially associated GAPDH at threonine 246 following I/R. Phosphorylated GAPDH promotes the accumulation of mitochondria at the periphery of LL structures, which coincides with increased mitochondrial permeability. Either inhibition of PKCδ or expression of a phosphorylation-defective GAPDH mutant during I/R promotes a reduction in mitochondrial mass and apoptosis, thus indicating rescued mitophagy. Taken together, we identified a GAPDH/PKCδ signaling switch, which is activated during oxidative stress to regulate the balance between cell survival by mitophagy and cell death due to accumulation of damaged mitochondria.     

Switching Akt: from survival signaling to deadly response

Akt, a protein kinase hyperactivated in many tumors, plays a major role in both cell survival and resistance to tumor therapy. A recent study, 1 along with other evidences, shows interestingly, that Akt is not a single‐function kinase, but may facilitate rather than inhibit cell death under certain conditions. This hitherto undetected function of Akt is accomplished by its ability to increase reactive oxygen species and to suppress antioxidant enzymes. The ability of Akt to down‐regulate antioxidant defenses uncovers a novel Achilles' heel, which could be exploited by oxidant therapies in order to selectively eradicate tumor cells that express high levels of Akt activity.     

Bcl-2 family proteins regulate mitochondrial reactive oxygen production and protect against oxidative stress

Bcl-2 family proteins protect against a variety of forms of cell death, including acute oxidative stress. Previous studies have shown that overexpression of the antiapoptotic protein Bcl-2 increases cellular redox capacity. Here we report that cell lines transfected with Bcl-2 paradoxically exhibit increased rates of mitochondrial H(2)O(2) generation. Using isolated mitochondria, we determined that increased H(2)O(2) release results from the oxidation of reduced nicotinamide adenine dinucleotide-linked substrates. Antiapoptotic Bcl-2 family proteins Bcl-xL and Mcl-1 also increase mitochondrial H(2)O(2) release when overexpressed. Chronic exposure of cells to low levels of the mitochondrial uncoupler carbonyl cyanide 4-(triflouromethoxy)phenylhydrazone reduced the rate of H(2)O(2) production by Bcl-xL overexpressing cells, resulting in a decreased ability to remove exogenous H(2)O(2) and enhanced cell death under conditions of acute oxidative stress. Our results indicate that chronic and mild elevations in H(2)O(2) release from Bcl-2, Bcl-xL, and Mcl-1 overexpressing mitochondria lead to enhanced cellular antioxidant defense and protection against death caused by acute oxidative stress.     

Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.     

Compartment-specific isoforms of TPI and GAPDH are imported into diatom mitochondria as a fusion protein: evidence in favor of a mitochondrial origin of the eukaryotic glycolytic pathway

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) are essential to glycolysis, the major route of carbohydrate breakdown in eukaryotes. In animals and other heterotrophic eukaryotes, both enzymes are localized in the cytosol; in photosynthetic eukaryotes, GAPDH and TPI exist as isoenzymes that function in the glycolytic pathway of the cytosol and in the Calvin cycle of chloroplasts. Here, we show that diatoms--photosynthetic protists that acquired their plastids through secondary symbiotic engulfment of a eukaryotic rhodophyte--possess an additional isoenzyme each of both GAPDH and TPI. Surprisingly, these new forms are expressed as an TPI-GAPDH fusion protein which is imported into mitochondria prior to its assembly into a tetrameric bifunctional enzyme complex. Homologs of this translational fusion are shown to be conserved and expressed also in nonphotosynthetic, heterokont-flagellated oomycetes. Phylogenetic analyses show that mitochondrial GAPDH and its N-terminal TPI fusion branch deeply within their respective eukaryotic protein phylogenies, suggesting that diatom mitochondria may have retained an ancestral state of glycolytic compartmentation that existed at the onset of mitochondrial symbiosis. These findings strongly support the view that nuclear genes for enzymes of glycolysis in eukaryotes were acquired from mitochondrial genomes and provide new insights into the evolutionary history (host-symbiont relationships) of diatoms and other heterokont-flagellated protists.     

Overexpression of midkine contributes to anti-apoptotic effects in human meningiomas

Meningiomas are the second most common intracranial tumours. Most meningiomas grow slowly; however, atypical and anaplastic meningiomas show an aggressive biological behaviour. Overexpression of growth factors is considered to be a cause of carcinogenesis. Midkine and pleiotrophin are heparin-binding growth factors that promote growth, survival, migration and differentiation of various target cells. Both molecules are highly expressed during human embryogenesis but are rarely seen in the adult. We show that in relation to normal dura and arachnoid tissues, midkine was overexpressed in meningiomas on the mRNA and protein level, whereas pleiotrophin was not. Thereby, not only the intact but also the truncated form of midkine could be observed. The expression of midkine receptors was variable in different samples. Midkine stimulation of cultured meningioma cells induced phosphorylation of Akt, whereas no increase in phosphorylation of p42/44 MAPK or p38 MAPK could be detected. Midkine did not influence the proliferation of meningioma cells in vitro, but it did protect meningioma cells from camptothecin-mediated apoptotic cell death through reduction in the amounts of active caspase-3. These findings provide evidence for the overexpression of midkine in meningiomas which contributes to protection from cell death in these second most common intracranial tumours.     

Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal

Numerous studies reveal that phosphatidylinositol (PI) 3-kinase and Akt protein kinase are important mediators of cell survival. However, the survival-promoting mechanisms downstream of these enzymes remain uncharacterized. Glycogen synthase kinase-3 beta (GSK-3 beta), which is inhibited upon phosphorylation by Akt, was recently shown to function during cell death induced by PI 3-kinase inhibitors. In this study, we tested whether GSK-3 beta is critical for the death of sympathetic neurons caused by the withdrawal of their physiological survival factor, the nerve growth factor (NGF). Stimulation with NGF resulted in PI 3-kinase-dependent phosphorylation of GSK-3 beta and inhibition of its protein kinase activity, indicating that GSK-3 beta is targeted by PI 3-kinase/Akt in these neurons. Expression of the GSK-3 beta inhibitor Frat1, but not a mutant Frat1 protein that does not bind GSK-3 beta, rescued neurons from death caused by inhibiting PI 3-kinase. Similarly, expression of Frat1 or kinase-deficient GSK-3 beta reduced death caused by inhibiting Akt. In NGF-maintained neurons, overexpression of GSK-3 beta caused a small but significant decrease in survival. However, expression of neither Frat1, kinase-deficient GSK-3 beta, nor GSK-3-binding protein inhibited NGF withdrawal-induced death. Thus, although GSK-3 beta function is required for death caused by inactivation of PI 3-kinase and Akt, neuronal death caused by NGF withdrawal can proceed through GSK-3 beta-independent pathways.     

Role of Translation Initiation Factor 2B in Control of Cell Survival by the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3β Signaling Pathway

The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling pathway is an important mediator of growth factor-dependent survival of mammalian cells. A variety of targets of the Akt protein kinase have been implicated in cell survival, including the protein kinase glycogen synthase kinase 3beta (GSK-3beta). One of the targets of GSK-3beta is translation initiation factor 2B (eIF2B), linking global regulation of protein synthesis to PI 3-kinase/Akt signaling. Because of the central role of protein synthesis, we have investigated the involvement of eIF2B, which is inhibited as a result of GSK-3beta phosphorylation, in programmed cell death. We demonstrate that expression of eIF2B mutants lacking the GSK-3beta phosphorylation or priming sites is sufficient to protect both Rat-1 and PC12 cells from apoptosis induced by overexpression of GSK-3beta, inhibition of PI 3-kinase, or growth factor deprivation. Consistent with these effects on cell survival, expression of nonphosphorylatable eIF2B prevented inhibition of protein synthesis following treatment of cells with the PI 3-kinase inhibitor LY294002. Conversely, cycloheximide induced apoptosis of PC12 and Rat-1 cells, further indicating that protein synthesis was required for cell survival. Inhibition of translation resulting from treatment with cycloheximide led to the release of cytochrome c from mitochondria, similar to the effects of inhibition of PI 3-kinase. Expression of nonphosphorylatable eIF2B prevented cytochrome c release resulting from PI 3-kinase inhibition but did not affect cytochrome c release or apoptosis induced by cycloheximide. Regulation of translation resulting from phosphorylation of eIF2B by GSK-3beta thus appears to contribute to the control of cell survival by the PI 3-kinase/Akt signaling pathway, acting upstream of mitochondrial cytochrome c release.