Selenium and GPx-1 overexpression protect mammalian cells against UV-induced DNA damage

Supplementation of the culture media of human MCF-7 breast carcinoma cells or mouse fibroblasts with low levels of selenium (30 nM) provided as sodium selenite was shown to protect these cells from ultraviolet (UV)-induced chromosome damage, as quantified by micronucleus assay. Selenium supplementation was also effective in reducing UV-induced gene mutations as measured in the lacI shuttle vector model. Protection was dependent on functional BRCA1 activity, a protein implicated in breast cancer risk and DNA damage repair. In addition, overexpression of GPx-1, a selenoprotein with antioxidant activity, also attenuated UV induced micronuclei formation in the absence of selenium supplementation. Combining selenium supplementation with GPx-1 overexpression further reduced UV-induced micronucleus frequency. These data provide evidence that the benefits of selenium supplementation might be either through the prevention or repair of DNA damage, and they implicate at least one selenoprotein (GPx-1) in the process.     

The effects of selenium and the GPx-1 selenoprotein on the phosphorylation of H2AX

Background

Significant data supports the health benefits of selenium although supplementation trials have yielded mixed results. GPx-1, whose levels are responsive to selenium availability, is implicated in cancer etiology by human genetic data. Selenium's ability to alter the phosphorylation of the H2AX, a histone protein that functions in the reduction of DNA damage by recruiting repair proteins to the damage site, following exposure to ionizing radiation and bleomycin was investigated.

Methods

Human cell lines that were either exposed to selenium or were transfected with a GPx-1 expression construct were exposed to ionizing radiation or bleomycin. Phosphorylation of histone H2AX was quantified by flow cytometry and survival by the MTT assay. Phosphorylation of the Chk1 and Chk2 checkpoint proteins was quantified by western blotting.

Results

In colon-derived cells, selenium increases GPx-1 and attenuated H2AX phosphorylation following genotoxic exposures while the viability of these cells was unaffected. MCF-7 cells and transfectants that express high GPx-1 levels were exposed to ionizing radiation and bleomycin, and H2AX phosphorylation and cell viability were assessed. GPx-1 increased H2AX phosphorylation and viability following the induction of DNA damage while enhancing the levels of activated Chk1 and Chk2.

Conclusions

Exposure of mammalian cells to selenium can alter the DNA damage response and do so by mechanisms that are dependent and independent of its effect on GPx-1.

General significance

Selenium and GPx-1 may stimulate the repair of genotoxic DNA damage and this may account for some of the benefits attributed to selenium intake and elevated GPx-1 activity.     

GPx-1 modulates Akt and P70S6K phosphorylation and Gadd45 levels in MCF-7 cells

Selenium has been shown to prevent cancer in animal models, and recent data indicate it is likely to be effective in humans as well. One selenium-containing protein, the cytoplasmic form of glutathione peroxidase (GPx-1), has been implicated in cancer risk and development by genetic studies identifying at-risk alleles and loss of heterozygosity in tumors. In order to evaluate the biological consequences of GPx-1 overexpression, human MCF-7 cells were stably transfected with a GPx-1 expression construct and the effects of GPx-1 on protein kinases associated with stress responses were determined. GPx-1 overexpression affected phosphorylation of p70S6K, whereas Erk1/2 and p38 MAPK were not affected. Site-specific phosphorylation of Akt declined and the levels of Gadd45, a DNA damage response protein, increased significantly as a consequence of elevated GPx-1 expression. Effects on p70S6K and Gadd45 after selenium supplementation have been reported, and given previous data demonstrating a role for GPx-1 in cancer etiology, these results support the concept that the chemopreventive properties of selenium may be due, at least in part, to its role in regulating GPx-1.     

Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT-737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia

Bcr-Abl is the cause of Philadelphia-positive (Ph(+)) leukemias and also constitutes their principal therapeutic target, as exemplified by dramatic effects of imatinib mesylate. However, mono-targeting of Bcr-Abl does not always achieve complete leukemia eradication, and additional strategies those enable complete elimination of leukemic cells are desired to develop. Here we demonstrate that INNO-406, a much more active Bcr-Abl tyrosine kinase inhibitor than imatinib, augments the activities of several proapoptotic Bcl-2 homology (BH)3-only proteins (Bim, Bad, Bmf and Bik) and induces apoptosis in Ph(+) leukemia cells via Bcl-2 family-regulated intrinsic apoptosis pathway. ABT-737, an inhibitor of antiapoptotic Bcl-2 and Bcl-X(L), greatly enhanced the apoptosis by INNO-406, even in INNO-406-less sensitive cells with Bcr-Abl point mutations except T315I mutation. In contrast, co-treatment with INNO-406 and other pharmacologic inducers of those BH3-only proteins, such as 17-allylaminogeldanamycin, an heat shock protein-90 inhibitor, or PS-341, a proteasome inhibitor, did not further increase the BH3-only protein levels or sensitize leukemic cells to INNO-406-induced apoptosis, suggesting a limit to how much expression levels of BH3-only proteins can be increased by anticancer agents. Thus, double-barrelled molecular targeting for Bcr-Abl-driven oncogenic signaling and the cell protection by antiapoptotic Bcl-2 family proteins may be the rational therapeutic approach for eradicating Ph(+) leukemic cells.     

Inhibition of protein-tyrosine phosphatase 1B (PTP1B) mediates ubiquitination and degradation of Bcr-Abl protein

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.     

Imatinib-resistant CML cells have low ENT activity but maintain sensitivity to gemcitabine

Philadelphia chromosome-positive chronic myelogenus leukemia (CML) is widely treated with imatinib mesylate (imatinib), a potent inhibitor of the Bcr-Abl tyrosine kinase. However, resistance to this compound remains a concern. Current treatment approaches include combinations of imatinib with nucleoside analogs such as gemcitabine, which requires equilibrative nucleoside transporters (ENTs) for uptake, to overcome this resistance. Here we report that imatinib treatment decreased ENT1-dependent activity and mRNA expression. Although, imatinib-resistant cells showed decreased levels of both ENT1 and ENT2 activity and expression, these cells remained sensitive to gemcitabine, suggesting that nucleoside analogs can be used as adjunctive therapy.     

Regulation of the extracellular antioxidant selenoprotein plasma glutathione peroxidase (GPx-3) in mammalian cells

Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing extracellular antioxidant protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides. Selenoprotein expression involves the alternate recognition of a UGA codon as a selenocysteine codon and requires signals in the 3′-untranslated region (UTR), including a selenocysteine insertion sequence (SECIS), as well as specific translational cofactors. To ascertain regulatory determinants of GPx-3 expression and function, we generated recombinant GPx-3 (rGPX-3) constructs with various 3′-UTR, as well as a Sec73Cys mutant. In transfected Cos7 cells, the Sec73Cys mutant was expressed at higher levels than the wild type rGPx-3, although the wild type rGPx-3 had higher specific activity, similar to plasma purified GPx-3. A 3′-UTR with only the SECIS was insufficient for wild type rGPx-3 protein expression. Selenocompound supplementation and co-transfection with SECIS binding protein 2 increased wild type rGPx-3 expression. These results demonstrate the importance of translational mechanisms in GPx-3 expression.     

Rapamycin-mediated mTOR inhibition attenuates survivin and sensitizes glioblastoma cells to radiation therapy

Survivin, an antiapoptotic protein, is elevated in most malignancies and attributes to radiation resistance in tumors including glioblastoma multiforme. The downregulation of survivin could sensitize glioblastoma cells to radiation therapy. In this study, we investigated the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), in attenuating survivin and enhancing the therapeutic efficacy for glioblastoma cells, and elucidated the underlying mechanisms. Here we tested various concentrations of rapamycin (1-8 nM) in combination with radiation dose 4 Gy. Rapamycin effectively modulated the protein kinase B (Akt)/mTOR pathway by inhibiting the phosphorylation of Akt and mTOR proteins, and this inhibition was further enhanced by radiation. The expression level of survivin was decreased in rapamycin pre-treatment glioblastoma cells followed by radiation; meanwhile, the phosphorylation of H2A histone family member X (H2AX) at serine-139 (γ-H2AX) was increased. p21 protein was also induced on radiation with rapamycin pre-treatment, which enhanced G1 arrest and the accumulation of cells at G0/subG1 phase. Furthermore, the clonogenic cell survival assay revealed a significant dose-dependent decrease in the surviving fraction for all three cell lines pre-treated with rapamycin. Our studies demonstrated that targeting survivin may be an effective approach for radiosensitization of malignant glioblastoma.     

Effect of Chronic Administration of Low Dose Rapamycin on Development and Immunity in Young Rats

Mammalian target of rapamycin (mTOR) regulates cell growth, cell differentiation and protein synthesis. Rapamycin, an inhibitor of mTOR, has been widely used as an immunosuppressant and anti-cancer drug. Recently, mTOR inhibitors have also been reported to be a potential anti-epileptic drug, which may be effective when used in young patients with genetic epilepsy. Thus, a suitable dose of rapamycin which can maintain the normal function of mTOR and has fewer side effects ideally should be identified. In the present study, we first detected changes in marker proteins of mTOR signaling pathway during development. Then we determined the dose of rapamycin by treating rats of 2 weeks of age with different doses of rapamycin for 3 days and detected its effect on mTOR pathway. Young rats were then treated with a suitable dose of rapamycin for 4 weeks and the effect of rapamycin on mTOR, development and immunity were investigated. We found that the expression of the marker proteins of mTOR pathway was changed during development in brain hippocampus and neocortex. After 3 days of treanent, 0.03 mg/kg rapamycin had no effect on phospho-S6, whereas 0.1, 0.3, 1.0 and 3.0 mg/kg rapamycin inhibited phospho-S6 in a dose-dependent manner. However, only 1.0 mg/kg and 3.0 mg/kg rapamycin inhibited phospho-S6 after 4 weeks treatment of rapamycin. Parallel to this result, rats treated with 0.1 and 0.3 mg/kg rapamycin had no obvious adverse effects, whereas rats treated with 1.0 and 3.0 mg/kg rapamycin showed significant decreases in body, spleen and thymus weight. Additionally, rats treated with 1.0 and 3.0 mg/kg rapamycin exhibited cognitive impairment and anxiety as evident by maze and open field experiments. Furthermore, the content of IL-1β, IL-2, IFN-γ, TNF-α in serum and cerebral cortex were significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. The expression of DCX was also significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. However, rats treated with 1.0 mg/ kg rapamycin exhibited fewer and milder side effects than those treated with 3.0 mg/kg. In summary, all these data suggest that there is not a rapamycin dose that can inhibit mTOR for epilepsy without causing any side effects, but 1 mg /kg may be the optimal dose for young rats for suppressing mTOR with relatively few side effects.     

Syntaxin13 Expression Is Regulated by Mammalian Target of Rapamycin (mTOR) in Injured Neurons to Promote Axon Regeneration

Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration.     

Phospholipid-hydroperoxide glutathione peroxidase (GPx-4) localization in resting platelets, and compartmental change during platelet activation

Seleno-glutathione peroxidases are an important family of antioxidant enzymes, that include the phospholipid hydroperoxide glutathione peroxidase (GPx-4), an enzyme that reduces lipid hydroperoxides in membranes. The essential characteristics of platelet GPx-4 were found to be the same as the GPx-4 from other tissues. To explore the subcellular expression of GPx-4 in human platelets, we first investigated both its activity and localization in subcellular fractions. About 47% of the total cell enzyme activity was found in the membrane fractions, 29% in the mitochondria and 23% in the cytosol fractions. The same subcellular distribution of GPx-4 protein was demonstrated in resting platelets. This distribution data was further established by confocal microscopy. Of major potential biological significance, this distribution changed when platelets were activated. Confocal immunofluorescence microscopy localized mainly GPx-4 to membranes in contrast to cytoplasm in the resting cells. Based on these results we propose that cytoplasmic GPx-4 could be moved to the membrane for protection during platelet activation. This enzyme would then be important to maintain the integrity of platelet function in vascular system stressed by oxidative reactions.     

Oncogenic stress induced by acute hyper-activation of Bcr-Abl leads to cell death upon induction of excessive aerobic glycolysis

In response to deregulated oncogene activation, mammalian cells activate disposal programs such as programmed cell death. To investigate the mechanisms behind this oncogenic stress response we used Bcr-Abl over-expressing cells cultivated in presence of imatinib. Imatinib deprivation led to rapid induction of Bcr-Abl activity and over-stimulation of PI3K/Akt-, Ras/MAPK-, and JAK/STAT pathways. This resulted in a delayed necrosis-like cell death starting not before 48 hours after imatinib withdrawal. Cell death was preceded by enhanced glycolysis, glutaminolysis, and amino acid metabolism leading to elevated ATP and protein levels. This enhanced metabolism could be linked to induction of cell death as inhibition of glycolysis or glutaminolysis was sufficient to sustain cell viability. Therefore, these data provide first evidence that metabolic changes induced by Bcr-Abl hyper-activation are important mediators of oncogenic stress-induced cell death.During the first 30 hours after imatinib deprivation, Bcr-Abl hyper-activation did not affect proliferation but resulted in cellular swelling, vacuolization, and induction of eIF2α phosphorylation, CHOP expression, as well as alternative splicing of XPB, indicating endoplasmic reticulum stress response. Cell death was dependent on p38 and RIP1 signaling, whereas classical death effectors of ER stress, namely CHOP-BIM were antagonized by concomitant up-regulation of Bcl-xL.Screening of 1,120 compounds for their potential effects on oncogenic stress-induced cell death uncovered that corticosteroids antagonize cell death upon Bcr-Abl hyper-activation by normalizing cellular metabolism. This protective effect is further demonstrated by the finding that corticosteroids rendered lymphocytes permissive to the transforming activity of Bcr-Abl. As corticosteroids are used together with imatinib for treatment of Bcr-Abl positive acute lymphoblastic leukemia these data could have important implications for the design of combination therapy protocols.In conclusion, excessive induction of Warburg type metabolic alterations can cause cell death. Our data indicate that these metabolic changes are major mediators of oncogenic stress induced by Bcr-Abl.     

Aclacinomycin A Sensitizes K562 Chronic Myeloid Leukemia Cells to Imatinib through p38MAPK-Mediated Erythroid Differentiation

Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and apoptosis. These results provided a potential management by which ACM might have a crucial impact on increasing sensitivity of CML cells to imatinib in the differentiation therapeutic approaches.     

mTORC1 inhibition increases neurotensin secretion and gene expression through activation of the MEK/ERK/c-Jun pathway in the human endocrine cell line BON

The mammalian target of rapamycin (mTOR) signaling exists in two complexes: mTORC1 and mTORC2. Neurotensin (NT), an intestinal hormone secreted by enteroendocrine (N) cells in the small bowel, has important physiological effects in the gastrointestinal tract. The human endocrine cell line BON abundantly expresses the NT gene and synthesizes and secretes NT in a manner analogous to that of N cells. Here, we demonstrate that the inhibition of mTORC1 by rapamycin (mTORC1 inhibitor), torin1 (both mTORC1 and mTORC2 inhibitor) or short hairpin RNA-mediated knockdown of mTOR, regulatory associated protein of mTOR (RAPTOR), and p70 S6 kinase (p70S6K) increased basal NT release via upregulating NT gene expression in BON cells. c-Jun activity was increased by rapamycin or torin1 or p70S6K knockdown. c-Jun overexpression dramatically increased NT promoter activity, which was blocked by PD98059, an mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, overexpression of MEK1 or extracellular signal-regulated kinase 1 (ERK1) increased c-Jun expression and NT promoter activity. More importantly, PD98059 blocked rapamycin- or torin1-enhanced NT secretion. Consistently, rapamycin and torin1 also increased NT gene expression in Hep3B cells, a human hepatoma cell line that, similar to BON, expresses high levels of NT. Phosphorylation of c-Jun and ERK1/2 was also increased by rapamycin and torin1 in Hep3B cells. Finally, we showed activation of mTOR in BON cells treated with amino acids, high glucose, or serum and, concurrently, the attenuation of ERK1/2 and c-Jun phosphorylation and NT secretion. Together, mTORC1, as a nutrient sensor, negatively regulates NT secretion via the MEK/ERK/c-Jun signaling pathway. Our results identify a physiological link between mTORC1 and MEK/ERK signaling in controlling intestinal hormone gene expression and secretion.     

Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells

While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation.     

Phosphomimetic substitution of heterogeneous nuclear ribonucleoprotein A1 at serine 199 abolishes AKT-dependent internal ribosome entry site-transacting factor (ITAF) function via effects on strand annealing and results in mammalian target of rapamycin complex 1 (mTORC1) inhibitor sensitivity

The relative activity of the AKT kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mammalian target of rapamycin (mTOR) complex 1 inhibitors. Our previous studies have shown that the multifunctional RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates a salvage pathway facilitating internal ribosome entry site (IRES)-dependent mRNA translation of critical cellular determinants in an AKT-dependent manner following mTOR inhibitor exposure. This pathway functions by stimulating IRES-dependent translation in cells with relatively quiescent AKT, resulting in resistance to rapamycin. However, the pathway is repressed in cells with elevated AKT activity, rendering them sensitive to rapamycin-induced G(1) arrest as a result of the inhibition of global eIF-4E-mediated translation. AKT phosphorylation of hnRNP A1 at serine 199 has been demonstrated to inhibit IRES-mediated translation initiation. Here we describe a phosphomimetic mutant of hnRNP A1 (S199E) that is capable of binding both the cyclin D1 and c-MYC IRES RNAs in vitro but lacks nucleic acid annealing activity, resulting in inhibition of IRES function in dicistronic mRNA reporter assays. Utilizing cells in which AKT is conditionally active, we demonstrate that overexpression of this mutant renders quiescent AKT-containing cells sensitive to rapamycin in vitro and in xenografts. We also demonstrate that activated AKT is strongly correlated with elevated Ser(P)(199)-hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 serine 199 regulates the AKT-dependent sensitivity of cells to rapamycin and functionally links IRES-transacting factor annealing activity to cellular responses to mTOR complex 1 inhibition.     

Inhibition of mammalian target of rapamycin activates apoptosis signal-regulating kinase 1 signaling by suppressing protein phosphatase 5 activity

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces a cellular stress response characterized by rapid and sustained activation of the apoptosis signal-regulating kinase 1 (ASK1) signaling pathway and selective apoptosis of cells lacking functional p53. Here we have investigated how mTOR regulates ASK1 signaling using p53-mutant rhabdomyosarcoma cells. In Rh30 cells, ASK1 was found to physically interact with protein phosphatase 5 (PP5), previously identified as a negative regulator of ASK1. Rapamycin did not affect either protein level of PP5 or association of PP5 with ASK1. Instead, rapamycin caused rapid dissociation of the PP2A-B" regulatory subunit (PR72) from the PP5-ASK1 complex, which was associated with reduced phosphatase activity of PP5. This effect was dependent on expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Down-regulation of PP5 activity by rapamycin coordinately activated ASK1, leading to elevated phosphorylation of c-Jun. Amino acid deprivation, which like rapamycin inhibits mTOR signaling, also inhibited PP5 activity, caused rapid dissociation of PR72, and activated ASK1 signaling. Overexpression of PP5, but not the PP2A catalytic subunit, blocked rapamycin-induced phosphorylation of c-Jun, and protected cells from rapamycin-induced apoptosis. The results suggest that PP5 is downstream of mTOR, and positively regulated by the mTOR pathway. The findings suggest that in the absence of serum factors, mTOR signaling suppresses apoptosis through positive regulation of PP5 activity and suppression of cellular stress.