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Involvement of general control nonderepressible kinase 2 in cancer cell apoptosis by posttranslational mechanisms.

ABSTRACT: General control nonderepressible kinase 2 (GCN2) is a promising target for cancer therapy. However, the role of GCN2 in cancer cell survival or death is elusive; further, small molecules targeting GCN2 signaling are not available. By using a GCN2 level-based drug screening assay, we found that GCN2 protein level critically determined the sensitivity of the cancer cells toward Na(+),K(+)-ATPase ligand-induced apoptosis both in vitro and in vivo, and this effect was largely dependent on C/EBP homologous protein (CHOP) induction. Further analysis revealed that GCN2 is a short-lived protein. In A549 lung carcinoma cells, cellular ?-arrestin1/2 associated with GCN2 and maintained the GCN2 protein level at a low level by recruiting the E3 ligase NEDD4L and facilitating consequent proteasomal degradation. However, Na(+),K(+)-ATPase ligand treatment triggered the phosphorylation of GCN2 at threonine 899, which increased the GCN2 protein level by disrupting the formation of GCN2-?-arrestin-NEDD4L ternary complex. The enhanced GCN2 level, in turn, aggravated Na(+),K(+)-ATPase ligand-induced cancer cell apoptosis. Our findings reveal that GCN2 can exert its proapoptotic function in cancer cell death by posttranslational mechanisms. Moreover, Na(+),K(+)-ATPase ligands emerge as the first identified small-molecule drugs that can trigger cancer cell death by modulating GCN2 signaling.

SUBMITTER: Wei C

PROVIDER: S-EPMC4357505 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Involvement of general control nonderepressible kinase 2 in cancer cell apoptosis by posttranslational mechanisms.

Wei Chen C Lin Ma M Jinjun Bian B Su Feng F Dan Cao C Yan Chen C Jie Yang Y Jin Zhang Z Zi-Chun Hua H Wu Yin Y

Molecular biology of the cell 20150114 6

General control nonderepressible kinase 2 (GCN2) is a promising target for cancer therapy. However, the role of GCN2 in cancer cell survival or death is elusive; further, small molecules targeting GCN2 signaling are not available. By using a GCN2 level-based drug screening assay, we found that GCN2 protein level critically determined the sensitivity of the cancer cells toward Na(+),K(+)-ATPase ligand-induced apoptosis both in vitro and in vivo, and this effect was largely dependent on C/EBP homo ...[more]

PMID: 25589675

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Project description:Hepatic cytochromes P450 3A (P450s 3A) are endoplasmic reticulum (ER)-proteins, responsible for xenobiotic metabolism. They are degraded by the ubiquitin-dependent 26S proteasome. Consistent with this, we have shown that proteasomal inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and N-benzoyloxycarbonyl-Leu-Leu-Leu-B(OH)(2) (MG262) stabilize CYP3A proteins. However, MG132 has been reported to suppress P450s 3A as a result of impaired nuclear factor-kappaB activation and consequently reduced CYP3A protein stability. Because the MG132 concentration used in those studies was 10-fold higher than that required for CYP3A stabilization, we examined the effect of MG132 (0-300 microM) concentration-dependent proteasomal inhibition on CYP3A turnover in cultured primary rat hepatocytes. We found a biphasic MG132 concentration effect on CYP3A turnover: Stabilization at 5 to 10 muM with marked suppression at >100 microM. Proteasomal inhibitors reportedly induce ER stress, heat shock, and apoptotic response. At these high MG132 concentrations, such CYP3A suppression could be due to ER stress induction, so we monitored the activity of PERK [PKR (RNA-dependent protein kinase)-like ER kinase (EIF2AK3)], the ER stress-activated eukaryotic initiation factor 2alpha (eIF2alpha) kinase. Indeed, we found a marked (approximately 4-fold) MG132 concentration-dependent PERK autophosphorylation, along with an 8-fold increase in eIF2alpha-phosphorylation. In parallel, MG132 also activated GCN2 [general control nonderepressible-2 (EIF2AK4)] eIF2alpha kinase in a concentration-dependent manner, but not the heme-regulated inhibitor eIF2alpha kinase [(EIF2AK1)]. Pulse-chase, immunoprecipitation/immunoblotting analyses documented the consequently dramatic translational shutoff of total hepatic protein, including but not limited to CYP3A and tryptophan 2,3-dioxygenase protein syntheses. These findings reveal that at high concentrations, MG132 is indeed cytotoxic and can suppress CYP3A synthesis, a result confirmed by confocal immunofluorescence analyses of MG132-treated hepatocytes.

Project description:We hypothesized that the general control nonderepressible 2 (GCN2)/eukaryotic initiation factor 2 (eIF2) signaling pathway and intracellular protein synthesis (PS) are regulated to maintain milk PS in primary bovine mammary epithelial cells (MECs) under essential amino acid (EAA) starvation conditions. We cultured MECs with 0%, 2% (depletion), and 100% (control) EAA for two exposure times (8 and 24 h), followed by three refeeding (RF) times with 100% EAA (0, 8, and 24 h). Subsequently, we measured cell viability, total protein concentration, and proliferation. Western blotting was used to quantify the levels of casein and the expression of total GCN2 and eIF2, as well as phosphorylated GCN2 (GCN2P) and eIF2 (eIF2P). The ISOQuant method was used to assess MEC proteomes, and the resultant data were analyzed using the Kruskal-Wallis test, nonpaired Wilcoxon rank post-hoc test, and ANOVA-Tukey test, as well as principal component analyses and multiple regressions models. Differences in cell viability were observed between the control versus the depleted and repleted MECs, respectively, where 97.2-99.8% viability indicated low cell death rates. Proliferation (range, 1.02-1.55 arbitrary units (AU)) was affected by starvation for 12 and 24 h and repletion for 24 h, but it was not increased compared with the control. Total protein expression was unaffected by both depletion and repletion treatments (median 3158 µg/mL). eIF2P expression was significantly increased (p < 0.05) after treatment with 2% EAA for 8 and 24 h compared with 2% EAA with 8 h + 24 h RF and 2% EAA with 24 h + 8 h RF. GCN2P also showed significantly increased expression (p < 0.05) after treatment with 2% EAA for 24 h compared with the control and 2% EAA with 24 h + 8 h RF. Intracellular casein/α-tubulin expression was unaffected by 2% EAA compared with control (0.073 ± 0.01 AU versus 0.086 ± 0.02 AU, respectively). We studied 30 of the detected 1180 proteins, 16 of which were differentially expressed in starved and refed MECs. Cells faced with EAA deficiency activated the GCN2P/eIF2P pathway, and the lack of change in the levels of casein and other milk proteins suggested that the EAA deficit was mitigated by metabolic flexibility to maintain homeostasis.

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Involvement of general control nonderepressible kinase 2 in cancer cell apoptosis by posttranslational mechanisms.

Publications

Involvement of general control nonderepressible kinase 2 in cancer cell apoptosis by posttranslational mechanisms.

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