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

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 β-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–β-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.