Phosphorylation of interleukin (IL)-24 is required for mediating its anti-cancer activity

Interleukin (IL)-24 is a tumor suppressor/cytokine gene that undergoes post-translational modifications (PTMs). Glycosylation and ubiquitination are important for IL-24 protein stabilization and degradation respectively. Little is known about IL-24 protein phosphorylation and its role in IL-24-mediated anti-tumor activities. In this study we conducted molecular studies to determine whether IL-24 phosphorylation is important for IL-24-mediated anti-cancer activity. Human H1299 lung tumor cell line that was stably transfected with a doxycycline (DOX)-inducible (Tet-on) plasmid vector carrying the cDNA of IL-24-wild-type (IL-24(wt)) or IL-24 with all five phosphorylation sites replaced (IL-24(mt)) was used in the present study. Inhibition of tumor cell proliferation, cell migration and invasion, and induction of G2/M cell cycle arrest was observed in DOX-induced IL-24(wt)-expressing cells but not in IL-24(mt)-expressing cells. Secretion of IL-24(mt) protein was greatly reduced compared to IL-24(wt) protein. Further, IL-24(wt) and IL-24(mt) proteins markedly differed in their subcellular organelle localization. IL-24(wt) but not IL-24(mt) inhibited the AKT/mTOR signaling pathway. SiRNA-mediated AKT knockdown and overexpression of myristolyated AKT protein confirmed that IL-24(wt) but not IL-24(mt) mediated its anti-cancer activity by inhibiting the AKT signaling pathway. Our results demonstrate that IL-24 phosphorylation is required for inhibiting the AKT/mTOR signaling pathway and exerting its anti-cancer activities.