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

Abstract

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-24wt) or IL-24 with all five phosphorylation sites replaced (IL-24mt) 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-24wt-expressing cells but not in IL-24mt-expressing cells. Secretion of IL-24mt protein was greatly reduced compared to IL-24wt protein. Further, IL-24wt and IL-24mt proteins markedly differed in their subcellular organelle localization. IL-24wt but not IL-24mt inhibited the AKT/mTOR signaling pathway. SiRNA-mediated AKT knockdown and overexpression of myristolyated AKT protein confirmed that IL-24wt but not IL-24mt 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.

Keywords: IL-24; cytokine; lung cancer; phosphorylation.

Figure 1. IL-24^wt and IL-24^mt have different protein banding patterns

A. Western blotting showed that IL-24^wt and IL-24^mt protein banding patterns differed following DOX treatment of H1299-IL-24^wt and H1299-IL-24^mt cells. Cells that did not receive DOX treatment served as controls. B. Cell lysates from DOX-treated H1299-IL24^wt and H1299-IL24^mt were immunoprecipitated (IP) with phosphorylated Serine or Threonine antibody and immunoblotted (IB) with human IL-24 antibody. IL-24 protein was detected in H1299-IL-24^mt cell lysate, but not in H1299-IL-24^mt cell lysate. This shows that only wild-type IL-24 protein is phosphorylated. IgG protein band served as internal protein loading control. C. Immunofluorescence studies showed that IL-24^wt protein was uniformly distributed in the cytoplasm, with some localized in the endoplasmic reticulum (ER) of the cell. In contrast, IL-24^mt protein was mostly localized in the ER, with little distributed in the cytoplasm of the cell. Magnification, x100. D. IL-24 protein level was markedly low in the culture supernatant collected from DOX-treated H1299-IL24^mt cells compared with the IL-24 protein level in the supernatant from DOX-treated H1299-IL24^wt cells, as determined by ELISA. Cell culture supernatant from untreated cells served as a negative control. The number above the bar indicates the protein concentration (ng/ml). E. Expression of IL-24^wt following DOX treatment greatly reduced cell viability of H1299 cells, compared with cells expressing IL-24^mt at 72 h. F. A colony formation assay on soft agar demonstrated that H1299-IL-24^wt cells formed fewer colonies than H1299-IL-24^mt when treated with DOX. G. Cell cycle analysis showed that only IL-24^wt induced G2/M cell-cycle arrest at 48 h after DOX treatment. H. IL-24^wt activated caspase-9, PARP and pJNK^{Thr183/Tyr185} in H1299 cells at 48 h after DOX treatment, while IL-24^mt did not. Beta actin was detected as protein loading control. *denotes P < 0.05. “NS” denotes Not Significant.

Figure 2. IL-24^wt, but not IL-24^mt, inhibits cell migration and invasion

A. Scratch assay showed that IL-24^wt-expressing H1299 cells migrated less than IL-24^mt-expressing H1299 cells. B. IL-24^wt produced a greater inhibitory effect on cell migration than did IL-24^mt at 24 h and 48 h after DOX treatment. C. Matrigel invasion assay showed that IL-24^wt inhibited cell invasion to a greater extent than IL-24^mt at 24 h and 48 h after DOX treatment. Cells that did not receive DOX treatment served as controls. Results shown are the means ± SD of three independent experiments. *P < 0.05.

Figure 3. IL-24^wt, but not IL-24^mt, inhibits the AKT-mTOR signaling pathway

Cell lysates prepared from DOX-treated H1299-IL-24^wt and H1299-IL-24^mt cells were analyzed for proteins associated with the AKT signaling pathway and changes in protein expression quantified by Western blotting. A. IL-24^wt, but not IL-24^mt, significantly reduced the expression of phosphorylated (p) AKT^S473, pPRAS40^T246, and pmTOR^S2448 proteins at the two time points tested. B. Analysis for changes in the expression of AKT isoforms demonstrated that IL-24^wt significantly reduced the expression of pAKT^T308, pAKT1^S473, and pAKT2^S474. Increased expression of all of the AKT isoforms was observed in IL-24^mt-expressing H1299 cells. Beta actin was detected as protein loading control. *denotes P < 0.05.

Figure 4. IL-24 mediates anti-tumor activity by suppressing AKT and AKT-mediated PRAS40 phosphorylation

H1299-IL-24^wt and H1299-IL-24^mt cells were transfected with AKT siRNA, or not transfected, followed by treatment with or without DOX (1 μg/ml). Then, cells were subjected to molecular analysis at 48 h after DOX treatment. A. Western blotting showed that IL-24^wt plus AKT siRNA produced a greater inhibitory effect on pAKT^S473 expression than IL-24^mt plus siAKT. B. Cell migration assay results correlated with Western blotting and showed that IL-24^wt plus siAKT produced inhibited cell migration more than IL-24^mt plus siAKT. C. H1299- IL-24^wt cells were either not transfected, or transfected with myr-AKT plasmid, followed by treatment with or without DOX (1 μg/ml) and subjected to molecular analysis at 48 h after treatment. Expression of myr-AKT abrogated the inhibitory effect of IL-24^wt on AKT and its downstream targets, PRAS40 and mTOR. D. IL-24^wt-mediated inhibition of cell migration was reduced when myr-AKT was expressed. E. Phosphorylation of PRAS40^T246 and AKT^S473 was significantly reduced in IL-24^wt-expressing and PRAS40^mt-expressing cells compared with controls. However, greater inhibition of pPRAS40^T246 and pAKT^S473 was observed when IL-24^wt and PRAS40^mt were co-expressed. Control cells did not receive any treatment. Beta actin was detected as protein loading control. *denotes P < 0.05. “NS” denotes Not Significant.

Figure 5. IL-24^wt modulates PRAS40 and Raptor interaction

Immunoprecipitation studies showed that Raptor protein bound to PRAS40 was markedly reduced in A. IL-24^wt-expressing cells and B. rapamycin-treated cells, compared with untreated controls. Total proteins for Raptor, PRAS40, and mTOR were detected in cell lysates to ensure that the reduced Raptor binding was not due to a reduction in total protein levels. Beta actin was detected as protein loading control.

Figure 6. IL-24^wt alters the expression of downstream targets of AKT

H1299-IL-24^wt and H1299-IL-24^mt cells treated with DOX (1 μg/ml) were subjected to molecular analysis at 48 h after treatment. Control cells did not receive any treatment. Only IL-24^wt reduced the protein expression of pGSK^S21/9, pFLNa^S2152, pPAK1^T423, and cyclin D1. Expression of pβ-catenin^S33/37/T41, however, was increased in IL-24^wt-, but not in IL-24^mt-, expressing cells. Beta actin was detected as protein loading control. *denotes P-value < 0.05.