It should be noted that DRAM1 has wide range of tissue expression , which suggests that DRAM1 might exert broad functions through inhibiting rpS6 in different tissues. human cancer cells Hela, SW480, and HCT116 were transfected with the FLAG-DRAM1 plasmid and phosphorylated rpS6 and rpS6 were detected with Western blot analysis. Results DRAM1 induced autophagy and inhibited rpS6 phosphorylation in an mTORC1-dependent manner in HEK293T cells. DRAM1 didnt affect ML604440 the phosphorylated and total levels of p53. Furthermore, DRAM1 inhibited the activation of the PI3K-Akt pathway stimulated with growth factors or serum. DRAM1 was localized at the plasma membrane and regulate the ML604440 phosphorylation of IGF-1 receptor. DRAM1 decreased cell viability and colony numbers upon serum starvation. Additionally, DRAM1 inhibited rpS6 phosphorylation in several human cancer cells. Conclusions Here we provided evidence that DRAM1 inhibited rpS6 ML604440 phosphorylation in multiple cell types. DRAM1 inhibited the phosphorylation of Akt and the activation of Akt-rpS6 pathway stimulated with growth factors and serum. Furthermore, DRAM1 regulated the activation of IGF-1 receptor. Thus, our results identify that the class I PI3K-Akt-rpS6 pathway is regulated by DRAM1 and may provide new insight into the potential role of DRAM1 in human cancers. 0.01?vs the indicated groups DRAM1 inhibits rpS6 phosphorylation in human cancer cells The previous study identified DRAM1 as a potential tumor-suppressor in human cancer . To investigate whether DRAM1 could inhibit the phosphorylation of rpS6 in human cancer cell lines, we overexpressed DRAM1 in human cancer cells. Using HEK293T cells as a positive control (Fig.?7a), we found that DRAM1 inhibited rpS6 phosphorylation in human colon cancer cells, SW480 (Fig.?7b) and HCT116 (Fig.?7c), with phosphorylation at Ser235/236 more significantly affected by DRAM1 than?the site at?Ser240/244 (Fig.?7d and e). These data demonstrated that DRAM1 inhibited rpS6 phosphorylation in human colon cancer cells. Open in a separate window Fig. 7 DRAM1 ML604440 inhibits rpS6 phosphorylation in human cancer cells. a, b and c HEK293T, SW480 and HCT116 cells were transfected with FLAG empty vector or FLAG-DRAM1 plasmids for 24?h. The protein levels of p-rpS6 (S235/236, S240/244), rpS6, FLAG and -actin were detected with immunoblotting. d and e Quantitative analysis of the optical densities of p-rpS6 (S235/236, S240/244) and rpS6. Data represent mean??SEM of combined data from three independent experiments. * em p /em ? ?0.05 and ** em p /em ? ?0.01 vs the indicated groups Discussion DRAM1 has been identified as the direct p53 target gene more than a decade ago [20, 25]. Initial study showed that DRAM1 induced autophagy and was necessary for p53-induced apoptosis . However, the signalling pathways involved in DRAM1-induced autophagy and apoptosis are still not clear. In this study, we demonstrated that DRAM1 inhibited the phosphorylation of rpS6 in multiple cell lines. Furthermore, DRAM1 inhibited the activation of the class I PI3K-Akt pathway ML604440 stimulated with growth factors and serum. Our results suggest that the class I PI3K-Akt-mTORC1-rpS6 pathway plays a key role in DRAM1-induced autophagy and apoptosis. DNAPK Early study found that DRAM1-inducible cells accumulated double-membraned autophagic vesicle under electron microscopy and induced GFP-LC3 from diffuse staining to small puncta structure . These data demonstrated that DRAM1 induced autophagy. We transfected HEK293T cells with DRAM1 plasmid and could also observed the turnover of LC3-II from LC3-I as well as the change from the diffuse pattern of GFP-LC3 to puncture structure in the presence of DRAM1, indicating that DRAM1 induced autophagy. We also observed some interesting results upon DRAM1-induced autophagy. For example, in Fig.?1c, Bafilomycin A1 induced accumulation of levels of p62 and LC3-II was blunted by overexpression of DRAM1. Our previous study.