DRAM1 通过抑制磷酸肌醇 3-激酶-Akt-mTOR-核糖体蛋白 S6 通路来调节自噬和细胞增殖。
DRAM1 regulates autophagy and cell proliferation via inhibition of the phosphoinositide 3-kinase-Akt-mTOR-ribosomal protein S6 pathway.
机构信息
Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Science, Soochow University, 199 Ren Ai Road, Suzhou, 215123, China.
Department of Pharmacology and Neurology, Emory University School of Medicine, Atlanta, GA, USA.
出版信息
Cell Commun Signal. 2019 Mar 22;17(1):28. doi: 10.1186/s12964-019-0341-7.
BACKGROUND
Macroautophagy (hereafter autophagy) is a tightly regulated process that delivers cellular components to lysosomes for degradation. Damage-regulated autophagy modulator 1 (DRAM1) induces autophagy and is necessary for p53-mediated apoptosis. However, the signalling pathways regulated by DRAM1 are not fully understood.
METHODS
HEK293T cells were transfected with FLAG-DRAM1 plasmid. Autophagic proteins (LC3 and p62), phosphorylated p53 and the phosphorylated proteins of the class I PI3K-Akt-mTOR-ribosomal protein S6 (rpS6) signalling pathway were detected with Western blot analysis. Cellular distribution of DRAM1 was determined with immunostaining. DRAM1 was knocked down in HEK293T cells using siRNA oligos which is confirmed by quantitative RT-PCR. Cells were serum starved for 18 h after overexpression or knockdown of DRAM1 to decrease the rpS6 activity to the basal level, and then the cells were stimulated with insulin growth factor, epidermal growth factor or serum. rpS6 phosphorylation and rpS6 were detected with Western blotting. Similarly, after overexpression or knockdown of DRAM1, phosphorylation of IGF-1Rβ and IGF-1R were examined with Western blotting. Cell viability was determined with CCK-8 assay and colony formation assay. Finally, 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 didn't affect 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 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 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.
背景
巨自噬（以下简称自噬）是一个严格调控的过程，它将细胞成分递送至溶酶体进行降解。损伤调节自噬调节剂 1（DRAM1）诱导自噬，并且是 p53 介导的细胞凋亡所必需的。然而，DRAM1 调节的信号通路尚未完全阐明。
方法
HEK293T 细胞用 FLAG-DRAM1 质粒转染。用 Western blot 分析检测自噬蛋白（LC3 和 p62）、磷酸化 p53 和 I 类 PI3K-Akt-mTOR-核糖体蛋白 S6（rpS6）信号通路的磷酸化蛋白。用免疫染色法确定 DRAM1 的细胞分布。用 siRNA 寡核苷酸敲低 HEK293T 细胞中的 DRAM1，并通过定量 RT-PCR 进行验证。转染 DRAM1 过表达或敲低质粒后，细胞饥饿培养 18 小时以降低 rpS6 活性至基础水平，然后用胰岛素生长因子、表皮生长因子或血清刺激细胞。用 Western blot 检测 rpS6 磷酸化和 rpS6。同样，用 Western blot 检测 DRAM1 过表达或敲低后 IGF-1Rβ 和 IGF-1R 的磷酸化。用 CCK-8 测定和集落形成测定检测细胞活力。最后，用 FLAG-DRAM1 质粒转染人癌细胞 Hela、SW480 和 HCT116，用 Western blot 分析检测磷酸化 rpS6 和 rpS6。
结果
DRAM1 在 HEK293T 细胞中以 mTORC1 依赖的方式诱导自噬并抑制 rpS6 磷酸化。DRAM1 不影响磷酸化和总 p53 水平。此外，DRAM1 抑制生长因子或血清刺激的 PI3K-Akt 通路的激活。DRAM1 位于质膜上，调节 IGF-1 受体的磷酸化。血清饥饿时，DRAM1 降低细胞活力和集落数。此外，DRAM1 抑制多种人癌细胞中 rpS6 的磷酸化。
结论
本研究提供了证据表明，DRAM1 在多种细胞类型中抑制 rpS6 磷酸化。DRAM1 抑制生长因子和血清刺激的 Akt 和 Akt-rpS6 通路的磷酸化。此外，DRAM1 调节 IGF-1 受体的激活。因此，我们的结果表明，I 类 PI3K-Akt-rpS6 通路受 DRAM1 调节，并可为 DRAM1 在人类癌症中的潜在作用提供新的见解。
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