Department of Life Science, College of Natural Science, Hanyang University, Seoul, Republic of Korea.
Exp Cell Res. 2013 Feb 1;319(3):122-33. doi: 10.1016/j.yexcr.2012.11.014. Epub 2012 Nov 30.
Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.
自噬，或自噬作用，是一种涉及细胞自身溶酶体装置的选择性细胞内降解过程。自噬是细胞发育、稳态、修复和应激抵抗的重要组成部分，可能导致细胞死亡或存活。细胞的靶向区域被隔离在膜结构中，即自噬体，以调节分解代谢过程。自噬体形成和自噬体成熟的关键因素是由紫外线照射抗性相关基因 (UVRAG) 编码的蛋白质。相反，丝氨酸/苏氨酸特异性蛋白激酶 B (PKB，也称为 Akt)，它调节各种癌症的存活，通过 mTOR 激活抑制自噬。我们发现 Akt1 也可以通过下调 293T 瞬时转染系统和稳定表达 Akt1 的乳腺癌细胞中的 UVRAG 来直接抑制自噬。在假定的 Akt1 磷酸化位点发生突变的 UVRAG 仍然被 Akt1 抑制，显性失活 Akt1 也抑制 UVRAG 表达，这表明 Akt1 通过非激酶活性依赖的机制下调 UVRAG。我们表明，在 MDA-MB-231 乳腺癌细胞中过表达 Akt1 会下调 UVRAG 转录。过表达 Akt1 的细胞比对照细胞对紫外线诱导的自噬更具抵抗力，并表现出相关的细胞活力降低。过表达 Akt1 的细胞中自噬体标志物蛋白 LC3B-II 和 mRFP-GFP-LC3 的水平降低。通过 siRNA 抑制 Akt1 或重新引入 UVRAG 基因可挽救紫外线照射后的 LC3B-II 水平。总的来说，这些数据表明 Akt1 可能通过降低 UVRAG 表达来抑制自噬，从而使癌细胞对紫外线照射更敏感。
