Department of Medical Biology and Genetics, Faculty of Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland.
Department of Molecular Biology, Faculty of Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland.
Cells. 2021 Apr 17;10(4):929. doi: 10.3390/cells10040929.
Autophagy is a specific macromolecule and organelle degradation process. The target macromolecule or organelle is first enclosed in an autophagosome, and then delivered along acetylated microtubules to the lysosome. Autophagy is triggered by stress and largely contributes to cell survival. We have previously shown that S6K1 kinase is essential for autophagic flux under stress conditions. Here, we aimed to elucidate the underlying mechanism of S6K1 involvement in autophagy. We stimulated autophagy in S6K1/2 double-knockout mouse embryonic fibroblasts by exposing them to different stress conditions. Transient gene overexpression or silencing, immunoblotting, immunofluorescence, flow cytometry, and ratiometric fluorescence analyses revealed that the perturbation of autophagic flux in S6K1-deficient cells did not stem from impaired lysosomal function. Instead, the absence of S6K1 abolished stress-induced tubulin acetylation and disrupted the acetylated microtubule network, in turn impairing the autophagosome-lysosome fusion. S6K1 overexpression restored tubulin acetylation and autophagic flux in stressed S6K1/2-deficient cells. Similar effect of S6K1 status was observed in prostate cancer cells. Furthermore, overexpression of an acetylation-mimicking, but not acetylation-resistant, tubulin variant effectively restored autophagic flux in stressed S6K1/2-deficient cells. Collectively, S6K1 controls tubulin acetylation, hence contributing to the autophagic flux induced by different stress conditions and in different cells.
自噬是一种特定的大分子和细胞器降解过程。目标大分子或细胞器首先被包裹在自噬体中，然后沿着乙酰化微管被递送到溶酶体。自噬是由应激触发的，在很大程度上有助于细胞存活。我们之前已经表明，S6K1 激酶在应激条件下对自噬通量是必不可少的。在这里，我们旨在阐明 S6K1 参与自噬的潜在机制。我们通过使 S6K1/2 双敲除鼠胚胎成纤维细胞暴露于不同的应激条件来刺激自噬。瞬时基因过表达或沉默、免疫印迹、免疫荧光、流式细胞术和比率荧光分析表明，S6K1 缺陷细胞中自噬通量的扰动不是源于溶酶体功能受损。相反，S6K1 的缺失消除了应激诱导的微管蛋白乙酰化，并破坏了乙酰化微管网络，从而损害了自噬体-溶酶体融合。S6K1 的过表达恢复了应激 S6K1/2 缺陷细胞中的微管蛋白乙酰化和自噬通量。S6K1 状态的类似效应也在前列腺癌细胞中观察到。此外，过表达模拟乙酰化但不模拟乙酰化抗性的微管蛋白变体可有效恢复应激 S6K1/2 缺陷细胞中的自噬通量。总之，S6K1 控制着微管蛋白的乙酰化，从而有助于不同应激条件下和不同细胞中的自噬通量。
