GSK-3β controls autophagy by modulating LKB1-AMPK pathway in prostate cancer cells.
GSK-3β controls autophagy by modulating LKB1-AMPK pathway in prostate cancer cells.
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DOI：
10.1002/pros.23106
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发表时间：
2016-02
期刊：
影响因子：
--
通讯作者：
Li B
中科院分区：
文献类型：
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作者：
Sun A;Li C;Chen R;Huang Y;Chen Q;Cui X;Liu H;Thrasher JB;Li B
糖原合成酶激酶3β（Glycogen synthase kinase 3β，GSK-3β）是一种参与多种细胞过程的多功能蛋白激酶，其活性在血清剥夺后升高。我们已经表明，GSK-3β活性的抑制在前列腺癌细胞中引发了深刻的自噬反应和随后的血清剥夺后的坏死细胞死亡。在这项研究中，我们剖析了GSK-3β抑制触发自噬的机制。前列腺癌PC-3和DU 145细胞用于研究。使用多种GSK-3β特异性抑制剂，包括小分子化学品TDZD 8、Tideglusib、TWS 119和肽L 803-mts。蛋白质印迹法结合磷酸化特异性抗体检测信号通路的激活。ATP水平用ATPLite试剂盒和HPLC法测定。通过在Western印迹分析中评价微管相关蛋白1A/1B轻链3B（LC 3B）加工和p62蛋白稳定性来确定自噬反应。免疫荧光显微镜检测LKB 1易位。GSK-3β活性的抑制导致细胞ATP产生的显著下降，导致AMP/ATP比率的显著增加，这是前列腺癌PC-3细胞中AMP活化蛋白激酶（AMPK）活化的强烈触发因素。与LC-3B生物合成增加和p62蛋白减少（自噬诱导的经典标志）平行，AMPK在GSK-3β活性抑制后被激活。进一步的分析表明，肝激酶B1（LKB 1）而不是钙/钙调素依赖性蛋白激酶激酶β（CaMKKβ）参与AMPK激活和GSK-3β抑制引发的自噬诱导。同时，GSK-3β抑制促进LKB 1从核到胞质的移位，并增强LKB 1与其调节配偶体Mouse protein-25（MO 25）和STE 20相关衔接子（STRAD）的相互作用。结论：GSK-3β可能通过调节LKB 1-AMPK通路的激活，在控制血清剥夺后的自噬诱导中发挥重要作用。
Glycogen synthase kinase 3β (GSK3B, GSK-3β) is a multi-functional protein kinase involved in various cellular processes and its activity elevates after serum deprivation. We have shown that inhibition of GSK-3β activity triggered a profound autophagic response and subsequent necrotic cell death after serum deprivation in prostate cancer cells. In this study, we dissected the mechanisms involved in GSK-3β inhibition-triggered autophagy. Prostate cancer PC-3 and DU145 cells were used in the study. Multiple GSK-3β specific inhibitors were used including small chemicals TDZD8, Tideglusib, TWS119, and peptide L803-mts. Western blot assay coupled with phospho-specific antibodies were used in detecting signal pathway activation. ATP levels were assessed with ATPLite kit and HPLC methods. Autophagy response was determined by evaluating Microtubule-associated proteins 1A/1B light chain 3B (LC3B) processing and p62 protein stability in Western blot assays. Immunofluorescent microscopy was used to detect LKB1 translocation. Inhibition of GSK-3β activity resulted in a significant decline of cellular ATP production, leading to a significant increase of AMP/ATP ratio, a strong trigger of AMP-activated protein kinase (AMPK) activation in prostate cancer PC-3 cells. In parallel with increased LC-3B biosynthesis and p62 protein reduction, the classical sign of autophagy induction, AMPK was activated after inhibition of GSK-3β activity. Further analysis revealed that Liver kinase B1 (LKB1) but not Calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) is involved in AMPK activation and autophagy induction triggered by GSK-3β inhibition. Meanwhile, GSK-3β inhibition promoted LKB1 translocation from nuclear to cytoplasmic compartment and enhanced LKB1 interaction with its regulatory partners Mouse protein-25 (MO25) and STE20-related adaptor (STRAD). In conclusion, our data suggest that GSK-3β plays an important role in controlling autophagy induction by modulating the activation of LKB1-AMPK pathway after serum deprivation.