Sun Aijing, Li Changlin, Chen Ruibao, Huang Yiling, Chen Qi, Cui Xiangjun, Liu Huafeng, Thrasher J Brantley, Li Benyi
Department of Pathology, Shaoxing University School of Medicine, Shaoxing, China.
Department of Urology, University of Kansas Medical Center, Kansas City, Kansas.
Prostate. 2016 Feb;76(2):172-83. doi: 10.1002/pros.23106. Epub 2015 Oct 6.
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.
糖原合酶激酶3β（GSK3B，GSK - 3β）是一种参与多种细胞过程的多功能蛋白激酶，血清剥夺后其活性升高。我们已经表明，抑制GSK - 3β活性会在前列腺癌细胞血清剥夺后引发深刻的自噬反应和随后的坏死性细胞死亡。在本研究中，我们剖析了GSK - 3β抑制引发自噬的机制。
本研究使用前列腺癌PC - 3和DU145细胞。使用了多种GSK - 3β特异性抑制剂，包括小分子化合物TDZD8、替德格鲁西、TWS119和肽L803 - mts。使用与磷酸化特异性抗体结合的蛋白质印迹法检测信号通路激活。使用ATPLite试剂盒和高效液相色谱法评估ATP水平。通过在蛋白质印迹分析中评估微管相关蛋白1A / 1B轻链3B（LC3B）加工和p62蛋白稳定性来确定自噬反应。使用免疫荧光显微镜检测LKB1易位。
抑制GSK - 3β活性导致细胞ATP产生显著下降，导致AMP / ATP比值显著增加，这是前列腺癌PC - 3细胞中AMP激活蛋白激酶（AMPK）激活的强烈触发因素。与自噬诱导的经典标志LC - 3B生物合成增加和p62蛋白减少同时发生，GSK - 3β活性抑制后AMPK被激活。进一步分析表明，肝激酶B1（LKB1）而非钙/钙调蛋白依赖性蛋白激酶激酶β（CaMKKβ）参与了GSK - 3β抑制引发的AMPK激活和自噬诱导。同时，GSK - 3β抑制促进LKB1从核区室向细胞质区室易位，并增强LKB1与其调节伙伴小鼠蛋白 - 25（MO25）和STE20相关衔接蛋白（STRAD）的相互作用。
总之，我们的数据表明，GSK - 3β在血清剥夺后通过调节LKB1 - AMPK途径的激活在控制自噬诱导中起重要作用。
