Gsk-3β Controls Autophagy by Modulating LKB1-AMPK Pathway in Prostate Cancer Cells

BACKGROUND. Glycogen synthase kinase 3 beta (GSK3 beta, GSK-3 beta) 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-3 beta 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-3 beta inhibition-triggered autophagy. METHODS. Prostate cancer PC-3 and DU145 cells were used in the study. Multiple GSK-3 beta 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. RESULTS. Inhibition of GSK-3 beta 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-3 beta activity. Further analysis revealed that Liver kinase B1 (LKB1) but not Calcium/calmodulin-dependent protein kinase kinase beta (CaMKK beta) is involved in AMPK activation and autophagy induction triggered by GSK-3 beta inhibition. Meanwhile, GSK-3 beta 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). CONCLUSIONS. In conclusion, our data suggest that GSK-3b plays an important role in controlling autophagy induction by modulating the activation of LKB1-AMPK pathway after serum deprivation. (C) 2015 Wiley Periodicals, Inc.