Toxicological sciences : an official journal of the Society of Toxicology

Serine 9 and tyrosine 216 phosphorylation of GSK-3β differentially regulates autophagy in acquired cadmium resistance.

PMID 23897984


Glycogen synthase kinase-3β (GSK-3β) plays an important role in the regulation of apoptosis. To investigate its involvement in acquired cadmium (Cd) resistance, Cd-resistant cells (RH460) were established from H460 lung carcinoma cells. Cd resistance led to interruption of apoptosis and autophagy, as determined by an apoptotic sub-G1 population, procaspase-3 clevage, and LC3-II induction. Cd-induced autophagy preceded apoptosis as determined by 3-methyladenine or zVAD and time-course experiments after Cd treatment. Despite β-catenin accumulation, phospho(p)-Ser/Tyr GSK-3α/β increased in the nucleus until 12h after treatment and then p-Ser partly translocated to the cytoplasm. The GSK-3 inhibitor lithium augmented Cd-induced p-Ser GSK-3α/β, which accumulated in the nucleus and cytoplasm, and increased autophagy. SB216763 inhibited p-Ser/p-Tyr GSK-3α/β and subsequent autophagy. GSK-3β knockdown decreased Cd-induced autophagy. Cd exposure to RH460 cells overexpressed with pcDNA-GSK-3β-HA strongly phosphorylated Ser(9)/Tyr(216) residues and decreased LC3-II. Constitutively active pcDNA-GSK-3β(S9A)-HA overexpression phosphorylated Tyr(216) and decreased LC3-II, suggesting that p-Tyr inhibits autophagy. PI3K inhibitors decreased Cd-induced p-Ser GSK-3αβ and LC3-II, whereas a Ser/Thr phosphatase inhibitor, okadaic acid, hyperphosphorylated Ser residues, which accumulated in the nucleus and cytosol, and enhanced LC3-II. The general tyrosine kinase inhibitor genistein suppressed Cd-induced p-Tyr/p-Ser GSK-3α/β and LC3-II. Mouse lung tissues respond to long-term Cd exposure increased p-Tyr, downregulated LC3-II, and accumulated full-length Bax and procaspase-3. Taken together, this study shows that acquired Cd resistance is regulated by GSK-3β phosphorylation state, but not activation state, and intracellular localization of p-Ser GSK-3 regulates Cd-induced autophagy and apoptosis.