Glycogen synthase kinase-3β induces neuronal cell death via direct phosphorylation of mixed lineage kinase 3

Abstract

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase member that activates the c-Jun N-terminal kinase (JNK) pathway. Aberrant activation of MLK3 has been implicated in neurodegenerative diseases. Similarly, glycogen synthase kinase (GSK)-3β has also been shown to activate JNK and contribute to neuronal apoptosis. Here, we show a functional interaction between MLK3 and GSK-3β during nerve growth factor (NGF) withdrawal-induced cell death in PC-12 cells. The protein kinase activities of GSK-3β, MLK3, and JNK were increased upon NGF withdrawal, which paralleled increased cell death in NGF-deprived PC-12 cells. NGF withdrawal-induced cell death and MLK3 activation were blocked by a GSK-3β-selective inhibitor, kenpaullone. However, the MLK family inhibitor, CEP-11004, although preventing PC-12 cell death, failed to inhibit GSK-3β activation, indicating that induction of GSK-3β lies upstream of MLK3. In GSK-3β-deficient murine embryonic fibroblasts, ultraviolet light was unable to activate MLK3 kinase activity, a defect that was restored upon ectopic expression of GSK-3β. The activation of MLK3 by GSK-3β occurred via phosphorylation of MLK3 on two amino acid residues, Ser789 and Ser793, that are located within the C-terminal regulatory domain of MLK3. Furthermore, the cell death induced by GSK-3β was mediated by MLK3 in a manner dependent on its phosphorylation of the specific residues within the C-terminal domain by GSK-3β. Taken together, our data provide a direct link between GSK-3β and MLK3 activation in a neuronal cell death pathway and identify MLK3 as a direct downstream target of GSK-3β. Inhibition of GSK-3 is thus a potential therapeutic strategy for neurodegenerative diseases caused by trophic factor deprivation.