Glycogen Synthase Kinase-3β Phosphorylates Bax and Promotes Its Mitochondrial Localization during Neuronal Apoptosis

Abstract

Glycogen synthase kinase-3β (GSK-3β) is a critical activator of neuronal apoptosis induced by a diverse array of neurotoxic insults. However, the downstream substrates of GSK-3β that ultimately induce neuronal death are unknown. Here, we show that GSK-3β phosphorylates and regulates the activity of Bax, a pro-apoptotic Bcl-2 family member that stimulates the intrinsic (mitochondrial) death pathway by eliciting cytochrome c release from mitochondria. In cerebellar granule neurons undergoing apoptosis, inhibition of GSK-3β suppressed both the mitochondrial translocation of an expressed green fluorescent protein (GFP)-Bax α fusion protein and the conformational activation of endogenous Bax. GSK-3β directly phosphorylated Bax α on Ser163, a residue found within a species-conserved, putative GSK-3β phosphorylation motif. Coexpression of GFP-Bax α with a constitutively active mutant of GSK-3β, GSK-3β(Ser9Ala), enhanced the in vivo phosphorylation of wild-type Bax α , but not a Ser163Ala mutant of Bax α , in transfected human embryonic kidney 293 (HEK293) cells. Moreover, cotransfection with constitutively active GSK-3β promoted the localization of Bax α to mitochondria and induced apoptosis in both transfected HEK293 cells and cerebellar granule neurons. In contrast, neither a Ser163Ala point mutant of Bax α nor a naturally occurring splice variant that lacks 13 amino acids encompassing Ser163 (Bax σ ) were driven to mitochondria in HEK293 cells coexpressing constitutively active GSK-3β. In a similar manner, either mutation or deletion of the identified GSK-3β phosphorylation motif prevented the localization of Bax to mitochondria in cerebellar granule neurons undergoing apoptosis. Our results indicate that GSK-3β exerts some of its pro-apoptotic effects in neurons by regulating the mitochondrial localization of Bax, a key component of the intrinsic apoptotic cascade.