Glycogen synthase kinase-3beta phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis.
Glycogen synthase kinase-3beta (GSK-3beta) is a critical activator of neuronal apoptosis induced by a diverse array of neurotoxic insults. However, the downstream substrates of GSK-3beta that ultimately induce neuronal death are unknown. Here, we show that GSK-3beta 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-3beta suppressed both the mitochondrial translocation of an expressed green fluorescent protein (GFP)-Bax(alpha) fusion protein and the conformational activation of endogenous Bax. GSK-3beta directly phosphorylated Bax(alpha) on Ser163, a residue found within a species-conserved, putative GSK-3beta phosphorylation motif. Coexpression of GFP-Bax(alpha) with a constitutively active mutant of GSK-3beta, GSK-3beta(Ser9Ala), enhanced the in vivo phosphorylation of wild-type Bax(alpha), but not a Ser163Ala mutant of Bax(alpha), in transfected human embryonic kidney 293 (HEK293) cells. Moreover, cotransfection with constitutively active GSK-3beta promoted the localization of Bax(alpha) to mitochondria and induced apoptosis in both transfected HEK293 cells and cerebellar granule neurons. In contrast, neither a Ser163Ala point mutant of Bax(alpha) nor a naturally occurring splice variant that lacks 13 amino acids encompassing Ser163 (Bax(sigma)) were driven to mitochondria in HEK293 cells coexpressing constitutively active GSK-3beta. In a similar manner, either mutation or deletion of the identified GSK-3beta phosphorylation motif prevented the localization of Bax to mitochondria in cerebellar granule neurons undergoing apoptosis. Our results indicate that GSK-3beta exerts some of its pro-apoptotic effects in neurons by regulating the mitochondrial localization of Bax, a key component of the intrinsic apoptotic cascade.
Linseman
Daniel A
DA
Department of Pharmacology, University of Colorado Health Sciences Center, and Denver Veterans Affairs Medical Center, Denver, Colorado 80262, USA.
Butts
Brent D
BD
Precht
Thomas A
TA
Phelps
Reid A
RA
Le
Shoshona S
SS
Laessig
Tracey A
TA
Bouchard
Ron J
RJ
Florez-McClure
Maria L
ML
Heidenreich
Kim A
KA
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D017124:Conserved Sequence; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D010766:Phosphorylation; D011487:Protein Conformation; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C494828:Bax protein, rat; D009419:Nerve Tissue Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein; D049452:Green Fluorescent Proteins; D012694:Serine; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3