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The Journal of Neuroscience, November 3, 2004, 24(44):9993-10002; doi:10.1523/JNEUROSCI.2057-04.2004
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Cellular/Molecular
Glycogen Synthase Kinase-3 Phosphorylates Bax and Promotes Its Mitochondrial Localization during Neuronal Apoptosis
Daniel A. Linseman, *
Brent D. Butts, *
Thomas A. Precht,
Reid A. Phelps,
Shoshona S. Le,
Tracey A. Laessig,
Ron J. Bouchard,
Maria L. Florez-McClure, and
Kim A. Heidenreich
Department of Pharmacology, University of Colorado Health Sciences Center, and Denver Veterans Affairs Medical Center, Denver, Colorado 80262
Glycogen synthase kinase-3 (GSK-3) is a critical activator of neuronal apoptosis induced by a diverse array of neurotoxic insults. However, the downstream substrates of GSK-3 that ultimately induce neuronal death are unknown. Here, we show that GSK-3 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-3 suppressed both the mitochondrial translocation of an expressed green fluorescent protein (GFP)-Bax fusion protein and the conformational activation of endogenous Bax. GSK-3 directly phosphorylated Bax on Ser163, a residue found within a species-conserved, putative GSK-3 phosphorylation motif. Coexpression of GFP-Bax with a constitutively active mutant of GSK-3, GSK-3(Ser9Ala), enhanced the in vivo phosphorylation of wild-type Bax, but not a Ser163Ala mutant of Bax, in transfected human embryonic kidney 293 (HEK293) cells. Moreover, cotransfection with constitutively active GSK-3 promoted the localization of Bax to mitochondria and induced apoptosis in both transfected HEK293 cells and cerebellar granule neurons. In contrast, neither a Ser163Ala point mutant of Bax nor a naturally occurring splice variant that lacks 13 amino acids encompassing Ser163 (Bax ) were driven to mitochondria in HEK293 cells coexpressing constitutively active GSK-3. In a similar manner, either mutation or deletion of the identified GSK-3 phosphorylation motif prevented the localization of Bax to mitochondria in cerebellar granule neurons undergoing apoptosis. Our results indicate that GSK-3 exerts some of its pro-apoptotic effects in neurons by regulating the mitochondrial localization of Bax, a key component of the intrinsic apoptotic cascade.
Key words: glycogen synthase kinase; cerebellar granule neuron; apoptosis; mitochondria; Bax; phosphorylation
Received Jan 21, 2004;
revised September 9, 2004;
accepted September 27, 2004.
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