Glycogen synthase kinase-3β phosphorylates bax and promotes its mitochondrial localization during neuronal apoptosis
Glycogen synthase kinase-3β phosphorylates bax and promotes its mitochondrial localization during neuronal apoptosis
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DOI：
10.1523/jneurosci.2057-04.2004
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发表时间：
2004-11-03
影响因子：
5.3
通讯作者：
Heidenreich, KA
中科院分区：
文献类型：
--
作者：
Linseman, DA;Butts, BD;Heidenreich, KA
糖原合成酶激酶-3 β（GSK-3 β）是一种重要的神经细胞凋亡激活因子。然而，最终诱导神经元死亡的GSK-3 β的下游底物是未知的。在这里，我们发现GSK-3 β磷酸化和调节Bax的活性，Bax是一种促凋亡Bcl-2家族成员，通过引发线粒体释放细胞色素c来刺激内在（线粒体）死亡途径。在经历凋亡的小脑颗粒神经元中，GSK-3 β的抑制抑制了表达的绿色荧光蛋白（GFP）-Bax（α）融合蛋白的线粒体易位和内源性Bax的构象激活。GSK-3 β直接磷酸化Bax（α）的Ser 163，一个在物种保守的，推定的GSK-3 β磷酸化基序中发现的残基。在转染的人胚肾293（HEK 293）细胞中，GFP-Bax（alpha）与GSK-3 β的组成型活性突变体GSK-3 β（Ser 9Ala）的共表达增强了野生型Bax（alpha）的体内磷酸化，但没有增强Bax（alpha）的Ser 163 Ala突变体的体内磷酸化。此外，与组成型活性GSK-3 β共转染促进Bax（α）定位于线粒体，并诱导转染的HEK 293细胞和小脑颗粒神经元的凋亡。相比之下，Bax（alpha）的Ser 163 Ala点突变体和天然存在的缺少13个氨基酸的包含Ser 163的剪接变体（Bax（sigma））在共表达组成型活性GSK-3 β的HEK 293细胞中均未被驱动至线粒体。以类似的方式，无论是突变或删除所确定的GSK-3 β磷酸化基序阻止Bax的本地化的线粒体在小脑颗粒神经元进行凋亡。我们的研究结果表明，GSK-3 β通过调节Bax的线粒体定位在神经元中发挥其促凋亡作用，Bax是内在凋亡级联反应的关键组分。
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.