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.

Summary Aberrant Wnt/β-catenin signaling following loss of the tumor suppressor adenomatous polyposis coli (APC) is thought to initiate colon adenoma formation. Using zebrafish and human cells, we show that homozygous loss of APC causes failed intestinal cell differentiation, but that this occurs in the absence of nuclear β-catenin and increased intestinal cell proliferation. Therefore, loss of APC is insufficient for causing β-catenin nuclear localization. APC mutation-induced intestinal differentiation defects instead depend on the transcriptional corepressor CtBP1 whereas proliferation defects, and nuclear accumulation of β-catenin, require the additional activation of KRAS. These findings suggest that, following APC loss, CtBP1 contributes to adenoma initiation as a first step, while KRAS activation and β-catenin nuclear localization promotes adenoma progression to carcinomas as a second step. Consistent with this model, human FAP adenomas showed robust upregulation of CtBP1 in the absence of detectable nuclear β-catenin, whereas nuclear β-catenin was detected in carcinomas.

Cerebellar granule neurons depend on insulin-like growth factor-I (IGF-I) for their survival. However, the mechanism underlying the neuroprotective effects of IGF-I is presently unclear. Here we show that IGF-I protects granule neurons by suppressing key elements of the intrinsic (mitochondrial) death pathway. IGF-I blocked activation of the executioner caspase-3 and the intrinsic initiator caspase-9 in primary cerebellar granule neurons deprived of serum and depolarizing potassium. IGF-I inhibited cytochrome c release from mitochondria and prevented its redistribution to neuronal processes. The effects of IGF-I on cytochrome c release were not mediated by blockade of the mitochondrial permeability transition pore, because IGF-I failed to inhibit mitochondrial swelling or depolarization. In contrast, IGF-I blocked induction of the BH3-only Bcl-2 family member, Bim (Bcl-2 interacting mediator of cell death), a mediator of Bax-dependent cytochrome c release. The suppression of Bim expression by IGF-I did not involve inhibition of the c-Jun transcription factor. Instead, IGF-I prevented activation of the forkhead family member, FKHRL1, another transcriptional regulator of Bim. Finally, adenoviral-mediated expression of dominant-negative AKT activated FKHRL1 and induced expression of Bim. These data suggest that IGF-I signaling via AKT promotes survival of cerebellar granule neurons by blocking the FKHRL1-dependent transcription of Bim, a principal effector of the intrinsic death-signaling cascade.

Cerebellar granule neurons (CGNs) require depolarization for their survival in culture. When deprived of this stimulus, CGNs die via an intrinsic apoptotic cascade involving Bim induction, Bax translocation, cytochrome c release, and caspase-9 and -3 activation. Opening of the mitochondrial permeability transition pore (mPTP) is an early event during intrinsic apoptosis; however, the precise role of mPTP opening in neuronal apoptosis is presently unclear. Here, we show that mPTP opening acts as an initiating event to stimulate Bax translocation to mitochondria. A C-terminal (a9 helix) GFP-Bax point mutant (T182A) that constitutively localizes to mitochondria circumvents the requirement for mPTP opening and is entirely sufficient to induce CGN apoptosis. Collectively, these data indicate that the major role of mPTP opening in CGN apoptosis is to trigger Bax translocation to mitochondria, ultimately leading to cytochrome c release and caspase activation.