25-hydroxycholesterol induces mitochondria-dependent apoptosis via activation of glycogen synthase kinase-3beta in PC12 cells

25-hydroxycholesterol (25-OH-chol) induces apoptosis in many cell types. The present study investigated the possible involvement of mitochondria-dependent apoptotic signalling molecules in the death of PC12 cells treated with 25-OH-chol. 25-OH-chol increased the production of reactive oxygen species and opened mitochondrial permeability transition pore, resulting in release of cytochrome c and subsequent activation of caspase-9 and -3. 25-OH-chol induced the activation of c-Jun N-terminal kinase (JNK) and glycogen synthase kinase-3beta (GSK-3beta). The JNK inhibitor SP600125 attenuated the activation of caspase-9 and -3 and reduced 25-OH-chol-induced cell death. GSK inhibitors SB415286 and SB216763 significantly down-regulated JNK activity and attenuated the cytotoxicity of 25-hydroxycholesterol. However, SP600125 did not alter the activity of GSK-3beta. The results indicate that 25-OH-chol induces cell death via activation of GSK-3beta and subsequent up-regulation of JNK. Pharmacological intervention of GSK-3beta-JNK-caspase signalling pathway may be useful for the reduction of cytotoxicity of oxysterols.     

Regulation of glycogen synthase activation in isolated hepatocytes

Molecular and Cellular Biochemistry - Glycogen synthase, the regulatory enzyme of glycogen synthesis undergoes multisite phosphorylation leading to its inactivation. The kinases responsible for...     

Nitric oxide induces tau hyperphosphorylation via glycogen synthase kinase-3beta activation

Nitric oxide is associated with neurofibrillary tangle, which is composed mainly of hyperphosphorylated tau in the brain of Alzheimer's disease (AD). However, the role of nitric oxide in tau hyperphosphorylation is unclear. Here we show that nitric oxide produced by sodium nitroprusside (SNP), a recognized donor of nitric oxide, induces tau hyperphosphorylation at Ser396/404 and Ser262 in HEK293/tau441 cells with a simultaneous activation of glycogen synthase kinase-3beta (GSK-3beta). Pretreatment of the cells with 10 mM lithium chloride (LiCl), an inhibitor of GSK-3, 1 h before SNP administration inhibits GSK-3beta activation and prevents tau from hyperphosphorylation. This is the first direct evidence demonstrating that nitric oxide induces AD-like tau hyperphosphorylation in vitro, and GSK-3beta activation is partially responsible for the nitric oxide-induced tau hyperphosphorylation. It is suggested that nitric oxide may be an upstream element of tau abnormal hyperphosphorylation in AD.     

多功能的蛋白:糖原合成酶激酶-3

糖原合成酶激酶-3(GSK-3)是一个多功能的丝氨酸／苏氨酸类激酶,在真核生物中普遍存在。在哺乳动物中包括两个亚型,即GSK-3a和GSK-3β。GSK-3至少在三条细胞通路上有作用：Wnt/wingless,P13-kinase以及Hedgehog信号通路,该酶的作用主要包括调节糖原的合成代谢,参与细胞的分化与增殖等。研究发现,GSK-3在某些疾病,如阿尔茨海默病和非胰岛素依赖型糖尿病(NIDDM)中,其活性会异常升高。现已发现了几种针对该酶的抑制剂,如aloisine,paullones和马来酰胺类化合物等。这些抑制剂的确在分子水平特异性地抑制GSK-3的活性,而对其他激酶几乎没有作用。关于这些抑制剂的研究工作也已经在细胞水平和动物模型上开展起来,为开发以GSK-3为靶点的新的治疗药物创造了良好的基础。     

Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation

Stress of the endoplasmic reticulum (ER), which is associated with many neurodegenerative conditions, can lead to the elimination of affected cells by apoptosis through only partially understood mechanisms. Thapsigargin, which causes ER stress by inhibiting the ER Ca(2+)-ATPase, was found to not only activate the apoptosis effector caspase-3 but also to cause a large and prolonged increase in the activity of glycogen synthase kinase-3beta (GSK3beta). Activation of GSK3beta was obligatory for thapsigargin-induced activation of caspase-3, because inhibition of GSK3beta by expression of dominant-negative GSK3beta or by the GSK3beta inhibitor lithium blocked caspase-3 activation. Thapsigargin treatment activated GSK3beta by inducing dephosphorylation of phospho-Ser-9 of GSK3beta, a phosphorylation that normally maintains GSK3beta inactivated. Caspase-3 activation induced by thapsigargin was blocked by increasing the phosphorylation of Ser-9-GSK3beta with insulin-like growth factor-1 or with the phosphatase inhibitors okadaic acid and calyculin A, but the calcineurin inhibitors FK506 and cyclosporin A were ineffective. Insulin-like growth factor-1, okadaic acid, calyculin A, and lithium also protected cells from two other inducers of ER stress, tunicamycin and brefeldin A. Thus, ER stress activates GSK3beta through dephosphorylation of phospho-Ser-9, a prerequisite for caspase-3 activation, and this process is amenable to pharmacological intervention.     

ERK1/2 antagonizes glycogen synthase kinase-3beta-induced apoptosis in cortical neurons

Inhibition of glycogen synthase kinase-3beta (GSK3beta) is one of the mechanisms by which phosphatidylinositol 3-kinase (PI3K) activation protects neurons from apoptosis. Here, we report that inhibition of ERK1/2 increased the basal activity of GSK3beta in cortical neurons and that both ERK1/2 and PI3K were required for brain-derived neurotrophic factor (BDNF) suppression of GSK3beta activity. Moreover, cortical neuron apoptosis induced by expression of recombinant GSK3beta was inhibited by coexpression of constitutively active MKK1 or PI3K. Activation of both endogenous ERK1/2 and PI3K signaling pathways was required for BDNF to block apoptosis induced by expression of recombinant GSK3beta. Furthermore, cortical neuron apoptosis induced by LY294002-mediated activation of endogenous GSK3beta was blocked by expression of constitutively active MKK1 or by BDNF via stimulation of the endogenous ERK1/2 pathway. Although both PI3K and ERK1/2 inhibited GSK3beta activity, neither had an effect on GSK3beta phosphorylation at Tyr-216. Interestingly, PI3K (but not ERK1/2) induced the inhibitory phosphorylation of GSK3beta at Ser-9. Significantly, coexpression of constitutively active MKK1 (but not PI3K) still suppressed neuronal apoptosis induced by expression of the GSK3beta(S9A) mutant. These data suggest that activation of the ERK1/2 signaling pathway protects neurons from GSK3beta-induced apoptosis and that inhibition of GSK3beta may be a common target by which ERK1/2 and PI3K protect neurons from apoptosis. Furthermore, ERK1/2 inhibits GSK3beta activity via a novel mechanism that is independent of Ser-9 phosphorylation and likely does not involve Tyr-216 phosphorylation.     

The glamour and gloom of glycogen synthase kinase-3

Glycogen synthase kinase-3 (GSK3) is now recognized as a key component of a surprisingly large number of cellular processes and diseases. Several mechanisms play a part in controlling the actions of GSK3, including phosphorylation, protein complex formation, and subcellular distribution. These are used to control and direct the far-reaching influences of GSK3 on cellular structure, growth, motility and apoptosis. Dysregulation of GSK3 is linked to several prevalent pathological conditions, such as diabetes and/or insulin resistance, and Alzheimer's disease. Therefore, much effort is currently directed towards understanding the functions and control of GSK3, and identifying methods capable of diminishing the deleterious impact of GSK3 in pathological conditions.     

Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II (glycogen synthase kinase-5)

Glycogen synthase kinase-3 was isolated from rabbit skeletal muscle by an improved procedure. The purification was estimated to be 67000-fold and 0.2 mg of enzyme was isolated from 5000 g muscle, corresponding to an overall yield of 7%. The preparation was homogeneous by ultracentrifugal and electrophoretic criteria. The enzyme had a relative molecular mass of 47 kDa by sedimentation equilibrium centrifugation and 51 kDa by SDS-polyacrylamide gel electrophoresis. These values demonstrate that glycogen synthase kinase-3 is monomeric. The Stokes radius of 37 nm suggests the molecule to be asymmetric. The activating factor of the Mg-ATP dependent form of protein phosphatase-1 coeluted with glycogen synthase kinase-3 activity at the final step, establishing that these two activities reside in the same protein. Glycogen synthase kinase-3 phosphorylates glycogen synthase at sites-3, while casein kinase-II phosphorylates site-5, just C-terminal to sites-3 (Picton, C., Aitken, A., Bilham, T. and Cohen, P. (1982) Eur. J. Biochem. 124, 37-45). The basis for the substrate specificities of these protein kinases was investigated using chymotryptic peptides that contain the sites phosphorylated by each enzyme. These studies showed that efficient phosphorylation of sites-3, required the presence of phosphate in site-5 and a region of polypeptide more than 20 residues C-terminal to site-5. In contrast, efficient phosphorylation by casein kinase-II does not require this C-terminal region, and the results are consistent with the view that the enzyme recognises acidic residues immediately C-terminal to site-5.     

Phospholipase C-independent activation of glycogen synthase kinase-3beta and C-terminal Src kinase by Galphaq

It is generally thought that activation of phospholipase Cbeta (PLCbeta) by Galphaq accounts for most of the effects of Gq-coupled receptors. Here we describe a novel effect of Galphaq that is independent of the PLCbeta pathway. Expression of the constitutively active Galphaq mutant Galphaq(Q209L) promoted an increase in glycogen synthase kinase-3beta (GSK-3beta) activity that was associated with increased phosphorylation of Tyr216 on GSK-3beta. Galphaq(Q209L)-AA, a mutant that cannot activate PLCbeta, also induced GSK-3beta activation and phosphorylation of Tyr216. We speculate that the protein-tyrosine kinase Csk (C-terminal Src kinase), which is also activated by Galphaq(Q209L) and Galphaq(Q209L)-AA, acts upstream of GSK-3beta. Expression of Csk accentuated the activation of GSK-3beta by Galphaq(Q209L), whereas catalytically inactive Csk blocked GSK-3beta activation by Galphaq(Q209L). Recombinant Csk phosphorylated and activated GSK-3beta in vitro, and GSK-3beta coprecipitated with Csk from cell lysates. These results suggest that activation of Csk and GSK-3beta by Galphaq may contribute to the physiological and pathological effects of Gq-coupled receptors.     

Regulation of angiogenesis by glycogen synthase kinase-3beta

Glycogen synthase kinase-3beta (GSK3beta) plays important roles in metabolism, embryonic development, and tumorigenesis. Here we investigated the role of GSK3beta signaling in vascular biology by examining its function in endothelial cells (ECs). In EC, the regulatory phosphorylation of GSK3beta was found to be under the control of phosphoinositide 3-kinase-, MAPK-, and protein kinase A-dependent signaling pathways. The transduction of a nonphosphorylatable constitutively active mutant of GSKbeta promoted apoptosis under the conditions of prolonged serum deprivation or the disruption of cell-matrix attachments. Conversely, the transduction of catalytically inactive GSK3beta promoted EC survival under the conditions of cellular stress. Under normal cell culture conditions, the activation of GSK3beta signaling inhibited the migration of EC to vascular endothelial growth factor or basic fibroblast growth factor. Angiogenesis was inhibited by GSK3beta activation in an in vivo Matrigel plug assay, whereas the inhibition of GSK3beta signaling enhanced capillary formation. These data suggest that GSK3beta functions at the nodal point of converging signaling pathways in EC to regulate vessel growth through its control of vascular cell migration and survival.     

Functional significance of glycogen synthase kinase-3 regulation by serotonin

Serotonin modulates brain physiology and behavior and has major roles in brain diseases involving abnormal mood and cognition. Enhancing brain serotonin has been found to regulate glycogen synthase Kinase-3 (GSK3), but the signaling mechanism and functional significance of this regulation remain to be determined. In this study, we tested the signaling mechanism mediating 5-HT1A receptor-regulated GSK3 in the hippocampus. Using mutant GSK3 knock-in mice, we also tested the role of GSK3 in the behavioral effects of 5-HT1A receptors and the serotonin reuptake inhibitor fluoxetine. The results showed that activation of 5-HT1A receptors by 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) increased phosphorylation of the N-terminal serine of both GSK3α and GSK3β in several areas of the hippocampus. The effect of 8-OH-DPAT was accompanied by an increase in the active phosphorylation of Akt, and was blocked by LY294002, an inhibitor of phosphoinositide 3-kinases (PI3K). Phosphorylation of GSK3β, but not GSK3α, was necessary for 5-HT1A receptors to suppress the hippocampus-associated contextual fear learning. Furthermore, acute fluoxetine treatment up-regulated both phospho-Ser21-GSK3α and phospho-Ser9-GSK3β in the hippocampus. Blocking phosphorylation of GSK3α and GSK3β diminished the anti-immobility effect of fluoxetine treatment in the forced swim test, wherein the effect of GSK3β was more prominent. These results together suggest that PI3K/Akt is a signaling mechanism mediating the GSK3-regulating effect of 5-HT1A receptors in the hippocampus, and regulation of GSK3 is an important intermediate signaling process in the behavioral functions of 5-HT1A receptors and fluoxetine.     

The effects of glycogen synthase kinase-3beta in serotonin neurons

Glycogen synthase kinase-3 (GSK3) is a constitutively active protein kinase in brain. Increasing evidence has shown that GSK3 acts as a modulator in the serotonin neurotransmission system, including direct interaction with serotonin 1B (5-HT1B) receptors in a highly selective manner and prominent modulating effect on 5-HT1B receptor activity. In this study, we utilized the serotonin neuron-selective GSK3β knockout (snGSK3β-KO) mice to test if GSK3β in serotonin neurons selectively modulates 5-HT1B autoreceptor activity and function. The snGSK3β-KO mice were generated by crossbreeding GSK3β-floxed mice and ePet1-Cre mice. These mice had normal growth and physiological characteristics, similar numbers of tryptophan hydroxylase-2 (TpH2)-expressing serotonin neurons, and the same brain serotonin content as in littermate wild type mice. However, the expression of GSK3β in snGSK3β-KO mice was diminished in TpH2-expressing serotonin neurons. Compared to littermate wild type mice, snGSK3β-KO mice had a reduced response to the 5-HT1B receptor agonist anpirtoline in the regulation of serotonergic neuron firing, cAMP production, and serotonin release, whereas these animals displayed a normal response to the 5-HT1A receptor agonist 8-OH-DPAT. The effect of anpirtoline on the horizontal, center, and vertical activities in the open field test was differentially affected by GSK3β depletion in serotonin neurons, wherein vertical activity, but not horizontal activity, was significantly altered in snGSK3β-KO mice. In addition, there was an enhanced anti-immobility response to anpirtoline in the tail suspension test in snGSK3β-KO mice. Therefore, results of this study demonstrated a serotonin neuron-targeting function of GSK3β by regulating 5-HT1B autoreceptors, which impacts serotonergic neuron firing, serotonin release, and serotonin-regulated behaviors.     

Regulation and function of glycogen synthase kinase-3 isoforms in neuronal survival

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase consisting of two isoforms, alpha and beta. The activities of GSK-3 are regulated negatively by serine phosphorylation but positively by tyrosine phosphorylation. GSK-3 inactivation has been proposed as a mechanism to promote neuronal survival. We used GSK-3 isoform-specific small interfering RNAs, dominant-negative mutants, or pharmacological inhibitors to search for functions of the two GSK-3 isoforms in regulating neuronal survival in cultured cortical neurons in response to glutamate insult or during neuronal maturation/aging. Surprisingly, RNA interference-induced depletion of either isoform was sufficient to block glutamate-induced excitotoxicity, and the resulting neuroprotection was associated with enhanced N-terminal serine phosphorylation in both GSK-3 isoforms. However, GSK-3beta depletion was more effective than GSK-3alpha depletion in suppressing spontaneous neuronal death in extended culture. This phenomenon is likely due to selective and robust inhibition of GSK-3beta activation resulting from GSK-3beta Ser9 dephosphorylation during the course of spontaneous neuronal death. GSK-3alpha silencing resulted in reduced tyrosine phosphorylation of GSK-3beta, suggesting that tyrosine phosphorylation is also a critical autoregulatory event. Interestingly, GSK-3 inhibitors caused a rapid and long-lasting increase in GSK-3alpha Ser21 phosphorylation levels, followed by a delayed increase in GSK-3beta Ser9 phosphorylation and a decrease in GSK-3alpha Tyr279 and GSK-3beta Tyr216 phosphorylation, thus implying additional levels of GSK-3 autoregulation. Taken together, our results underscore important similarities and dissimilarities of GSK-3alpha and GSK-3beta in the roles of cell survival as well as their distinct modes of regulation. The development of GSK-3 isoform-specific inhibitors seems to be warranted for treating GSK-3-mediated pathology.     

Lithium inhibits glycogen synthase kinase-3 by competition for magnesium

The mechanism by which lithium (Li(+)) inhibits the protein kinase glycogen synthase kinase-3 (GSK-3) is unknown. Here, we demonstrate that Li(+) is a competitive inhibitor of GSK-3 with respect to magnesium (Mg(2+)), but not to substrate or ATP. This mode of inhibition is conserved between mammalian and Dictyostelium GSK-3 isoforms, and is not experienced with other group I metal ions. As a consequence, the potency of Li(+) inhibition is dependent on Mg(2+) concentration. We also found that GSK-3 is sensitive to chelation of free Mg(2+) by ATP and is progressively inhibited when ATP concentrations exceed that of Mg(2+). Given the cellular concentrations of ATP and Mg(2+), our results indicate that Li(+) will have a greater effect on GSK-3 activity in vivo than expected from in vitro studies and this may be a factor relevant to its use in the treatment of depression.     

A Proteasome-regulated glycogen synthase kinase-3 modulates disease response in plants

Glycogen synthase kinase-3 (GSK-3) is a key player in various important signaling pathways in animals. The activity of GSK-3 is known to be modulated by protein phosphorylation and differential complex formation. However, little information is available regarding the function and regulation of plant GSK-3/shaggy-like kinases (GSKs). Analysis of the in vivo kinase activity of MsK1, a GSK from Medicago sativa, revealed that MsK1 is active in healthy plants and that MsK1 activity is down-regulated by the elicitor cellulase in a time- and dose-dependent manner. Surprisingly, cellulase treatment triggered the degradation of the MsK1 protein in a proteasome-dependent manner suggesting a novel mechanism of GSK-3 regulation. Inhibition of MsK1 kinase activity and degradation of the protein were two successive processes that could be uncoupled. In a transgenic approach, stimulus-induced inhibition of MsK1 was impeded by constant replenishment of MsK1 by a strong constitutive promoter. MsK1 overexpressing plants exhibited enhanced disease susceptibility to the virulent bacterial pathogen Pseudomonas syringae. MAP kinase activation in response to pathogen infection was compromised in plants with elevated MsK1 levels. These data strongly suggest that tight regulation of the plant GSK-3, MsK1, may be important for innate immunity to limit the severity of virulent bacterial infection.