Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3 beta

The goal of this study was to determine whether the intracellular distribution of the proapoptotic enzyme glycogen synthase kinase-3 beta (GSK-3 beta) is dynamically regulated by conditions that activate apoptotic signaling cascades. In untreated human neuroblastoma SH-SY5Y cells, GSK-3 beta was predominantly cytosolic, although a low level was also detected in the nucleus. The nuclear level of GSK-3 beta was rapidly increased after exposure of cells to serum-free media, heat shock, or staurosporine. Although each of these conditions caused changes in the serine 9 and/or tyrosine phosphorylation of GSK-3 beta, neither of these modifications was correlated with nuclear accumulation of GSK-3 beta. Heat shock and staurosporine treatments increased nuclear GSK-3 beta prior to activation of caspase-9 and caspase-3, and this nuclear accumulation of GSK-3 beta was unaltered by pretreatment with a general caspase inhibitor. The GSK-3 beta inhibitor lithium did not alter heat shock-induced nuclear accumulation of GSK-3 beta but increased the nuclear level of cyclin D1, indicating that cyclin D1 is a substrate of nuclear GSK-3 beta. Thus, the intracellular distribution of GSK-3 beta is dynamically regulated by signaling cascades, and apoptotic stimuli cause increased nuclear levels of GSK-3 beta, which facilitates interactions with nuclear substrates.     

Inhibition of glycogen synthase kinase-3beta activity is sufficient to stimulate myogenic differentiation

Skeletal muscle atrophy is a prominent and disabling feature of chronic wasting diseases. Prevention or reversal of muscle atrophy by administration of skeletal muscle growth (hypertrophy)-stimulating agents such as insulin-like growth factor I (IGF-I) could be an important therapeutic strategy in these diseases. To elucidate the IGF-I signal transduction responsible for muscle formation (myogenesis) during muscle growth and regeneration, we applied IGF-I to differentiating C₂C₁₂ myoblasts and evaluated the effects on phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase-3 (GSK-3) signaling and myogenesis. IGF-I caused phosphorylation and inactivation of GSK-3 activity via signaling through the PI3K/Akt pathway. We assessed whether pharmacological inhibition of GSK-3 with lithium chloride (LiCl) was sufficient to stimulate myogenesis. Addition of IGF-I or LiCl stimulated myogenesis, evidenced by increased myotube formation, muscle creatine kinase (MCK) activity, and troponin I (TnI) promoter transactivation during differentiation. Moreover, mRNAs encoding MyoD, Myf-5, myogenin, TnI-slow, TnI-fast, MCK, and myoglobin were upregulated in myoblasts differentiated in the presence of IGF-I or LiCl. Importantly, blockade of GSK-3 inhibition abrogated IGF-I- but not LiCl-dependent stimulation of myogenic mRNA accumulation, suggesting that the promyogenic effects of IGF-I require GSK-3 inactivation and revealing an important negative regulatory role for GSK-3 in myogenesis. Therefore, this study identifies GSK-3 as a potential target for pharmacological stimulation of muscle growth. insulin-like growth factor I; muscle hypertrophy     

Inhibition of alanyl-aminopeptidase suppresses the activation-dependent induction of glycogen synthase kinase-3beta (GSK-3beta) in human T cells

Inhibition of alanyl-aminopeptidase (APN, CD13) gene expression or enzymatic activity compromises T cell proliferation and function. Molecular mechanisms mediating these effects are not known as yet. Recently, we found the expression of the proto-oncogen Wnt-5a to be strongly affected by APN-inhibition. Wnt-5a and other members of the Wnt family of secreted factors are implicated in cell growth and differentiation. Here, we analyzed by quantitative RT-PCR and immunoblotting the expression in mitogen-activated T cells of a major constituent of the Wnt-5a pathway, glycogen synthase kinase-3beta (GSK-3beta). T cell activation by phytohaemagglutinin or pokeweed mitogen results in a strong increase of GSK-3beta mRNA amounts. At the protein level, we observed an up-regulation of both GSK-3beta and phosphorylated GSK-3beta. This induction-dependent increase of GSK-3beta is markedly reduced in response to inhibitors of alanyl-aminopeptidase, actinonin, leuhistin, and RB3014. These findings may provide a rational for the growth inhibition resulting from a diminished expression or activity of alanyl aminopeptidase.     

Calcineurin dephosphorylates glycogen synthase kinase-3 beta at serine-9 in neuroblast-derived cells

This study examined the role of calcineurin, a major calcium-dependent protein phosphatase, in dephosphorylating Ser-9 and activating glycogen synthase kinase-3β (GSK-3β). Treatment with calcineurin inhibitors increased phosphorylation of GSK-3β at Ser-9 in SH-SY5Y human neuroblastoma cells. The over-expression of a constitutively active calcineurin mutant, calcineurin A beta (1–401), led to a significant decrease in phosphorylation at Ser-9, an increase in the activity of GSK-3β, and an increase in the phosphorylation of tau. K_m of calcineurin for a GSK-3β phosphopeptide was 469.3 μM, and specific activity of calcineurin was 15.2 nmol/min/mg. In addition, calcineurin and GSK-3β were co-immunoprecipitated in neuron-derived cells and brain tissues, and calcineurin formed a complex only with dephosphorylated GSK-3β. We conclude that in vitro, calcineurin can dephosphorylate GSK-3β at Ser-9 and form a stable complex with GSK-3β, suggesting the possibility that calcineurin regulates the dephosphorylation and activation of GSK-3β in vivo .     

A role for glycogen synthase kinase-3beta in the mammalian circadian clock

The Drosophila shaggy gene product is a mammalian glycogen synthase kinase-3beta (GSK-3beta) homologue that contributes to the circadian clock of the Drosophila through TIMELESS phosphorylation, and it regulates nuclear translocation of the PERIOD/TIMELESS heterodimer. We found that mammalian GSK-3beta is expressed in the suprachiasmatic nucleus and liver of mice and that GSK-3beta phosphorylation exhibits robust circadian oscillation. Rhythmic GSK-3beta phosphorylation is also observed in serum-shocked NIH3T3 cells. Exposing serum-shocked NIH3T3 cells to lithium chloride, a specific inhibitor of GSK-3beta, increases GSK-3beta phosphorylation and delays the phase of rhythmic clock gene expression. On the other hand, GSK-3beta overexpression advances the phase of clock gene expression. We also found that GSK-3beta interacts with PERIOD2 (PER2) in vitro and in vivo. Recombinant GSK-3beta can phosphorylate PER2 in vitro. GSK-3beta promotes the nuclear translocation of PER2 in COS1 cells. The present data suggest that GSK-3beta plays important roles in mammalian circadian clock.     

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.     

Redox modulation of long-term potentiation in the hippocampus via regulation of the glycogen synthase kinase-3beta pathway

Alzheimer disease (AD) is an age-related neurodegenerative disorder. Many observations indicate that impaired redox regulation is implicated in AD with synaptic failure. The aim of the current investigation was to characterize the role of redox-active agents on long-term potentiation (LTP) in the CA1 region of rat hippocampal slices and to elucidate the molecular sequence of events leading to these changes. The results presented here indicate that the membrane-permeable oxidizing agent chloramine-T (CH-T) inhibits the induction of LTP, whereas the membrane-permeable reducing agent dithiothreitol (DTT) enhances the induction of LTP. In contrast, neither the membrane-impermeable oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) nor the membrane-impermeable reducing agent tris-(2-carboxyethyl) phosphine (TCEP) can affect the induction of LTP. The inhibition of LTP by CH-T can be restored by pretreatment with DTT but not with TCEP, whereas the enhancement of LTP by DTT can be reversed by pretreatment with CH-T but not with DTNB. We also provide evidence that the CH-T-evoked inhibition of LTP is mediated via activation of glycogen synthase kinase-3beta (GSK-3beta), whereas the DTT-evoked enhancement of LTP is mediated via inactivation of GSK-3beta. These findings will benefit the understanding of the redox contribution to the mechanisms underlying synaptic plasticity and AD pathogenesis.     

The regulation of glycogen synthase kinase-3 nuclear export by Frat/GBP

Previous studies have shown that nuclear levels of glycogen synthase kinase-3 (GSK-3) are dynamically regulated and may affect access of GSK-3 to its substrates. In this study we show that the GSK-3-binding protein Frat/GBP regulates the nuclear export of GSK-3. We show that Frat/GBP contains a nuclear export sequence that promotes its own nuclear export and that of associated GSK-3. Treating cells with leptomycin B increased nuclear levels of endogenous GSK-3 suggesting that an endogenous process targets GSK-3 for nuclear export. To investigate this further, we used two approaches to disrupt the interaction between GSK-3 and endogenous Frat. First we isolated mutants of GSK-3 that selectively interfered with Frat binding and found that these mutants were poorly exported. Second we expressed a peptide that competes with Frat for GSK-3 binding and found that it caused endogenous GSK-3 to accumulate in the nucleus. Together these data suggest that Frat may be the endogenous factor that targets GSK-3 for nuclear export. The dynamic expression patterns of Frat mRNAs together with the role of Frat in mediating GSK-3 nuclear export have important implications for the control of the substrate access of GSK-3 in several signaling pathways.     

New insights into the autoinhibition mechanism of glycogen synthase kinase-3beta

It has been suggested that phosphorylation at serine 9 near the N-terminus of glycogen synthase kinase-3β (GSK-3β) mimics the prephosphorylation of its substrate and, therefore, the N-terminus functions as a pseudosubstrate. The molecular basis for the pseudosubstrate's binding to the catalytic core and autoinhibition has not been fully defined. Here, we combined biochemical and computational analyses to identify the potential residues within the N-terminus and the catalytic core engaged in autoinhibition of GSK-3β. Bioinformatic analysis found Arg4, Arg6, and Ser9 in the pseudosubstrate sequence to be extremely conserved through evolution. Mutations at Arg4 and Arg6 to alanine enhanced GSK-3β kinase activity and impaired its ability to autophosphorylate at Ser9. In addition, and unlike wild-type GSK-3β, these mutants were unable to undergo autoinhibition by phosphorylated Ser9. We further show that Gln89 and Asn95, located within the catalytic core, interact with the pseudosubstrate. Mutation at these sites prevented inhibition by phosphorylated Ser9. Furthermore, the respective mutants were not inhibited by a phosphorylated pseudosubstrate peptide inhibitor. Finally, computational docking of the pseudosubstrate into the catalytic active site of the kinase suggested specific interactions between Arg6 and Asn95 and of Arg4 to Asp181 (apart from the interaction of phosphorylated serine 9 with the “phosphate binding pocket”). Altogether, our study supports a model of GSK-3-pseudosubstrate autoregulation that involves phosphorylated Ser9, Arg4, and Arg6 within the N-terminus and identified the specific contact sites within the catalytic core.     

Wogonin inhibits cell cycle progression by activating the glycogen synthase kinase-3 beta in hepatocellular carcinoma

BackgroundWogonin has been reported to exhibit various biological activities such as anti-inflammation, anti-microbial, and anti-tumor. Previous studies have demonstrated that wogonin could down-regulate Cyclin D1 activity on multiple cancers. However, the related mechanisms have not been fully elucidated so far.PurposeThe aim of the current study was to explore whether wogonin can suppress hepatocellular carcinoma (HCC) progression and the mechanism of wogonin in inhibiting Cyclin D1 expression.MethodsHerein, we assessed the anti-tumor activity of wogonin against hepatocellular carcinoma (HCC) by MTT assay, clonogenic assay, cell cycle analysis and orthotopic xenograft mouse models. Western blot, immunofluoscence assay, co-immunoprecipitation assay, docking program, surface plasmon resonance, site-directed mutagenesis assay and immunohistochemical assay were performed for exploring the underlying mechanisms of wogonin-induced growth inhibition in HCC.ResultsOur results showed that non-toxic dosage of wogonin (10, 20 µM) could inhibit cells proliferation and suppress cells cycle progression in MHCC97L and HepG2 cell. Moreover, the findings from the western blot and immunofluoscence assay confirmed the inhibition action of wogonin (10, 20 µM) on Cyclin D1 expression in MHCC97L cells, and wogonin (10, 20 µM) pre-treatment was capable of promoting Cyclin D1 ubiquitination and degradation in MHCC97L cell. In addition, wogonin promoted phosphorylation of Cyclin D1 on threonine-286 site, the mutation of threonine-286 to alanine-286A blocked Cyclin D1 proteolysis induced by wogonin. Wogonin-promoted Cyclin D1 phosphorylation and subsequent proteolysis may associate with the activation of GSK3beta in cancer cells. The phosphorylated form of GSK3beta (active form) expression was significantly increased after wogonin (20 µM) exposure. Molecular docking study and Biacore SPR analysis of GSK3beta mutant further validated the high-affinity wogonin binding site on GSK3beta. Moreover, in vivo studies further confirmed that phospho-GSK3beta Tyr216 was over-expressed in HCC specimens after wogonin treatment while the amount of Cyclin D1 was significantly decreased.ConclusionIn summary, our data reveal a novel molecular mechanism by which wogonin induces HCC cells cycle arrest and suppresses tumor proliferation.     

Regulation of glycogen synthase kinase-3beta by products of lipid peroxidation in human neuroblastoma cells

The potential role of 4-hydroxynonenal (HNE), a major product of membrane lipid peroxidation, in regulating glycogen synthase kinase-3beta (GSK3beta) activity was examined in human neuroblastoma IMR-32 cells. The inhibition of GSK3beta activity by HNE was observed by in vitro kinase assays with two substrates, the synthetic glycogen synthase peptide-2 and the human recombinant tau. GSK3beta activity is regulated by Ser9 (inhibitory) and Tyr216 (stimulatory) phosphorylation. By using specific activity-dependent phospho-antibodies, immunoblot analysis revealed that HNE induces an increase in phosphorylation of GSK3beta in Ser9, enhancing basal phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase 2 (ERK2) signalling pathways. Ser9-GSK3beta phosphorylation induced by HNE was abolished by treatment with LY294002 or U0126, two inhibitors of PI3K/AKT and ERK pathways, respectively. These experiments provide evidence for a crucial role of the PI3K/AKT and ERK2 pathways as intracellular targets of HNE that mediate the inhibition of GSK3beta activity in regulating cellular response to HNE in viable cells under conditions in which membrane lipid peroxidation occurs. These data support a key role for GSK3beta as a mediator of the signalling pathways activated by oxidative stress, and therefore it may be included among the redox-sensitive enzymes.     

Regulation of type 1 protein phosphatase/inhibitor-2 complex by glycogen synthase kinase-3beta in intact cells

Inhibitor 2 (I-2) is a ubiquitous regulator of type 1 protein phosphatase (PP1). Previous in vitro studies suggested that its inhibitory activity towards PP1 is regulated by phosphorylation at Thr72 by glycogen synthase kinase-3beta (GSK-3beta), and at Ser86, Ser120, and Ser121 by casein kinase 2 (CK2). Here we report that GSK-3beta expressed in COS-7 cells phosphorylates wild-type I-2 but not an I-2 mutant carrying a T to A substitution at residue 72, showing that GSK-3beta phosphorylates I-2 at T72 in vivo as well. Co-immunoprecipitation study demonstrated that HA-GSK-3beta and I-2-FLAG co-exist in a same complex in the intact cells, but they do not bind directly. It is noteworthy that co-expression of Myc-PP1C significantly increased co-precipitation of HA-GSK-3beta with I-2-FLAG, showing a complex formation of HA-GSK-3beta/Myc-PP1C / I-2-FLAG in vivo. Further studies using a GSK-3beta kinase-dead mutant and LiCl, an inhibitor of GSK-3beta, showed that the enzyme activity of GSK-3beta is required for co-precipitation. IP-Western study using several I-2 mutants substituted at phosphorylation sites (T72, S86, S120, and S121) suggested that phosphorylation of I-2 by CK2 is also involved in enhancement of association between GSK-3beta and I-2 in vivo. This study is the first demonstration that GSK-3beta associates with PP1C/I-2 complex and phosphorylates I-2 at T72 in the intact cells.     

Fibroblast growth factor 1 regulates signaling via the glycogen synthase kinase-3beta pathway. Implications for neuroprotection

We hypothesize that in neurodegenerative disorders such as Alzheimer's disease and human immunodeficiency virus encephalitis the neuroprotective activity of fibroblast growth factor 1 (FGF1) against several neurotoxic agents might involve regulation of glycogen synthase kinase-3beta (GSK3beta), a pathway important in determining cell fate. In primary rat neuronal and HT22 cells, FGF1 promoted a time-dependent inactivation of GSK3beta by phosphorylation at serine 9. Blocking FGF1 receptors with heparinase reduced this effect. The effects of FGF1 on GSK3beta were dependent on phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) because inhibitors of this pathway or infection with dominant negative Akt adenovirus blocked inactivation. Furthermore, treatment of neuronal cells with FGF1 resulted in ERK-independent Akt phosphorylation and beta-catenin translocation into the nucleus. On the other hand, infection with wild-type GSK3beta recombinant adenovirus-associated virus increased activity of GSK3beta and cell death, both of which were reduced by FGF1 treatment. Moreover, FGF1 protection against glutamate toxicity was dependent on GSK3beta inactivation by the PI3K-Akt but was independent of ERK. Taken together these results suggest that neuroprotective effects of FGF1 might involve inactivation of GSK3beta by a pathway involving activation of the PI3K-Akt cascades.     

Unrestrained glycogen synthase kinase-3 beta activity leads to activated T cell death and can be inhibited by natural adjuvant

Activated T cell death (ATCD) after peak clonal expansion is required for effective homeostasis of the immune system. Using a mouse model of T cell clonal expansion and contraction, we found that regulation of the proapoptotic kinase glycogen synthase kinase (GSK)-3beta plays a decisive role in determining the extent to which T cells are eliminated after activation. Involvement of GSK-3beta in ATCD was tested by measuring T cell survival after GSK-3beta inhibition, either ex vivo with chemical and pharmacological inhibitors or in vivo by retroviral expression of a dominant-negative form of GSK-3. We also measured amounts of inactivating phosphorylation of GSK-3beta (Ser9) in T cells primed in the presence or absence of LPS. Our results show that GSK-3beta activity is required for ATCD and that its inhibition promoted T cell survival. Adjuvant treatment in vivo maintained GSK-3beta (Ser9) phosphorylation in activated T cells, whereas with adjuvant-free stimulation it peaked and then decayed as the cells became susceptible to ATCD. We conclude that the duration of GSK-3beta inactivation determines activated T cell survival and that natural adjuvant stimulation decreases the severity of clonal contraction in part by keeping a critical proapoptotic regulatory factor, GSK-3beta, inactivated.