• Molecules and Cells
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• v.26 no.1
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• pp.100-105
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• 2008

Glycogen synthase kinase $3{\beta}$ (GSK $3{\beta}$) is a serine/threonine kinase that phosphorylates substrates such as ${\beta}$-catenin and is involved in a variety of biological processes, including embryonic development, metabolism, tumorigenesis, and cell death. Here, we present evidence that human GSK $3{\beta}$ is associated with Fe65, which has the characteristics of an adaptor protein, possessing a WW domain, and two phosphotyrosine interaction domains, PID1 and PID2. The GSK $3{\beta}$ catalytic domain also contains a putative WW domain binding motif ($^{371}PPLA^{374}$), and we observed, using a pull down approach and co-immunoprecipitation, that it interacts physically with Fe65 via this motif. In addition, we detected co-localization of GSK $3{\beta}$ and Fe65 by confocal microscopy, and this co-localization was disrupted by mutation of the putative WW domain binding motif of GSK $3{\beta}$. Finally, in transient transfection assays interaction of GSK $3{\beta}$ (wt) with Fe65 induced substantial cell apoptosis, whereas interaction with the GSK $3{\beta}$ AALA mutant ($^{371}AALA^{374}$) did not, and we noted that phosphorylation of the Tyr 216 residue of the GSK $3{\beta}$ AALA mutant was significantly reduced compared to that of GSK $3{\beta}$ wild type. Thus, our observations indicate that GSK $3{\beta}$ binds to Fe65 through its $^{371}PPLA^{374}$ motif and that this interaction regulates apoptosis and phosphorylation of Tyr 216 of GSK $3{\beta}$.

• IMMUNE NETWORK
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• v.10 no.3
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• pp.99-103
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• 2010

Background: Glycogen synthase kinase $3{\beta}$ ($GSK3{\beta}$) is a ubiquitous serine/threonine kinase that is regulated by serine phosphorylation at 9. Recent studies have reported the beneficial effects of a number of the pharmacological $GSK3{\beta}$ inhibitors in rodent models of septic shock. Since most of the $GSK3{\beta}$ inhibitors are targeted at the ATP-binding site, which is highly conserved among diverse protein kinases, the development of novel non-ATP competitive $GSK3{\beta}$ inhibitors is needed. Methods: Based on the unique phosphorylation motif of $GSK3{\beta}$, we designed and generated a novel class of $GSK3{\beta}$ inhibitor (GSK3i) peptides. In addition, we investigated the effects of a GSK3i peptide on lipopolysaccharide (LPS)-stimulated cytokine production and septic shock. Mice were intraperitoneally injected with GSK3i peptide and monitored over a 7-day period for survival. Results: We first demonstrate its effects on LPS-stimulated pro-inflammatory cytokine production including interleukin (IL)-6 and IL-12p40. LPS-induced IL-6 and IL-12p40 production in macrophages was suppressed when macrophages were treated with the GSKi peptide. Administration of the GSK3i peptide potently suppressed LPS-mediated endotoxin shock. Conclusion: Collectively, we present a rational strategy for the development of a therapeutic GSK3i peptide. This peptide may serve as a novel template for the design of non-ATP competitive GSK3 inhibitors.

• Molecules and Cells
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• v.37 no.10
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• pp.747-752
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• 2014

Protein kinase C (PKC) family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. However, the role of PKC in receptor activator of NF-${\kappa}B$ ligand (RANKL) signaling has remained elusive. We now demonstrate that $PKC{\beta}$ acts as a positive regulator which inactivates glycogen synthase kinase-$3{\beta}$ (GSK-$3{\beta}$) and promotes NFATc1 induction during RANKL-induced osteoclastogenesis. Among PKCs, $PKC{\beta}$ expression is increased by RANKL. Pharmacological inhibition of $PKC{\beta}$ decreased the formation of osteoclasts which was caused by the inhibition of NFATc1 induction. Importantly, the phosphorylation of GSK-$3{\beta}$ was decreased by $PKC{\beta}$ inhibition. Likewise, down-regulation of $PKC{\beta}$ by RNA interference suppressed osteoclast differentiation, NFATc1 induction, and GSK-$3{\beta}$ phosphorylation. The administration of PKC inhibitor to the RANKL-injected mouse calvaria efficiently protected RANKL-induced bone destruction. Thus, the $PKC{\beta}$ pathway, leading to GSK-$3{\beta}$ inactivation and NFATc1 induction, has a key role in the differentiation of osteoclasts. Our results also provide a further rationale for $PKC{\beta}$'s therapeutic targeting to treat inflammation-related bone diseases.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.2
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• pp.155-161
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• 2014

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer's disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta ($GSK3{\beta}$) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the $GSK3{\beta}$ kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.

• Anatomy and Cell Biology
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• v.50 no.3
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• pp.207-213
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• 2017

Glycogen synthase kinase $(GSK)-3{\beta}$ and related enzymes are associated with various forms of neuroinflammation, including spinal cord injury (SCI). Our aim was to evaluate whether lithium, a non-selective inhibitor of $GSK-3{\beta}$, ameliorated SCI progression, and also to analyze whether lithium affected the expression levels of two representative $GSK-3{\beta}$-associated molecules, nuclear factor erythroid 2-related factor-2 (Nrf-2) and heme oxygenase-1 (HO-1) (a target gene of Nrf-2). Intraperitoneal lithium chloride (80 mg/kg/day for 3 days) significantly improved locomotor function at 8 days post-injury (DPI); this was maintained until 14 DPI (P<0.05). Western blotting showed significantly increased phosphorylation of $GSK-3{\beta}$ (Ser9), Nrf-2, and the Nrf-2 target HO-1 in the spinal cords of lithium-treated animals. Fewer neuropathological changes (e.g., hemorrhage, inflammatory cell infiltration, and tissue loss) were observed in the spinal cords of the lithium-treated group compared with the vehicle-treated group. Microglial activation (evaluated by measuring the immunoreactivity of ionized calcium-binding protein-1) was also significantly reduced in the lithium-treated group. These findings suggest that $GSK-3{\beta}$ becomes activated after SCI, and that a non-specific enzyme inhibitor, lithium, ameliorates rat SCI by increasing phosphorylation of $GSK-3{\beta}$ and the associated molecules Nrf-2 and HO-1.

• Journal of Physiology & Pathology in Korean Medicine
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• v.29 no.1
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• pp.18-26
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• 2015

Akebiae Caulis is a galenical originated from Akebia quinata Decaisne species. It is commonly used in the treatment of oposiuria, inflammation, nociceptive and fever. Here, we investigated the effect of Akebiae Caulis extract (ACE) to protect hepatocyte against the malfunction of mitochondria and apoptosis. Arachidonic acid (AA)+iron promoted excessive reactive oxygen species (ROS) production and exerted a deleterious effect on mitochondria. Treatment with ACE protected hepatocytes from AA+iron-induced cytotoxicity, as shown by alterations in the protein levels related with apoptosis such as poly(ADP-ribose) polymerase, pro-caspase 3, Bcl-XL and Bcl-2. Moreover, AA+iron-induced $H_2O_2$ production, GSH depletion and mitochondrial dysfunction were alleviated by ACE pretreatment. As a potential molecular mechanism for the ACE-mediated cytoprotection, phosphorylation of AMP-activated protein kinase (AMPK), a key regulator in determining cell survival or death, was increased by ACE. Moreover, ACE treatment enhanced inactive phosphorylation of glycogen synthase kinase-$3{\beta}$ ($GSK3{\beta}$), downstream substrate kinase of AMPK. More importantly, ACE prevented a decrease in the $GSK3{\beta}$ phosphorylation derived by AA+iron, which might contribute to mitohondiral protection and cell survival. To further identify essential compounds in Akebiae Caulis for the protection of AA+iron-mediated cytotoxicity, we found that betulin in combination with hederagenin protected from AA+iron-induced mitochondrial dysfunction. Betulin+hederagenin treatment also increased inactive phosphorylation of $GSK3{\beta}$ in common with ACE. These results suggest that ACE protected hepatocytes against oxidative stress and mitochondrial dysfunction, which is mediated with inactive $GSK3{\beta}$ phosphorylation downstream of AMPK.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.10a
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• pp.68-68
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• 2019

In this study, we evaluated the effect of Rodgersia podophylla leave extracts (RPL) on ${\beta}$-catenin level in human cancer cells. RPL dose-dependently inhibited cell proliferation in SW480, A549, MDA-MB-231, PC-3 and AsPC-1 cells. RPL dramatically decreased ${\beta}$-catenin protein level in all cancer cells. However, decreased level of ${\beta}$-catenin mRNA expression was observed in A549 and AsPC-1 cells. In addition, RPL dramatically attenuated cyclin D1 mRNA expression in all cancer cells. MG132 decreased the downregulation of ${\beta}$-catenin protein level induced by RPL in all cancer cells, while RPL-induced downregulation of ${\beta}$-catenin was inhibited by the inhibition of $GSK-3{\beta}$ by LiCl in MDA-MB-231 cells. RPL phosphorylated ${\beta}$-catenin and $GSK-3{\beta}$. In addition, the inhibition of $GSK-3{\beta}$ by LiCl attenuated RPL-induced ${\beta}$-catenin phosphorylation. Based on these findings, RPL may be a potential candidate for the development of chemopreventive or therapeutic agents for human cancer.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.93-93
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• 2019

In this study, we evaluated the anti-cancer activity and potential molecular mechanism of 70% ethanol extracts of branches from Taxillus yadoriki parasitic to Neolitsea sericea (TN-NS-B) against human lung cancer cells, A549. TY-NS-B dose-dependently suppressed the growth of A549 cells. TY-NS-B decreased ${\beta}$-catenin protein level, but not mRNA level in A549 cells. The downregulation of ${\beta}$-catenin protein level by TY-NS-B was attenuated in the presence of MG132. Although TY-NS-B phosphorylated ${\beta}$-catenin protein, the inhibition of $GSK3{\beta}$ by LiCl did not blocked the reduction of ${\beta}$-catenin by TY-NS-B. In addition, TY-NS-B decreased ${\beta}$-catenin protein in A549 cells transfected with Flag-tagged wild type ${\beta}$-catenin or Flag-tagged S33/S37/T41 mutant ${\beta}$-catenin construct. Our results suggested that TN-NS-B may downregulate ${\beta}$-catenin protein level independent on GSK3${\beta}$-induced ${\beta}$-catenin phosphorylation. Based on these findings, TY-NS-B may be a potential candidate for the development of chemopreventive or therapeutic agents for human lung cancer.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.1
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• pp.53-61
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• 2018

Ethyl linoleate is an unsaturated fatty acid used in many cosmetics for its various attributes, such as antibacterial and anti-inflammatory properties and clinically proven to be an effective anti-acne agent. In this study, we investigated the effect of ethyl linoleate on the melanogenesis and the mechanism underlying its action on melanogenesis in B16F10 murine melanoma cells. Our results revealed that ethyl linoleate significantly inhibited melanin content and intracellular tyrosinase activity in ${\alpha}$-MSH-induced B16F10 cells, but it did not directly inhibit activity of mushroom tyrosinase. Ethyl linoleate inhibited the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase related protein 1 (TRP1) in governing melanin pigment synthesis. We observed that ethyl linoleate inhibited phosphorylation of Akt and glycogen synthase kinase $3{\beta}$ ($GSK3{\beta}$) and reduced the level of ${\beta}-catenin$, suggesting that ethyl linoleate inhibits melanogenesis through $Akt/GSK3{\beta}/{\beta}-catenin$ signal pathway. Therefore, we propose that ethyl linoleate may be useful as a safe whitening agent in cosmetic and a potential therapeutic agent for reducing skin hyperpigmentation in clinics.

• Biomolecules & Therapeutics
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• v.31 no.1
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• pp.127-138
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• 2023

Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer's disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3β from Enamine's screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (Tau_RD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3β activities ranged from 36.1% to 90.0% were detected at the IC₅₀ of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 Tau_RD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 Tau_RD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3β Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogenactivated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/ Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 Tau_RD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3β kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.