• Molecules and Cells
• /
• v.26 no.1
• /
• pp.100-105
• /
• 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
• /
• v.10 no.3
• /
• pp.99-103
• /
• 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.

• Tuberculosis and Respiratory Diseases
• /
• v.57 no.5
• /
• pp.449-460
• /
• 2004

Background : PS-341 is a novel, highly selective and potent proteasome inhibitor, which showed cytotoxicity against some tumor cells. Its anti-tumor activity has been suggested to be associated with modulation of the expression of apoptosis-associated proteins, such as p53, $p21^{WAF/CIP1}$, $p27^{KIP1}$, NF-${\kappa}B$, Bax and Bcl-2. c-Jun N-terminal kinase (JNK) and glycogen synthase kinase-$3{\beta}$ (GSK-$3{\beta}$) are important modulators of apoptosis. However, their role in PS-341-induced apoptosis is unclear. This study was undertaken to elucidate the role of JNK and GSK-$3{\beta}$ in the PS-341-induced apoptosis in lung cancer cells. Method : NCI-H157 and A549 cells were used in the experiments. The cell viability was assayed using the MTT assay and apoptosis was evaluated by proteolysis of PARP. The JNK activity was measured by an in vitro immuno complex kinase assay and by phosphorylation of endogenous c-Jun. The protein expression was evaluated by Western blot analysis. Dominant negative JNK1 (DN-JNK1) and GSK-$3{\beta}$ were overexpressed using plasmid and adenovirus vectors, respectively. Result : PS-341 reduced the cell viability via apoptosis, activated JNK and increased the c-Jun expression. Blocking of the JNK activation by overexpression of DN-JNK1, or pretreatment with SP600125, suppressed the apoptosis induced by PS-341. The activation of caspase 3 was mediated by JNK activation. Blocking of the caspase 3 activation suppressed PS-341-induced apoptosis. PS-341 activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but its blockade enhanced the PS-341-induced cell death via apoptosis. GSK-$3{\beta}$ was inactivated by PS-341 via the PI3K/Akt pathway. Overexpression of constitutively active GSK-$3{\beta}$ enhanced PS-341-induced apoptosis; in contrast, this was suppressed by dominant negative GSK-$3{\beta}$ (DN-GSK-$3{\beta}$). Inactivation of GSK-$3{\beta}$ by pretreatment with lithium chloride or the overexpression of DN-GSK-$3{\beta}$ suppressed both the JNK activation and c-Jun up-regulation induced by PS-341. Conclusion : The JNK/caspase pathway is involved in PS-341-induced apoptosis, which is negatively regulated by the PI3K/Akt-mediated inactivation of GSK-$3{\beta}$ in lung cancer cells.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.7
• /
• pp.1170-1176
• /
• 2015

Ginsenosides, the major active component of ginseng, are traditionally used to treat various diseases, including cancer, inflammation, and obesity. Among these, compound K (CK), an intestinal bacterial metabolite of the ginsenosides Rb₁, Rb₂, and Rc from Bacteroides JY-6, is reported to inhibit cancer cell growth by inducing cell-cycle arrest or cell death, including apoptosis and necrosis. However, the precise effect of CK on breast cancer cells remains unclear. MCF-7 cells were treated with CK ($0-70{\mu}M$) for 24 or 48 h. Cell proliferation and death were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Changes in downstream signaling molecules involved in cell death, including glycogen synthase kinase $3\beta$ ($GSK3\beta$), $GSK3\beta$, $\beta$-catenin, and cyclin D1, were analyzed by western blot assay. To block $GSK3\beta$ signaling, MCF-7 cells were pretreated with $GSK3\beta$ inhibitors 1 h prior to CK treatment. Cell death and the expression of $\beta$-catenin and cyclin D1 were then examined. CK dose- and time-dependently inhibited MCF-7 cell proliferation. Interestingly, CK induced programmed necrosis, but not apoptosis, via the $GSK3\beta$ signaling pathway in MCF-7 cells. CK inhibited $GSK3\beta$ phosphorylation, thereby suppressing the expression of $\beta$-catenin and cyclin D1. Our results suggest that CK induces programmed necrosis in MCF-7 breast cancer cells via the $GSK3\beta$ signaling pathway.

• Journal of the Korean Magnetic Resonance Society
• /
• v.9 no.1
• /
• pp.38-47
• /
• 2005

Axin is a scaffold protein of the APC/axin/GSK complex, binding to all of the other signalling components. Axin interacts with Glycogen synthase kinase 3$\beta$ (GSK 3$\beta$) and functions as a negative regulator of Wnt signalling pathways. To determine the solution structure of the GSK3$\beta$ binding regions of the axin, we initiated NMR study of axin fragment comprising residues 3$Val^{388} - Arg^{401}$using circular dichroism (CD) and two-dimensional NMR spectroscopy. The CD spectra of 3$axin^{pep}$ in the presence of 30% TFE displayed a standard 3$\alpha$-helical conformation, exhibiting the bound structure of 3$axin^{pep}$ to GSK3$\bata$. On the basis of experimental restraints including $NOE_s$, and $^3J_{HN\alpha} $ coupling constants, the solution conformation of $axin^{pep}$ was determined with program CNS. The 20 lowest energy structures were selected out of 50 final simulated-annealing structures in both water and TFE environment, respectively. The $RMSD_s$ for the 20 structures in TFE solution were 0.086 nm for backbone atoms and 0.195 nm for all heavy atoms, respectively. The Ramachandran plot indicates that the $\varphi$, $\psi$ angles of the 20 final structures is properly distributed in energetically acceptable regions. $Axin^pep$ in aqueous solutions consists of a stable $\alpha$-helix spanning residues form $Glu^{391}$ to $Val^{391} $, which is an interacting motif with GSK3$\beta$.

• Molecules and Cells
• /
• v.27 no.1
• /
• pp.15-19
• /
• 2009

Notch signaling is controlled at multiple levels. In particular, stabilized Notch receptor activation directly affects the transcriptional activations of Notch target genes. Although some progress has been made in terms of defining the regulatory mechanism that alters Notch stability, it has not been determined whether Notch1/NICD stability is regulated by $GSK-3{\alpha}$. Here, we show that Notch1/NICD levels are significantly regulated by $GSK-3{\beta}$ and by $GSK-3{\alpha}$. Treatment with LiCl (a specific GSK-3 inhibitor) or the overexpression of the kinase-inactive forms of $GSK-3{\alpha}/{\beta}$ significantly increased Notch1/NICD levels. Endogenous NICD levels were also increased by either $GSK-3{\alpha}/{\beta}$- or $GSK-3{\alpha}$-specific siRNA. Furthermore, it was found that $GSK-3{\alpha}$ binds to Notch1. Deletion analysis showed that at least three Thr residues in Notch1 (Thr-1851, 2123, and 2125) are critical for its response to LiCl, which increased not only the transcriptional activity of endogenous NICD but also Hes1 mRNA levels. Taken together, our results indicate that $GSK-3{\alpha}$ is a negative regulator of Notch1/NICD.

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

• Anatomy and Cell Biology
• /
• v.50 no.3
• /
• pp.207-213
• /
• 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 Pharmacopuncture
• /
• v.22 no.4
• /
• pp.248-252
• /
• 2019

Objectives: The aim of the present work is evaluating the special effects of Urtica Dioica and Lamium Album on the serum level of K-Ras and GSK-3 beta in diabetic rats. Methods: In the present experimental study, 32 male Wistar rats randomly divided into 4 groups (Group I: normal control rats; receiving daily PBS, Group 2: diabetic control rats; receiving single dose of streptozotocin (60 mg/kg) and daily PBS, Group 3: Diabetic rats treated with 100 mg/kg of hydroalcoholic extract of the U. dioica, Group 4: Diabetic rats treated with 100 mg/kg of hydroalcoholic extract of L. Album. Diabetes-induced by an intraperitoneal injection of streptozotocin (60 mg/ kg). On the 14 th day of treatment, the weight, fasting blood sugar (FBS) and on 28 th day blood glucose, K-Ras and GSK3 beta was measured. Results: In diabetic group blood GSK- 3 beta increase in comparison to control group (P < 0.05), also blood K-Ras decrease in the diabetic group (P < 0.05). Both extracts reduced GSK-3 beta level, however, this reduction was only statistically significant by U.dioica (P < 0.05). Compared to diabetic group, blood K-Ras level increased by both extract (P < 0.05). Also diabetes induction increase blood glucose levels and both extracts decrease its level significantly (P < 0.05).there is no significant differences among both extract effects on blood glucose, and K-Ras. Conclusion: For the first time shown that both extracts by regulating GSK-3 beta and K-Ras improve blood glucose level. More studies are needed to determine all the effects of these herbs.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.46 no.4
• /
• pp.417-425
• /
• 2017

Cnidium monnieri (L.) Cusson is distributed in China and Korea, and the fruit of C. monnieri is used as traditional Chinese medicine to treat carbuncle and pain in female genitalia. In this study, we examined the anti-proliferation and cell cycle arrest effects of ethanol extracts from C. monnieri (CME) in AGS gastric cancer cells. Our results show that CME suppressed cell proliferation and induced release of lactate dehydrogenase (LDH) in AGS cells by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and LDH assay. Cell morphology was altered by CME in a dose-dependent manner. In order to identify the cell cycle arrest effects of CME, we investigated cell cycle analysis after CME treatment. In our results, CME induced cell cycle arrest at G1 phase. Protein kinase B (Akt) plays a major role in cell survival mechanisms such as growth, division, and metastasis. Akt protein regulates various downstream proteins such as glycogen synthase kinase-$3{\beta}$ (GSK-$3{\beta}$) and tumor protein p53 (p53). Expression levels of p-Akt, p-GSK-$3{\beta}$, p53, p21, cyclin E, and cyclin-dependent kinase 2 (CDK2) were determined by Western blot analysis. Protein levels of p-Akt, p-GSK-$3{\beta}$, and cyclin E were reduced while those of p53, p21, and p-CDK2 (T14/Y15) were elevated by CME. Moreover, treatment with CME, LY294002 (phosphoinositide 3-kinase/Akt inhibitor), BIO (GSK-$3{\beta}$ inhibitor), and Pifithrin-${\alpha}$ (p53 inhibitor) showed that cell cycle arrest effects were mediated through regulation of the Akt/GSK-$3{\beta}$/p53 signaling pathway. These results suggest that CME induces cell cycle arrest at G1 phase via the Akt/GSK-$3{\beta}$/p53 signaling pathway in AGS gastric cancer cells.