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Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 posttranslational regulation. Here, we characterize a phosphorylation site at Ser325 situated C terminal to the catalytic domain of PTPN22 and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by glycogen synthase kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9-mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p, and Moira domain binding proteins, and next-generation sequencing revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein’s properties by deuterium exchange mass spectrometry and NMR spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22. [ABSTRACT FROM AUTHOR]

A structural protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), nucleocapsid (N) protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3β constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), found in many endogenous GSK-3β binding proteins, such as Axins, FRATs, WWOX, and GSKIP. Indeed, N interacts with GSK-3β similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. The N phosphorylation is also required for its structural loading in a virus-like particle (VLP). Compared to other coronaviruses, N of Sarbecovirus lineage including bat RaTG13 harbors a CDK1-primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3β. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allow increased abundance and hyper-phosphorylation of N. Our observations suggest that GID and mutations for increased phosphorylation in N may have contributed to the evolution of variants. [ABSTRACT FROM AUTHOR]

Pah1 phosphatidate (PA) phosphatase plays a major role in triacylglycerol synthesis in Saccharomyces cerevisiae by producing its precursor diacylglycerol and concurrently regulates de novo phospholipid synthesis by consuming its precursor PA. The function of Pah1 requires its membrane localization, which is controlled by its phosphorylation state. Pah1 is dephosphorylated by the Nem1-Spo7 protein phosphatase, whereas its phosphorylation occurs by multiple known and unknown protein kinases. In this work, we show that Rim11, a yeast homolog of mammalian glycogen synthase kinase-3β, is a protein kinase that phosphorylates Pah1 on serine (Ser12, Ser602, and Ser818) and threonine (Thr163, Thr164, Thr522) residues. Enzymological characterization of Rim11 showed that its Km for Pah1 (0.4 μM) is similar to those of other Pah1-phosphorylating protein kinases, but its Km for ATP (30 μM) is significantly higher than those of these same kinases. Furthermore, we demonstrate Rim11 phosphorylation of Pah1 does not require substrate prephosphorylation but was increased ~2-fold upon its prephosphorylation by the Pho85-Pho80 protein kinase. In addition, we show Rim11-phosphorylated Pah1 was a substrate for dephosphorylation by Nem1-Spo7. Finally, we demonstrate the Rim11 phosphorylation of Pah1 exerted an inhibitory effect on its PAphosphatase activity by reduction of its catalytic efficiency. Mutational analysis of the major phosphorylation sites (Thr163, Thr164, and Ser602) indicated that Rim11-mediated phosphorylation at these sites was required to ensure Nem1-Spo7-dependent localization of the enzyme to the membrane. Overall, these findings advance our understanding of the phosphorylation-mediated regulation of Pah1 function in lipid synthesis. [ABSTRACT FROM AUTHOR]

Phosphorylation levels of glycogen synthase kinase 3ß (GSK3ß) negatively correlated with psychomotor stimulant-induced locomotor activity. Locomotor sensitization induced by psychomotor stimulants was previously shown to selectively accompany the decrease of GSK3ß phosphorylation in the nucleus accumbens (NAcc) core, suggesting that intact GSK3ß activity in this region is necessary for psychomotor stimulants to produce locomotor sensitization. Similarly, GSK3ß in the NAcc was also implicated in mediating the conditioned effects formed by the associations of psychomotor stimulants. However, it remains undetermined whether GSK3ß plays a differential role in the two sub-regions (core and shell) of the NAcc in the expression of drug-conditioned behaviors. In the present study, we found that GSK3ß phosphorylation was significantly lower in the NAcc shell obtained from rats expressing amphetamine (AMPH)-induced conditioned locomotor activity. Further, we demonstrated that these effects were normalized by treatment with lithium chloride, a GSK3ß inhibitor. These results suggest that the behavior produced by AMPH itself and a conditioned behavior formed by associations with AMPH are differentially mediated by the two sub-regions of the NAcc. [ABSTRACT FROM AUTHOR]

Alterations in prolyl oligopeptidase (PREP) activity have been connected, for example, with bipolar and major depressive disorder, and several studies have reported that lack or inhibition of PREP blocks the effects of lithium on inositol 1,4,5‐triphosphate (IP3) levels. However, the impact of PREP modulation on other intracellular targets of lithium, such as glycogen synthase kinase 3 beta (GSK3b) or protein kinase B (Akt), has not been studied. We recently found that PREP regulates protein phosphatase 2A (PP2A), and because GSK3b and Akt are PP2A substrates, we studied if PREP‐related lithium insensitivity is dependent on PP2A. To assess this, HEK‐293 and SH‐SY5Y cells with PREP deletion or PREP inhibition (KYP‐2047) were exposed to lithium, and thereafter, the phosphorylation levels of GSK3b and Akt were measured by Western blot. As expected, PREP deletion and inhibition blocked the lithium‐induced phosphorylation on GSK3b and Akt in both cell lines. When lithium exposure was combined with okadaic acid, a PP2A inhibitor, KYP‐2047 did not have effect on lithium‐induced GSK3b and Akt phosphorylation. Therefore, we conclude that PREP deletion or inhibition blocks the intracellular effects of lithium on GSK3b and Akt via PP2A activation. [ABSTRACT FROM AUTHOR]

Interleukin (IL)‐37 diminishes a variety of inflammatory responses through ligation to its receptor IL‐1R8/Sigirr. Sigirr is a Toll like receptor/IL‐1R family member. We have shown that Sigirr is not stable in response to IL‐37 treatment. IL‐37‐induced Sigirr degradation is mediated by the ubiquitin‐proteasome system, and the process is reversed by a deubiquitinase, USP13. However, the molecular mechanisms by which USP13 regulates Sigirr stability have not been revealed. In this study, we investigate the roles of glycogen synthesis kinase 3β (GSK3β) in Sigirr phosphorylation and stability. IL‐37 stimulation induced Sigirr phosphorylation and degradation, as well as activation of GSK3β. Inhibition of GSK3β attenuated IL‐37‐induced Sigirr phosphorylation, while exogenous expressed GSK3β promoted Sigirr phosphorylation at threonine (T)372 residue. Sigirr association with GSK3β was detected. Amino acid residues 51–101 in GSK3β were identified as the Sigirr binding domain. These data indicate that GSK3β mediates IL‐37‐induced threonine phosphorylation of Sigirr. Further, we investigated the role of GSK3β‐mediated phosphorylation of Sigirr in Sigirr degradation. Inhibition of GSK3β attenuated IL‐37‐induced Sigirr degradation, while T372 mutant of Sigirr was resistant to IL‐37‐mediated degradation. Furthermore, inhibition of Sigirr phosphorylation prevented Sigirr internalization and association with USP13, suggesting GSK3β promotes Sigirr degradation through disrupting Sigirr association with USP13. [ABSTRACT FROM AUTHOR]

Aim: Typical features of human osteosarcoma are highly invasive and migratory capacities. Our study aimed to investigate the roles of glycogen synthase kinase 3β (GSK3β) in human osteosarcoma metastasis. Methods: GSK3β expressions in clinical osteosarcoma tissues with or without metastasis were examined by immunohistochemical staining. The expressions of GSK3β, p-GSK3βSer9, and p-GSK3βTyr216 in human osteoblast cells (hFOB1.19) and human osteosarcoma cells (MG63, SaOS-2, and U2-OS) were detected by Western blotting. The GSK3β activity was measured by non-radio isotopic in vitro kinase assay. Migration and invasion abilities of MG-63 cells treated with small-molecular GSK3β inhibitors were respectively examined by monolayer-based wound-healing assay and transwell assay. The mRNA expressions of GSK3β, matrix metalloproteinase-2 (MMP-2), MMP-9, phosphatase with tensin homology (PTEN), and focal adhesion kinase (FAK) were detected after siRNA transfection for 72 h. Meanwhile, protein expressions of GSK3β, FAK, p-FAKY397, PTEN, MMP-2, and MMP-9 were measured by Western blotting. Results: Clinical osteosarcoma tissues with metastasis showed higher GSK3β expressions. MG63 and U2-OS cells that were easy to occur metastasis showed significantly higher expressions and activities of GSK3β than SaOS-2 cells. Inhibition of GSK3β with small-molecular GSK3β inhibitors in MG63 cells significantly attenuated cell migration and invasion. These effects were associated with reduced expressions of MMP-2 and MMP-9. Moreover, increased PTEN and decreased p-FAKY397 expressions were observed following GSK3β knockdown by siRNA transfection. Conclusion: GSK3β might promote osteosarcoma invasion and migration via pathways associated with PTEN and phosphorylation of FAK. [ABSTRACT FROM AUTHOR]

In Alzheimer's disease (AD), the enzyme acetylcholinesterase (AChE) co‐localizes with hyperphosphorylated tau (P‐tau) within neurofibrillary tangles. Having demonstrated that AChE expression is increased in the transgenic mouse model of tau Tg‐VLW, here we examined whether modulating phosphorylated tau levels by over‐expressing wild‐type human tau and glycogen synthase kinase‐3β (GSK3β) influences AChE expression. In SH‐SY5Y neuroblastoma cells expressing higher levels of P‐tau, AChE activity and protein increased by (20% ± 2%) and (440% ± 150%), respectively. Western blots and qPCR assays showed that this increment mostly corresponded to the cholinergic ACHE‐T variant, for which the protein and transcript levels increased ~60% and ~23%, respectively. Moreover, in SH‐SY5Y cells differentiated into neurons by exposure to retinoic acid (10 µM), over‐expression of GSK3β and tau provokes an imbalance in cholinergic activity with a decrease in the neurotransmitter acetylcholine in the cell (45 ± 10%). Finally, we obtained cerebrospinal fluid (CSF) from AD patients enrolled on a clinical trial of tideglusib, an irreversible GSK3β inhibitor. In CSF of patients that received a placebo, there was an increase in AChE activity (35 ± 16%) respect to basal levels, probably because of their treatment with AChE inhibitors. However, this increase was not observed in tideglusib‐treated patients. Moreover, CSF levels of P‐tau at the beginning measured by commercially ELISA kits correlated with AChE activity. In conclusion, this study shows that P‐tau can modulate AChE expression and it suggests that AChE may possibly increase in the initial phases of AD. [ABSTRACT FROM AUTHOR]

HtrA2, a proapoptotic mitochondrial serine protease, promotes cellular protection against oxidative damage. Literature reports show positive correlation between loss of HtrA2 protease activity and Parkinson's Disease (PD) susceptibility. Homozygous loss-of-function mutations in murine-HtrA2, and when they rarely occur in humans result in severe neurodegeneration and infantile death. Here, we report a novel heterozygous pathogenic HTRA2 variant, c.725C > T (p.T242M) in Indian PD patients. Although, this mutation exhibits no significant conformational changes compared to the wild-type, functional studies with HtrA2-T242M transfected neurons reveal common features of PD pathogenesis such as dysfunction, altered morphology and mitochondrial membrane depolarization. Despite exhibiting two-fold decrease in enzyme activity, observation of excessive cell-death due to over-expression of the mutant has been correlated with it being constitutively active. This interesting behavioral anomaly has been attributed to the loss of phosphorylation-mediated regulatory checkpoint at the T242M mutation site that is otherwise controlled by glycogen synthase kinase-3β (GSK-3β). This study, with seamless amalgamation of biophysical and biomedical research unravels a mechanistic pathway of HtrA2 regulation and delineates its biological role in PD. Therefore, this investigation will not only prove beneficial toward devising therapeutic strategies against HtrA2-associated diseases mediated by GSK-3β but also suggest new avenues for treatment of Parkinsonian phenotype. [ABSTRACT FROM AUTHOR]

Serotonin (5‐HT) transporter (SERT) plays a crucial role in serotonergic transmission in the central nervous system, and any aberration causes serious mental illnesses. Nevertheless, the cellular mechanisms that regulate SERT function and trafficking are not entirely understood. Growing evidence suggests that several protein kinases act as modulators. Here, we delineate the molecular mechanisms by which glycogen synthase kinase‐3ß (GSK3ß) regulates SERT. When mouse striatal synaptosomes were treated with the GSK3α/ß inhibitor CHIR99021, we observed a significant increase in SERT function, Vmax, surface expression with a reduction in 5‐HT Km and SERT phosphorylation. To further study how the SERT molecule is affected by GSK3α/ß, we used HEK‐293 cells as a heterologous expression system. As in striatal synaptosomes, CHIR99021 treatment of cells expressing wild‐type hSERT (hSERT‐WT) resulted in a time and dose‐dependent elevation of hSERT function with a concomitant increase in the Vmax and surface transporters because of reduced internalization and enhanced membrane insertion; silencing GSK3α/ß in these cells with siRNA also similarly affected hSERT. Converting putative GSK3α/ß phosphorylation site serine at position 48 to alanine in hSERT (hSERT‐S48A) completely abrogated the effects of both the inhibitor CHIR99021 and GSK3α/ß siRNA. Substantiating these findings, over‐expression of constitutively active GSK3ß with hSERT‐WT, but not with hSERT‐S48A, reduced SERT function, Vmax, surface density, and enhanced transporter phosphorylation. Both hSERT‐WT and hSERT‐S48A were inhibited similarly by PKC activation or by inhibition of Akt, CaMKII, p38 MAPK, or Src kinase. These findings provide new evidence that GSK3ß supports basal SERT function and trafficking via serine‐48 phosphorylation. [ABSTRACT FROM AUTHOR]

Outer membrane vesicles (OMVs) are nanosized vesicles produced by the gut microbiota (GM). The GM is well‐known to be involved in the pathological process of Alzheimer's disease (AD). However, the mechanism of OMVs is not clear. In the present study, we demonstrated the involvement of OMVs in the development of cognitive (learning and memory) dysfunction induced by blood–brain barrier (BBB) disruption. More important, further study showed that OMVs induced tau phosphorylation by activating glycogen synthase kinase 3β (GSK‐3β) in the hippocampus. OMVs activated astrocytes and microglia, increased secretion of inflammatory cytokines (nuclear factor κB, interleukin‐1β, and tumour necrosis factor‐α) in the hippocampus. Therefore, OMVs increase the permeability of the BBB and promote the activation of astrocytes and microglia, inducing an inflammatory response and tau hyperphosphorylation by activating the GSK‐3β pathway and finally leading to cognitive impairment. [ABSTRACT FROM AUTHOR]

Objective: Acute carbon monoxide （CO）poisoning can cause delayed neurological sequelae (DNS). Glycogen synthase kinase 3β (GSK-3β) /Tau protein pathway is reported to play a key role in neurological abnormalities. In the present study, we aimed to determine the role of GSK-3β/Tau in DNS following acute CO poisoning. Methods: 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), a specific non-competitive inhibitor of GSK-3β, was used to inhibit GSK-3β. Twenty-four male Sprague-Dawley rats were randomly assigned to the three groups: Control group, CO group and CO-TDZD-8 group. Rats breathed 1000 ppm CO for 40 minutes and then 3000 ppm for up to 20 minutes until they lost consciousness. TDZD-8 (1 mg/kg) was administered intravenously three times after the end of CO exposure at 0, 24, 48 hours late. Learning and memory abilities were observed using the Morris Water Maze (MWM). Brain histological changes were evaluated by hematoxylin-eosin staining. Moreover, the expression levels of Tau and GSK-3β were detected after acute carbon monoxide poisoning. Results: TDZD-8 significantly attenuated the learning and memory dysfunction induced by acute CO poisoning, ameliorated the histology structure of damaged neural cells in cortex and hippocampus CA1 area. TDZD-8 clearly decreased p-Tau expression, reversed the reduction of p-GSK-3β induced by acute CO poisoning. Conclusions: The therapeutic effect of TDZD-8 in alleviating DNS caused by acute CO poisoning is related to the inactivation of Tau by intensifying the level of GSK-3β phosphorylation. [ABSTRACT FROM AUTHOR]

[Display omitted] Glycogen synthase kinase 3 (GSK-3) is a highly conserved protein serine/threonine kinase that plays a central role in a wide variety of cellular processes to coordinate catabolic and anabolic pathways and regulate cell growth and fate. There is increasing evidence showing that abnormal glycogen synthase kinase 3 (GSK-3) is associated with the pathogenesis and progression of many disorders, such as cancer, diabetes, psychiatric diseases, and neurodegenerative diseases. In this review, we summarize recent findings about the regulatory role of GSK-3 in the occurrence and development of multiple neurodegenerative diseases, mainly focusing on Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The aim of this study is to provide new insight into the shared working mechanism of GSK-3 as a therapeutic target of multiple neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease (AD). It is shown that apoE4 preferentially undergoes aberrant cleavage in neurons, yielding neurotoxic C-terminal-truncated apoE4 fragment. Endoplasmic reticulum (ER) stress has also been known to be involved in the pathogenesis of AD. However, little is known about the contribution of ER stress to the neurotoxicity of apoE4 fragment. In the present study, we established the neuron-specific expression human C-terminal-truncated apoE4(1-272) fragment transgenic mice and also transfected apoE4(1-272) fragment in neuroblastoma N2a cells. We found that human apoE4(1-272) fragment could trigger ER stress as evidenced by increasing the expression of ER stress markers both in vivo and in vitro. Meanwhile, the apoE4(1-272) transgenic mice presented obviously AD-like neuropathological changes, including the impairment of spatial learning and memory, prominent axonal morphological changes, and hyperphosphorylation of tau. At the same time, we also found that glycogen synthase kinase-3 activities were significantly increased. Furthermore, these neuropathological changes, especially tau hyperphosphorylation and axonal transport impairment, were significantly rescued by the ER stress protector 4-phenylbutyric acid (4-PBA) in apoE4(1-272)-transfected N2a cells. Pretreatment with 4-PBA not only decreased the protein expression of immunoglobulin binding protein (BiP) and C/EBP-homologous protein (CHOP), but also significantly reversed these defects in axonal transport. These results suggested that the neurotoxic effects of apoE4(1-272) fragment found in AD subjects, at least in part, through triggering ER stress and inducing tau hyperphosphorylation, led to the enduring impairment of axonal transport. [ABSTRACT FROM AUTHOR]

Lipid homeostasis is critically dependent on the liver. Hepatic genes involved in lipid biosynthesis are controlled by combinatorial actions of multiple transcription factors that include three sterol regulatory element binding proteins (SREBPs), carbohydrate responsive element binding protein, liver X receptors, and others. SREBP-1c, a seminal regulator of de novo lipogenesis, resides in the endoplasmic reticulum as a transcriptionally inert precursor and must undergo a regulated intramembrane proteolysis (RIP) prior to its nuclear translocation as a bone fide transcription factor. The regulation of biosynthesis, turnover and actions of SREBP-1c and lipogenesis are mechanistically linked to signaling kinases, canonically induced by macronutrients and insulin. Here, we briefly review the evidence showing that phosphorylation of SREBP-1c and its interacting partners, catalyzed by phosphatidyl inositol-3-kinase, protein kinase B, mechanistic target of rapamycin complex 1 and 2, mitogen activated protein kinases, glycogen synthase kinase-3β, protein kinase A and 5′ adenosine monophosphate-activated protein kinase regulates the mechanisms of RIP and stability of SREBP-1c and de novo lipogenesis. • Sterol response element binding proteins (SREBPs) are key regulators of lipid homeostasis • Biosynthesis, proteolytic maturation and nuclear actions of SREBPs are dynamically regulated by macronutrients and insulin • Phosphorylation-dependent proteostasis of SREBPs and hepatic lipogenesis are inextricably linked • Kinases controlling proteostasis of SREBPs represent attractive drug targets for dyslipidemia and type-2 diabetes mellitus [ABSTRACT FROM AUTHOR]

Portal hypertension is the primary cause of complications in patients with chronic liver diseases, and markedly impacts metabolism within the nervous system. Until recently, the role of portal hypertension in hepatocellular metabolism was unclear. The present study demonstrated that an increase in extracellular pressure significantly decreased hepatocellular glycogen concentrations in HepG2 and HL-7702 cells. In addition, it reduced glycogen synthase activity, by inhibiting the phosphorylation of glycogen synthase 1. RNA-seq analysis revealed that mechanical pressure suppressed glycogen synthesis by activating the p53/phosphatase and tensin homolog pathway, further suppressing glycogen synthase activity. The present study revealed an association between mechanical pressure and hepatocellular glycogen metabolism, and identified the regulatory mechanism of glycogen synthesis under pressure. [ABSTRACT FROM AUTHOR]

Living fungal mycelium with abolished ability to form fruiting bodies is a self-healing substance, which is particularly valuable for further engineering and development as materials sensing environmental changes and secreting signals. Suppression of fruiting body formation is also a useful tool for maintaining the stability of a mycelium-based material with ease and lower cost. The objective of this study was to provide a biochemical solution to regulate the fruiting body formation, which may replace heat killing of mycelium in practice. The concentrations of glycogen synthase kinase-3 (GSK-3) inhibitors, such as lithium chloride or CHIR99021 trihydrochloride, were found to directly correlate with the development of fruiting bodies in the mushroom forming fungi such as Coprinopsis cinerea and Pleurotus djamor. Sensitive windows to these inhibitors throughout the fungal life cycle were also identified. We suggest the inclusion of GSK-3 inhibitors in the cultivation recipes for inhibiting fruiting body formation and regulating mycelium growth. This is the first report of using a GSK-3 inhibitor to suppress fruiting body formation in living fungal mycelium-based materials. It provides an innovative strategy for easy, reliable, and low cost maintenance of materials containing living fungal mycelium. [ABSTRACT FROM AUTHOR]

The biological relation between ubiquitin ligases and their substrates has been largely unclear. We previously developed a method—differential proteomics‐based identification of ubiquitylation substrates (DiPIUS)—for the comprehensive identification of substrates for a given ubiquitin ligase. We have now applied DiPIUS to the F‐box protein Fbxw7 in three cell lines (mHepa, Neuro2A and C2C12) and thereby identified Krüppel‐like factor 7 (KLF7) as a candidate substrate of the SCFFbxw7 ubiquitin ligase complex. KLF7 was shown to interact with Fbxw7 and to undergo Fbxw7‐mediated polyubiquitylation. The stability of KLF7 was increased by depletion of Fbxw7, mutation of a putative Cdc4 phosphodegron (CPD) of KLF7 or exposure to inhibitors of glycogen synthase kinase‐3 (GSK‐3). Over‐expression of Fbxw7 in Neuro2A cells down‐regulated expression of the p21Cip1 gene, which is a transcriptional target of KLF7 in neuronal differentiation and maintenance. Despite the presence of an almost identical CPD sequence in KLF6, the closest paralog of KLF7, mutation of this sequence affected neither the interaction of KLF6 with Fbxw7 nor its half‐life. Our results suggest that KLF7, but not KLF6, is a bona fide substrate of SCFFbxw7, and that control of KLF7 abundance by SCFFbxw7 might contribute to the regulation of neuronal differentiation and maintenance. [ABSTRACT FROM AUTHOR]

The sequencing of the zebrafish genome, the emergence of powerful gene-editing tools, and the development of in vivo imaging techniques have propelled the economical zebrafish into prominence as a biomedical research model. Neurodegenerative disorders with a cholinergic component, such as Alzheimer's and Parkinson's diseases, are currently modeled using zebrafish. Still, the utility of zebrafish as a research model will not be fully realized until their neurophysiological properties are thoroughly characterized. In mammals, the coupling of cholinergic receptors to the phosphorylation of glycogen synthase kinase-3 β (GSK3β) and extracellular signal-regulated kinase 1/2 (ERK1/2) is of critical importance to cognitive processes and imparts protection against neuropathogenic events. Similarly, it is known that cholinergic receptors are required for learning and memory in zebrafish and that in vivo activation of cholinergic receptors induces transient changes in evoked synaptic transmission in the telencephalon. However, the intracellular events mediating cholinergic processes in zebrafish have yet to be elucidated. In the current study, an ex vivo drug treatment assay was used to demonstrate that carbachol (CCh)-mediated cholinergic stimulation of the intact adult zebrafish brain induces phosphorylation of GSK3β and ERK1/2 in the zebrafish telencephalon. These findings suggest GSK3β and ERK1/2 may underly cognitive processes in zebrafish. [ABSTRACT FROM AUTHOR]

Glycogen synthase kinase-3 (GSK3) is over-expressed and hyperactivated in non-small cell lung carcinoma (NSCLC) and plays a role in ensuring the correct alignment of chromosomes on the metaphase plate during mitosis through regulation of microtubule stability. This makes the enzyme an attractive target for cancer therapy. We examined the effects of a selective cell-permeant GSK3 inhibitor (CHIR99021), used alone or in combination with paclitaxel, using an in vitro cell growth assay, a quantitative chromosome alignment assay, and a tumor xenograft model. CHIR99021 inhibits the growth of human H1975 and H1299 NSCLC cell lines in a synergistic manner with paclitaxel. CHIR99021 and paclitaxel promoted a synergistic defect in chromosomal alignment when compared to each compound administered as monotherapy. Furthermore, we corroborated our in vitro findings in a mouse tumor xenograft model. Our results demonstrate that a GSK3 inhibitor and paclitaxel act synergistically to inhibit the growth of NSCLC cells in vitro and in vivo via a mechanism that may involve converging modes of action on microtubule spindle stability and thus chromosomal alignment during metaphase. Our findings provide novel support for the use of the GSK3 inhibitor, CHIR99021, alongside taxol-based chemotherapy in the treatment of human lung cancer. [ABSTRACT FROM AUTHOR]

Lymphatic vessels play an important role in health and in disease. In this study, we evaluated the effects of GSK3-β inhibition on lung lymphatic endothelial cells in vitro. Pharmacological inhibition and silencing of GSK3-β resulted in increased lymphangiogenesis of lung lymphatic endothelial cells. To investigate mechanisms of GSK3-β-mediated lymphangiogenesis, we interrogated the mammalian/mechanistic target of rapamycin pathway and found that inhibition of GSK3-β resulted in PTEN activation and subsequent decreased activation of AKT, leading to decreased p-P70S6kinase levels, indicating inhibition of the mTOR pathway. In addition, consistent with a negative role of GSK3-β in β-catenin stability through protein phosphorylation, we found that GSK3-β inhibition resulted in an increase in β-catenin levels. Simultaneous silencing of β-catenin and inhibition of GSK3-β demonstrated that β-catenin is required for GSK3-β-induced lymphangiogenesis. [ABSTRACT FROM AUTHOR]

The p63 transcription factor, a p53 family protein, regulates genes involved in various cellular processes, including cell growth and differentiation. We previously showed that RNAbinding motif protein (Rbm38) is a p63 target and, in turn, regulates p63αmRNA stability by binding to the AU/U-rich element in its 3'UTR. Interestingly, Rbm38 can be phosphorylated at serine 195, altering its ability to regulate mRNA translation. However, whether the Ser-195 phosphorylation affects Rbm38's ability to destabilize p63 mRNA remains unclear. Here, using MCF7 and HaCaT cells, we showed that ectopic expression of phosphomimetic Rbm38-S195D increases, whereasWTRbm38 and nonphosphorylatable Rbm38-S195A decrease p63αprotein and transcript levels. We also found that upon activation of glycogen synthase kinase 3 (GSK3β), phosphorylation of Rbm38 at Ser-195 is increased, enhancing p63αexpression in an Rbm38-dependent manner. To confirm this, we generated mouse embryo fibroblasts (MEFs) in which Ser-193 in mouse Rbm38 (equivalent to Ser-195 in human Rbm38) was substituted with aspartic acid (Rbm38S193D/S193D) or alanine (Rbm38S193A/S193A). We observed that the p63 transcript level was increased in Rbm38S193D/S193D MEFs, but decreased in Rbm38S193A/S193A MEFs. Mechanistically, we found that WT Rbm38, but not Rbm38-S195D, is required for p63 mRNA degradation mediated by microRNA 203 (miR203). Furthermore, we noted that Argonaute 2 (Ago2), a key regulator in microRNA-mediatedmRNAdecay, associates withWTRbm38, and this association was reduced by Ser-195 phosphorylation. Together, our results reveal a critical mechanism by which Ser- 195 phosphorylation in Rbm38 increases p63 expression by attenuating the association of Rbm38 with the Ago2-miR203 complex. [ABSTRACT FROM AUTHOR]

Abstract Background Drinking water from copper vessels and aerobic exercise have been the known facts for cardioprotection. Our previous report explored the significant cardioprotective potential of copper and exercise training by increasing phosphorylation of GSK-3β and anti-oxidant potential. Objective Present study focuses the therapeutic potential of CuNP and exercise training through their molecular interaction with GSK-3β, inflammatory cytokinin, oxidative stress and necrosis. Methods The Myocardial damage was assessed by estimating the serum nitrite/nitrate concentration, increased CKMB, LDH, cTnI level, oxidative stress, inflammatory cytokinin and structural abnormalities in I/R insulted rats. Expression of Akt/pAkt and GSK-3β/pGSK-3β was measured by western blotting. Result Treatment with CuNP (1 mg/kg/day, p.o., 4 weeks) and exercise training (swimming, 90 min/4 weeks) either alone or in combination markedly reduced I/R induced myocardial damage by attenuating biochemical and structural alteration. A significant reduction in oxidative stress and inflammatory mediators were observed in CuNP and exercise training treatment against I/R insulted rats. Moreover, improved serum NO bioavailability was observed in CuNP and exercise treated rats. Wortmannin associated blockage of cardioprotection induced by CuNP and exercise training and up-regulation of pAkt and pGSK-3β in I/R insulted heart confirmed the GSK-3β phosphorylation potential of CuNP and exercise training and -associated cardioprotection. Conclusion Treatment with CuNP and exercise training either alone or in combination favourably phosphorylate GSK-3β kinase pathways and further diminish oxidative stress, inflammatory cytokines, apoptosis and increase serum bioavailability of NO in the I/R insulted rats which tends to protect myocardial damage. Graphical abstract Unlabelled Image Highlights • Copper nanoparticles (CuNP) provide cardioprotection against I/R -induced myocardial infarction. • This myocardial protection is associated with the inhibition of GSK-3 β. • Swimming exercise training also shows the cardioprotection against I/R -induced myocardial infarction. • Swimming exercise training associated with preconditioning may be a pivotal mechanism of protection. • Moreover, both CuNP and exercise training treatment attenuates myocardial oxidative stress and structural abnormalities. [ABSTRACT FROM AUTHOR]

Recent studies have revealed a positive therapeutic effect of dihydroartemisinin (DHA) on tumor cells. However, the underlying mechanism of this has not yet been elucidated. The present study examined the potential therapeutic role and mechanism of DHA in T-cell lymphoma cells. It was revealed that DHA inhibited the proliferation of Jurkat and HuT-78 T-cell lymphoma cells in a concentration- and time-dependent manner. Furthermore, DHA reduced c-Myc protein expression at the transcriptional level, and induced the phosphorylation of c-Myc and the degradation of c-Myc oncoprotein levels. DHA treatment resulted in decreased phosphorylation of protein kinase B (Akt) and glycogen synthase 3β (GSK3β) in T-cell lymphoma cells. In addition, DHA treatment induced cell apoptosis, which was accompanied by an increased ratio of Bax/Bcl-2. Taken together, the results of the present study suggested that DHA may exert its antitumor role by accelerating c-Myc proteolysis and inhibiting the Akt/GSK3β pathway in T-cell lymphoma cells. [ABSTRACT FROM AUTHOR]

Myofibril breakdown is a fundamental cause of muscle wasting and inevitable sequel of aging and disease. We demonstrated that myofibril loss requires depolymerization of the desmin cytoskeleton, which is activated by phosphorylation. Here, we developed a mass spectrometry-based kinase-trap assay and identified glycogen synthase kinase 3-β (GSK3-β) as responsible for desmin phosphorylation. GSK3-β inhibition in mice prevented desmin phosphorylation and depolymerization and blocked atrophy upon fasting or denervation. Desmin was phosphorylated by GSK3-β 3 d after denervation, but depolymerized only 4 d later when cytosolic Ca2+levels rose. Mass spectrometry analysis identified GSK3-β and the Ca2+-specific protease, calpain-1, bound to desmin and catalyzing its disassembly. Consistently, calpain-1 down-regulation prevented loss of phosphorylated desmin and blocked atrophy. Thus, phosphorylation of desmin filaments by GSK3-β is a key molecular event required for calpain-1-mediated depolymerization, and the subsequent myofibril destruction. Consequently, GSK3-β represents a novel drug target to prevent myofibril breakdown and atrophy. [ABSTRACT FROM AUTHOR]

Frozen-thawed boar sperm have less motility and fertility capacity in comparison to fresh sperm. Glycogen Synthase Kinase 3 (GSK3) contributes to sperm motility in fresh semen. In addition, GSK3 inhibition in boar spermatozoa in fresh semen improves motility variables. The role of GSK3 on boar cryopreserved sperm, however, is still unknown. The hypothesis in the present study was that GSK3 pathway inhibition by alsterpaullone (AST) could result in enhancement of the quality of sperm afer cryopreservation. Two different strategies were evaluated: i) AST supplementation to the freezing medium (AST + Cryo); ii) AST supplementation after sperm thawing (AST + Thaw). Sperm motility was evaluated using the CASA system and different sperm quality variables were evaluated using flow cytometry, as well as amount of GSK3 phosphorylation of thawed spermatozoa after 30 and 90 min incubation at 38.5 °C. Results indicate that AST supplementation had detrimental effects on sperm viability (live spermatozoa) and mitochondrial membrane potential when it was added after thawing ( P  <  0.05) The AST supplementation after thawing, however, had a protective effect on plasma membrane lipid disorganization ( P  <  0.05). The percentage of motile spermatozoa was not modified by AST supplementation. Nonetheless, after 30 min post-thawing, STR and LIN variables (related to straightness of the movement) as well as the percentage of rapid lineal spermatozoa were increased with both AST supplementation protocols. The GSK3α phosphorylation was not modified through the incubation time in boar thawed sperm. In summary, results do not support the idea of adding AST to the cryopreservation/thawing medium to improve boar sperm quality after cryopreservation. [ABSTRACT FROM AUTHOR]

The multifaceted glycogen synthase kinase (GSK3) has an essential role in sperm and male fertility. Since cyclic AMP (cAMP) plays an important role in sperm function, we investigated whether GSK3 and cAMP pathways may be interrelated. We used GSK3 and soluble adenylyl cyclase (sAC) knockout mice and pharmacological modulators to examine this relationship. Intracellular cAMP levels were found to be significantly lower in sperm lacking GSK3α or GSK3β. A similar outcome was observed when sperm cells were treated with SB216763, a GSK3 inhibitor. This reduction of cAMP levels was not due to an effect on sperm adenylyl cyclase but was caused by elevated phosphodiesterase (PDE) activity. The PDE4 inhibitor RS25344 or the general PDE inhibitor IBMX could restore cAMP levels in sperm lacking GSK3α or β‐isoform. PDE activity assay also showed that hyperactivated PDE4 contributes in lowering of cAMP levels in GSK3α null sperm suggesting that in wild‐type sperm PDE4 activity is kept in check by GSK3. Conversely, PKA being triggered by cAMP, affected GSK3 activity through increasing its phosphorylation. Increased GSK3 phosphorylation also occurred by inhibition of sperm specific protein phosphatase type 1, PP1γ2. The relationship between cAMP, GSK3, and PP1γ2 activities was also confirmed in sperm from sAC null mice. Pull‐down assay using recombinant PP1γ2 indicated that PKA, GSK3, and PP1γ2 could exist as a complex. Pharmacological inhibition of GSK3 in mature spermatozoa resulted in significantly reduced fertilization of eggs in vitro. Our results show that cAMP, PKA, and GSK3 are interrelated in regulation of sperm function. [ABSTRACT FROM AUTHOR]

Amyloid β (Aβ) peptide can cause neurotoxicity in Alzheimer's disease (AD). The main purpose of the present study is to investigate the protective role of asiatic acid (AA) against Aβ 25–35 -induced neurotoxicity in neuronally differentiated PC12 cells. Differentiated PC12 cells were pretreated with 5, 10 or 20 μM AA before treatment with 20 μM Aβ 25–35 . The viability and apoptosis of differentiated PC12 cells were determined by MTT assay and Annexin V-FITC/PI double staining, respectively. The mitochondrial membrane potential (MMP) of differentiated PC12 cells was analyzed by JC-1 staining. The expression levels of proteins were detected by western blot analysis. We found that AA significantly increased the viability of differentiated PC12 cells but attenuated the mitochondria-mediated apoptosis dose-dependently when challenging with Aβ 25–35 . Besides, the results of western blot analysis showed that AA prevented IκBα degradation and p65 nuclear translocation, and promoted the phosphorylation of Akt and GSK-3β in Aβ 25–35 -treated differentiated PC12 cells. Moreover, LY294002, a specific PI3K inhibitor, was found to abolish the beneficial effects of AA on Aβ 25–35 -induced apoptosis and tau protein hyperphosphorylation. Our findings demonstrated that AA protects differentiated PC12 cells from Aβ 25–35 -induced apoptosis and tau protein hyperphosphorylation, which might be partially mediated by the activation of the PI3K/Akt/GSK-3β signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract: Breast cancer was the highest incidence of tumor in women, which seriously threaten women's health. Our previous study found that the expression of IQUB (IQ motif and ubiquitin domain containing) was significantly increased in the development of breast cancer by transcriptome sequencing. However, there were no studies on the mechanism of IQUB in tumorigenesis. Further study showed that IQUB expression was significantly increased in breast cancer, which had a significantly positive correlation with pathological differentiation of breast cancer by tissue microarray analysis. Furthermore, we also discovered that IQUB overexpression could obviously promote the proliferation and migration of MCF‐7 cells and increase the proportion of MCF‐7 cells in S and G2/M phase in vitro study, while knockdown of IQUB caused inhibition of cell proliferation and migration in MDA‐MB‐231 cells and increased the proportion of MDA‐MB‐231 cells in G1 phase. Furthermore, IQUB overexpression or knockdown combined with treatment of Licl or MG‐132 showed that IQUB activated Akt to promote GSK3β phosphorylation, which in turn activated Wnt/β‐catenin signaling pathway in breast cancer cells. Taken together, these results indicated that upregulated IQUB promoted breast cancer cell proliferation and migration via activating Akt/GSK3β/β‐catenin signaling pathway, which played an important part in the tumorigenesis and development of breast cancer. [ABSTRACT FROM AUTHOR]

Abstract: Endoplasmic reticulum (ER) stress is involved in Alzheimer's disease (AD), but the mechanism is not fully understood. Here, we injected tunicamycin (TM), a recognized ER stress inducer, into the brain ventricle of Sprague‐Dawley (SD) rats to induce the unfolded protein response (UPR), demonstrated by the enhanced phosphorylation of pancreatic ER kinase (PERK), inositol‐requiring enzyme‐1 (IRE‐1) and activating transcription factor‐6 (ATF‐6). We observed that UPR induced spatial memory deficits and impairments of synaptic plasticity in the rats. After TM treatment, GSK‐3β was activated and phosphorylation of cAMP response element binding protein at Ser129 (pS129‐CREB) was increased with an increased nuclear co‐localization of pY126‐GSK‐3β and pS129‐CREB. Simultaneous inhibition of GSK‐3β by hippocampal infusion of SB216763 (SB) attenuated TM‐induced UPR and spatial memory impairment with restoration of pS129‐CREB and synaptic plasticity. We concluded that UPR induces AD‐like spatial memory deficits with mechanisms involving GSK‐3β/pS129‐CREB pathway. [ABSTRACT FROM AUTHOR]

Neurofibrillary pathology contributes to neuronal dysfunction and correlates with the clinical progression of Alzheimer’s disease (AD). Tau phosphorylation is mainly regulated by a balance of glycogen synthase kinase-3β (GSK-3β) and protein phosphatase 2A (PP2A) activities. Cornel iridoid glycoside (CIG) is a main component extracted from Cornus officinalis. The purpose of this study was to investigate the effects of CIG on GSK-3β and PP2A, thus to explore the mechanisms of CIG to inhibit tau hyperphosphorylation. The rat model of tau hyperphosphorylation was established by intraventricular injection of wortmannin and GF-109203X (GFX) to activate GSK-3β. The results showed that intragastrical administration of CIG inhibited tau hyperphosphorylation in the brain of rats induced by wortmannin/GFX. The results in vivo and in vitro exhibited that CIG inhibited tau hyperphosphorylation and GSK-3β over-activation. In the mechanism of action, CIG’s attenuating GSK-3β activity was found to be dependent on PI3K/AKT signaling pathway. PP2A catalytic C subunit (PP2Ac) siRNA abrogated the effect of CIG on PI3K/AKT/GSK-3β. Additionally and crucially, we also found that CIG inhibited the demethylation of PP2Ac at Leu309 in vivo and in vitro. It enhanced PP2A activity, decreased tau hyperphosphorylation, and protected cell morphology in okadaic acid (OA)-induced cell model in vitro. PP2Ac siRNA abated the inhibitory effect of CIG on tau hyperphosphorylation. Moreover, CIG inhibited protein phosphatase methylesterase-1 (PME-1) and demethylation of PP2Ac, enhanced PP2A activity, and decreased tau hyperphosphorylation in PME-1-transfectd cells. Taken together, CIG inhibited GSK-3β activity via promoting P13K/AKT and PP2A signaling pathways. In addition, CIG also elevated PP2A activity via inhibiting PME-1-induced PP2Ac demethylation to inhibit GSK-3β activity, thus regulated the cross-talk between GSK-3β and PP2A signaling and consequently inhibited tau hyperphosphorylation. These results suggest that CIG may be a promising agent for AD therapy. [ABSTRACT FROM AUTHOR]

Reduced hepatic expression levels of bromodomain-containing protein 7 (BRD7) have been suggested to play a role in the development of glucose intolerance in obesity. However, the molecular mechanism by which BRD7 regulates glucose metabolism has remained unclear. Here, we show that BRD7 increases phosphorylation of glycogen synthase kinase 3β (GSK3β) in response to activation of the insulin receptor-signaling pathway shortly after insulin stimulation and the nutrient-sensing pathway after feeding. BRD7 mediates phosphorylation of GSK3β at the Serine 9 residue and this effect on GSK3β occurs even in the absence of AKT activity. Using both in vitro and in vivo models, we further demonstrate that BRD7 mediates phosphorylation of ribosomal protein S6 kinase (S6K) and leads to increased phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and, therefore, relieves its inhibition of the eukaryotic translation initiation factor 4E (eIF4E). However, the increase in phosphorylation of 4E-BP1 with BRD7 overexpression is blunted in the absence of AKT activity. In addition, using liver-specific BRD7 knockout (LBKO) mice, we show that BRD7 is required for mTORC1 activity on its downstream molecules. These findings show a novel basis for understanding the molecular dynamics of glucose metabolism and suggest the unique function of BRD7 in the regulation of glucose homeostasis. [ABSTRACT FROM AUTHOR]

Past studies have linked intracellular pathways related to psychotic disorders to the GSK3B enzyme. This study aimed to investigate GSK3B protein expression and phosphorylation in drug-naïve first-episode psychosis patients (n=43) at baseline and following symptom remission, and in healthy controls (n=77). At baseline GSK3B total level was higher in patients (p<0.001). In schizophrenia spectrum patients (n=25) GSK3B total and phosphorylated levels were higher than in controls and patients with other non-affective psychotic disorders (n=18) (p<0.001; p=0.027; p=0.05 respectively). No enzyme changes were found after clinical remission. The implication of this finding for the biology of psychoses warrants further studies to clarify whether increased GSK3B may be useful as a biomarker for psychosis in general, and schizophrenia in particular. [ABSTRACT FROM AUTHOR]

Glycogen synthase kinase 3β (GSK‐3β) is a pivotal signaling node that regulates a myriad of cellular functions and is deregulated in many pathological conditions, making it an attractive therapeutic target. Inhibitory Ser‐9 phosphorylation of GSK3β by AKT is an important mechanism for negative regulation of GSK3β activity upon insulin stimulation. Here, we report that Thr‐7 and Thr‐8 residues located in the AKT/PKB substrate consensus sequence on GSK3β are essential for insulin‐stimulated Ser‐9 phosphorylation in vivo and for GSK3β inactivation. Intestinal cell kinase (ICK) phosphorylates GSK3β Thr‐7 in vitro and in vivo. Thr‐8 phosphorylation partially inhibits GSK3β, but Thr‐7 phosphorylation promotes GSK3β activity and blocks phospho‐Ser‐9‐dependent GSK3β autoinhibition. Our findings uncover novel mechanistic and signaling inputs involved in the autoinhibition of GSK3β. [ABSTRACT FROM AUTHOR]

This etiologic model proposes that Alzheimers Disease (AD) arises when an unusually rapid increase in ventricle volume triggers axon stretch that culminates in the physical separation of trans-synaptic proteins. As a result, these proteins, such as neurexin, neuroligin, N-Cadherin and Amyloid Precursor Protein (APP), experience a change in the configuration of their cytoplasmic tail, so that instead of transmitting signals to create and maintain synaptic structure they activate enzymes, and generate molecules, that stimulate neurite growth; for example, the transformation of the N-Cadherin tail dissolves its complex with presenilin and β-catenin triggering activation of glycogen synthase kinase 3 beta (GSK3β) and cytoskeletal disruption. This disruption leads to an increase in pro nerve growth factor (proNGF), a molecule that stimulates neurite growth via the p75 neurotrophin receptor (p75). GSK3β contributes to this growth by increasing microtubule instability through the phosphorylation of tau. Separation of trans-synaptic APPs leads to their cis dimerization and this stimulates production of amyloid beta (Aβ), an autocrine growth factor that interacts with both the p75 and alpha 7 nicotinic acetylcholine receptors. Cis dimerization of APPs may also allow the autophosphorylation of Y682 and T668 in the APP cytoplasmic tail, triggering activation of c-Jun N terminal kinase, Abelson kinase and cyclin dependent kinase 5, all of which play a role in neurite growth. ProNGF, Aβ and the kinase cascades work together to transform synapses into growth cones and stimulate sprouting of neuropil threads in an attempt to reconnect axons and dendrites. Neurofibrillary tangles, located in neural cell soma, consist of neurofilaments and microtubules needed to fuel this renewal of neurite growth. The model suggests that the best way to treat AD is to prevent synaptic separation by identifying individuals experiencing unusually high rates of ventricle growth and reducing this to more normal levels by shunting or some other technique. [ABSTRACT FROM AUTHOR]

Glycogen Synthase Kinase-3 (GSK-3) is a serine/threonine kinase which is ubiquitously expressed and is regarded as a regulator for various cellular events and signalling pathways. It exists in two isoforms, GSK-3α and GSK-3β and can phosphorylate a wide range of substrates. Aberrancy in the GSK-3 activity can lead to various diseases like Alzheimer's, diabetes, cancer, neurodegeneration etc., rendering it an attractive target to develop potent and specific inhibitors. The present review focuses on the recent developments in the area of GSK-3 inhibitors and also enlightens its therapeutic applicability in various disease conditions. [ABSTRACT FROM AUTHOR]

The whitefly-transmitted geminiviruses induce severe developmental abnormalities in plants. Geminivirus-encoded C4 protein functions as one of viral symptom determinants that could induce abnormal cell division. However, the molecular mechanism by which C4 contributes to cell division induction remains unclear. Here we report that tomato leaf curl Yunnan virus (TLCYnV) C4 interacts with a glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase, designed NbSKη, in Nicotiana benthamiana. Pro32, Asn34 and Thr35 of TLCYnV C4 are critical for its interaction with NbSKη and required for C4-induced typical symptoms. Interestingly, TLCYnV C4 directs NbSKη to the membrane and reduces the nuclear-accumulation of NbSKη. The relocalization of NbSKη impairs phosphorylation dependent degradation on its substrate-Cyclin D1.1 (NbCycD1;1), thereby increasing the accumulation level of NbCycD1;1 and inducing the cell division. Moreover, NbSKη-RNAi, 35S::NbCycD1;1 transgenic N. benthamiana plants have the similar phenotype as 35S::C4 transgenic N. benthamiana plants on callus-like tissue formation resulted from abnormal cell division induction. Thus, this study provides new insights into mechanism of how a viral protein hijacks NbSKη to induce abnormal cell division in plants. [ABSTRACT FROM AUTHOR]

Glycogen Synthase Kinase-3 (GSK3) was originally reported as a key enzyme of glucose homeostasis through regulation of the rate of glycogen synthesis. It has subsequently been found to influence most cellular processes, including growth, differentiation and death, as part of its role in modulating response to hormonal, nutritional and cellular stress stimuli. More than 100 protein targets for GSK3 have been proposed although only a small fraction of these have been convincingly validated in physiological cell systems. The effects of GSK3 phosphorylation on substrates include alteration of enzyme activity, protein localisation, protein:protein interaction and protein stability. This latter form of regulation of GSK3 substrates is the focus of this review. There is an ever-growing list of GSK3 substrates that upon phosphorylation are targeted to the beta-transducin repeat containing protein (β-TrCP), thereby allowing ubiquitination of bound protein by cullin-1 and so initiating destruction at the proteasome. We propose the existence of a GSK3-β-TrCP ‘destruction hit-list’ that allows co-ordinated removal (or stabilisation) of a set of proteins with a common physiological purpose, through control of GSK3. We identify 29 proteins where there is relatively strong evidence for regulation by a GSK3-β-TrCP axis and note common features of regulation and pathophysiology. Furthermore, we assess the potential of pre-phosphorylation (priming) of these targets (normally a prerequisite for GSK3 recognition) to provide a second layer of regulation delineated by the priming kinase that allows GSK3 to mark them for destruction. Finally, we discuss whether this knowledge improves options for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Chronic rhinosinusitis without nasal polyps (CRSsNP) is one of the most common otorhinolaryngologic diseases worldwide. However, the underlying mechanism remains unclear. In this study, the expression of glycogen synthase kinase 3 (GSK-3) was quantitatively evaluated in patients with CRSsNP ( n = 20) and healthy controls ( n = 20). The mRNA levels of GSK-3α and GSK-3β were examined by qPCR, the immunoreactivities of GSK-3β and nuclear factor-κB (NF-κB) were examined by immunohistochemistry (IHC) staining, and the protein levels of GSK-3β, phospho-GSK-3β (p-GSK-3β, s9) and NF-κB were examined using Western blot analysis. We found that GSK-3 was highly expressed in both CRSsNP and control groups without significant difference in both GSK-3β mRNA and protein levels. However, when compared with healthy control group, the GSK-3β activation index, defined as the ratio of GSK-3β over p-GSK-3β, was significantly decreased, whereas the NF-κB protein abundance was significantly increased in CRSsNP group ( P < 0.05). Strikingly, the GSK-3β activation index, was highly correlated with NF-κB protein level, as well as CT scores in CRSsNP group ( P < 0.05). It was also highly correlated with the mRNA expressions of inflammation-related genes, including T-bet, IFN-γ and IL-4 in CRSsNP group ( P < 0.05). Our findings suggest that GSK-3β activation index, reflecting the inhibitory levels of GSK-3β through phosphorylation, may be a potential indicator for recurrent inflammation of CRSsNP, and that the insufficient inhibitory phosphorylation of GSK-3β may play a pivotal role in the pathogenesis of CRSsNP. [ABSTRACT FROM AUTHOR]

Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3-dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ~2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3-dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3-dependent phosphorylation of multiple splicing factors and regulators of RNAbiosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3~ phosphorylated these proteins in vitro. RNASeq of WT and Gsk3 DKO ESCs identified ~190 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MSdata also identified posttranscriptional regulation of protein abundance by GSK-3, with ~47 proteins (1.4%) whose levels increased and~78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing. [ABSTRACT FROM AUTHOR]

Background A recent study found that Salvia miltiorrhiza extract substantially increased amniotic fluid index in women with oligohydramnios. Abnormal aquaporin (AQP) expression is associated with oligohydramnios. This study aims to investigate the effect of tanshinone IIA, the major lipophilic component in the root of Salvia miltiorrhiza , on the function and expression of AQPs in human amniotic epithelial WISH cells and the molecular mechanism underlying the effect. Methods WISH cells were treated with tanshinone IIA. Cell proliferation and swelling were determined by cell counting kit-8 assay and measurement of cell size, respectively. A human phospho-MAPK antibody array was used to screen tanshinone IIA-associated signaling molecules. The protein expression of AQPs, glycogen synthase kinase 3β (GSK-3β), and phosphorylated GSK-3β (p-GSK-3β) was analyzed by Western blot. Result Tanshinone IIA significantly inhibited WISH cell proliferation at ≥ 50 μM ( P < 0.05) and the IC 50 was 35 μM. Cell size was significantly increased by the treatment with 35 μM tanshinone IIA for 24 h ( P < 0.05). WISH cells expressed AQP1, AQP3, AQP8, and AQP9 predominantly in cytoplasm. The dose of tanshinone IIA to maximally induce AQP1 and AQP3 expression was 35 μM; for AQP8 and AQP9, the dose of maximal stimulation was 5 μM (All P < 0.05). Tanshinone IIA (35 μM)-mediated up-regulation of AQP8, AQP9, AQP1, and AQP3 peaked at 6, 12, 24, and 24 hours of treatment, respectively. Tanshinone IIA increased p-GSK-3β level significantly ( P < 0.05). Blockade of GSK-3β expression by siRNA and inhibition of GSK-3β activity by lithium chloride significantly reduced tanshinone IIA-induced AQP1 and AQP3 expression (All P < 0.05). Conclusion Tanshinone IIA increases AQPs expression in WISH cells by stimulating p-GSK-3β. AQP8 responds to tanshinone IIA the most rapidly compared with AQP9, AQP1, and AQP3 in WISH cells. [ABSTRACT FROM AUTHOR]

Oxidative stress impacts normal cellular function leading to the pathogenesis of various diseases including pulmonary illnesses. Protein arginine methyltransferase 4 (PRMT4) is critical for normal lung alveolar epithelial cell development; however, the regulation of PRMT4 within such pulmonary diseases has yet to be elucidated. Using biochemical approaches, we uncovered that peroxide (H2O2) treatment decreases PRMT4 protein stability in murine lung epithelial (MLE12) cells to impede cell migration. Protein kinase glycogen synthase kinase 3β (GSK-3β) interacts with PRMT4 and catalyzes PRMT4 T132 phosphorylation that protects PRMT4 from ubiquitin proteasomal degradation. H2O2 downregulates GSK-3β to reduce PRMT4 at protein level. PRMT4 promotes cell migration and H2O2 degrades PRMT4 to inhibit lung epithelial cell migration. These observations demonstrate that oxidative stress destabilizes PRMT4 via GSK-3β signaling to impede lung epithelial cell migration that may hinder the lung repair and regeneration process. [ABSTRACT FROM AUTHOR]

Glycogen synthase kinase 3β (GSK-3β) is a key downstream protein in the PI3K/Akt pathway. Phosphorylation of serine 9 of GSK-3β (GSK-3β activity inhibition) promotes cell survival. In this study, we examined changes in expressions of GSK-3β and phosphorylation of GSK-3β (p-GSK-3β) in the gerbil hippocampal CA1 area after 5 min of transient cerebral ischemia. GSK-3β immunoreactivity in the CA1 area was increased in pyramidal cells at 6 h after ischemia-reperfusion. It was decreased in CA1 pyramidal cells from 12 h after ischemia-reperfusion, and hardly detected in the CA1 pyramidal cells at 5 days after ischemia-reperfusion. p-GSK-3β immunoreactivity was slightly decreased in CA1 pyramidal cells at 6 and 12 h after ischemia-reperfusion. It was significantly increased in these cells at 1 and 2 days after ischemia-reperfusion. Five days after ischemia-reperfusion, p-GSK-3β immunoreactivity was hardly found in CA1 pyramidal cells. However, p-GSK-3β immunoreactivity was strongly expressed in astrocytes primarily distributed in strata oriens and radiatum. In conclusion, GSK-3β and p-GSK-3β were significantly changed in pyramidal cells and/or astrocytes in the gerbil hippocampal CA1 area following 5 min of transient cerebral ischemia. This finding indicates that GSK-3β and p-GSK-3β are closely related to delayed neuronal death. [ABSTRACT FROM AUTHOR]

Signaling networks that regulate cellular proliferation often involve complex interactions between several signaling pathways. In this manuscript we review the crosstalk between the Casein Kinase II (CK2) and Glycogen Synthase Kinase-3 (GSK-3) pathways that plays a critical role in the regulation of cellular proliferation in leukemia. Both CK2 and GSK-3 are potential targets for anti-leukemia treatment. Previously published data suggest that CK2 and GSK-3 act synergistically to promote the phosphatidylinositol-3 kinase (PI3K) pathway via phosphorylation of PTEN. More recent data demonstrate another mechanism through which CK2 promotes the PI3K pathway – via transcriptional regulation of PI3K pathway genes by the newly-discovered CK2-Ikaros axis. Together, these data suggest that the CK2 and GSK-3 pathways regulate AKT/PI3K signaling in leukemia via two complementary mechanisms: a) direct phosphorylation of PTEN and b) transcriptional regulation of PI3K-promoting genes. Functional interactions between CK2, Ikaros and GSK3 define a novel signaling network that regulates proliferation of leukemia cells. This regulatory network involves both direct posttranslational modifications (by CK and GSK-3) and transcriptional regulation (via CK2-mediated phosphorylation of Ikaros). This information provides a basis for the development of targeted therapy for leukemia. [ABSTRACT FROM AUTHOR]

Glycogen Synthase Kinase-3 (GSK3 or GSK-3) is a promiscuous protein kinase and its phosphorylation of its diverse substrates has major influences on many areas of physiology and pathology, including cellular metabolism, lineage commitment and neuroscience. GSK3 was one of the first identified substrates of the heavily studied oncogenic kinase AKT, phosphorylation by which inhibits GSK3 activity via the formation of an autoinhibitory pseudosubstrate sequence. This has led to investigation of the role of GSK3 inhibition as a key component of the cellular responses to growth factors and insulin, which stimulate the class I PI 3-Kinases and in turn AKT activity and GSK3 phosphorylation. GSK3 has been shown to phosphorylate several upstream and downstream components of the PI3K/AKT/mTOR signalling network, including AKT itself, RICTOR, TSC1 and 2, PTEN and IRS1 and 2, with the potential to apply feedback control within the network. However, it has been clear for some time that functionally distinct, insulated pools of GSK3 exist which are regulated independently, so that for some GSK3 substrates such as β-catenin, phosphorylation by GSK3 is not controlled by input from PI3K and AKT. Instead, as almost all GSK3 substrates require a priming phosphorylated residue to be 4 amino acids C-terminal to the Ser/Thr phosphorylated by GSK3, the predominant form of regulation of the activity of GSK3 often appears to be through control over these priming events, specific to individual substrates. Therefore, a major role of GSK3 can be viewed as an amplifier of the electrostatic effects on protein function which are caused by these priming phosphorylation events. Here we discuss these different aspects to GSK3 regulation and function, and the functions of GSK3 as it integrates with signalling through the PI3K-AKT-mTOR signalling axis. [ABSTRACT FROM AUTHOR]

Diabetes is an independent risk factor for myocardial ischemia, and many epidemiological data and laboratory studies have revealed that diabetes significantly exacerbated myocardial ischemia/reperfusion injury and ameliorated protective effects. The present study aimed to determine whether pharmacological postconditioning with atorvastatin calcium lessened diabetic myocardial ischemia/reperfusion injury, and investigated the role of glycogen synthase kinase (GSK3β) in this. A total of 72 streptozotocin-induced diabetic rats were randomly divided into six groups, and 24 age-matched male non-diabetic Sprague-Dawley rats were randomly divided into two groups. Rats all received 40 min myocardial ischemia followed by 180 min reperfusion, except sham-operated groups. Compared with the non-diabetic ischemia/reperfusion model group, the diabetic ischemia/reperfusion group had a comparable myocardial infarct size, but a higher level of serum cardiac troponin I (cTnI) and morphological alterations to their myocardial cells. Compared with the diabetic ischemia/reperfusion group, the group that received pharmacological postconditioning with atorvastatin calcium had smaller myocardial infarct sizes, lower levels of cTnI, reduced morphological alterations to myocardial cells, higher levels of p-GSK3β, heat shock factor (HSF)-1 and heat shock protein (HSP)70. The cardioprotective effect conferred by atorvastatin calcium did not attenuate myocardial ischemia/reperfusion injury following application of TDZD-8, which phosphorylates and inactivates GSK3β. Pharmacological postconditioning with atorvastatin calcium may attenuate diabetic heart ischemia/reperfusion injury in the current context. The phosphorylation of GSK3β serves a critical role during the cardioprotection in diabetic rats, and p-GSK3β may accelerate HSP70 production partially by activating HSF-1 during myocardial ischemic/reperfusion injury. [ABSTRACT FROM AUTHOR]

The selective and neuronal activity-dependent degradation of synaptic proteins appears to be crucial for long-term synaptic plasticity. One such protein is activity-regulated cytoskeleton-associated protein (Arc), which regulates the synaptic content of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), excitatory synapse strength and dendritic spine morphology. The levels of Arc protein are tightly regulated, and its removal occurs via proteasome-mediated degradation that requires prior ubiquitination. Glycogen synthase kinases α and β (GSK3α, GSKβ; collectively named GSK3α/β) are serine-threonine kinases with abundant expression in the central nervous system. Both GSK3 isozymes are tonically active under basal conditions, but their activity is regulated by intra- and extracellular factors, intimately involved in neuronal activity. Similar to Arc, GSK3α and GSK3β contribute to synaptic plasticity and the structural plasticity of dendritic spines. The present study identified Arc as a GSK3α/β substrate and showed that GSKβ promotes Arc degradation under conditions that induce de novo Arc synthesis. We also found that GSK3a/b inhibition potentiated spine head thinning that was caused by the prolonged stimulation of N-methyl-D-aspartate receptors (NMDAR). Furthermore, overexpression of Arc mutants that were resistant to GSK3β-mediated phosphorylation or ubiquitination resulted in a stronger reduction of dendritic spine width than wildtype Arc overexpression. Thus, GSK3b terminates Arc expression and limits its effect on dendritic spine morphology. Taken together, the results identify GSK3α/β-catalyzed Arc phosphorylation and degradation as a novel mechanism for controlling the duration of Arc expression and function. [ABSTRACT FROM AUTHOR]

Objectives: Pharmacological treatment of reperfusion injury using insulin and GSK3β inhibition has been shown to be cardioprotective, however, their interaction with the endogenous cardioprotective strategy, ischemic postconditioning, is not known.Design: Langendorff perfused ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. For the first 15 min of reperfusion hearts received either vehicle (Ctr), insulin (Ins) or a GSK3β inhibitor (SB415286; SB41), with or without interruption of ischemic postconditioning (IPost; 3 × 30 s of global ischemia). In addition, the combination of insulin and SB41 for 15 min was assessed.Results: Insulin, SB41 or IPost significantly reduced infarct size versus vehicle treated controls (IPost 33.5 ± 3.3%, Ins 33.5 ± 3.4%, SB41 30.5 ± 3.0% vs. Ctr 54.7 ± 6.8%, p < 0.01). Combining insulin and SB415286 did not confer additional cardioprotection compared to the treatments given alone (SB41 + Ins 26.7 ± 3.5%, ns). Conversely, combining either of the pharmacological reperfusion treatments with IPost completely abrogated the cardioprotection afforded by the treatments separately (Ins + IPost 59.5 ± 3.4% vs. Ins 33.5 ± 3.4% and SB41 + IPost 50.2 ± 6.6% vs. SB41 30.5 ± 3.0%, both p < 0.01), and was associated with blunted Akt, GSK3β and STAT3 phosphorylation.Conclusion: Pharmacological reperfusion treatment with insulin and SB41 interferes with the cardioprotection afforded by ischemic postconditioning. [ABSTRACT FROM AUTHOR]

While TGF-β1 is known to induce epithelial–mesenchymal transition (EMT), a major factor in the pathogenesis of proliferative vitreoretinopathy (PVR), in ARPE-19 cells. The molecular pathways involved in EMT formation have not yet to be fully characterized. In this study, we have found that TGF-β1-mediated induction of EMT in ARPE-19 cells varied in a dose- and time-dependent manner. Specifically, TGF-β1 inhibited GSK-3β by accelerating phosphorylation at ser9. GSK-3β inhibitor or knockdown of GSK-3β resulted in enhanced TGF-β1-mediated EMT, migration and collagen contraction in ARPE-19 cells, which were then abrogated by GSK-3β overexpression and PI3K/AKT inhibitor. Importantly, GSK-3β also mediated metabolic reprogramming in TGF-β1-treated cells. Our results indicate that GSK-3β plays a pivotal role in TGF-β1-mediated EMT in ARPE-19 cells. [ABSTRACT FROM AUTHOR]

Glycogen synthase kinase 3 ( GSK3) is a serine-threonine kinase that regulates mammalian circadian rhythms at the behavioral, molecular and neurophysiological levels. In the central circadian pacemaker, the suprachiasmatic nucleus ( SCN), inhibitory phosphorylation of GSK3 exhibits a rhythm across the 24 h day. We have recently shown that GSK3 is capable of influencing both the molecular clock and SCN neuronal activity rhythms. However, it is not known whether GSK3 regulates the response to environmental cues such as light. The goal of this study was to test the hypothesis that GSK3 activation mediates light-induced SCN excitability and photic entrainment. Immunofluorescence staining in the SCN of mice showed that late-night light exposure significantly increased GSK3 activity (decreased pGSK3β levels) 30-60 min after the light-pulse. In addition, pharmacological inhibition of GSK3 blocked the expected light-induced excitability in SCN neurons; however, this effect was not associated with changes in resting membrane potential or input resistance. Behaviorally, mice with constitutively active GSK3 ( GSK3- KI) re-entrained to a 6-h phase advance in the light-dark cycle in significantly fewer days than WT control animals. Furthermore, the behavioral and SCN neuronal activity of GSK3- KI mice was phase-advanced compared to WT, in both normal and light-exposed conditions. Finally, GSK3- KI mice exhibited normal negative-masking behavior and electroretinographic responses to light, suggesting that the enhanced photic entrainment is not due to an overall increased sensitivity to light in these animals. Taken together, these results provide strong evidence that GSK3 activation contributes to light-induced phase-resetting at both the neurophysiological and behavioral levels. [ABSTRACT FROM AUTHOR]