Glycogen Synthase Kinase-3 Substrate Research Articles

GSK3 and lamellipodin balance lamellipodial protrusions and focal adhesion maturation in mouse neural crest migration

Neural crest cells are multipotent cells that delaminate from the neuroepithelium, migrating throughout the embryo. Aberrant migration causes developmental defects. Animal models are improving our understanding of neural crest anomalies, but invivo migration behaviors are poorly understood. Here, we demonstrate that murine neural crest cells display actin-based lamellipodia and filopodia invivo. Using neural crest-specific knockouts or inhibitors, we show that the serine-threonine kinase glycogen synthase kinase-3 (GSK3) and the cytoskeletal regulator lamellipodin (Lpd) are required for lamellipodia formation while preventing focal adhesion maturation. Lpd is a substrate of GSK3, and phosphorylation of Lpd favors interactions with the Scar/WAVE complex (lamellipodia formation) at the expense of VASP and Mena interactions (adhesion maturation and filopodia formation). This improved understanding of cytoskeletal regulation in mammalian neural crest migration has general implications for neural crest anomalies and cancer.

Inhibition of glycogen synthase kinase 3 by lithium, a mechanism in search of specificity.

Inhibition of Glycogen synthase kinase 3 (GSK3) is a popular explanation for the effects of lithium ions on mood regulation in bipolar disorder and other mental illnesses, including major depression, cyclothymia, and schizophrenia. Contribution of GSK3 is supported by evidence obtained from animal and patient derived model systems. However, the two GSK3 enzymes, GSK3α and GSK3β, have more than 100 validated substrates. They are thus central hubs for major biological functions, such as dopamine-glutamate neurotransmission, synaptic plasticity (Hebbian and homeostatic), inflammation, circadian regulation, protein synthesis, metabolism, inflammation, and mitochondrial functions. The intricate contributions of GSK3 to several biological processes make it difficult to identify specific mechanisms of mood stabilization for therapeutic development. Identification of GSK3 substrates involved in lithium therapeutic action is thus critical. We provide an overview of GSK3 biological functions and substrates for which there is evidence for a contribution to lithium effects. A particular focus is given to four of these: the transcription factor cAMP response element-binding protein (CREB), the RNA-binding protein FXR1, kinesin subunits, and the cytoskeletal regulator CRMP2. An overview of how co-regulation of these substrates may result in shared outcomes is also presented. Better understanding of how inhibition of GSK3 contributes to the therapeutic effects of lithium should allow for identification of more specific targets for future drug development. It may also provide a framework for the understanding of how lithium effects overlap with those of other drugs such as ketamine and antipsychotics, which also inhibit brain GSK3.

Abstract 152: Discovery and characterization of GSK3 allosteric activators

Abstract The intestinal epithelium undergoes constant regeneration and is tightly regulated. Disruption of its homeostasis can result in the development of colorectal cancer (CRC). Glycogen synthase kinase 3 (GSK3) is a kinase that plays a central role in multiple signaling pathways, including cellular proliferation, where it inhibits the activity of its substrates. GSK3 dynamics can be described as a multiple input multiple output system. Numerous upstream growth factors can regulate and thus inhibit GSK3 from acting on its many substrates. Mutations in these growth signaling pathways (e.g., Wnt and KRAS) have been well established to have a role in CRC progression. Therefore, aberrantly activated signaling due to these proteins are presumed to result in the constitutive inhibition of GSK3 activity in CRC, thus making it an ideal therapeutic target. The aim of this study is to discover an allosteric GSK3 small molecule activator that can restore GSK3 kinase activity within CRC. Method: We screened 50,000 small molecule compounds using a luciferase-based in-vitro kinase assay. We are further confirming our top hits using both chemical and cell-based assays. For the cell-based assays, we test the compounds in human colonic epithelial cells (HCEC) and its three isogenic cell lines, which mimic CRC progression, and monitor changes in GSK3 kinase activity indirectly, via changes in its substrate protein levels, and directly via a live-cell GSK3 specific reporter. In addition, the top hits are also tested in both normal colon and CRC organoids. Results: Prior to running our large screen, we performed various optimization steps to determine the ideal condition for our screening assay. Our screening assay scored a Z-Score of 0.5 and higher, which indicated that it was a high-quality screening assay. Additionally, we have developed two cell-based orthogonal HTS assays. One using immunofluorescence and single-cell quantification of 24 GSK3 substrates from various pathways in our cell lines. Second, we engineered a GSK3 live-cell reporter and expressed it in the HCEC cells and three isogenic cell lines and organoid models. Impact: There is a need for advancements in preventative or therapeutic targets against CRC. Research indicates that suppression of GSK3 enzymatic activity decreases sensitivity to certain chemotherapeutic and targeted therapies. Therefore, restoring GSK3 enzymatic activity in CRC may re-sensitize tumors to other targeted therapies and establish GSK3 as a potential therapeutic target. Citation Format: Reeba P. Varghese, Curtis A. Thorne. Discovery and characterization of GSK3 allosteric activators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 152.

GSK3α, not GSK3β, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring.

Glycogen synthase kinase-3 (GSK3) is an important signalling protein in the brain and modulates different forms of synaptic plasticity. Neuronal functions of GSK3 are typically attributed to one of its two isoforms, GSK3β, simply because of its prevalent expression in the brain. Consequently, the importance of isoform-specific functions of GSK3 in synaptic plasticity has not been fully explored. We now directly address this question for NMDA receptor-dependent long-term depression (LTD) in the hippocampus. Here, we specifically target the GSK3 isoforms with shRNA knock-down in mouse hippocampus and with novel isoform-selective drugs to dissect their roles in LTD. Using electrophysiological and live imaging approaches, we find that GSK3α, but not GSK3β, is required for LTD. The specific engagement of GSK3α occurs via its transient anchoring in dendritic spines during LTD induction. We find that the major GSK3 substrate, the microtubule-binding protein tau, is required for this spine anchoring of GSK3α and mediates GSK3α-induced LTD. These results link GSK3α and tau in a common mechanism for synaptic depression and rule out a major role for GSK3β in this process.

Identification of a novel selective and potent inhibitor of glycogen synthase kinase-3.

Glycogen synthase kinase-3 (GSK-3) is a multitasking protein kinase that regulates numerous critical cellular functions. Not surprisingly, elevated GSK-3 activity has been implicated in a host of diseases including pathological inflammation, diabetes, cancer, arthritis, asthma, bipolar disorder, and Alzheimer's. Therefore, reagents that inhibit GSK-3 activity provide a means to investigate the role of GSK-3 in cellular physiology and pathophysiology and could become valuable therapeutics. Finding a potent inhibitor of GSK-3 that can selectively target this kinase, among over 500 protein kinases in the human genome, is a significant challenge. Thus there remains a critical need for the identification of selective inhibitors of GSK-3. In this work, we introduce a novel small organic compound, namely COB-187, which exhibits potent and highly selective inhibition of GSK-3. Specifically, this study 1) utilized a molecular screen of 414 kinase assays, representing 404 unique kinases, to reveal that COB-187 is a highly potent and selective inhibitor of GSK-3; 2) utilized a cellular assay to reveal that COB-187 decreases the phosphorylation of canonical GSK-3 substrates indicating that COB-187 inhibits cellular GSK-3 activity; and 3) reveals that a close isomer of COB-187 is also a selective and potent inhibitor of GSK-3. Taken together, these results demonstrate that we have discovered a region of chemical design space that contains novel GSK-3 inhibitors. These inhibitors will help to elucidate the intricate function of GSK-3 and can serve as a starting point for the development of potential therapeutics for diseases that involve aberrant GSK-3 activity.

Abstract 1310: Abrogation of KRas-addicted tumors by GSK3 suppression-mediated upregulation of β-catenin and c-myc

Abstract The significant involvement of mutant KRas in human cancer development underscores the need to develop approaches that disable mutant KRas-driven tumors. Targeting KRas directly is challenging, and such identifying vulnerabilities specific for mutant KRas tumors is an important alternative approach. In this study, we discovered that glycogen synthase kinase 3 (GSK3) is required for the in vitro and in vivo growth and survival of human mutant KRas-dependent tumors but not for mutant KRas-independent tumors. Pharmacological inhibition with the GSK3 inhibitor SB as well as siRNA depletion of GSK3 lead to tumor suppression that is mediated at least in part by inhibition of the phosphorylation of the GSK3 substrates c-Myc on T58 and β-catenin on S33/S37/T41. CRISPR/Cas9 targeted knock out studies demonstrated that c-Myc and β-catenin upregulation mediate the antitumor activity of SB. Importantly, GSK3 blockade inhibits the in vivo growth of G12D, G12V, and G12C mutant KRas primary and metastatic patient-derived xenografts from pancreatic cancer patients who progressed on chemo- and radiation therapies. This discovery warrants advanced pre-clinical and clinical investigations of GSK3 inhibitors in mutant KRas-dependent cancers. Note: This abstract was not presented at the meeting. Citation Format: Aslamuzzaman Kazi, Shengyan Xiang, Hua Yang, Daniel Delitto, Jose Trevino, Rays H. Jiang, Muhammad Ayaz, Harshani Lawrence, Perry Kennedy, Said M. Sebti. Abrogation of KRas-addicted tumors by GSK3 suppression-mediated upregulation of β-catenin and c-myc [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1310.

GSK3 suppression upregulates \u03b2-catenin and c-Myc to abrogate KRas-dependent tumors

Mutant KRas is a significant driver of human oncogenesis and confers resistance to therapy, underscoring the need to develop approaches that disable mutant KRas-driven tumors. Because targeting KRas directly has proven difficult, identifying vulnerabilities specific for mutant KRas tumors is an important alternative approach. Here we show that glycogen synthase kinase 3 (GSK3) is required for the in vitro and in vivo growth and survival of human mutant KRas-dependent tumors but is dispensable for mutant KRas-independent tumors. Further, inhibiting phosphorylation of GSK3 substrates c-Myc on T58 and β-catenin on S33/S37/T41 and their subsequent upregulation contribute to the antitumor activity of GSK3 inhibition. Importantly, GSK3 blockade inhibits the in vivo growth of G12D, G12V, and G12C mutant KRas primary and metastatic patient-derived xenografts from pancreatic cancer patients who progressed on chemo- and radiation therapies. This discovery opens new avenues to target mutant KRas-dependent cancers.

POLAR-guided signalling complex assembly and localization drive asymmetric cell division.

Stomatal cell lineage is an archetypal example of asymmetric cell division (ACD), which is necessary for plant survival1-4. In Arabidopsis thaliana, the GLYCOGEN SYNTHASE KINASE3 (GSK3)/SHAGGY-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) phosphorylates both the mitogen-activated protein kinase (MAPK) signalling module5,6 and its downstream target, the transcription factor SPEECHLESS (SPCH)7, to promote and restrict ACDs, respectively, in the same stomatal lineage cell. However, the mechanisms that balance these mutually exclusive activities remain unclear. Here we identify the plant-specific protein POLAR as a stomatal lineage scaffold for a subset of GSK3-like kinases that confines them to the cytosol and subsequently transiently polarizes them within the cell, together with BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), before ACD. As a result, MAPK signalling is attenuated, enabling SPCH to drive ACD in the nucleus. Moreover, POLAR turnover requires phosphorylation on specific residues, mediated by GSK3. Our study reveals a mechanism by which the scaffolding protein POLAR ensures GSK3 substrate specificity, and could serve as a paradigm for understanding regulation of GSK3 in plants.

Disruption of the psychiatric risk gene Ankyrin 3 enhances microtubule dynamics through GSK3/CRMP2 signaling

The ankyrin 3 gene (ANK3) is a well-established risk gene for psychiatric illness, but the mechanisms underlying its pathophysiology remain elusive. We examined the molecular effects of disrupting brain-specific Ank3 isoforms in mouse and neuronal model systems. RNA sequencing of hippocampus from Ank3+/− and Ank3+/+ mice identified altered expression of 282 genes that were enriched for microtubule-related functions. Results were supported by increased expression of microtubule end-binding protein 3 (EB3), an indicator of microtubule dynamics, in Ank3+/− mouse hippocampus. Live-cell imaging of EB3 movement in primary neurons from Ank3+/− mice revealed impaired elongation of microtubules. Using a CRISPR-dCas9-KRAB transcriptional repressor in mouse neuro-2a cells, we determined that repression of brain-specific Ank3 increased EB3 expression, decreased tubulin acetylation, and increased the soluble:polymerized tubulin ratio, indicating enhanced microtubule dynamics. These changes were rescued by inhibition of glycogen synthase kinase 3 (GSK3) with lithium or CHIR99021, a highly selective GSK3 inhibitor. Brain-specific Ank3 repression in neuro-2a cells increased GSK3 activity (reduced inhibitory phosphorylation) and elevated collapsin response mediator protein 2 (CRMP2) phosphorylation, a known GSK3 substrate and microtubule-binding protein. Pharmacological inhibition of CRMP2 activity attenuated the rescue of EB3 expression and tubulin polymerization in Ank3-repressed cells by lithium or CHIR99021, suggesting microtubule instability induced by Ank3 repression is dependent on CRMP2 activity. Taken together, our data indicate that ANK3 functions in neuronal microtubule dynamics through GSK3 and its downstream substrate CRMP2. These findings reveal cellular and molecular mechanisms underlying brain-specific ANK3 disruption that may be related to its role in psychiatric illness.

Systematic analysis of GSK-3 signaling pathways in aging of cerebral tissue.

Glycogen synthase kinase-3 (GSK-3) is a constitutively active kinase, involved in regulation of multiple physiological processes. In brain, changes in GSK-3 signaling are related to neurodegenerative issues, including Alzheimer's disease. Due to the wide range of GSK-3 cellular targets, a therapeutic use of the enzyme inhibitors entails significant risk of side effects. Thus, altering the ratio of specific pool of GSK-3 or specific substrates instead of changing the global activity of GSK-3 in brains might be a more appropriate strategy. This paper provides a comprehensive data on abundances of proteins involved in GSK-3 signaling in three regions of young and old mouse brains. It might help to identify novel protein targets with the highest therapeutic potential for treatment of age-related neurodegenerative diseases.

Phosphodiesterase PdeD, dynacortin, and a Kelch repeat-containing protein are direct GSK3 substrates in Dictyostelium that contribute to chemotaxis towards cAMP.

The migration of cells according to a diffusible chemical signal in their environment is called chemotaxis, and the slime mold Dictyostelium discoideum is widely used for the study of eukaryotic chemotaxis. Dictyostelium must sense chemicals, such as cAMP, secreted during starvation to move towards the sources of the signal. Previous work demonstrated that the gskA gene encodes the Dictyostelium homologue of glycogen synthase kinase 3 (GSK3), a highly conserved serine/threonine kinase, which plays a major role in the regulation of Dictyostelium chemotaxis. Cells lacking the GskA substrates Daydreamer and GflB exhibited chemotaxis defects less severe than those exhibited by gskA- (GskA null) cells, suggesting that additional GskA substrates might be involved in chemotaxis. Using phosphoproteomics we identify the GskA substrates PdeD, dynacortin and SogA and characterize the phenotypes of their respective null cells in response to the chemoattractant cAMP. All three chemotaxis phenotypes are defective, and in addition, we determine that carboxylesterase D2 is a common downstream effector of GskA, its direct substrates PdeD, GflB and the kinases GlkA and YakA, and that it also contributes to cell migration. Our findings identify new GskA substrates in cAMP signalling and break down the essential role of GskA in myosin II regulation.

Rab-GTPase binding effector protein 2 (RABEP2) is a primed substrate for Glycogen Synthase kinase-3 (GSK3)

Glycogen synthase kinase-3 (GSK3) regulates many physiological processes through phosphorylation of a diverse array of substrates. Inhibitors of GSK3 have been generated as potential therapies in several diseases, however the vital role GSK3 plays in cell biology makes the clinical use of GSK3 inhibitors potentially problematic. A clearer understanding of true physiological and pathophysiological substrates of GSK3 should provide opportunities for more selective, disease specific, manipulation of GSK3. To identify kinetically favourable substrates we performed a GSK3 substrate screen in heart tissue. Rab-GTPase binding effector protein 2 (RABEP2) was identified as a novel GSK3 substrate and GSK3 phosphorylation of RABEP2 at Ser200 was enhanced by prior phosphorylation at Ser204, fitting the known consensus sequence for GSK3 substrates. Both residues are phosphorylated in cells while only Ser200 phosphorylation is reduced following inhibition of GSK3. RABEP2 function was originally identified as a Rab5 binding protein. We did not observe co-localisation of RABEP2 and Rab5 in cells, while ectopic expression of RABEP2 had no effect on endosomal recycling. The work presented identifies RABEP2 as a novel primed substrate of GSK3, and thus a potential biomarker for GSK3 activity, but understanding how phosphorylation regulates RABEP2 function requires more information on physiological roles of RABEP2.

Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing

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 RNA biosynthesis 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-3β phosphorylated these proteins in vitro RNA-Seq 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 MS data 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.

The GSK3 kinase inhibitor lithium produces unexpected hyperphosphorylation of β-catenin, a GSK3 substrate, in human glioblastoma cells.

ABSTRACTLithium salt is a classic glycogen synthase kinase 3 (GSK3) inhibitor. Beryllium is a structurally related inhibitor that is more potent but relatively uncharacterized. This study examined the effects of these inhibitors on the phosphorylation of endogenous GSK3 substrates. In NIH-3T3 cells, both salts caused a decrease in phosphorylated glycogen synthase, as expected. GSK3 inhibitors produce enhanced phosphorylation of Ser9 of GSK3β via a positive feedback mechanism, and both salts elicited this enhancement. Another GSK3 substrate is β-catenin, which has a central role in Wnt signaling. In A172 human glioblastoma cells, lithium treatment caused a surprising increase in phospho-Ser33/Ser37-β-catenin, which was quantified using an antibody-coupled capillary electrophoresis method. The β-catenin hyperphosphorylation was unaffected by p53 RNAi knockdown, indicating that p53 is not involved in the mechanism of this response. Lithium caused a decrease in the abundance of axin, a component of the β-catenin destruction complex that has a role in coordinating β-catenin ubiquitination and protein turnover. The axin and phospho-β-catenin results were reproduced in U251 and U87MG glioblastoma cell lines. These observations run contrary to the conventional view of the canonical Wnt signaling pathway, in which a GSK3 inhibitor would be expected to decrease, not increase, phospho-β-catenin levels.This article has an associated First Person interview with the first author of the paper.

Insensitivity of PI3K/Akt/GSK3 signaling in peripheral blood mononuclear cells of age-related macular degeneration patients.

Our recent studies with cultured retinal pigment epithelium cells suggested that overexpression of interleukin 17 receptor C (IL-17RC), a phenomenon observed in peripheral blood and chorioretinal tissues with age-related macular degeneration (AMD), was associated with altered activation of phosphatidylinositide 3-kinase (PI3K), Akt, and glycogen synthase kinase 3 (GSK3). We wondered whether or not altered PI3K, Akt, and GSK3 activities could be detected in peripheral blood mononuclear cells (PBMC) obtained from AMD patients. In the patients' PBMC, absent or reduced serine-phosphorylation of GSK3α or GSK3β was observed, which was accompanied with increased phosphorylation of GSK3 substrates (e.g. CCAAT enhancer binding protein α, insulin receptor substrate 1, and TAU), indicative of enhanced GSK3 activation. In addition, decreased protein mass of PI3K85α and tyrosine-phosphorylation of PI3K50α was present in PBMC of the AMD patients, suggesting impaired PI3K activation. Moreover, abnormally lowered molecular weight forms of Akt and GSK3 were detected in PBMC of the AMD patients. These data demonstrate that despite the presence of high levels of IL-17RC, Wnt-3a and vascular endothelial growth factor, the PI3K/Akt/GSK3 signaling pathway is insensitive to these stimuli in PBMC of the AMD patients. Thus, measurement of PI3K/Akt/GSK3 expression and activity in PBMC may serve as a surrogate biomarker for AMD.

Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor signaling.

Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment.

Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis.

Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

Natural Functions of PLIN2 Mediating Wnt/LiCl Signaling and Glycogen Synthase Kinase 3 (GSK3)/GSK3 Substrate-Related Effects Are Modulated by Lipid.

Belonging to the PLIN family, PLIN2 associates with lipid storage droplets (LSDs), but other functions of PLIN2 remain unclear. Here, we suggest that PLIN2 mediates Wnt signaling because PLIN2 small interfering RNA (siRNA) suppresses activation of Wnt/coreceptor pathways. The mediation in the Wnt/Frizzled pathway seems to occur from Dishevelleds to axin/glycogen synthase kinase 3(GSK3)/β-catenin complexes (AGβC) as Wnt decreases Dishevelled/PLIN2 but increases AGβC/PLIN2 associations. Augmenting cellular LSDs that affect PLIN2 associations with these proteins, oleic acid (OA) treatment inhibits Wnt-increased AGβC/PLIN2 associations and β-catenin T-cell factor signaling (β-CTS). Revealing that PLIN2 is a GSK3-associated protein, the study explored PLIN2-mediated effects on GSK3/GSK3 substrates. PLIN2 siRNA reduces inhibitory GSK3 levels and lithium chloride (LiCl)-upregulated β-catenin or CCAAT/enhancer binding protein α (c/EBPα) expression. OA treatment decreases LiCl-increased c/EBPα via PLIN2-c/EBPα dissociation. In addition to PLIN2 overexpression increasing β-CTS, PLIN2 depletion or overexpression drops or adds expression of GSK3 substrates, such as β-catenin, c/EBPα,c-Myc, cyclin D1, and insulin receptor substrate 1, and cell growth/survival. PLIN2 N or C terminus overexpression that is associated with higher levels of the substrates suggests that those substrates bind to specific regions of PLIN2. Mimicking the possible high lipid concentrations in cells in the human body under conditions of hyperlipidemia/obesity, OA-treated cells gain or reduce GSK3 substrate expression in parallel with a decrease (a Wnt-like effect) or increase in GSK3 activity, likely regulated by GSK3/PLIN2/GSK3 substrate associations.

A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP.

The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death. KSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human protein microarray for ERK-primed GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.

Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation

BackgroundGlycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC.MethodsThe expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture.ResultsExpression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes.ConclusionNSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC.