Huntington’s disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of polyglutamine repeats in the N-terminal of huntingtin. The amount of aggregate-prone protein is controlled by various mechanisms, including molecular chaperones. Vaccinia-related kinase 2 (VRK2) is known to negatively regulate chaperonin TRiC, and VRK2-facilitated degradation of TRiC increases polyQ protein aggregation, which is involved in HD. We found that VRK2 activity was negatively controlled by glycogen synthase kinase 3β (GSK3β). GSK3β directly bound to VRK2 and inhibited the catalytic activity of VRK2 in a kinase activity-independent manner. Furthermore, GSK3β increased the stability of TRiC and decreased the formation of HttQ103-GFP aggregates by inhibiting VRK2. These results indicate that GSK3β signaling may be a regulatory mechanism of HD progression and suggest targets for further therapeutic trials for HD.
Huntington’s disease (HD) is a neurodegenerative disease caused by abnormal CAG triplet repeats (>35 residues) in the huntingtin (Htt) gene exon 1 at the N-terminal1. This expanded polyglutamine repeat causes protein aggregation and progressive cell death2. The number of glutamine residues correlates with the severity of HD symptoms and the age of disease onset3,4. Because the pathological hallmark of HD is the formation of polyQ-containing Htt aggregates, it is important to prevent this process. The level of aggregated protein is controlled by diverse mechanisms such as molecular chaperones4,5. In particular, the eukaryotic chaperonin TRiC (TCP-1 Ring Complex, also known as CCT for chaperonin containing TCP-1) attenuates Htt-polyQ protein aggregation and reduces cytotoxicity6.
The Vaccinia-related kinase (VRK) family is a serine/threonine kinases that is related to the casein kinase I family7. VRK2 has two isoforms: VRK2A and VRK2B. VRK2A has a transmembrane domain and localizes mainly in the endoplasmic reticulum, whereas VRK2B lacks a transmembrane domain and localizes mainly in the cytosol and nucleus8. So far, identified substrates of VRK2 include NFAT-1 and USP259,10. However, because VRK2 substrates are rarely identified, VRK2 function remains largely unknown. Meanwhile, several reports indicate that VRK2 is associated with neurological disorders such as epilepsy11, schizophrenia12,13, and HD9,14. We previously found that VRK2 downregulates CCT4, which results in increased polyQ aggregation9,14.
Glycogen synthase kinase 3 (GSK3) is a constitutively active serine/threonine kinase with two isoforms: GSK3α and GSK3β15. Its kinase activity is regulated by inhibitory phosphorylation sites at serine-21 and serine-9 in GSK3α and GSK3β, respectively16. GSK3β localizes predominantly in the cytoplasm17 but is sometimes found in the nucleus18, and its subcellular localization changes in response to binding partners or stimuli. GSK3β has several substrates and is involved in many cellular processes, including cell development19, proliferation, cell migration20, glucose regulation, and apoptosis18.
Consistent with its involvement in a variety of signaling pathways, GSK3β is associated with many diseases such as Alzheimer’s disease21, cancer22,23, bipolar disorder24, diabetes22, and HD. Decreased GSK3β levels and activity are observed in the brains of R6/1 mice, an animal model of HD25, and reduced GSK3β levels are also found in the brains of human patients with HD26. However, the precise role of GSK3β in HD has not been elucidated.
In this study, we show that GSK3β directly interacted with VRK2 and inhibited VRK2 catalytic activity in vitro. Subsequently, GSK3β reduced VRK2-mediated degradation of TRiC, thereby suppressing polyQ-expanded Htt aggregation.
GSK3β directly interacts with VRK2
We previously found that VRK2 indirectly affects the folding of polyQ proteins involved in HD9,14. To examine whether other proteins are involved in the regulation of VRK2-mediated polyQ protein aggregation, we carried out immunoprecipitation analysis. We found that GSK3β was a VRK2 binding partner. To confirm the interaction between VRK2 and GSK3β, HEK293T cells were transfected with Flag-tagged human VRK2 and HA-tagged GSK3β followed by immunoprecipitation with α-Flag antibody. We found that Flag-VRK2 interacted with HA-GSK3β (Fig. 1a). To verify that GSK3β directly binds to VRK2 in vitro, recombinant GSK3β and VRK2 proteins were purified as GST (GST-GSK3β)- and Flag (Flag-VRK2)-fused proteins in E. coli, respectively, and immunoprecipitation assays were performed with α-Flag antibody or control IgG. We found that VRK2 directly formed a complex with GSK3β (Fig. 1b). Furthermore, immunocytochemical analysis showed that EGFP-VRK2 colocalized with HA-GSK3β in the same subcellular compartments in SH-SY5Y (Fig. 1c) and HEK293A (Supplementary Fig. S1a) cells.
To study the structural relationship between VRK2 and GSK3β, we performed in silico docking analysis. VRK2 and GSK3β structures found in the RCSB Protein Data Bank (PDB) were computationally docked into a 3D model using PatchDock. We found three binding interfaces with high scores in the protein-protein docking model (Fig. 1d). According to the crystal structure of VRK2 (PDB entry: 2V62) and GSK3β (PDB entry: 1I09), residues D269, L271, Q290, H296, K311, and H316 of VRK2 and D49, R50, D90, Y117, S119, G120, and K122 of GSK3β (Interface 1); residues Y191, K200, N205, R207, and D256 of VRK2 and R96, G202, Q206, E211, and E279 of GSK3β (Interface 2); and residues E66, G113, and S115 of VRK2 and D233, E279, and R282 of GSK3β (Interface 3) are crucial for the interaction (Fig. 1e). Furthermore, the electrostatic interaction model of Interface 1 showed that interactions occurred between acidic residues on GSK3β and basic residues on VRK2 (Supplementary Fig. S1b). To understand the influence of surface charges on binding, we conducted point mutations of VRK2. K311, D256 and E66 were selected as key amino acid residues for binding to GSK3β because they have high affinity binding by hydrogen bond pairing. We observed that K311A point mutant (Interface 1), D256A point mutant (Interface 2), E66A point mutant (Interface 3) and K311/D256A/E66A triple mutant had weaker binding affinity for GSK3β compared with wild-type (WT) VRK2 (Supplementary Fig. S1c). Taken together, these results indicate that VRK2 interacts with GSK3β in vitro, in vivo, and in silico.
GSK3β inhibits VRK kinase activity in vitro
As GSK3β and VRK2 directly interact and are both serine/threonine-protein kinases, we tested whether they phosphorylate each other in an in vitro kinase assay. To determine whether VRK2 is a substrate of GSK3β, recombinant His-VRK2 kinase-dead (KD) mutant protein was incubated with recombinant GST-GSK3β protein. We found that GSK3β did not phosphorylate VRK2 (Fig. 2a). Therefore, we repeated in vitro kinase assay to determine whether VRK2 phosphorylates GSK3β. We found that VRK2 did not phosphorylate GSK3β or affect its kinase activity (Fig. 2b). However, VRK2 autophosphorylation was decreased in the presence of GSK3β regardless of GSK3β kinase activity (Fig. 2b,c). These results suggest that VRK2 kinase activity is inhibited by GSK3β without protein phosphorylation and that GSK3β could act upstream of VRK2. Our previous study demonstrates that USP25 is phosphorylated and inhibited by VRK2. Among three fragments of UPS25, the fragment containing the regulatory domain between amino acids 655 to 780 from the N-terminal was phosphorylated by VRK29. To clarify whether GSK3β inhibits the effect of VRK2 kinase activity on USP25 phosphorylation, we performed in vitro kinase assays with His-VRK2 protein, USP25 fragment, and GST-GSK3β. We found that VRK2-mediated phosphorylation of the USP25 fragment was reduced in the presence of WT or KD GSK3β (Fig. 2d). Furthermore, we confirmed that GSK3β decreased VRK2-mediated histone H3 phosphorylation (Supplementary Fig. S2a). These results suggest that VRK2 kinase activity is inhibited by the presence of GSK3β protein regardless of its kinase activity.
GSK3β suppresses VRK2-mediated degradation of TRiC
The chaperonin TRiC is composed of eight homologous subunits, from CCT1 to CCT8. We previously found that VRK2 facilitates CCT4 polyubiquitination, which downregulates CCT414. Based on the interaction between GSK3β and VRK2 and the observation that GSK3β is an upstream regulator of VRK2, we tested whether GSK3β regulates VRK2-mediated CCT4 downregulation. First, we examined the localization of GSK3β, VRK2, and CCT4. Fluorescence imaging showed that EGFP-VRK2 colocalized with Flag-GSK3β and HA-CCT4 in the same subcellular compartments of HEK293A cells (Fig. 3a). Next, we examined changes in the endogenous level of CCT4 with GSK3β overexpression. For this experiment, we used constitutively VRK2-overexpressing U2OS cells for visualizing prominent polyQ protein aggregates when polyQ-containing protein was overexpressed (Supplementary Fig. S3a). We found that the level of CCT4 protein was significantly increased when WT or KD GSK3β was overexpressed (Fig. 3b,c). Next, we further examined CCT4 protein stability in two different cell lines with overexpression of Flag-VRK2 with or without HA-GSK3β. We found that GSK3β rescued CCT4 protein levels, whereas VRK2 overexpression decreased CCT4 protein levels (Fig. 3d,e). Taken together, these results suggest that GSK3β acts as a positive regulator of CCT4 by inhibiting VRK2.
GSK3β reduces VRK2-mediated polyQ aggregation
We previously found that VRK2 reduces CCT4 protein levels, resulting in the accumulation of HttQ103 aggregation14. To investigate the effect of GSK3β on VRK2-mediated aggregation of polyQ-containing protein, we evaluated polyQ aggregates using a GFP-fused polyQ-expanded Htt fragment. We coexpressed HttQ103-GFP and HA-GSK3β in HEK293A cells with or without overexpression of Flag-VRK2. Interestingly, HttQ103 insoluble aggregates were decreased in HA-GSK3β-overexpressing cells (Fig. 4a). Because GSK3β inhibits degradation of TRiC protein levels by inhibiting VRK2 kinase activity, this suggests that functional TRiC plays a role in inhibiting pathogenic HttQ103 aggregation. In addition to the stabilization of TRiC, the amount of insoluble HttQ103 aggregates was also reduced by overexpression of GSK3β WT or KD (Supplementary Fig. S4a). We further confirmed that GSK3β reduced HttQ103 aggregation by filter-trap assay (Fig. 4b,c, Supplementary Fig. S4b). Consistent with the results of Western blotting and filter-trap assay, HttQ103 aggregates did not increase significantly in GSK3β overexpression with co-overexpression of VRK2 condition while VRK2 alone overexpression increased aggregates (Fig. 4d,e). Collectively, these results suggest that GSK3β-mediated VRK2 inhibition reduces HttQ103 aggregation through the stabilization of chaperonin.
HD is a neurodegenerative disorder caused by an expanded CAG repeat sequence (encoding a poly-glutamine stretch) in the Htt gene27, which causes protein aggregation in brain cells and progressive cell death, especially in the striatum and cortex1,2. Accumulation of mutant Htt protein is toxic and affects important cellular functions such as transcription, axonal transport, synaptic transmission, and mitochondrial function4. Mutant Htt interacts with several cellular proteins and sequesters into nuclear aggregates or forms cytoplasmic inclusions that cause progressive neuronal degeneration in HD mouse models. The number of glutamine residues correlates with the severity of HD symptoms, and the pathological hallmark of HD is the formation of polyQ-expanded aggregates3,4. Therefore, it is important to prevent this process. Therapeutic strategies for HD and other neurodegenerative diseases have focused on elevating molecular chaperones5 or activating various cleaning mechanisms, including proteolytic systems28 and autophagy29. Chaperones promote refolding or degradation of misfolded proteins and prevent protein aggregation. A previous study reports that eukaryotic chaperonin TRiC attenuates the accumulation of Htt-polyQ protein aggregates and their cytotoxicity6. Therefore, functional TRiC is involved in decreasing pathogenic HttQ103 aggregate formation.
Recent studies indicate that TRiC directly interacts with and regulates the transcriptional activity of HSF130. Also, GSK3β phosphorylates HSF1 on residue serine 30331. Thus, we performed co-immunoprecipitation to test whether GSK3β binds to HSF1 in the presence of VRK2. Binding between GSK3β and HSF1 was not significantly changed by VRK2 overexpression (Supplementary Fig. S5a), suggesting that HSF1 constitutively binds to GSK3β in the presence or absence of VRK2. Next, we examined whether GSK3β-mediated inhibition of VRK2-induced TRiC destabilization affects HSF1 activity by examining transcription levels of the HSF1 target gene hsp70 using real-time quantitative PCR. We found no change in HSF1 transcriptional activity under our experimental conditions (Supplementary Fig. S5b).
We previously found that VRK2 specifically binds to TRiC/CCT subunits, causing chaperonin downregulation through the ubiquitin-proteasome system14. This results in a lack of sufficient chaperonin action to mediate proper protein folding in VRK2-overexpressing cells. The level of active VRK2 enzymes may be an important indicator of HD onset and progression. To prove this, alternatively, we investigated VRK2 protein levels in a Huntington’s disease model, R6/2 transgenic mouse, which is a well-studied transgenic animal and most widely used as HD mouse model. The amount of VRK2 protein was relatively increased in the R6/2 transgenic mouse brain lysates compared to wild-type mouse brain lysates at 4 weeks (Supplementary Fig. S6a). In support of this concept, recently, many genome-wide association studies suggest that VRK2 is a risk factor for neurological disorders including schizophrenia12,13 and epilepsy11. VRK2 mRNA levels are upregulated in schizophrenic patient brains compared with normal control brains12, and VRK2 gene rs3732136 polymorphism is associated with schizophrenia in the worldwide population32. Therefore, genetic variations could affect expression of the VRK2 gene, and malfunctions in VRK2 kinase activity might contribute to susceptibility to neurodegenerative diseases.
In the present study, we demonstrated that GSK3β and VRK2 directly interact and co-localize in cells using in vitro and in vivo binding assays. Structure-based docking analysis showed the presence of many hydrogen bonds and high-affinity interactions between GSKβ and VRK2. This docking analysis suggests that binding Interfaces 2 and 3 of GSK3β are interacting sites that bind to VRK2 outside of the active site. GSK3β is positioned near the catalytic domain of VRK2, and the binding of GSK3β to VRK2 altered protein flexibility in regions involved in substrate binding and turnover, suggesting their importance to kinase activity. Thus, GSK3β may inhibit VRK2 catalytic activity by disrupting its flexibility. The inhibition of VRK2 catalytic activity by GSK3β may also inhibit VRK2-induced degradation of TRiC, which could suppress polyQ-expanded Htt aggregation. However, a previous study demonstrates that GSK3β inhibitor protects polyglutamine protein-induced cell death but had inconsistent effects on the number of Htt aggregates in cells33. GSK3β inhibitor also has variable effects in a transgenic mouse model of HD34 and has beneficial effects when combined with other drugs35,36. However, the main reason why GSK3β inhibitors do not have reliable effects may be due to markedly reduced levels and activity of GSK3β in the striatum and other brain regions in R6/1 HD mouse models and the frontal cortex of postmortem HD patients25. Our findings support previous observations of decreased levels and reduced activity of GSK3β in HD mouse models and postmortem human brains. Therefore, the level of GSK3β in the brain may be critical for controlling HD progression.
In conclusion, we propose that GSK3β has positive effects on HD by regulating VRK2 and the stability of chaperonin TRiC. GSK3β appears to inhibit VRK2 kinase activity via protein-protein interaction in a kinase activity-independent manner, thereby reducing ubiquitination-proteosomal degradation of TRiC and suppressing polyQ protein aggregation (Fig. 5). Our results could aid in the development of efficient ways for preventing or treating HD by regulating GSK3β levels.
Materials and Methods
All plasmids used in this study were described in earlier studies9,14. VRK2 kinase-dead mutant was generated by site-directed mutagenesis (Lys61 to Ala). Flag-hVRK2-mutants (K311A (Interface 1), D256A (Interface 2), and E66A (Interface 3)) were amplified from Flag-hVRK2-WT plasmid using a forward primer (5′-AAGCGGCCGCAATGCCACCAAAAAGAAATG-3′) and reverse primer (5′-AATCTAGATCAGAGAAAAAATAAAGCAAGAA-3′). The first round of PCR, using forward and reverse primer (5′-GGTTCAAAATTGCCTTGAGGGC-3′ for Interface 1, 5′-AGCCACAGGTGCCTTCAGGTT-3′ for Interface 2, and 5′-CGGGCCATTTGCTTGATATTC-3′ for Interface 3) and another forward (5′-GCCCTCAAGGCAATTTTGAAC-3′ for Interface 1, 5′-AACCTGAAGGCACCTGTGGCT-3′ for Interface 2, and 5′-GAATATCAAGCAAATGGCCCG-3′ for Interface 3) and reverse primer, created two fragments. The two PCR products were mixed together for a second round of PCR. The full-length coding sequence of hVRK2 was digested with NotI/XbaI and cloned into pFlag-CMV2 (Sigma, St. Louis, MO) control vector. The GSK3β constructs were gift from Sang Ki Park (POSTECH, Pohang, South Korea). The HA-tagged cDNA of human GSK3β wild-type and two mutants, catalytically inactive GSK3β (GSK3β K85A) and constitutively active GSK3β (GSK3β S9A), were cloned into pGEX-4T-3 (Amersham, Piscataway, NJ).
Antibodies used in this study included: anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and anti-GST (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HA (Bethyl, Montgomery, TX, USA), anti-CCT4 (Abcam, Cambridge, United Kingdom), and anti-FLAG (Sigma, St. Louis, MO). All fluorescence-conjugated secondary antibodies were from Invitrogen.
Cell culture and transfection
HEK293A, HEK293T, U2OS, and SH-SY5Y cells were propagated in Dulbecco’s Modified Eagle’s Medium or Minimum Essential Media supplemented with 10% fetal bovine serum and 100 U/ml each of penicillin G and streptomycin in 5% CO2 at 37 °C. For transient transfection with plasmids, lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) or Microporator (Invitrogen) was used according to the manufacturer’s instructions.
Cell extract and protein preparation, immunoprecipitation, and Western blotting were performed as previously described9,14. Protein bands were visualized using a SUPEX ECL solution kit (Neuronex, Daegu, South Korea) and a LAS-4000 chemiluminescence detection system (Fujifilm, Tokyo, Japan).
In vitro kinase assay
In vitro kinase assay with recombinant proteins was performed as previously described9.
Cells were lysed in RIPA buffer, and sonicated lysates were diluted in 1% SDS-PBS and boiled at 95 °C for 5 min. The membrane was pre-equilibrated with 1% SDS in PBS. Proteins were loaded onto a 0.22-μm nitrocellulose membrane. A dot blotter apparatus (Bio-Rad, Hercules, CA) was used for the application of samples, and anti-GFP antibody (Santa Cruz, CA) was used for detection.
Transfected cells were maintained for 24–48 h, fixed with 4% paraformaldehyde for 20 min, and immunostained with appropriate primary and fluorescence-conjugated secondary antibodies. Coverslips were mounted onto slides using Fluoromount (Sigma). All images were obtained using a laser scanning confocal microscope (model FV1000; OLYMPUS), and FV10-ASW2.0 fluoviewer software was used for image analysis.
Structural modeling and analysis
PatchDock was used to study the structural relationship between GSK3β and VRK237. According to the crystal structure of GSK3β (PDB entry: 1I09)38 and VRK2 (PDB entry: 2V62)39, the best structure for docking was selected and further analyzed using PyMOL.
Establishment of stable cell lines
Stable cell lines were generated by G418 selection. Briefly, pNTAP-mock or pNTAP-hVRK2 vector was transfected into the normal U2OS cell line using electroporation and incubated for 24 h. Cells were selected by 800 μg/ml G418 with media changes every 2 days until single colonies formed. After single colony isolation, stable cells were maintained with 200 μg/ml G418-containing media.
Quantitative real-time reverse transcription PCR
Total RNA was isolated using TRI reagent (Molecular Research Center, Cincinnati, OH) and reverse transcribed using ImProm-II (Promega) according to the manufacturer’s instructions. For detection and quantification, the StepOnePlus real-time PCR system (Applied Biosystems) was used.
Approval of the study protocol was obtained from the Pohang University of Science and Technology Institutional Animal Care and Use Committee (POSTECH IACUC) (Approval ID: 2013-03-004). All animal experiments were carried out according to the provisions of the Animal Welfare Act, PHS Animal Welfare Policy, and the principles of the NIH Guide for the Care and Use of Laboratory Animals. All mice were maintained under conventional conditions at the POSTECH animal facility under institutional guidelines.
Quantitative data are shown as mean ± standard error of the mean (SEM). Statistical analyses were performed using Student’s t-test and one-way analysis of variance (ANOVA), two-way ANOVA and post-hoc Tukey’s multiple comparison tests using GraphPad software.
How to cite this article: Lee, E. et al. Glycogen synthase kinase 3β suppresses polyglutamine aggregation by inhibiting Vaccinia-related kinase 2 activity. Sci. Rep. 6, 29097; doi: 10.1038/srep29097 (2016).
A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. The Huntington’s Disease Collaborative Research Group. Cell 72, 971–983 (1993).
Michalik, A. & Van Broeckhoven, C. Pathogenesis of polyglutamine disorders: aggregation revisited. Hum Mol Genet 12 Spec No 2, R173–R186, doi: 10.1093/hmg/ddg295 (2003).
Ross, C. A. Polyglutamine pathogenesis: emergence of unifying mechanisms for Huntington’s disease and related disorders. Neuron 35, 819–822 (2002).
Zoghbi, H. Y. & Orr, H. T. Glutamine repeats and neurodegeneration. Annu Rev Neurosci 23, 217–247, doi: 10.1146/annurev.neuro.23.1.217 (2000).
Tyedmers, J., Mogk, A. & Bukau, B. Cellular strategies for controlling protein aggregation. Nat Rev Mol Cell Biol 11, 777–788, doi: 10.1038/nrm2993 (2010).
Kitamura, A. et al. Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state. Nat Cell Biol 8, 1163–1170, doi: 10.1038/ncb1478 (2006).
Manning, G., Whyte, D. B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. Science 298, 1912–1934, doi: 10.1126/science.1075762 (2002).
Blanco, S., Klimcakova, L., Vega, F. M. & Lazo, P. A. The subcellular localization of vaccinia-related kinase-2 (VRK2) isoforms determines their different effect on p53 stability in tumour cell lines. FEBS J 273, 2487–2504, doi: 10.1111/j.1742-4658.2006.05256.x (2006).
Kim, S. et al. Vaccinia-Related Kinase 2 Controls the Stability of the Eukaryotic Chaperonin TRiC/CCT by Inhibiting the Deubiquitinating Enzyme USP25. Mol Cell Biol 35, 1754–1762, doi: 10.1128/MCB.01325-14 (2015).
Vazquez-Cedeira, M. & Lazo, P. A. Human VRK2 (vaccinia-related kinase 2) modulates tumor cell invasion by hyperactivation of NFAT1 and expression of cyclooxygenase-2. J Biol Chem 287, 42739–42750, doi: 10.1074/jbc.M112.404285 (2012).
Consortium, E. et al. Genome-wide association analysis of genetic generalized epilepsies implicates susceptibility loci at 1q43, 2p16.1, 2q22.3 and 17q21.32. Hum Mol Genet 21, 5359–5372, doi: 10.1093/hmg/dds373 (2012).
Li, M. et al. Meta-analysis and brain imaging data support the involvement of VRK2 (rs2312147) in schizophrenia susceptibility. Schizophr Res 142, 200–205, doi: 10.1016/j.schres.2012.10.008 (2012).
Steinberg, S. et al. Common variants at VRK2 and TCF4 conferring risk of schizophrenia. Hum Mol Genet 20, 4076–4081, doi: 10.1093/hmg/ddr325 (2011).
Kim, S. et al. Vaccinia-related kinase 2 mediates accumulation of polyglutamine aggregates via negative regulation of the chaperonin TRiC. Mol Cell Biol 34, 643–652, doi: 10.1128/MCB.00756-13 (2014).
Woodgett, J. R. Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J 9, 2431–2438 (1990).
Doble, B. W. & Woodgett, J. R. GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci 116, 1175–1186 (2003).
Fraser, E. et al. Identification of the Axin and Frat binding region of glycogen synthase kinase-3. J Biol Chem 277, 2176–2185, doi: 10.1074/jbc.M109462200 (2002).
Meares, G. P. & Jope, R. S. Resolution of the nuclear localization mechanism of glycogen synthase kinase-3: functional effects in apoptosis. J Biol Chem 282, 16989–17001, doi: 10.1074/jbc.M700610200 (2007).
Hoeflich, K. P. et al. Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation. Nature 406, 86–90, doi: 10.1038/35017574 (2000).
Kobayashi, T. et al. Glycogen synthase kinase 3 and h-prune regulate cell migration by modulating focal adhesions. Mol Cell Biol 26, 898–911, doi: 10.1128/MCB.26.3.898-911.2006 (2006).
Hanger, D. P., Hughes, K., Woodgett, J. R., Brion, J. P. & Anderton, B. H. Glycogen synthase kinase-3 induces Alzheimer’s disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localisation of the kinase. Neurosci Lett 147, 58–62 (1992).
Amar, S., Belmaker, R. H. & Agam, G. The possible involvement of glycogen synthase kinase-3 (GSK-3) in diabetes, cancer and central nervous system diseases. Curr Pharm Des 17, 2264–2277 (2011).
Manoukian, A. S. & Woodgett, J. R. Role of glycogen synthase kinase-3 in cancer: regulation by Wnts and other signaling pathways. Adv Cancer Res 84, 203–229 (2002).
Li, X., Liu, M., Cai, Z., Wang, G. & Li, X. Regulation of glycogen synthase kinase-3 during bipolar mania treatment. Bipolar Disord 12, 741–752, doi: 10.1111/j.1399-5618.2010.00866.x (2010).
Fernandez-Nogales, M. et al. Decreased glycogen synthase kinase-3 levels and activity contribute to Huntington’s disease. Hum Mol Genet 24, 5040–5052, doi: 10.1093/hmg/ddv224 (2015).
Lim, N. K. et al. Localized changes to glycogen synthase kinase-3 and collapsin response mediator protein-2 in the Huntington’s disease affected brain. Hum Mol Genet 23, 4051–4063, doi: 10.1093/hmg/ddu119 (2014).
Ha, A. D. & Fung, V. S. Huntington’s disease. Curr Opin Neurol 25, 491–498, doi: 10.1097/WCO.0b013e3283550c97 (2012).
Lecker, S. H., Goldberg, A. L. & Mitch, W. E. Protein degradation by the ubiquitin-proteasome pathway in normal and disease states. J Am Soc Nephrol 17, 1807–1819, doi: 10.1681/ASN.2006010083 (2006).
Jeong, H. et al. Acetylation targets mutant huntingtin to autophagosomes for degradation. Cell 137, 60–72, doi: 10.1016/j.cell.2009.03.018 (2009).
Neef, D. W. et al. A direct regulatory interaction between chaperonin TRiC and stress-responsive transcription factor HSF1. Cell Rep 9, 955–966, doi: 10.1016/j.celrep.2014.09.056 (2014).
Chu, B., Soncin, F., Price, B. D., Stevenson, M. A. & Calderwood, S. K. Sequential phosphorylation by mitogen-activated protein kinase and glycogen synthase kinase 3 represses transcriptional activation by heat shock factor-1. J Biol Chem 271, 30847–30857 (1996).
Zhang, B. et al. Association of the VRK2 gene rs3732136 polymorphism with schizophrenia in a Northwest Chinese Han population. Genet Mol Res 14, 9404–9411, doi: 10.4238/2015.August.14.4 (2015).
Carmichael, J., Sugars, K. L., Bao, Y. P. & Rubinsztein, D. C. Glycogen synthase kinase-3beta inhibitors prevent cellular polyglutamine toxicity caused by the Huntington’s disease mutation. J Biol Chem 277, 33791–33798, doi: 10.1074/jbc.M204861200 (2002).
Wood, N. I. & Morton, A. J. Chronic lithium chloride treatment has variable effects on motor behaviour and survival of mice transgenic for the Huntington’s disease mutation. Brain Res Bull 61, 375–383 (2003).
Chiu, C. T., Liu, G., Leeds, P. & Chuang, D. M. Combined treatment with the mood stabilizers lithium and valproate produces multiple beneficial effects in transgenic mouse models of Huntington’s disease. Neuropsychopharmacology 36, 2406–2421, doi: 10.1038/npp.2011.128 (2011).
Scheuing, L., Chiu, C. T., Liao, H. M., Linares, G. R. & Chuang, D. M. Preclinical and clinical investigations of mood stabilizers for Huntington’s disease: what have we learned? Int J Biol Sci 10, 1024–1038, doi: 10.7150/ijbs.9898 (2014).
Schneidman-Duhovny, D., Inbar, Y., Nussinov, R. & Wolfson, H. J. PatchDock and SymmDock: servers for rigid and symmetric docking. Nucleic Acids Res 33, W363–W367, doi: 10.1093/nar/gki481 (2005).
ter Haar, E. et al. Structure of GSK3beta reveals a primed phosphorylation mechanism. Nat Struct Biol 8, 593–596, doi: 10.1038/89624 (2001).
Scheeff, E. D., Eswaran, J., Bunkoczi, G., Knapp, S. & Manning, G. Structure of the pseudokinase VRK3 reveals a degraded catalytic site, a highly conserved kinase fold, and a putative regulatory binding site. Structure 17, 128–138, doi: 10.1016/j.str.2008.10.018 (2009).
We are also grateful to Dr. Sang Ki Park (POSTECH, South Korea) for providing GSK3β constructs. This work was supported by grants from “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01121602)” from Rural Development Administration, BK21 Plus funded by the Ministry of Education [10Z20130012243], and POSCO (AP-TP, Green science), Korea. H.G.R. is also supported by a KT&G Scholarship Foundation (KT&G, Korea).
The authors declare no competing financial interests.
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Lee, E., Ryu, H., Kim, S. et al. Glycogen synthase kinase 3β suppresses polyglutamine aggregation by inhibiting Vaccinia-related kinase 2 activity. Sci Rep 6, 29097 (2016). https://doi.org/10.1038/srep29097
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