GSK-3β: a bifunctional role in cell death pathwaysKeith M Jacobs1, Sandeep R Bhave2, Daniel J Ferraro1, Jerry J Jaboin1, 4, Dennis E Hallahan1, 3 4 , DineshThotala1,4,*1 Department of Radiation Oncology, Washington University, St. Louis, MO2 , Vanderbilt University School of Medicine, Nashville, TN3 Mallinckrodt Institute of Radiology, St. Louis, MO4 Siteman Cancer Center, St. Louis, MOCorresponding author: Dinesh Thotala, Ph.D., Department of Radiation Oncology, Washington Universityin St. Louis, 4511 Forest Park, St. Louis, MO, 63108 Tel.: 314-747-5456, e-mail:firstname.lastname@example.org.
ABSTRACT:Although Glycogen synthase kinase-3 beta (GSK-3β) was originally named for its ability to phosphorylateglycogen synthase and regulate glucose metabolism, this multifunctional kinase is presently known to bea key regulator of a wide range of cellular functions. GSK-3β is involved in modulating a variety offunctions including cell signaling, growth metabolism and various transcription factors that determinesthe survival or death of the organism. Secondary to the role of GSK-3β in various diseases includingAlzheimer’s disease, inflammation, diabetes and cancer, small molecule inhibitors of GSK-3β are gainingsignificant attention. This review is primarily focused on addressing the bifunctional or conflicting rolesof GSK-3β in both the promotion of cell survival and of apoptosis. GSK-3β has emerged as an importantmolecular target for drug development.
INTRODUCTION:Glycogen synthase kinase-3 is a ubiquitously expressed protein kinase that exists in two isoforms, α andβ. Originally identified based on its role in glycogen biosynthesis based on its inactivatingphosphorylation of glycogen synthase, it has since been found to regulate a myriad of functions throughWnt and other signaling pathways. The two isoforms are strongly conserved within their kinasedomain but differ greatly at the C-terminus, while the α isoform additionally contains a glycine-rich N-terminus extension. Our review will focus on the β isoform due to its more established role in cellsurvival and viability. Glycogen synthase kinase-3 beta (GSK-3β) is involved in the regulation of a widerange of cellular functions including differentiation, growth, proliferation, motility, cell cycleprogression, embryonic development, apoptosis, and insulin response [1-8]. It has emerged as animportant regulator of neuronal, endothelial, hepatocyte, fibroblast, and astrocyte cell death inresponse to various stimuli [6, 7, 9].GSK-3β is comprised of 12 exons in humans and 11 exons in mice with the ATG start codon locatedwithin exon 1 and the TAG stop codon found in the terminal exon. The gene product is a 46 kDa proteinconsisting of 433 amino acids in the human and 420 amino acids in the mouse. Figure 1 shows theoverall structure of GSK-3It is similar to other Ser/Thr kinases [10, 11]. The N-terminal domain iscomprised of the first 135 residues and forms a 7-strand -barrel motif. A small linker region connectsthe N-terminal domain to the central α-helical domain formed by residues 139 through 342. The ATPbinding site lies at the interface of the N-terminal and α-helical domains. Residues 343 through 433 formthe C-terminal domain, which is outside of the classical Ser/Thr kinase core fold. These residues form ahelix/loop domain that interacts with the core α-helical domain. The N-terminal amino acids 78 through92 are necessary for association with p53 (Figure 1). The activity of GSK-3 can be reduced byphosphorylation at Ser-9. Several kinases are able to mediate this modification, including p70S6 kinase,
p90RSK, PKC and Akt [12, 13]. In opposition to the inhibitory phosphorylation of GSK3 at Ser-9,phosphorylation of GSK-3 at Tyr-216 by ZAK1 or Fyn increases its enzyme activity  (Figure 2).Dysregulation of GSK-3β expression leads to many pathological conditions, including diabetes (or insulinresistance), neuronal dysfunction, Alzheimer’s disease [15-18], schizophrenia , Dopamine-associatedbehaviors , bipolar disorders , Parkinsons disease  and cancer. Of special interest is theinvolvement of GSK-3β in cancer with data supporting a role as a tumor suppressor and tumorpromoter, a discrepancy that at least in part depends on both cell type and signaling environment. Forexample, GSK-3β has been shown to inhibit androgen receptor-stimulated cell growth in prostatecancer, thus acting as a tumor suppressor . In contrast, GSK-3β is highly expressed in colorectalcancer [24, 25] and has been shown to participate in nuclear factor-κB (NF-κB)-mediated cell survival inpancreatic cancer , thus behaving as a tumor promoter. Moreover, the kinase has dual functions inthe regulation of cell survival, where it can either activate or inhibit apoptosis [3, 27], furthercomplicating its involvement in cancer. This review will focus on how GSK-3β can both activate as wellas protect from apoptosis with a focus on oncology.Regulation of -catenin levels is a critical step in Wnt signaling. β-Catenin is phosphorylated by GSK-3βand then degraded through the ubiquitin-proteasome system [28-30]. Inhibition of GSK-3β activity leadsto stabilization and accumulation of β-catenin in the cytosol, which is shuttled into the nucleus andregulates gene expression (Figure 2). GSK-3β is also involved in cell cycle regulation through thephosphorylation of cyclin D1, which results in the rapid proteolytic turnover of cyclin D1 protein [1, 31](Figure 2). Direct overexpression of wild-type GSK-3 is known to induce apoptosis in various cell typesin culture, and specific inhibitors of GSK-3 are able to stop this apoptotic signaling [6, 7, 9, 32]. Thedetailed molecular mechanism of GSK-3’s pro-apoptotic effect is as yet unknown, but it involvesregulation of metabolic and signaling proteins, transcription factors and gene expression [4, 33].
GSK-3β is required for proper development  and is ubiquitously expressed in the animal kingdom.GSK-3β protein was originally isolated from skeletal muscle, but though widely expressed, the protein ismost abundant in brain tissue, especially neurons. The high level of expression in brain tissue is likelydue to its vital role in neuronal signaling. In neuronal cells, GSK-3β is required for dendrite extension andsynapse formation in newborns.Regulation of Apoptosis by GSK-3GSK-3β has been shown to induce apoptosis in a wide variety of conditions including DNA damage ,hypoxia , endoplasmic reticulum stress , and Huntington’s disease-associated polyglutaminetoxicity . In cell culture studies, apoptosis was either attenuated or fully abrogated by inhibitingGSK-3 in primary neurons , HT-22 cells , PC12 cells , and human SH-SY5Y neuroblastomacells [36, 41].GSK-3 promotes apoptosis by inhibiting pro-survival transcription factors, such as CREB and heat shockfactor-1 , and facilitating pro-apoptotic transcription factors such as p53 . A list of somealternative conditions where GSK-3β facilitates apoptosis is given in Table 1. A large number of proteinshave been shown to interact with the tumor suppressor transcription factor p53 to regulate its actions[43, 44], which has been implicated in the pro-apoptotic actions of GSK-3β in several studies. FollowingDNA damage, the normally short-lived p53 protein is stabilized and modified by a complex array ofposttranslational modifications, such as phosphorylation, acetylation, methylation, ubiquitination,sumoylation, glycosylation, and neddylation. One of these regulatory proteins is GSK-3β, which forms acomplex with nuclear p53 to promote p53-induced apoptosis [34, 45, 46]. GSK-3β binds directly to p53and the C-terminal region of p53 is necessary for this interaction . GSK-3β was shown to directlyphosphorylate p53 at Ser-33 , and to mediate p53 phosphorylation at Ser-315 and Ser-376 [48, 49].GSK-3β also promotes p53-mediated transcription of specific genes and regulates the intracellular
localization of p53 [45, 46, 49]. In addition to GSK-3β regulating p53, GSK-3β is also regulated by p53.The activity of GSK-3β is increased by a phosphorylation-independent mechanism of direct binding ofp53 to GSK-3β . Nuclear localization of GSK-3β may also be regulated by binding of activated p53.In addition to direct interaction, GSK-3β can regulate p53 levels through the phosphorylation of thep53-specific E3 ubiquitin ligase MDM2 . Regulation of p53 by MDM2 is multifaceted. In the classicalmodel, N-terminal phosphorylation of p53 at Ser-15 (mouse Ser-18) and Ser-20 (mouse Ser-23) inhibitsthe interaction with MDM2 and thereby prevents MDM2-mediated ubiquitination and the resultingproteasomal degradation of p53  (Figure 3). Stabilized p53 then enters a complex regulatorynetwork to induce DNA binding and transcriptional activation of p53 target genes, in part through therecruitment of coactivators and corepressors. This determines the specific cellular response, which caninclude survival, growth arrest, DNA repair or apoptosis . Inhibition of GSK-3 in hippocampalneurons protected it from radiation-induced apoptosis [9, 52]. Similar protection from GSK-3β inhibitionhas been seen in primary neurons . The mechanism of protection from radiation-induced apoptosisin these cells involves subcellular localization and interaction of GSK3, p53, and MDM2. GSK-3inhibition blocks radiation-induced accumulation of p53 by upregulating levels of MDM2 thatsubsequently result in decreased radiation-dependent apoptosis . In addition to abrogation ofradiation-induced p53 phosphorylation, accumulation, and nuclear translocation, GSK-3 inhibitionresults in the accumulation of MDM2 and sequestration of GSK-3, p53, and MDM2 in the cytoplasmwhere p53 cannot act on its target genes . The role of attenuated p53 function in the pro-survivaleffects of the GSK-3β inhibitors, have also been previously described [34, 46, 52, 54, 55].In regulation of the apoptotic response, mammalian cells employ multiple pro-survival proteins from theBcl-2 family (Bcl-2, Bcl-XL, Bcl-w, Mcl1 and A1) that antagonize the pro-apoptotic function of Bax and Bak
[34, 56]. Bax and Bak localize to the mitochondrial outer membrane and trigger death signals leading tocytochrome c release to the cytosol [56, 57]. Apoptosis requires a group of effector caspases todismantle the cells. Cytochrome c activates caspase-9, which subsequently activates caspase-3 . Theactivation of caspase-3 is an essential step leading to cleavage of the DNA repair enzyme, poly (ADP-ribose) polymerase (PARP), resulting in genomic DNA fragmentation. Bax protein levels and cleavage(activation) of caspase-3 were increased due to radiation and were abrogated by GSK-3β inhibitors (Figure 3). GSK-3β was also found to be associated with mitochondrial apoptotic signaling. Inhibition ofGSK-3β prevented mitochondrial release of cytochrome c, which is known to activate caspase -3 andinitiate apoptosis . Phosphatidylinositol 3-kinase (PI3-kinase) and its downstream effector, theprotein-serine/threonine kinase Akt, a negative regulator of GSK-3β, play an important role inpreventing apoptosis by blocking activation of the caspase cascade.Survival-Promoting Effects of GSK-3βGSK-3β is involved in multiple signaling pathways and has many phosphorylation targets. It shouldtherefore not be surprising that GSK-3β has both pro- and anti-apoptotic roles. The overall effect ofGSK-3β on cell survival varies depending on cell type, transformation status, and the specific signalingpathway being activated. For example, despite evidence for a substantial pro-apoptotic role of GSK-3β,it is the inhibition of GSK-3β that promotes apoptosis and decreases viability in neuroblastoma cells .Several examples of pro-survival roles of GSK-3β not mentioned here are summarized in Table 2 [62-66].Additionally, while GSK-3β has been typically identified as an activator of p53-mediated apoptosis ,conflicting reports suggest an inhibitory effect of GSK-3β signaling on p53 activation. Inhibition ofGSK-3β blocks activation of MDM2 by reducing Ser-254 phosphorylation. This prevents p53 degradationand promotes apoptosis despite the induction of p53 ubiqui tination . Similarly, ionizing radiation wasfound to induce an inactivating phosphorylation at Ser-9 of GSK-3β, corresponding to
hypophosphorylation of MDM2 and accumulation of p53 . In contrast to its pro-apoptotic effects,this data suggests that GSK-3β inhibits apoptosis under basal conditions through MDM2-dependentdegradation of p53. Overexpression of β-catenin, a downstream signaling factor negatively regulated byGSK-3β, was found to increase basal p53 levels by blocking both MDM2-dependent and independentdegradation in neuroblastoma cells , providing additional supporting evidence for an inhibitoryeffect of GSK-3β on p53 mediated apoptosis. Interestingly, a negative feedback loop exists betweenβ-catenin and p53; while β-catenin upregulates p53 levels the activation of p53 results in degradation ofβ-catenin through GSK-3β . While the majority of publications suggest a pro-apoptotic role for GSK-3β in p53 signaling, it is clear that more comprehensive studies are needed in order to fully understandthe p53-GSK-3β relationship.GSK-3β is specifically required for hepatocyte survival in normal embryos, and GSK-3β knockout mice areembryonically lethal between E13.15-14.5. Hepatocyte apoptosis in GSK-3β knockout mice and mouseembryonic fibroblasts result only after exposure to tumor necrosis factor (TNF), while inhibition ofGSK-3β in wild-type cells with lithium increases TNF sensitivity. GSK-3β loss in these cells has adetrimental effect on the action of NF-κB, which protects against TNF-induced apoptosis . Otherstudies have shown that GSK-3β directly promotes NF-κB stability and activation through both thedegradation of p105 and activation of the p65 subunit, suggesting a likely mechanism forlithium-induced TNF hypersensitivity [70, 71] (Figure 3). The role of GSK-3β on NF-κB may also bemediated indirectly through inhibition of β-catenin, as cancer cells with high β-catenin levels areespecially sensitive to TNF-induced death .Despite the abundance of evidence implicating GSK-3β in protection from TNF-mediated apoptosis, afew conflicting reports further complicate our understanding of the pathway. A more recent studyclaims that GSK-3 inhibition does indeed reduce NF-κB activity but does not result in TNF-mediated
apoptosis, potentially due to the activation of pro-survival genes through Wnt signaling . Similarly,TNF sensitization by lithium in multiple sarcoma cell lines was found to be independent of both GSK-3βand NF-B  while GSK-3β inhibition in prostate cancer and HEK cells actually increased NF-B activitydespite promoting TNF-induced apoptosis .The specifics of apoptosis regulation by GSK-3β remain both ambiguous and complex, requiring furtherresearch in order to determine the mechanisms of action responsible for differential control of cellsurvival. In addition to variations in cell signaling and proliferation status, the effect of GSK-3β onapoptosis may depend on cellular localization. Only cytosolic GSK-3β was found to inhibit TNF-mediatedapoptosis  while apoptosis enhances nuclear localization , suggesting a potential localization-based mechanism for differential apoptotic regulation. Insufficient data is available to explain thecontradictory effects proposed for GSK-3β on p53-mediated apoptosis and a more detailed study isrequired in order to determine the reasons for these observed differences, but differential localizationof p53, MDM2 and GSK-3β may help define the regulatory role of GSK-3β in various systems.Positive Regulators of GSK-3βSeveral molecules are known to potentiate the downstream effects of GSK-3β (Table 3). Positiveregulators ofGSK-3β are often utilized for enhancing the pro-apoptotic effects of GSK-3β in the contextof chemotherapy for cancer treatment (reviewed in ). These regulators typically operate through anindirect mechanism, actually serving as inhibitors for upstream negative regulators. For example, GSK-3βactivity is increased upon inhibition of PI3-Kinase with wortmannin or LY294002 [78-80]. Many GSK-3βregulators act to inhibit Akt by blocking its activation or kinase activity. The kinase inhibitorstaurosporine and the COX-2 inhibitor Celecoxib block the activating phosphorylation of Akt by PDK [81-85]. Additionally, curcumin dephosphorylates Akt to prevent its downstream inactivation of GSK-3β ,as does the histone deacetylase inhibitor Trichostatin A, in a PP1-dependent manner . Akt/protein
kinase B signaling inhibitor-2 (API-2) appears to suppress both Akt activation and kinase activityindependent of any upstream inhibitor effects .Alternative GSK-3β regulators have less defined and more indirect mechanisms. The mTOR inhibitorrapamycin has been shown to activate GSK-3β with some studies suggesting a potential influence of themTOR pathway on GSK-3β regulation through phosphorylation by s6 kinase [88, 89]. Other moleculestarget the ability of GSK-3β to degrade cyclin D1. Vitamin A derived retinoids and multipledifferentiation-inducing factors (DIFs) enhance GSK-3β activation and kinase activity [90-93] as a meansfor cyclin D inhibition to promote cell cycle arrest and differentiation.Inhibitors of GSK-3While a potential therapeutic role of GSK-3β inhibitors has been suggested for some time, they havegained significant interest as a clinical tool over the past decade. GSK-3β inhibitors are currently beingutilized for the treatment of various diseases including Alzheimer’s disease [94, 95] and otherneurodegenerative diseases, diabetes, inflammatory disorders , radiation damage, and cancer. Various pharmaceutical companies have these inhibitors in clinical trials . A classical example ofa non-specific GSK-3β inhibitor is lithium , which has been shown to inhibit GSK-3β with an IC50 ofapproximately 2 mM in an uncompetitive manner with respect to peptide substrate. Lithium was foundto inhibit GSK-3β in a competitive manner by binding directly to magnesium binding sites of the enzyme, thus providing evidence for a molecular mechanism for enzyme inactivation by lithium ions. Fourdistinct regions of GSK-3 have been targeted for inhibition: the Mg2+ATP-binding active site, a separateMg2+-binding site, the substrate-binding groove and the scaffold-binding region [33, 99]. Severalinhibitors compete with Mg2+ and/or ATP to occupy its binding site. However, the specificity of theseinhibitors towards GSK-3 relative to other kinases varies significantly (Table 4). Structural studies have
further elucidated molecular mechanisms for substrate selection and GSK3-inhibition [100-106].Beryllium was shown to compete with both ATP and Mg 2+, while lithium competed only with Mg2+ .The small molecule inhibitors of GSK-3 SB-216763 and SB-415286 are structurally distinct maleimidesthat inhibit GSK-3/ in vitro, with Kis of 9 nM and 31 nM respectively, in an ATP competitive manner. Hymenialdisine  and paullones  also inhibit GSK-3β in a ATP competitive manner.Indirubins inhibit GSK-3β in a ATP competitive manner with a IC 50 of 50-100 nM [111-113]. Smallmolecule inhibitors like TZDZ8 that are thiadiazolidinones inhibit GSK-3β with a IC50 of 2µM in a noncompetitive manner [114, 115]. The other type of GSK-3 inhibitors is represented by cell-permeable,phosphorylated substrate-competitive peptides which interact with the phospho-recognition motifcomprising R96, R180 and K205 to prevent substrate access to the active site. There are alsoGSK-3-inhibiting peptides that contain GSK-3 interacting domains and block the interaction betweenAxin and GSK-3 and prevent β-catenin phosphorylation . In the recent decade small moleculeinhibitors of GSK-3β are emerging as a promising drug for treatments against neurodegenerativediseases, radiation damage, Alzheimer’s disease, diabetes and cancer.Exploiting the GSK-3β ConundrumGSK-3β signaling is a complex process influenced not only by cellular type and transformation status, butby environmental and cellular conditions. Survival signals have been mainly determined by studiesinvolving GSK-3β inhibition, through gene silencing or pharmacologic inhibition. The resulting inhibitionof apoptosis is complex, and requires further elucidation. However several studies suggest that theeffects may at least in part be mediated by the effect of GSK-3β on NF-κB levels. In addition, it is clearthat subcellular localization is important, as only cytosolic GSK-3β seems to be able to mediate thesurvival signals.
Notably, the role in promotion of apoptosis by GSK-3β has been more clearly delineated. It performs thistask by both facilitating pro-apoptotic signals while inhibiting anti-apoptotic molecules. This signalinterplay occurs mostly at the level of the mitochondria, and combined with the association withprimarily nuclear GSK-3β, suggests a downstream role of GSK-3β in modulation.So how do we exploit these paradoxical roles of GSK-3β? In healthy cells, the shift to pro-survival modesis important for cell survival under conditions of cellular stress. In these cases, the upstream signalsseem to override the mitochondrial-based apoptotic machinery to allow the cells to escape potentiallylethal damage. There have been attempts to exploit these pro-survival roles in neurodegenerativediseases, which are typified by high apoptosis rates. Reduction of disease -associated apoptosis byGSK-3β modulating agents can restore balance to off-kilter apoptotic machinery, resulting in decreasedcellular turnover and the resultant protection of the at-risk neuronal population. In addition to diabetes,and neurodegenerative disorders, we believe that GSK-3β inhibition may play a promising role inpatients receiving irradiation.While radiation dose-escalation has been important for the treatment of multiple cranial tumors (e.g.brain metastases, primary gliomas) and benign disorders (e.g. vestibular schwannoma, meningioma),the treatment is limited by the effects of irradiation on healthy surrounding neurons. It has beendemonstrated that GSK-3β inhibition can protect hippocampal neurons (in primary culture and murinepups) from irradiation-induced damage [9, 52]. Thotala et al. demonstrated improved survival ofintestinal crypt cells, and increased latency to murine GI-related death from irradiation . This reportsuggested that GSK-3β inhibitors could reduce deleterious consequences of intestinal irradiation, andpossibly improve patient quality of life measures. It would be worthwhile to explore their utility insyngenic murine models of neural cancer, murine tumor xenografts, as well as human clinical trials of
patients in the setting of re-irradation (e.g. recurrent glioma). Reports of radiation protection have alsobeen demonstrated with small molecular inhibitors of GSK-3β in the gastrointestinal system.In cancer, however, the apoptotic machinery is often defective allowing cells to undergo unregulatedproliferation. In this case, negative regulation of GSK-3β can serve to tip the balance in favor ofapoptosis. Dickey et al. demonstrated the ability of GSK-3β inhibition to effectively enhance cell death ofneuroblastoma cells in vitro and in a murine xenograft model . Similar findings have beendemonstrated in glioma [63, 64]. The interplay between GSK-3β regulation and other cell death stimuli isbeing carefully studied across a wide variety of cancer types, and there is promising data suggesting astrong role for this form of therapy in the near future. The bifunctional role of GSK-3β as a facilitator ofapoptosis and a mediator of pro-survival signals has important implications in both the generation ofnovel therapies, and the understanding of complex disease states.The use of both positive and negative regulators of GSK-3β offer exciting treatment possibilities for amultitude of diseases. The complexity of the GSK-3β network requires careful examination howeverwhen considering modulating its function in a clinical setting. More studies are required to clearlyunderstand the effects of regulating GSK-3β on the multiple signaling pathways involved in growth,development and metabolism. The effect of GSK-3β on cell survival and apoptosis appears to be contextdependent, and the required mode of action will likely depend on the specific pathway, cell type anddisease being targeted. While the vast network of GSK-3β offers a treatment option for multiplediseases it also requires careful consideration of all the factors involved in order to prepare againstpotential side effects.
Table 1. Conditions where GSK-3β facilitates apoptosis.System or Stimulus MechanismC(2) Ceramide-associated Inhibits the phosphorylation of AKT and ERK pathways anddamage through the dephosphorylation of GSK-3β. GSK-3β inhibitors have been shown to inhibit apoptosis through inhibiting dephosphorylation of AKT and GSK-3β .LPS mediated endotoxic shock While specific apoptotic studies have not been performed, LPS has been shown to stabilize apoptotic signal-regulating kinase-1 (ASK-1), a serine-threonine kinase associated with stress-induced apoptosis Immune System Regulates in apoptosis of activated T-Cells HIV-mediated neuronal damage Inhibits NF-κB [121-123]Neurodegenerative Neuronal or oligodendrocyte injury or toxicity (including priondisease-related toxicity and peptide) is associated with increased activity of GSK-3β. [117, 124-130]Oxidative stress Negative regulators of GSK-3B are associated with increased survival factors [117, 124-130] and neuroprotection[9, 38]ER Stress ER-Stress can lead to dephosphorylation of pGSK-3β(S9), leading to stress-induced apoptosis through activated caspase-3[12-14, 26, 28]Hypoxia/Ischemia Activates mitochondrial death pathway [35, 131-134]
Table 2. Other Pro-survival roles of GSK-3β.System MechanismER stress Reduces expression of the pro-apoptotic transcription factor CHOP/GADD153 Glioblastoma differentiation Promotes self-renewal through interaction with Bmi1 Death receptor complex Inhibits apoptotic signaling and caspase activation Chemotherapy Targeting by death-inducing drugs suggesting an inhibitory role Oncogenic activation Inhibits apoptotic activation by c-myc Glucose Metabolism Prevents apoptosis through mitochondrial stabilization 
Table 3. List of known positive regulators of GSK-3βActivator Activation Potency Mode of Activation Notes Inhibits PDKCelecoxib IC50 = 3.5µM phosphorylation of Akt COX-2 inhibitor  General kinase Inhibits PDK inhibitor (includingStaurosporine IC50 = 0.22µM phosphorylation of Akt PKA/PKC) [82, 84, 85] Induces Akt HDAC inhibitor, actsTrichostatin A Unknown dephosphorylation through PP1  Direct target notCircumin Unknown AKT dephosphorylation known Akt/protein kinase B Does not affectsignaling inhibitor-2 Suppresses Akt kinase upstream Akt(API-2) Unknown activity and activation activators  Indirect effect onWortmannin IC50 = 5nM Inhibits PI3-Kinase GSK-3B [78, 79] Likely affects ATPLY294002 IC50 = 1.4µM Inhibits PI3-Kinase binding to kinase [79, 80] mTOR pathway canRapamycin Unknown Potentially inhibits S6K1 also inhibit GSK3 [88, 89] Reduces inhibitory Enhances GSK-3β kinase phosphorylation andDifferentiation- activity and promotes enhances activatinginducing factors (DIFs) Unknown nuclear localization phosphorylation [92, 93] Promotes Reduces inhibitory GSK-3β-dependent phosphorylation of cyclin D1 degradation [62, 90]Retinoids Unknown GSK-3β
Table 4. Selected list of known GSK-3 inhibitors.Inhibitor Inhibition potency Mode of Inhibition NotesBeryllium IC50 = 6 mM Mg competitor Also inhibits cdc2Lithium Ki=2 mM Mg competitor Does not inhibit aAnilino maleimides (SB216763, Ki = 10-30 nM ATP competitor range of otherSB415286) kinasesArylpyrazolopyridazines (e.g. 6-aryl Also inhibits IC50 = 0.8-150 nM ATP competitorpyrazole [3,4-b] byridine 4) CDK2Bisindole maleimides (e.g. Ro IC50 = 5-170 nM ATP competitor Also inhibits PKC31-8220, GF 109203x)Indirubins (6-bromoindirubin-3’- Also inhibits IC50 = 5-50 nM ATP competitoroxime, aka BIO) CDKs Also inhibitsPaullones (alsterpaullone) IC50 = 4-80 nM ATP competitor CDKs SubstratePseudosubstrate peptide Ki = 0.7 mM Specific competitor
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Basic Domain S9 Y216NH2 COOH p53 AssociationFigure 1: Glycogen synthase kinase-3β (GSK-3β) structure. Above: GSK-3β is a 433 residue proteinconsisting of 3 distinct structural domains. The N-terminal domain (yellow) consists of the first 134residues and forms a 7-strand β-barrel. A short linker from the N-terminal domain, residues 135-151connect the N-terminal domain to the α-helical domain (magenta). The α-helical domain is composed ofresidues 152-342. Sandwiched between the N-terminal and -helical domain is the ATP binding site. TheC- terminal domain consists of residues 343-433 (blue). Below: A strand diagram of GSK-3.Phosphorylation of Ser-9 inactivates the enzyme, while phosphorylation of Tyr-216 activates. The p53association region and basic domain region are both located in the N-terminal domain. Image was madeusing PyMol Molecular Graphics Software version 1.3 with the PDB structure 1UV5.
Figure 2: Regulation of GSK-3β. GSK-3β is a multifunctional kinase that has a role in various signalingpathway that regulate cell fate. ZAK1 or Fyn can phosphorylate Tyr-216 which increases the GSK-3βactivity. GSK-3β can phosphorylate downstream targets like β-catenin and degrade it through theubiquitin-proteasome system. Akt and PKC on the other hand can attenuate GSK-3β enzymatic activityby phosphorylating Ser-9. Inhibition of GSK-3β activity therefore leads to stabilization and accumulationof β-catenin in the cytosol, which is shuttled into the nucleus where it functions to regulate geneexpression. GSK-3β is also involved in cell cycle regulation through the phosphorylation of cyclin D1,which results in the rapid proteolytic turnover of cyclin D1 protein.
Figure 3: GSK-3β’s role in apoptosis signaling. The above schematic shows the role of activated GSK-3βand its role in regulating apoptosis. Active GSK-3β inhibits MDM2 regulation of p53, leading to DNArepair and growth arrest, and in some cases the activation of the caspase cascade through Bax topromote apoptosis. Active GSK-3β also positively regulates NFB by activating IKK, IB and p65, leadingto the inhibition of TNF-mediated apoptosis. These actions inhibit the initiation of apoptosis through theTNF signaling caspase. Conversely, inactivation of GSK-3β leads to accumulation of β-catenin that istransported into the nucleus for WNT signaling to further promote cell survival.