International Journal of Cell Biology System ER stress Glioblastoma diﬀerentiation Death receptor complex Chemotherapy Oncogenic activation Glucose metabolism activation of the p65 subunit, suggesting a likely mech- anism for lithium-induced TNF hypersensitivity [89, 90] (Figure 3). The role of GSK-3β on NF-κB activation may also be mediated indirectly through inhibition of β-catenin, as cancer cells with high β-catenin levels are especially sensitive to TNF-induced death [91]. Despite the abundance of evidence implicating GSK- 3β in protection from TNF-mediated apoptosis, a few conﬂicting reports further complicate our understanding of the pathway. A more recent study claims 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 [92]. Similarly, TNF sensitization by lithium in multiple sarcoma cell lines was found to be independent of both GSK-3β and NF-κB [93] while GSK-3β inhibition in prostate cancer and HEK cells actually increased NF-κB activity despite promoting TNF-induced apoptosis [94]. The speciﬁcs of apoptosis regulation by GSK-3β remain both ambiguous and complex, requiring further research in order to determine the mechanisms of action responsible for diﬀerential control of cell survival. In addition to variations in cell signaling and proliferation status, the eﬀect of GSK-3β on apoptosis may depend on cellular localization. Only cytosolic GSK-3β was found to inhibit TNF-mediated apoptosis [80] while apoptosis enhances nuclear localization [95], suggesting a potential localization-based mechanism for diﬀerential apoptotic regulation. Insuﬃcient data is available to explain the contradictory eﬀects proposed for GSK-3β on p53-mediated apoptosis, and a more detailed study is required in order to determine the reasons for these observed diﬀerences, but diﬀerential localization of p53, MDM2, and GSK-3β may help deﬁne the regulatory role of GSK-3β in various systems. 4. Positive Regulators of GSK-3β Several molecules are known to potentiate the downstream eﬀects of GSK-3β (Table 3). Positive regulators of GSK-3β are often utilized for enhancing the proapoptotic eﬀects of GSK-3β in the context of chemotherapy for cancer treatment (reviewed in [96]). These regulators typically operate through an indirect mechanism, actually serving as inhibitors for upstream negative regulators. For example, GSK-3β activity is increased upon inhibition of PI3-Kinase with wortmannin or LY294002 [97–99]. Many GSK-3β reg- ulators act to inhibit Akt by blocking its activation or kinase 5 Table 2: Other pro survival roles of GSK-3β. Mechanism Reduces expression of the proapoptotic transcription factor CHOP/GADD153 [87]. Promotes self-renewal through interaction with Bmi1 [81]. Inhibits apoptotic signaling and caspase activation [83]. Targeted by death-inducing drugs suggesting an inhibitory role [84]. Inhibits apoptotic activation by c-myc [82]. Prevents apoptosis through mitochondrial stabilization [82]. activity. The kinase inhibitor staurosporine and the COX-2 inhibitor Celecoxib block the activating phosphorylation of Akt by PDK [100–104]. Additionally, curcumin dephospho- rylates Akt to prevent its downstream inactivation of GSK-3β [102], as does the histone deacetylase inhibitor Trichostatin A, in a PP1-dependent manner [105]. Akt/protein kinase B signaling inhibitor-2 (API-2) appears to suppress both Akt activation and kinase activity independent of any upstream inhibitor eﬀects [106]. Alternative GSK-3β regulators have less deﬁned and more indirect mechanisms. The mTOR inhibitor rapamycin has been shown to activate GSK-3β with some studies suggesting a potential inﬂuence of the mTOR pathway on GSK-3β regulation through phosphorylation by s6 kinase [107, 108]. Other molecules target the ability of GSK-3β to degrade cyclin D1. Vitamin A derived retinoids and multiple diﬀerentiation-inducing factors (DIFs) enhance GSK-3β activation and kinase activity [109–112] as a means for cyclin D inhibition to promote cell cycle arrest and diﬀerentiation. 5. Inhibitors of GSK-3β While a potential therapeutic role of GSK-3β inhibitors has been suggested for some time, they have gained signiﬁcant interest as a clinical tool over the past decade. GSK-3β inhibitors are currently being utilized for the treatment of various diseases including Alzheimer’s disease [113, 114] and other neurodegenerative diseases [18], diabetes, inﬂamma- tory disorders [115], radiation damage, and cancer [116]. Various pharmaceutical companies have these inhibitors in clinical trials [116]. A classical example of a nonspeciﬁc GSK-3β inhibitor is lithium [21], which has been shown to inhibit GSK-3β with an IC50 of approximately 2 mM in an uncompetitive manner with respect to peptide substrate. Lithium was found to inhibit GSK-3β in a competitive manner by binding directly to magnesium-binding sites of the enzyme [117], thus providing evidence for a molecular mechanism for enzyme inactivation by lithium ions. Four distinct regions of GSK-3β have been targeted for inhibi- tion: the Mg2+ ATP-binding active site, a separate Mg2+- binding site, the substrate-binding groove, and the scaﬀold- binding region [33, 118]. Several inhibitors compete with Mg2+ and/or ATP to occupy its binding site. However, the speciﬁcity of these inhibitors towards GSK-3β relative to other kinases varies signiﬁcantly (Table 4). Structural studies have further elucidated molecular mechanisms for substrate