GSK-3: Functional Insights from Cell Biology and Animal Models

The Hedgehog (Hh) signalling pathway has a crucial role in several developmental processes and is aberrantly activated in a variety of cancers. In Drosophila, many of the canonical Hh pathway components are phosphorylated, yet the precise role of these phosphorylation events in the regulation of Hh signal transduction is unclear. Furthermore, the Hh pathway receives input from several kinases that have well-described roles in other cellular functions, some of which have both positive and negative effects on Hh signalling. Several recent studies have characterized the role of specific phosphorylation events in the Hh pathway, and have begun to shed light on how phosphorylation of Hh signalling components affects their subcellular location, stability and activity to mediate the transcriptional response to the Hh gradient.

Regulation of GSK-3: A Cellular Multiprocessor

Oncoprotein Networks

Insulin regulates the phosphorylation and activities of Akt and glycogen synthase kinase-3 (GSK3) in peripheral tissues, but in the brain it is less clear how this signaling pathway is regulated in vivo and whether it is affected by diabetes. We found that Akt and GSK3 are sensitive to glucose, because fasting decreased and glucose administration increased by severalfold the phosphorylation of Akt and GSK3 in the cerebral cortex and hippocampus of non-diabetic mice. Brain Akt and GSK3 phosphorylation also increased after streptozotocin administration (3 days), which increased blood glucose and depleted blood insulin, indicating regulation by glucose availability even with deficient insulin. Changes in Akt and GSK3 phosphorylation and activities in epididymal fat were opposite to those of brain after streptozotocin treatment. Streptozotocin-induced hyperglycemia and increased brain Akt and GSK3 phosphorylation were reversed by lowering blood glucose with insulin administration. Long term hyperglycemia also increased brain Akt and GSK3 phosphorylation, both 4 weeks after streptozotocin and in db/db insulin-resistant mice. Thus, the Akt-GSK3 signaling pathway is regulated in mouse brain in vivo in response to physiological and pathological changes in insulin and glucose.

Catecholamines, acting through adrenergic receptors, play an important role in modulating the effects of insulin on glucose metabolism. Insulin activation of glycogen synthesis is mediated in part by the inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3). In this study, catecholamine regulation of GSK-3beta was investigated in Rat-1 fibroblasts stably expressing the alpha1A-adrenergic receptor. Treatment of these cells with either insulin or phenylephrine (PE), an alpha1-adrenergic receptor agonist, induced Ser-9 phosphorylation of GSK-3beta and inhibited GSK-3beta activity. Insulin-induced GSK-3beta phosphorylation is mediated by the phosphatidylinositol 3-kinase/Akt signaling pathway. PE treatment does not activate phosphatidylinositol 3-kinase or Akt (Ballou, L. M., Cross, M. E., Huang, S., McReynolds, E. M., Zhang, B. X., and Lin, R. Z. (2000) J. Biol. Chem. 275, 4803-4809), but instead inhibits insulin-induced Akt activation and GSK-3beta phosphorylation. Experiments using protein kinase C (PKC) inhibitors suggest that phorbol ester-sensitive novel PKC and Gö 6983-sensitive atypical PKC isoforms are involved in the PE-induced phosphorylation of GSK-3beta. Indeed, PE treatment of Rat-1 cells increased the activity of atypical PKCzeta, and expression of PKCzeta in COS-7 cells stimulated GSK-3beta Ser-9 phosphorylation. In addition, PE-induced GSK-3beta phosphorylation was reduced in Rat-1 cells treated with a cell-permeable PKCzeta pseudosubstrate peptide inhibitor. These results suggest that the alpha1A-adrenergic receptor regulates GSK-3beta through two signaling pathways. One pathway inhibits insulin-induced GSK-3beta phosphorylation by blocking insulin activation of Akt. The second pathway stimulates Ser-9 phosphorylation of GSK-3beta, probably via PKC.

There are 518 protein kinases in the human genome, many of which act as important nodes in signal transduction pathways (1). Members of this class of important regulatory enzymes are also, unsurprisingly, frequently mutated or dysregulated in cancer and many other diseases. They have thus become the target of therapeutic interventions, with many successes. What is often overlooked, however, is that there are fewer than 15 signaling pathways for transmitting extracellular information into the cell. This paucity of communication routes is responsible for extracting appropriate cellular responses to a myriad of external cues. If it sounds as though too many eggs are in a limited number of baskets, the situation is further exacerbated by sharing of several transduction components between pathways. The most egregious example is that of glycogen synthase kinase-3 (GSK-3), a protein kinase first identified as a regulator of glycogen synthesis (2). This innocuously named protein is anything but because it plays a central role in at least four of these signaling pathways—the Wnt, Notch, Hedgehog, and nuclear factor-kB (NF-kB) pathways—with important roles in at least six more—the ras/mitogen-associated protein kinase (RAS/MAPK), cyclic-AMP, transforming growth factor-b/activin (TGF-b), phosphatidylinositol -3-kinase (PI3K), jun kinase/stressactivated protein kinase (JNK/SAPK), and janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. There are two highly related isoforms of GSK-3 (termed a and b) encoded by distinct genes, but that is still a substantial responsibility assigned to a particular protein kinase begging the question of why and how pathways maintain the authenticity of their signals if relying on the same molecules (3). Only the cyclic GMP, p38 mitogen-activated protein kinase (p38 MAPK), Ca 2+ , calmodulin, and Hippo pathways, and the intracellular DNA damage response and unfolded protein response pathways currently lack known roles for GSK-3. In this issue of the Journal, Tang et al. (4) report that suppression of GSK-3b interferes with the growth and viability of osteosarcoma cells, likely by inhibition of NF-kB. This transcription factor plays a critical role in inflammatory responses and has been directly implicated in the promotion of tumorigenesis (5). An important connection between GSK-3 and NF-kB was revealed upon the generation of mice that were genetically engineered to lack GSK-3b (6). GSK-3b null embryos frequently died before birth of extensive apoptosis in the liver, a phenotype reminiscent of mice that were engineered to lack RelA, a subunit of NF-kB (7). Mouse embryo fibroblasts derived from the GSK-3 b null mice displayed a substantial deficit in the ability of tumor necrosis factor alpha (TNF-a) to induce activation of NF-kB and its regulation of anti-apoptotic gene expression. (TNF-a-mediated induction of the caspase pathway was unaffected; hence, the hepatocytes were sensitized to programmed cell death.) Interestingly, mice engineered to lack GSK-3a are viable and NF-kB signaling is normal (8). The molecular mechanism by which GSK-3b selectively regulates and is required for NF-kB function remains unclear, but this kinase modifies the spectrum of genes regulated by NF- kB (9). Both isoforms of GSK-3 are negatively regulated by serine phosphorylation (ie, Serine 21 of GSK-3a and Serine 9 of GSK-3b), and this is one mechanism by which the cyclic-AMP and phosphatidylinositol-3- kinase pathways suppress GSK-3 activity. Tang et al. (4) observed that the level of inhibitory phosphorylation of GSK-3b at Serine 9 was low in several osteosarcoma lines compared with that in a normal osteoblast cell line, suggesting that GSK-3 activity was higher than normal, although this was not directly measured. They also found that b-catenin levels (a target of the Wnt pathway) were increased in some lines, but this finding is unlikely to be related to GSK-3b phosphorylation for several reasons. First, agonists that induce serine phosphorylation of GSK-3 do not typically affect b-catenin (10,11), probably because

Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease.

the wnt signaling system consists of ancient pathways with highly conserved core components tracing back more than 700 million years including insects and nematodes ([20][1]). Wnt signals classically play a crucial role in embryonic development concerning cell fate, motility, and polarity, as well

The ability of glycogen synthase kinase-3 (GSK-3) to phosphorylate insulin receptor substrate-1 (IRS-1) is a potential inhibitory mechanism for insulin resistance in type 2 diabetes. However, the serine site(s) phosphorylated by GSK-3 within IRS-1 had not been yet identified. Using an N-terminal deleted IRS-1 mutant and two IRS-1 fragments, PTB-1 1-320 and PTB-2 1-350, we localized GSK-3 phosphorylation site(s) within amino acid sequence 320-350. Mutations of serine 332 or 336, which lie in the GSK-3 consensus motif (SXXXS) within PTB-2 or IRS-1, to alanine abolished their phosphorylation by GSK-3. This suggested that Ser332 is a GSK-3 phosphorylation site and that Ser336 serves as the "priming" site typically required for GSK-3 action. Indeed, dephosphorylation of IRS-1 prevented GSK-3 phosphorylation. Furthermore, the phosphorylated peptide derived from the IRS-1 sequence was readily phosphorylated by GSK-3, in contrast to the nonphosphorylated peptide, which was not phosphorylated by the enzyme. When IRS-1 mutants S332A(IRS-1), S336A(IRS-1), or S332A/336A(IRS-1) were expressed in Chinese hamster ovary cells overexpressing insulin receptors, their insulin-induced tyrosine phosphorylation levels increased compared with that of wild-type (WT) IRS-1. This effect was stronger in the double mutant S332A/336A(IRS-1) and led to enhanced insulin-mediated activation of protein kinase B. Finally, immunoblot analysis with polyclonal antibody directed against IRS-1 phosphorylated at Ser332 confirmed IRS-1 phosphorylation in cultured cells. Moreover, treatment with the GSK-3 inhibitor lithium reduced Ser332 phosphorylation, whereas overexpression of GSK-3 enhanced this phosphorylation. In summary, our studies identify Ser332 as the GSK-3 phosphorylation target in IRS-1, indicating its physiological relevance and demonstrating its novel inhibitory role in insulin signaling.

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.

Glycogen synthase kinase 3 (GSK3) would have to be a contender for the "most-studied molecule" award. Once the subject of intense investigation for its role in control of metabolism, it is now recognized to have critical roles in the Wnt signaling pathway and diverse cellular processes, including development and tumorigenesis (see the Wnt Connections Map Pathway ). Now, Jia et al. add a new role for the kinase in control of development. They find that GSK3 inhibits signaling through the Hedgehog pathway in Drosophila . Hedgehog is a secreted protein that acts in patterning during development, and disrupted Hh signaling appears to contribute to human diseases, including cancer. Hh signaling promotes processing of the transcription factor Ci (Cubitus interruptus) to a form that activates target promoters. Such processing of Ci is regulated by phosphorylation, and Jia et al. 's results indicate that Ci, once primed by phosphorylation by protein kinase A, is phosphorylated by GSK3 and that full phosphorylation is required for processing. Lack of the Drosophila GSK3 gene causes developmental abnormalities similar to those seen with excessive Hh signaling. Clearly, GSK3 is engaged in many signaling pathways, and unwanted cross talk seems to be prevented by localization of GSK3 in protein complexes with its signaling partners. Nevertheless, the authors note that their findings raise the possibility of coordinate regulation of both the Hh and Wnt pathways--two major regulatory pathways controlling development--through GSK3. R. T. Moon, Wnt/beta-catenin pathway. Science's STKE (Connections Map, as seen April 2002), http://stke.sciencemag.org/cgi/cm/CMP_5533. [Pathway] J. Jia, K. Amanai, G. Wang, J. Tang, B. Wang, J. Jiang, Shaggy/GSK3 antagonizes Hedgehog signalling by regulating Cubitus interruptus. Nature 416 , 548-552 (2002). [Online Journal]

Essential Roles for GSK-3s and GSK-3-Primed Substrates in Neurotrophin-Induced and Hippocampal Axon Growth

Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-beta peptide (Abeta) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of Abeta. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of beta-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of Abeta by increasing the beta-cleavage of amyloid precursor protein, generating more substrate for an unmodified gamma-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of beta-catenin and slightly decreased Abeta production. It is concluded that the LiCl-mediated increase in Abeta production is not related to GSK3 inhibition.

Excessive neuroinflammation contributes to many neurological disorders and is poorly controlled therapeutically. The signal transducer and activator of transcription (STAT) family of transcription factors has a central role in inflammatory reactions, being stimulated by multiple cytokines and interferons and regulating the expression of many proteins involved in inflammation. We found that STAT3 activation is highly dependent on glycogen synthase kinase-3 (GSK3). Inhibitors of GSK3 greatly reduced (>75%) the activating STAT3 tyrosine phosphorylation in mouse primary astrocytes, microglia, and macrophage-derived RAW264.7 cells induced by interferon-gamma (IFNgamma), IFNalpha, interleukin-6, or insulin. GSK3 inhibitors blocked STAT3 DNA binding activity and the expression of STAT3-induced GFAP and Bcl-3. GSK3 dependence was selective for activation of STAT3 and STAT5, whereas STAT1 and STAT6 activation were GSK3-independent. Knockdown of the two GSK3 isoforms showed STAT3 and STAT5 activation were dependent on GSK3beta, but not GSK3alpha. The regulatory mechanism involved GSK3beta binding STAT3 and promoting its association with the IFNgamma receptor-associated intracellular signaling complex responsible for activating STAT3. Furthermore, GSK3beta associated with the IFNgamma receptor and was activated by stimulation with IFNgamma. Thus, inhibitors of GSK3 reduce the activation of STAT3 and STAT5, providing a mechanism to differentially regulate STATs to modulate the inflammatory response.

Despite what its namesake suggests, Glycogen Synthase Kinase 3 (GSK3) is a kinase that is implicated in a myriad of signalling pathways and has recently received considerable interest due to its conflicting roles oncogenesis. Though GSK3 has classically been regarded as a tumour suppressor due to in role in supressing WNT signalling, emerging evidence suggests that GSK3 also functions as a tumor promoter by facilitating tumor cell survival and conferring drug resistance in cancer. These tumor promoting effects have been partly linked to the expression of certain NFκB target genes, whose expression in some cancers are influenced by direct GSK3 phosphorylation of NFκB's p65 subunit. Given the complex interplay between GSK3 and the androgen receptor in prostate cancer (PCa), and the inverse correlation between the androgen receptor status and NFκB, we investigate the potential of GSK3 inhibitors to treat and overcome drug resistance in PCa. A kinase inhibitor screen for 80 compounds and additional chemical inhibitors for GSK3 was carried out using the JANUS Automated Workstation at the robotics facility at NUI Galway, Ireland. The cellular fractions for treated cells were separated and assessed for differences in expression between the cytoplasmic and nuclear compartments using western blotting. Flow cytometry and western blotting using antibodies for markers of apoptosis were used to assess for mode of cell death. Differences in gene expression of NFκB target genes were assessed using gene specific primers for SYBR green RT-PCR. RNAi using siRNA directed against each of the GSK3 isoforms, α and β, was utilised for target validation The kinase inhibitor screen demonstrated significant growth inhibition among all the tested PCa cell lines to the GSK3 inhibitor, BIO. This result was validated using two additional chemical GSK3 inhibitors, LiCl and CHIR99021. Expression analysis demonstrated aberrant NFκB and GSK3 expression in the cytoplasmic and nuclear compartments of the cell lines that varied upon treatment with GSK3 inhibitors. siRNA directed against GSK3 confirmed that the effects of the inhibitors were GSK3 specific and demonstrated that both isoforms distinguished between the effects of each of the two isoforms. Our results suggest that GSK3 has a role in the pathogenesis of PCa. This notion is supported by the findings that GSK3 inhibition reduces the viability of PCa cells via a mechanism that is consistent with apoptosis. Furthermore, both NFκB and GSK3 are shown to be active and aberrantly co-expressed in our PCa model. Collectively, these results suggest that GSK3 has an effect on NFκB that may be independent of WNT signalling. Citation Format: Husnain Ali, Amy Burke, Enda O'Connell, Frank Sullivan, Frank Giles, Sharon Glynn. GSK3α/β inhibition as a drug target in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4370. doi:10.1158/1538-7445.AM2015-4370