Glycogen Synthase Kinase-3 Levels Research Articles

Regulation of the Interaction between Glycogen Synthase Kinase 3 and the Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein stabilizes beta-catenin by the novel mechanism of binding to the negative regulator, glycogen synthase kinase 3 (GSK-3), and depleting cytoplasmic GSK-3 levels. The two domains of LANA required for interaction with GSK-3 were further characterized. Evidence for similarity between the C-terminal LANA interaction domain and the axin GSK-3 interaction domain was obtained using GSK-3 and LANA mutants. GSK-3(F291L), which does not interact with axin, also failed to bind to LANA, and a mutation in the axin homology domain of LANA, L1132P, destroyed binding to GSK-3. The N-terminal LANA interaction domain was found to mediate interaction by acting as a substrate for GSK-3. GSK-3(R96A), a priming pocket mutant, did not bind to LANA, suggesting that LANA was a primed GSK-3 substrate. Phosphorylation of endogenous LANA precipitated from primary effusion lymphoma cells was inhibited by the GSK-3 inhibitor LiCl. GST-LANA(1-340) was phosphorylated by GSK-3, and mitogen-activated protein kinase (MAPK) and casein kinase I functioned as priming kinases in vitro. Mutation of consensus GSK-3 sites revealed that sites between LANA amino acids 219 and 268 were important for GSK-3 phosphorylation. Immunoprecipitation assays revealed that loss of GSK-3 phosphorylation of this N-terminal domain correlated with loss of GSK-3 interaction. Although LANA-associated GSK-3 actively phosphorylated LANA, GSK-3 coprecipitated with LANA was unable to phosphorylate an exogenous peptide substrate. LANA sequestration of GSK-3 may explain the ability of KSHV-infected cells to tolerate increased levels of nuclear GSK-3.

Acute anoxia induces tau dephosphorylation in rat brain slices and its possible underlying mechanisms

Abnormal phosphorylation of microtubule-associated protein tau plays a critical role in Alzheimer's disease (AD), together with a distinct decrease of energy metabolism in the affected brain regions. To explore the effect of acute energy crisis on tau phosphorylation and the underlying mechanisms, we incubated rat brain slices in artificial cerebrospinal fluid (aCSF) at 37 degrees C with or without an oxygen supply, or in aCSF with low glucose concentrations. Then, the levels of total, phosphorylated and unphosphorylated tau, as well as the activities and levels of protein phosphatase (PP)-1, PP-2A, glycogen synthase kinase 3 (GSK-3), extracellular signal-regulated protein kinase (ERK) and C-jun amino terminal kinase (JNK), were measured. It was found, unexpectedly, that tau was significantly dephosphorylated at Ser396/Ser404 (PHF-1), Ser422 (R145), Ser199/Ser202 (Tau-1), Thr181 (AT270), Ser202/Thr205 (AT8) and Thr231 (AT180) by acute anoxia for 30 min or 120 min. The activity of PP-2A and the level of dephosphorylated PP-2A catalytic subunit at tyrosine 307 (Tyr307) were simultaneously increased. The active forms of ERK1/2 and JNK1/2 were decreased under anoxic incubation. The PP-2A inhibitor, okadaic acid (OA, 0.75 microm), completely prevented tau from acute anoxia-induced dephosphorylation and restored the active forms of ERK1/2 and JNK1/2 to the control level. The activities and protein levels of GSK-3 and PP-1 showed no change during acute anoxia. These data suggest that acute anoxia induces tau dephosphorylation, and that PP-2A may play a key role in tau dephosphorylation induced by acute anoxia.

In vivo regulation of GSK3 phosphorylation by cholinergic and NMDA receptors

Glycogen synthase kinase-3 (GSK3), which is inhibited by serine-phosphorylation, is involved in the neuropathology of Alzheimer's disease (AD). We tested if the two therapeutic strategies used for AD, inhibition of acetylcholinesterase and of N-methyl- d-aspartate (NMDA) receptors, modulate the phosphorylation state of the two isoforms of GSK3 in mouse brain. Large, rapid increases in the levels of phospho-Ser21-GSK3α and phospho-Ser9-GSK3β occurred in mouse hippocampus, cerebral cortex, and striatum after treatment of mice with the muscarinic agonist pilocarpine or the acetylcholinesterase inhibitor physostigmine. Treatment with memantine, an NMDA receptor antagonist, also increased the serine-phosphorylation of both GSK3 isoforms in mouse brain. Co-administration of physostigmine and memantine increased serine-phosphorylated GSK3 levels equally to that achieved by either agent alone, indicating that the actions of these two drugs converge on overlapping pools of GSK3. Thus, drugs in each class of therapeutic agents used for AD have the common property of increasing the regulatory serine-phosphorylation of GSK3 within common pools of the enzyme.

Hepatocyte CYP2E1 Overexpression and Steatohepatitis Lead to Impaired Hepatic Insulin Signaling

Insulin resistance and increased cytochrome P450 2E1 (CYP2E1) expression are both associated with and mechanistically implicated in the development of nonalcoholic fatty liver disease. Although currently viewed as distinct factors, insulin resistance and CYP2E1 expression may be interrelated through the ability of CYP2E1-induced oxidant stress to impair hepatic insulin signaling. To test this possibility, the effects of in vitro and in vivo CYP2E1 overexpression on hepatocyte insulin signaling were examined. CYP2E1 overexpression in a hepatocyte cell line decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 in response to insulin. CYP2E1 overexpression was also associated with increased inhibitory serine 307 and 636/639 IRS-1 phosphorylation. In parallel, the effects of insulin on Akt activation, glycogen synthase kinase 3, and FoxO1a phosphorylation, and glucose secretion were all significantly decreased in CYP2E1 overexpressing cells. This inhibition of insulin signaling by CYP2E1 overexpression was partially c-Jun N-terminal kinase dependent. In the methionine- and choline-deficient diet mouse model of steatohepatitis with CYP2E1 overexpression, insulin-induced IRS-1, IRS-2, and Akt phosphorylation were similarly decreased. These findings indicate that increased hepatocyte CYP2E1 expression and the presence of steatohepatitis result in the down-regulation of insulin signaling, potentially contributing to the insulin resistance associated with nonalcoholic fatty liver disease.

Anesthesia and post‐mortem interval profoundly influence the regulatory serine phosphorylation of glycogen synthase kinase‐3 in mouse brain

Glycogen synthase kinase-3 (GSK3) is a crucial enzyme contributing to the regulation of neuronal structure, plasticity and survival, is implicated as a contributory factor in prevalent diseases such as Alzheimer's disease and mood disorders and is regulated by a wide range of signaling systems and pharmacological agents. Therefore, factors regulating GSK3 in vivo are currently of much interest. GSK3 is inhibited by phosphorylation of serine-9 or serine-21 in GSK3beta and GSK3alpha, respectively. This study found that accurate measurements of phospho-Ser-GSK3 in brain are confounded by a rapid post-mortem dephosphorylation, with approximately 90% dephosphorylation of both GSK3 isoforms occurring within 2 min post-mortem. Furthermore, three anesthetics, pentobarbital, halothane and chloral hydrate, each caused large in vivo increases in the serine phosphorylation of both GSK3beta and GSK3alpha in several regions of mouse brain. Thus, studies of the phosphorylation state of GSK3 in brain, and perhaps in other tissues, need to take into account post-mortem changes and the effects of anesthetics and there is a direct correlation between anesthesia and high levels of serine-phosphorylated GSK3.

Development of glucose intolerance in male transgenic mice overexpressing human glycogen synthase kinase-3β on a muscle-specific promoter

Glycogen synthase kinase-3 (GSK-3) protein levels and activity are elevated in skeletal muscle in type 2 diabetes, and inversely correlated with both glycogen synthase activity and insulin-stimulated glucose disposal. To explore this relationship, we have produced transgenic mice that overexpress human GSK-3β in skeletal muscle. GSK-3β transgenic mice were heavier, by up to 20% ( P < .001), than their age-matched controls due to an increase in fat mass. The male GSK-3β transgenic mice had significantly raised plasma insulin levels and by 24 weeks of age became glucose-intolerant as determined by a 50% increase in the area under their oral glucose tolerance curve ( P < .001). They were also hyperlipidemic with significantly raised serum cholesterol (+90%), nonesterified fatty acids (NEFAs) (+55%), and triglycerides (+170%). At 29 weeks of age, GSK-3β protein levels were 5-fold higher, and glycogen synthase activation (−27%), glycogen levels (−58%) and insulin receptor substrate-1 (IRS-1) protein levels (−67%) were significantly reduced in skeletal muscle. Hepatic glycogen levels were significantly increased 4-fold. Female GSK-3β transgenic mice did not develop glucose intolerance despite 7-fold overexpression of GSK-3β protein and a 20% reduction in glycogen synthase activation in skeletal muscle. However, plasma NEFAs and muscle IRS-1 protein levels were unchanged in females. We conclude that overexpression of human GSK-3β in skeletal muscle of male mice resulted in impaired glucose tolerance despite raised insulin levels, consistent with the possibility that elevated levels of GSK-3 in type 2 diabetes are partly responsible for insulin resistance.

P3-265 Casein kinase 1 phosphorylates tau and colocalizes with lesions in Alzheimer's disease and inclusion body myositis

Brain protein kinase B (Akt) and glycogen synthase kinase-3 (GSK-3) activities are adaptable to changes of peripheral blood glucose level in vivo. GSK-3 phosphorylates microtube-associated protein tau at multiple sites, which can be antagonized by protein phosphatase-2A (PP-2A). The imbalance among these enzymes might have potential connections with diabetes mellitus (DM) and Alzheimer's disease (AD). In this study hyperglycemia rat DM model was achieved by streptozotocin (STZ) treatment. The phosphorylation of tau in the rat hippocampus was detected with specific antibodies. Insulin and Li2CO3 administration were also employed to find out the regulatory efforts of the kinases. We observed that rat hippocampus tau was hyperphosphorylated at Ser396/Ser404 (PHF-1 sites) in STZ-induced DM model, accompanied by lowered phosphorylation levels of Akt, GSK-3 and PP-2A. Lithium, a specific GSK-3 inhibitor, nearly reversed all phosphorylation of tau at above sites in 30 days. Insulin administration restored the blood glucose level in DM rats but suppressed PP-2A activity, resulting in the PHF-1 sites of tau not being dephosphorylated. These findings strongly suggest that STZ-induced hyperglycemia may cause disorder of Akt/GSK-3/PP-2A regulations in rat brain and further lead to abnormal phosphorylation of hippocampus tau.

Glycogen synthase kinase 3: a drug target for CNS therapies.

Abstract Glycogen synthase kinase3 (GSK3) is emerging as a prominent drug target in the CNS. The most exciting of the possibilities of GSK3 lies within the treatment of Alzheimer's disease (AD) where abnormal increases in GSK3 levels and activity have been associated with neuronal death, paired helical filament tau formation and neurite retraction as well as a decline in cognitive performance. Abnormal activity of GSK3 is also implicated in stroke. Lithium, a widely used drug for affective disorders, inhibits GSK3 at therapeutically relevant concentrations. Thus while the rationale remains testable, pharmaceutical companies are investing in finding a selective inhibitor of GSK3. In the present review, we summarize the properties of GSK3, and discuss the potential for such a therapy in AD, and other CNS disorders.

Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo.

Insulin resistance plays a central role in the development of type 2 diabetes, but the precise defects in insulin action remain to be elucidated. Glycogen synthase kinase 3 (GSK-3) can negatively regulate several aspects of insulin signaling, and elevated levels of GSK-3 have been reported in skeletal muscle from diabetic rodents and humans. A limited amount of information is available regarding the utility of highly selective inhibitors of GSK-3 for the modification of insulin action under conditions of insulin resistance. In the present investigation, we describe novel substituted aminopyrimidine derivatives that inhibit human GSK-3 potently (K(i) < 10 nmol/l) with at least 500-fold selectivity against 20 other protein kinases. These low molecular weight compounds activated glycogen synthase at approximately 100 nmol/l in cultured CHO cells transfected with the insulin receptor and in primary hepatocytes isolated from Sprague-Dawley rats, and at 500 nmol/l in isolated type 1 skeletal muscle of both lean Zucker and ZDF rats. It is interesting that these GSK-3 inhibitors enhanced insulin-stimulated glucose transport in type 1 skeletal muscle from the insulin-resistant ZDF rats but not from insulin-sensitive lean Zucker rats. Single oral or subcutaneous doses of the inhibitors (30-48 mg/kg) rapidly lowered blood glucose levels and improved glucose disposal after oral or intravenous glucose challenges in ZDF rats and db/db mice, without causing hypoglycemia or markedly elevating insulin. Collectively, our results suggest that these selective GSK-3 inhibitors may be useful as acute-acting therapeutics for the treatment of the insulin resistance of type 2 diabetes.

The Regulation of Glycogen Synthase Kinase-3 Nuclear Export by Frat/GBP

Previous studies have shown that nuclear levels of glycogen synthase kinase-3 (GSK-3) are dynamically regulated and may affect access of GSK-3 to its substrates. In this study we show that the GSK-3-binding protein Frat/GBP regulates the nuclear export of GSK-3. We show that Frat/GBP contains a nuclear export sequence that promotes its own nuclear export and that of associated GSK-3. Treating cells with leptomycin B increased nuclear levels of endogenous GSK-3 suggesting that an endogenous process targets GSK-3 for nuclear export. To investigate this further, we used two approaches to disrupt the interaction between GSK-3 and endogenous Frat. First we isolated mutants of GSK-3 that selectively interfered with Frat binding and found that these mutants were poorly exported. Second we expressed a peptide that competes with Frat for GSK-3 binding and found that it caused endogenous GSK-3 to accumulate in the nucleus. Together these data suggest that Frat may be the endogenous factor that targets GSK-3 for nuclear export. The dynamic expression patterns of Frat mRNAs together with the role of Frat in mediating GSK-3 nuclear export have important implications for the control of the substrate access of GSK-3 in several signaling pathways.

Compartmentalized tau hyperphosphorylation and increased levels of kinases in transgenic mice.

The formation of neurofibrillary tangles in Alzheimer's disease is preceded by a pretangle stage of hyperphosphorylated tau. To characterize pretangle tau in vivo, we correlated, in human tau transgenic mice, levels of kinases known to phosphorylate tau in vitro with the phosphorylation of tau at specific epitopes. Levels of cyclin-dependent kinase-5 were increased in axons of CA1 pyramidal neurons, where tau was phosphorylated specifically at the AD2 epitope Ser396/Ser404. The 12E8 epitope serine262/serine356 and the AT180 epitope threonine231/serine235 were phosphorylated in dendrites, and colocalized with increased levels of glycogen synthase kinase-3. CA1 neurons phosphorylated tau at more epitopes than dentate gyrus neurons, suggesting that tau phosphorylation is cell type-specific, a possible explanation for the spatial distribution of neurofibrillary tangles.

Distribution, levels, and activity of glycogen synthase kinase-3 in the Alzheimer disease brain.

A number of studies have implicated a proline-directed protein kinase, glycogen synthase kinase-3 (GSK-3) in the hyperphosphorylation of tau in Alzheimer's disease (AD). Toward understanding the role of GSK-3 in the abnormal hyperphosphorylation of tau in AD we have found that GSK-3 is prominently present in neuronal cell bodies and their processes and co-localizes with neurofibrillary changes in AD brain. Furthermore, the levels of GSK-3 as determined by indirect ELISA are approximately 50% increased in the postsynaptosomal supernatant from AD brains as compared to the controls. However, no increase in GSK-3 enzyme activity was detected. In AD brain, with its reduced phosphatase activity, even normal levels of GSK-3 activity might be sufficient for the hyperphosphorylation of tau.

Glycogen synthase kinase 3 alteration in Alzheimer disease is related to neurofibrillary tangle formation.

Highly phosphorylated tau protein is the main component of paired helical filaments (PHF), which comprise the neurofibrillary tangles (NFT) in some neurons of patients with Alzheimer disease (AD). Glycogen synthase kinase 3 (GSK3) phosphorylates tau in vitro at several sites also found to be phosphorylated in PHF-tau; tau is phosphorylated at these sites in both AD and normal control (NC) brains, although the extent of phosphorylation is far greater in tau from AD. If GSK3 levels are increased in AD, then tau phosphorylation and perhaps PHF formation may occur. To quantify GSK3, blots of AD and NC brain supernatant and particulate fractions were probed with antibodies to GSK3. In particulate fractions of AD compared to NC, GSK3 alpha immunoreactivity did not increase, but in fact, decreased 40%, and GSK3 beta immunoreactivity decreased 30%. GSK3 alpha and GSK3 beta levels correlated well with each other. GSK3 levels correlated negatively with numbers of NFT.