PS1 activates PI3K thus inhibiting GSK-3 activity and tau overphosphorylation: effects of FAD mutations

Phosphatidylinositol 3-kinase (PI3K) promotes cell survival and communication by activating its downstream effector Akt kinase. Here we show that PS1, a protein involved in familial Alzheimer's disease (FAD), promotes cell survival by activating the PI3K/Akt cell survival signaling. This function of PS1 is unaffected by gamma-secretase inhibitors. Pharmacological and genetic evidence indicates that PS1 acts upstream of Akt, at or before PI3K kinase. PS1 forms complexes with the p85 subunit of PI3K and promotes cadherin/PI3K association. Furthermore, conditions that inhibit this association prevent the PS1-induced PI3K/Akt activation, indicating that PS1 stimulates PI3K/Akt signaling by promoting cadherin/PI3K association. By activating PI3K/Akt signaling, PS1 promotes phosphorylation/inactivation of glycogen synthase kinase-3 (GSK-3), suppresses GSK-3-dependent phosphorylation of tau at residues overphosphorylated in AD and prevents apoptosis of confluent cells. PS1 FAD mutations inhibit the PS1-dependent PI3K/Akt activation, thus promoting GSK-3 activity and tau overphosphorylation at AD-related residues. Our data raise the possibility that PS1 may prevent development of AD pathology by activating the PI3K/Akt signaling pathway. In contrast, FAD mutations may promote AD pathology by inhibiting this pathway.     

Insulin neuroprotection against oxidative stress is mediated by Akt and GSK-3beta signaling pathways and changes in protein expression

Previously we demonstrated that insulin protects against neuronal oxidative stress by restoring antioxidants and energy metabolism. In this study, we analysed how insulin influences insulin-(IR) and insulin growth factor-1 receptor (IGF-1R) intracellular signaling pathways after oxidative stress caused by ascorbate/Fe2+ in rat cortical neurons. Insulin prevented oxidative stress-induced decrease in tyrosine phosphorylation of IR and IGF-1R and Akt inactivation. Insulin also decreased the active form of glycogen synthase kinase-3beta (GSK-3beta) upon oxidation. Since phosphatidylinositol 3-kinase (PI-3K)/Akt-mediated inhibition of GSK-3beta may stimulate protein synthesis and decrease apoptosis, we analysed mRNA and protein expression of "candidate" proteins involved in antioxidant defense, glucose metabolism and apoptosis. Insulin prevented oxidative stress-induced increase in glutathione peroxidase-1 and decrease in hexokinase-II expression, supporting previous findings of changes in glutathione redox cycle and glycolysis. Moreover, insulin precluded Bcl-2 decrease and caspase-3 increased expression. Concordantly, insulin abolished caspase-3 activity and DNA fragmentation caused by oxidative stress. Thus, insulin-mediated activation of IR/IGF-1R stimulates PI-3K/Akt and inhibits GSK-3beta signaling pathways, modifying neuronal antioxidant defense-, glucose metabolism- and anti-apoptotic-associated protein synthesis. These and previous data implicate insulin as a promising neuroprotective agent against oxidative stress associated with neurodegenerative diseases.     

Role of connexin-43 in protective PI3K-Akt-GSK-3β signaling in cardiomyocytes

Sarcolemmal connexin-43 (Cx43) and mitochondrial Cx43 play distinct roles: formation of gap junctions and production of reactive oxygen species (ROS) for redox signaling. In this study, we examined the hypothesis that Cx43 contributes to activation of a major cytoprotective signal pathway, phosphoinositide 3-kinase (PI3K)-Akt-glycogen synthase kinase-3β (GSK-3β) signaling, in cardiomyocytes. A δ-opioid receptor agonist {[d-Ala(2),d-Leu(5)]enkephalin acetate (DADLE)}, endothelin-1 (ET-1), and insulin-like growth factor-1 (IGF-1) induced phosphorylation of Akt and GSK-3β in H9c2 cardiomyocytes. Reduction of Cx43 protein to 20% of the normal level by Cx43 small interfering RNA abolished phosphorylation of Akt and GSK-3β induced by DADLE or ET-1 but not that induced by IGF-1. DADLE and IGF-1 protected H9c2 cells from necrosis after treatment with H(2)O(2) or antimycin A. The protection by DADLE or ET-1, but not that by IGF-1, was lost by reduction of Cx43 protein expression. In contrast to Akt and GSK-3β, PKC-ε, ERK and p38 mitogen-activated protein kinase were phosphorylated by ET-1 in Cx43-knocked-down cells. Like diazoxide, an activator of the mitochondrial ATP-sensitive K(+) channel, DADLE and ET-1 induced significant ROS production in mitochondria, although such an effect was not observed for IGF-1. Cx43 knockdown did not attenuate the mitochondrial ROS production by DADLE or ET-1. Cx43 was coimmunoprecipitated with the β-subunit of G protein (Gβ), and knockdown of Gβ mimicked the effect of Cx43 knockdown on ET-1-induced phosphorylation of Akt and GSK-3β. These results suggest that Cx43 contributes to activation of class I(B) PI3K in PI3K-Akt-GSK-3β signaling possibly as a cofactor of Gβ in cardiomyocytes.     

Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal

Numerous studies reveal that phosphatidylinositol (PI) 3-kinase and Akt protein kinase are important mediators of cell survival. However, the survival-promoting mechanisms downstream of these enzymes remain uncharacterized. Glycogen synthase kinase-3 beta (GSK-3 beta), which is inhibited upon phosphorylation by Akt, was recently shown to function during cell death induced by PI 3-kinase inhibitors. In this study, we tested whether GSK-3 beta is critical for the death of sympathetic neurons caused by the withdrawal of their physiological survival factor, the nerve growth factor (NGF). Stimulation with NGF resulted in PI 3-kinase-dependent phosphorylation of GSK-3 beta and inhibition of its protein kinase activity, indicating that GSK-3 beta is targeted by PI 3-kinase/Akt in these neurons. Expression of the GSK-3 beta inhibitor Frat1, but not a mutant Frat1 protein that does not bind GSK-3 beta, rescued neurons from death caused by inhibiting PI 3-kinase. Similarly, expression of Frat1 or kinase-deficient GSK-3 beta reduced death caused by inhibiting Akt. In NGF-maintained neurons, overexpression of GSK-3 beta caused a small but significant decrease in survival. However, expression of neither Frat1, kinase-deficient GSK-3 beta, nor GSK-3-binding protein inhibited NGF withdrawal-induced death. Thus, although GSK-3 beta function is required for death caused by inactivation of PI 3-kinase and Akt, neuronal death caused by NGF withdrawal can proceed through GSK-3 beta-independent pathways.     

Varicella-zoster virus requires a functional PI3K/Akt/GSK-3alpha/beta signaling cascade for efficient replication

Successful replication of Varicella-zoster virus (VZV) relies upon strategies to counteract host defense mechanisms. This can be achieved by modulating host cell signaling pathways, which regulate apoptosis and cell survival. The Akt cascade is crucial for the regulation of cell survival since it controls factors such as Bad, FOXO1, mTor and GSK-3alpha/beta. These factors are involved in the regulation of cell death, cell cycle and translation. Here, we report i) that the VZV infection of MeWo cells caused a 9 to 18-fold increased phosphorylation of Akt. This phosphorylation was independent from PI3K inasmuch as the PI3K phosphorylation pattern differed strongly from the one of Akt. Bad, FOXO1 and mTor showed also variations in their phosphorylation patterns: phosphorylation of Bad (ser-136) decreased during the infection while phosphorylation of ser-2448 of mTor and of ser-256 of FOXO1 increased. The phosphorylation of GSK-3alpha/beta remained relatively stable during the infection. ii) Inhibition of PI3K, Akt or GSK-3alpha/beta prior to infection resulted in a severe decline of viral replication. The inhibition of Akt resulted also in an increased apoptotic response. iii) Transfection studies using plasmids coding for functional or inactive VZV protein kinases, pORFs 47 and 66, demonstrated an increase in Akt phosphorylation. Infection of MeWo cells with VZVDelta47 and VZVDelta66 resulted in a decline of Akt and GSK-3alpha/beta phosphorylation. These results suggest i) an essential role of PI3K/Akt/GSK-3alpha/beta signaling for a successful replication of VZV and ii) a key function of VZV kinases pORFs 47 and 66 to activate this pathway.     

Bilobalide regulates soluble amyloid precursor protein release via phosphatidyl inositol 3 kinase-dependent pathway

Bilobalide (BB) is a sesquiterpenoid extracted from Ginkgo biloba leaves. An increasing number of studies have demonstrated its neuroprotective effects. The neuroprotective mechanisms may be associated with modulation of intracellular signaling cascades such as the phosphatidyl inositol 3-kinase (PI3K) pathway. Using differentiated SH-SY5Y cells, this study investigated whether BB modulation of intracellular signaling pathways, such as the protein kinase C (PKC) and PI3K pathways, contributes to amyloid precursor protein (APP) metabolism, a key event in the pathogenesis of Alzheimer’s disease (AD). We demonstrated in this study that BB enhanced the secretion of α-secretase-cleaved soluble amyloid precursor protein (sAPPα, a by-product of non-amyloidogenic processing of APP) and decreased the β amyloid protein (Aβ, a by-product of amyloidogenic processing of APP) via PI3K-dependent pathway. The PI3K pathway mediated the rapid effect of BB on APP processing possibly via regulation of intracellular APP trafficking. After longer time BB incubation (12 h), this effect was reinforced by PI3K pathway-mediated up-regulation of disintegrin and metalloproteinase domain-containing protein 10 (ADAM10, an α-secretase candidate). Given the strong association between APP metabolism and AD pathogenesis, the ability of BB to regulate APP processing suggests its potential use in AD prevention.     

(−)-Epigallocatechin-3-gallate alleviates spatial memory impairment in APP/PS1 mice by restoring IRS-1 signaling defects in the hippocampus

Alzheimer’s disease (AD) fundamentally represents a metabolic disease associated with brain insulin resistance. TNF-α/c-Jun N-terminal kinase (JNK) signaling plays a central role in serine phosphorylation of insulin receptor substrate-1 (IRS-1). (?)-Epigallocatechin-3-gallate (EGCG), a potent antioxidant, has been verified to attenuate peripheral insulin resistance by reducing IRS-1 signaling blockage. This study aimed to investigate the effects and possible mechanisms of EGCG on central IRS-1 signaling in vivo. APP/PS1 mice were treated with EGCG, and spatial memory was assessed by the Morris water maze test. Levels of soluble and insoluble Aβ42 in the hippocampus were determined by ELISA. The activation of NF-α/JNK and IRS signaling was detected by immunohistochemistry and Western blot analysis. Our results showed that EGCG ameliorated the impaired learning and memory in APP/PS1 mice. Notably, we found a significant reduction of IRS-1pS636 level accompanied with decreased Aβ42 levels in the hippocampus of 13-month-old female APP/PS1 mice after treatment with EGCG (2 or 6 mg/kg/day) for 4 weeks. Furthermore, EGCG treatment inhibited TNF-α/JNK signaling and increased the phosphorylation of Akt and glycogen synthase kinase-3β in the hippocampus of APP/PS1 mice. In conclusion, our study provides evidence that long-term consumption of EGCG may alleviate AD-related cognitive deficits by effectively attenuating central insulin resistance.     

Defective neurite extension is caused by a mutation in amyloid β/A4 (Aβ) protein precursor found in Familial Alzheimer's Disease

Clonal central nervous system neuronal cells, B103, do not synthesize detectable endogenous APP or APLP. B103 cells transfected with both wild-type (B103/APP) and mutant APP construct (B103/APPΔNL) secreted comparable amounts of soluble forms of APP (sAPP). B103/APP cells produced sAPP and cleaved at amyloid β/A4 (Aβ) 16, the α-secretase site, and B103/APPΔNL cells produced sAPPβ cleaved at Aβ 1, the β-secretase site. B103/APPΔNL cells developed fewer neurites than B103/APP cells in a serum-free defined medium. Neurite numbers of parent B103 cells were increased by the 50% conditioned medium (CM) from B103/APP cells but reduced by the CM from B103/APPΔNL cells. Chemically synthesized Aβ at concentration levels higher than 1 nM reduced numbers of neurites from B103 or B103/APPΔNL cells. However, Aβ at 1–100 nM could not reduce the neurite number of B103/APP cells. The protective activity against Aβ's deleterious effect to reduce neurite numbers was attributed to sAPPα in the CM. Although sAPPα could block the effect of Aβ, sAPPβ could not do so under the identical condition, suggesting the importance of the C-terminal 15-amino acid sequence in sAPPα. Nevertheless, sAPPα's protective activity required the N-terminal sequence around RERMS, previously identified to be the active domain of sAPPβ. The overall effect of APP mutation which overproduced Aβ and sAPPβ and underproduced sAPPα was a marked decline in the neurotrophic effect of APP. We suggest that the disruption of balance between the detrimental effect of Aβ and the trophic effect of sAPP may be important in the pathogenesis of AD caused by this pathogenic APP mutation © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 469–480, 1997     

Amyloid-β Protein Modulates Insulin Signaling in Presynaptic Terminals

Synaptic loss is a major neuropathological correlate of memory decline as a result of Alzheimer's disease (AD). This phenomenon appears to be aggravated by soluble amyloid-β (Aβ) oligomers causing presynaptic terminals to be particularly vulnerable to damage. Furthermore, insulin is known to participate in synaptic plasticity through the activation of the insulin receptor (IR) and the PI3K signaling pathway, while low concentrations of soluble Aβ and Aβ oligomers aberrantly modulate IR function in cultured neurons. To further examine how Aβ and insulin interact in the pathology of AD, the present work analyzes the effect of insulin and Aβ in the activation of the IR/PI3K pathway in synaptosomes. We found that insulin increased mitochondrial activity and IR/Akt phosphorylation in synaptosomes taken from both hippocampus and cortex. Also, pretreatment with Aβ antagonized insulin's effect on hippocampal synaptosomes, but not vice versa. These results show that Aβ can reduce responsiveness to insulin. Combined with evidence that insulin desensitization can increase the risk of developing AD, our results suggest that the initial mechanism that impairs synaptic maintenance in AD might start with Aβ changes in insulin sensitivity.     

Comparative signaling pathways of insulin-like growth factor-1 and brain-derived neurotrophic factor in hippocampal neurons and the role of the PI3 kinase pathway in cell survival

Insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor (BDNF) are trophic factors required for the viability and normal functions of various neuronal cells. However, the detailed intracellular mechanism(s) involved in these effects in neuronal cells remains to be fully elucidated. In present study, the respective intracellular signaling pathway induced by IGF-1 and BDNF and their possible role in neuronal survival were investigated. Both IGF-1 and BDNF protected hippocampal neurons from serum deprivation-induced death with IGF-1 apparently being more potent. Western blot analyses showed that both IGF-1 and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3)/Akt (protein kinase B) kinase and the mitogen-activated protein kinase (MAPK) pathways. The phosphorylation of Akt and its downstream target, FKHRL1, induced by IGF-1 was rapid and sustained while that of MAPK was transient. The reverse situation was observed for BDNF. Moreover, IGF-1 potently induced the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with PI3 kinase while BDNF was weak in these assays. In contrast, the tyrosine phosphorylation of Shc proteins was dramatically stimulated by BDNF, with IGF-1 having only a minimal effect. Most interestingly, only the inhibitor of the PI3K/Akt pathway, LY294002, was able to block the survival effects of both IGF-1 and BDNF; an inhibitor of the MAPK pathway inhibitor, PD98059, being ineffective. Taken together, these data reveal that the survival properties of both IGF-1 and BDNF against serum deprivation are mediated by the activation of the PI3K/Akt, but not the MAPK, pathway in hippocampal neurons.     

PI3 kinase/Akt activation mediates estrogen and IGF-1 nigral DA neuronal neuroprotection against a unilateral rat model of Parkinson's disease

Recently, using the medial forebrain bundle (MFB) 6-hydroxydopmaine (6-OHDA) lesion rat model of Parkinson's disease (PD), we have demonstrated that blockade of central IGF-1 receptors (IGF-1R) attenuated estrogen neuroprotection of substantia nigra pars compacta (SNpc) DA neurons, but exacerbated 6-OHDA lesions in IGF-1 only treated rats (Quesada and Micevych [2004]: J Neurosci Res 75:107-116). This suggested that the IGF-1 system is a central mechanism through which estrogen acts to protect the nigrostriatal DA system. Moreover, these results also suggest that IGF-1R-induced intracellular signaling pathways are involved in the estrogen mechanism that promotes neuronal survival. In vitro, two convergent intracellular signaling pathways used by estrogen and IGF-1, the mitogen-activated protein kinase (MAPK/ERK), and phosphatidyl-inositol-3-kinase/Akt (PI3K/Akt), have been demonstrated to be neuroprotective. Continuous central infusions of MAPK/ERK and PI3K/Akt inhibitors were used to test the hypothesis that one or both of these signal transduction pathways mediates estrogen and/or IGF-1 neuroprotection of SNpc DA neurons after a unilateral administration of 6-OHDA into the MFB of rats. Motor behavior tests and tyrosine hydroxylase immunoreactivity revealed that the inhibitor of the PI3K/Akt pathway (LY294002) blocked the survival effects of both estrogen and IGF-1, while an inhibitor of the MAPK/ERK signaling (PD98059) was ineffective. Western blot analyses showed that estrogen and IGF-1 treatments increased PI3K/Akt activation in the SN; however, MAPK/ERK activation was decreased in the SN. Indeed, continuous infusions of inhibitors blocked phosphorylation of PI3K/Akt and MAPK/ERK. These findings indicate that estrogen and IGF-1-mediated SNpc DA neuronal protection is dependent on PI3K/Akt signaling, but not on the MAPK/ERK pathway.     

Insulin and IGF-1 signalling: longevity, protein homoeostasis and Alzheimer's disease

The quality control of protein homoeostasis deteriorates with aging, causing the accumulation of misfolded proteins and neurodegeneration. Thus, in AD (Alzheimer's disease), soluble oligomers, protofibrils and fibrils of the Aβ (amyloid β-peptide) and tau protein accumulate in specific brain regions. This is associated with the progressive destruction of synaptic circuits controlling memory and higher mental function. The primary signalling mechanisms that (i) become defective in AD to alter the normal proteostasis of Aβ and tau, and (ii) initiate a pathophysiological response to cause cognitive decline, are unclear. The IIS [insulin/IGF-1 (insulin-like growth factor 1)-like signalling] pathway is mechanistically linked to longevity, protein homoeostasis, learning and memory, and is emerging to be central to both (i) and (ii). This pathway is aberrantly overactivated in AD brain at the level of increased activation of the serine/threonine kinase Akt and the phosphorylation of its downstream targets, including mTOR (mammalian target of rapamycin). Feedback inhibition of normal insulin/IGF activation of the pathway also occurs in AD due to inactivation of IRS-1 (insulin receptor substrate 1) and decreased IRS-1/2 levels. Pathogenic forms of Aβ may induce aberrant sustained activation of the PI3K (phosphoinositide 3-kinase)/Akt signal in AD, also causing non-responsive insulin and IGF-1 receptor, and altered tau phosphorylation, conformation and function. Reducing IIS activity in animal models by decreasing IGF-1R levels or inhibiting mTOR activity alters Aβ and tau protein homoeostasis towards less toxic protein conformations, improves cognitive function and extends healthy lifespan. Thus normalizing IIS dysfunction may be therapeutically relevant in abrogating Aβ and tau proteotoxicity, synaptic dysfunction and cognitive decline in AD.     

TRAF6-mediated regulation of the PI3 kinase (PI3K)-Akt-GSK3beta cascade is required for TNF-induced cell survival

We recently demonstrated that the tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) helps maintenance of cell survival by regulating glycogen synthase kinase 3β (GSK3β) activity during TNF signaling. However, the molecular linkage between TRAF6 and GSK3β signaling is unknown. Herein, we showed that TRAF6 positively regulated cell survival by modulating PI3K-Akt-GSK3β cascades. In 3T3 cells lacking TRAF6, but not those lacking TRAF2, TNF stimulation led to prolonged hyperphosphorylation of Akt, which coincided with the activation of upstream PI3K. Pharmacologically blocking PI3K significantly inhibited Akt and GSK3β phosphorylation. Importantly, PI3K inhibition rescued cell death in TRAF6-null 3T3 cells. These data suggested TRAF6 regulates TNF-mediated cell survival through PI3K-Akt-GSK3β cascades.     

Convergence of multiple signaling cascades at glycogen synthase kinase 3: Edg receptor-mediated phosphorylation and inactivation by lysophosphatidic acid through a protein kinase C-dependent intracellular pathway

Lysophosphatidic acid (LPA) is a natural phospholipid with multiple biological functions. We show here that LPA induces phosphorylation and inactivation of glycogen synthase kinase 3 (GSK-3), a multifunctional serine/threonine kinase. The effect of LPA can be reconstituted by expression of Edg-4 or Edg-7 in cells lacking LPA responses. Compared to insulin, LPA stimulates only modest phosphatidylinositol 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt) that does not correlate with the magnitude of GSK-3 phosphorylation induced by LPA. PI3K inhibitors block insulin- but not LPA-induced GSK-3 phosphorylation. In contrast, the effect of LPA, but not that of insulin or platelet-derived growth factor (PDGF), is sensitive to protein kinase C (PKC) inhibitors. Downregulation of endogenous PKC activity selectively reduces LPA-mediated GSK-3 phosphorylation. Furthermore, several PKC isotypes phosphorylate GSK-3 in vitro and in vivo. To confirm a specific role for PKC in regulation of GSK-3, we further studied signaling properties of PDGF receptor beta subunit (PDGFRbeta) in HEK293 cells lacking endogenous PDGF receptors. In clones expressing a PDGFRbeta mutant wherein the residues that couple to PI3K and other signaling functions are mutated with the link to phospholipase Cgamma (PLCgamma) left intact, PDGF is fully capable of stimulating GSK-3 phosphorylation. The process is sensitive to PKC inhibitors in contrast to the response through the wild-type PDGFRbeta. Therefore, growth factors, such as PDGF, which control GSK-3 mainly through the PI3K-PKB/Akt module, possess the ability to regulate GSK-3 through an alternative, redundant PLCgamma-PKC pathway. LPA and potentially other natural ligands primarily utilize a PKC-dependent pathway to modulate GSK-3.     

Role of phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) in intracellular amyloid precursor protein (APP) processing and amyloid plaque pathogenesis

One of the pathological hallmarks of Alzheimer disease is the accumulation of amyloid plaques in the extracellular space in the brain. Amyloid plaques are primarily composed of aggregated amyloid β peptide (Aβ), a proteolytic fragment of the transmembrane amyloid precursor protein (APP). For APP to be proteolytically cleaved into Aβ, it must be internalized into the cell and trafficked to endosomes where specific protease complexes can cleave APP. Several recent genome-wide association studies have reported that several single nucleotide polymorphisms (SNPs) in the phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) gene were significantly associated with Alzheimer disease, suggesting a role in APP endocytosis and Aβ generation. Here, we show that PICALM co-localizes with APP in intracellular vesicles of N2a-APP cells after endocytosis is initiated. PICALM knockdown resulted in reduced APP internalization and Aβ generation. Conversely, PICALM overexpression increased APP internalization and Aβ production. In vivo, PICALM was found to be expressed in neurons and co-localized with APP throughout the cortex and hippocampus in APP/PS1 mice. PICALM expression was altered using AAV8 gene transfer of PICALM shRNA or PICALM cDNA into the hippocampus of 6-month-old APP/PS1 mice. PICALM knockdown decreased soluble and insoluble Aβ levels and amyloid plaque load in the hippocampus. Conversely, PICALM overexpression increased Aβ levels and amyloid plaque load. These data indicate that PICALM, an adaptor protein involved in clathrin-mediated endocytosis, regulates APP internalization and subsequent Aβ generation. PICALM contributes to amyloid plaque load in brain likely via its effect on Aβ metabolism.     

Dual regulation of glycogen synthase kinase-3beta by the alpha1A-adrenergic receptor

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 G? 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.     

Deoxycholyltaurine rescues human colon cancer cells from apoptosis by activating EGFR-dependent PI3K/Akt signaling

Recent studies indicate that secondary bile acids promote colon cancer cell proliferation but their role in maintaining cell survival has not been explored. We found that deoxycholyltaurine (DCT) markedly attenuated both unstimulated and TNF-alpha-stimulated programmed cell death in colon cancer cells by a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. To examine the role of bile acids and PI3K signaling in maintaining colon cancer cell survival, we explored the role of signaling downstream of bile acid-induced activation of the epidermal growth factor receptor (EGFR) in regulating both apoptosis and proliferation of HT-29 and H508 human colon cancer cells. DCT caused dose- and time-dependent Akt (Ser(473)) phosphorylation, a commonly used marker of activated PI3K/Akt signaling. Both EGFR kinase and PI3K inhibitors attenuated DCT-induced Akt phosphorylation and Akt activation, as demonstrated by reduced phosphorylation of a GSK-3-paramyosin substrate. Transfection of HT-29 cells with kinase-dead EGFR (K721M) reduced DCT-induced Akt phosphorylation. In HT-29 cells, EGFR and PI3K inhibitors as well as transfection with dominant negative AKT attenuated DCT-induced cell proliferation. DCT-induced PI3K/Akt activation resulted in downstream phosphorylation of GSK-3 (Ser(21/9)) and BAD (Ser(136)), and nuclear translocation (activation) of NF-kappaB, thereby confirming that DCT-induced activation of PI3K/Akt signaling regulates both proproliferative and prosurvival signals. Collectively, these results indicate that DCT-induced activation of post-EGFR PI3K/Akt signaling stimulates both colon cancer cell survival and proliferation.     

Mitochondrial mechanisms in amyloid beta peptide-induced cerebrovascular degeneration

Prevailing evidence suggests that amyloid beta peptide (Aβ), a key mediator in age-dependent neuronal and cerebrovascular degeneration, activates death signaling processes leading to neuronal as well as non-neuronal cell death in the central nervous system. A major cellular event in Aβ-induced death of non-neuronal cells, including cerebral endothelial cells, astrocytes and oligodendrocytes, is mitochondrial dysfunction. The death signaling cascade upstream of mitochondria entails Aβ activation of neutral sphingomyelinase, resulting in the release of ceramide from membrane sphingomyelin. Ceramide then activates protein phosphatase 2A (PP2A), a member in the ceramide-activated protein phosphatase (CAPP) family. PP2A dephosphorylation of Akt and FKHRL1 plays a pivotal role in Aβ-induced Bad translocation to mitochondria and transactivation of Bim. Bad and Bim are pro-apoptotic proteins that cause mitochondrial dysfunction characterized by excessive ROS formation, mitochnondrial DNA (mtDNA) damage, and release of mitochondrial apoptotic proteins including cytochrome c, apoptosis inducing factor (AIF), endonuclease G and Smac. The cellular events activated by Aβ to induce death of non-neuronal cells are complex. Understanding these death signaling processes will aid in the development of more effective strategies to slow down age-dependent cerebrovascular degeneration caused by progressive cerebrovascular Aβ deposition.     

Serine Phosphorylation of the Insulin-like Growth Factor I (IGF-1) Receptor C-terminal Tail Restrains Kinase Activity and Cell Growth

Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3β in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3β knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the (1248)SFYYS(1252) motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3β restrains kinase activity and regulates receptor trafficking and signaling.