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.     

Reelin signals through phosphatidylinositol 3-kinase and Akt to control cortical development and through mTor to regulate dendritic growth

Reelin is an extracellular matrix protein with various functions during development and in the mature brain. It activates different signaling cascades in target cells, one of which is the phosphatidylinositol 3-kinase (PI3K) pathway, which we investigated further using pathway inhibitors and in vitro brain slice and neuronal cultures. We show that the mTor (mammalian target of rapamycin)-S6K1 (S6 kinase 1) pathway is activated by Reelin and that this depends on Dab1 (Disabled-1) phosphorylation and activation of PI3K and Akt (protein kinase B). PI3K and Akt are required for the effects of Reelin on the organization of the cortical plate, but their downstream partners mTor and glycogen synthase kinase 3beta (GSK3beta) are not. On the other hand, mTor, but not GSK3beta, mediates the effects of Reelin on the growth and branching of dendrites of hippocampal neurons. In addition, PI3K fosters radial migration of cortical neurons through the intermediate zone, an effect that is independent of Reelin and Akt.     

Age-dependent accumulation of soluble amyloid beta (Abeta) oligomers reverses the neuroprotective effect of soluble amyloid precursor protein-alpha (sAPP(alpha)) by modulating phosphatidylinositol 3-kinase (PI3K)/Akt-GSK-3beta pathway in Alzheimer mouse model

Neurotrophins, activating the PI3K/Akt signaling pathway, control neuronal survival and plasticity. Alterations in NGF, BDNF, IGF-1, or insulin signaling are implicated in the pathogenesis of Alzheimer disease. We have previously characterized a bigenic PS1×APP transgenic mouse displaying early hippocampal Aβ deposition (3 to 4 months) but late (17 to 18 months) neurodegeneration of pyramidal cells, paralleled to the accumulation of soluble Aβ oligomers. We hypothesized that PI3K/Akt/GSK-3β signaling pathway could be involved in this apparent age-dependent neuroprotective/neurodegenerative status. In fact, our data demonstrated that, as compared with age-matched nontransgenic controls, the Ser-9 phosphorylation of GSK-3β was increased in the 6-month PS1×APP hippocampus, whereas in aged PS1×APP animals (18 months), GSK-3β phosphorylation levels displayed a marked decrease. Using N2a and primary neuronal cell cultures, we demonstrated that soluble amyloid precursor protein-α (sAPPα), the predominant APP-derived fragment in young PS1×APP mice, acting through IGF-1 and/or insulin receptors, activated the PI3K/Akt pathway, phosphorylated the GSK-3β activity, and in consequence, exerted a neuroprotective action. On the contrary, several oligomeric Aβ forms, present in the soluble fractions of aged PS1×APP mice, inhibited the induced phosphorylation of Akt/GSK-3β and decreased the neuronal survival. Furthermore, synthetic Aβ oligomers blocked the effect mediated by different neurotrophins (NGF, BDNF, insulin, and IGF-1) and sAPPα, displaying high selectivity for NGF. In conclusion, the age-dependent appearance of APP-derived soluble factors modulated the PI3K/Akt/GSK-3β signaling pathway through the major neurotrophin receptors. sAPPα stimulated and Aβ oligomers blocked the prosurvival signaling. Our data might provide insights into the selective vulnerability of specific neuronal groups in Alzheimer disease.     

Exogenous zinc protects cardiac cells from reperfusion injury by targeting mitochondrial permeability transition pore through inactivation of glycogen synthase kinase-3beta

The purpose of this study was to determine whether exogenous zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via glycogen synthase kinase-3beta (GSK-3beta). The treatment of cardiac H9c2 cells with ZnCl2 (10 microM) in the presence of zinc ionophore pyrithione for 20 min significantly enhanced GSK-3beta phosphorylation at Ser9, indicating that exogenous zinc can inactivate GSK-3beta in H9c2 cells. The effect of zinc on GSK-3beta activity was blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 but not by the mammalian target of rapamycin (mTOR) inhibitor rapamycin or the PKC inhibitor chelerythrine, implying that PI3K but not mTOR or PKC accounts for the action of zinc. In support of this interpretation, zinc induced a significant increase in Akt but not mTOR phosphorylation. Further experiments found that zinc also increased mitochondrial GSK-3beta phosphorylation. This may indicate an involvement of the mitochondria in the action of zinc. The effect of zinc on mitochondrial GSK-3beta phosphorylation was not altered by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. Zinc applied at reperfusion reduced cell death in cells subjected to simulated ischemia/reperfusion, indicating that zinc can prevent reperfusion injury. However, zinc was not able to exert protection in cells transfected with the constitutively active GSK-3beta (GSK-3beta-S9A-HA) mutant, suggesting that zinc prevents reperfusion injury by inactivating GSK-3beta. Cells transfected with the catalytically inactive GSK-3beta (GSK-3beta-KM-HA) also revealed a significant decrease in cell death, strongly supporting the essential role of GSK-3beta inactivation in cardioprotection. Moreover, zinc prevented oxidant-induced mPTP opening through the inhibition of GSK-3beta. Taken together, these data suggest that zinc prevents reperfusion injury by modulating the mPTP opening through the inactivation of GSK-3beta. The PI3K/Akt signaling pathway is responsible for the inactivation of GSK-3beta by zinc.     

Constitutive activation of phosphatidylinositol 3-kinase signaling pathway down-regulates TLR4-mediated tumor necrosis factor-alpha release in alveolar macrophages from asymptomatic HIV-positive persons in vitro

Alveolar macrophages represent critical effector cells of innate immunity to infectious challenge in the lungs and recognize bacterial pathogens through pattern recognition receptors such as Toll-like receptors (TLRs). Phosphatidylinositol 3-kinase (PI3K) regulates TLR-mediated cytokine release, but whether HIV infection influences PI3K signaling pathway and alters TLR4-mediated macrophage response has not been investigated. In the current study, surface TLR4 expression were similar but TLR4 activation (lipid A, 10 microg/ml) resulted in lower TNF-alpha release by HIV+ human macrophages compared with healthy cells. Pharmacological inhibition of PI3K (LY294002) normalized TNF-alpha release in HIV+ macrophages and augments ERK1/2 mitogen-activated protein kinase phosphorylation in response to lipid A. Importantly, HIV+ macrophages demonstrated increased constitutive phosphatidylinositol 3,4,5-trisphosphate formation, increased phosphorylation of downstream signaling molecules Akt and glycogen synthase kinase-3beta (GSK-3beta) at Ser9, and reduced PTEN protein expression. As a functional assessment of GSK-3beta phosphorylation, TLR4-mediated interleukin-10 release was significantly higher in HIV+ human macrophages compared with healthy cells. Incubation of human macrophages with exogenous HIV Nef protein induced phosphorylation of Akt and GSK-3beta (whereas phosphorylation was reduced by PI3K inhibition) and promoted interleukin-10 release. Taken together, these data demonstrate increased constitutive activation of the PI3K signaling pathway in HIV+ macrophages and support the concept that PI3K activation (by HIV proteins such as Nef) may contribute to reduced TLR4-mediated TNF-alpha release in HIV+ human macrophages and impair host cell response to infectious challenge.     

Role of Translation Initiation Factor 2B in Control of Cell Survival by the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3β Signaling Pathway

The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling pathway is an important mediator of growth factor-dependent survival of mammalian cells. A variety of targets of the Akt protein kinase have been implicated in cell survival, including the protein kinase glycogen synthase kinase 3beta (GSK-3beta). One of the targets of GSK-3beta is translation initiation factor 2B (eIF2B), linking global regulation of protein synthesis to PI 3-kinase/Akt signaling. Because of the central role of protein synthesis, we have investigated the involvement of eIF2B, which is inhibited as a result of GSK-3beta phosphorylation, in programmed cell death. We demonstrate that expression of eIF2B mutants lacking the GSK-3beta phosphorylation or priming sites is sufficient to protect both Rat-1 and PC12 cells from apoptosis induced by overexpression of GSK-3beta, inhibition of PI 3-kinase, or growth factor deprivation. Consistent with these effects on cell survival, expression of nonphosphorylatable eIF2B prevented inhibition of protein synthesis following treatment of cells with the PI 3-kinase inhibitor LY294002. Conversely, cycloheximide induced apoptosis of PC12 and Rat-1 cells, further indicating that protein synthesis was required for cell survival. Inhibition of translation resulting from treatment with cycloheximide led to the release of cytochrome c from mitochondria, similar to the effects of inhibition of PI 3-kinase. Expression of nonphosphorylatable eIF2B prevented cytochrome c release resulting from PI 3-kinase inhibition but did not affect cytochrome c release or apoptosis induced by cycloheximide. Regulation of translation resulting from phosphorylation of eIF2B by GSK-3beta thus appears to contribute to the control of cell survival by the PI 3-kinase/Akt signaling pathway, acting upstream of mitochondrial cytochrome c release.     

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.     

PGF2alpha-associated vascular smooth muscle hypertrophy is ROS dependent and involves the activation of mTOR, p70S6k, and PTEN

Prostaglandin F2alpha (PGF2alpha) increases reactive oxygen species (ROS) and induces vascular smooth muscle cell (VSMC) hypertrophy by largely unknown mechanism(s). To investigate the signaling events governing PGF2alpha-induced VSMC hypertrophy we examined the ability of the PGF2alpha analog, fluprostenol to elicit phosphorylation of Akt, the mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6k), glycogen synthase kinase-3beta (GSK-3beta), phosphatase and tensin homolog (PTEN), extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) in growth arrested A7r5 VSMC. Fluprostenol-induced hypertrophy was associated with increased ROS, mTOR translocation from the nucleus to the cytoplasm, along with Akt, mTOR, GSK-3beta, PTEN and ERK1/2 but not JNK phosphorylation. Whereas inhibition of phosphatidylinositol 3-kinase (PI3K) by LY-294002 blocked fluprostenol-induced changes in total protein content, pre-treatment with rapamycin or with the MEK1/2 inhibitor U0126 did not. Taken together, these findings suggest that fluprostenol-induced changes in A7r5 hypertrophy involve mTOR translocation and occur through PI3K-dependent mechanisms.     

Mechanism of zinc-induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3beta in SH-SY5Y neuroblastoma cells

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)-3beta, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK-3beta phosphorylation is unknown. In the current study, 100 microM zinc sulfate was used, and influences of various components of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on p70S6K and GSK-3beta phosphorylation have been investigated in serum-deprived SH-SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p-PKB, p-GSK-3beta, p-ERK1/2, p-JNK and p-p38, especially in long-term treatment (4-8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK-3beta is regulated by rapamycin-dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK-3beta affected levels of tau unphosphorylated at the Tau-1 site and phosphorylated at the PHF-1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 microM zinc might activate PKB, GSK-3beta, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH-SY5Y cells.     

AD认知功能障碍的代谢紊乱机制:脑胰岛素抵抗及其介导的PI3K/Akt信号通路受损

阿尔茨海默病(Alzheimer’s disease, AD)是一种以进行性痴呆为主要特征的中枢神经系统退行性疾病,其认知功能障碍可能与Ⅱ型糖尿病(type 2 diabetes, T2DM)诱发的胰岛素抵抗所损伤的PI3K/Akt胰岛素信号级联通路相关。胰岛素是调节机体新陈代谢的重要激素,通过与神经细胞表面的胰岛素受体结合激活PI3K/Akt信号通路,以调控葡萄糖、脂质的代谢。任何中间媒介功能紊乱所导致的脑胰岛素水平和胰岛素敏感性的降低都会损坏PI3K/Akt信号通路,诱发脑能量代谢障碍、Aβ沉积、Tau蛋白过度磷酸化,引起并加重AD认知功能障碍。因此,本文以PI3K/Akt胰岛素信号通路为主线,揭示了T2DM中脑胰岛素抵抗(insulin resistance, IR)与AD之间的复杂机制,旨在加深对脑IR介导的AD病理过程的系统性理解,借此为延缓或治疗AD的认知功能障碍提供理论基础。     

Protein phosphatase-2C alpha as a positive regulator of insulin sensitivity through direct activation of phosphatidylinositol 3-kinase in 3T3-L1 adipocytes

During differentiation, expression of protein phosphatase-2Calpha (PP2Calpha) is increased in 3T3-L1 adipocytes. To elucidate the role of PP2Calpha in insulin signaling, we overexpressed wild-type (WT) PP2Calpha by adenovirus-mediated gene transfer in 3T3-L1 adipocytes. Overexpression of PP2Calpha-WT enhanced the insulin sensitivity of glucose uptake without any changes in the early steps of insulin signaling. Infection with adenovirus 5 expressing PP2Calpha-WT increased phosphatidylinositol 3-kinase (PI3K) activities in the immunoprecipitate using antibody against the p85 or p110 subunit under both basal and insulin-stimulated conditions, followed by activation of downstream steps in the PI3K pathway, such as phosphorylation of Akt, glycogen synthase kinase-3, and atypical protein kinase C. In contrast, overexpression of the phosphatase-defective mutant PP2Calpha(R174G) did not produce such effects. Furthermore, overexpression of PP2Calpha-WT (but not PP2Calpha(R174G)) decreased the (32)P-labeled phosphorylation state as well as the gel mobility shift of the p85 subunit, suggesting that dephosphorylation of the p85 subunit by PP2Calpha activation might stimulate PI3K catalytic activity. Moreover, knockdown of PP2Calpha by transfection of small interfering RNA led to a significant decrease in Akt phosphorylation. In addition, microinjection of anti-PP2Calpha antibody or PP2Calpha small interfering RNA led to decreased insulin-stimulated GLUT4 translocation. In conclusion, PP2Calpha is a new positive regulator of insulin sensitivity that acts through a direct activation of PI3K in 3T3-L1 adipocytes.     

Phosphorylation of serine 468 by GSK-3beta negatively regulates basal p65 NF-kappaB activity

The activity of NF-kappaB is controlled at several levels including the phosphorylation of the strongly transactivating p65 (RelA) subunit. However, the overall number of phosphorylation sites, the signaling pathways and protein kinases that target p65 NF-kappaB and the functional role of these phosphorylations are still being uncovered. Using a combination of peptide arrays with in vitro kinase assays we identify serine 468 as a novel phosphorylation site of p65 NF-kappaB. Serine 468 lies within a GSK-3beta consensus site, and recombinant GSK-3beta specifically phosphorylates a GST-p65-(354-551) fusion protein at Ser(468) in vitro. In intact cells, phosphorylation of endogenous Ser(468) of p65 is induced by the PP1/PP2A phosphatase inhibitor calyculin A and this effect is inhibited by the GSK-3beta inhibitor LiCl. Reconstitution of p65-deficient cells with a p65 protein where serine 468 was mutated to alanine revealed a negative regulatory role of serine 468 for NF-kappaB activation. Collectively our results suggest that a GSK-3beta-PP1-dependent mechanism regulates phosphorylation of p65 NF-kappaB at Ser(468) in unstimulated cells and thereby controls the basal activity of NF-kappaB.     

四氢紫堇萨明对AD细胞模型Tau蛋白磷酸化的影响及其可能机制研究

研究四氢紫堇萨明对Aβ25-35诱导的阿尔茨海默病细胞模型Tau蛋白磷酸化的影响及其可能作用机理。以神经细胞PC-12为载体,Aβ25-35诱导48h建立AD细胞模型,MTS试剂盒检测细胞活力,激光共聚焦显微镜观察细胞核和细胞微管变化情况,Westernblot法检测蛋白表达水平。结果显示,四氢紫堇萨明可显著增强模型细胞活力,改善微管形态,降低p-Tau(Ser396)和p-GSK-3β(Tyr216)表达水平,升高p-GSK-3β(Ser9)和p-AKT表达水平(P<0.05);PI3K抑制剂LY294002可部分阻断四氢紫堇萨明对模型细胞的上述改善作用。以上结果表明四氢紫堇萨明可显著改善AD细胞模型Tau蛋白过度磷酸化,从而改善细胞骨架微管形态,增强细胞活力,其机理可能与激活PI3K/Akt信号通路,降低GSK-3β活性,改善蛋白激酶/蛋白磷酸酯酶系统失衡相关。     

Functional studies of shaggy/glycogen synthase kinase 3 phosphorylation sites in Drosophila melanogaster

Early studies of glycogen synthase kinase 3 (GSK-3) in mammalian systems focused on its pivotal role in glycogen metabolism and insulin-mediated signaling. It is now recognized that GSK-3 is central to a number of diverse signaling systems. Here, we show that the major form of the kinase Shaggy (Sgg), the GSK-3 fly ortholog, is negatively regulated during insulin-like/phosphatidylinositol 3-kinase (PI3K) signaling in vivo. Since genetic studies of Drosophila melanogaster had previously shown that Wingless (Wg) signaling also acts to antagonize Sgg, we investigate how the kinase might integrate, or else discriminate, signaling inputs by Wg and insulin. Using Drosophila cell line assays, we found, in contrast to previous reports, that Wg induces accumulation of its transducer Armadillo (Arm)/beta-catenin without significant alteration of global Sgg-specific activity. In agreement with a previous study using human GSK-3beta, Wg did not cause phosphorylation changes of the Ser9 or Tyr214 regulatory phosphorylated sites of Sgg. Conversely, as shown in mammalian systems, insulin-induced inhibition of Sgg-specific activity by phosphorylation at the N-terminal pseudosubstrate site (Ser9) did not induce Arm/beta-catenin accumulation, showing selectivity in response to the different signaling pathways. Interestingly, a minigene bearing a Ser9-to-Ala change rescued mutant sgg without causing abnormal development, suggesting that the regulation of Sgg via the inhibitory pseudosubstrate domain is dispensable for many aspects of its function. Our studies of Drosophila show that Wg and insulin or PI3K pathways do not converge on Sgg but that they exhibit cross-regulatory interactions.     

Human serum and glucocorticoid-inducible kinase-like kinase (SGKL) phosphorylates glycogen syntheses kinase 3 beta (GSK-3beta) at serine-9 through direct interaction

Serum and glucocorticoid-inducible kinase-like kinase (SGKL) has been identified as a new integrator that decodes lipid signals produced by the activation of phosphoinositide 3-kinase (PI3K). SGKL is activated via its lipid-binding domain (phox homology domain) in response to PI3K signaling. However, downstream targets of SGKL as well as the role of SGKL as a mediator in PI3K signaling in human tissues remain to be established. In this study, we identified human glycogen synthase kinase 3 beta (GSK-3beta) as a specific interacting partner with SGKL in a yeast two-hybrid screening of human brain cDNA library. The association between these two proteins is confirmed independently in human embryonic kidney (HEK293) cells by co-immunoprecipitation. Furthermore, the kinase activity of wild-type SGKL was required for the in vitro phosphorylation of a GSK-3 crosstide fusion protein at serine-21/9 as demonstrated with a Phospho-GSK-3alpha/beta (Ser21/9) specific antibody. The present results provide strong evidences that SGKL could utilize GSK-3beta as a direct downstream target by phosphorylating GSK-3beta at serine-9.     

GSK-3beta regulation in skeletal muscles by adrenaline and insulin: evidence that PKA and PKB regulate different pools of GSK-3

We have recently shown that while adrenaline alone has no effect on the activation of Protein Kinase B (PKB) in rat soleus muscle, it greatly potentiates the effects of insulin (Brennesvik et al., Cellular Signalling 17: 1551-1559, 2005). In the current study we went on to investigate whether this was paralleled by a similar effect on GSK-3, which is a major PKB target. Surprisingly adrenaline alone increased phosphorylation of GSK-3beta Ser9 and GSK-3alpha Ser21 and adrenaline's effects were additive with those of insulin but did not synergistically potentiate insulin action. Dibutyryl-cAMP (5 mM) and the PKA specific cAMP analogue N6-Benzoyl-cAMP (2 mM) increased GSK-3beta Ser9 phosphorylation, whereas the Epac specific cAMP analogue 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (1 mM) did not. Wortmannin (PI 3-kinase inhibitor; 1 microM) blocked insulin-stimulated GSK-3 phosphorylation completely, but adrenaline increased GSK-3beta Ser9 phosphorylation in the presence of wortmannin. The PKA inhibitor H89 (50 microM) reduced adrenaline-stimulated GSK-3beta Ser9 phosphorylation but did not influence the effects of insulin. Insulin-stimulated GSK-3 Ser9 phosphorylation was paralleled by decreased glycogen synthase phosphorylation at the sites phosphorylated by GSK-3 as expected. However, adrenaline-stimulated GSK-3 Ser9 phosphorylation was paralleled by increased glycogen synthase phosphorylation indicating this pool of GSK-3 may not be directly involved in phosphorylation of glycogen synthase. Our results indicate the existence of at least two distinct pools of GSK-3beta in soleus muscle, one phosphorylated by PKA and another by PKB. Further, we hypothesise that each of these pools is involved in the control of different cellular processes.     

Alpha-synuclein overexpression negatively regulates insulin receptor substrate 1 by activating mTORC1/S6K1 signaling

Alpha-synuclein (α-Syn) is a major component of Lewy bodies, a pathological feature of Parkinson's and other neurodegenerative diseases collectively known as synucleinopathies. Among the possible mechanisms of α-Syn-mediated neurotoxicity is interference with cytoprotective pathways such as insulin signaling. Insulin receptor substrate (IRS)-1 is a docking protein linking IRs to downstream signaling pathways such as phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K)1; the latter exerts negative feedback control on insulin signaling, which is impaired in Alzheimer's disease. Our previous study found that α-Syn overexpression can inhibit protein phosphatase (PP)2A activity, which is involved in the protective mechanism of insulin signaling. In this study, we found an increase in IRS-1 phosphorylation at Ser636 and decrease in tyrosine phosphorylation, which accelerated IRS-1 turnover and reduced insulin-Akt signaling in α-Syn-overexpressing SK-N-SH cells and transgenic mice. The mTOR complex (C)1/S6K1 blocker rapamycin inhibited the phosphorylation of IRS-1 at Ser636 in cells overexpressing α-Syn, suggesting that mTORC1/S6K1 activation by α-Syn causes feedback inhibition of insulin signaling via suppression of IRS-1 function. α-Syn overexpression also inhibited PP2A activity, while the PP2A agonist C2 ceramide suppressed both S6K1 activation and IRS-1 Ser636 phosphorylation upon α-Syn overexpression. Thus, α-Syn overexpression negatively regulated IRS-1 via mTORC1/S6K1 signaling while activation of PP2A reverses this process. These results provide evidence for a link between α-Syn and IRS-1 that may represent a novel mechanism for α-Syn-associated pathogenesis.     

Regulation of type 1 protein phosphatase/inhibitor-2 complex by glycogen synthase kinase-3beta in intact cells

Inhibitor 2 (I-2) is a ubiquitous regulator of type 1 protein phosphatase (PP1). Previous in vitro studies suggested that its inhibitory activity towards PP1 is regulated by phosphorylation at Thr72 by glycogen synthase kinase-3beta (GSK-3beta), and at Ser86, Ser120, and Ser121 by casein kinase 2 (CK2). Here we report that GSK-3beta expressed in COS-7 cells phosphorylates wild-type I-2 but not an I-2 mutant carrying a T to A substitution at residue 72, showing that GSK-3beta phosphorylates I-2 at T72 in vivo as well. Co-immunoprecipitation study demonstrated that HA-GSK-3beta and I-2-FLAG co-exist in a same complex in the intact cells, but they do not bind directly. It is noteworthy that co-expression of Myc-PP1C significantly increased co-precipitation of HA-GSK-3beta with I-2-FLAG, showing a complex formation of HA-GSK-3beta/Myc-PP1C / I-2-FLAG in vivo. Further studies using a GSK-3beta kinase-dead mutant and LiCl, an inhibitor of GSK-3beta, showed that the enzyme activity of GSK-3beta is required for co-precipitation. IP-Western study using several I-2 mutants substituted at phosphorylation sites (T72, S86, S120, and S121) suggested that phosphorylation of I-2 by CK2 is also involved in enhancement of association between GSK-3beta and I-2 in vivo. This study is the first demonstration that GSK-3beta associates with PP1C/I-2 complex and phosphorylates I-2 at T72 in the intact cells.     

Glycogen synthase kinase-3 couples AKT-dependent signaling to the regulation of p21Cip1 degradation.

Signaling via the phosphoinositide 3-kinase (PI3K)/AKT pathway is crucial for the regulation of endothelial cell (EC) proliferation and survival, which involves the AKT-dependent phosphorylation of the DNA repair protein p21(Cip1) at Thr-145. Because p21(Cip1) is a short-lived protein with a high proteasomal degradation rate, we investigated the regulation of p21(Cip1) protein levels by PI3K/AKT-dependent signaling. The PI3K inhibitors Ly294002 and wortmannin reduced p21(Cip1) protein abundance in human umbilical vein EC. However, mutation of the AKT site Thr-145 into aspartate (T145D) did not increase its protein half-life. We therefore investigated whether a kinase downstream of AKT regulates p21(Cip1) protein levels. In various cell types, AKT phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). Upon serum stimulation of EC, GSK-3beta was phosphorylated at Ser-9. Site-directed mutagenesis revealed that GSK-3 in vitro phosphorylated p21(Cip1) specifically at Thr-57 within the Cdk binding domain. Overexpression of GSK-3beta decreased p21(Cip1) protein levels in EC, whereas the specific inhibition of GSK-3 with lithium chloride interfered with p21(Cip1) degradation and increased p21(Cip1) protein about 10-fold in EC and cardiac myocytes (30 mm, p < 0.001). These data indicate that GSK-3 triggers p21(Cip1) degradation. In contrast, stimulation of AKT increases p21(Cip1) via inhibitory phosphorylation of GSK-3.