GSK-3beta Phosphorylation Research Articles

Phosphatidylinositol 3‐kinase/Akt plays a role in sphingosine 1‐phosphate‐stimulated HSP27 induction in osteoblasts

We previously reported that p38 mitogen-activated protein (MAP) kinase plays a part in sphingosine 1-phosphate-stimulated heat shock protein 27 (HSP27) induction in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) is involved in the induction of HSP27 in these cells. Sphingosine 1-phosphate time dependently induced the phosphorylation of Akt. Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, reduced the HSP27 induction stimulated by sphingosine 1-phosphate. The sphingosine 1-phosphate-induced phosphorylation of GSK-3beta was suppressed by Akt inhibitor. The sphingosine 1-phosphate-induced HSP27 levels were attenuated by LY294002 or wortmannin, PI3K inhibitors. Furthermore, LY294002 or Akt inhibitor did not affect the sphingosine 1-phosphate-induced phosphorylation of p38 MAP kinase and SB203580, a p38 MAP kinase inhibitor, had little effect on the phosphorylation of Akt. These results suggest that PI3K/Akt plays a part in the sphingosine 1-phosphate-stimulated induction of HSP27, maybe independently of p38 MAP kinase, in osteoblasts.

Abeta and tau form soluble complexes that may promote self aggregation of both into the insoluble forms observed in Alzheimer's disease.

To date, there is no reasonable explanation as to why plaques and tangles simultaneously accumulate in Alzheimer's disease (AD). We demonstrate here by Western blotting and ELISA that a stable complex can form between tau and amyloid-beta protein (Abeta). This complex enhances tau phosphorylation by GSK3beta, but the phosphorylation then promotes dissociation of the complex. We have localized the sites of this interaction by using peptide membrane arrays. Abeta binds to multiple tau peptides, especially those in exons 7 and 9. This binding is sharply reduced or abolished by phosphorylation of specific serine and threonine residues. Conversely, tau binds to multiple Abeta peptides in the mid to C-terminal regions of Abeta. This binding is also significantly decreased by GSK3beta phosphorylation of tau. We used surface plasmon resonance to determine the binding affinity of Abeta for tau and found it to be in the low nanomolar range and almost 1,000-fold higher than tau for itself. In soluble extracts from AD and control brain tissue, we detected Abeta bound to tau in ELISAs. We also found by double immunostaining of AD brain tissue that phosphorylated tau and Abeta form separate insoluble complexes within the same neurons and their processes. We hypothesize that in AD, an initial step in the pathogenesis may be the intracellular binding of soluble Abeta to soluble nonphosphorylated tau, thus promoting tau phosphorylation and Abeta nucleation. Blocking the sites where Abeta initially binds to tau might arrest the simultaneous formation of plaques and tangles in AD.

Implication of the Phosphatidylinositol-3 Kinase/Protein Kinase B Signaling Pathway in the Neuroprotective Effect of Estradiol in the Striatum of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Mice

The present experiments sought to determine the implication of estrogen receptors (ERalpha and ERbeta) and their interaction with insulin-like growth factor receptor (IGF-IR) signaling pathways in neuroprotection by estradiol against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity. C57BL/6 male mice were pretreated for 5 days with 17beta-estradiol, an estrogen receptor alpha (ERalpha) agonist, 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)tris-phenol (PPT), or an estrogen receptor beta (ERbeta) agonist, 5-androsten-3beta, 17beta-diol (Delta5-diol). On day 5, mice received MPTP (9 mg/kg) or saline injections, and estrogenic treatments were continued for 5 more days. MPTP decreased striatal dopamine, measured by high-performance liquid chromatography, to 59% of control values; 17beta-estradiol and PPT but not Delta5-diol protected against this depletion. MPTP increased IGF-IR measured by Western blot, which was prevented by PPT. The phosphorylation of protein kinase B (Akt) (at serine 473), an essential mediator of IGF-I neuroprotective actions, increased after 17beta-estradiol and tended to increase with PPT but not with Delta5-diol treatments in MPTP mice. Glycogen synthase kinase 3beta (GSK3beta) phosphorylation (at serine 9) was greatly reduced in MPTP mice; this was completely prevented by PPT, whereas 17beta-estradiol and Delta5-diol treatments were less effective. The ratio between the levels of striatal Bcl-2 and BAD proteins, two apoptotic regulators, decreased after MPTP treatment. This effect was effectively prevented only in the animals treated with PPT. In nonlesioned mice, 17beta-estradiol and PPT increased phosphorylation of striatal Akt and GSK3beta, whereas the other markers measured remained unchanged. Delta5-Diol increased GSK3beta phosphorylation less than the PPT treatment. These results suggest that a pretreatment with estradiol promoted dopamine neuron survival by activating ERalpha and increasing Akt and GSK3beta phosphorylation.

Irs1 and Irs2 signaling is essential for hepatic glucose homeostasis and systemic growth

Insulin receptor substrates, including Irs1 and Irs2, integrate insulin and IGF receptor signals with heterologous pathways to coordinate growth and metabolism. Since Irs2 is thought to be especially important in hepatic nutrient homeostasis, we deleted Irs2 [corrected] from hepatocytes of WT mice (called LKO) or genetically insulin-resistant Irs1-/- mice (called LKO::Irs1-/-). Viable LKO::Irs1-/- mice were 70% smaller than WT or LKO mice, and 40% smaller than Irs1-/- mice. Hepatic insulin receptors were functional in all the mice, but insulin signaling via the Akt-FoxO1 pathway was reduced in Irs1-/- and LKO liver, and undetected in LKO::Irs1-/- liver; however, Gsk3beta phosphorylation (Ser9) and hepatic glycogen stores were nearly normal in all of the mice. LKO and Irs1-/- mice developed insulin resistance and glucose intolerance that never progressed to diabetes, whereas LKO::Irs1-/- mice developed hyperglycemia and hyperinsulinemia immediately after birth. Regardless, few hepatic genes changed expression significantly in Irs1-/- or LKO mice, whereas hundreds of genes changed in LKO::Irs1-/- mice--including elevated levels of Pck1, G6pc, Ppargc1, Pparg, and Igfbp1. Thus, signals delivered by Irs1 or Irs2 regulate hepatic gene expression that coordinates glucose homeostasis and systemic growth.

Regulation of insulin signalling, glucose uptake and metabolism in rat skeletal muscle cells upon prolonged exposure to resistin

Debate exists regarding the role of resistin in the pathophysiology of insulin resistance. The aim of this study was to directly assess the effects of resistin (0-24 h) on basal and insulin-stimulated glucose uptake and metabolism in skeletal muscle cells and to investigate the mechanisms responsible for the effects of resistin. We used L6 rat skeletal muscle cells and examined [(3)H]2-deoxyglucose uptake, GLUT4 translocation and GLUT protein content. We assessed glucose metabolism by measuring the incorporation of D-[U-(14)C]glucose into glycogen, (14)CO(2) and lactate production, as well as the phosphorylation level and total protein content of insulin signalling proteins, including insulin receptor beta-subunit (IRbeta), insulin receptor substrate (IRS), Akt and glycogen synthase kinase-3beta (GSK-3beta). Treatment of L6 rat skeletal muscle cells with recombinant resistin (50 nmol/l, 0-24 h) reduced levels of basal and insulin-stimulated 2-deoxyglucose uptake and decreased insulin-stimulated GLUT4myc content at the cell surface, with no alteration in the production of GLUT4 or GLUT1. Resistin also decreased glycogen synthesis and GSK-3beta phosphorylation. Insulin-stimulated oxidation of glucose via the Krebs cycle was reduced by resistin, whereas lactate production was unaltered. Although insulin receptor protein level and phosphorylation were unaltered by resistin, production of IRS-1, but not IRS-2, was downregulated and a decreased tyrosine phosphorylation of IRS-1 was detected. Reduced phosphorylation of Akt on T308 and S473 was observed, while total Akt and Akt1, but not Akt2 or Akt3, production was decreased. Our data show that resistin regulates the function of IRS-1 and Akt1 and decreases GLUT4 translocation and glucose uptake in response to insulin. Selective decreases in insulin-stimulated glucose metabolism via oxidation and conversion to glycogen were also induced by resistin. These observations highlight the potential role of resistin in the pathophysiology of type 2 diabetes in obesity.

A New Strategy for Studying Protein Kinase B and Its Three Isoforms. Role of Protein Kinase B in Phosphorylating Glycogen Synthase Kinase-3, Tuberin, WNK1, and ATP Citrate Lyase

Protein kinase B appears to play a key role in insulin signaling and in the control of apoptosis, although the precise targets of PKB are incompletely understood. PKB exists as three isoforms (alpha, beta, and gamma) that may have unique as well as common functions within the cell. To facilitate understanding the precise roles of PKB and its isoforms, novel tools of widespread applicability are described. These tools are antisense oligonucleotide probes that enable the specific and potent knock down of endogenous PKB alpha, beta, or gamma isoforms, individually or in various combinations, including concurrent removal of all three isoforms. The probes were applied to dissect the role of PKB in phosphorylating glycogen synthase kinase-3 (GSK-3), a critical mediator in multiple responses, and other potentially key targets. Triple antisense knock down of PKB alpha, beta, and gamma so that total PKB was <6% blocked insulin-stimulated phosphorylation of endogenous GSK-3alpha and GSK-3beta isoforms by 67% and 45%, respectively, showing that GSK-3alpha and GSK-3beta are controlled by endogenous PKB. Each PKB isoform contributed to GSK-3alpha and GSK-3beta phosphorylation, with PKBbeta having the predominant role. Knock down of total PKB incompletely blocked insulin-stimulated phosphorylation of GSK-3alpha and GSK-3beta, and a pathway involving atypical PKCs, zeta/lambda, was shown to contribute to the signal. Triple antisense knock down of PKB alpha, beta, and gamma abrogated the insulin-stimulated phosphorylation of WNK1, ATP citrate lyase, and tuberin. However, antisense-mediated knock down of PKB alpha, beta, and gamma had no effect on insulin-stimulated DNA synthesis in 3T3-L1 adipocytes, indicating that pathways other than PKB mediate this response in these cells. Finally, our PKB antisense strategy provides a method of general usefulness for further dissecting the precise targets and roles of PKB and its isoforms.

Glycogen Synthase Kinase 3β Modulates Synphilin-1 Ubiquitylation and Cellular Inclusion Formation by SIAH: IMPLICATIONS FOR PROTEASOMAL FUNCTION AND LEWY BODY FORMATION

alpha-Synuclein is known to play a major role in the pathogenesis of Parkinson disease. We previously identified synphilin-1 as an alpha-synuclein-interacting protein and more recently found that synphilin-1 also interacts with the E3 ubiquitin ligases SIAH-1 and SIAH-2. SIAH proteins ubiquitylate synphilin-1 and promote its degradation through the ubiquitin proteasome system. Inability of the proteasome to degrade synphilin-1 promotes the formation of ubiquitylated inclusion bodies. We now show that synphilin-1 is phosphorylated by GSK3beta within amino acids 550-659 and that this phosphorylation is significantly decreased by pharmacological inhibition of GSK3beta and suppression of GSK3beta expression by small interfering RNA duplex. Mutation analysis showed that Ser556 is a major GSK3beta phosphorylation site in synphilin-1. GSK3beta co-immunoprecipitated with synphilin-1, and protein 14-3-3, an activator of GSK3beta activity, increased synphilin-1 phosphorylation. GSK3beta decreased the in vitro and in vivo ubiquitylation of synphilin-1 as well as its degradation promoted by SIAH. Pharmacological inhibition and small interfering RNA suppression of GSK3beta greatly increased ubiquitylation and inclusion body formation by SIAH. Additionally, synphilin-1 S556A mutant, which is less phosphorylated by GSK3beta, formed more inclusion bodies than wild type synphilin-1. Inhibition of GSK3beta in primary neuronal cultures decreased the levels of endogenous synphilin-1, indicating that synphilin-1 is a physiologic substrate of GSK3beta. Using GFPu as a reporter to measure proteasome function in vivo, we found that synphilin-1 S556A is more efficient in inhibiting the proteasome than wild type synphilin-1, raising the possibility that the degree of synphilin-1 phosphorylation may regulate the proteasome function. Activation of GSK3beta during endoplasmic reticulum stress and the specific phosphorylation of synphilin-1 by GSK3beta place synphilin-1 as a possible mediator of endoplasmic reticulum stress and proteasomal dysfunction observed in Parkinson disease.

A novel mass spectrometry-based assay for GSK-3β activity

BackgroundAs a component of the progression from genomic to proteomic analysis, there is a need for accurate assessment of protein post-translational modifications such as phosphorylation. Traditional kinase assays rely heavily on the incorporation of γ-P32 radiolabeled isotopes, monoclonal anti-phospho-protein antibodies, or gel shift analysis of substrate proteins. In addition to the expensive and time consuming nature of these methods, the use of radio-ligands imposes restrictions based on the half-life of the radionucleotides and pose potential health risks to researchers. With the shortcomings of traditional assays in mind, the aim of this study was to develop a high throughput, non-radioactive kinase assay for screening Glycogen Synthase Kinase-3beta (GSK-3β) activity.ResultsSynthetic peptide substrates designed with a GSK-3β phosphorylation site were assayed with both recombinant enzyme and GSK-3β immunoprecipitated from NIH 3T3 fibroblasts. A molecular weight shift equal to that of a single phosphate group (80 Da.) was detected by surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) in a GSK-3β target peptide (2B-Sp). Not only was there a dose-dependent response in molecular weight shift to the amount of recombinant GSK-3β used in this assay, this shift was also inhibited by lithium chloride (LiCl), in a dose-dependent manner.ConclusionWe present here a novel method to sensitively measure peptide phosphorylation by GSK-3β that, due to the incorporation of substrate controls, is applicable to either purified enzyme or cell extracts. Future studies using this method have the potential to elucidate the activity of GSK-3β in vivo, and to screen enzyme activity in relation to a variety of GSK-3β related disorders.

NMDA neuroprotection against a phosphatidylinositol‐3 kinase inhibitor, LY294002 by NR2B‐mediated suppression of glycogen synthase kinase‐3β‐induced apoptosis

To identify the intracellular signaling pathways that mediate the pro-survival activity of NMDA receptors (NMDARs), we studied effects of exogenous NMDA on cultured rat cortical and hippocampal neurons that were treated with a phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002. NMDA at 5 or 10 microm protected against LY294002-induced apoptosis, suggesting NMDAR-mediated activation of a survival signaling pathway that is PI3K-independent. NR2B-specific NMDAR blockers antagonized anti-apoptotic effects of NMDA, indicating a critical role of NR2B NMDARs in the neuroprotection. NMDA at 10 microm suppressed LY294002-induced activation of a pro-apoptotic kinase, glycogen synthase kinase 3beta (GSK3beta). GSK3beta activation by LY294002 was associated with decreased levels of inhibitory GSK3beta phosphorylation at the Ser9 residue. However, NMDA did not prevent the LY294002-mediated decline of phospho-Ser9 levels. In addition, NMDA inhibited cortical neuron apoptosis induced by the overexpression of either wild type (wt) or Ser9Ala mutant form of GSK3beta, suggesting that NMDA suppressed GSK3beta in a Ser9-independent manner. Finally, inhibition of NR2B NMDARs reduced the NMDA protection against overexpression of GSK3betawt. These data indicate that moderate stimulation of NR2B NMDAR protects against inhibition of PI3K by a Ser9-independent inhibition of the pro-apoptotic activity of GSK3beta. Hence, the activation of NR2B and the Ser9-independent inhibition of GSK3beta are two newly identified elements of the signaling network that mediates the pro-survival effects of NMDA.

The PKC- Inhibitor Enzastaurin Inhibits MM Cell Growth, Survival and Migration in the Bone Marrow Microenvironment.

Members of the protein kinase C (PKC) family of serine- threonine protein kinases mediate multiple physiological functions including differentiation, growth and survival, invasiveness, angiogenesis and drug efflux. Dysregulation of PKC signaling has been implicated in tumor progression and prompted the development of novel anticancer therapeutics. In multiple myeloma (MM) PKC isoforms are: (1) involved in MM cell apoptosis; (2) associated with VEGF- and Wnt- induced MM cell migration; and (3) controlling shedding of IL-6 receptor alpha. However, to date the potential of targeting PKC signaling sequelae in MM has not been evaluated. Here we investigated the novel orally available protein- kinase C (PKC) inhibitor Enzastaurin (Eli Lilly and Company) for its therapeutic efficacy in MM. We first tested the ability of Enzastaurin to suppress MM cell proliferation in a wide array of MM cell lines. Our data show that Enzastaurin inhibits 3H[dT] uptake in all cell lines tested in a low micromolar range equivalent to the concentration range achieved in the patient plasma during clinical trials. Importantly, Enzastaurin also abrogates MM cell proliferation in a BMSC-MM coculture system. We next sought to determine whether Enzastaurin can inhibit cell survival and found dose- dependent induction of MM cell apoptosis in MM cell lines MM.1S, MM.1R, OPM-1, OPM-2, RPMI-8226, and RPMI-dox40. Moreover, Enzastaurin significantly inhibited VEGF- induced MM cell migration on fibronectin. Importantly, IGF-1- induced MM cell migration was abrogated by Enzastaurin, demonstrating the requirement of PKC. Signaling pathways mediating these effects were next examined: Our data show that Enzastaurin abrogates phosphorylation of Akt and GSK3beta, which is required for MM cell growth and migration. Furthermore, ongoing studies are evaluating the efficacy of Enzastaurin in a murine model of human MM. Taken together, these studies show for the first time the preclinical efficacy of the orally available PKC inhibitor Enzastaurin providing the basis for its clinical evaluation to improve patient outcome in MM.

The inhibition of glycogen synthase kinase 3β by a metabotropic glutamate receptor 5 mediated pathway confers neuroprotection to Aβ peptides

Activation of glycogen synthase kinase 3beta (Gsk3beta) has been shown to be a key component in signaling pathways that underlie neurodegeneration and neurodegenerative disease. Conversely, inactivation of Gsk3beta by phosphoinositide 3-kinase (PI3K)/Akt is an important neuroprotective mechanism. Previous studies have shown that agonist activation of group I metabotropic glutamate receptors (mGluRs) can increase neuronal survival and prevent apoptosis. However, little is known about the signaling pathways that couple mGluR5 to neuroprotection. In this report, we investigated whether activation of the PI3K/Akt/Gsk3beta pathway, which has been shown to have an important neuroprotective mechanism, is required for mGluR5 activation mediated neuroprotection against beta-amyloid. We found that brief incubations of mouse hippocampal slices with (R,S)-3,5-dihydroxyphenylglycine (DHPG) resulted in increased phosphorylation of Akt and Gsk3beta. The PI3K inhibitors, LY294002 and wortmannin, blocked the DHPG-induced increased phosphorylation of Akt and Gsk3beta. Similar results were observed in rat primary hippocampal cultures. Finally, we found that the PI3K inhibitor LY294002 can block (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) mediated neuroprotection against beta-amyloid. Thus, these findings suggest that mGluR5 can modulate the PI3K/Akt/Gsk3beta pathway in the hippocampus, and that modulation of this signaling pathway can reverse beta-amyloid-induced neuronal toxicity.

Corneal cell survival in adenovirus type 19 infection requires phosphoinositide 3-kinase/Akt activation.

Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the PI3K/Akt pathway to maintain corneal fibroblast viability in acute infection. We demonstrate phosphorylation of GSK-3beta and nuclear translocation of the p65 subunit of NF-kappaB, both downstream targets of the PI3K/Akt pathway, in adenovirus-infected corneal fibroblasts in a PI3K-dependent manner. Inhibition of PI3K had no effect on early viral gene expression, suggesting normal viral internalization, but pretreatment with the PI3K inhibitor LY294002 or overexpression of dominant negative Akt induced early cytopathic effect and caspase-mediated cell death in adenovirus-infected cells. Early cell death could be circumvented despite LY294002 by overexpression of constitutively active Akt. Furthermore, we show an interaction between cSrc and the p85 regulatory subunit of PI3K in infected cells through a phosphorylation-dependent mechanism. The results presented in this paper provide the first direct evidence that PI3K-mediated Akt activation in adenovirus-infected corneal cells may contribute to viral pathogenesis by the prolongation of cell viability.

Decreased apoptosis in polyamine depleted IEC-6 cells depends on Akt-mediated NF-κB activation but not GSK3β activity

The PI3-kinase/Akt pathway promotes cell survival in many different cell types including intestinal epithelial cells. Increased AKT activation in polyamine depleted intestinal epithelial cells correlated well with the decrease in TNF-alpha-induced apoptosis. Increased Akt activation and GSK3beta (Ser 9) phosphorylation without significant effect on Bad (Ser136) phosphorylation indicate that Akt-mediated protection is independent of Bad phosphorylation but may depend on GSK3beta. Pretreatment of polyamine-depleted cells with LY294002 increased caspase-9 and caspase-3 activation and decreased basal levels of GSK-3beta phosphorylation. Inhibition of GSK3beta activity using AR-A014418 or lithium chloride or siRNA-mediated downregulation of its expression had no effect on apoptosis. Inhibition of PI3-kinase and over-expression of dominant negative Akt (DN-AKT), significantly increased apoptosis in polyamine depleted cells. DN-Akt expression reversed the protective effect of polyamine depletion on apoptosis. DN-Akt, as well as the PI3-kinase inhibitors, prevented Akt activation and subsequent translocation of NF-kappaB to the nucleus. Constitutively active Akt (CA-AKT) expression increased resistance to TNF-alpha-induced apoptosis. Constitutively active-Akt expression increased nuclear staining of NF-kappaB. Moreover, polyamine depletion of DN-Akt cells prevented basal and TNF-alpha-induced IkappaBalpha phosphorylation. Prevention of NF-kappaB activation in DN-IkappaBalpha-transfected cells increased apoptosis in control cells and restored it in polyamine-depleted cells to control levels. These data indicate that Akt regulates the mitochondrial pathway, preventing activation of caspase-9 and thereby caspase-3 via NF-kappaB and these effects are independent of GSK-3beta activity.

Tau and GSK3β Dephosphorylations are Required for Regulating Pin1 Phosphorylation

Pin1 binds mitotically phosphorylated Thr231-Pro232 and Thr212-Pro213 sites on tau, and a Pin1 deficiency in mice leads to tau hyperphosphorylation. The aim of this study was to determine if the dephosphorylation or inhibition of tau and GSK3beta phosphorylation induces the Pin1 phosphorylation. To test this, human SK-N-MC cells were stably transfected with a fusion gene containing neuron-specific enolase (NSE)-controlled APPsw gene(NSE/APPsw), to induce Abeta-42. The stable transfectants were then transiently transfected with NSE/Splice, lacking human tau (NSE/Splice), or NSE/hTau, containing human tau, into the cells. The NSE/Splice- and NSE/hTau-cells were then treated with lithium. We concluded that (i) there was more C99-beta APP accumulation than C83-betaAPP in APPsw-tansfectant and thereby promoted Abeta-42 production in transfectants. (ii) the inhibition of tau and GSK3beta phosphorylations correlated with increase in Pin1 activation in NSE/hTau- cells. Thus, these observations suggest that Pin1 might have an inhibitive role in phosphorylating tau and GSK3beta for protecting against Alzheimer's disease.

Alternate Paths from Epidermal Growth Factor Receptor to Akt in Malignant Versus Nontransformed Lung Epithelial Cells

In many human lung adenocarcinoma cell lines, a pathway involving epidermal growth factor receptor (EGFR), ErbB2 and ErbB3 receptors, phosphatidyl inositol 3-kinase (PI3K), Akt, glycogen synthase kinase 3-beta (GSK3-beta), and cyclin D1 controls cell growth, survival, and invasiveness. We have investigated this pathway in paired transformed/nontransformed cell lines from murine peripheral lung epithelium, E9/E10 and A5/C10. The E9 and A5 carcinoma lines expressed ErbB3 and transforming growth factor-alpha (TGF-alpha) and responded to TGF-alpha stimulation with protein complex formation including the p85 regulatory subunit of PI3K, activation of Akt, phosphorylation of GSK3-beta, and increased cyclin D1 protein and the cell cycle. ErbB3 and TGF-alpha were not detected in the nontransformed E10 and C10 cell lines. Nevertheless, exposure of E10 or C10 cells to TGF-alpha activated PI3K and Akt and increased cyclin D1 and cell growth. The effector pathway from the EGFR to PI3K in these nontransformed cells included the adaptor Grb2, the docking protein Gab1, and the phosphatase Shp2. Gab1 was highly expressed in E10 and C10 cells but not in the malignant E9 and A5 sister lines. Complexes of EGFR/Grb2/Gab1/Shp2 after TGF-alpha stimulation were prominent only in E10 and C10 cells. Thus, alternate pathways downstream of EGFR regulate mitosis in these paired malignant versus nontransformed lung cell lines.

Oncogenic K- ras Stimulates Wnt Signaling in Colon Cancer Through Inhibition of GSK-3β

Two key genetic events underlying the development of colon cancer are activation of the K-ras and Wnt signaling pathways. We have previously shown that these 2 pathways can cooperate to regulate vascular endothelial growth factor (VEGF) gene expression. The goal of this study was to define the molecular basis for this interaction. The effects of K-ras(Val12) on VEGF and T-cell factor 4 (TCF-4) promoter activity, nuclear levels of beta-catenin and beta-catenin/TCF-4 complexes, glycogen synthase kinase 3beta (GSK-3beta) phosphorylation, and GSK-3beta kinase activity were measured. LY294002 and PD98059 were used to define the role of specific ras effector pathways. Oncogenic K-ras up-regulated the activity of the VEGF promoter, and selective mutagenesis of TCF-4 binding sites significantly blocked this induction. K-ras(Val12) also induced the activity of a heterologous TCF-4 reporter construct in Caco-2 and HeLa cells. LY294002 and dominant negative phosphatidylinositol 3-kinase nearly completely blocked this induction. K-ras(Val12) increased the stability of beta-catenin, the levels of nuclear beta-catenin, and the formation of nuclear beta-catenin/TCF-4 complexes, and these effects were also blocked by LY294002. Finally, K-ras(Val12) inhibited the kinase activity of total cellular GSK-3beta and GSK-3beta complexed with Axin. This effect was not mediated through phosphorylation at serine 9 but did depend on phosphatidylinositol 3-kinase. Our results suggest a unique cooperative interaction between 2 critical oncogenic pathways in colorectal tumorigenesis and highlight the pivotal role of GSK-3beta.

Compensatory recovery of liver mass by Akt-mediated hepatocellular hypertrophy in liver-specific STAT3-deficient mice

Liver regeneration following hepatectomy is complicated and involves a variety of interacting factors. The present study was designed to study the roles of proliferation and hypertrophy of hepatocytes in liver regeneration following hepatectomy in liver-specific STAT3-knockout (LS3-KO) mice lacking mitogenic activity. Partial hepatectomy was performed in LS3-KO and control mice. Liver regeneration was estimated by the liver weight, cell proliferation and cell size, and the related cellular signals were analyzed. Proliferation of hepatocytes following PH was markedly suppressed in LS3-KO mice with reduced cyclinD1 transcript. However, liver mass recovered sufficiently following PH in LS3-KO mice almost equal to that of control mice. Analysis of hepatocellular growth revealed that cell size following hepatectomy was significantly larger in LS3-KO mice than in control mice. Hepatectomy induced immediate but transient phosphorylation of Akt, p70S6K, mTOR and GSK-3beta in LS3-KO mice much more than in control mice. Additionally, adenoviral transfection of dominant negative mutant of Akt to control and LS3-KO mice led to insufficient liver regeneration following hepatectomy. PI3-K/Akt-mediated responsive hepatocellular hypertrophy may be essential for liver regeneration following hepatectomy and sufficiently compensated liver regeneration even in STAT3-deficient liver, in which cell proliferation is impaired.

Increased phosphorylation of Ser473-Akt, Ser9-GSK-3β and Ser133-CREB in the rat frontal cortex after MK-801 intraperitoneal injection

GSK-3beta is regarded as playing an important part in the pathogenesis of schizophrenia and the action of psychotomimetic agents. We observed phosphorylation of molecules associated with the GSK-3beta signalling pathway in the rat brain after MK-801 injection, which induces a schizophrenia-like state in humans. Ser9-GSK-3beta phosphorylation was increased after injection of 1 mg/kg MK-801 in the rat frontal cortex but not in the hippocampus or cerebellum. This increase peaked at 30 min and was maintained until 90 min after injection. The phosphorylation showed a dose-dependent increase up to 1 mg/kg MK-801, followed by a decrease at higher dosage. Furthermore, phosphorylation of Ser473-Akt and Ser133-CREB showed similar temporal, dose-dependent and regionally specific patterns with those of Ser9-GSK-3beta. However, phosphorylation of Dvl and Ser33-beta-catenin was not affected by MK-801. These results suggest that GSK-3beta phosphorylation by MK-801 may be associated with the Akt-GSK-3beta pathway rather than with the Wnt-Dvl-GSK3beta pathway.

Urokinase-Induced Smooth Muscle Cell Migration Requires PI3-K and Akt Activation 1,2

To examine the role of the phospho-inositol-3'-kinase (PI3-K)-akt signaling axis during smooth muscle cell (SMC) migration in response to the aminoterminal fragment of urokinase (ATF). Urokinase (uPA) is involved in vessel remodeling and mediates smooth muscle cell migration. Migration in response to urokinase is dependent on ATF. The role of PI3-K/akt signaling during migration in response to the uPA fragments is not understood. Murine arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of ATF with and without the PI3-K inhibitors (Wortmannin, Wn [10 nm] and LY294002, LY [10 microm]) and an akt inhibitor (aktI, [10 microm]). Western blotting was performed for akt, ERK1/2, and GSK3beta phosphorylation after cells were stimulated with ATF in the presence and absence of the inhibitors. Statistics were analyzed by one-way ANOVA. Both PI3-K and akt inhibitors blocked the migratory response to ATF in both assays. ATF induced time-dependent increases in akt phosphorylation at both S472 and T308 sites and ERK1/2 phosphorylation. Activation of akt and ERK1/2 was inhibited by Wn and LY. Manumycin A, a ras inhibitor, did not inhibit activation of akt but did inhibit ERK1/2 activation. Activation of akt and the dephosphorylation of its downstream kinase GSK3beta were inhibited by the akt inhibitor. Direct inhibition of akt did not influence ERK1/2 activation and inhibition of ERK1/2 did not influence akt activation. ATF mediated migration is PI3-K dependent and activates two separate pathways: ERK1/2 and akt. ATF induces akt phosphorylation through a PI3K-mediated but ras-independent mechanism while both ras and PI3K are required for ERK1/2 activation. Defining key signaling pathways is vital to regulate vessel remodeling.

14‐3‐3 Proteins regulate glycogen synthase 3β phosphorylation and inhibit cardiomyocyte hypertrophy

14-3-3 proteins are dimeric phophoserine-binding molecules that participate in important cellular processes such as cell proliferation, cell-cycle control and the stress response. In this work, we report that several isoforms of 14-3-3s are expressed in neonatal rat cardiomyocytes. To understand their function, we utilized a general 14-3-3 peptide inhibitor, R18, to disrupt 14-3-3 functions in cardiomyocytes. Cardiomyocytes infected with adenovirus-expressing YFP-R18 (AdR18) exhibited markedly increased protein synthesis and atrial natriuretic peptide production and potentiated the responses to norepinephrine stimulation. This response was blocked by the pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Consistent with a role of PI3K in the R18 effect, R18 induced phosphorylation of a protein cloned from the vakt oncogene of retrovirus AKT8 (Akt - also called protein kinase B, PKB) at Ser473 and glycogen synthase 3beta (GSK3beta) at Ser9, but not extracellular signal-regulated kinase 1/2 (ERK1/2). AdR18-induced PKB and GSK3beta phosphorylation was completely blocked by LY294002. In addition, a member of the nuclear factor of activated T cells (NFAT) family, NFAT3, was converted into faster mobility forms and translocated into the nucleus upon the treatment of AdR18. These results suggest that 14-3-3s inhibits cardiomyocytes hypertrophy through regulation of the PI3K/PKB/GSK3beta and NFAT pathway.