Single-unit activity of cerebellar nuclear cells in the awake genetically dystonic rat

We have established a cell culture system that reproduces morphogenic processes in the developing mammary gland. EpH4 mouse mammary epithelial cells cultured in matrigel form branched tubules in the presence of hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the c-met tyrosine kinase receptor. In contrast, alveolar structures are formed in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. These distinct morphogenic responses can also be observed with selected human mammary carcinoma tissue in explant culture. HGF/SF-induced branching was abrogated by the PI3 kinase inhibitors wortmannin and LY294002. In contrast, neuregulin- induced alveolar morphogenesis was inhibited by the MAPK kinase inhibitor PD98059. The c-met-mediated response could also be evoked by transfection of a c-met specific substrate, Gab1, which can activate the PI3 kinase pathway. An activated hybrid receptor that contained the intracellular domain of c-erbB2 receptor suffices to induce alveolar morphogenesis, and was observed in the presence of tyrosine residues Y1028, Y1144, Y1201, and Y1226/27 in the substrate-binding domain of c-erbB2. Our data demonstrate that c-met and c-erbB2 signaling elicit distinct morphogenic programs in mammary epithelial cells: formation of branched tubules relies on a pathway involving PI3 kinase, whereas alveolar morphogenesis requires MAPK kinase.     

AKT2, a member of the protein kinase B family, is activated by growth factors, v-Ha-ras, and v-src through phosphatidylinositol 3-kinase in human ovarian epithelial cancer cells

Three members have been identified in the protein kinase B (PKB) family, i.e., Akt/PKB alpha, AKT2/PKB beta, and AKT3/PKB gamma. Previous studies have demonstrated that only AKT2 is predominantly involved in human malignancies and has oncogenic activity. However, the mechanism of transforming activity of AKT2 is still not well understood. Here, we demonstrate the activation of AKT2 with several growth factors, including epidermal growth factor, insulin-like growth factor 1, insulin-like growth factor II, basic fibroblast growth factor, platelet-derived growth factor, and insulin, in human ovarian epithelial cancer cells. The kinase activity and the phosphorylation of AKT2 were induced by the growth factors and blocked by the phosphatidylinositol (PI) 3-kinase inhibitor, wortmannin, and dominant-negative Ras (N17Ras). Moreover, the activated Ras and v-Src, two proteins that transduce growth factor-generated signals, also activated AKT2, and this activation was not significantly enhanced by growth factor stimulation but was abrogated by wortmannin. These results indicate that AKT2 is a downstream target of PI 3-kinase and that Ras and Src function upstream of PI 3-kinase and mediate the activation of AKT2 by growth factors. The findings also provide further evidence that AKT2, in cooperation with Ras and Src, is important in the development of some human malignancies.     

Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.     

Effects of excitatory amino acids on cerebral oxygen consumption and blood flow in rat

The oncogene Tpr-Met is a constitutively active form of the hepatocyte growth factor/scatter factor (HGF/SF) receptor Met. It comprises the intracellular moiety of Met linked to the dimerization domain of the nuclear envelope protein Tpr, thus functioning as a constitutively activated Met. HGF/SF is responsible for various biological processes including angiogenesis and wound healing, in which secreted serine protease urokinase-type plasminogen activator (uPA) is implicated. The action of HGF/SF on cells is mediated by the autophosphorylation of Met on two carboxyterminal tyrosine residues, Y1349VHVNATVY1356VNV. The two tyrosine residues provide docking sites for various effector molecules, suggesting that multiple signaling pathways are activated to exert biological effects of HGF/SF [Ponzetto et al., Cell (1994) 77: 261]. We found that Tpr-Met efficiently activates the uPA gene via a SOS/Ras/extracellular signal regulated kinase (ERK)-dependent signaling pathway. Mutation of Y1356, which abrogates GRB2 binding, reduced the induction to half of the control level, while mutation of Y1349 showed little effect on uPA induction, suggesting an important but partly replaceable role for GRB2 in Met-dependent uPA gene induction. Mutation of both Y1349VHV and Y1356VNV into optimal PI 3-kinase sites resulted in a residual induction of about one quarter of the control level, suggesting a potential role for PI 3-kinase. Dose-response analysis of the Tpr-Met showed a biphasic curve. These results suggest that the interplay among different signaling molecules on the receptor is important for full induction of the pathway leading to the activation of the uPA gene.     

Identification and characterization of mutations in Ha-Ras that selectively decrease binding to cRaf-1

The oncoprotein Ras transforms cells by binding to one or more effector proteins. Effector proteins have been identified by their ability to bind to Ras in the GTP but not GDP form, and by their requirement for the Ras effector domain for binding. The best understood Ras effectors are serine/threonine kinases of the Raf family, but other candidate Ras effectors, including a Ral guanine nucleotide dissociation stimulator and phosphatidylinositol 3-kinase (PI3 kinase) have also been identified. To investigate the mechanism of binding of cRaf-1 to Ras, and to investigate the roles of other candidate Ras effectors in transformation, we have isolated and characterized mutants of activated Ras with decreased binding to cRaf-1 relative to other candidate effectors. Examination of these mutants indicates that surface-exposed residues of Ras outside the minimal effector domain interact differentially with cRaf-1 and other Ras-binding proteins, and that fibroblast transformation correlates with cRaf-1 binding and mitogen-activated protein (MAP) kinase activation. Furthermore, activation of PI3 kinase can occur in the absence of significant MAP kinase activation, suggesting that PI3 kinase activation is a primary effect of Ras.     

Stimulation of IRS-1-associated phosphatidylinositol 3-kinase and Akt/protein kinase B but not glucose transport by beta1-integrin signaling in rat adipocytes

The signal transduction pathway by which insulin stimulates glucose transport is not understood, but a role for complexes of insulin receptor substrate (IRS) proteins and phosphatidylinositol (PI) 3-kinase as well as for Akt/protein kinase B (PKB) has been proposed. Here, we present evidence suggesting that formation of IRS-1/PI 3-kinase complexes and Akt/PKB activation are insufficient to stimulate glucose transport in rat adipocytes. Cross-linking of beta1-integrin on the surface of rat adipocytes by anti-beta1-integrin antibody and fibronectin was found to cause greater IRS-1 tyrosine phosphorylation, IRS-1-associated PI 3-kinase activity, and Akt/PKB activation, detected by anti-serine 473 antibody, than did 1 nM insulin. Clustering of beta1-integrin also significantly potentiated stimulation of insulin receptor and IRS-1 tyrosine phosphorylation, IRS-associated PI 3-kinase activity, and Akt/PKB activation caused by submaximal concentrations of insulin. In contrast, beta1-integrin clustering caused neither a change in deoxyglucose transport nor an effect on the ability of insulin to stimulate deoxyglucose uptake at any concentration along the entire dose-response relationship range. The data suggest that (i) beta1-integrins can engage tyrosine kinase signaling pathways in isolated fat cells, potentially regulating fat cell functions and (ii) either formation of IRS-1/PI 3-kinase complexes and Akt/PKB activation is not necessary for regulation of glucose transport in fat cells or an additional signaling pathway is required.     

Insulin-stimulated glycogen synthesis in cultured hepatoma cells: differential effects of inhibitors of insulin signaling molecules

In rat HTC hepatoma cells overexpressing human insulin receptors, insulin stimulated glycogen synthesis by 55-70%. To study postreceptor signaling events leading to insulin-stimulated glycogen synthesis in these cells, we have employed pathway-specific chemical inhibitors such as LY294002, rapamycin and PD98059 to inhibit phosphatidylinositol-3-kinase (PI3K), p70 ribosomal S6 kinase and mitogen-activated protein kinase (MAPK) kinase/MAPK, respectively. LY294002 (50 microM) completely abolished insulin-stimulated glycogen synthesis whereas rapamycin (2-20 nM) partially inhibited it. Neither LY294002 nor rapamycin significantly affected the basal glycogen synthesis. However, PD98059 (100 microM) significantly inhibited the basal glycogen synthesis without affecting insulin-stimulated glycogen synthesis. In these cells, insulin at 100 nM decreased glycogen synthase kinase 3 alpha (GSK3 alpha) activity by 30-35%. LY294002, but neither rapamycin nor PD98059, abolished insulin-induced inactivation of GSK3 alpha. These data suggest that insulin-stimulated glycogen synthesis in rat HTC hepatoma cells is mediated mainly by PI3K-dependent mechanism. In these cells, inactivation of GSK3 alpha, downstream of PI3K, may play a role in insulin-stimulated glycogen synthesis.     

Insulin receptor substrate-1 and phosphatidylinositol 3-kinase regulate extracellular signal-regulated kinase-dependent and -independent signaling pathways during myogenic differentiation

Activation of the insulin-like growth factor (IGF) autocrine loop is required for myogenic differentiation and results in sustained activation of extracellular signal-regulated kinases-1 and -2 (ERK-1 and -2). We show here that insulin receptor substrate-1 (IRS-1) phosphorylation on tyrosine and serine residues and association with phosphatidylinositol 3-kinase (PI 3-kinase) are also associated with IGF-dependent myogenic differentiation. Down-regulation of IRS-1 is linked to its serine phosphorylation dependent on PI 3-kinase activity and appears required for differentiation to occur, as IRS-1 is not modified and continues to accumulate in a nondifferentiating myoblast cell line. Furthermore, inhibition of PI 3-kinase activity with LY294002 blocks differentiation, as demonstrated by inhibition of myogenin and myosin heavy chain expression and ERK activation. Blocking the Raf/MEK/ERK cascade with PD98059 does not block myogenic differentiation; however, myotubes do not survive. Thus, PI 3-kinase, in association with IRS-1, is involved in an ERK-independent signaling pathway in myoblasts required for IGF-dependent myogenic differentiation and in inducing sustained activation of ERKs necessary for later stages of differentiation.     

Listeria monocytogenes invasion of epithelial cells requires the MEK-1/ERK-2 mitogen-activated protein kinase pathway

PD98059, a specific inhibitor of MEK-1 mitogen-activated protein (MAP) kinase kinase, blocked Listeria monocytogenes invasion into HeLa epithelial cells. The effects of PD98059 were reversible, as adherent extracellular bacteria were internalized upon removal of the drug. Previously, we reported that L. monocytogenes could activate ERK-1 and ERK-2 MAP kinases through the action of listeriolysin O (LLO) on the host cell (P. Tang, I. Rosenshine, P. Cossart, and B. B. Finlay, Infect. Immun. 64:2359-2361, 1996). We have now found that two other MAP kinase pathways, those of p38 MAP kinase and c-Jun N-terminal kinase, are also activated by wild-type L. monocytogenes. Mutants lacking functional LLO (hly mutants) were still invasive but only activated ERK-2 and only activated it at later (90-min) postinfection times. Two inhibitors of L. monocytogenes invasion, cytochalasin D, which disrupts actin polymerization, and wortmannin, which blocks phosphatidylinositol (PI) 3-kinase activity, did not block ERK-2 activation by wild-type L. monocytogenes and hly mutants. However, genistein, an inhibitor of tyrosine kinases, and PD98059 both blocked invasion and decreased ERK-2 activation. These results suggest that MEK-1 and ERK-2 activities are essential for L. monocytogenes invasion into host epithelial cells. This is the first report to show that a MAP kinase pathway is required for bacterial invasion.     

Ligation of the T cell co-stimulatory receptor CD28 activates the serine-threonine protein kinase protein kinase B

The intracellular signaling pathways activated upon ligation of the co-stimulatory receptor CD28 remain relatively ill-defined, although CD28 ligation does result in the strong association with, and activation of, phosphatidylinositol (PI) 3-kinase. The downstream effector targets of the CD28-activated PI 3-kinase-dependent signaling pathway remain poorly defined, but recent evidence from other systems has shown that Akt/protein kinase B (PKB) is a major target of PI 3-kinase and have indicated that a major function of PKB is the regulation of cell survival events. Given the strong coupling of CD28 to PI 3-kinase and the known protective effects of both CD28 and PI 3-kinase against apoptosis in different cell models, we investigated the effects of CD28 on PKB activation. We demonstrate that ligation of CD28 by either anti-CD28 monoclonal antibodies or the natural ligand B7.1, results in the marked activation of PKB in both the leukemic T cell line Jurkat and freshly isolated human peripheral blood-derived normal T lymphocytes. Our data suggest therefore, that PKB may be an important intracellular signal involved in CD28 signal transduction and demonstrate CD28 coupling to downstream elements of a signaling cascade known to promote cell survival.     

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.     

Kinase suppressor of Ras inhibits the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase by growth factors, activated Ras, and Ras effectors

Kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. Genetic and biochemical studies suggest that KSR is a positive regulator of Ras signaling that functions between Ras and Raf or in a pathway parallel to Raf. We examined the effect of mammalian KSR expression on the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase in fibroblasts. Ectopic expression of KSR inhibited the activation of ERK MAP kinase by insulin, phorbol ester, or activated alleles of Ras, Raf, and mitogen and extracellular-regulated kinase. Expression of deletion mutants of KSR demonstrated that the KSR kinase domain was necessary and sufficient for the inhibitory effect of KSR on ERK MAP kinase activity. KSR inhibited cell transformation by activated RasVal-12 but had no effect on the ability of RasVal-12 to induce membrane ruffling. These data indicate that KSR is a potent modulator of a signaling pathway essential to normal and oncogenic cell growth and development.     

Analysis of signal transduction pathways regulating cytokine-mediated Fc receptor activation on human eosinophils

Igs can be potent stimulants of eosinophil activation since interaction with IgA or IgG-coated particles can lead to eosinophil degranulation. We have investigated the comparative roles of mitogen-activated protein (MAP) kinases (MAPKs; ERK1/2 and p38) and phosphatidylinositol-3 kinase (PI3K) in the priming and regulation of Fc receptor functioning on human eosinophils utilizing a MAPK kinase (MEK) inhibitor (PD98059), a p38 inhibitor SB203580, and the widely used PI3K inhibitors wortmannin and LY294002. We demonstrate that priming of human eosinophils with Th2-derived cytokines, IL-4 and IL-5, differentially activate phosphotyrosine-associated PI3K and ERK and p38 MAP kinases. This activation can be inhibited by pre-incubation with wortmannin or LY294002, PD98059, and SB203580, respectively. Analysis of the effects of the inhibitors on rosette formation between human eosinophils and IgA- or IgG-coated beads revealed that activation of MEK was not required for IgA binding after priming with IL-4 or IL-5. However, inhibition of MEK did inhibit IL-5-primed binding of IgG-beads. The rosette formation of primed eosinophils with IgA-beads could be completely inhibited by wortmannin and LY294002 treatment, demonstrating a critical role for PI3K. Interestingly, inhibition of the p38 pathway also resulted in a complete blockade of IgA rosette formation. This work demonstrates regulatory control by inside-out signaling of Fc receptors by various cytokines on human eosinophils. Thus in vivo the local production of Th2-derived cytokines will regulate the effector functions of Fc receptors.     

Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons

Recent studies have suggested a role for phosphatidylinositol (PI) 3-kinase in cell survival, including the survival of neurons. We used rat sympathetic neurons maintained in vitro to characterize the potential survival signals mediated by PI 3-kinase and to test whether the Akt protein kinase, a putative effector of PI 3-kinase, functions during nerve growth factor (NGF)-mediated survival. Two PI 3-kinase inhibitors, LY294002 and wortmannin, block NGF-mediated survival of sympathetic neurons. Cell death caused by LY294002 resembles death caused by NGF deprivation in that it is blocked by a caspase inhibitor or a cAMP analog and that it is accompanied by the induction of c-jun, c-fos, and cyclin D1 mRNAs. Treatment of neurons with NGF activates endogenous Akt protein kinase, and LY294002 or wortmannin blocks this activation. Expression of constitutively active Akt or PI 3-kinase in neurons efficiently prevents death after NGF withdrawal. Conversely, expression of dominant negative forms of PI 3-kinase or Akt induces apoptosis in the presence of NGF. These results demonstrate that PI 3-kinase and Akt are both necessary and sufficient for the survival of NGF-dependent sympathetic neurons.     

Vanadyl sulfate-stimulated glycogen synthesis is associated with activation of phosphatidylinositol 3-kinase and is independent of insulin receptor tyrosine phosphorylation

Salts of the trace element vanadium, such as sodium orthovanadate and vanadyl sulfate (VS), exhibit a myriad of insulin-like effects, including stimulation of glycogen synthesis and improvement of glucose homeostasis in type I and type II animal models of diabetes mellitus. However, the cellular mechanism by which these effects are mediated remains poorly characterized. We have shown earlier that different vanadium salts stimulate the MAP kinase pathway and ribosomal-S-6-kinase (p70s6k) in chinese hamster ovary cells overexpressing human insulin receptor (CHO-HIR cells) [Pandey, S. K., Chiasson, J.-L., and Srivastava, A. K. (1995) Mol. Cell. Biochem. 153, 69-78]. In the present studies, we have investigated if similar to insulin, VS also activates phosphatidylinositol 3-kinase (PI3-k) activity, and whether VS-induced activation of the PI3-k, MAP kinase, and p70s6k pathways contributes to glycogen synthesis. Treatment of CHO-HIR cells with VS resulted in increased glycogen synthesis and PI3-k activity which were blocked by pretreatment of the cells with wortmannin and LY294002, two specific inhibitors of PI3-k. On the other hand, PD98059 and rapamycin, specific inhibitors of the MAP kinase pathway and p70s6k, respectively, were unable to inhibit VS-stimulated glycogen synthesis. Moreover, VS-stimulated glycogen synthesis and PI3-k were observed without any change in the tyrosine phosphorylation of insulin receptor (IR) beta-subunit but were associated with increased tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In addition, PI3-k activation was detected in IRS-1 immunoprecipitates from VS-stimulated cells, indicating that tyrosine-phosphorylated IRS-1 was able to interact and thereby activate PI3-k in response to VS. Taken together, these results provide evidence that tyrosine phosphorylation of IRS-1 and activation of PI3-k play a key role in mediating the insulinomimetic effect of VS on glycogen synthesis independent of IR-tyrosine phosphorylation.     

PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells

PTEN/MMAC1/TEP1 is a tumor suppressor that possesses intrinsic phosphatase activity. Deletions or mutations of its encoding gene are associated with a variety of human cancers. However, very little is known about the molecular mechanisms by which this important tumor suppressor regulates cell growth. Here, we show that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN was mediated by its ability to block cell cycle progression in the G1 phase. Such an arrest correlated with a significant increase of the cell cycle kinase inhibitor p27(KIP1) and a concomitant decrease in the activities of the G1 cyclin-dependent kinases. PTEN expression also led to the inhibition of Akt/protein kinase B, a serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27(KIP1) and the cell cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, our studies suggest that the PTEN tumor suppressor modulates G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase inhibitor p27(KIP1).