Interferon-gamma engages the p70 S6 kinase to regulate phosphorylation of the 40S S6 ribosomal protein

The signals generated by the IFNgamma receptor to initiate mRNA translation and generation of protein products that mediate IFNgamma responses are largely unknown. In the present study, we provide evidence for the existence of an IFNgamma-dependent signaling cascade activated downstream of the phosphatidylinositol (PI) 3'-kinase, involving the mammalian target of rapamycin (mTOR) and the p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated and activated during engagement of the IFNgamma receptor in sensitive cell lines. Such activation of p70 S6 kinase is blocked by pharmacological inhibitors of the PI 3' kinase and mTOR, and is abrogated in double-knockout mouse embryonic fibroblasts for the alpha and beta isoforms of the p85 regulatory subunit of the PI 3'-kinase. The IFNgamma-activated p70 S6 kinase subsequently phosphorylates the 40S S6 ribosomal protein on serines 235/236, to regulate IFNgamma-dependent mRNA translation. In addition to phosphorylation of 40S ribosomal protein, IFNgamma also induces phosphorylation of the 4E-BP1 repressor of mRNA translation on threonines 37/46, threonine 70, and serine 65, sites whose phosphorylation is required for the inactivation of 4E-BP1 and its dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. Thus, engagement of the PI 3'-kinase and mTOR by the IFNgamma receptor results in the generation of two distinct signals that play roles in the initiation of mRNA translation, suggesting an important role for this pathway in IFNgamma signaling.     

PERK is responsible for the increased phosphorylation of eIF2alpha and the severe inhibition of protein synthesis after transient global brain ischemia

Reperfusion after global brain ischemia results initially in a widespread suppression of protein synthesis in neurons that is due to inhibition of translation initiation as a result of the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2). To address the role of the eIF2alpha kinase RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) in the reperfused brain, transgenic mice with a targeted disruption of the Perk gene were subjected to 20 min of forebrain ischemia followed by 10 min of reperfusion. In wild-type mice, phosphorylated eIF2alpha was detected in the non-ischemic brain and its levels were elevated threefold after 10 min of reperfusion. Conversely, there was no phosphorylated eIF2alpha detected in the non-ischemic transgenic mice and there was no sizeable rise in phosphorylated eIF2alpha levels in the forebrain after ischemia and reperfusion. Moreover, there was a substantial rescue of protein translation in the reperfused transgenic mice. Neither group showed any change in total eIF2alpha, phosphorylated eukaryotic elongation factor 2 or total eukaryotic elongation factor 2 levels. These data demonstrate that PERK is responsible for the large increase in phosphorylated eIF2alpha and the suppression of translation early in reperfusion after transient global brain ischemia.     

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.     

Parallel increase in p70 kinase activation and tau phosphorylation (S262) with Abeta overproduction

This study set out to search for a link between overproduction of Abeta and p70S6 kinase (p70S6K) phosphorylation/activation. Results showed that levels of p-p70S6K at T421/S424 and T389 are significantly increased in mouse N2a neuroblastoma cells carrying human APP with Swedish mutation (APPswe), and in transgenic APPswe/PS1 (A246E) mice as compared with respective controls, corresponding to the increase of tau phosphorylation at S262. This parallel increase in p70S6K activation and tau phosphorylation could be demonstrated by treating wild-type N2a cells with Abeta25-35. Our results suggest that the Abeta deposition in senile plaques in Alzheimer disease brains might be a primary event that activates p70S6K and phosphorylates tau at S262, resulting in microtubule disruption.     

Ganglioside GM1 Activates the Mitogen-Activated Protein Kinase Erk2 and p70 S6 Kinase in U-1242 MG Human Glioma Cells

Abstract: Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U-1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [³H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U-1242 MG cells with GM1 caused activation of the mitogen-activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1-stimulated Erk2 activation and GM1-stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70^s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1-stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet-derived growth factor also activated both Erk2 and p70^s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1-mediated DNA synthesis.     

Activation of Microtubule-Associated Protein Kinase (Erk) and p70 S6 Kinase by D2 Dopamine Receptors

Abstract: The ability of human and rat D_2(short) and D_2(long) dopamine receptors to activate microtubule-associated protein (MAP) kinase (Erk1/2) and p70 S6 kinase has been investigated in recombinant cells expressing these receptors. In cells expressing the D_2(short) receptor, dopamine activated both enzymes in a transient manner but with very different time courses, with activation of Erk being much quicker. Activation of both enzymes by dopamine was dose-dependent and could be prevented by a range of selective dopamine antagonists. Excellent correlations were observed between the potencies of the antagonists for blocking enzyme activation and their affinities for the D₂ dopamine receptor. Activation of Erk and of p70 S6 kinase via the D₂ dopamine receptors was prevented by pretreatment of the cells with pertussis toxin, indicating the involvement of G proteins of the G_i or G_o family. Inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) were found to block substantially, but not completely, activation of p70 S6 kinase by dopamine, suggesting the involvement of PI 3-kinase-dependent and -independent signalling pathways in its control by dopamine. p70 S6 kinase activation was completely blocked by rapamycin. In the case of Erk, activation was partially blocked by wortmannin or LY294002, indicating a possible link with PI 3-kinase.     

P70 S6 kinase mediates tau phosphorylation and synthesis

Currently, we found that the 70-kDa p70 S6 kinase (p70S6K) directly phosphorylates tau at S262, S214, and T212 sites in vitro. By immunoprecipitation, p-p70S6K (T421/S424) showed a close association with p-tau (S262 and S396/404). Zinc-induced p70S6K activation could only upregulate translation of total S6 and tau but not global proteins in SH-SY5Y cells. The requirement of p70S6K activation was confirmed in the SH-SY5Y cells that overexpress wild-type htau40. Level of p-p70S6K (T421/S424) was only significantly correlated with p-tau at S262, S214, and T212, but not T212/S214, in Alzheimer's disease (AD) brains. These suggested that p70S6K might contribute to tau related pathologies in AD brains.     

胰岛素和佛波酯在蛋白质合成中经不同途径激活p70 S6激酶

为研究佛波酯 (PMA)和胰岛素在蛋白质合成中的信号传递 ,应用激酶活性测定和Western印迹等方法 ,分别检测mTOR(mammaliantargetofrapamycin)特异性抑制剂rapamycin或磷脂酰肌醇 3激酶 (PI3K)的特异性抑制剂LY2 94 0 0 2预处理、PMA或胰岛素处理的血清饥饿的中国仓鼠肺成纤维细胞 (CHL)中p70S6激酶 (p70S6K)和蛋白激酶B(PKB)的活性及表达 .结果显示 ,PMA或胰岛素刺激促进p70S6K的活化和表达 .而rapamycin预处理可阻断PMA和胰岛素对p70S6K的激活作用 ,表明PMA和胰岛素可能是通过mTOR 依赖性途径激活p70S6K .结果还显示 ,胰岛素刺激促进PKB的活化和表达 ,而PMA对PKB的活性和表达无影响 .LY2 94 0 0 2预处理可阻断胰岛素对p70S6K和PKB的激活作用 ,但不能抑制PMA刺激引起的p70S6K的活化 .表明胰岛素和PMA介导p70S6K活化的信号途径有所不同 ,胰岛素介导p70S6K的活化可能依赖于PI3K途径 ,而PMA介导p70S6K的活化不通过PI3K途径     

Heat shock protein 70 promotes coxsackievirus B3 translation initiation and elongation via Akt‐mTORC1 pathway depending on activation of p70S6K and Cdc2

We previously demonstrated that coxsackievirus B3 (CVB3) infection upregulated heat shock protein 70 (Hsp70) and promoted CVB3 multiplication. Here, we report the underlying mechanism by which Hsp70 enhances viral RNA translation. By using an Hsp70‐overexpressing cell line infected with CVB3, we found that Hsp70 enhanced CVB3 VP1 translation at two stages. First, Hsp70 induced upregulation of VP1 translation at the initiation stage via upregulation of internal ribosome entry site trans‐acting factor lupus autoantigen protein and activation of eIF4E binding protein 1, a cap‐dependent translation suppressor. Second, we found that Hsp70 increased CVB3 VP1 translation by enhancing translation elongation. This was mediated by the Akt‐mammalian target of rapamycin complex 1 signal cascade, which led to the activation of eukaryotic elongation factor 2 via p70S6K‐ and cell division cycle protein 2 homolog (Cdc2)‐mediated phosphorylation and inactivation of eukaryotic elongation factor 2 kinase. We also determined the position of Cdc2 in this signal pathway, indicating that Cdc2 is regulated by mammalian target of rapamycin complex 1. This signal transduction pathway was validated using a number of specific pharmacological inhibitors, short interfering RNAs (siRNAs) and a dominant negative Akt plasmid. Because Hsp70 is a central component of the cellular network of molecular chaperones enhancing viral replication, these data may provide new strategies to limit this viral infection.     

Leucine induces phosphorylation and activation of p70S6K in cortical neurons via the system L amino acid transporter

p70S6 kinase is a multipotent kinase that phosphorylates substrates in response to extracellular stimuli. This kinase activity inhibits apoptosis, regulates cell size and controls translation. In the CNS, p70S6K also participates in synaptic plasticity. In this study, we report that leucine, a branched-chain amino acid, induces phosphorylation and activation of p70S6 kinase in cortical neurons. Leucine also induces phosphorylation of S6 protein, a substrate of p70S6K. These effects of leucine are completely inhibited by rapamycin, consistent with mammalian target of rapamycin mediating p70S6 phosphorylation. Finally, we demonstrate that the action of leucine on cortical neurons is mediated by the system L amino acid transporter. Neurons express components of system L amino acid transporter LAT1, LAT2, and CD98. Leucine uptake and its effect on p70S6 kinase are both inhibited by a specific inhibitor of system L amino acid transporter. We propose that leucine plays important roles in regulating signaling by p70S6 kinase by acting as an intercellular communicator in the CNS.     

Elongation Factor 2 in the Liver and Skeletal Muscle of Mice is Decreased by Starvation

We examined whether starvation affected the amount of EF-2 protein as well as the level of its mRNA in the liver and skeletal muscle of mice, to understand the molecular mechanism for nutritional adaptation of protein-turnover. Although the amount of EF-2 was diminished by starvation in each of the tissues examined, the amount of EF-2 mRNA did not decrease in parallel with the protein.     

Vimentin intermediate filament reorganization by Cdc42: involvement of PAK and p70 S6 kinase

Rho family GTPases play a major role in actin cytoskeleton reorganization. Recent studies have shown that the activation of Rho family GTPases also induces collapse of the vimentin intermediate filament (IF) network in fibroblasts. Here, we report that Cdc42V12 induces the reorganization of vimentin IFs in Hela cells, and such reorganization is independent of actin and microtubule status. We analyzed the involvement of three serine/threonine kinase effectors, MRCK, PAK and p70 S6K in the Cdc42-induced vimentin reorganization. Surprisingly, the ROK-related MRCK is not involved in this IF reorganization. We detected phosphorylation of vimentin Ser72, a site phosphorylated by PAK, after Cdc42 activation. PAK inhibition partially blocked Cdc42-induced vimentin IF collapse suggesting the involvement of other effectors. We report that p70 S6 kinase (S6K)1 participates in this IF rearrangement since the inhibitor rapamycin or a dominant inhibitory S6K could reduce the Cdc42V12 or bradykinin-induced vimentin collapse. Further, inhibition of PAK and S6K in combination very effectively prevents Cdc42-induced vimentin IF collapse. Conversely, only in combination active PAK and S6K could induce a vimentin IF rearrangement that mimics the Cdc42 effect. Thus, Cdc42-induced vimentin reorganization involves PAK and, in a novel cytoskeletal role, p70 S6K.     

Arsenite induces cell transformation by reactive oxygen species, AKT, ERK1/2, and p70S6K1

Arsenic is naturally occurring element that exists in both organic and inorganic formulations. The inorganic form arsenite has a positive association with development of multiple cancer types. There are significant populations throughout the world with high exposure to arsenite via drinking water. Thus, human exposure to arsenic has become a significant public health problem. Recent evidence suggests that reactive oxygen species (ROS) mediate multiple changes to cell behavior after acute arsenic exposure, including activation of proliferative signaling and angiogenesis. However, the role of ROS in mediating cell transformation by chronic arsenic exposure is unknown. We found that cells chronically exposed to sodium arsenite increased proliferation and gained anchorage-independent growth. This cell transformation phenotype required constitutive activation of AKT, ERK1/2, mTOR, and p70S6K1. We also observed these cells constitutively produce ROS, which was required for the constitutive activation of AKT, ERK1/2, mTOR, and p70S6K1. Suppression of ROS levels by forced expression of catalase also reduced cell proliferation and anchorage-independent growth. These results indicate cell transformation induced by chronic arsenic exposure is mediated by increased cellular levels of ROS, which mediates activation of AKT, ERK1/2, and p70S6K1.     

Osteoprotegerin inhibit osteoclast differentiation and bone resorption by enhancing autophagy via AMPK/mTOR/p70S6K signaling pathway in vitro

Osteoclasts are highly differentiated terminal cells formed by fusion of hematopoietic stem cells. Previously, osteoprotegerin (OPG) inhibit osteoclast differentiation and bone resorption by blocking receptor activator of nuclear factor-κB ligand (RANKL) binding to RANK indirect mechanism. Furthermore, autophagy plays an important role during osteoclast differentiation and function. However, whether autophagy is involved in OPG-inhibited osteoclast formation and bone resorption is not known. To elucidate the role of autophagy in OPG-inhibited osteoclast differentiation and bone resorption, we used primary osteoclast derived from mice bone marrow monocytes/macrophages (BMM) by induced M-CSF and RANKL. The results showed that autophagy-related proteins expression were upregulated; tartrate-resistant acid phosphatase-positive osteoclast number and bone resorption activity were decreased; LC3 puncta and autophagosomes number were increased and activated AMPK/mTOR/p70S6K signaling pathway. In addition, chloroquine (as the autophagy/lysosome inhibitor, CQ) or rapamycin (as the autophagy/lysosome inhibitor, Rap) attenuated osteoclast differentiation and bone resorption activity by OPG treatment via AMPK/mTOR/p70S6K signaling pathway. Our data demonstrated that autophagy plays a critical role in OPG inhibiting osteoclast differentiation and bone resorption via AMPK/mTOR/p70S6K signaling pathway in vitro.     

The binding between p67 and eukaryotic initiation factor 2 plays important roles in the protection of eIF2alpha from phosphorylation by kinases

Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 is the major regulatory step in the initiation of protein synthesis in mammals. P67, a cellular glycoprotein, protects phosphorylation of eIF2alpha from kinases. Previously, we reported that the D6/2 mutant of p67 has higher levels of protection of eIF2alpha phosphorylation (POEP) activity. In this study, we report that the D6/2 mutant and its double mutants containing second-site alanine substitutions at the five conserved amino acid residues (D251, D262, H331, E364, and E459) show increased POEP activity in serum-starved rat tumor hepatoma cells. Serum-restoration to those cells did not abolish their increased POEP activity except the D6/2+H331A double mutant. The latter mutant shows slight inhibition of POEP activity during serum starvation and this inhibition increased significantly during serum restoration. KRC-7 cells constitutively expressing the D6/2 mutant showed slightly decreased levels of PKR phosphorylation and significantly low level of phosphorylation of ERKs 1 and 2. The D6/2 mutant also showed increased binding with eIF2alpha and eIF2gamma and almost similar binding with ERKs 1 and 2 as compared to wild type p67. Altogether, our data demonstrate that the increased binding of the D6/2 mutant with the subunits of eIF2 may be in part the cause for its high POEP activity.     

Coupling of Serotonin 5-HT1B Receptors to Activation of Mitogen-Activated Protein Kinase (ERK-2) and p70 S6 Kinase Signaling Systems

Abstract: Little is known about the coupling of serotonin 5-HT_1B receptors to cellular signals other than cyclic AMP. In the present studies, the activation by 5-HT_1B receptors of p70 S6 kinase and the mitogen-activated protein kinase (MAP kinase) ERK-2 was investigated. Studies were performed by using both nontransfected Chinese hamster ovary (CHO) cells, which express endogenous receptors at a very low density, and a stable transfected CHO cell line expressing 5-HT_1B receptors at 230 fmol/mg of membrane protein, a density similar to that expressed in cortex. In nontransfected cells, 5-HT was found to stimulate a greater than twofold increase in MAP kinase activity with an EC₅₀ of 20 n M . Reflecting increased density of receptors, 5-HT caused a greater than eightfold activation of ERK-2 in transfected cells with an EC₅₀ of 2 n M . 5-HT was found to also stimulate p70 S6 kinase in both nontransfected and transfected cells. The stimulation was sixfold in both types of cells, but the EC₅₀ for 5-HT was fourfold lower in transfected cells. The coupling of 5-HT_1B receptors to ERK-2 and to p70 S6 kinase was inhibited by pertussis toxin, inhibitors of phosphatidylinositol 3-kinase, and by the inhibitor of MAP kinase kinase PD098059. Activation of p70 S6 kinase, but not ERK-2, was also inhibited by rapamycin. These findings demonstrate that 5-HT_1B receptors couple to ERK-2 and p70 S6 kinase through overlapping, but nonidentical, pathways.     

mTOR is the rapamycin-sensitive kinase that confers mechanically-induced phosphorylation of the hydrophobic motif site Thr(389) in p70(S6k)

Mechanical stretch induces phosphorylation of the hydrophobic motif site Thr(389) in p70(S6k) through a rapamycin-sensitive (RS) pathway that involves a unique PI3K-independent mechanism. Rapamycin is considered to be a highly specific inhibitor of the protein kinase mTOR; however, mTOR is also considered to be a PI3K-dependent signaling molecule. Thus, questions remain as to whether mTOR is the RS element that confers mechanically-induced signaling to p70(S6k)(389). In this study, rapamycin-resistant mutants of mTOR along with mechanical stretch were used to address this question. The results indicate that mTOR is the RS element and reveal that mTOR signaling can be activated through a PI3K-independent mechanism.     

Akt2, but not Akt1 or Akt3 mediates pressure-stimulated serum-opsonized latex bead phagocytosis through activating mTOR and p70 S6 kinase

Monocytes and macrophages play critical roles in innate host defense and are sensitive to mechanical stimuli. Tissue pressure is often altered in association with inflammation or infection. Low pressure (20 mmHg), equivalent to normal tissue pressure, increases phagocytosis by primary monocytes and PMA-differentiated THP-1 macrophages, in part by FAK and ERK inhibition and p38 activation. PI-3K is required for macrophage phagocytosis, but whether PI-3K mediates pressure-stimulated phagocytosis is not known. Furthermore, little is known about the role played by the PI-3K downstream Kinases, Akt, and p70 S6 kinase (p70S6K) in modulating macrophage phagocytosis. Thus, we studied the contribution of PI-3K, Akt, and p70S6K to pressure-increased serum-opsonized bead phagocytosis. Pressure-induced p85 PI-3K translocation from cytosolic to membrane fractions and increased Akt activation by 36.1 +/- 12.0% in THP-1 macrophages. LY294002 or Akt inhibitor IV abrogated pressure-stimulated but not basal phagocytosis. Basal Akt activation was inhibited 90% by LY294002 and 70% by Akt inhibitor IV. Each inhibitor prevented Akt activation by pressure. SiRNA targeted to Akt1, Akt2, or Akt3 reduced Akt1, Akt2, and Akt3 expression by 50%, 45%, and 40%, respectively. However, only Akt2SiRNA abrogated the pressure-stimulated phagocytosis without affecting basal. Pressure also activated mTOR and p70S6K. mTORSiRNA and p70S6K inhibition by rapamycin or p70S6KSiRNA blocked pressure-induced, but not basal, phagocytosis. Changes in tissue pressure during inflammation may regulate macrophage phagocytosis by activation of PI-3K, which activates Akt2, mTOR, and p70S6K.