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1

P70 S6 kinase mediates tau phosphorylation and synthesis  

Microsoft Academic Search

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

Jin-Jing Pei; Wen-Lin An; Xin-Wen Zhou; Takeshi Nishimura; Jan Norberg; Eirikur Benedikz; Jürgen Götz; Bengt Winblad

2006-01-01

2

P70 S6 kinase mediates tau phosphorylation and synthesis.  

PubMed

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. PMID:16364302

Pei, Jin-Jing; An, Wen-Lin; Zhou, Xin-Wen; Nishimura, Takeshi; Norberg, Jan; Benedikz, Eirikur; Götz, Jürgen; Winblad, Bengt

2005-12-06

3

Structural Basis of Human p70 Ribosomal S6 Kinase-1 Regulation by Activation Loop Phosphorylation  

SciTech Connect

p70 ribosomal S6 kinase (p70S6K) is a downstream effector of the mTOR signaling pathway involved in cell proliferation, cell growth, cell-cycle progression, and glucose homeostasis. Multiple phosphorylation events within the catalytic, autoinhibitory, and hydrophobic motif domains contribute to the regulation of p70S6K. We report the crystal structures of the kinase domain of p70S6K1 bound to staurosporine in both the unphosphorylated state and in the 3{prime}-phosphoinositide-dependent kinase-1-phosphorylated state in which Thr-252 of the activation loop is phosphorylated. Unphosphorylated p70S6K1 exists in two crystal forms, one in which the p70S6K1 kinase domain exists as a monomer and the other as a domain-swapped dimer. The crystal structure of the partially activated kinase domain that is phosphorylated within the activation loop reveals conformational ordering of the activation loop that is consistent with a role in activation. The structures offer insights into the structural basis of the 3{prime}-phosphoinositide-dependent kinase-1-induced activation of p70S6K and provide a platform for the rational structure-guided design of specific p70S6K inhibitors.

Sunami, Tomoko; Byrne, Noel; Diehl, Ronald E.; Funabashi, Kaoru; Hall, Dawn L.; Ikuta, Mari; Patel, Sangita B.; Shipman, Jennifer M.; Smith, Robert F.; Takahashi, Ikuko; Zugay-Murphy, Joan; Iwasawa, Yoshikazu; Lumb, Kevin J.; Munshi, Sanjeev K.; Sharma, Sujata (Banyu); (Merck)

2010-03-04

4

Cdc2-cyclin B phosphorylates p70 S6 kinase on Ser411 at mitosis.  

PubMed

The carboxyl terminus of p70 S6 kinase (p70(s6k)) has a set of Ser and Thr residues (Ser411, Ser418, Ser424, and Thr421) phosphorylated in vivo by an unidentified kinase(s). These Ser/Thr sites are immediately followed by proline, a motif that is commonly seen in the substrates of cyclin-dependent kinases (Cdk) and mitogen-activated protein kinases. A previous study has shown that Cdc2 (Cdk1) indeed phosphorylates these p70(s6k) Ser/Thr residues in vitro. Here, we demonstrate that Cdc2-cyclin B complex phosphorylates Ser411 in the KIRSPRR sequence, whereas other Cdk-cyclin complexes including those containing Cdk2, Cdk4, or Cdk6 do not. Additionally, Ser411 phosphorylation in vivo was increased at mitosis in parallel with Cdc2 activation, and it was suppressed by a dominant negative form of Cdc2. These data indicate that p70(s6k) is a physiological substrate of Cdc2-cyclin B in mitosis. Since the activity of p70(s6k) is low during mitosis, Cdc2-cyclin B may play a role in inactivating p70(s6k) during mitosis, where protein synthesis is suppressed. PMID:9614117

Papst, P J; Sugiyama, H; Nagasawa, M; Lucas, J J; Maller, J L; Terada, N

1998-06-12

5

Involvement of Phosphatidylinositol 3Kinase in the Mediation of Erythropoietin-Induced Activation of p70 S6k  

Microsoft Academic Search

We have previously shown that, in HCD-57 cells, erythropoietin (EPO) induces a biphasic activation of the ribosomal S6 kinase p70S6k, an enzyme playing a key role in the regulation of cell cycle progression. Here we present evidence that p70S6k is activated through both phosphatidylinositol (PI) 3-kinase-dependent and -independent pathways: whereas the early phase of EPO-dependent stimulation of p70S6k activity was

Robert Jaster; Thomas Bittorf; Josef Brock

1997-01-01

6

Tuberin Regulates p70 S6 Kinase Activation and Ribosomal Protein S6 Phosphorylation  

Microsoft Academic Search

Although the cellular functions of TSC2 and its pro- tein product, tuberin, are not known, somatic mutations in the TSC2 tumor suppressor gene are associated with tumor development in lymphangioleiomyomatosis (LAM). We found that ribosomal protein S6 (S6), which exerts translational control of protein synthesis and is required for cell growth, is hyperphosphorylated in the smooth muscle-like cell lesions of

Elena A. Goncharova; Dmitry A. Goncharov; Andrew Eszterhas; Deborah S. Hunter; Marilyn K. Glassberg; Raymond S. Yeung; Cheryl L. Walker; Daniel Noonan; David J. Kwiatkowski; Margaret M. Chou; Reynold A. Panettieri; Vera P. Krymskaya

7

Vascular tumors have increased p70 S6-kinase activation and are inhibited by topical rapamycin.  

PubMed

Vascular tumors are endothelial cell neoplasms whose cellular and molecular mechanisms, leading to tumor formation, are poorly understood, and current therapies have limited efficacy with significant side effects. We have investigated mechanistic (mammalian) target of rapamycin (mTOR) signaling in benign and malignant vascular tumors, and the effects of mTOR kinase inhibitor as a potential therapy for these lesions. Human vascular tumors (infantile hemangioma and angiosarcoma) were analyzed by immunohistochemical stains and western blot for the phosphorylation of p70 S6-kinase (S6K) and S6 ribosomal protein (S6), which are activated downstream of mTOR complex-1 (mTORC1). To assess the function of S6K, tumor cells with genetic knockdown of S6K were analyzed for cell proliferation and migration. The effects of topical rapamycin, an mTOR inhibitor, on mTORC1 and mTOR complex-2 (mTORC2) activities, as well as on tumor growth and migration, were determined. Vascular tumors showed increased activation of S6K and S6. Genetic knockdown of S6K resulted in reduced tumor cell proliferation and migration. Rapamycin fully inhibited mTORC1 and partially inhibited mTORC2 activities, including the phosphorylation of Akt (serine 473) and PKC?, in vascular tumor cells. Rapamycin significantly reduced vascular tumor growth in vitro and in vivo. As a potential localized therapy for cutaneous vascular tumors, topically applied rapamycin effectively reduced tumor growth with limited systemic drug absorption. These findings reveal the importance of mTOR signaling pathways in benign and malignant vascular tumors. The mTOR pathway is an important therapeutic target in vascular tumors, and topical mTOR inhibitors may provide an alternative and well-tolerated therapy for the treatment of cutaneous vascular lesions. PMID:23938603

Du, Wa; Gerald, Damien; Perruzzi, Carole A; Rodriguez-Waitkus, Paul; Enayati, Ladan; Krishnan, Bhuvaneswari; Edmonds, Joseph; Hochman, Marcelo L; Lev, Dina C; Phung, Thuy L

2013-08-12

8

p70 S6 kinase limits tumor necrosis factor-alpha-induced interleukin-6 synthesis in osteoblast-like cells.  

PubMed

Our previous study demonstrated that tumor necrosis factor-alpha (TNF-alpha) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether p70 S6 kinase is involved in TNF-alpha-stimulated IL-6 synthesis in MC3T3-E1 cells. TNF-alpha time dependently induced the phosphorylation of p70 S6 kinase. Rapamycin, an inhibitor of p70 S6 kinase, which attenuated the phosphorylation of p70 S6 kinase induced by TNF-alpha, significantly amplified the TNF-alpha-stimulated IL-6 synthesis. TNF-alpha-induced phosphorylations of both p44/p42 MAP kinase and Akt were markedly enhanced by rapamycin. The amplification by rapamycin of TNF-alpha-induced IL-6 synthesis was reduced by PD98059, a specific inhibitor of MEK1/2, or Akt inhibitor. Rapamycin enhanced the IL-6 synthesis and the phosphorylation of Akt induced by TNF-alpha also in human osteoblasts. Taken together, these results strongly suggest that p70 S6 kinase limits the TNF-alpha-stimulated IL-6 synthesis at a point upstream from p44/p42 MAP kinase and Akt in osteoblast-like cells. PMID:19879324

Minamitani, Chiho; Tokuda, Haruhiko; Adachi, Seiji; Matsushima-Nishiwaki, Rie; Yamauchi, Junichi; Kato, Kenji; Natsume, Hideo; Mizutani, Jun; Kozawa, Osamu; Otsuka, Takanobu

2009-10-29

9

p70S6 Kinase Phosphorylates AMPK on Serine 491 to Mediate Leptin's Effect on Food Intake  

PubMed Central

SUMMARY The PI3K-AKT, mTOR-p70S6 kinase and AMPK pathways play distinct and critical roles in metabolic regulation. Each pathway is necessary for leptin's anorexigenic effects in the hypothalamus. Here we show that these pathways converge in an integrated phosphorylation cascade to mediate leptin action in the hypothalamus. We identify serine491 on ?2AMPK as the site of convergence and show that p70S6 kinase forms a complex with ?2AMPK, resulting in phosphorylation on serine491. Blocking ?2AMPK-serine491 phosphorylation increases hypothalamic AMPK activity, food intake, and body weight. Serine491 phosphorylation is necessary for leptin's effects on hypothalamic ?2AMPK activity, neuropeptide expression, food intake, and body weight. These results identify an inhibitory AMPK kinase, p70S6 kinase, and demonstrate that AMPK is a substrate for mTOR-p70S6 kinase. This discovery has broad biologic implications since mTOR-p70S6 kinase and AMPK have multiple, fundamental and generally opposing cellular effects that regulate metabolism, cell growth, and development.

Dagon, Yossi; Hur, Elizabeth; Zheng, Bin; Wellenstein, Kerry; Cantley, Lewis C.; Kahn, Barbara B.

2012-01-01

10

An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/? mice  

PubMed Central

PTEN phosphatase acts as a tumor suppressor by negatively regulating the phosphoinositide 3-kinase (PI3K) signaling pathway. It is unclear which downstream components of this pathway are necessary for oncogenic transformation. In this report we show that transformed cells of PTEN+/? mice have elevated levels of phosphorylated Akt and activated p70/S6 kinase associated with an increase in proliferation. Pharmacological inactivation of mTOR/RAFT/FRAP reduced neoplastic proliferation, tumor size, and p70/S6 kinase activity, but did not affect the status of Akt. These data suggest that p70/S6K and possibly other targets of mTOR contribute significantly to tumor development and that inhibition of these proteins may be therapeutic for cancer patients with deranged PI3K signaling.

Podsypanina, Katrina; Lee, Richard T.; Politis, Chris; Hennessy, Ian; Crane, Allison; Puc, Janusz; Neshat, Mehran; Wang, Hong; Yang, Lin; Gibbons, Jay; Frost, Phil; Dreisbach, Valley; Blenis, John; Gaciong, Zbigniew; Fisher, Peter; Sawyers, Charles; Hedrick-Ellenson, Lora; Parsons, Ramon

2001-01-01

11

A Heat-Sensitive Arabidopsis thaliana Kinase Substitutes for Human p70s6k Function In Vivo  

PubMed Central

In mammalian cells, mitogen-induced phosphorylation of ribosomal protein S6 by p70s6k has been implicated in the selective translational upregulation of 5?TOP mRNAs. We demonstrate here that the homologous Arabidopsis thaliana protein, AtS6k2, ectopically expressed in human 293 cells or isolated from plant cells, phosphorylates specifically mammalian and plant S6 at 25°C but not at 37°C. When Arabidopsis suspension culture cells are shifted from 25 to 37°C, the kinase becomes rapidly inactivated, consistent with the observation that heat shock abrogates S6 phosphorylation in plants. Treatment with potato acid phosphatase reduced the specific activity of immunoprecipitated AtS6k2 threefold, an effect which was blocked in the presence of 4-nitrophenyl phosphate. In quiescent mammalian cells, AtS6k2 is activated by serum stimulation, a response which is abolished by the fungal metabolite wortmannin but is resistant to rapamycin. Treatment of mammalian cells with rapamycin abolishes in vivo S6 phosphorylation by p70s6k; however, ectopic expression of AtS6k2 rescues the rapamycin block. Collectively, the data demonstrate that AtS6k2 is the functional plant homolog of mammalian p70s6k and identify a new signalling pathway in plants.

Turck, Franziska; Kozma, Sara C.; Thomas, George; Nagy, Ferenc

1998-01-01

12

Hydrogen peroxide mediates arsenite activation of p70 s6k and extracellular signal-regulated kinase  

Microsoft Academic Search

To define the mechanism of arsenite-induced tumor promotion, we examined the role of reactive oxygen species (ROS) in the signaling pathways of cells exposed to arsenite. Arsenite treatment resulted in the persistent activation of p70s6k and extracellular signal-regulated kinase 1\\/2 (ERK1\\/2) which was accompanied by an increase in intracellular ROS production. The predominant produced appeared to be H2O2, because the

Dong Keun Jung; Gyu-Un Bae; Yong Kee Kim; Seung-Hee Han; Wahn Soo Choi; Hyeog Kang; Dong Wan Seo; Hoi Young Lee; Eun-Jung Cho; Hyang-Woo Lee; Jeung-Whan Han

2003-01-01

13

Hydrophobic motif phosphorylation is not required for activation loop phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1).  

PubMed

p70 ribosomal protein S6 kinase 1 (S6K1) is regulated by multiple phosphorylation events. Three of these sites are highly conserved among AGC kinases (cAMP dependent Protein Kinase, cGMP dependent Protein Kinase, and Protein Kinase C subfamily): the activation loop in the kinase domain, and two C-terminal sites, the turn motif and the hydrophobic motif. The common dogma has been that phosphorylation of the hydrophobic motif primes S6K1 for the phosphorylation at the activation loop by phosphoinositide-dependent protein kinase 1 (PDK1). Here, we show that the turn motif is, in fact, phosphorylated first, the activation loop second, and the hydrophobic motif is third. Specifically, biochemical analyses of a construct of S6K1 lacking the C-terminal autoinhibitory domain as well as full-length S6K1, reveals that S6K1 is constitutively phosphorylated at the turn motif when expressed in insect cells and becomes phosphorylated in vitro by purified PDK1 at the activation loop. Only the species phosphorylated at the activation loop by PDK1 gets phosphorylated at the hydrophobic motif by mammalian target of rapamycin (mTOR) in vitro. These data are consistent with a previous model in which constitutive phosphorylation of the turn motif provides the key priming step in the phosphorylation of S6K1. The data provide evidence for regulation of S6K1, where hydrophobic motif phosphorylation is not required for PDK1 to phosphorylate S6K1 at the activation loop, but instead activation loop phosphorylation of S6K1 is required for mTOR to phosphorylate the hydrophobic motif of S6K1. PMID:21561857

Keshwani, Malik M; von Daake, Sventja; Newton, Alexandra C; Harris, Thomas K; Taylor, Susan S

2011-05-11

14

INTERLEUKIN 10 MODULATION OF TUMOUR NECROSIS FACTOR RECEPTORS REQUIRES TYROSINE KINASES BUT NOT THE PI 3KINASE\\/p70 S6 KINASE PATHWAY  

Microsoft Academic Search

We have previously shown that interleukin (IL-)10-induced proliferation of the murine mast cell line D36, was dependent upon the activation of PI 3-kinase and p70 S6 kinase. Conversely, we were able to show that this pathway was not involved in the signal transduction pathway mediating IL-10 inhibition of pro-inflammatory cytokine release from monocytes. We have extended these studies to investigate

Lynn Williams; Ferdinand Lali; Christopher Clarke; Fionula Brennan; Brian Foxwell

2000-01-01

15

Insulin stimulation of SREBP-1c processing in transgenic rat hepatocytes requires p70 S6-kinase  

PubMed Central

Insulin activates sterol regulatory element-binding protein-1c (SREBP-1c) in liver, thereby increasing fatty acid and triglyceride synthesis. We created a line of transgenic rats that produce epitope-tagged human SREBP-1c in liver under control of the constitutive apolipoprotein E promoter/enhancer. This system allows us to dissect the pathway by which insulin stimulates SREBP-1c processing without interference by the insulin-mediated increase in SREBP-1c mRNA. Rats are used because freshly isolated rat hepatocytes respond much more robustly to insulin than do mouse hepatocytes. The data reveal that insulin-mediated stimulation of SREBP-1c processing requires the mechanistic target of rapamycin complex 1 (mTORC1), which also is required for insulin-mediated SREBP-1c mRNA induction. However, in contrast to mRNA induction, insulin stimulation of SREBP-1c processing is blocked by an inhibitor of p70 S6-kinase. The data indicate that the pathways for insulin enhancement of SREBP-1c mRNA and proteolytic processing diverge after mTORC1. Stimulation of processing requires the mTORC1 target p70 S6-kinase, whereas induction of mRNA bypasses this enzyme. Insulin stimulation of both processes is blocked by glucagon. The transgenic rat system will be useful in further defining the molecular mechanism for insulin stimulation of lipid synthesis in liver in normal and diabetic states.

Owen, Joshua L.; Zhang, Yuanyuan; Bae, Soo-Han; Farooqi, Midhat S.; Liang, Guosheng; Hammer, Robert E.; Goldstein, Joseph L.; Brown, Michael S.

2012-01-01

16

Resveratrol Attenuates Doxorubicin-Induced Cardiomyocyte Death via Inhibition of p70 S6 Kinase 1-Mediated Autophagy  

PubMed Central

Resveratrol is a plant-derived polyphenol that can attenuate the cardiotoxic effects of doxorubicin (DOX), a powerful antibiotic widely used in cancer chemotherapy. However, the underlying protective mechanisms of resveratrol remain elusive. Here, we show that resveratrol inhibited DOX-induced autophagy and cardiomyocyte death, and autophagy suppression is an important mechanism that mediates the ability of resveratrol to protect against DOX cardiotoxicity. Indeed, resveratrol, 3-methyladenine (3-MA), and a short hairpin RNA directed against autophagy gene beclin 1 (shBCN1) each was able to attenuate DOX-induced autophagy and cardiomyocyte death, but resveratrol did not provide additional protection in the presence of 3-MA or shBCN1. In contrast, up-regulation of autophagy by beclin 1 overexpression not only exacerbated DOX cardiotoxicity but also abolished the protective effects of resveratrol. Intriguingly, p70 S6 kinase 1 (S6K1) was activated by DOX, which was prevented by resveratrol. Knocking down S6K1 with small interfering RNA diminished DOX-induced autophagy and cardiotoxicity, but resveratrol failed to exert an additive effect. In addition, S6K1 overexpression impaired the ability of resveratrol to antagonize DOX-induced autophagy and cardiomyocyte death. Taken together, our data indicate that the protective effect of resveratrol against DOX cardiotoxicity largely depends on its ability to suppress DOX-induced autophagy via the inhibition of S6K1.

Xu, Xianmin; Chen, Kai; Kobayashi, Satoru; Timm, Derek

2012-01-01

17

TAK1 regulates autophagic cell death by suppressing the phosphorylation of p70 S6 kinase 1.  

PubMed

There is growing interest in identifying regulators of autophagy. The molecular mechanism underlying transforming growth factor-? activated kinase 1 (TAK1)-induced autophagy is poorly understood. We found that TAK1 inhibits p70 S6 kinase1 (S6K1) phosphorylation by interfering interaction of raptor with S6K1, thus inducing autophagy. The factors that determine whether autophagy is cytoprotective or cytotoxic have not been fully elucidated. In Drosophila, TAK1 overexpression leads to an impaired eye phenotype despite inhibition of apoptosis, indicating that the phenotype was mainly due to autophagy. Also, TAK1 overexpression increases lactate dehydrogenase (LDH) level in mammalian cells. When treated with autophagy inhibitors, the level of TAK1-induced cytotoxicity or cell death was significantly attenuated, indicating that TAK1 induces cytotoxic autophagic cell death. This study provides the first in vitro and in vivo evidence of TAK1-induced autophagy and we believe that our findings significantly contribute to the understanding of the mechanisms underlying the induction of autophagy. PMID:23532117

Shin, Ju Hyun; Min, Sang-Hyun; Kim, Seong-Jin; Kim, Young-Il; Park, Junsoo; Lee, Heung Kyu; Yoo, Ook Joon

2013-01-01

18

Requirement for phosphoinositide 3-OH kinase in growth hormone signalling to the mitogen-activated protein kinase and p70s6k pathways.  

PubMed Central

Pituitary growth hormone (GH) co-ordinately stimulates three distinct signalling pathways in 3T3-F442A preadipocytes, the STAT (signal transducer and activator of transcription) pathway, the mitogen-activated protein (MAP) kinase cascade and p70s6k. The mechanisms linking the GH receptor to these signals have not been fully identified. In this study we have examined the role of phosphoinositide 3-OH kinase (PI 3-kinase). Pretreatment of cells with wortmannin, a specific inhibitor of PI 3-kinase, prevented the activation of p70s6k and partially inhibited the activation of p42 and p44 MAP kinases by GH. In contrast, wortmannin failed to appreciably affect the GH-stimulated tyrosyl phosphorylation of JAK-2 or STAT-1. GH transiently increased the activity of PI 3-kinase recovered in antiphosphotyrosine immunoprecipitates. In addition, several tyrosyl-phosphorylated proteins were specifically adsorbed from lysates of cells exposed to GH by a glutathione S-transferase fusion protein containing the 85 kDa regulatory subunit of PI 3-kinase. GH also induced an increase in the PI 3-kinase activity associated with both JAK-2 and insulin receptor substrate-1 (IRS-1) immunoprecipitates. These results establish PI 3-kinase as an important mediator of GH signalling to the MAP kinase and p70s6k pathways and suggest that PI 3-kinase is activated by a mechanism involving JAK-2 and IRS-1.

Kilgour, E; Gout, I; Anderson, N G

1996-01-01

19

A Heat-Sensitive Arabidopsis thaliana Kinase Substitutes for Human p70 s6k Function In Vivo  

Microsoft Academic Search

Received 13 October 1997\\/Returned for modification 10 December 1997\\/Accepted 7 January 1998 In mammalian cells, mitogen-induced phosphorylation of ribosomal protein S6 by p70 s6k has been impli- cated in the selective translational upregulation of 5*TOP mRNAs. We demonstrate here that the homologous Arabidopsis thaliana protein, AtS6k2, ectopically expressed in human 293 cells or isolated from plant cells, phosphorylates specifically mammalian

FRANZISKA TURCK; SARA C. KOZMA; GEORGE THOMAS; FERENC NAGY

1998-01-01

20

Insulin activates a PD 098059-sensitive kinase that is involved in the regulation of p70 S6K and PHAS-I  

Microsoft Academic Search

Incubating either Chinese hamster ovary (CHO) cells or 3T3-L1 adipocytes with insulin increased the phosphorylation of the eIF-4E-binding protein, PHAS-I. Insulin also activated p70S6K and the Erk-1 and Erk-2 isoforms of mitogen-activated protein kinase (MAP kinase). However, the concentrations of the hormone needed to activate MAP kinase were 10–100 times higher than those needed to increase PHAS-I phosphorylation and p70S6K

Pamela H Scott; John C Lawrence

1997-01-01

21

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

Microsoft Academic Search

Mechanical stretch induces phosphorylation of the hydrophobic motif site Thr(389) in p70S6k 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

Troy Alan Hornberger; Kunal Balu Sukhija; Xiao-Rong Wang; Shu Chien

2007-01-01

22

Insulin-induced phosphorylation and activation of phosphodiesterase 3B in rat adipocytes: possible role for protein kinase B but not mitogen-activated protein kinase or p70 S6 kinase.  

PubMed

Insulin stimulation of adipocytes results in serine phosphorylation/activation of phosphodiesterase 3B (PDE 3B) and activation of a kinase that phosphorylates PDE 3B in vitro, key events in the antilipolytic action of this hormone. We have investigated the role for p70 S6 kinase, mitogen-activated protein kinases (MAP kinases), and protein kinase B (PKB) in the insulin signaling pathway leading to phosphorylation/activation of PDE 3B in adipocytes. Insulin stimulation of adipocytes resulted in increased activity of p70 S6 kinase, which was completely blocked by pretreatment with rapamycin. However, rapamycin had no effect on the insulin-induced phosphorylation/activation of PDE 3B or the activation of the kinase that phosphorylates PDE 3B. Stimulation of adipocytes with insulin or phorbol myristate acetate induced activation of MAP kinases. Pretreatment of adipocytes with the MAP kinase kinase inhibitor PD 98059 was without effect on the insulin-induced activation of PDE 3B. Furthermore, phorbol myristate acetate stimulation did not result in phosphorylation/activation of PDE 3B or activation of the kinase that phosphorylates PDE 3B. Using Mono Q and Superdex chromatography, the kinase that phosphorylates PDE 3B was found to co-elute with PKB, but not with p70 S6 kinase or MAP kinases. Furthermore, both PKB and the kinase that phosphorylates PDE 3B were found to translocate to membranes in response to peroxovanadate stimulation of adipocytes in a wortmannin-sensitive way. Whereas these results suggest that p70 S6 kinase and MAP kinases are not involved in the insulin-induced phosphorylation/activation of PDE 3B in rat adipocytes, they are consistent with PKB being the kinase that phosphorylates PDE 3B. PMID:9421418

Wijkander, J; Landström, T R; Manganiello, V; Belfrage, P; Degerman, E

1998-01-01

23

Stem cell factor/c-kit up-regulates cyclin D3 and promotes cell cycle progression via the phosphoinositide 3-kinase/p70 S6 kinase pathway in spermatogonia.  

PubMed

Stem cell factor (SCF)/c-kit plays an important role in the regulation of hematopoiesis, melanogenesis, and spermatogenesis. In the testis, the SCF/c-kit system is believed to regulate germ cell proliferation, meiosis, and apoptosis. Studies with type A spermatogonia in vivo and in vitro have indicated that SCF induces DNA synthesis and proliferation. However, the signaling pathway for this function of SCF/c-kit has not been elucidated. We now demonstrate that SCF activates phosphoinositide 3-kinase (PI3-K) and p70 S6 kinase (p70S6K) and that rapamycin, a FRAP/mammalian target of rapamycin-dependent inhibitor of p70S6K, completely inhibited bromodeoxyuridine incorporation induced by SCF in primary cultures of spermatogonia. SCF induced cyclin D3 expression and phosphorylation of the retinoblastoma protein through a pathway that is sensitive to both wortmannin and rapamycin. Furthermore, AKT, but not protein kinase C-zeta, is used by SCF/c-kit/PI3-K to activate p70S6K. Dominant negative AKT-K179M completely abolished p70S6K phosphorylation induced by the constitutively active PI3-K catalytic subunit p110. Constitutively active v-AKT highly phosphorylated p70S6K, which was totally inhibited by rapamycin. Thus, SCF/c-kit uses a rapamycin-sensitive PI3-K/AKT/p70S6K/cyclin D3 pathway to promote spermatogonial cell proliferation. PMID:10849422

Feng, L X; Ravindranath, N; Dym, M

2000-08-18

24

NECA at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase  

Microsoft Academic Search

The A1\\/A2 adenosine agonist 5?-(N-ethylcarboxamido) adenosine (NECA) limits infarction when administered at reperfusion. The present study investigated whether\\u000a p70S6 kinase is involved in this anti-infarct effect. Adult rat ventricular myocytes were isolated and incubated in tetramethylrhodamine\\u000a ethyl ester (TMRE, 100 nM), which causes cells to fluoresce in proportion to their mitochondrial membrane potential. A reduction\\u000a in TMRE fluorescence serves as

Karina Förster; Ina Paul; Natalia Solenkova; Alexander Staudt; Michael V. Cohen; James M. Downey; Stephan B. Felix; Thomas Krieg

2006-01-01

25

Rosiglitazone inhibits insulin-like growth factor?1-induced polycystic kidney disease cell growth and p70S6 kinase activation.  

PubMed

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic kidney disorders. Thiazolidinediones (TZDs) are anti-diabetic drugs that have been shown to suppress polycystic kidney diseases (PKD) development. However, their underlying mechanism of action remains largely unknown. Insulin-like growth factor?1 (IGF-1) expression increases with the progression of cystic lesions in ADPKD and murine PKD, thus the increased expression of IGF-1 may contribute to the progression of cystic lesions. p70S6 kinase (p70S6K) is an important downstream signaling molecule of IGF-1 and is implicated in the regulation of cell cycle progression and cell proliferation. In the present study, we found that IGF-1 increased the growth of cyst?lining epithelial cells by 15-20% in a dose-dependent manner, while no effect on the proliferation of normal renal cortical tubular epithelial cells (RCTEC) was observed. Rosiglitazone, a TZD, was found to inhibit the IGF-1?induced growth of cyst?lining epithelial cells when applied at a dose of 50-200 µM. However, the IGF-1?induced growth of immortalized epithelial cells from >30 individual renal cysts obtained from 11 ADPKD patients (WT9-12 cells) was inhibited with a 12.5-µM dose of rosiglitazone. Moreover, rosiglitazone (at the same concentration) was shown to inhibit the IGF-1-induced activation of p70S6K. TZDs are known to exert antitumor properties via peroxisome proliferator?activated receptor (PPAR)?-dependent and -independent mechanisms. The present study showed that PPAR? small interfering RNA (siRNA) did not block the effect of rosiglitazone in inhibiting the IGF-1-induced phosphorylation of p70S6K. In conclusion, cyst?lining epithelial cells were found to be more sensitive to IGF-1 compared with normal cells. Rosiglitazone inhibited the proliferation of cyst?lining epithelial cells; more specifically, it inhibited the proliferation-promoting activity of IGF-1 in these cells. This effect of rosiglitazone was demonstrated to be partially due to the inhibition of IGF-1-induced activation of p70S6K. Increased IGF-1 expression was identified in early?stage PKD, indicating that rosiglitazone is more suitable for the treatment of early?stage PKD. PMID:23864113

Liu, Chunyan; Zhang, Yi; Yuan, Li; Fu, Lili; Mei, Changlin

2013-07-16

26

Activation of S6K1 (p70 ribosomal protein S6 kinase 1) requires an initial calcium-dependent priming event involving formation of a high-molecular-mass signalling complex.  

PubMed Central

The mitogen-stimulated protein kinase p70 ribosomal protein S6 kinase 1 (S6K1) is a key enzyme in the regulation of cell growth and proliferation. Activation of S6K1 requires a complex, ordered series of conformational changes and phosphorylation reactions. While the role of sequential, multi-site phosphorylation has been extensively detailed, characterization of the priming step required to initiate this cascade has remained elusive. In the present study we show for the first time that this priming process is dependent on calcium. Calcium-dependent regulation of S6K1 did not specifically target Thr-229 and Thr-389, the key regulatory phosphorylation sites; rather, calcium chelation resulted in a global inhibition of S6K1 phosphorylation. Mutation of individual phosphorylation sites in the auto-inhibitory and hydrophobic domains to acidic residues (to mimic phosphorylation) yields a kinase that remains sensitive to calcium chelation, while the combined mutations alleviate the requirement for calcium. Furthermore, deletion of the C-terminal residues (398-502) also renders the kinase insensitive to calcium. We hypothesize that the initial calcium-dependent process is required to release an inhibitory interaction between the C- and N-termini of S6K1, thus allowing phosphorylation of these key domains. The requirement for this priming step can only be overcome by mutations mimicking the phosphorylation of both the auto-inhibitory and hydrophobic domains. We further propose that the priming event involves formation of a calcium-dependent protein complex that releases the interaction between the N- and C-termini. S6K1 is then accessible for activation by the kinases that target the known regulatory phosphorylation sites. Consistent with this hypothesis, serum stimulation of S6K1 activity is associated with its incorporation into a calcium-dependent high-molecular-mass complex.

Hannan, Katherine M; Thomas, George; Pearson, Richard B

2003-01-01

27

Alterations of Akt1 (PKB?) and p70 S6K in Transient Focal Ischemia  

Microsoft Academic Search

The serine-threonine kinase Akt1 promotes cell survival through inhibition of apoptosis. One of the potential downstream targets of Akt1 is p70 S6 kinase, p70S6K, an enzyme implicated in the regulation of protein synthesis. In this study, we investigated the changes in total and phosphorylated levels of Akt1 and p70S6K during transient focal ischemia. Male Wistar rats were subjected to 2

Shorena Janelidze; Bing-Ren Hu; Peter Siesjö; Bo K. Siesjö

2001-01-01

28

Autocrine Phosphorylation of p70 S6k in Response to Acute Stretch in Myotubes  

Microsoft Academic Search

Phosphorylation of 70-KDa S6 kinase (p70S6k) is correlated with in vivo skeletal muscle hypertrophy. Experiments tested whether mechanical stretch is sufficient to increase p70S6k phosphorylation in skeletal myotubes. Immediately following stretch, there was a small increase in p70S6k phosphorylation (63.2 ± 8.5%) with maximal phosphorylation at 3 h (129.5 ± 22.2%) and it remained elevated through 24 h (46.0 ±

Keith Baar; Carol E. Torgan; William E. Kraus; Karyn Esser

2000-01-01

29

Insulin stimulation of glycogen synthesis and glycogen synthase activity is blocked by wortmannin and rapamycin in 3T3-L1 adipocytes: evidence for the involvement of phosphoinositide 3-kinase and p70 ribosomal protein-S6 kinase.  

PubMed Central

We have investigated the involvement of phosphoinositide (PI) 3-kinase and p70 ribosomal protein-S6 kinase (p70s6k) in mediating insulin stimulation of glycogen synthesis in 3T3-L1 adipocytes using specific inhibitors. Wortmannin inhibited PI 3-kinase activity (IC50 approximately 10 nM), inhibition being complete at 100 nm. Wortmannin (100 nM) completely blocked the ability of insulin to activate glycogen synthase in 3T3-L1 adipocytes and the ability of insulin to stimulate glucose incorporation into glycogen in 3T3-L1 fibroblasts. Rapamycin, which blocks insulin-stimulated activation of p70s6k, decreased insulin activation of glycogen synthase in a dose-dependent manner (IC50 approximately 0.8 ng/ml), with a maximum approx. 75% inhibition of insulin's stimulatory effect. Rapamycin inhibited insulin-stimulated glucose incorporation into glycogen to a similar extent and with similar dose-dependency, while having no effect on insulin-stimulated glucose transport. We conclude that PI 3-kinase and p70s6k are involved in the signalling pathways by which insulin stimulates glycogen synthase in 3T3-L1 adipocytes.

Shepherd, P R; Nave, B T; Siddle, K

1995-01-01

30

TBC1D3, a Hominoid-Specific Gene, Delays IRS-1 Degradation and Promotes Insulin Signaling by Modulating p70 S6 Kinase Activity  

PubMed Central

Insulin/IGF-1 signaling plays a pivotal role in the regulation of cellular homeostasis through its control of glucose metabolism as well as due to its effects on cell proliferation. Aberrant regulation of insulin signaling has been repeatedly implicated in uncontrolled cell growth and malignant transformations. TBC1D3 is a hominoid specific gene previously identified as an oncogene in breast and prostate cancers. Our efforts to identify the molecular mechanisms of TBC1D3-induced oncogenesis revealed the role of TBC1D3 in insulin/IGF-1 signaling pathway. We document here that TBC1D3 intensifies insulin/IGF-1-induced signal transduction through intricate, yet elegant fine-tuning of signaling mechanisms. We show that TBC1D3 expression substantially delayed ubiquitination and degradation of insulin receptor substrate-1 (IRS-1). This effect is achieved through suppression of serine phosphorylation at S636/639, S307 and S312 of IRS-1, which are key phosphorylation sites required for IRS-1 degradation. Furthermore, we report that the effect of TBC1D3 on IRS-1:S636/639 phosphorylation is mediated through TBC1D3-induced activation of protein phosphatase 2A (PP2A), followed by suppression of T389 phosphorylation on p70 S6 kinase (S6K). TBC1D3 specifically interacts with PP2A regulatory subunit B56?, indicating that TBC1D3 and PP2A B56? operate jointly to promote S6K:T389 dephosphorylation. These findings suggest that TBC1D3 plays an unanticipated and potentially unique role in the fine-tuning of insulin/IGF-1 signaling, while providing novel insights into the regulation of tumorigenesis by a hominoid-specific protein.

Kong, Chen; Srikanth, Priya; Samovski, Dmitri; Su, Xiong; Stahl, Philip D.

2012-01-01

31

Ro 31-6045, the inactive analogue of the protein kinase C inhibitor Ro 31-8220, blocks in vivo activation of p70 s6k\\/p85 s6k: implications for the analysis of S6K signalling  

Microsoft Academic Search

The mitogen-stimulated protein kinase p70s6k\\/p85s6k (S6K) plays an essential role in cell proliferation and growth, with inhibitors of the S6K signalling pathway showing promise as anti-tumour therapeutics. Here, we report that the bisindolylmaleimide derivative Ro 31-6045, previously reported to be inactive as a kinase inhibitor, inhibited S6K activity in vivo with an IC50=8 ?M. Structure\\/function analysis using mutant forms of

Nelly Marmy-Conus; Katherine M Hannan; Richard B Pearson

2002-01-01

32

HGF and KGF-induced Activation of PI3K\\/p70 S6 Kinase Pathway in Corneal Epithelial Cells: its Relevance in Wound Healing  

Microsoft Academic Search

In this study we have investigated the involvement of PI-3K and its downstream target p70 S6K in the signaling response of corneal epithelial cells after HGF and KGF stimulation. HGF induced three- to five-fold increase in PI-3K activity in 5–10min, whereas KGF stimulation resulted in two- to three-fold increase in activity in 2–10min. Both growth factors also caused the phosphorylation

Gudiseva Chandrasekher; Azucena H Kakazu; Haydee E. P Bazan

2001-01-01

33

Serotonin-Induced Growth of Pulmonary Artery Smooth Muscle Requires Activation of Phosphatidylinositol 3-Kinase/Serine-Threonine Protein Kinase B/Mammalian Target of Rapamycin/p70 Ribosomal S6 Kinase 1  

PubMed Central

We have previously found that both mitogen-activated protein kinase (MAPK)- and Rho kinase (ROCK)-related signaling pathways are necessary for the induction of pulmonary artery smooth muscle cell (SMC) proliferation by serotonin (5-hydroxytryptamine [5-HT]). In the present study, we investigated the possible additional participation of a phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (S6K1) pathway in this growth response. We found transient activation of Akt (Ser473) and more prolonged activation of S6K1 by 5-HT. Inhibition of PI3K with Wortmannin and LY294002 completely blocked these activations, but not that of MAPK or the ROCK substrate myosin phosphatase targeting subunit. Similarly, inhibition of MAPK and ROCK failed to block the Akt activation. Inhibition of Akt with NL-71–101 and downregulation of Akt expression with Akt small interfering RNA blocked 5-HT–induced S6K1 phosphorylation. Wortmannin, LY294002, and NL-71–101 dose-dependently inhibited 5-HT–induced SMC proliferation. 5-HT stimulated mTOR phosphorylation and the mTOR inhibitor, rapamycin, blocked activations of S6K1 and S6 ribosomal protein, and inhibited 5-HT–induced SMC proliferation. Akt phosphorylation and cell proliferation were also blocked by the antioxidants, N-acetyl-l-cysteine, Ginko biloba 501, and tiron, the reduced nicotinamide adenine dinucleotide phosphate oxidase inhibitor, diphenyleneiodonium, and the 5-HT2 receptor antagonists ketanserin and mianserin, but not by the 5-HT serotonin transporter or 5-HT 1B/1D receptor antagonists. We conclude from these studies that a parallel PI3K- and reactive oxygen species–dependent Akt/mTOR/S6K1 pathway participates independently from MAPK and Rho/ROCK in the mitogenic effect of 5-HT on pulmonary artery SMCs. From these and other studies, we postulate that independent signaling pathways leading to 5-HT–induced SMC proliferation are initiated through multiple 5-HT receptors and serotonin transporter at the cell surface.

Liu, Yinglin; Fanburg, Barry L.

2006-01-01

34

Role of the p70 S6K pathway in regulating the actin cytoskeleton and cell migration  

Microsoft Academic Search

We have examined the role of endogenous 70-kDa S6 kinase (p70S6K) in actin cytoskeletal organization and cell migration in Swiss 3T3 fibroblasts. Association of p70S6K with the actin cytoskeleton was demonstrated by cosedimentation of p70S6K with F-actin and by subcellular fractionation in which p70S6K activity was measured in the F-actin cytoskeletal fraction. Immunocytochemical studies showed that p70S6K, Akt1, PDK1, and

Leise A Berven; Francis S Willard; Michael F Crouch

2004-01-01

35

Activation of a PKC is required for vanadate-induced phosphorylation of protein kinase B (Akt), but not p70 S6k in mouse epidermal JB6 cells  

Microsoft Academic Search

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms by which vanadate mediates adverse effects are not well understood. The present study investigated the vanadate-induced phosphorylation of Akt and p70S6K, two kinases known to be vital for cell survival, growth, transformation, and transition of

Jingxia Li; Sujatha Dokka; Liying Wang; Xianglin Shi; Vincent Castranova; Yan Yan; Max Costa; Chuanshu Huang

2004-01-01

36

Increased activity of MAP, p70S6 and p90rs kinases is associated with AP-1 activation in spontaneous liver tumours, but not in adjacent tissue in mice  

PubMed Central

Growth factor-responsive protein kinases regulate expression of genes involved in cell cycle control, cell proliferation and differentiation. To better understand the role of these kinases in the abnormal proliferation of malignant cells, we examined basal and epidermal growth factor (EGF)-inducible mitogen-activated protein kinase (MAPK), p70S6k and p90rsk activities in spontaneous hepatocellular neoplasms (adenomas and carcinomas) from CBA-T6 mice and in L1 sarcoma tumours implanted in livers of BALB/c mice. In spontaneous and implanted hepatic tumours, basal cytoplasmic and nuclear MAPK, p70S6k and p90rsk activities were significantly higher compared to the activities found in the part of the liver uninvolved by the tumour. Interestingly, the activities of these enzymes in the uninvolved tissue of the livers harbouring the tumour were higher compared to the livers from control mice. Basal kinase activities correlated with tumour morphology; they were lower in adenomas than in carcinomas and sarcomas. In contrast to basal activities, EGF-triggered kinase responses in normal livers and hepatic tumours were indistinguishable. Activating protein-1 (AP-1) DNA-binding activity was detected in tumours but not in the adjacent tissues. Constitutively activated kinases and AP-1 transcription factor found in hepatic malignancies are reminiscent of cells activated by EGF, suggesting that EGF and its intracellular effectors play a role in these malignancies. © 2000 Cancer Research Campaign

Ostrowski, J; Woszczynski, M; Kowalczyk, P; Wocial, T; Hennig, E; Trzeciak, L; Janik, P; Bomsztyk, K

2000-01-01

37

Genistein promotes insulin action through adenosine monophosphate-activated protein kinase activation and p70 ribosomal protein S6 kinase 1 inhibition in the skeletal muscle of mice fed a high energy diet.  

PubMed

Genistein (GEN), a soy isoflavone, exerts insulin-sensitizing actions in animals; however, the underlying mechanisms have not been determined. Because GEN is a known activator of adenosine monophosphate-activated protein kinase (AMPK), we hypothesize that GEN activates insulin signaling through AMPK activation. To test this hypothesis, a high fat-high fructose diet (HFFD)-fed mice model of insulin resistance was administered GEN, and the insulin signaling pathway proteins in the skeletal muscle were examined. Hyperglycemia and hyperinsulinemia observed in HFFD-fed mice were significantly lowered by GEN. GEN increased insulin-stimulated tyrosine phosphorylation of insulin receptor-? and insulin receptor substrate (IRS) 1 but down-regulated IRS-1 serine phosphorylation in the skeletal muscle of HFFD-fed mice. Furthermore, GEN treatment improved muscle IRS-1-associated phospatidylinositol-3 kinase expression, phosphorylation of Akt at Ser(473), and translocation of glucose transporter subtype 4. Phosphorylation of AMPK at Thr(172) and acetyl coenzyme A carboxylase (ACC) at Ser(79) was augmented, whereas phosphorylation of p70 ribosomal protein S6 kinase 1 at Thr(389) was significantly decreased after GEN treatment in the skeletal muscle of HFFD-fed mice. These results suggest that GEN might improve insulin action in the skeletal muscle by targeting AMPK. PMID:22935345

Arunkumar, Elumalai; Anuradha, Carani Venkatraman

2012-07-25

38

Regulation of ERK\\/JNK\\/p70 S6K in two rat models of liver injury and fibrosis  

Microsoft Academic Search

Background\\/Aims: The regulation of three major intracellular signalling protein kinases was investigated in two models of liver injury leading to hepatic fibrosis, dimethylnitrosamine administration (DMN) and bile duct ligation (BDL).Methods: Extracellular signal-regulated kinases (ERK)1\\/2, c-Jun terminal kinase (JNK) and p70S6-kinase (p70S6K) were studied in vivo in the whole liver, in liver sections and in isolated hepatocytes, cholangiocytes and hepatic stellate

Gianluca Svegliati-Baroni; Francesco Ridolfi; Zaira Caradonna; Domenico Alvaro; Marco Marzioni; Stefania Saccomanno; Cinzia Candelaresi; Luciano Trozzi; Giampiero Macarri; Antonio Benedetti; Franco Folli

2003-01-01

39

Constitutive activation of p70 S6k in cancer cells  

Microsoft Academic Search

The mitogen-stimulated serine\\/threonine kinase p70S6k plays an important role in the progression of cells from Go\\/G1, to S phase of the cell cycle by translational up-regulation of a family of mRNA transcripts family of mRNA transcripts which\\u000a contain polypyrimidine tract at their 5 transcriptional start site. Here, we report that p70S6k was constitutively phosphorylated and activated to various degrees in

Hyoung-Keun Kwon; Gyu-Un Bae; Jong-Woo Yoon; Yong Kee Kim; Hoi-Young Lee; Hyang-Woo Lee; Jeung-Whan Han

2002-01-01

40

Phospholipase D2-derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR  

Microsoft Academic Search

The product of phospholipase D (PLD) enzymatic action in cell membranes, phosphatidic acid (PA), regulates kinases implicated in NADPH oxidase activation, as well as the mammalian target of rapamy- cin (mTOR) kinase. However, other protein targets for this lipid second messenger must exist in order to explain other key PA-mediated cellular functions. In this study, PA was found to specifically

Nicholas Lehman; Bill Ledford; Mauricio Di Fulvio; Kathleen Frondorf; Linda C. McPhail; Julian Gomez-Cambronero

2007-01-01

41

Differential regulation of eEF2 and p70S6K by AMPKalpha2 in heart.  

PubMed

Eukaryotic elongation factor 2 (eEF-2) and mammalian target of rapamycin (mTOR)-p70 ribosomal protein S6 kinase (p70S6K) signaling pathways control protein synthesis and are inhibited during myocardial ischemia. Intracellular acidosis and AMP-activated protein kinase (AMPK) activation, both occurring during ischemia, have been proposed to participate in this inhibition. We evaluated the contribution of AMPK?2, the main cardiac AMPK catalytic subunit isoform, in eEF2 and mTOR-p70S6K regulation using AMPK?2 KO mice. Hearts were perfused ex vivo with or without insulin, and then submitted or not to ischemia. Insulin pre-incubation was necessary to activate mTOR-p70S6K and evaluate their subsequent inhibition by ischemia. Ischemia decreased insulin-induced mTOR-p70S6K phosphorylation in WT and AMPK?2 KO mice to a similar extent. This AMPK?2-independent p70S6K inhibition correlated well with the inhibition of PKB/Akt, located upstream of mTOR-p70S6K and can be mimicked in cardiomyocytes by decreasing pH. By contrast, ischemia-induced inhibitory phosphorylation of eEF-2 was drastically reduced in AMPK?2 KO mice. Interestingly, AMPK?2 also played a role under normoxia. Its deletion increased the insulin-induced p70S6K stimulation. This p70S6K over-stimulation was associated with a decrease in inhibitory phosphorylation of Raptor, an mTOR partner identified as an AMPK target. In conclusion, AMPK?2 controls cardiac p70S6K under normoxia and regulates eEF-2 but not the mTOR-p70S6K pathway during ischemia. This challenges the accepted notion that mTOR-p70S6K is inhibited by myocardial ischemia mainly via an AMPK-dependent mechanism. PMID:23466593

Demeulder, Bénédicte; Zarrinpashneh, Elham; Ginion, Audrey; Viollet, Benoit; Hue, Louis; Rider, Mark H; Vanoverschelde, Jean-Louis; Beauloye, Christophe; Horman, Sandrine; Bertrand, Luc

2013-03-04

42

A hexane fraction of guava Leaves (Psidium guajava L.) induces anticancer activity by suppressing AKT/mammalian target of rapamycin/ribosomal p70 S6 kinase in human prostate cancer cells.  

PubMed

This study was carried out to evaluate the anticancer effects of guava leaf extracts and its fractions. The chemical compositions of the active extracts were also determined. In the present study, we set out to determine whether the anticancer effects of guava leaves are linked with their ability to suppress constitutive AKT/mammalian target of rapamycin (mTOR)/ribosomal p70 S6 kinase (S6K1) and mitogen-activated protein kinase (MAPK) activation pathways in human prostate cancer cells. We found that guava leaf hexane fraction (GHF) was the most potent inducer of cytotoxic and apoptotic effects in PC-3 cells. The molecular mechanism or mechanisms of GHF apoptotic potential were correlated with the suppression of AKT/mTOR/S6K1 and MAPK signaling pathways. This effect of GHF correlated with down-regulation of various proteins that mediate cell proliferation, cell survival, metastasis, and angiogenesis. Analysis of GHF by gas chromatography and gas chromatography-mass spectrometry tentatively identified 60 compounds, including ?-eudesmol (11.98%), ?-copaene (7.97%), phytol (7.95%), ?-patchoulene (3.76%), ?-caryophyllene oxide (CPO) (3.63%), caryophylla-3(15),7(14)-dien-6-ol (2.68%), (E)-methyl isoeugenol (1.90%), ?-terpineol (1.76%), and octadecane (1.23%). Besides GHF, CPO, but not phytol, also inhibited the AKT/mTOR/S6K1 signaling pathway and induced apoptosis in prostate cancer cells. Overall, these findings suggest that guava leaves can interfere with multiple signaling cascades linked with tumorigenesis and provide a source of potential therapeutic compounds for both the prevention and treatment of cancer. PMID:22280146

Ryu, Nae Hyung; Park, Kyung-Ran; Kim, Sung-Moo; Yun, Hyung-Mun; Nam, Dongwoo; Lee, Seok-Geun; Jang, Hyeung-Jin; Ahn, Kyoo Seok; Kim, Sung-Hoon; Shim, Bum Sang; Choi, Seung-Hoon; Mosaddik, Ashik; Cho, Somi K; Ahn, Kwang Seok

2012-01-26

43

The modular phosphorylation and activation of p70 s6k  

Microsoft Academic Search

The activation of p70s6k is accompanied by a complex series of phosphorylation events. In this review we propose a model of activation which divides p70s6k into four functional modules that cooperate in leading to full enzyme activity. In the light of the model, we suggest how candidate effectors of p70s6k activation might function by directing the phosphorylation of specific sites.

Nicholas Pullen; George Thomas

1997-01-01

44

Rapamycin inhibits constitutive p70s6k phosphorylation, cell proliferation, and colony formation in small cell lung cancer cells.  

PubMed

The serine/threonine kinase p70s6k was found to be constitutively phosphorylated in H 69, H 345, and H 510 small cell lung cancer cells as judged by the retarded electrophoretic mobility of both isoforms of this kinase. Pretreatment of H 69, H 345, and H 510 cells with the potent immunosuppressant rapamycin led to p70s6k dephosphorylation in a concentration-dependent manner; half-maximum and maximum effects were achieved at 0.3 and 3 nM rapamycin, respectively. Rapamycin inhibited growth of H 69, H 345, and H 510 cells in liquid culture at similar concentrations to those required for inducing dephosphorylation of p70s6k. Furthermore, rapamycin markedly reduced the basal colony forming ability of H 69, H 345, and H 510 cells in semisolid media. Thus, constitutively phosphorylated/active p70s6k plays an important role in promoting the growth of small cell lung cancer cells. Furthermore, the rapamycin-sensitive p70s6k pathway may provide a novel target for therapeutic intervention in small cell lung cancer. PMID:8752154

Seufferlein, T; Rozengurt, E

1996-09-01

45

Mechanical stimuli of skeletal muscle: implications on mTOR\\/p70s6k and protein synthesis  

Microsoft Academic Search

The skeletal muscle is a tissue with adaptive properties which are essential to the survival of many species. When mechanically\\u000a stimulated it is liable to undergo remodeling, namely, changes in its mass\\/volume resulting mainly from myofibrillar protein\\u000a accumulation. The mTOR pathway (mammalian target of rapamycin) via its effector p70s6k (ribosomal protein kinase S6) has been\\u000a reported to be of importance

Nelo Eidy Zanchi; Antonio Herbert Lancha

2008-01-01

46

Activation of p70s6k is Associated with Phosphorylation of Four Clustered Sites Displaying Ser\\/Thr-Pro Motifs  

Microsoft Academic Search

Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70s6k (M_r 70,000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were associated with p70s6k activation, the kinase was labeled to high specific activity with 32P_i in Swiss mouse 3T3 cells. By sequential cleavage with CNBr

Stefano Ferrari; Willi Bannwarth; Simon J. Morley; Nicholas F. Totty; George Thomas

1992-01-01

47

The mTOR\\/p70 S6K Signal Transduction Pathway Plays a Role in Cardiac Hypertrophy and Influences Expression of Myosin Heavy Chain Genes in vivo  

Microsoft Academic Search

Objective: Rapamycin (R) inhibits p70 S6 kinase (p70S6K) activity and hypertrophy of cultured neonatal rat cardiac myocytes. The purpose of the present study was to determine whether rapamycin inhibits left ventricular (L V) hypertrophy in intact rats and whether it alters cardiac gene expression.

Marvin O. Boluyt; Zhao Bo Li; Amy M. Loyd; Antony F. Scalia; Georgina M. Cirrincione; Rebecca R. Jackson

2004-01-01

48

Inhibition of PI3K\\/p70 S6K and p38 MAPK cascades increases osteoblastic differentiation induced by BMP2  

Microsoft Academic Search

Bone morphogenetic proteins (BMPs) transdifferentiate C2C12 cells from the myogenic to the osteogenic lineage. In this work we examine the role of the phosphatidylinositol 3-kinase\\/p70 S6 kinase (PI3K\\/p70 S6K) and p38 mitogen-activated protein kinase (p38 MAPK) cascades in the osteogenic effects of BMP-2. BMP-2 stimulated both cascades transiently (maximal at 1 h and decreasing thereafter). In contrast, BMP-2 had no

Francesc Viñals; Teresa López-Rovira; Jose Luis Rosa; Francesc Ventura

2002-01-01

49

Homocysteine thiolactone inhibits insulin-stimulated DNA and protein synthesis: possible role of mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3) and p70 S6K phosphorylation  

Microsoft Academic Search

Hyperhomocysteinemia and insulin resistance are independent factors for cardiovascular disease. Most of the angiotoxic effects of homocysteine are related to the formation of homocysteine thiolactone and the consequent increase in oxidative stress. We have recently found that homocysteine thiolactone inhibits insulin receptor tyrosine kinase activity, which results in decreased phosphatidylinositol 3-kinase (PI3K) activity and inhibition of glycogen synthesis. Oxidative stress

S Najib; V Sánchez-Margalet

2005-01-01

50

Carbachol induces p70S6K1 activation through an ERK-dependent but Akt-independent pathway in human colonic epithelial cells  

PubMed Central

Stimulation of human colonic epithelial T84 cells with the muscarinic receptor agonist carbachol, a stable analog of acetylcholine, induced Akt, p70S6K1 and ERK activation. Treatment of T84 cells with the selective inhibitor of EGF receptor (EGFR) tyrosine kinase AG1478 abrogated Akt phosphorylation on Ser473 induced by either carbachol or EGF, indicating that carbachol-induced Akt activation is mediated through EGFR transactivation. Surprisingly, AG1478 did not suppress p70S6K1 phosphorylation on Thr389 in response to carbachol, indicating the G protein-coupled receptor (GPCR) stimulation induces p70S6K1 activation, at least in part, via an Akt-independent pathway. In contrast, treatment with the selective MEK inhibitor U0126 (but not with the inactive analog U0124) inhibited carbachol-induced p70S6K1 activation, indicating that the MEK/ERK/RSK pathway plays a critical role in p70S6K1 activation in GPCR-stimulated T84 cells. These findings imply that GPCR activation induces p70S6K1 via ERK rather than through the canonical PI 3-kinase/Akt/TSC/mTORC1 pathway in T84 colon carcinoma cells.

Jiang, Xiaohua; Sinnett-Smith, James; Rozengurt, Enrique

2009-01-01

51

PGF 2?-associated vascular smooth muscle hypertrophy is ROS dependent and involves the activation of mTOR, p70 S6k, and PTEN  

Microsoft Academic Search

Prostaglandin F2? (PGF2?) increases reactive oxygen species (ROS) and induces vascular smooth muscle cell (VSMC) hypertrophy by largely unknown mechanism(s). To investigate the signaling events governing PGF2?-induced VSMC hypertrophy we examined the ability of the PGF2? analog, fluprostenol to elicit phosphorylation of Akt, the mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6k), glycogen synthase kinase-3? (GSK-3?), phosphatase and

K. M. Rice; S. Uddemarri; D. H. Desai; R. G. Morrison; R. Harris; G. L. Wright; E. R. Blough

2008-01-01

52

Regulation of insulin signalling by hyperinsulinaemia: role of IRS-1\\/2 serine phosphorylation and the mTOR\\/p70 S6K pathway  

Microsoft Academic Search

Aim\\/hypothesis  Several epidemiological studies have suggested an association between chronic hyperinsulinaemia and insulin resistance. However, the causality of this relationship remains uncertain.Methods  We performed chronic hyperinsulinaemic–euglycaemic clamps and delineated, by western blotting, an IR\\/IRSs\\/phosphatidylinositol 3-kinase(PI[3]K)\\/Akt pathway in insulin-responsive tissues of hyperinsulinaemic rats. IRS-1\\/2 serine phosphorylation, IR\\/protein tyrosine phosphatase 1B (PTP1B) association, and mammalian target of rapamycin (mTOR)\\/p70 ribosomal S6 kinase (p70 S6K)

M. Ueno; J. B. C. Carvalheira; R. C. Tambascia; R. M. N. Bezerra; M. E. Amaral; E. M. Carneiro; F. Folli; K. G. Franchini; M. J. A. Saad

2005-01-01

53

Role of ERK1\\/ERK2 and p70 S6K pathway in insulin signalling of protein synthesis  

Microsoft Academic Search

The signalling pathways by which insulin triggers protein synthesis were studied using an antisense strategy to deplete ERK1\\/ERK2 and rapamycin to inhibit the p70S6K pathway. The results indicated that ERK1\\/ERK2 principally regulated the amount of the protein synthesis machinery available in the cell while the p70S6K pathway contributed to modulating its activation in response to insulin. ERK1\\/ERK2 also mediated in

Elizabeth M. Sale; Peter P. G. Atkinson; Caroline H. Arnott; John E. Chad; Graham J. Sale

1999-01-01

54

Effects of rapamycin on cell proliferation and phosphorylation of mTOR and p70 S6K in HepG2 and HepG2 cells overexpressing constitutively active Akt\\/PKB  

Microsoft Academic Search

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that plays an important role in the regulation of cell proliferation and protein synthesis through the activation of its downstream target ribosomal p70 S6 kinase (p70S6K). The levels of p-mTOR are regulated by the protein kinase B (Akt\\/PKB). Therefore, the effects of insulin and rapamycin (an inhibitor of mTOR) on the

Shailly Varma; Ramji L. Khandelwal

2007-01-01

55

MiR-128 Inhibits Tumor Growth and Angiogenesis by Targeting p70S6K1  

PubMed Central

MicroRNAs are a class of small noncoding RNAs that function as critical gene regulators through targeting mRNAs for translational repression or degradation. In this study, we showed that miR-128 expression levels were decreased in glioma, and identified p70S6K1 as a novel direct target of miR-128. Overexpression of miR-128 suppressed p70S6K1 and its downstream signaling molecules such as HIF-1 and VEGF expression, and attenuated cell proliferation, tumor growth and angiogenesis. Forced expression of p70S6K1 can partly rescue the inhibitory effect of miR-128 in the cells. Taken together, these findings will shed light to the role and mechanism of miR-128 in regulating glioma tumor angiogenesis via miR-128/p70S6K1 axis, and miR-128 may serve as a potential therapeutic target in glioma in the future.

Yan, Zhiping; You, Yong-ping; Li, Chong-yong; Qian, Xu; Yin, Yu; Zhao, Peng; Wang, Ying-ying; Wang, Xie-feng; Li, Ming-na; Liu, Ling-Zhi; Liu, Ning; Jiang, Bing-Hua

2012-01-01

56

Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size.  

PubMed

Myostatin is a negative regulator of skeletal muscle size, previously shown to inhibit muscle cell differentiation. Myostatin requires both Smad2 and Smad3 downstream of the activin receptor II (ActRII)/activin receptor-like kinase (ALK) receptor complex. Other transforming growth factor-beta (TGF-beta)-like molecules can also block differentiation, including TGF-beta(1), growth differentiation factor 11 (GDF-11), activins, bone morphogenetic protein 2 (BMP-2) and BMP-7. Myostatin inhibits activation of the Akt/mammalian target of rapamycin (mTOR)/p70S6 protein synthesis pathway, which mediates both differentiation in myoblasts and hypertrophy in myotubes. Blockade of the Akt/mTOR pathway, using small interfering RNA to regulatory-associated protein of mTOR (RAPTOR), a component of TOR signaling complex 1 (TORC1), increases myostatin-induced phosphorylation of Smad2, establishing a myostatin signaling-amplification role for blockade of Akt. Blockade of RAPTOR also facilitates myostatin's inhibition of muscle differentiation. Inhibition of TORC2, via rapamycin-insensitive companion of mTOR (RICTOR), is sufficient to inhibit differentiation on its own. Furthermore, myostatin decreases the diameter of postdifferentiated myotubes. However, rather than causing upregulation of the E3 ubiquitin ligases muscle RING-finger 1 (MuRF1) and muscle atrophy F-box (MAFbx), previously shown to mediate skeletal muscle atrophy, myostatin decreases expression of these atrophy markers in differentiated myotubes, as well as other genes normally upregulated during differentiation. These findings demonstrate that myostatin signaling acts by blocking genes induced during differentiation, even in a myotube, as opposed to activating the distinct "atrophy program." In vivo, inhibition of myostatin increases muscle creatine kinase activity, coincident with an increase in muscle size, demonstrating that this in vitro differentiation measure is also upregulated in vivo. PMID:19357233

Trendelenburg, Anne Ulrike; Meyer, Angelika; Rohner, Daisy; Boyle, Joseph; Hatakeyama, Shinji; Glass, David J

2009-04-08

57

Lengthening contractions differentially affect p70 s6k phosphorylation compared to isometric contractions in rat skeletal muscle  

Microsoft Academic Search

The purpose of this investigation was to determine if p70s6k phosphorylation is dependent on the mode of resistance exercise (e.g. isometric vs. lengthening). Two groups (n = 5 each) of Female Sprague Dawley rats, ?12 weeks old, were tested. Rats were anesthetized and indwelling electrodes used\\u000a to stimulate the right hind limb muscles via the sciatic nerve. The tibialis anterior (TA) muscle of

Martin Burry; David Hawkins; Espen E. Spangenburg

2007-01-01

58

AKT/mTOR substrate P70S6K is frequently phosphorylated in gallbladder cancer tissue and cell lines  

PubMed Central

Background Gallbladder carcinoma is a highly malignant tumor and a public health problem in some parts of the world. It is characterized by a poor prognosis and its resistance to radio and chemotherapy. There is an urgent need to develop novel therapeutic alternatives for the treatment of gallbladder carcinoma. The mammalian target of the rapamycin (mTOR) signaling pathway is activated in about 50% of human malignancies, and its role in gallbladder carcinoma has previously been suggested. In the present study, we investigated the phosphorylation status of the mTOR substrate p70S6K in preneoplastic and neoplastic gallbladder tissues and evaluated the effect of three mTOR inhibitors on cell growth and migration in gallbladder carcinoma cell lines. Methods Immunohistochemical staining of phospho-p70S6K was analyzed in 181 gallbladder carcinoma cases, classified according to lesion type as dysplasia, early carcinoma, or advanced carcinoma. Protein expression of AKT/mTOR members was also evaluated in eight gallbladder carcinoma cell lines by Western blot analysis. We selected two gallbladder carcinoma cell lines (G415 and TGBC-2TKB) to evaluate the effect of rapamycin, RAD001, and AZD8055 on cell viability, cell migration, and protein expression. Results Our results showed that phospho-p70S6K is highly expressed in dysplasia (66.7%, 12/18), early cancer (84.6%, 22/26), and advanced cancer (88.3%, 121/137). No statistical correlation was observed between phospho-p70S6K status and any clinical or pathological features, including age, gender, ethnicity, wall infiltration level, or histological differentiation (P < 0.05). In vitro treatment with rapamycin, RAD001, and AZD8055 reduced cell growth, cell migration, and phospho-p70S6K expression significantly in G-415 and TGBC-2TKB cancer cells (P < 0.001). Conclusion Our findings confirm the upregulation of this signaling pathway in gallbladder carcinoma and provide a rationale for the potential use of mTOR inhibitors as a therapeutic strategy for human gallbladder carcinoma.

Leal, Pamela; Garcia, Patricia; Sandoval, Alejandra; Buchegger, Kurt; Weber, Helga; Tapia, Oscar; Roa, Juan C

2013-01-01

59

Low Amount of Salinomycin Greatly Increases Akt Activation, but Reduces Activated p70S6K Levels  

PubMed Central

The present study identified a novel salinomycin (Sal)-sensitization mechanism in cancer cells. We analyzed the signal proteins Akt, Jnk, p38, Jak, and Erk1/2 in cancer cell lines that had arrested growth following low amounts of Sal treatment. We also tested the signal molecules PI3K, PDK1, GSK3?, p70S6K, mTOR, and PTEN to analyze the PI3K/Akt/mTOR pathway. The results showed that Sal sensitization positively correlates with large reductions in p70S6K activation. Interestingly, Akt was the only signal protein to be significantly activated by Sal treatment. The Akt activation appeared to require the PI3K pathway as its activation was abolished by the PI3K inhibitors LY294002 and wortmannin. The Akt activation by Sal was conserved in the other cell lines analyzed, which originated from other organs. Both Akt activation and C-PARP production were proportionally increased with increased doses of Sal. In addition, the increased levels of pAkt were not reduced over the time course of the experiment. Co-treatment with Akt inhibitors sensitized the Sal-treated cancer cells. The results thereby suggest that Akt activation is increased in cells that survive Sal treatment and resist the cytotoxic effect of Sal. Taken together; these results indicate that Akt activation may promote the resistance of cancer cells to Sal.

Kim, Ju-Hwa; Choi, Ae-Ran; Kim, Yong Kee; Kim, Hyung Sik; Yoon, Sungpil

2013-01-01

60

PI3K/Akt and mTOR/p70S6K pathways mediate neuroprotectin D1-induced retinal pigment epithelial cell survival during oxidative stress-induced apoptosis.  

PubMed

The initiation and progression of several forms of retinal degenerations involve excessive, repetitive, and/or sustained oxidative stress that, in turn, mediate photoreceptor cell damage and death. Since phosphatidylinositol 3-kinase (PI3K)/Akt and mTOR/p70S6-kinase pathways are part of survival signaling in cells confronted with oxidative stress, we asked whether or not docosahexaenoic acid-derived neuroprotectin D1 (NPD1) mediates survival upon single-dose and/or repetitive oxidative stress through this pathway. For this purpose, we used human retinal pigment epithelial (ARPE-19) cells challenged by exposure to hydrogen peroxide (H(2)O(2)) plus tumor necrosis factor alpha (TNF-alpha). We found that in single-dose oxidative stress-induced apoptosis, phosphorylation of Akt, mTOR, and p70S6K was both time- and dose- dependent. Inhibition of PI3K or mTOR/p70S6K by wortmannin and rapamycin, respectively, increased apoptosis and inhibited phosphorylation of Akt and p70S6K induced by single-dose oxidative stress. While two exposures of a low dose, non-damaging oxidation induced apoptosis and upregulation of Akt, mTOR, and p70S6K, longer treatment of the cells with three exposures of low dose to low-dose stress showed no changes in the levels of Akt, mTOR, or p70S6K, and resulted in enhanced apoptosis compared to higher doses. Removing the oxidative stress-inducing agents following the single-dose or short term repetitive oxidative stress at the peak of Akt, mTOR, and p70S6K phosphorylation (i.e., 30 min after induction) led to recovery, with no apoptosis after 16 h of incubation. Cells that were induced with three low doses of stress did not show recovery when oxidative stress was removed 30 min after the last exposure. NPD1 protected the RPE cells against both single-dose and repetitive oxidative stress-induced apoptosis and promoted higher levels of phosphorylated Akt, mTOR, and p70S6K. Together, our results show that a) repetitive oxidative stress is dose dependent and may not be recovered by removing the oxidative stress-inducing agents, b) PI3K/Akt and mTOR/p70S6K pathways play a major role in the protection against oxidative stress-induced apoptosis in ARPE-19 cells, and c) NPD1 exerts protection under these conditions by inducing PI3K/Akt and mTOR/p70S6K pathways. PMID:20230819

Faghiri, Zahra; Bazan, Nicolas G

2010-03-15

61

Basal activation of p70S6K results in adipose-specific insulin resistance in protein-tyrosine phosphatase 1B -/- mice.  

PubMed

Although protein-tyrosine phosphatase 1B (PTP-1B) is a negative regulator of insulin action, adipose tissue from PTP-1B-/- mice does not show enhanced insulin-stimulated insulin receptor phosphorylation. Investigation of glucose uptake in isolated adipocytes revealed that the adipocytes from PTP-1B-/- mice have a significantly attenuated insulin response as compared with PTP-1B+/+ adipocytes. This insulin resistance manifests in PTP-1B-/- animals older than 16 weeks of age and could be partially rescued by adenoviral expression of PTP-1B in null adipocytes. Examination of adipose signaling pathways found that the basal p70S6K activity was at least 50% higher in adipose from PTP-1B-/- mice compared with wild type animals. The increased basal activity of p70S6K in PTP-1B-/- adipose correlated with decreases in IR substrate-1 protein levels and insulin-stimulated Akt/protein kinase B activity, explaining the decrease in insulin sensitivity even as insulin receptor phosphorylation was unaffected. The insulin resistance of the of the PTP-1B-/- adipocytes could also be rescued by treatment with rapamycin, suggesting that in adipose the loss of PTP-1B results in basal activation of mTOR (mammalian target of rapamycin) complex 1 leading to a tissue-specific insulin resistance. PMID:17664276

Ruffolo, Salvatore C; Forsell, Pontus K A; Yuan, Xiling; Desmarais, Sylvie; Himms-Hagen, Jean; Cromlish, Wanda; Wong, Kenny K; Kennedy, Brian P

2007-07-30

62

Overexpression of 4EBP1, p70S6K, Akt1 or Akt2 differentially promotes Coxsackievirus B3-induced apoptosis in HeLa cells.  

PubMed

Our previous studies have shown that the inhibition of phosphatidylinositol 3-kinase (PI3K) or mTOR complex 1 can obviously promote the Coxsackievirus B3 (CVB3)-induced apoptosis of HeLa cells by regulating the expression of proapoptotic factors. To further illustrate it, Homo sapiens eIF4E-binding protein 1 (4EBP1), p70S6 kinase (p70S6K), Akt1 and Akt2 were transfected to HeLa cells, respectively. And then, we established the stable transfected cell lines. Next, after CVB3 infection, apoptosis in different groups was determined by flow cytometry; the expressions of Bim, Bax, caspase-9 and caspase-3 were examined by real-time fluorescence quantitative PCR and western blot analysis; the expression of CVB3 mRNA and viral capsid protein VP1 were also analyzed by real-time fluorescence quantitative PCR, western blot analysis and immunofluorescence, respectively. At the meantime, CVB3 replication was observed by transmission electron microscope. We found that CVB3-induced cytopathic effect and apoptosis in transfected groups were more obvious than that in controls. Unexpectedly, apoptosis rate in Akt1 group was higher than others at the early stage after viral infection and decreased with the viral-infected time increasing, which was opposite to other groups. Compared with controls, the expression of CVB3 mRNA was increased at 3, 6, 12 and 24?h postinfection (p. i.) in all groups. At the meantime, VP1 expression in 4EBP1 group was higher than control during the process of infection, while the expressions in the other groups were change dynamically. Moreover, overexpression of 4EBP1 did not affect the mRNA expressions of Bim, Bax, caspase-9 and caspase-3; while protein expressions of Bim and Bax were decreased, the self-cleavages of caspase-9 and caspase-3 were stimulated. Meanwhile, overexpression of p70S6K blocked the CVB3-induced Bim, Bax and caspase-9 expressions but promoted the self-cleavage of caspase-9. In the Akt1 group, it is noteworthy that the expressions of Bim protein were higher than controls at 3 and 6?h p. i. but lower at 24?h p. i., and the expression of Bax protein were higher at 6 and 24?h p. i., while their mRNA expressions were all decreased. Furthermore, overexpression of Akt1 stimulated the procaspase-9 and procaspase-3 expression but blocked their self-cleavages. Overexpression of Akt2, however, had little effect on Bim, Bax and caspase-3, while prevented caspase-9 from self-cleavage at the late stage of CVB3 infection. As stated above, our results demonstrated that overexpression of 4EBP1, p70S6K, Akt1 or Akt2 could promote the CVB3-induced apoptosis in diverse degree via different mediating ways in viral replication and proapoptotic factors in BcL-2 and caspase families. As 4EBP1, p70S6K and Akt are the important substrates of PI3K and mammalian target of rapamycin (mTOR), we further illustrated the role of PI3K/Akt/mTOR signaling pathway in the process of CVB3-induced apoptosis. PMID:24030155

Li, X; Li, Z; Zhou, W; Xing, X; Huang, L; Tian, L; Chen, J; Chen, C; Ma, X; Yang, Z

2013-09-12

63

Overexpression of 4EBP1, p70S6K, Akt1 or Akt2 differentially promotes Coxsackievirus B3-induced apoptosis in HeLa cells  

PubMed Central

Our previous studies have shown that the inhibition of phosphatidylinositol 3-kinase (PI3K) or mTOR complex 1 can obviously promote the Coxsackievirus B3 (CVB3)-induced apoptosis of HeLa cells by regulating the expression of proapoptotic factors. To further illustrate it, Homo sapiens eIF4E-binding protein 1 (4EBP1), p70S6 kinase (p70S6K), Akt1 and Akt2 were transfected to HeLa cells, respectively. And then, we established the stable transfected cell lines. Next, after CVB3 infection, apoptosis in different groups was determined by flow cytometry; the expressions of Bim, Bax, caspase-9 and caspase-3 were examined by real-time fluorescence quantitative PCR and western blot analysis; the expression of CVB3 mRNA and viral capsid protein VP1 were also analyzed by real-time fluorescence quantitative PCR, western blot analysis and immunofluorescence, respectively. At the meantime, CVB3 replication was observed by transmission electron microscope. We found that CVB3-induced cytopathic effect and apoptosis in transfected groups were more obvious than that in controls. Unexpectedly, apoptosis rate in Akt1 group was higher than others at the early stage after viral infection and decreased with the viral-infected time increasing, which was opposite to other groups. Compared with controls, the expression of CVB3 mRNA was increased at 3, 6, 12 and 24?h postinfection (p. i.) in all groups. At the meantime, VP1 expression in 4EBP1 group was higher than control during the process of infection, while the expressions in the other groups were change dynamically. Moreover, overexpression of 4EBP1 did not affect the mRNA expressions of Bim, Bax, caspase-9 and caspase-3; while protein expressions of Bim and Bax were decreased, the self-cleavages of caspase-9 and caspase-3 were stimulated. Meanwhile, overexpression of p70S6K blocked the CVB3-induced Bim, Bax and caspase-9 expressions but promoted the self-cleavage of caspase-9. In the Akt1 group, it is noteworthy that the expressions of Bim protein were higher than controls at 3 and 6?h p. i. but lower at 24?h p. i., and the expression of Bax protein were higher at 6 and 24?h p. i., while their mRNA expressions were all decreased. Furthermore, overexpression of Akt1 stimulated the procaspase-9 and procaspase-3 expression but blocked their self-cleavages. Overexpression of Akt2, however, had little effect on Bim, Bax and caspase-3, while prevented caspase-9 from self-cleavage at the late stage of CVB3 infection. As stated above, our results demonstrated that overexpression of 4EBP1, p70S6K, Akt1 or Akt2 could promote the CVB3-induced apoptosis in diverse degree via different mediating ways in viral replication and proapoptotic factors in BcL-2 and caspase families. As 4EBP1, p70S6K and Akt are the important substrates of PI3K and mammalian target of rapamycin (mTOR), we further illustrated the role of PI3K/Akt/mTOR signaling pathway in the process of CVB3-induced apoptosis.

Li, X; Li, Z; Zhou, W; Xing, X; Huang, L; Tian, L; Chen, J; Chen, C; Ma, X; Yang, Z

2013-01-01

64

Age-related differences in the des IGF-I-mediated activation of Akt1 and p70 S6K in mouse skeletal muscle  

Microsoft Academic Search

The ability of des IGF-I to activate Akt-1 and p70 S6K in skeletal muscle with or without acute endurance exercise was examined in young and old mice. Mice were sacrificed 12 h after a moderate intensity treadmill run following an interperitoneal injection of des-IGF-I or saline. Blood and skeletal muscle were collected and IGF-I receptor, Akt-1 and p70 S6K protein

Minghua Li; Chunmei Li; Wade S. Parkhouse

2003-01-01

65

Nitric Oxide Donors Selectively Potentiate Thrombin-Stimulated p70 S6k Activity and Morphological Changes in Swiss 3T3 Cells  

Microsoft Academic Search

Thrombin stimulates both DNA synthesis and cell morphological changes in Swiss 3T3 cells, although the mechanism of signal coordination leading to these responses is unknown. We report here that nitric oxide (NO) donors selectively enhance thrombin-stimulated p70S6k activity by 40–60%, an effect that was sustained for 24 h. Potentiation of p70S6k also was observed with cGMP analogues indicating that this

Leise A. Berven; Ian J. Frew; Michael F. Crouch

1999-01-01

66

Liquiritigenin inhibits serum-induced HIF-1? and VEGF expression via the AKT/mTOR-p70S6K signalling pathway in HeLa cells.  

PubMed

Liquiritigenin (LQ) is a non-toxic dietary flavonoid with chemopreventive and anticancer properties. However, the mechanism of its antiangiogenesis remains unclear. Hypoxia-inducible factor-1? (HIF-1?) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in tumour angiogenesis and represent an attractive chemotherapeutic target. In this study, we investigated the effect of LQ on the molecular mechanism of angiogenesis. We found that LQ inhibited VEGF expression at both mRNA and protein levels. Liquiritigenin did not affect HIF-1? expression at the mRNA level, but it dramatically inhibited both serum- and mimicked hypoxic-induced HIF-1? protein accumulation in HeLa cells. Furthermore, we showed that LQ inhibited serum-induced expression of HIF-1? by reducing its stability and decreased the synthesis in a dose-dependent manner. Mechanistically, we demonstrated that LQ inhibited HIF-1? and VEGF expression involved in blocking the protein kinase B (PKB/Akt) signalling pathway, and the mechanisms correlated with dephosphorylation of the mammalian target of rapamycin (mTOR) and its effector ribosomal protein S6 kinase (p70S6K). In addition, LQ inhibited VEGF-induced formation of capillary-like structures in human umbilical vein endothelial cells (HUVEC). Taken together, our study provided valuable insights into the mechanism of antiangiogenic effect of LQ. PMID:22170854

Xie, Si-Rou; Wang, Yu; Liu, Chang-Wei; Luo, Kang; Cai, Yun-Qing

2011-12-14

67

Mitogen-responsive S6 kinase.  

PubMed

Many cell lines respond to mitogenic stimuli (serum, growth factors) with rapid phosphorylation of the ribosomal protein S6 at several serine sites. We have tried to identify the protein kinase(s) mediating this effect of growth stimuli. Examining post-DEAE chromatography fractions of S49 kin- cell extracts, we could detect a highly active effector-independent S6 kinase with specificity for serine residues. The study was extended to the presumably homologous human enzyme, using HeLa S3 cells as model system. Activity yields increased up to sevenfold when exhausted HeLa cells were supplied with fresh medium plus serum. The enzyme uses ATP, not GTP, as cosubstrate, 40-S or 80-S (reassociated from subunits) ribosomal particles being substrate. The optimal K+ concentration, measured at 3 mM Mg2+, is 35 mM. Under optimized assay conditions S6 phosphorylation proceeded faster in vitro than it appeared to do in vivo. The apparent Mr of the enzyme, as estimated by gel filtration on Sephadex G-100, is 56,000 (determination in the presence of 200 mM KCl in 25 mM phosphate buffer). Tighter binding to DEAE-Sephacel and higher specificity for S6 distinguishes this enzyme from the following S6-phosphorylating protein kinases: protein kinase C, protease-activated kinase II, histone-4 phosphotransferase and an enzyme with the properties of casein kinase I. In published summaries of observations shown here and in a follow-up study with chick embryo fibroblasts, the enzyme(s) has been referred to as mitogen-responsive S6 kinase(s) [Martini, O. H. W. and Lawen, A. (1985) in Hormones and cell regulation (Dumont, J. E., Hamprecht, B. and Nunez, J., eds) vol. 9, pp. 411-412, Elsevier Company, North-Holland, Amsterdam; Lawen, A. and Martini, O. H. W. (1985) FEBS Lett. 185, 272-276]. PMID:2547604

Lawen, A; Burger, M; Martini, O H

1989-08-01

68

Protein kinases: Getting NEKed for S6K activation  

Microsoft Academic Search

The 70\\/85 kDa ribosomal protein S6 kinase is regulated through the concerted actions of multiple phosphatases and kinases. A newly identified S6 kinase kinase, NEK6, appears to provide the penultimate activation step.

Dennis J Templeton

2001-01-01

69

Overexpression of the lily p70(s6k) gene in Arabidopsis affects elongation of flower organs and indicates TOR-dependent regulation of AP3, PI and SUP translation.  

PubMed

The p70 ribosomal S6 kinase (p70(s6k)) signaling pathway plays a key role in regulating the cell cycle via translational regulation of specific 5'TOP mRNAs. However, the function of this signaling pathway is still poorly understood in plants. Ectopic expression of the lily putative p70(s6k) gene, LS6K1, resulted in up-regulation of NAP (NAC-LIKE, ACTIVATED BY AP3/PI) and PISTILLATA (PI) expression, and significantly inhibited cell expansion for petals and stamens, resulting in the male sterility phenotype in transgenic Arabidopsis. Sequence analysis revealed that the genes involved in petal and stamen development, such as APETALA3 (AP3), PI and SUPERMAN (SUP), probably encode 5'TOP mRNAs. Green fluorescent protein (GFP), fused to oligopyrimidine tract sequences that were identified in the 5'-untranslated region (UTR) of AP3, PI and SUP, was translationally regulated in human cells in response to mitogen stimulation and inhibition by the macrolide antibiotic rapamycin. Furthermore, 35S::LS6K1 significantly up-regulated beta-glucuronidase (GUS) activity in the flower buds of transgenic plants carrying the GUS transgene fused to the AP3 promoter and the 5' UTR. These results have identified a novel role for the p70(s6k) gene in regulating cell division and the expansion of petals and stamens by translational regulation of the 5'TOP mRNAs once ectopically expressed in Arabidopsis. PMID:19651701

Tzeng, Tsai-Yu; Kong, Lih-Ren; Chen, Chun-Hung; Shaw, Chih-Chi; Yang, Chang-Hsien

2009-08-03

70

Differential effects of des IGF-1 on ERKs, AKT1 and P70 S6K activation in mouse skeletal and cardiac muscle  

Microsoft Academic Search

Alterations in the degree of the phosphorylation of ERK1\\/2, Akt-1 and p70 S6K in mouse skeletal and cardiac muscle was examined in vivo following an intraperitoneal injection of des IGF-I. Plasma levels of insulin, IGF-I and glucose were measured. The administration of des IGF-I had no effect on plasma levels of insulin, or IGF-I, but plasma glucose levels were decreased

M. Li; C. Li; W. S. Parkhouse

2002-01-01

71

Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K pathway  

PubMed Central

Robust production of type I interferon (IFN-?/?) in plasmacytoid dendritic cells (pDCs) is crucial for antiviral immunity. Here we show involvement of the mammalian target of rapamycin (mTOR) pathway in regulating interferon production by pDCs. Inhibition of mTOR or its ‘downstream’ mediators, the p70 ribosomal S6 protein kinases p70S6K1 and p70S6K2, during pDC activation by Toll-like receptor 9 (TLR9) blocked the interaction of TLR9 with the adaptor MyD88 and subsequent activation of the interferon-regulatory factor IRF7, which resulted in impaired IFN-?/? production. Microarray analysis confirmed that inhibition of mTOR by the immunosuppressive drug rapamycin suppressed antiviral and anti-inflammatory gene expression. Consistent with this, targeting rapamycin-encapsulated microparticles to antigen-presenting cells in vivo resulted in less IFN-?/? production in response to CpG DNA or the yellow fever vaccine virus strain 17D. Thus, mTOR signaling is crucial in TLR-mediated IFN-?/? responses by pDCs.

Cao, Weiping; Manicassamy, Santhakumar; Tang, Hua; Kasturi, Sudhir Pai; Pirani, Ali; Murthy, Niren; Pulendran, Bali

2013-01-01

72

Receptor association and tyrosine phosphorylation of S6 kinases  

Microsoft Academic Search

Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser\\/Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as

Heike Rebholz; Ganna Panasyuk; Timothy Fenton; Ivan Nemazanyy; Taras Valovka; Marc Flajolet; Lars Ronnstrand; Len Stephens; Andrew West; Ivan T Gout

2006-01-01

73

Exercise training reduces insulin resistance and upregulates the mTOR/p70S6k pathway in cardiac muscle of diet-induced obesity rats.  

PubMed

Obesity and insulin resistance are rapidly expanding public health problems. These disturbances are related to many diseases, including heart pathology. Acting through the Akt/mTOR pathway, insulin has numerous and important physiological functions, such as the induction of growth and survival of many cell types and cardiac hypertrophy. However, obesity and insulin resistance can alter mTOR/p70S6k. Exercise training is known to induce this pathway, but never in the heart of diet-induced obesity subjects. To evaluate the effect of exercise training on mTOR/p70S6k in the heart of obese Wistar rats, we analyzed the effects of 12 weeks of swimming on obese rats, induced by a high-fat diet. Exercise training reduced epididymal fat, fasting serum insulin and plasma glucose disappearance. Western blot analyses showed that exercise training increased the ability of insulin to phosphorylate intracellular molecules such as Akt (2.3-fold) and Foxo1 (1.7-fold). Moreover, reduced activities and expressions of proteins, induced by the high-fat diet in rats, such as phospho-JNK (1.9-fold), NF-kB (1.6-fold) and PTP-1B (1.5-fold), were observed. Finally, exercise training increased the activities of the transduction pathways of insulin-dependent protein synthesis, as shown by increases in Raptor phosphorylation (1.7-fold), p70S6k phosphorylation (1.9-fold), and 4E-BP1 phosphorylation (1.4-fold) and a reduction in atrogin-1 expression (2.1-fold). Results demonstrate a pivotal regulatory role of exercise training on the Akt/mTOR pathway, in turn, promoting protein synthesis and antagonizing protein degradation. PMID:20717955

Medeiros, Cleber; Frederico, Marisa J; da Luz, Gabrielle; Pauli, José R; Silva, Adelino S R; Pinho, Ricardo A; Velloso, Lício A; Ropelle, Eduardo R; De Souza, Cláudio T

2011-03-01

74

Erythropoietin induces biphasic activation of p70 S6k: Evidence for a different regulation of early and late phase of activation  

Microsoft Academic Search

The murine erythroleukaemia cell line HCD-57 proliferates in response to erythropoietin. Stimulation of erythropoietin-deprived cells with the cytokine induces the phosphorylation and biphasic activation of the 70,000 Mr S6 kinase. Two peaks of enzyme activity were observed after 30 and 120 min, respectively. Early and late phase of activation differ in their sensitivity to the protein kinase C inhibitor staurosporine

Robert Jaster; Thomas Bittorf; Michael Markewitz; Gesine Selig; Josef Brock

1995-01-01

75

Regulation of Ribosomal Protein S6 Phosphorylation by Casein Kinase 1 and Protein Phosphatase 1*  

PubMed Central

Ribosomal protein S6 (rpS6) is a critical component of the 40 S ribosomal subunit that mediates translation initiation at the 5?-m7GpppG cap of mRNA. In response to mitogenic stimuli, rpS6 undergoes ordered C-terminal phosphorylation by p70 S6 kinases and p90 ribosomal S6 kinases on four conserved Ser residues (Ser-235, Ser-236, Ser-240, and Ser-244) whose modification potentiates rpS6 cap binding activity. A fifth site, Ser-247, is also known to be phosphorylated, but its function and regulation are not well characterized. In this study, we employed phospho-specific antibodies to show that Ser-247 is a target of the casein kinase 1 (CK1) family of protein kinases. CK1-dependent phosphorylation of Ser-247 was induced by mitogenic stimuli and required prior phosphorylation of upstream S6 kinase/ribosomal S6 kinase residues. CK1-mediated phosphorylation of Ser-247 also enhanced the phosphorylation of upstream sites, which implies that bidirectional synergy between C-terminal phospho-residues is required to sustain rpS6 phosphorylation. Consistent with this idea, CK1-dependent phosphorylation of rpS6 promotes its association with the mRNA cap-binding complex in vitro. Additionally, we show that protein phosphatase 1 (PP1) antagonizes rpS6 C terminus phosphorylation and cap binding in intact cells. These findings further our understanding of rpS6 phospho-regulation and define a direct link between CK1 and translation initiation.

Hutchinson, John A.; Shanware, Naval P.; Chang, Haeyoon; Tibbetts, Randal S.

2011-01-01

76

Regulation of ribosomal protein S6 phosphorylation by casein kinase 1 and protein phosphatase 1.  

PubMed

Ribosomal protein S6 (rpS6) is a critical component of the 40 S ribosomal subunit that mediates translation initiation at the 5'-m(7)GpppG cap of mRNA. In response to mitogenic stimuli, rpS6 undergoes ordered C-terminal phosphorylation by p70 S6 kinases and p90 ribosomal S6 kinases on four conserved Ser residues (Ser-235, Ser-236, Ser-240, and Ser-244) whose modification potentiates rpS6 cap binding activity. A fifth site, Ser-247, is also known to be phosphorylated, but its function and regulation are not well characterized. In this study, we employed phospho-specific antibodies to show that Ser-247 is a target of the casein kinase 1 (CK1) family of protein kinases. CK1-dependent phosphorylation of Ser-247 was induced by mitogenic stimuli and required prior phosphorylation of upstream S6 kinase/ribosomal S6 kinase residues. CK1-mediated phosphorylation of Ser-247 also enhanced the phosphorylation of upstream sites, which implies that bidirectional synergy between C-terminal phospho-residues is required to sustain rpS6 phosphorylation. Consistent with this idea, CK1-dependent phosphorylation of rpS6 promotes its association with the mRNA cap-binding complex in vitro. Additionally, we show that protein phosphatase 1 (PP1) antagonizes rpS6 C terminus phosphorylation and cap binding in intact cells. These findings further our understanding of rpS6 phospho-regulation and define a direct link between CK1 and translation initiation. PMID:21233202

Hutchinson, John A; Shanware, Naval P; Chang, Haeyoon; Tibbetts, Randal S

2011-01-13

77

The mTORC1 signaling repressors REDD1/2 are rapidly induced and activation of p70S6K1 by leucine is defective in skeletal muscle of an immobilized rat hindlimb.  

PubMed

Limb immobilization, limb suspension, and bed rest cause substantial loss of skeletal muscle mass, a phenomenon termed disuse atrophy. To acquire new knowledge that will assist in the development of therapeutic strategies for minimizing disuse atrophy, the present study was undertaken with the aim of identifying molecular mechanisms that mediate control of protein synthesis and mechanistic target of rapamycin complex 1 (mTORC1) signaling. Male Sprague-Dawley rats were subjected to unilateral hindlimb immobilization for 1, 2, 3, or 7 days or served as nonimmobilized controls. Following an overnight fast, rats received either saline or L-leucine by oral gavage as a nutrient stimulus. Hindlimb skeletal muscles were extracted 30 min postgavage and analyzed for the rate of protein synthesis, mRNA expression, phosphorylation state of key proteins in the mTORC1 signaling pathway, and mTORC1 signaling repressors. In the basal state, mTORC1 signaling and protein synthesis were repressed within 24 h in the soleus of an immobilized compared with a nonimmobilized hindlimb. These responses were accompanied by a concomitant induction in expression of the mTORC1 repressors regulated in development and DNA damage responses (REDD) 1/2. The nutrient stimulus produced an elevation of similar magnitude in mTORC1 signaling in both the immobilized and nonimmobilized muscle. In contrast, phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) on Thr(229) and Thr(389) in response to the nutrient stimulus was severely blunted. Phosphorylation of Thr(229) by PDK1 is a prerequisite for phosphorylation of Thr(389) by mTORC1, suggesting that signaling through PDK1 is impaired in response to immobilization. In conclusion, the results show an immobilization-induced attenuation of mTORC1 signaling mediated by induction of REDD1/2 and defective p70S6K1 phosphorylation. PMID:23193052

Kelleher, Andrew R; Kimball, Scot R; Dennis, Michael D; Schilder, Rudolf J; Jefferson, Leonard S

2012-11-27

78

ERK1/2-dependent activation of mTOR/mTORC1/p70S6K regulates thrombin-induced RPE cell proliferation.  

PubMed

Epithelial-mesenchymal transition (EMT), proliferation and migration of RPE cells characterize the development of proliferative vitreoretinopathy (PVR) and other fibro-proliferative eye diseases leading to blindness. A common event in these pathologies is the alteration of the BRB which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Thrombin promotion of cytoskeletal reorganization, proliferation, and migration has been reported in different cell types, although the molecular mechanisms involved in these processes remain poorly understood. Our previous work demonstrated that thrombin promotes RPE cell proliferation, cytoskeletal remodeling and migration, hallmark processes in the development of PVR. Thrombin induction of RPE cell proliferation requires PI3K, PDK1, and Akt/PKB (Akt) signaling leading to cyclin D1 gene expression. Since Akt functions as an upstream activator of mechanistic target of rapamycin complex 1 (mTORC1) and is also a downstream target for mTORC2, the aim of this work was to determine whether mTOR is involved in thrombin-induced RPE cell proliferation by regulating cyclin D1 expression in immortalized rat RPE-J cell line. Results demonstrate that thrombin-induced cyclin D1 expression and cell proliferation require Akt-independent phosphorylation/activation of mTOR at Ser 2448 mediated by PI3K/PKC-?/ERK1/2 signaling, concomitant to Akt-dependent activation of p70S6K carried by mTORC1. PMID:23291002

Parrales, Alejandro; López, Edith; Lee-Rivera, Irene; López-Colomé, Ana María

2013-01-03

79

Activation of myelin basic protein and S6 peptide kinases in phorbol ester- and PAF-treated sheep platelets.  

PubMed

The involvement of myelin basic protein (MBP) kinases and ribosomal S6 peptide kinases in sheep platelet signal transduction was investigated. Treatment of platelets with 200 nM 12-O-tetradecanoylphorbol-13-acetate (PMA) led to 5-fold stimulations of cytosolic MBP and S6 peptide kinase activities within 1 min. Immunoblotting analysis of phenyl-Superose-fractionated cytosol from PMA-treated platelets with a panel of mitogen-activated protein (MAP) kinase anti-peptide antibodies revealed that one of the activated MBP kinases was p42mapk. This MAP kinase isoform was also stimulated to a lesser extent (approximately 2-fold) when platelets were exposed to 200 microM platelet-activating factor (PAF) for 3 min. The pathways of PAF-activation of p42mapk also involved a protein kinase C-independent route, since the staurosporin analog compound 3 reduced PAF-induced activation by approximately 30% under conditions in which it inhibited PMA-activation of p42mapk by approximately 80%. Another MAP kinase isoform of 44 kDa, most probably p44erk1, was also detected in platelet cytosol, but it was only marginally modulated in response to PMA or PAF. The predominant PMA- and PAF-activated MBP kinase detected after MonoQ fractionation of platelet cytosol did not appear to correspond to a MAP kinase. MonoQ chromatography of platelet cytosol also resolved two PMA- and PAF-activated S6 peptide kinases, which appeared to coelute on phenyl-Sepharose. Western blotting analysis of the MonoQ fractions with antibodies raised against peptide sequences in the S6 kinases p90rsk and p70S6K revealed immunoreactive proteins of approximately 75 kDa and approximately 95 kDa that coincided with the first S6 peptide kinase peak. These proteins probably corresponded to the 502 and 525 amino-acid-length forms of p70S6K. Only the second peak of S6 peptide kinase activity from MonoQ was appreciably stimulated in response to PAF-treatment of platelets, and this was largely abolished by compound 3. It is more likely that the novel MBP and S6 peptide kinases described here, rather than p42mapk and p70S6K, play a significant role in PAF signal transduction in the platelet. PMID:8385998

Samiei, M; Sanghera, J S; Pelech, S L

1993-04-16

80

Novel Role for SHP-2 in Nutrient-Responsive Control of S6 Kinase 1 Signaling  

PubMed Central

Amino acids are required for the activation of the mammalian target of rapamycin complex 1 (mTORC1), which plays a critical role in cell growth, proliferation, and metabolism. The branched-chain amino acid leucine is an essential nutrient that stimulates mTORC1 to promote protein synthesis by activating p70 S6 kinase 1 (S6K1). Here we show that the protein tyrosine phosphatase SHP-2 is required for leucine-induced activation of S6K1 in skeletal myoblasts. In response to leucine, S6K1 activation is inhibited in myoblasts either lacking SHP-2 expression or overexpressing a catalytically inactive mutant of SHP-2. Activation of S6K1 by leucine requires the mobilization of intracellular calcium (Ca2+), which we show is mediated by SHP-2 in an inositol-1,4,5-trisphosphate-dependent manner. Ectopic Ca2+ mobilization rescued the S6K1 activation defect in SHP-2-deficient myoblasts. SHP-2 was identified to act upstream of phospholipase C ?4, linking it to the generation of nutrient-induced Ca2+ release and S6K1 phosphorylation. Consistent with these results, SHP-2-deficient myoblasts exhibited impaired leucine sensing, leading to defective autophagy and reduced myoblast size. These data define a new role for SHP-2 as a nutrient-sensing regulator in skeletal myoblasts that is required for the activation of S6K1.

Mercan, Fatih; Lee, Hojin; Kolli, Sivanagarani

2013-01-01

81

Fibronectin Stimulates Non-Small Cell Lung Carcinoma Cell Growth through Activation of Akt\\/Mammalian Target of Rapamycin\\/S6 Kinase and Inactivation of LKB1\\/AMP Activated Protein Kinase Signal Pathways  

Microsoft Academic Search

The Akt\\/mammalian target of rapamycin (mTOR)\\/ribosomal protein S6 kinase (p70S6K) pathway is considered a central regulator of protein synthesis and of cell proliferation, diffe- rentiation, and survival. However, the role of the Akt\\/mTOR\\/ p70S6K pathway in lung carcinoma remains unknown. We previously showed that fibronectin, a matrix glycoprotein highly expressed in tobacco-related lung disease, stimulates non-small cell lung carcinoma (NSCLC)

ShouWei Han; Fadlo R. Khuri; Jesse Roman

82

Antisense Inhibition of S6 Kinase 1 Produces Improved Glucose Tolerance and Is Well Tolerated for 4 Weeks of Treatment in Rats  

Microsoft Academic Search

p70 ribosomal S6 kinase 1 (S6K1) is implicated in the pathogenesis of type 2 diabetes as knockout mice are hypoinsulinemic, hypersensitive to insulin treatment and are less susceptible to obesity-induced insulin resistance. Although S6K1 knockout mice provide important information on the biology of this target, the therapeutic relevance of S6K1 inhibition in adult animals is unknown. Thus, this research evaluated

H. S. Younis; B. Hirakawa; W. Scott; P. Tran; G. Bhat; T. Affolter; J. Chapman; J. Heyen; K. Chakravarty; G. Alton

2011-01-01

83

Stimulation of MAP kinase and S6 kinase by vanadium and selenium in rat adipocytes  

Microsoft Academic Search

To explore the mechanism underlying the insulin-mimetic actions of vanadium and selenium we examined their effects on the mitogen activated protein\\/myelin basic protein kinases (MAPK) and ribosomal S6 protein kinases, which are among the best characterized of the kinases that comprise the phosphorylation cascade in insulin signal transduction. We observed a transient activation of MAPK and S6 kinases by insulin

Yong-jiang Hei; Sepehr Farahbakhshian; Xunsheng Chen; Mary L. Battell; John H. McNeill

1998-01-01

84

Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin.  

PubMed Central

The proliferation of most normal cells depends on the co-operation of several growth factors and hormones, each with a specific role, but the key events involved in the action of each necessary stimulant remain largely uncharacterized. In the present study, the pathways involved in the mechanism(s) of co-operation have been investigated in primary cultures of dog thyroid epithelial cells. In this physiologically relevant system, thyroid stimulating hormone (TSH) acting through cAMP, epidermal growth factor (EGF) and phorbol esters (such as PMA) induce DNA synthesis. Their effect requires stimulation of the insulin-like growth factor-1 (IGF-1) receptor by either IGF-1 or insulin, which are not themselves mitogenic agents. In contrast, hepatocyte growth factor (HGF) is itself fully mitogenic. The results of the study demonstrate that cAMP, EGF, HGF and PMA stimulate p70 ribosomal S6 kinase (p70 S6 kinase). However, insulin/IGF-1 also stimulate p70 S6 kinase. Thus stimulation of p70 S6 kinase might be necessary, but is certainly not sufficient, for the induction of DNA synthesis and is not specific for any stimulated pathway. In contrast, phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB) activation by insulin and HGF is strong and sustained, whereas it is weak and transient with EGF and absent in the presence of TSH or PMA. These findings suggest that: (i) stimulation of PI 3-kinases and/or PKB is not involved in the cAMP-dependent pathways leading to thyrocyte proliferation, or in the action of PMA, (ii) the stimulation of the PI 3-kinase/PKB pathway may account for the permissive action of insulin/IGF-1 in the proliferation of these cells, and (iii) the stimulation of this pathway by HGF may explain why this agent does not require insulin or IGF-1 for its mitogenic action.

Coulonval, K; Vandeput, F; Stein, R C; Kozma, S C; Lamy, F; Dumont, J E

2000-01-01

85

Ribosomal S6 Kinase Cooperates with Casein Kinase 2 to Modulate the Drosophila Circadian Molecular Oscillator  

PubMed Central

There is a universal requirement for post-translational regulatory mechanisms in circadian clock systems. Previous work in Drosophila has identified several kinases, phosphatases and an E3 ligase that are critical for determining the nuclear translocation and/or stability of clock proteins. The present study evaluated the function of p90 ribosomal S6 kinase (RSK) in the Drosophila circadian system. In mammals, RSK1 is a light- and clock-regulated kinase known to be activated by the MAPK pathway, but there is no direct evidence that it functions as a component of the circadian system. Here, we show that Drosophila S6KII RNA displays rhythms in abundance, indicative of circadian control. Importantly, an S6KII null mutant exhibits a short-period circadian phenotype that can be rescued by expression of the wild-type gene in clock neurons, indicating a role for S6KII in the molecular oscillator. Peak PER clock protein expression is elevated in the mutant, indicative of enhanced stability, whereas per mRNA level is decreased, consistent with enhanced feedback repression. Gene reporter assays show that decreased S6KII is associated with increased PER repression. Surprisingly, we demonstrate a physical interaction between S6KII and the Casein Kinase 2 regulatory subunit (CK2?), suggesting a functional relationship between the two kinases. In support of such a relationship, there are genetic interactions between S6KII and CK2 mutations, in vivo, which indicate that CK2 activity is required for S6KII action. We propose that the two kinases cooperate within clock neurons to fine-tune circadian period, improving the precision of the clock mechanism.

Akten, Bikem; Tangredi, Michelle M.; Jauch, Eike; Roberts, Mary A.; Ng, Fanny; Raabe, Thomas; Jackson, F. Rob

2009-01-01

86

Inhibition of S6 kinase suppresses the apoptotic effect of eIF4E ablation by inducing TGF-?-dependent G1 cell cycle arrest.  

PubMed

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of cap-dependent translation through its direct activation of ribosomal protein p70 S6 kinase (S6 kinase) and indirect activation of eukaryotic initiation factor 4E (eIF4E). We recently reported that inhibition of eIF4E expression caused apoptosis in cancer cells in the absence of serum. This was indicated by treatment with the mTORC1 inhibitor rapamycin, which suppressed both S6 kinase and 4E-BP1 phosphorylation (dephosphorylated 4E-BP1 binds and inactivates eIF4E), or by knockdown of eIF4E. We report here that knockdown of eIF4E also causes apoptosis in the presence of serum. This was unexpected because rapamycin induces G1 cell cycle arrest in the presence of serum. Upon investigation, we have found that inactivated S6 kinase prevents the apoptotic effect observed by singular knockdown of eIF4E and results in G1 cell cycle arrest. This effect is dependent on TGF-? (transforming growth factor-?) signaling which contributes to G1 cell cycle arrest. Suppression of S6 kinase phosphorylation alone is insufficient to mediate cell cycle arrest, indicating that complete G1 cell cycle arrest is due to suppression of both S6 kinase and eIF4E. These data indicate that the cytostatic effect of rapamycin is suppression of both S6 kinase and eIF4E, while the cytotoxic effects are due suppression of eIF4E in the absence of S6 kinase-dependent activation of TGF-? signals. Our findings place an importance on the evaluating the activity/expression level of S6 kinase and eIF4E as readouts for rapamycin/rapalog efficacy. PMID:23376634

Yellen, Paige; Chatterjee, Amrita; Preda, Angela; Foster, David A

2013-01-29

87

Ca(2+)-independent protein kinase C activity is required for alpha1-adrenergic-receptor-mediated regulation of ribosomal protein S6 kinases in adult cardiomyocytes.  

PubMed

The alpha(1)-adrenergic agonist, phenylephrine (PE), exerts hypertrophic effects in the myocardium and activates protein synthesis. Both Ca(2+)-dependent protein kinase C (PKC, PKCalpha) and Ca(2+)-independent PKC isoforms (PKCdelta and epsilon ) are detectably expressed in adult rat cardiomyocytes. Stimulation of the alpha(1)-adrenergic receptor by PE results in activation of Ca(2+)-independent PKCs, as demonstrated by translocation of the delta and epsilon isoenzymes from cytosol to membrane fractions. PE also induces activation of p70 ribosomal protein S6 kinases (S6K1 and 2) in adult cardiomyocytes. We have studied the role of Ca(2+)-independent PKCs in the regulation of S6K activity by PE. Activation of S6K1/2 by PE was blocked by the broad-spectrum PKC inhibitor bisindolylmaleimide (BIM) I, whereas Gö6976, a compound that only inhibits Ca(2+)-dependent PKCs, did not inhibit S6K activation. Rottlerin, which selectively inhibits PKCdelta, also prevented PE-induced S6K activation. The isoform-specific PKC inhibitors had similar effects on the phosphorylation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1, a translation repressor that, like the S6Ks, lies downstream of the mammalian target of rapamycin (mTOR). Infection of cells with adenoviruses encoding dominant-negative PKCdelta or epsilon inhibited the activation of extracellular-signal-regulated kinase (ERK) by PE, and also inhibited the activation and/or phosphorylation of S6Ks 1 and 2. The PE-induced activation of protein synthesis was abolished by BIM I and markedly attenuated by rottlerin. Our data thus suggest that Ca(2+)-independent PKC isoforms play an important role in coupling the alpha(1)-adrenergic receptor to mTOR signalling and protein synthesis in adult cardiomyocytes. PMID:12720544

Wang, Lijun; Rolfe, Mark; Proud, Christopher G

2003-07-15

88

p42 mitogen-activated protein kinase and p90 ribosomal S6 kinase are selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation.  

PubMed Central

Human platelets provide an excellent model system for the study of phosphorylation events during signal transduction and cell adhesion. Platelets are terminally differentiated cells that exhibit rapid phosphorylation of many proteins upon agonist-induced activation and aggregation. We have sought to identify the kinases as well as the phosphorylated substrates that participate in thrombin-induced signal transduction and platelet aggregation. In this study, we have identified two forms of mitogen-activated protein kinase (MAPK), p42mapk and p44mapk, in platelets. The data demonstrate that p42mapk but not p44mapk becomes phosphorylated on serine, threonine, and tyrosine during platelet activation. Immune complex kinase assays, gel renaturation assays, and a direct assay for MAPK activity in platelet extracts all support the conclusion that p42mapk but not p44mapk shows increased kinase activity during platelet activation. The activation of p42mapk, independently of p44mapk, in platelets is unique since in other systems, both kinases are coactivated by a variety of stimuli. We also show that platelets express p90rsk, a ribosomal S6 kinase that has previously been characterized as a substrate for MAPK. p90rsk is phosphorylated on serine in resting platelets, and this phosphorylation is enhanced upon thrombin-induced platelet activation. Immune complex kinase assays demonstrate that the activity of p90rsk is markedly increased during platelet activation. Another ribosomal S6 protein kinase, p70S6K, is expressed by platelets but shows no change in kinase activity upon platelet activation with thrombin. Finally, we show that the increased phosphorylation and activity of both p42mapk and p90rsk does not require integrin-mediated platelet aggregation. Since platelets are nonproliferative cells, the signal transduction pathways that include p42mapk and p90rsk cannot lead to a mitogenic signal and instead may regulate cytoskeletal or secretory changes during platelet activation. Images

Papkoff, J; Chen, R H; Blenis, J; Forsman, J

1994-01-01

89

Development of Monoclonal Antibodies Specific to Ribosomal Protein S6 Kinase 2  

PubMed Central

Ribosomal protein S6 kinase 2 (S6K2) is a serine/threonine kinase that belongs to the family of AGC kinases, which includes PKB/Akt, PKC, PDK1, and SGK1. Mammalian cells express two isoforms of S6K, termed S6K1 and S6K2. Each of these has nuclear and cytoplasmic spicing variants, which originate from different initiation start codons. Nuclear isoforms of S6K1 and S6K2 are slightly longer, as they possess additional sequences at the N-terminus with nuclear localization signals. Biochemical and genetic studies implicated S6Ks in the regulation of cell size, growth, and energy metabolism. Deregulation of S6K signaling has been linked to various human pathologies, making them excellent targets for drug discovery. The aim of this study was to produce monoclonal antibodies directed at the N-terminal regulatory region of S6K2, which shows very low homology to S6K1 or other members of the AGC family. To achieve this goal, two S6K2 fragments covering 1–64aa and 14–64aa N-terminal sequences were expressed in bacteria as GST/6His fusion proteins. Affinity purified recombinant proteins were used as antigens for immunization, hybridoma screening, and analysis of generated clones. We produced a panel of S6K2-specific antibodies, which recognized recombinant S6K2 proteins in ELISA and Western blot analysis. Further analysis of selected clones revealed that three clones, termed B1, B2, and B4, specifically recognized not only recombinant, but also endogenous S6K2 in Western blot analysis of HEK293 cell lysates. Specificity of B2 clone has been confirmed in additional commonly used immunoassays, including immunoprecipitation and immunocytochemistry. These properties make B2 MAb particularly valuable for elucidating signal transduction pathways involving S6K2 signaling under physiological conditions and in human pathologies.

Savinska, Lilia; Skorokhod, Oleksandr; Klipa, Olga; Gout, Ivan

2012-01-01

90

S6 kinase 1 is required for rapamycin-sensitive liver proliferation after mouse hepatectomy  

PubMed Central

Rapamycin is an antibiotic inhibiting eukaryotic cell growth and proliferation by acting on target of rapamycin (TOR) kinase. Mammalian TOR (mTOR) is thought to work through 2 independent complexes to regulate cell size and cell replication, and these 2 complexes show differential sensitivity to rapamycin. Here we combine functional genetics and pharmacological treatments to analyze rapamycin-sensitive mTOR substrates that are involved in cell proliferation and tissue regeneration after partial hepatectomy in mice. After hepatectomy, hepatocytes proliferated rapidly, correlating with increased S6 kinase phosphorylation, while treatment with rapamycin derivatives impaired regeneration and blocked S6 kinase activation. In addition, genetic deletion of S6 kinase 1 (S6K1) caused a delay in S phase entry in hepatocytes after hepatectomy. The proliferative defect of S6K1-deficient hepatocytes was cell autonomous, as it was also observed in primary cultures and hepatic overexpression of S6K1-rescued proliferation. We found that S6K1 controlled steady-state levels of cyclin D1 (Ccnd1) mRNA in liver, and cyclin D1 expression was required to promote hepatocyte cell cycle. Notably, in vivo overexpression of cyclin D1 was sufficient to restore the proliferative capacity of S6K-null livers. The identification of an S6K1-dependent mechanism participating in cell proliferation in vivo may be relevant for cancer cells displaying high mTOR complex 1 activity and cyclin D1 accumulation.

Espeillac, Catherine; Mitchell, Claudia; Celton-Morizur, Severine; Chauvin, Celine; Koka, Vonda; Gillet, Cynthia; Albrecht, Jeffrey H.; Desdouets, Chantal; Pende, Mario

2011-01-01

91

ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase  

PubMed Central

It has been proposed that cell growth and autophagy are coordinated in response to cellular nutrient status, but the relationship between them is not fully understood. Here, we have characterized the fly mutants of Autophagy-specific gene 1 (ATG1), an autophagy-regulating kinase, and found that ATG1 is a negative regulator of the target of rapamycin (TOR)/S6 kinase (S6K) pathway. Our Drosophila studies have shown that ATG1 inhibits TOR/S6K-dependent cell growth and development by interfering with S6K activation. Consistently, overexpression of ATG1 in mammalian cells also markedly inhibits S6K in a kinase activity-dependent manner, and short interfering RNA-mediated knockdown of ATG1 induces ectopic activation of S6K and S6 phosphorylation. Moreover, we demonstrated that ATG1 specifically inhibits S6K activity by blocking phosphorylation of S6K at Thr 389. Taken together, our genetic and biochemical results strongly indicate crosstalk between autophagy and cell growth regulation.

Lee, Sung Bae; Kim, Sunhong; Lee, Jiwoon; Park, Jeehye; Lee, Gina; Kim, Yongsung; Kim, Jin-Man; Chung, Jongkyeong

2007-01-01

92

CDNA cloning and characterization of S6 kinase and its effect on yolk protein gene expression in the oriental fruit fly Bactrocera dorsalis (Hendel).  

PubMed

p70 S6 kinase (S6K), a serine/threonine protein kinase, is a downstream target of target of rapamycin (TOR) gene and an important regulator of protein synthesis responsible for cell growth and reproduction. In this study, a S6K gene, named BdS6K (GenBank Accession No. GQ203802), was isolated from the oriental fruit fly Bactrocera dorsalis (Hendel). Quantitative RT-PCR showed that BdS6K mRNA is expressed at a higher level in egg than in other developmental stages, as well as in ovary than in fat body. Downregulation of BdS6K activity by rapamycin treatment in larval stage resulted in the developmental defects of larvae, pupae, and adults, with a reduced yolk protein (YP) expression in the fat body throughout the first reproductive cycle with a substantial reduction in ovary size, and also repressed the egg development in female fruit fly. Knockdown of BdS6K gene by RNA interference in the adult significantly decreased the YP expression. These observations support the involvement of BdS6K signaling in the regulation of the YP synthesis and egg development in B. dorsalis. PMID:22105664

Suganya, R; Chen, Shiu-Ling; Lu, Kuang-Hui

2011-12-01

93

Phytohormones Participate in an S6 Kinase Signal Transduction Pathway in Arabidopsis1  

PubMed Central

Addition of fresh medium to stationary cells of Arabidopsis suspension culture induces increased phosphorylation of the S6 ribosomal protein and activation of its cognate kinase, AtS6k. Analysis of the activation response revealed that medium constituents required for S6 kinase activation were the phytohormones 1-naphthylacetic acid (auxin) and kinetin. Pretreatment of cells with anti-auxin or PI3-kinase drugs inhibited this response. Consistent with these findings, LY294002, a PI3-kinase inhibitor, efficiently suppressed phytohormone-induced S6 phosphorylation and translational up-regulation of ribosomal protein S6 and S18A mRNAs without affecting global translation. These data indicate that (1) activation of AtS6k is regulated by phytohormones, at least in part, via a lipid kinase-dependent pathway, that (2) the translational regulation of ribosomal proteins appears to be conserved throughout the plant and animal kingdom, and that (3) these events are hallmarks of a growth-related signal transduction pathway novel in plants.

Turck, Franziska; Zilbermann, Frederic; Kozma, Sara C.; Thomas, George; Nagy, Ferenc

2004-01-01

94

Role of the phosphatidylinositol 3-kinase\\/Akt and mTOR\\/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma  

Microsoft Academic Search

Multiple myeloma (MM) is a plasma cell malignancy preliminary localized in the bone marrow and characterized by its capacity to disseminate. IL-6 and IGF-1 have been shown to mediate proliferative and anti-apoptotic signals in plasmocytes. However, in primary plasma-cell leukemia (PCL) and in end-stage aggressive extramedullar disease, the cytokine requirement for both effects may be not mandatory. This suggests that

Frédéric Pene; Yann-Erick Claessens; Odile Muller; Franck Viguié; Patrick Mayeux; François Dreyfus; Catherine Lacombe; Didier Bouscary

2002-01-01

95

A membrane-bound protein kinase from rabbit reticulocytes is an active form of multipotential S6 kinase  

Microsoft Academic Search

An active ribosomal protein S6 kinase has been highly purified from the membranes of rabbit reticulocytes by chromatography of the Triton X-100 extract on DEAE-cellulose, SP-Sepharose Fast Flow, and by FPLC on Mono Q and Superose-12. The S6 kinase elutes around 40 000 daltons upon gel filtration on Superose-12 or Sephacryl S-200. It has a subunit molecular weight of 40–43

Eric S. Bensen; Jason L. Umphress; Jolinda A. Traugh; Lorenzo A. Pinna; Polygena T. Tuazon

1996-01-01

96

Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1  

SciTech Connect

The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added at a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector.

Park, In-Hyun [Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States)]. E-mail: ihpark@uiuc.edu; Erbay, Ebru [Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Nuzzi, Paul [Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Chen Jie [Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States)

2005-09-10

97

Genetic ablation of S6-kinase does not prevent processing of SREBP1.  

PubMed

The SREBP family of transcription factors regulates the expression of genes involved in fatty acid and cholesterol biosynthesis. The activation of SREBP transcription factors requires proteolytic cleavage of the inactive precursor and nuclear translocation of the mature form of the protein. It has been shown that nuclear accumulation of the mature form of SREBP1 is induced in response to activation of the serine/threonine kinase Akt, an important effector of the Ras/PI3-kinase signalling pathway. Activation of SREBP by Akt depends on the mammalian target of rapamycin complex 1 (mTORC1) but the exact mechanism of this activation remains unclear. We have investigated whether ablation of different signalling molecules downstream of mTORC1 affects expression of SREBP targets genes. We could show that inhibition of S6-kinases 1 and 2 expression using RNA interference did not block induction of expression of fatty acid synthase (FASN) or ATP-citrate lyase (ACLY) following activation of Akt in human retinal pigment epithelial cells. Furthermore, accumulation of mature SREBP1 was not inhibited after combined silencing of S6-kinases 1 and 2. Genetic ablation of both kinases also did not prevent the formation of mature SREBP1 in mouse embryonic fibroblasts. Taken together, these results suggest that S6-kinases 1 and 2 are dispensable for the induction of SREBP processing in the experimental systems used here. PMID:21093473

Lewis, Caroline A; Griffiths, Beatrice; Santos, Claudio R; Pende, Mario; Schulze, Almut

2010-11-18

98

S6K in geroconversion.  

PubMed

Markers of cellular senescence depend in part on the MTOR (mechanistic target of rapamycin) pathway. MTOR participates in geroconversion, a conversion from reversible cell cycle arrest to irreversible senescence. Recently we demonstrated that hyper-induction of cyclin D1 during geroconversion was mostly dependent on MEK, whereas rapamycin only partially inhibited cyclin D1 accumulation. Here we show that, while not affecting cyclin D1, siRNA for p70S6K partially prevented loss of RP (replicative/regenerative potential) during p21-induced cell cycle arrest. Similarly, an inhibitor of p70 S6 kinase (PF-4708671) partially inhibited phosphorylation of S6 and preserved RP, while only marginally prevented cyclin D1 induction. Thus S6K and MEK play different roles in geroconversion. PMID:24036549

Leontieva, Olga V; Demidenko, Zoya N; Blagosklonny, Mikhail V

2013-09-09

99

CARDIAC OVEREXPRESSION OF METALLOTHIONEIN RESCUES CHRONIC ALCOHOL INTAKE-INDUCED CARDIOMYOCYTE DYSFUNCTION: ROLE OF AKT, MAMMALIAN TARGET OF RAPAMYCIN AND RIBOSOMAL P70S6 KINASE  

Microsoft Academic Search

Aims: Reduced insulin sensitivity following alcohol intake plays a role in alcohol-induced organ damage although its precise mechanism is undefined. This study was designed to examine the effect of cardiac overexpression of the antioxidant metalloth- ionein on alcohol-induced cardiac contractile dysfunction and post-receptor insulin signaling. Methods: FVB and metallothionein mice were fed a 4% alcohol diet for 16 weeks. Cardiomyocyte

QUN LI; JUN REN

100

Constitutively active Akt1 expression in mouse pancreas requires S6 kinase 1 for insulinoma formation  

PubMed Central

Factors that promote pancreatic ? cell growth and function are potential therapeutic targets for diabetes mellitus. In mice, genetic experiments suggest that signaling cascades initiated by insulin and IGFs positively regulate ? cell mass and insulin secretion. Akt and S6 kinase (S6K) family members are activated as part of these signaling cascades, but how the interplay between these proteins controls ? cell growth and function has not been determined. Here, we found that although transgenic mice overexpressing the constitutively active form of Akt1 under the rat insulin promoter (RIP-MyrAkt1 mice) had enlarged ? cells and high plasma insulin levels, leading to improved glucose tolerance, a substantial proportion of the mice developed insulinomas later in life, which caused decreased viability. This oncogenic transformation tightly correlated with nuclear exclusion of the tumor suppressor PTEN. To address the role of the mammalian target of rapamycin (mTOR) substrate S6K1 in the MyrAkt1-mediated phenotype, we crossed RIP-MyrAkt1 and S6K1-deficient mice. The resulting mice displayed reduced insulinemia and glycemia compared with RIP-MyrAkt1 mice due to a combined effect of improved insulin secretion and insulin sensitivity. Importantly, although the increase in ? cell size in RIP-MyrAkt1 mice was not affected by S6K1 deficiency, the hyperplastic transformation required S6K1. Our results therefore identify S6K1 as a critical element for MyrAkt1-induced tumor formation and suggest that it may represent a useful target for anticancer therapy downstream of mTOR.

Alliouachene, Samira; Tuttle, Robyn L.; Boumard, Stephanie; Lapointe, Thomas; Berissi, Sophie; Germain, Stephane; Jaubert, Francis; Tosh, David; Birnbaum, Morris J.; Pende, Mario

2008-01-01

101

Neutral Sphingomyelinase-2 Mediates Growth Arrest by Retinoic Acid through Modulation of Ribosomal S6 Kinase*  

PubMed Central

All-trans-retinoic acid (ATRA) induces growth arrest of many cell types. Previous studies have reported that ATRA can modulate cellular sphingolipids, but the role of sphingolipids in the ATRA response is not clear. Using MCF-7 cells as a model system, we show that ATRA stimulates an increase in ceramide levels followed by G0/G1 growth arrest. Notably, induction of nSMase2 was the primary effect of ATRA on the sphingolipid network and was both time- and dose-dependent. Importantly, pretreatment with nSMase2 siRNA significantly inhibited ATRA effects on ceramide levels and growth arrest. In contrast, nSMase2 overexpression was sufficient to increase ceramide levels and induce G0/G1 growth arrest of asynchronous MCF-7 cells. Surprisingly, neither ATRA stimulation nor nSMase2 overexpression had significant effects on classical cell cycle regulators such as p21/WAF1 or retinoblastoma. In contrast, ATRA suppressed phosphorylation of ribosomal S6 kinase (S6K) and its downstream targets S6 and eIF4B. Importantly, these effects were significantly inhibited by nSMase2 siRNA. Reciprocally, nSMase2 overexpression was sufficient to suppress S6K phosphorylation and signaling. Notably, neither ATRA effects nor nSMase2 effects on S6K phosphorylation required the ceramide-activated protein phosphatase PP2A, previously identified as important for S6K regulation. Finally, nSMase2 overexpression was sufficient to decrease translation as measured by methionine incorporation and analysis of polyribosome profiles. Taken together, these results implicate nSMase2 as a major component of ATRA-induced growth arrest of MCF-7 cells and identify S6K as a novel downstream target of nSMase2.

Clarke, Christopher J.; Mediwala, Krutika; Jenkins, Russell W.; Sutton, Che A.; Tholanikunnel, Baby G.; Hannun, Yusuf A.

2011-01-01

102

Insulin activates a 70-kDa S6 kinase through serine/threonine-specific phosphorylation of the enzyme polypeptide  

SciTech Connect

The dominant insulin-stimulated ribosomal protein S6 kinase activity was purified to near homogeneity from insulin-treated {sup 32}P-labeled rat H4 hepatoma cells and found to copurify with a 70-kDa {sup 32}P-labeled polypeptide. The dominant S6 kinase purified from livers of cycloheximide-treated rats is also a 70-kDa polypeptide. Antiserum raised against rat liver S6 kinase specifically immunoprecipitates the purified {sup 32}P-labeled H4 hepatoma insulin-stimulated S6 kinase. Immune complexes prepared from the cytosol of {sup 32}P-labeled H4 cells contain several {sup 32}P-labeled polypeptides. Insulin treatment increases the {sup 32}P content of the immunoprecipitated 70-kDa S6 kinase polypeptide 3- to 4-fold over basal levels. Tryptic peptide maps indicate that the insulin-stimulated S6 kinase purified from {sup 32}P-labeled H4 cells is phosphorylated at multiple sites distinct from those which participate in autophosphorylation in vitro. The S6 kinases purified from liver of cycloheximide-treated rat and H4 hepatoma insulin-stimulated enzyme are each completely deactivated by incubation with protein phosphatase type 2A in both autophosphorylating and 40S S6 phosphorylating activities. Thus insulin activates the 70-kDa S6 kinase by promoting phosphorylation of specific serine/threonine residues on the enzyme polypeptide, probably through activating an as-yet-unidentified serine/threonine protein kinase distinct from microtubule-associated protein 2 kinase.

Price, D.J.; Gunsalus, J.R.; Avruch, J. (Harvard Medical School, Boston, MA (USA))

1990-10-01

103

Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation.  

PubMed Central

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network. Images

Cheatham, B; Vlahos, C J; Cheatham, L; Wang, L; Blenis, J; Kahn, C R

1994-01-01

104

Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1-E2F pathway activity  

PubMed Central

The 40S ribosomal protein S6 kinase (S6K) is a conserved component of signalling pathways controlling growth in eukaryotes. To study S6K function in plants, we isolated single- and double-knockout mutations and RNA-interference (RNAi)-silencing lines in the linked Arabidopsis S6K1 and S6K2 genes. Hemizygous s6k1s6k2/++ mutant and S6K1 RNAi lines show high phenotypic instability with variation in size, increased trichome branching, produce non-viable pollen and high levels of aborted seeds. Analysis of their DNA content by flow cytometry, as well as chromosome counting using DAPI staining and fluorescence in situ hybridization, revealed an increase in ploidy and aneuploidy. In agreement with this data, we found that S6K1 associates with the Retinoblastoma-related 1 (RBR1)–E2FB complex and this is partly mediated by its N-terminal LVxCxE motif. Moreover, the S6K1–RBR1 association regulates RBR1 nuclear localization, as well as E2F-dependent expression of cell cycle genes. Arabidopsis cells grown under nutrient-limiting conditions require S6K for repression of cell proliferation. The data suggest a new function for plant S6K as a repressor of cell proliferation and required for maintenance of chromosome stability and ploidy levels.

Henriques, Rossana; Magyar, Zoltan; Monardes, Antonia; Khan, Safina; Zalejski, Christine; Orellana, Juan; Szabados, Laszlo; de la Torre, Consuelo; Koncz, Csaba; Bogre, Laszlo

2010-01-01

105

The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells  

Microsoft Academic Search

Background: Protein kinase B (PKB), and the p70 and p90 ribosomal S6 kinases (p70 S6 kinase and p90 Rsk, respectively), are activated by phosphorylation of two residues, one in the ‘T-loop’ of the kinase domain and, the other, in the hydrophobic motif carboxy terminal to the kinase domain. The 3-phosphoinositide-dependent protein kinase 1 (PDK1) activates many AGC kinases in vitro

Michayla R. Williams; J. Simon C. Arthur; Anudharan Balendran; Jeroen van der Kaay; Valeria Poli; Philip Cohen; Dario R. Alessi

2000-01-01

106

Growth hormone activates mitogen-activated protein kinase and S6 kinase and promotes intracellular tyrosine phosphorylation in 3T3-F442A preadipocytes.  

PubMed Central

Physiological concentrations of growth hormone induced a rapid and transient activation of mitogen-activated protein kinase (MAP kinase) and S6 kinase in 3T3-F442A preadipocytes. These effects were abrogated by staurosporine and in cells chronically pretreated with phorbol esters, suggesting that protein kinase C is involved in the mechanism of activation. In addition, three cytosolic proteins exhibited a growth-hormone-dependent increase in tyrosine phosphorylation. Images Fig. 2.

Anderson, N G

1992-01-01

107

Novel 5'TOPmRNAs regulated by ribosomal S6 kinase are important for cardiomyocyte development: S6 kinase suppression limits cardiac differentiation and promotes pluripotent cells toward a neural lineage.  

PubMed

Moving stem cells from bench to bedside has been a challenging task. Undermining this task is comprehending and optimizing the underlying regulatory mechanisms that drive differentiation of stem cells into desired cell and tissue types. Here we present evidence that ribosomal S6 kinase (S6K) is among the proteins upregulated as embryonic stem cells (ESCs) and human induced pluripotent stem cells differentiate into beating cardiomyocytes. We hypothesized that S6K plays a pivotal role in cardiomyogenesis, primarily because it regulates the translation of 3 cardiac-involved genes recently shown to have 5' terminal oligopyrimidine (5'TOP) sequences: connexin 43 (Cx43), desmoplakin (Dsp), and phosphatase and tensin homolog (PTEN). Along with another independent laboratory, we confirmed that S6K is indeed upregulated in beating ESC-derived cardiomyocytes compared to the surrounding nonbeating, differentiated cells. S6K short interfering RNA-transfected stem cell cultures indicate that inhibition of S6K strongly hinders development of cardiomyocyte beating and translation of Cx43, Dsp, and PTEN; these cardiac 5'TOP mRNAs were only properly translated in cells with S6K, supporting our hypothesis. An unexpected discovery took the role of S6K one step further: S6K-knockdown stem cell cultures developed significantly more neurons than seen in embryoid bodies subjected to a typical cardiac differentiation protocol. These results introduced the novel idea that in addition to its critical cardiac roles, S6K may be a significant factor that prevents stem cells from pursuing a neuronal pathway. Overall, results have indicated the necessity of S6K for normal stem cell cardiomyogenesis, as well as lowered S6K expression for stem cell neurogenesis. PMID:22165977

Li, LeeAnn; Larabee, Shannon M; Chen, Shenglin; Basiri, Ladan; Yamaguchi, Seiji; Zakaria, Asif; Gallicano, G Ian

2012-02-08

108

S6 kinase in quiescent Swiss mouse 3T3 cells is activated by phosphorylation in response to serum treatment  

SciTech Connect

To investigate the role of phosphorylation in the activation of S6 kinase, the enzyme was isolated from {sup 32}P-labeled Swiss mouse 3T3 cells before and after stimulation with serum. The kinase activity was followed through several purification steps, and a radioactive protein of M{sub r} 70,000 was obtained from the stimulated cells. This band was not detected in resting cells. The M{sub r} 70,000 protein exhibited the same size upon NaDodSO{sub 4}/PAGE as the homogeneous kinase, and it comigrated with the in vitro autophosphorylated form of the enzyme. Treatment of the in vivo-labeled material with phosphatase 2A led to a loss of kinase activity concomitant with a release of {sup 32}P{sub i} from the M{sub r} 70,000 protein. The partially dephosphorylated protein migrated faster during PAGE, displaying distinct species of M{sub r} 69,000 and 68,000. Most importantly, phospho amino acid analysis of the labeled S6 kinase showed only phosphoserine and phosphothreonine. These results argue that the S6 kinase is phosphorylated at multiple sites in vivo and that it is activated by serine/threonine phosphorylation.

Ballou, L.M.; Siegmann, M.; Thomas, G. (Friedrich Miescher-Institut, Basel (Switzerland))

1988-10-01

109

Activation of pp70/85 S6 kinases in interleukin-2-responsive lymphoid cells is mediated by phosphatidylinositol 3-kinase and inhibited by cyclic AMP.  

PubMed Central

Activation of phosphatidylinositol 3-kinase (PI3K) and activation of the 70/85-kDa S6 protein kinases (alpha II and alpha I isoforms, referred to collectively as pp70S6k) have been independently linked to the regulation of cell proliferation. We demonstrate that these kinases lie on the same signalling pathway and that PI3K mediates the activation of pp70 by the cytokine interleukin-2 (IL-2). We also show that the activation of pp70S6k can be blocked at different points along the signalling pathway by using specific inhibitors of T-cell proliferation. Inhibition of PI3K activity with structurally unrelated but highly specific PI3K inhibitors (wortmannin or LY294002) results in inhibition of IL-2-dependent but not phorbol ester (conventional protein kinase C [cPKC])-dependent pp70S6k activation. The T-cell immunosuppressant rapamycin potently antagonizes IL-2-(PI3K)- and phorbol ester (cPKC)-mediated activation of pp70S6k. Thus, wortmannin and rapamycin antagonize IL-2-mediated activation of pp70S6k at distinct points along the PI3K-regulated signalling pathway, or rapamycin antagonizes another pathway required for pp70S6k activity. Agents that raise the concentration of intracellular cyclic AMP (cAMP) and activate cAMP-dependent protein kinase (PKA) also inhibit IL-2-dependent activation of pp70S6k. In this case, inhibition appears to occur at least two points in this signalling path. Like rapamycin, PKA appears to act downstream of cPKC-mediated pp70S6k activation, and like wortmannin, PKA antagonizes IL-2-dependent activation of PI3K. The results with rapamycin and wortmannin are of added interest since the yeast and mammalian rapamycin targets resemble PI3K in the catalytic domain.

Monfar, M; Lemon, K P; Grammer, T C; Cheatham, L; Chung, J; Vlahos, C J; Blenis, J

1995-01-01

110

Cloning of the mitogen-activated S6 kinase from rat liver reveals an enzyme of the second messenger subfamily  

SciTech Connect

Recently the authors reported the purification of a mitogen-activated S6 kinase from Swiss mouse 3T3 fibroblasts and rat liver. The rat liver protein was cleaved with cyanogen bromide or trypsin and 17 of the resulting peptides were sequenced. DNA primers were generated from 3 peptides that had homology to sequences of the conserved catalytic domain of protein kinases. These primers were used in the polymerase chain reaction to obtain a 0.4-kilobase DNA fragment. This fragment was either radioactively labeled and hybridized to Northern blots of poly(A){sup {sup plus}} mRNA or used to screen a rat liver cDNA library. Northern blot analysis revealed four transcripts of 2.5, 3.2, 4.0, and 6.0 kilobases, and five S6 kinase clones were obtained by screening the library. Only two of the clones, which were identical, encoded a full-length protein. This protein had a molecular weight of 56,160, which correlated closely to that of the dephosphorylated kinase determined by SDS/PAGE. The catalytic domain of the kinase resembles that of other serine/threonine kinases belonging to the second messenger subfamily of protein kinases.

Kozma, S.C.; Ferrari, S. Bassand, P.; Siegmann, M.; Thomas, G. (Friedrich Miescher Institute, Basel (Switzerland)); Totty, N. (Ludwig Institute for Cancer Research, London (United Kingdom))

1990-10-01

111

Identical M sub r 70,000 S6 kinase is activated biphasically by epidermal growth factor: A phosphopeptide that characterizes the late phase  

SciTech Connect

Mitogenic stimulation of quiescent mouse 3T3 cells with epidermal growth factor leads to biphasic S6 kinase activation. The kinases present in both phases of the response have been purified from {sup 32}P-labeled cells and shown to contain a phosphoprotein of equivalent M{sub r} 70,000. Chromatographic analysis of the purified S6 kinases on a Mono Q column reveals that (1) all {sup 32}P-labeled protein coelutes with S6 kinase activity, (2) only those fractions containing S6 kinase autophosphorylate, (3) autophosphorylation is restricted to a single M{sub r} 70,000 protein, and (4) the extent of autophosphorylation directly parallels the degree of S6 kinase activation. Analysis of the two autophosphorylated S6 kinases by two-dimensional tryptic phosphopeptide mapping indicates that they are the same protein. Both in vivo {sup 32}P-labeled S6 kinase contain phosphoserine and phosphothreonine but no detectable phosphotyrosine. Two-dimensional tryptic peptide maps of the in vivo {sup 32}P-labeled S6 kinases are essentially identical, except for a single qualitative change in the late-phase S6 kinase.

Susa, M.; Thomas, G. (Friedrich Miescher Inst., Basel (Switzerland))

1990-09-01

112

Endotoxicosis induced by Coxiella burnetii lipopolysaccharide stimulates a ribosomal protein S6 kinase: some properties of the partially purified enzyme.  

PubMed Central

Guinea pig endotoxicosis induced by lipopolysaccharide from Coxiella burnetii Nine Mile phase I stimulates phosphorylation of liver ribosomal protein S6, with a 50% increase at 12 h postinoculation. The responsible protein kinase (S6PK) has been partially purified from liver; its activity is independent of cyclic AMP and of Ca2+ plus phosphatidyl serine or diacylglycerol. The preparation has an apparent optimum concentration of 20 mM Mg2+, while Ca2+ and Mn2+ are each inhibitory at 2 mM. The apparent Km for ATP is 30 microM with intact ribosomes. Because of the central role of phosphorylation in metabolic regulation and a purported role of phosphorylated S6 in protein synthesis, the lipopolysaccharide-induced stimulation of S6PK suggests a significant regulatory role of such enzymes in the pathobiochemistry of Q fever infection and endotoxicosis. Images

Picking, W D; Hackstadt, T; Paretsky, D

1989-01-01

113

The unusual mechanism of inhibition of the p90 ribosomal S6 kinase (RSK) by flavonol rhamnosides.  

PubMed

All known protein kinases share a bilobal kinase domain with well conserved structural elements. Because of significant structural similarities of nucleotide binding pocket, the development of highly selective kinase inhibitors is a very challenging task. Flavonols, naturally occurring plant metabolites, have long been known to inhibit kinases by mimicking the adenine moiety. Interestingly, recent data show that some flavonol glycosides are more selective, although underlying mechanisms were unknown. Crystallographic data from our laboratory revealed that the N-terminal kinase domain of p90 ribosomal S6 kinase, isoform 2, binds three different flavonol rhamnosides in a highly unusual manner, distinct from other kinase inhibitor interactions. The kinase domain undergoes a reorganization of several structural elements in response to the binding of the inhibitors. Specifically, the main ?-sheet of the N-lobe undergoes a twisting rotation by ~56° around an axis passing through the N- and C-lobes, leading to the restructuring of the canonical ATP-binding pocket into pockets sterically adapted to the inhibitor shape. The flavonol rhamnosides appear to adopt compact, but strained conformations with the rhamnose moiety swept under the B-ring of flavonol, unlike the structure of the free counterparts in solution. These data suggest that the flavonol glycoside scaffold could be used as a template for new inhibitors selective for the RSK family. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012). PMID:23541530

Utepbergenov, Darkhan; Derewenda, Zygmunt S

2013-03-27

114

Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis.  

PubMed Central

A phosphorothioate-oligonucleotide-based antisense strategy for depleting MAP kinase was developed. The 17mer antisense probe, EAS 1, caused a potent and concentration-dependent decrease in the steady state expression of p42 and p44 MAP kinase in 3T3 L1 fibroblasts and adipocytes with submicromolar concentrations effective. Antisense EAS 1 elicited a dose-dependent inhibition of insulin- and serum-stimulated DNA synthesis. Elimination of p42 MAP kinase by > 95% and p44 MAP kinase to levels undetected blocked the ability of serum in 3T3 L1 fibroblasts and insulin in 3T3 L1 adipocytes to stimulate DNA synthesis by 87-95%. The differentiation of 3T3 L1 fibroblasts into adipocytes was prevented by 1 microM antisense EAS 1. The corresponding sense, scrambled or sense plus antisense EAS 1 phosphorothioate oligonucleotides did not deplete the p42 or p44 MAP kinase from either cell type, did not inhibit stimulation of DNA synthesis and did not interfere with differentiation. Two kinases on different MAP kinase activation pathways were not depleted by antisense EAS 1 whereas the ability of insulin to activate p90 S6 kinase was > 90% eliminated in 3T3 L1 adipocytes by 4.5 microM antisense EAS 1. In conclusion these results show that MAP kinase is required for insulin and serum stimulation of DNA synthesis, for insulin stimulation of p90 S6 kinase activity and for differentiation of 3T3 L1 cells. Moreover, the development of the antisense probe EAS 1 against a target sequence of p42 MAP kinase that is conserved in p44 MAP kinase and across a range of species provides a molecular tool of general applicability for further dissecting the precise targets and roles of MAP kinase. Images

Sale, E M; Atkinson, P G; Sale, G J

1995-01-01

115

Involvement of 90-kuD ribosomal S6 kinase in collagen type I expression in rat hepatic fibrosis  

Microsoft Academic Search

AIM: To investigate the relationship between 90-kuD ribosomal S6 kinase (p90RSK) and collagen type ? expression during the development of hepatic fibrosis in vivo and in vitro . METHODS: Rat hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. The protein expression and cell location of p90RSK and their relationship with collagen type ? were determined by co-immunofluoresence and confocal

Miao-Fang Yang; Jun Xie; Xiao-Yi Gu; Xiao-Hua Zhang; Andrew K Davey; Shuang-Jie Zhang; Ji-Ping Wang; Ren-Min Zhu

2009-01-01

116

S6 kinase localizes to the presynaptic active zone and functions with PDK1 to control synapse development  

PubMed Central

The dimensions of neuronal dendrites, axons, and synaptic terminals are reproducibly specified for each neuron type, yet it remains unknown how these structures acquire their precise dimensions of length and diameter. Similarly, it remains unknown how active zone number and synaptic strength are specified relative the precise dimensions of presynaptic boutons. In this paper, we demonstrate that S6 kinase (S6K) localizes to the presynaptic active zone. Specifically, S6K colocalizes with the presynaptic protein Bruchpilot (Brp) and requires Brp for active zone localization. We then provide evidence that S6K functions downstream of presynaptic PDK1 to control synaptic bouton size, active zone number, and synaptic function without influencing presynaptic bouton number. We further demonstrate that PDK1 is also a presynaptic protein, though it is distributed more broadly. We present a model in which synaptic S6K responds to local extracellular nutrient and growth factor signaling at the synapse to modulate developmental size specification, including cell size, bouton size, active zone number, and neurotransmitter release.

Cheng, Ling; Locke, Cody

2011-01-01

117

The C-terminal Kinase and ERK-binding Domains of Drosophila S6KII (RSK) Are Required for Phosphorylation of the Protein and Modulation of Circadian Behavior*  

PubMed Central

A detailed structure/function analysis of Drosophila p90 ribosomal S6 kinase (S6KII) or its mammalian homolog RSK has not been performed in the context of neuronal plasticity or behavior. We previously reported that S6KII is required for normal circadian periodicity. Here we report a site-directed mutagenesis of S6KII and analysis of mutants, in vivo, that identifies functional domains and phosphorylation sites critical for the regulation of circadian period. We demonstrate, for the first time, a role for the S6KII C-terminal kinase that is independent of its known role in activation of the N-terminal kinase. Both S6KII C-terminal kinase activity and its ERK-binding domain are required for wild-type circadian period and normal phosphorylation status of the protein. In contrast, the N-terminal kinase of S6KII is dispensable for modulation of circadian period and normal phosphorylation of the protein. We also show that particular sites of S6KII phosphorylation, Ser-515 and Thr-732, are essential for normal circadian behavior. Surprisingly, the phosphorylation of S6KII residues, in vivo, does not follow a strict sequential pattern, as implied by certain cell-based studies of mammalian RSK protein.

Tangredi, Michelle M.; Ng, Fanny S.; Jackson, F. Rob

2012-01-01

118

Structural Diversity of the Active N-Terminal Kinase Domain of p90 Ribosomal S6 Kinase 2  

PubMed Central

The p90 ribosomal protein kinase 2 (RSK2) is a highly expressed Ser/Thr kinase activated by growth factors and is involved in cancer cell proliferation and tumor promoter-induced cell transformation. RSK2 possesses two non-identical kinase domains, and the structure of its N-terminal domain (NTD), which is responsible for phosphorylation of a variety of substrates, is unknown. The crystal structure of the NTD RSK2 was determined at 1.8 Å resolution in complex with AMP-PNP. The N-terminal kinase domain adopted a unique active conformation showing a significant structural diversity of the kinase domain compared to other kinases. The NTD RSK2 possesses a three-stranded ?B-sheet inserted in the N-terminal lobe, resulting in displacement of the ?C-helix and disruption of the Lys-Glu interaction, classifying the kinase conformation as inactive. The purified protein was phosphorylated at Ser227 in the T-activation loop and exhibited in vitro kinase activity. A key characteristic is the appearance of a new contact between Lys216 (?B-sheet) and the ?-phosphate of AMP-PNP. Mutation of this lysine to alanine impaired both NTDs in vitro and full length RSK2 ex vivo activity, emphasizing the importance of this interaction. Even though the N-terminal lobe undergoes structural re-arrangement, it possesses an intact hydrophobic groove formed between the ?C-helix, the ?4-strand, and the ?B-sheet junction, which is occupied by the N-terminal tail. The presence of a unique ?B-sheet insert in the N-lobe suggests a different type of activation mechanism for RSK2.

Liu, Kangdong; Zheng, Duo; D'Angelo, Igor; Shim, Jung-Hyun; Steinman, Valerie; Bode, Ann M.; Dong, Zigang

2009-01-01

119

Structural Diversity of the Active N-Terminal Kinase Domain of p90 Ribosomal S6 Kinase 2  

SciTech Connect

The p90 ribosomal protein kinase 2 (RSK2) is a highly expressed Ser/Thr kinase activated by growth factors and is involved in cancer cell proliferation and tumor promoter-induced cell transformation. RSK2 possesses two non-identical kinase domains, and the structure of its N-terminal domain (NTD), which is responsible for phosphorylation of a variety of substrates, is unknown. The crystal structure of the NTD RSK2 was determined at 1.8 {angstrom} resolution in complex with AMP-PNP. The N-terminal kinase domain adopted a unique active conformation showing a significant structural diversity of the kinase domain compared to other kinases. The NTD RSK2 possesses a three-stranded {beta}B-sheet inserted in the N-terminal lobe, resulting in displacement of the {alpha}C-helix and disruption of the Lys-Glu interaction, classifying the kinase conformation as inactive. The purified protein was phosphorylated at Ser227 in the T-activation loop and exhibited in vitro kinase activity. A key characteristic is the appearance of a new contact between Lys216 ({beta}B-sheet) and the {beta}-phosphate of AMP-PNP. Mutation of this lysine to alanine impaired both NTDs in vitro and full length RSK2 ex vivo activity, emphasizing the importance of this interaction. Even though the N-terminal lobe undergoes structural re-arrangement, it possesses an intact hydrophobic groove formed between the {alpha}C-helix, the {beta}4-strand, and the {beta}B-sheet junction, which is occupied by the N-terminal tail. The presence of a unique {beta}B-sheet insert in the N-lobe suggests a different type of activation mechanism for RSK2.

Malakhova, Margarita; Kurinov, Igor; Liu, Kangdong; Zheng, Duo; D'Angelo, Igor; Shim, Jung-Hyun; Steinman, Valerie; Bode, Ann M.; Dong, Zigang; (Cornell); (CLS); (UMM); (CSB)

2010-10-08

120

Potent and selective thiophene urea-templated inhibitors of S6K  

Microsoft Academic Search

S6K1 (p70 S6 kinase-1) is thought to play a critical role in the development of obesity and insulin resistance, thus making it an attractive target in developing medicines for the treatment of these disorders. We describe a novel thiophene urea class of S6K inhibitors. The lead matter for the development of these inhibitors came from mining the literature for reports

Ping Ye; Cyrille Kuhn; Miret Juan; Rahul Sharma; Brendan Connolly; Gordon Alton; Hu Liu; Robert Stanton; Natasha M. Kablaoui

2011-01-01

121

Identification of the general transcription factor Yin Yang 1 as a novel and specific binding partner for S6 kinase 2.  

PubMed

S6 kinase is a member of the AGC family of serine/threonine kinases and plays a key role in diverse cellular processes including cell growth and metabolism. Although, the high degree of homology between S6K family members (S6K1 and S6K2) in kinase and kinase-extension domains, the two proteins are highly divergent in the N- and C-terminal regulatory regions, hinting at differential regulation, downstream signalling and cellular function. Deregulated signalling via S6Ks has been linked to various human pathologies, such as diabetes and cancer. Therefore, S6K has emerged as a promising target for drug development. Much of what we know about S6K signalling in health and disease comes from studies of S6K1, as molecular cloning of this isoform was reported a decade earlier than S6K2. In this study, we report for the first time, the identification of the general transcription factor Yin Yang 1 (YY1) as a novel and specific binding partner of S6K2, but not S6K1. The interaction between YY1 and S6K2 was demonstrated by co-immunoprecipitation of transiently overexpressed and endogenous proteins in a number of cell lines, including HEK293, MCF7 and U937. Furthermore, direct association between S6K2 and YY1 was demonstrated by GST pull-down assay using recombinant proteins. A panel of deletion mutants was used to show that the C-terminal regulatory region of S6K2 mediates the interaction with YY1. Interestingly, the complex formation between S6K2 and YY1 can be detected in serum-starved cells, but the interaction is strongly induced in response to mitogenic stimulation. The induction of S6K2/YY1 complex formation in response to serum stimulation is abolished by pre-treatment of cells with the mTOR inhibitor, rapamycin. Furthermore, mTOR is also detected in complex with YY1 and S6K2 in serum-stimulated cells. We utilized size exclusion chromatography along with co-immunoprecipitation analysis to demonstrate the existence of the mTOR/S6K2/YY1 complex in high molecular weight fractions, which might also involve other cellular proteins. The physiological significance of the mTOR/S6K2/YY1 complex, which is induced in response to mitogenic stimulation, remains to be further investigated. PMID:23403125

Ismail, Heba M S; Myronova, Olena; Tsuchiya, Yugo; Niewiarowski, Andrew; Tsaneva, Irina; Gout, Ivan

2013-02-10

122

Arabidopsis TARGET OF RAPAMYCIN Interacts with RAPTOR, Which Regulates the Activity of S6 Kinase in Response to Osmotic Stress Signals  

PubMed Central

TARGET OF RAPAMYCIN (TOR) kinase controls many cellular functions in eukaryotic cells in response to stress and nutrient availability and was shown to be essential for embryonic development in Arabidopsis thaliana. We demonstrated that Arabidopsis RAPTOR1 (a TOR regulatory protein) interacts with the HEAT repeats of TOR and that RAPTOR1 regulates the activity of S6 kinase (S6K) in response to osmotic stress. RAPTOR1 also interacts in vivo with Arabidopsis S6K1, a putative substrate for TOR. S6K1 fused to green fluorescent protein and immunoprecipitated from tobacco (Nicotiana tabacum) leaves after transient expression was active in phosphorylating the Arabidopsis ribosomal S6 protein. The catalytic domain of S6K1 could be phosphorylated by Arabidopsis 3-phosphoinositide-dependent protein kinase-1 (PDK1), indicating the involvement of PDK1 in the regulation of S6K. The S6K1 activity was sensitive to osmotic stress, while PDK1 activity was not affected. However, S6K1 sensitivity to osmotic stress was relieved by co-overexpression of RAPTOR1. Overall, these observations demonstrated the existence of a functional TOR kinase pathway in plants. However, Arabidopsis seedlings do not respond to normal physiological levels of rapamycin, which appears to be due its inability to bind to the Arabidopsis homolog of FKBP12, a protein that is essential for the binding of rapamycin with TOR. Replacement of the Arabidopsis FKBP12 with the human FKBP12 allowed rapamycin-dependent interaction with TOR. Since homozygous mutation in TOR is lethal, it suggests that this pathway is essential for integrating the stress signals into the growth regulation.

Mahfouz, Magdy M.; Kim, Sunghan; Delauney, Ashton J.; Verma, Desh Pal S.

2006-01-01

123

Thiazolidinediones Inhibit Insulin-Like Growth Factor-I-Induced Activation of p70S6 Kinase and Suppress Insulin-Like Growth Factor-I Tumor-Promoting Activity  

Microsoft Academic Search

Thiazolidinediones are a novel class of antidiabetic drugs that improve insulin sensitivity in type 2 diabetic patients. Recently, these compounds have also been shown to suppress tumor development in several animal models. The molecular basis for their antitumor action, however, is largely unknown. We report here that oral administration of thiazolidinediones (rosiglitazone and troglitazone) remarkably inhibited insulin- like growth factor-I

Guobin He; John DiGiovanni; Susan M. Fischer

2006-01-01

124

Phosphatidylinositol3 kinase\\/Akt\\/p70 S6K\\/AP1 signaling pathway mediated benzo(a)pyrene-induced cell cycle alternation via cell cycle regulatory proteins in human embryo lung fibroblasts  

Microsoft Academic Search

Benzo(a)pyrene (B(a)P), a potent environmental procarcinogen, has been shown to cause cell cycle alternation. However, the mechanisms involved in this effect are not well understood yet. Our current results demonstrated that B(a)P exposure led to cell proliferation and a 33.5% increase in S phase cells as well as a 26.8% decrease in G1 phase cells in human embryo lung fibroblasts

Ai Gao; Bingci Liu; Xianglin Shi; Xiaowei Jia; Meng Ye; Shi Jiao; Baorong You; Chuanshu Huang

2007-01-01

125

Nickel Compounds Act through Phosphatidylinositol3-kinase\\/Akt-Dependent, p70S6k-Independent Pathway to Induce Hypoxia Inducible Factor Transactivation and Cap43 Expression in Mouse Epidermal Cl41 Cells  

Microsoft Academic Search

Nickel compounds are a somewhat unique class of carcinogens. Previ- ous studies have demonstrated that NiCl2 exposure leads to marked induction of hypoxia inducible factor 1 (HIF-1) in human osteosarcoma and BALB\\/c 3T3 cells, a transcription factor that has been considered to play an important role in tumor promotion and progression. However, the signal transduction pathways leading to HIF-1 induction

Jingxia Li; Gerard Davidson; Yi Huang; Bing-Hua Jiang; Xianglin Shi; Max Costa; Chuanshu Huang

126

Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1).  

PubMed

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in (1)H-(15)N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D(2)ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D(2)ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions. PMID:17980619

Ragan, Timothy J; Ross, Duncan B; Keshwani, Malik M; Harris, Thomas K

2007-10-01

127

p90 ribosomal S6 kinase 3 contributes to cardiac insufficiency in ?-tropomyosin Glu180Gly transgenic mice.  

PubMed

Myocardial interstitial fibrosis is an important contributor to the development of heart failure. Type 3 p90 ribosomal S6 kinase (RSK3) was recently shown to be required for concentric myocyte hypertrophy under in vivo pathological conditions. However, the role of RSK family members in myocardial fibrosis remains uninvestigated. Transgenic expression of ?-tropomyosin containing a Glu180Gly mutation (TM180) in mice of a mixed C57BL/6:FVB/N background induces a cardiomyopathy characterized by a small left ventricle, interstitial fibrosis, and diminished systolic and diastolic function. Using this mouse model, we now show that RSK3 is required for the induction of interstitial fibrosis in vivo. TM180 transgenic mice were crossed to RSK3 constitutive knockout (RSK3(-/-)) mice. Although RSK3 knockout did not affect myocyte growth, the decreased cardiac function and mild pulmonary edema associated with the TM180 transgene were attenuated by RSK3 knockout. The improved cardiac function was consistent with reduced interstitial fibrosis in the TM180;RSK3(-/-) mice as shown by histology and gene expression analysis, including the decreased expression of collagens. The specific inhibition of RSK3 should be considered as a potential novel therapeutic strategy for improving cardiac function and the prevention of sudden cardiac death in diseases in which interstitial fibrosis contributes to the development of heart failure. PMID:23913705

Passariello, Catherine L; Gayanilo, Marjorie; Kritzer, Michael D; Thakur, Hrishikesh; Cozacov, Zoharit; Rusconi, Francesca; Wieczorek, David; Sanders, Michael; Li, Jinliang; Kapiloff, Michael S

2013-08-02

128

Identification and characterization of a constitutively T-loop phosphorylated and active recombinant S6K1: Expression, purification, and enzymatic studies in a high capacity non-radioactive TR-FRET Lance assay  

Microsoft Academic Search

The p70 S6 ribosomal protein kinase 1 (S6K) is a substrate and effector of the mammalian target of rapamycin (mTOR). The mTOR\\/S6K pathway is implicated in cancer and metabolic disorders. To study the molecular regulation of S6K and identify specific inhibitors, availability of active recombinant S6K and robust enzyme assays are critically needed. To date, however, expression of active recombinant

Wei-Guo Zhang; Boris Shor; Ker Yu

2006-01-01

129

Involvement of 90-kuD ribosomal S6 kinase in collagen type I expression in rat hepatic fibrosis  

PubMed Central

AIM: To investigate the relationship between 90-kuD ribosomal S6 kinase (p90RSK) and collagen type?I?expression during the development of hepatic fibrosis in vivo and in vitro. METHODS: Rat hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. The protein expression and cell location of p90RSK and their relationship with collagen type?I?were determined by co-immunofluoresence and confocal microscopy. Subsequently, RNAi strategy was employed to silence p90RSK mRNA expression in HSC-T6, an activated hepatic stellate cell (HSC) line. The expression of collagen type?I?in HSC-T6 cells was assessed by Western blotting and real-time polymerase chain reaction. Furthermore, HSCs were transfected with expression vectors or RNAi constructs of p90RSK to increase or decrease the p90RSK expression, then collagen type?I?promoter activity in the transfected HSCs was examined by reporter assay. Lastly HSC-T6 cells transfected with p90RSK siRNA was treated with or without platelet-derived growth factor (PDGF)-BB at a final concentration of 20 ?g/L and the cell growth was determined by MTS conversion. RESULTS: In fibrotic liver tissues, p90RSK was over-expressed in activated HSCs and had a significant positive correlation with collagen type?I?levels. In HSC-T6 cells transfected with RNAi targeted to p90RSK, the expression of collagen type?I?was down-regulated (61.8% in mRNA, P < 0.01, 89.1% in protein, P < 0.01). However, collagen type?I?promoter activity was not increased with over-expression of p90RSK and not decreased with low expression either, compared with controls in the same cell line (P = 0.076). Furthermore, p90RSK siRNA exerted the inhibition of HSC proliferation, and also abolished the effect of PDGF on the HSC proliferation. CONCLUSION: p90RSK is over-expressed in activated HSCs and involved in regulating the abnormal expression of collagen type?I?through initiating the proliferation of HSCs.

Yang, Miao-Fang; Xie, Jun; Gu, Xiao-Yi; Zhang, Xiao-Hua; Davey, Andrew K; Zhang, Shuang-Jie; Wang, Ji-Ping; Zhu, Ren-Min

2009-01-01

130

Age- and Diet-Specific Effects of Variation at S6 Kinase on Life History, Metabolic, and Immune Response Traits in Drosophila melanogaster  

PubMed Central

Life history theory hypothesizes that genetically based variation in life history traits results from alleles that alter age-specific patterns of energy allocation among the competing demands of reproduction, storage, and maintenance. Despite the important role that alleles with age-specific effects must play in life history evolution, few naturally occurring alleles with age-specific effects on life history traits have been identified. A recent mapping study identified S6 kinase (S6k) as a candidate gene affecting lipid storage in Drosophila. S6k is in the target of rapamycin pathway, which regulates cell growth in response to nutrient availability and has also been implicated to influence many life history traits from fecundity to life span. In this article, we used quantitative complementation tests to examine the effect of allelic variation at S6k on a range of phenotypes associated with metabolism and fitness in an age-, diet-, and sex-specific manner. We found that alleles of S6k have pleiotropic effects on total protein levels, glycogen storage, life span, and the immune response and demonstrate that these allelic effects are age, diet, and sex specific. As many of the genes in the target of rapamycin pathway are evolutionarily conserved, our data suggest that genes in this pathway could play a pivotal role in life history evolution in a wide range of taxa.

Cho, Irene; Horn, Lucas; Felix, Tashauna M.; Foster, Leanne; Gregory, Gwendolyn; Starz-Gaiano, Michelle; Chambers, Michelle M.

2010-01-01

131

Tumor Suppressor PDCD4 Represses Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Proteins and Is Regulated by S6 Kinase 2  

PubMed Central

Apoptosis can be regulated by extracellular signals that are communicated by peptides such as fibroblast growth factor 2 (FGF-2) that have important roles in tumor cell proliferation. The prosurvival effects of FGF-2 are transduced by the activation of the ribosomal protein S6 kinase 2 (S6K2), which increases the expression of the antiapoptotic proteins X chromosome-linked Inhibitor of Apoptosis (XIAP) and Bcl-xL. We now show that the FGF-2–S6K2 prosurvival signaling is mediated by the tumor suppressor programmed cell death 4 (PDCD4). We demonstrate that PDCD4 specifically binds to the internal ribosome entry site (IRES) elements of both the XIAP and Bcl-xL messenger RNAs and represses their translation by inhibiting the formation of the 48S translation initiation complex. Phosphorylation of PDCD4 by activated S6K2 leads to the degradation of PDCD4 and thus the subsequent derepression of XIAP and Bcl-xL translation. Our results identify PDCD4 as a specific repressor of the IRES-dependent translation of cellular mRNAs (such as XIAP and Bcl-xL) that mediate FGF-2–S6K2 prosurvival signaling and provide further insight into the role of PDCD4 in tumor suppression.

Liwak, Urszula; Thakor, Nehal; Jordan, Lindsay E.; Roy, Rajat; Lewis, Stephen M.; Pardo, Olivier E.; Seckl, Michael

2012-01-01

132

Central administration of metformin into the third ventricle of C57BL/6 mice decreases meal size and number and activates hypothalamic S6 kinase.  

PubMed

Administration of metformin is known to reduce both body weight and food intake. Although the hypothalamus is recognized as a critical regulator of energy balance and body weight, there is currently no evidence for an effect of metformin in the hypothalamus. Therefore, we sought to determine the central action of metformin on energy balance and body weight, as well as its potential involvement with key hypothalamic energy sensors, including adenosine monophosphate-activated protein kinase (AMPK) and S6 kinase (S6K). We used meal pattern analysis and a conditioned taste aversion (CTA) test and measured energy expenditure in C56BL/6 mice administered metformin (0, 7.5, 15, or 30 ?g) into the third ventricle (I3V). Furthermore, we I3V-administered either control or metformin (30 ?g) and compared the phosphorylation of AMPK and S6K in the mouse mediobasal hypothalamus. Compared with the control, I3V administration of metformin decreased body weight and food intake in a dose-dependent manner and did not result in CTA. Furthermore, the reduction in food intake induced by I3V administration of metformin was accomplished by decreases in both nocturnal meal size and number. Compared with the control, I3V administration of metformin significantly increased phosphorylation of S6K at Thr(389) and AMPK at Ser(485/491) in the mediobasal hypothalamus, while AMPK phosphorylation at Thr(172) was not significantly altered. Moreover, I3V rapamycin pretreatment restored the metformin-induced anorexia and weight loss. These results suggest that the reduction in food intake induced by the central administration of metformin in the mice may be mediated by activation of S6K pathway. PMID:23824960

Kim, Hyun-Ju; Park, Eun-Young; Oh, Mi-Jeong; Park, Sung-Soo; Shin, Kyung-Ho; Choi, Sang-Hyun; Chun, Boe-Gwun; Kim, Dong-Hoon

2013-07-03

133

A Gene Encoding a Mitogen-Activated Protein Kinase Kinase Kinase is Induced Simultaneously with Genes for a Mitogen-Activated Protein Kinase and an S6 Ribosomal Protein Kinase by Touch, Cold, and Water Stress in Arabidopsis thaliana  

Microsoft Academic Search

We describe here the cloning and characterization of a cDNA encoding a protein kinase that has high sequence homology to members of the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK or MEKK) family; this cDNA is named cATMEKK1 (\\\\underline{A}rabidopsis \\\\underline{t}haliana \\\\underline{M}AP kinase or \\\\underline{E}RK \\\\underline{k}inase \\\\underline{k}inase \\\\underline{1}). The catalytic domain of the putative ATMEKK1 protein shows ≈ 40% identity with

Tsuyoshi Mizoguchi; Kenji Irie; Takashi Hirayama; Nobuaki Hayashida; Kazuko Yamaguchi-Shinozaki; Kunihiro Matsumoto; Kazuo Shinozaki

1996-01-01

134

Identification of p122RhoGAP (deleted in liver cancer-1) Serine 322 as a substrate for protein kinase B and ribosomal S6 kinase in insulin-stimulated cells.  

PubMed

Protein kinase B (PKB or Akt) plays an essential role in the actions of insulin, cytokines, and growth factors, although the substrates for PKB that are relevant to many of its actions require identification. In this study, we have reported the identification of p122RhoGAP, a GTPase-activating protein selective for RhoA and rodent homologue of the tumor suppressor deleted in liver cancer (DLC1) as a novel insulin-stimulated phosphoprotein in primary rat adipocytes. We have demonstrated that Ser-322 is phosphorylated upon insulin stimulation of intact cells and that this site is directly phosphorylated in vitro by PKB and ribosomal S6 kinase, members of the AGC (protein kinases A, G, and C) family of insulin-stimulated protein kinases. Furthermore, expression of constitutively active mutants of PKB or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) stimulates Ser-322 phosphorylation in intact cells, demonstrating that activation of the PKB or MEK pathway is sufficient for Ser-322 phosphorylation in vivo. Indeed, in primary adipocytes, insulin-stimulated Ser-322 phosphorylation was almost exclusively regulated by the phosphatidylinositol 3-kinase/PKB pathway, whereas in immortalized cells, insulin-stimulated phosphorylation was predominantly regulated by the MEK/extracellular signal-regulated kinase/ribosomal S6 kinase pathway, with the phosphatidylinositol 3-kinase/PKB pathway playing a minor role. These results demonstrate that p122RhoGAP Ser-322 acts as an integrator of signal transduction in a manner dependent on the cellular context. PMID:16338927

Hers, Ingeborg; Wherlock, Matthew; Homma, Yoshimi; Yagisawa, Hitoshi; Tavaré, Jeremy M

2005-12-06

135

The mechanism by which epidermal growth factor inhibits glycogen synthase kinase 3 in A431 cells.  

PubMed Central

Glycogen synthase kinase 3 (GSK3) was inhibited by 50% within 5 min when A431 cells were stimulated with epidermal growth factor (EGF). The inhibition was unaffected by rapamycin at concentrations which blocked the activation of p70 S6 kinase, and reversed by incubation with protein phosphatase-1. EGF stimulation of A431 cells inhibited GSK3 alpha and GSK3 beta to a similar extent, and inhibition was accompanied by phosphorylation of the tryptic peptides containing the serine residues phosphorylated in vitro by p70 S6 kinase or MAP kinase-activated protein (MAPKAP) kinase-1 beta (also termed Rsk-2). These results demonstrate that EGF inhibits GSK3 by inducing phosphorylation of a serine residue and that GSK3 is not phosphorylated in vivo by either p70 S6 kinase or protein kinase C. Images Figure 2 Figure 3

Saito, Y; Vandenheede, J R; Cohen, P

1994-01-01

136

Relationship between multiple biologic effects of rapamycin and the inhibition of pp70S6 protein kinase activity. Analysis in mutant clones of a T cell lymphoma.  

PubMed

Rapamycin (RAP) inhibits several biologic responses in the YAC-1 T cell lymphoma, including the serum-driven proliferation and cyclin A mRNA expression, the induction of Ly-6E Ag expression by IFN, and the induction of IFN-gamma production by IL-1. RAP also suppresses the enzymatic activity of the 70 kDa S6 protein kinase (pp70s6k). To define the mechanistic relationship between these multiple effects of RAP, we have generated stable somatic mutants with altered sensitivities to this drug. A first series of mutants, represented by the R19, 4R16, and 10R13 clones, showed markedly reduced sensitivity to the inhibitory effect of RAP on all biologic responses tested and on pp70s6k activity. Two other mutant types, R103 and R125, were both highly sensitive to RAP-mediated suppression of proliferation, of IL-1-induced IFN-gamma production, and of pp70s6k activity but differed in their Ly-6E response. This response was not affected by RAP in the R125 clone and was enhanced in the R103 clone. Therefore, the inhibitory effects of RAP on proliferation and IL-1-mediated IFN-gamma induction both appear associated with the inhibition of pp70s6k activity, whereas the modulation of Ly-6E induction is independent from the latter. Moreover, the cellular binding of [3H]dihydro-FK-506 was found to be blocked by RAP in all mutant types to the same extent as in wild-type YAC-1 cells, suggesting that the altered sensitivity to the effects of RAP in these mutants is not due to an inability of the drug to enter the cells or to interact with FKBP. Further biochemical characterization of the mutant cells described here is expected to help clarify the mechanisms of RAP action. PMID:8301150

Dumont, F J; Altmeyer, A; Kastner, C; Fischer, P A; Lemon, K P; Chung, J; Blenis, J; Staruch, M J

1994-02-01

137

Cell size reduction induced by inhibition of the mTOR\\/S6K-signaling pathway protects Jurkat cells from apoptosis  

Microsoft Academic Search

In Jurkat cells, the decreased cell growth rate associated with a long-lasting deactivation of the mammalian target of rapamycin (mTOR)\\/p70 ribosomal S6 kinase (S6K)-signaling pathway generates a cell population of progressively reduced cellular mass and size. When promoted by rapamycin as prototype inhibitor, the mTOR deactivation-dependent cell size reduction was associated with slowed, but not suppressed, proliferation. Small-size cells were

C Fumarola; S La Monica; R R Alfieri; E Borra; G G Guidotti

2005-01-01

138

Hypoxia-Induced Invadopodia Formation Involves Activation of NHE-1 by the p90 Ribosomal S6 Kinase (p90RSK)  

PubMed Central

The hypoxic and acidic microenvironments in tumors are strongly associated with malignant progression and metastasis, and have thus become a central issue in tumor physiology and cancer treatment. Despite this, the molecular links between acidic pH- and hypoxia-mediated cell invasion/metastasis remain mostly unresolved. One of the mechanisms that tumor cells use for tissue invasion is the generation of invadopodia, which are actin-rich invasive plasma membrane protrusions that degrade the extracellular matrix. Here, we show that hypoxia stimulates the formation of invadopodia as well as the invasive ability of cancer cells. Inhibition or shRNA-based depletion of the Na+/H+ exchanger NHE-1, along with intracellular pH monitoring by live-cell imaging, revealed that invadopodia formation is associated with alterations in cellular pH homeostasis, an event that involves activation of the Na+/H+ exchange rate by NHE-1. Further characterization indicates that hypoxia triggered the activation of the p90 ribosomal S6 kinase (p90 RSK), which resulted in invadopodia formation and site-specific phosphorylation and activation of NHE-1. This study reveals an unsuspected role of p90RSK in tumor cell invasion and establishes p90RS kinase as a link between hypoxia and the acidic microenvironment of tumors.

Lucien, Fabrice; Brochu-Gaudreau, Karine; Arsenault, Dominique; Harper, Kelly; Dubois, Claire M.

2011-01-01

139

Mutation of the PDK1 PH Domain Inhibits Protein Kinase B\\/Akt, Leading to Small Size and Insulin Resistance  

Microsoft Academic Search

PDK1 activates a group of kinases, including protein kinase B (PKB)\\/Akt, p70 ribosomal S6 kinase (S6K), and serum and glucocorticoid-induced protein kinase (SGK), that mediate many of the effects of insulin as well as other agonists. PDK1 interacts with phosphoinositides through a pleckstrin homology (PH) domain. To study the role of this interaction, we generated knock-in mice expressing a mutant

Jose R. Bayascas; Stephan Wullschleger; Kei Sakamoto; J. M. Garcia-Martinez; Carol Clacher; David Komander; Daan M. F. van Aalten; Krishna M. Boini; Florian Lang; Christopher Lipina; Lisa Logie; Calum Sutherland; John A. Chudek; Janna A. van Diepen; Peter J. Voshol; John M. Lucocq; Dario R. Alessi

2008-01-01

140

PI 3-kinase, mTOR, protein synthesis and cancer  

Microsoft Academic Search

The mTOR kinase is an essential mediator of growth signals that originate from PI3-kinase. Downstream targets of mTOR, p70 S6 kinase and 4E binding protein are important regulators of protein synthesis. Inhibition of mTOR by rapamycin interferes with oncogenic transformation and with tumor development by gain-of-function in PI 3-kinase signaling.

Peter K Vogt

2001-01-01

141

Ribosomal S6 Kinase 2 (RSK2) Maintains Genomic Stability by Activating the Atm/p53-Dependent DNA Damage Pathway  

PubMed Central

Ribosomal S6 Kinase 2 (RSK2) is a member of the p90RSK family of serine/threonine kinases, which are widely expressed and respond to many growth factors, peptide hormones, and neurotransmitters. Loss-of function mutations in the RPS6KA3 gene, which encodes the RSK2 protein, have been implicated in Coffin-Lowry Syndrome (CLS), an X-linked mental retardation disorder associated with cognitive deficits and behavioral impairments. However, the cellular and molecular mechanisms underlying this neurological disorder are not known. Recent evidence suggests that defective DNA damage signaling might be associated with neurological disorders, but the role of RSK2 in the DNA damage pathway remains to be elucidated. Here, we show that Adriamycin-induced DNA damage leads to the phosphorylation of RSK2 at Ser227 and Thr577 in the chromatin fraction, promotes RSK2 nuclear translocation, and enhances RSK2 and Atm interactions in the nuclear fraction. Furthermore, using RSK2 knockout mouse fibroblasts and RSK2-deficient cells from CLS patients, we demonstrate that ablation of RSK2 impairs the phosphorylation of Atm at Ser1981 and the phosphorylation of p53 at Ser18 (mouse) or Ser15 (human) in response to genotoxic stress. We also show that RSK2 affects p53-mediated downstream cellular events in response to DNA damage, that RSK2 knockout relieves cell cycle arrest at the G2/M phase, and that an increased number of ?H2AX foci, which are associated with defects in DNA repair, are present in RSK2-deficient cells. Taken together, our findings demonstrated that RSK2 plays an important role in the DNA damage pathway that maintains genomic stability by mediating cell cycle progression and DNA repair.

Lim, Han Chi; Xie, Li; Zhang, Wei; Li, Rong; Chen, Zhong-Can; Wu, Guang-Zhi; Cui, Shu-Sen; Tan, Eng King; Zeng, Li

2013-01-01

142

Ribosomal S6 Kinase 2 (RSK2) Maintains Genomic Stability by Activating the Atm/p53-Dependent DNA Damage Pathway.  

PubMed

Ribosomal S6 Kinase 2 (RSK2) is a member of the p90(RSK) family of serine/threonine kinases, which are widely expressed and respond to many growth factors, peptide hormones, and neurotransmitters. Loss-of function mutations in the RPS6KA3 gene, which encodes the RSK2 protein, have been implicated in Coffin-Lowry Syndrome (CLS), an X-linked mental retardation disorder associated with cognitive deficits and behavioral impairments. However, the cellular and molecular mechanisms underlying this neurological disorder are not known. Recent evidence suggests that defective DNA damage signaling might be associated with neurological disorders, but the role of RSK2 in the DNA damage pathway remains to be elucidated. Here, we show that Adriamycin-induced DNA damage leads to the phosphorylation of RSK2 at Ser227 and Thr577 in the chromatin fraction, promotes RSK2 nuclear translocation, and enhances RSK2 and Atm interactions in the nuclear fraction. Furthermore, using RSK2 knockout mouse fibroblasts and RSK2-deficient cells from CLS patients, we demonstrate that ablation of RSK2 impairs the phosphorylation of Atm at Ser1981 and the phosphorylation of p53 at Ser18 (mouse) or Ser15 (human) in response to genotoxic stress. We also show that RSK2 affects p53-mediated downstream cellular events in response to DNA damage, that RSK2 knockout relieves cell cycle arrest at the G2/M phase, and that an increased number of ?H2AX foci, which are associated with defects in DNA repair, are present in RSK2-deficient cells. Taken together, our findings demonstrated that RSK2 plays an important role in the DNA damage pathway that maintains genomic stability by mediating cell cycle progression and DNA repair. PMID:24086335

Lim, Han Chi; Xie, Li; Zhang, Wei; Li, Rong; Chen, Zhong-Can; Wu, Guang-Zhi; Cui, Shu-Sen; Tan, Eng King; Zeng, Li

2013-09-23

143

Basic anatomy and tumor biology of the RPS6KA6 gene that encodes the p90 ribosomal S6 kinase-4  

PubMed Central

The RPS6KA6 gene encodes the p90 ribosomal S6 kinase-4 (RSK4) that is still largely uncharacterized. In this study we identified a new RSK4 transcription initiation site and several alternative splice sites with a 5’RACE approach. The resulting mRNA variants encompass four possible first start codons. The first 15 nucleotides (nt) of exon 22 in mouse and the penultimate exon in both human (exon 21) and mouse (exon 24) RSK4 underwent alternative splicing, although the penultimate exon deleted variant appeared mainly in cell clines, but not in most normal tissues. Demethylation agent 5-azacytidine inhibited the deletion of the penultimate exon whereas two indolocarbazole-derived inhibitors of cyclin dependent kinase 4 or 6 induced deletion of the first 39 nt from exon 21 of human RSK4. In all human cancer cell lines studied, the 90-kD wild type RSK4 was sparse but, surprisingly, several isoforms at or smaller than 72-kD were expressed as detected by seven different antibodies. On immunoblots, each of these smaller isoforms often appeared as a duplet or triplet and the levels of these isoforms varied greatly among different cell lines and culture conditions. Cyclin D1 inhibited RSK4 expression and serum starvation enhanced the inhibition, whereas c-Myc and RSK4 inhibited cyclin D1. The effects of RSK4 on cell growth, cell death and chemoresponse depended on the mRNA variant or the protein isoform expressed, on the specificity of the cell lines, as well as on the anchorage-dependent or -independent growth conditions and the in vivo situation. Moreover, we also observed that even a given cDNA might be expressed to multiple proteins; therefore, when using a cDNA, one needs to exclude this possibility before attribution of the biological results from the cDNA to the anticipated protein. Collectively, our results suggest that whether RSK4 is oncogenic or tumor suppressive depends on many factors.

Sun, Yuan; Cao, Shousong; Yang, Min; Wu, Sihong; Wang, Zhe; Lin, Xiukun; Song, Xiangrang; Liao, D.J.

2012-01-01

144

p90 Ribosomal S6 Kinase Regulates Activity of the Renin-Angiotensin System: a Pathogenic Mechanism for Ischemia/Reperfusion Injury  

PubMed Central

Increasing evidence suggests that local Renin-Angiotensin System (RAS) plays an important role in cardiac diseases. Elevated p90 ribosomal S6 Kinase (RSK) activity has been observed in diabetic animal, as well as in human failing hearts. We hypothesize that RSK mediates cardiac dysfunction by up regulating local RAS signaling. In the present study, we show that the prorenin mRNA level was significantly increased (~5.6-fold) in transgenic mouse hearts with cardiac specific expression of RSK (RSK-Tg). The RSK-Tg mice were more vulnerable to ischemia/reperfusion (I/R) injury than non-transgenic littermate controls (NLC). To further understand the direct contribution of cardiac renin to I/R injury, we used a Langendorff system to evaluate the effect of renin inhibition by aliskiren in RSK-Tg mouse hearts. In the vehicle-perfused group, I/R significantly decreased left ventricular developed pressure (LVDP) in RSK-Tg hearts compared to NLC (7% versus 60% of the baseline). However, aliskiren perfusion significantly increased LVDP in RSK-Tg (7% to 61%, p<0.01) but not in NLC hearts (60% to 62%, n.s.). The protective effect of aliskiren in RSK-Tg hearts was further demonstrated with positive (contraction) dp/dt (6.5% to 63%, p<0.01) and rate pressure product (RPP) (5% to 51%, p<0.01). Moreover, aliskiren significantly decreased I/R induced infarction in RSK-Tg (60% to 32%, p<0.01), compared to NLC hearts (37% to 32%, n.s.). These results suggest that RSK plays a crucial role in regulating local cardiac renin, which contributes to I/R induced cardiac injury and dysfunction. Thus, renin inhibition may provide an alternative therapeutic strategy under conditions of increased RAS.

Shi, Xi; Yan, Chen; Nadtochiy, Sergiy M; Abe, Jun-Ichi; Brookes, Paul S; Berk, Bradford C.

2011-01-01

145

Phosphatidylinositol 3-kinase and mTOR mediate lipopolysaccharide-stimulated nitric oxide production in macrophages via interferon-b  

Microsoft Academic Search

Bacterial lipopolysaccharide (LPS) elic- its responses by macrophages that help the body repel infections. Recent evidence indicates that phosphatidylinositol 3-kinase (PI 3-kinase) may mediate some of these responses. Here, we show that exposing macrophages to LPS rapidly in- creased membrane-associated PI 3-kinase activity and also elevated p70 S6 kinase activity. Inhibitors of PI 3-kinase or the mammalian target of rapamy-

Steven L. Weinstein; Alexander J. Finn; Shaival H. Dave; Fanying Meng; Clifford A. Lowell; Jasbinder S. Sanghera; Anthony L. DeFranco

146

Crystal structures of S6K1 provide insights into the regulation mechanism of S6K1 by the hydrophobic motif.  

PubMed

The activity of S6K1 (p70 ribosomal protein subunit 6 kinase 1) is stimulated by phosphorylation of Thr389 in the hydrophobic motif by mTORC1 (mammalian target of rapamycin complex 1) and phosphorylation of Thr229 in the activation loop by PDK1 (phosphoinositide-dependent kinase 1); however, the order of the two events is still ambiguous. In the present paper we report six crystal structures of the S6K1 kinase domain alone or plus the hydrophobic motif in various forms, in complexes with a highly specific inhibitor. The structural data, together with the biochemical data, reveal in vivo phosphorylation of Thr389 in the absence of Thr229 phosphorylation and demonstrate the importance of two conserved residues, Gln140 and Arg121, in the establishment of a hydrogen-bonding network between the N-lobe (N-terminal lobe) and the hydrophobic motif. Phosphorylation of Thr389 or introduction of a corresponding negatively charged group leads to reinforcement of the network and stabilization of helix ?C. Furthermore, comparisons of S6K1 with other AGC (protein kinase A/protein kinase G/protein kinase C) family kinases suggest that the structural and sequence differences in the hydrophobic motif and helix ?C account for their divergence in PDK1 dependency. Taken together, the results of the present study indicate that phosphorylation of the hydrophobic motif in S6K1 is independent of, and probably precedes and promotes, phosphorylation of the activation loop. PMID:23731517

Wang, Jianchuan; Zhong, Chen; Wang, Fang; Qu, Fangfang; Ding, Jianping

2013-08-15

147

Mitigation of off-target adrenergic binding and effects on cardiovascular function in the discovery of novel ribosomal S6 kinase 2 inhibitors.  

PubMed

We previously reported the discovery of a novel ribosomal S6 kinase 2 (RSK2) inhibitor, (R)-5-Methyl-1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a] indole-8-carboxylic acid [1-(3-dimethylamino-propyl)-1H-benzoimidazol-2-yl]-amide (BIX 02565), with high potency (IC(50) = 1.1 nM) targeted for the treatment of heart failure. In the present study, we report that despite nanomolar potency at the target, BIX 02565 elicits off-target binding at multiple adrenergic receptor subtypes that are important in the control of vascular tone and cardiac function. To elucidate in vivo the functional consequence of receptor binding, we characterized the cardiovascular (CV) profile of the compound in an anesthetized rat CV screen and telemetry-instrumented conscious rats. Infusion of BIX 02565 (1, 3, and 10 mg/kg) in the rat CV screen resulted in a precipitous decrease in both mean arterial pressure (MAP; to -65 ± 6 mm Hg below baseline) and heart rate (-93 ± 13 beats/min). In telemetry-instrumented rats, BIX 02565 (30, 100, and 300 mg/kg p.o. QD for 4 days) elicited concentration-dependent decreases in MAP after each dose (to -39 ± 4 mm Hg on day 4 at T(max)); analysis by Demming regression demonstrated strong correlation independent of route of administration and influence of anesthesia. Because of pronounced off-target effects of BIX 02565 on cardiovascular function, a high-throughput selectivity screen at adrenergic ?(1A) and ?(2A) was performed for 30 additional RSK2 inhibitors in a novel chemical series; a wide range of adrenergic binding was achieved (0-92% inhibition), allowing for differentiation within the series. Eleven lead compounds with differential binding were advanced to the rat CV screen for in vivo profiling. This led to the identification of potent RSK2 inhibitors (cellular IC(50) <0.14 nM) without relevant ?(1A) and ?(2A) inhibition and no adverse cardiovascular effects in vivo. PMID:22128344

Fryer, Ryan M; Muthukumarana, Akalushi; Chen, Rong Rhonda; Smith, James D; Mazurek, Suzanne Nodop; Harrington, Kyle E; Dinallo, Roger M; Burke, Jennifer; DiCapua, Frank M; Guo, Xin; Kirrane, Thomas M; Snow, Roger J; Zhang, Yunlong; Soleymanzadeh, Fariba; Madwed, Jeffrey B; Kashem, Mohammed A; Kugler, Stanley Z; O'Neill, Margaret M; Harrison, Paul C; Reinhart, Glenn A; Boyer, Stephen J

2011-11-29

148

Role of the Phosphoinositide 3-Kinase-Akt-Mammalian Target of the Rapamycin Signaling Pathway in Long-Term Potentiation and Trace Fear Conditioning Memory in Rat Medial Prefrontal Cortex  

ERIC Educational Resources Information Center

Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt (also known as protein kinase B, PKB), mammalian target of rapamycin (mTOR), the 70-kDa ribosomal S6 kinase (p70S6k), and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), may play important roles in long-term synaptic plasticity and memory in many…

Sui, Li; Wang, Jing; Li, Bao-Ming

2008-01-01

149

Role of the Phosphoinositide 3-Kinase-Akt-Mammalian Target of the Rapamycin Signaling Pathway in Long-Term Potentiation and Trace Fear Conditioning Memory in Rat Medial Prefrontal Cortex  

ERIC Educational Resources Information Center

|Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt (also known as protein kinase B, PKB), mammalian target of rapamycin (mTOR), the 70-kDa ribosomal S6 kinase (p70S6k), and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), may play important roles in long-term synaptic plasticity and memory in…

Sui, Li; Wang, Jing; Li, Bao-Ming

2008-01-01

150

Haloperidol Regulates the State of Phosphorylation of Ribosomal Protein S6 via Activation of PKA and Phosphorylation of DARPP-32  

PubMed Central

Administration of typical antipsychotic drugs, such as haloperidol, promotes cAMP-dependent signaling in the medium spiny neurons (MSNs) of the striatum. In this study, we have examined the effect of haloperidol on the state of phosphorylation of the ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit. We found that haloperidol increases the phosphorylation of rpS6 at the dual site Ser235/236, which is involved in the regulation of mRNA translation. This effect was exerted in the MSNs of the indirect pathway, which express specifically dopamine D2 receptors (D2Rs) and adenosine A2 receptors (A2ARs). The effect of haloperidol was decreased by blockade of A2ARs or by genetic attenuation of the G?olf protein, which couples A2ARs to activation of adenylyl cyclase. Moreover, stimulation of cAMP-dependent protein kinase A (PKA) increased Ser235/236 phosphorylation in cultured striatal neurons. The ability of haloperidol to promote rpS6 phosphorylation was abolished in knock-in mice deficient for PKA activation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32?kDa. In contrast, pharmacological or genetic inactivation of p70 rpS6 kinase 1, or extracellular signal-regulated kinases did not affect haloperidol-induced rpS6 phosphorylation. These results identify PKA as a major rpS6 kinase in neuronal cells and suggest that regulation of protein synthesis through rpS6 may be a potential target of antipsychotic drugs.

Valjent, Emmanuel; Bertran-Gonzalez, Jesus; Bowling, Heather; Lopez, Sebastien; Santini, Emanuela; Matamales, Miriam; Bonito-Oliva, Alessandra; Herve, Denis; Hoeffer, Charles; Klann, Eric; Girault, Jean-Antoine; Fisone, Gilberto

2011-01-01

151

Regulation of p70 S6k , GSK-3?, and calcineurin in rat striated muscle during aging  

Microsoft Academic Search

In this study we compared the content and phosphorylation levels of several molecules believed to regulate muscle hypertrophy and fiber type changes in the extensor digitorum longus (EDL), soleus, diaphragm, and heart of adult (6 months), aged (30 months), and very aged (36 months) Fischer 344 × Brown Norway rats. With aging, the mass of the EDL and soleus decreased significantly (~38% and

R. S. Kinnard; D. B. Mylabathula; S. Uddemarri; K. M. Rice; G. L. Wright; E. R. Blough

2005-01-01

152

Glutamate-dependent translational regulation in cultured Bergmann glia cells: Involvement of p70 S6K  

Microsoft Academic Search

Glutamate, the main excitatory amino acid transmitter in the vertebrate brain is involved in the dynamic changes in protein repertoire that underlie synaptic plasticity. Activity-dependent differential expression patterns occur not only in neurons but also in glial cells. In fact, a membrane to nuclei signaling has been described after ionotropic glutamate receptor stimulation in cultured chick cerebellar Bergmann glia cells.

M. E. González-Mejia; M. Morales; L. C. R. Hernández-Kelly; R. C. Zepeda; A. Bernabé; A. Ortega

2006-01-01

153

GPx-1 modulates Akt and P70 S6K phosphorylation and Gadd45 levels in MCF7 cells  

Microsoft Academic Search

Selenium has been shown to prevent cancer in animal models, and recent data indicate it is likely to be effective in humans as well. One selenium-containing protein, the cytoplasmic form of glutathione peroxidase (GPx-1), has been implicated in cancer risk and development by genetic studies identifying at-risk alleles and loss of heterozygosity in tumors. In order to evaluate the biological

Mohamed A Nasr; Mark J Fedele; Karyn Esser; Alan M Diamond

2004-01-01

154

Impaired insulin secretion and decreased expression of the nutritionally responsive ribosomal kinase protein S6K-1 in pancreatic islets from malnourished rats  

Microsoft Academic Search

Low protein diet has been shown to affect the levels and activities of several enzymes from pancreatic islets. To further extend the knowledge on how malnutrition affects insulin secretion pathway, we investigated in this work the insulin release induced by glucose or leucine, an insulin secretagogue, and the expression of insulin receptor (IR), insulin receptor substrate 1 (IRS1), phosphatidylinositol 3-kinase

Eliane Filiputti; Fabiano Ferreira; Kleber L. A. Souza; Luiz F. Stoppiglia; Vanessa C. Arantes; Antonio C. Boschero; Everardo M. Carneiro

2008-01-01

155

The degree of p70 S6k and S6 phosphorylation in human skeletal muscle in response to resistance exercise depends on the training volume  

Microsoft Academic Search

Regular performance of resistance exercise induces an increase in skeletal muscle mass, however, the molecular mechanisms\\u000a underlying this effect are not yet fully understood. The purpose of the present investigation was to examine acute changes\\u000a in molecular signalling in response to resistance exercise involving different training volumes. Eight untrained male subjects\\u000a carried out one, three and five sets of 6 repetition

Gerasimos Terzis; Konstantinos Spengos; Henrik Mascher; Giorgos Georgiadis; Panagiota Manta; Eva Blomstrand

2010-01-01

156

Inactivation of glycogen synthase kinase-3 beta by phosphorylation: new kinase connections in insulin and growth-factor signalling.  

PubMed Central

The beta-isoform of glycogen synthase kinase-3 (GSK3 beta) isolated from rabbit skeletal muscle was inactivated 90-95% following incubation with MgATP and either MAP kinase-activated protein kinase-1 (MAPKAP kinase-1, also termed RSK-2) or p70 S6 kinase (p70S6K), and re-activated with protein phosphatase 2A. MAPKAP kinase-1 and p70S6K phosphorylated the same tryptic peptide on GSK3 beta, and the site of phosphorylation was identified as the serine located nine residues from the N-terminus of the protein. The inhibitory effect of Ser-9 phosphorylation on GSK3 beta activity was observed with three substrates, (inhibitor-2, c-jun and a synthetic peptide), and also with glycogen synthase provided that 0.15 M KCl was added to the assays. The results suggest that Ser-9 phosphorylation underlies the reported inhibition of GSK3 beta by insulin and that GSK3 may represent a point of convergence of two major growth-factor-stimulated protein kinase cascades. Images Figure 1 Figure 2

Sutherland, C; Leighton, I A; Cohen, P

1993-01-01

157

The p90 ribosomal S6 kinase (RSK) inhibitor BI-D1870 prevents gamma irradiation-induced apoptosis and mediates senescence via RSK- and p53-independent accumulation of p21(WAF1/CIP1.).  

PubMed

The p90 ribosomal S6 kinase (RSK) family is a group of highly conserved Ser/Thr kinases that promote cell proliferation, growth, motility and survival. As they are almost exclusively activated downstream of extracellular signal-regulated kinases 1 and 2 (ERK1/2), therapeutic intervention by RSK inhibition is less likely to produce such severe side effects as those observed following inhibition of the upstream master regulators Raf, MEK and ERK1/2. Here, we report that BI-D1870, a potent small molecule inhibitor of RSKs, induces apoptosis, although preferentially, in a p21-deficient background. On the other hand, BI-D1870 also induces a strong transcription- and p53-independent accumulation of p21 protein and protects cells from gamma irradiation (?IR)-induced apoptosis, driving them into senescence even in the absence of ?IR. Although we identified p21 in in vitro kinase assays as a novel RSK substrate that specifically becomes phosphorylated by RSK1-3 at Ser116 and Ser146, RNA-interference, overexpression and co-immunoprecipitation studies as well as the use of SL0101, another specific RSK inhibitor, revealed that BI-D1870 mediates p21 accumulation via a yet unknown pathway that, besides its off-site targets polo-like kinase-1 and AuroraB, also does also not involve RSKs. Thus, this novel off-target effect of BI-D1870 should be taken into serious consideration in future studies investigating the role of RSKs in cellular signaling and tumorigenesis. PMID:24136223

Neise, D; Sohn, D; Stefanski, A; Goto, H; Inagaki, M; Wesselborg, S; Budach, W; Stühler, K; Jänicke, R U

2013-10-17

158

Insights into the Inhibition of the p90 Ribosomal S6 Kinase (RSK) by the Flavonol Glycoside SL0101 from the 1.5 Å Crystal Structure of the N-Terminal Domain of RSK2 with Bound Inhibitor  

SciTech Connect

The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-a-l-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 {angstrom} resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the aD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.

Utepbergenov, Darkhan; Derewenda, Urszula; Olekhnovich, Natalya; Szukalska, Gabriela; Banerjee, Budhaditya; Hilinski, Michael K.; Lannigan, Deborah A.; Stukenberg, P. Todd; Derewenda, Zygmunt S. (Lodz - Poland); (UV)

2012-09-11

159

Ischemic preconditioning protects by activating prosurvival kinases at reperfusion.  

PubMed

Pharmacological activation of the prosurvival kinases Akt and ERK-1/2 at reperfusion, after a period of lethal ischemia, protects the heart against ischemia-reperfusion injury. We hypothesized that ischemic preconditioning (IPC) protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion. In isolated perfused Sprague-Dawley rat hearts subjected to 35 min of lethal ischemia, the phosphorylation states of Akt, ERK-1/2, and p70 S6 kinase (p70S6K) were determined after 15 min of reperfusion, and infarct size was measured after 120 min of reperfusion. IPC induced a biphasic response in Akt and ERK-1/2 phosphorylation during the preconditioning and reperfusion phases after the period of lethal ischemia. IPC induced a fourfold increase in Akt, ERK-1/2, and p70S6K phosphorylation at reperfusion and reduced the infarct risk-to-volume ratio (56.9 +/- 5.7 and 20.9 +/- 3.6% for control and IPC, respectively, P < 0.01). Inhibiting the IPC-induced phosphorylation of Akt, ERK-1/2, and p70S6K at reperfusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 or the MEK-1/2 inhibitor PD-98059 abrogated IPC-induced protection (46.3 +/- 5.8, 49.2 +/- 4.0, and 20.9 +/- 3.6% for IPC + LY-294002, IPC + PD-98059, and IPC, respectively, P < 0.01), demonstrating that the phosphorylation of these kinases at reperfusion is required for IPC-induced protection. In conclusion, we demonstrate that the reperfusion phase following sustained ischemia plays an essential role in mediating IPC-induced protection. Specifically, we demonstrate that IPC protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion. PMID:15358610

Hausenloy, Derek J; Tsang, A; Mocanu, Mihaela M; Yellon, Derek M

2004-09-09

160

The mTOR kinase inhibitor Everolimus decreases S6 kinase phosphorylation but fails to reduce mutant huntingtin levels in brain and is not neuroprotective in the R6\\/2 mouse model of Huntington's disease  

Microsoft Academic Search

BACKGROUND: Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion within the huntingtin gene. Mutant huntingtin protein misfolds and accumulates within neurons where it mediates its toxic effects. Promoting mutant huntingtin clearance by activating macroautophagy is one approach for treating Huntington's disease (HD). In this study, we evaluated the mTOR kinase inhibitor and macroautophagy promoting

Jonathan H Fox; Teal Connor; Vanita Chopra; Kate Dorsey; Jibrin A Kama; Dorothee Bleckmann; Claudia Betschart; Daniel Hoyer; Stefan Frentzel; Marian DiFiglia; Paolo Paganetti; Steven M Hersch

2010-01-01

161

AMP-activated protein kinase counteracts brain-derived neurotrophic factor-induced mammalian target of rapamycin complex 1 signaling in neurons.  

PubMed

Growth factors and nutrients, such as amino acids and glucose, regulate mammalian target of rapamycin complex 1 (mTORC1) signaling and subsequent translational control in a coordinated manner. Brain-derived neurotrophic factor (BDNF), the most prominent neurotrophic factor in the brain, activates mTORC1 and induces phosphorylation of its target, p70S6 kinase (p70S6K), at Thr389 in neurons. BDNF also increases mammalian target of rapamycin-dependent novel protein synthesis in neurons. Here, we report that BDNF-induced p70S6K activation is dependent on glucose, but not amino acids, sufficiency in cultured cortical neurons. AMP-activated protein kinase (AMPK) is the molecular background to this specific nutrient dependency. Activation of AMPK, which is induced by glucose deprivation, treatment with pharmacological agents such as 2-Deoxy-d-glucose, metformin, and 5-aminoimidazole-4-carboxamide ribonucleoside or forced expression of a constitutively active AMPK? subunit, counteracts BDNF-induced phosphorylation of p70S6K and enhanced protein synthesis in cortical neurons. These results indicate that AMPK inhibits the effects of BDNF on mTORC1-mediated translation in neurons. Here, we report that growth factor-induced mTORC1 activity is dependent on glucose sufficiency, but not on amino acids in neurons. The mechanism underlying this phenomenon is AMP-activated protein kinase (AMPK) activation. AMPK activation inhibits BDNF-induced mTORC1 activity through TSC2 and raptor. PMID:23841933

Ishizuka, Yuta; Kakiya, Naomasa; Witters, Lee A; Oshiro, Noriko; Shirao, Tomoaki; Nawa, Hiroyuki; Takei, Nobuyuki

2013-07-29

162

Changes in growth-related kinases in head, neck and limb muscles with age  

PubMed Central

Sarcopenia coincides with declines in several systemic processes that signal through the MAP kinase and Akt-mTOR-p70S6k cascades typically associated with muscle growth. Effects of aging on these pathways have primarily been examined in limb muscles, which experience substantial activity and neural changes in addition to systemic hormonal and metabolic changes. Head and neck muscles are reported to undergo reduced sarcopenia and disuse with age relative to limb muscles, suggesting muscle activity may contribute to maintaining mass with age. However many head and neck muscles derive from embryonic branchial arches, rather than the somites from which limb muscles originate, suggesting that developmental origin may be important. This study compares the expression and phosphorylation of MAP kinase and mTOR networks in head, neck, tongue, and limb muscles from 8- and 26-month old F344 rats to test the hypothesis that physical activity and developmental origin contribute to preservation of muscle mass with age. Phosphorylation of p38 was exaggerated in aged branchial arch muscles. Phosphorylation of ERK and p70S6k T421/S424 declined with age only in the biceps brachii. Expression of p70S6k declined in all head and neck, tongue and limb muscles although no change in phosphorylation of p70S6k on T389 could be resolved. A systemic change that results in a loss of p70S6k protein expression may reduce the capacity to respond to acute hypertrophic stimuli, while the exaggerated p38 signaling in branchial arch muscles may reflect more active muscle remodeling.

Rahnert, Jill A.; Luo, Qingwei; Balog, Edward M.; Sokoloff, Alan J.; Burkholder, Thomas J.

2010-01-01

163

Novel indole ?-methylene-?-lactones as potent inhibitors for AKT-mTOR signaling pathway kinases  

Microsoft Academic Search

In an effort to generate novel anticancer agents, a series of hybrids of ?-methylene-?-lactones and 2-phenyl indoles has been synthesized and evaluated for inhibition activities on the phosphorylation of AKT, mTOR, p70S6 kinase, and 4E-BP1. The results indicate that substitutes on the ?-position of lactones have a rather significant influence on inhibition activities.

Huasheng Ding; Chao Zhang; Xihan Wu; Chunhao Yang; Xiongwen Zhang; Jian Ding; Yuyuan Xie

2005-01-01

164

Bacteriophage P70: unique morphology and unrelatedness to other Listeria bacteriophages.  

PubMed

Listeria monocytogenes is an important food-borne pathogen, and its bacteriophages find many uses in detection and biocontrol of its host. The novel broad-host-range virulent phage P70 has a unique morphology with an elongated capsid. Its genome sequence was determined by a hybrid sequencing strategy employing Sanger and PacBio techniques. The P70 genome contains 67,170 bp and 119 open reading frames (ORFs). Our analyses suggest that P70 represents an archetype of virus unrelated to other known Listeria bacteriophages. PMID:22993158

Schmuki, Martina M; Erne, Doris; Loessner, Martin J; Klumpp, Jochen

2012-09-19

165

Bacteriophage P70: Unique Morphology and Unrelatedness to Other Listeria Bacteriophages  

PubMed Central

Listeria monocytogenes is an important food-borne pathogen, and its bacteriophages find many uses in detection and biocontrol of its host. The novel broad-host-range virulent phage P70 has a unique morphology with an elongated capsid. Its genome sequence was determined by a hybrid sequencing strategy employing Sanger and PacBio techniques. The P70 genome contains 67,170 bp and 119 open reading frames (ORFs). Our analyses suggest that P70 represents an archetype of virus unrelated to other known Listeria bacteriophages.

Schmuki, Martina M.; Erne, Doris; Loessner, Martin J.

2012-01-01

166

Removal of S6K1 and S6K2 Leads to Divergent Alterations in Learning, Memory, and Synaptic Plasticity  

ERIC Educational Resources Information Center

|Protein synthesis is required for the expression of enduring memories and long-lasting synaptic plasticity. During cellular proliferation and growth, S6 kinases (S6Ks) are activated and coordinate the synthesis of de novo proteins. We hypothesized that protein synthesis mediated by S6Ks is critical for the manifestation of learning, memory, and…

Antion, Marcia D.; Merhav, Maayan; Hoeffer, Charles A.; Reis, Gerald; Kozma, Sara C.; Thomas, George; Schuman Erin M.; Rosenblum, Kobi; Klann, Eric

2008-01-01

167

Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway  

Microsoft Academic Search

Ovarian cancer is the leading cause of death from gynecological malignancy for women. The amplification of the PI3K catalytic subunit (p110?) and the lost function of PTEN are frequently detected in ovarian cancer cells. PI3K plays an important role in tumorigenesis. To specifically inhibit PI3K activity in ovarian cancer cells, we constructed small interfering RNA (siRNA) against p110?. The expression

Qiao Meng; Chang Xia; Jing Fang; Yon Rojanasakul; Bing-Hua Jiang

2006-01-01

168

High-frequency electrically stimulated skeletal muscle contractions increase p70 s6k phosphorylation independent of known IGF-I sensitive signaling pathways  

Microsoft Academic Search

Insulin-like growth factor (IGF-I) is hypothesized to be a critical upstream regulator of mammalian target of rapamycin (mTOR)-regulated protein synthesis with muscle contraction. We utilized a mouse model that expresses a skeletal muscle specific dominant-negative IGF-I receptor to investigate the role of IGF-I signaling of protein synthesis in response to unilateral lengthening contractions (10 sets, 6 repetitions, 100Hz) at 0

Sarah Witkowski; Richard M. Lovering; Espen E. Spangenburg

2010-01-01

169

Reversine stimulates adipocyte differentiation and downregulates Akt and p70 s6k signaling pathways in 3T3-L1 cells  

Microsoft Academic Search

In this study, we investigate the ability of reversine to stimulate adipocyte differentiation and its effect on cellular signaling pathways associated with adipocyte differentiation. Our data show that reversine treatment of 3T3-L1 cells under differentiation conditions synergistically enhances adipocyte differentiation and the expression of adipogenic marker genes such as aP2, PPAR-?, resistin, C\\/EBP?, and adiponectin. In parallel, reversine treatment leads

Yong Kee Kim; Hye-Young Choi; Nam Hyun Kim; Woojung Lee; Dong-Wan Seo; Dong-Won Kang; Hoi Young Lee; Jeung-Whan Han; Sahng Wook Park; Su-Nam Kim

2007-01-01

170

Increased p70 s6k phosphorylation during intake of a protein–carbohydrate drink following resistance exercise in the fasted state  

Microsoft Academic Search

The present study aimed at comparing the responses of myogenic regulatory factors and signaling pathways involved in muscle\\u000a protein synthesis after a resistance training session performed in either the fasted or fed state. According to a randomized\\u000a crossover study design, six young male subjects participated in two experimental sessions separated by 3 weeks. In each session,\\u000a they performed a standardized resistance

Louise Deldicque; Katrien De Bock; Michael Maris; Monique Ramaekers; Henri Nielens; Marc Francaux; Peter Hespel

2010-01-01

171

SU5416 inhibited VEGF and HIF-1? expression through the PI3K\\/AKT\\/p70S6K1 signaling pathway  

Microsoft Academic Search

Ovarian cancer has the highest mortality rate of any gynecological disease affecting women in Western countries. VEGF is a crucial inducer of angiogenesis both in vivo and in vitro. VEGF is commonly upregulated in ovarian cancer and is regulated by HIF-1. SU5416 is known to inhibit various stages of tumor growth. In this study, we show that SU5416 inhibited VEGF

Xiao-Song Zhong; Jenny Z. Zheng; Eddie Reed; Bing-Hua Jiang

2004-01-01

172

LKB1 is required for adiponectin-mediated modulation of AMPK-S6K axis and inhibition of migration and invasion of breast cancer cells  

PubMed Central

Adiponectin is widely known as an adipocytokine with therapeutic potential for its markedly protective function in the pathogenesis of obesity-related disorders, metabolic syndrome, systemic insulin resistance, cardiovascular disease and more recently carcinogenesis. In the present study, we show that adiponectin inhibits adhesion, invasion and migration of breast cancer cells. Further analysis of the underlying molecular mechanisms revealed that adiponectin treatment increased AMP-activated protein kinase (AMPK) phosphorylation and activity as evident by increased phosphorylation of downstream target of AMPK, acetyl-coenzyme A carboxylase and inhibition of p70S6 kinase (S6K). Intriguingly, we discovered that adiponectin treatment increases the expression of tumor suppressor gene LKB1 in breast cancer cells. Overexpression of LKB1 in breast cancer cells further increased adiponectin-mediated phosphorylation of AMPK. Using isogenic LKB1 knockdown cell line pair, we found that LKB1 is required for adiponectin-mediated modulation of AMPK–S6K axis and more importantly, inhibition of adhesion, migration and invasion of breast cancer cells. Taken together these data present a novel mechanism involving specific upregulation of tumor suppressor gene LKB1 by which adiponectin inhibits adhesion, invasion and migration of breast cancer cells. Our findings indicate the possibility of using adiponectin analogues to inhibit invasion and migration of breast cancer cells.

Taliaferro-Smith, L; Nagalingam, A; Zhong, D; Zhou, W; Saxena, NK; Sharma, D

2010-01-01

173

Notch- and Transducin-like Enhancer of Split (TLE)-dependent Histone Deacetylation Explain Interleukin 12 (IL-12) p70 Inhibition by Zymosan*  

PubMed Central

The fungal analog zymosan induces IL-23 and low amounts of IL-12 p70. This study addresses the molecular mechanisms underlying this cytokine pattern in human monocyte-derived dendritic cells. The transcriptional regulation of il23a, one of the chains of IL-23, depended on the activation of c-Rel and histone H3 phosphorylation, as judged from the association of c-Rel with the il23a promoter and the correlation between IL-23 production and Ser-10-histone H3 phosphorylation. Consistent with its reduced ability to produce IL-12 p70, zymosan induced a transient occupancy of the il12a promoter by c-Rel, blocked the production of IL-12 p70 and the transcription of il12a induced by other stimuli, and triggered the expression and nuclear translocation of the transcriptional repressors of the Notch family hairy and enhancer of split (Hes)-1, Hes5, hairy/enhancer-of-split related with YRPW motif protein (Hey)-1, and transducin-like enhancer of split (TLE). Zymosan also induced the interaction of Hes1 and TLE with histone H3 phosphorylated on Ser-10 and deacetylated on Lys-14. Inhibition of class III histone deacetylases increased the production of IL-12 p70 and partially blunted the inhibitory effect of zymosan on the production of IL-12 p70 elicited by LPS and IFN-?. These results indicate that the selective induction of IL-23 by ?-glucans is explained by the activation of c-Rel associated with Ser-10-histone H3 phosphorylation in the il23a promoter mediated by mitogen- and stress-activated kinase and/or protein kinase A and inhibition of il12a transcription by a mechanism involving activation of several corepressors with the ability to bind TLE and to promote histone deacetylation.

Alvarez, Yolanda; Municio, Cristina; Hugo, Etzel; Zhu, Jimmy; Alonso, Sara; Hu, Xiaoyu; Fernandez, Nieves; Crespo, Mariano Sanchez

2011-01-01

174

[Protein kinases role in adaptive phenomenon of heart ischemic postconditioning development].  

PubMed

Authors submitted an analysis of papers given up an involvement of protein kinases in heart ischemic postconditioning. This analysis of literature source allowed to authors affirms that signaling system of postconditioning can involve kinases: PKC, PI3K, Akt, MEKl/2, ERK1/2, MTOR, p70s6K, GSK3b, PKG and also eNOS, NO, GC, motoKATP channel, ROS, MPT pore. At the same time it is unclear a real contributions of kinases mTOR, p70s6, AMPK and GSK3b in the mechanism of infarct limiting impact of postconditioning. It is required a further study of the chain of signaling events following JAK2 and p38 kinase activation. The knowledge of Ras and Raf-1 role in postconditioning has hypothetical character. The tyrosine kinase significance in postcondi-tioning is unclear, particular Src kinase, which plays an important role in the regulation of cardiac tolerance to an impact of ischemia and reperfusion. PMID:23862384

Maslov, L N; Mrochek, A G; Shchepetkin, I A; Headrick, J P; Hanus, L; Barzakh, E I; Lishmanov, A Iu; Gorbunov, A S; Tsybul'nikov, S Iu; Ba?kov, A N

2013-04-01

175

IRS-4 mediates protein kinase B signaling during insulin stimulation without promoting antiapoptosis.  

PubMed

Insulin receptor substrate (IRS) proteins are tyrosine phosphorylated and mediate multiple signals during activation of the receptors for insulin, insulin-like growth factor 1 (IGF-1), and various cytokines. In order to distinguish common and unique functions of IRS-1, IRS-2, and IRS-4, we expressed them individually in 32D myeloid progenitor cells containing the human insulin receptor (32D(IR)). Insulin promoted the association of Grb-2 with IRS-1 and IRS-4, whereas IRS-2 weakly bound Grb-2; consequently, IRS-1 and IRS-4 enhanced insulin-stimulated mitogen-activated protein kinase activity. During insulin stimulation, IRS-1 and IRS-2 strongly bound p85alpha/beta, which activated phosphatidylinositol (PI) 3-kinase, protein kinase B (PKB)/Akt, and p70(s6k), and promoted the phosphorylation of BAD. IRS-4 also promoted the activation of PKB/Akt and BAD phosphorylation during insulin stimulation; however, it weakly bound or activated p85-associated PI 3-kinase and failed to mediate the activation of p70(s6k). Insulin strongly inhibited apoptosis of interleukin-3 (IL-3)-deprived 32D(IR) cells expressing IRS-1 or IRS-2 but failed to inhibit apoptosis of cells expressing IRS-4. Consequently, 32D(IR) cells expressing IRS-4 proliferated slowly during insulin stimulation. Thus, the activation of PKB/Akt and BAD phosphorylation might not be sufficient to inhibit the apoptosis of IL-3-deprived 32D(IR) cells unless p85-associated PI 3-kinase or p70(s6k) are strongly activated. PMID:10594015

Uchida, T; Myers, M G; White, M F

2000-01-01

176

Mutation of the PDK1 PH domain inhibits protein kinase B/Akt, leading to small size and insulin resistance.  

PubMed

PDK1 activates a group of kinases, including protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), and serum and glucocorticoid-induced protein kinase (SGK), that mediate many of the effects of insulin as well as other agonists. PDK1 interacts with phosphoinositides through a pleckstrin homology (PH) domain. To study the role of this interaction, we generated knock-in mice expressing a mutant of PDK1 incapable of binding phosphoinositides. The knock-in mice are significantly small, insulin resistant, and hyperinsulinemic. Activation of PKB is markedly reduced in knock-in mice as a result of lower phosphorylation of PKB at Thr308, the residue phosphorylated by PDK1. This results in the inhibition of the downstream mTOR complex 1 and S6K1 signaling pathways. In contrast, activation of SGK1 or p90 ribosomal S6 kinase or stimulation of S6K1 induced by feeding is unaffected by the PDK1 PH domain mutation. These observations establish the importance of the PDK1-phosphoinositide interaction in enabling PKB to be efficiently activated with an animal model. Our findings reveal how reduced activation of PKB isoforms impinges on downstream signaling pathways, causing diminution of size as well as insulin resistance. PMID:18347057

Bayascas, Jose R; Wullschleger, Stephan; Sakamoto, Kei; García-Martínez, Juan M; Clacher, Carol; Komander, David; van Aalten, Daan M F; Boini, Krishna M; Lang, Florian; Lipina, Christopher; Logie, Lisa; Sutherland, Calum; Chudek, John A; van Diepen, Janna A; Voshol, Peter J; Lucocq, John M; Alessi, Dario R

2008-03-17

177

Cocoa polyphenols suppress TNF-?-induced vascular endothelial growth factor expression by inhibiting phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase kinase-1 (MEK1) activities in mouse epidermal cells.  

PubMed

Cocoa polyphenols have antioxidant and anti-inflammatory effects. TNF-? is a pro-inflammatory cytokine that has a vital role in the pathogenesis of inflammatory diseases such as cancer and psoriasis. Vascular endothelial growth factor (VEGF) expression is associated with tumorigenesis, CVD, rheumatoid arthritis and psoriasis. We tested whether cocoa polyphenol extract (CPE) inhibited TNF-?-induced VEGF expression in promotion-sensitive JB6 mouse epidermal cells. CPE significantly inhibited TNF-?-induced up-regulation of VEGF via reducing TNF-?-induced activation of the nuclear transcription factors activator protein-1 (AP-1) and NF-?B, which are key regulators of VEGF expression. CPE also inhibited TNF-?-induced phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase. CPE blocked activation of their downstream kinases, p70 kDa ribosomal protein S6 kinase and p90 kDa ribosomal protein S6 kinase. CPE suppressed phosphoinositide 3-kinase (PI3K) activity via binding PI3K directly. CPE did not affect TNF-?-induced phosphorylation of mitogen-activated protein kinase kinase-1 (MEK1) but suppressed TNF-?-induced MEK1 activity. Collectively, these results indicate that CPE reduced TNF-?-induced up-regulation of VEGF by directly inhibiting PI3K and MEK1 activities, which may contribute to its chemopreventive potential. PMID:20550744

Kim, Jong-Eun; Son, Joe Eun; Jung, Sung Keun; Kang, Nam Joo; Lee, Chang Yong; Lee, Ki Won; Lee, Hyong Joo

2010-06-16

178

The role of S6K1 in ER-positive breast cancer  

PubMed Central

The 40S ribosomal S6 kinase 1 (S6K1) is a conserved serine/threonine protein kinase that belongs to the AGC family of protein kinases, which also includes Akt and many others. S6K1 is the principal kinase effector downstream of the mammalian target of rapamycin complex 1 (mTORC1). S6K1 is sensitive to a wide range of signaling inputs, including growth factors, amino acids, energy levels and hypoxia. S6K1 relays these signals to regulate a growing list of substrates and interacting proteins in control of oncogenic processes, such as cell growth and proliferation, cell survival and apoptosis and cell migration and invasion. Several lines of evidence suggest an important role for S6K1 in estrogen receptor (ER)-positive breast cancer. S6K1 directly phosphorylates and activates ER?. Furthermore, S6K1 expression is estrogenically regulated. Therefore, hyperactivation of mTORC1/S6K1 signaling may be closely related to ER-positive status in breast cancer and may be utilized as a marker for prognosis and a therapeutic target.

Holz, Marina K.

2012-01-01

179

Mitogen inactivation of glycogen synthase kinase-3 beta in intact cells via serine 9 phosphorylation.  

PubMed Central

Glycogen synthase kinase-3 (GSK-3), a protein-serine kinase implicated in cell-fate determination and differentiation, phosphorylates several regulatory proteins that are activated by dephosphorylation in response to hormones or growth factors. GSK-3 beta is phosphorylated in vitro at serine 9 by p70 S6 kinase and p90rsk-1, resulting in its inhibition [Sutherland, Leighton, and Cohen (1993) Biochem. J. 296, 15-19]. Using HeLa cells expressing GSK-3 beta or a mutant containing alanine at residue 9, we demonstrate that serine 9 is modified in intact cells and is targeted specifically by p90rsk-1, and that phosphorylation leads to loss of activity. Since p90rsk-1 is directly activated by mitogen-activated protein kinases, agonists of this pathway, such as insulin, repress GSK-3 function. Images Figure 1 Figure 2

Stambolic, V; Woodgett, J R

1994-01-01

180

Haloperidol promotes mTORC1-dependent phosphorylation of ribosomal protein S6 via dopamine- and cAMP-regulated phosphoprotein of 32 kDa and inhibition of protein phosphatase-1.  

PubMed

The ribosomal protein S6 (rpS6) is a component of the small 40S ribosomal subunit, involved in multiple physiological functions. Here, we examined the effects produced by haloperidol, a typical antipsychotic drug, on the phosphorylation of rpS6 at Ser240/244 in the striatum, a brain region involved in neurodegenerative and neuropsychiatric disorders. We found that administration of haloperidol increased Ser240/244 phosphorylation in a subpopulation of GABA-ergic medium spiny neurons (MSNs), which preferentially express dopamine D2 receptors (D2Rs). This effect was abolished by rapamycin, an inhibitor of the mammalian target of rapamycin complex 1 (mTORC1), or by PF470867, a selective inhibitor of the p70 ribosomal S6 kinase 1 (S6K1). We also found that the effect of haloperidol on Ser240/244 phosphorylation was prevented by functional inactivation of dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), an endogenous inhibitor of protein phosphatase-1 (PP-1). In line with this observation, incubation of striatal slices with okadaic acid and calyculin A, two inhibitors of PP-1, increased Ser240/244 phosphorylation. These results show that haloperidol promotes mTORC1- and S6K1-dependent phosphorylation of rpS6 at Ser240/244, in a subpopulation of striatal MSNs expressing D2Rs. They also indicate that this effect is exerted by suppressing dephosphorylation at Ser240/244, through PKA-dependent activation of DARPP-32 and inhibition of PP-1. PMID:23643747

Bonito-Oliva, Alessandra; Pallottino, Simone; Bertran-Gonzalez, Jesus; Girault, Jean-Antoine; Valjent, Emmanuel; Fisone, Gilberto

2013-05-03

181

Macrophage produced IL-12p70 mediates hemorrhage-induced damage in a complement dependent manner  

PubMed Central

Hemorrhage and hemorrhagic shock instigate intestinal damage and inflammation. Multiple components of the innate immune response, including complement and neutrophil infiltration, are implicated in this pathology. To investigate the interaction of complement activation and other components of the innate immune response during hemorrhage, we treated mice post-hemorrhage with CR2-fH, a targeted inhibitor of the alternative complement pathway and assessed intestinal damage and inflammation 2 h after hemorrhage. In wildtype mice, CR2-fH attenuated hemorrhage-induced, mid-jejunal damage and inflammation as determined by decreased mucosal damage, macrophage infiltration, LTB4, IL-12p40, and TNF-? production. The critical nature of intestinal macrophage infiltration and activation in the response to hemorrhage was further determined using mice pre-treated with clodronate containing liposomes. The absence of either macrophages or IL-12p70 attenuated intestinal damage. These data suggest that complement activation and macrophage infiltration with IL-12p70 production are critical to hemorrhage induced mid-jejunal damage and inflammation.

Hylton, Diana J.; Hoffman, Sara M.; Van Rooijen, N.; Tomlinson, Stephen; Fleming, Sherry D.

2010-01-01

182

Wortmannin inhibits the effects of insulin and serum on the activities of glycogen synthase kinase-3 and mitogen-activated protein kinase.  

PubMed Central

We have previously shown that insulin causes inactivation of glycogen synthase kinase-3 (GSK-3) in Chinese hamster ovary cells over-expressing the human insulin receptor (CHO.T cells). We now show that serum and phorbol ester also cause rapid inactivation of GSK-3, both in CHO.T cells and in the nontransfected parental cell line, CHO.K1 cells. Rapamycin was without effect on the inactivation of GSK-3 by insulin, serum or phorbol ester, indicating that the p70 S6 kinase pathway is not involved. In contrast, wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, blocked the effects of both insulin and serum on GSK-3 activity, and also substantially reduced the activation of both p90 S6 kinase (by insulin) and mitogen-activated protein (MAP) kinase (by insulin and serum). These findings imply (i) that GSK-3 activity is regulated by a cascade involving MAP kinase and p90 S6 kinase and (ii) that wortmannin affects an early step in the MAP kinase pathway. One can infer from this that GSK-3 may be an important regulatory enzyme for the control of several biosynthetic pathways, key enzymes in which are regulated by GSK-3-mediated phosphorylation. Wortmannin had a smaller effect on the activation of MAP kinase by phorbol ester, indicating that phorbol esters may stimulate MAP kinase by a different or additional mechanism to that employed by insulin or serum. Wortmannin had very little effect on the inactivation of GSK-3 by phorbol ester: possible reasons for this are discussed. Images Figure 4

Welsh, G I; Foulstone, E J; Young, S W; Tavare, J M; Proud, C G

1994-01-01

183

Specific interaction between S6K1 and CoA synthase: a potential link between the mTOR\\/S6K pathway, CoA biosynthesis and energy metabolism  

Microsoft Academic Search

Ribosomal protein S6 kinase (S6K) is a key regulator of cell size and growth. It is regulated via phosphoinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR) signaling pathways. We demonstrate for the first time that CoA synthase associates specifically with S6K1. The association was observed between native and transiently overexpressed proteins in vivo, as well as by BIAcore

Ivan Nemazanyy; Ganna Panasyuk; Alexander Zhyvoloup; George Panayotou; Ivan T. Gout; Valeriy Filonenko

2004-01-01

184

IL-8-INDUCED NEUTROPHIL CHEMOTAXIS IS MEDIATED BY JANUS KINASE 3 (JAK3)  

PubMed Central

Janus kinase 3 (JAK3) is a non-receptor tyrosine kinase vital to the regulation of T-cells. We report that JAK3 is a mediator of IL-8 stimulation of a different class of hematopoietic relevant cells: human neutrophils. IL-8 induced a time- and concentration-dependent activation of JAK3 activity in neutrophils and differentiated HL-60 leukemic cells. JAK3 was more robustly activated by IL-8 than other kinases: p70S6K, mTOR, MAPK or PKC. JAK3 silencing severely inhibited IL-8-mediated chemotaxis. Thus, IL-8 stimulates chemotaxis through a mechanism mediated by JAK3. Further, JAK3 activity and chemotaxis were inhibited by the flavonoid apigenin (4,5,7-trihydroxyflavone) at ~5 nM IC50. These new findings lay the basis for understanding the molecular mechanism of cell migration as it relates to neutrophil-mediated chronic inflammatory processes.

Henkels, Karen M.; Frondorf, Kathleen; Gonzalez-Mejia, M. Elba; Doseff, Andrea L.; Gomez-Cambronero, Julian

2010-01-01

185

PHLPP-Mediated Dephosphorylation of S6K1 Inhibits Protein Translation and Cell Growth ?  

PubMed Central

PHLPP is a family of Ser/Thr protein phosphatases that contains PHLPP1 and PHLPP2 isoforms. We have shown previously that PHLPP functions as a tumor suppressor by negatively regulating Akt signaling in cancer cells. Here we report the identification of ribosomal protein S6 kinase 1 (S6K1) as a novel substrate of PHLPP. Overexpression of both PHLPP isoforms resulted in a decrease in S6K1 phosphorylation in cells, and this PHLPP-mediated dephosphorylation of S6K1 was independent of its ability to dephosphorylate Akt. Conversely, S6K1 phosphorylation was increased in cells depleted of PHLPP expression. Furthermore, we showed that the insulin receptor substrate 1 (IRS-1) expression and insulin-induced Akt phosphorylation were significantly decreased as the result of activation of the S6K-dependent negative feedback loop in PHLPP knockdown cells. Functionally, the phosphorylation of ribosomal protein S6 (rpS6) and the amount of phosphorylated rpS6 bound to the translation initiation complex were increased in PHLPP-knockdown cells. This correlated with increased cell size, protein content, and rate of cap-dependent translation. Taken together, our results demonstrate that loss of PHLPP expression activates the S6K-dependent negative feedback loop and that PHLPP is a novel player involved in regulating protein translation initiation and cell size via direct dephosphorylation of S6K1.

Liu, Jianyu; Stevens, Payton D.; Li, Xin; Schmidt, Micheal D.; Gao, Tianyan

2011-01-01

186

Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway.  

PubMed

Brief intermittent episodes of ischemia and reperfusion, at the onset of reperfusion after a prolonged period of ischemia, confer cardioprotection, a phenomenon termed "ischemic postconditioning" (Postcond). We hypothesized that this phenomenon may just represent a modified form of reperfusion that activates the reperfusion injury salvage kinase (RISK) pathway. Isolated perfused rat hearts were subjected to: (a) 35 minutes of ischemia and 120 minutes of reperfusion, and infarct size was determined by tetrazolium staining; or (b) 35 minutes of ischemia and 7 minutes of reperfusion, and the phosphorylation states of Akt, endothelial NO synthase (eNOS), and p70S6K were determined. Postcond reduced infarct size from 51.2+/-3.4% to 31.5+/-4.1% (P<0.01), an effect comparable with ischemic preconditioning (IPC; 27.5+/-2.3%; P<0.01). Of interest, the combined protective effects of IPC and Postcond were not additive (30.1+/-4.8% with IPC+Postcond; P=NS). Inhibiting phosphatidylinositol 3-kinase (PI3K) at reperfusion using LY or Wortmannin (Wort) during the first 15 minutes of reperfusion completely abolished Postcond-induced protection (31.5+/-4.1% with Postcond versus 51.7+/-4.5% with Postcond+LY, P<0.01; 56.2+/-10.1% with Postcond+ Wort; P<0.01), suggesting that Postcond protects the heart by activating PI3K-Akt. Western blot analysis demonstrated that Postcond induced a significant increase in phosphorylation of Akt, eNOS, and p70S6K in an LY- and Wort-sensitive manner. In conclusion, we show for the first time that ischemic Postcond protects the myocardium by activating the prosurvival kinases PI3K-Akt, eNOS, and p70S6K in accordance with the RISK pathway. PMID:15242972

Tsang, Andrew; Hausenloy, Derek J; Mocanu, Mihaela M; Yellon, Derek M

2004-07-08

187

Rapamycin augments human DC IL-12p70 and IL-27 secretion to promote allogeneic Type 1 polarization modulated by NK cells.  

PubMed

Mammalian target of rapamycin kinase inhibitor (mTORi) rapamycin (RAPA) use in transplantation can lead to inflammatory complications in some patients. Our goal was to better understand how mTORi-exposed human monocyte-derived dendritic cells (DC) stimulated with pro-inflammatory cytokines shape T cell allo-immunity. RAPA-conditioned-DC (RAPA-DC) displayed a more immature phenotype than untreated, control (CTRL)-DC. However, subsequent exposure of RAPA-DC to an inflammatory cytokine cocktail (ICC) plus IFN-? induced a mature Type-1 promoting phenotype, consisting of elevated HLA-DR and co-stimulatory molecules, augmented IL-12p70 and IL-27 production, but decreased IL-10 secretion compared to CTRL-DC. Co-culture of mature (m)RAPA-DC with allogeneic peripheral blood mononuclear cells resulted in significantly increased Type-1 (IFN-?) responses by T cells. Moreover, NK cells acted as innate modulators that conveyed activating cell-to-cell contact signals in addition to helper (IFN-?) and/or regulatory (IL-10) soluble cytokines. We conclude that production of IL12-p70, IL-27 and low IL-10 by RAPA-DC allowed us to elucidate how these cytokines as well as NK-DC interaction shapes T cell allo-immunity. Thus, lack of inhibitory NK cell function during allo-specific T cell activation by human ICC + IFN-?-stimulated RAPA-DC may represent an unwanted effector mechanism that may underlie RAPA-induced inflammatory events in transplant patients undergoing microbial infection or allograft rejection. PMID:24034707

Macedo, C; Turnquist, H R; Castillo-Rama, M; Zahorchak, A F; Shapiro, R; Thomson, A W; Metes, D

2013-08-22

188

Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity  

SciTech Connect

Anti-Ki (Ku, p70/p80) autoantibodies, named after the prototype patient Kikuta by Tojo et al., occur in approximately 10% of patients with SLE, often in association with anti-Sm autoantibodies. Anti-Ki sera specifically immunoprecipitated two protein antigens, Ki/sub 86/ (M/sub r/ 86,000) and Ki/sub 66/ (M/sub r/ 66,000), from radiolabeled cell extracts. The Ki system was found to be immunologically identical to the Ku system described by Mimori et al. and the p70/p80 system described by Reeves. The Ki primary in vitro translation products were identified and proved similar in size to the cellular antigens. The Ki antigens were purified from human spleen by immunoaffinity chromatography followed by SDS-PAGE. The purified Ki antigens proved to be closely related by amino acid composition, and did not appear to be phosphorylated, glycosylated, or associated with RNA. The Ki antigens were found to bind to DNA, in agreement with the observations on the Ku and p70/p80 antigens. They were found to be widely conserved in mammals and were coordinately expressed in all tissues tested.

Francoeur, A.M.; Peebles, C.L.; Gompper, P.T.; Tan, E.M.

1986-03-01

189

Signaling from Akt to FRAP\\/TOR Targets both 4EBP and S6K in Drosophila melanogaster  

Microsoft Academic Search

The eIF4E-binding proteins (4E-BPs) interact with translation initiation factor 4E to inhibit translation. Their binding to eIF4E is reversed by phosphorylation of several key Ser\\/Thr residues. In Drosophila, S6 kinase (dS6K) and a single 4E-BP (d4E-BP) are phosphorylated via the insulin and target of rapamycin (TOR) signaling pathways. Although S6K phosphorylation is independent of phosphoinositide 3-OH kinase (PI3K) and serine\\/threonine

Mathieu Miron; Paul Lasko; Nahum Sonenberg

2003-01-01

190

Insulin signal transduction in rat small intestine: role of MAP kinases in expression of mucosal hydrolases.  

PubMed

The postreceptor events regulating the signal of insulin downstream in rat intestinal cells have not yet been analyzed. Our objectives were to identify the nature of receptor substrates and phosphorylated proteins involved in the signaling of insulin and to investigate the mechanism(s) by which insulin enhances intestinal hydrolases. In response to insulin, the following proteins were rapidly phosphorylated on tyrosine residues: 1) insulin receptor substrates-1 (IRS-1), -2, and -4; 2) phospholipase C-isoenzyme-gamma; 3) the Ras-GTPase-activating protein (GAP) associated with Rho GAP and p62(Src); 4) the insulin receptor beta-subunit; 5) the p85 subunits of phosphatidylinositol 3-kinase (PI 3-kinase); 6) the Src homology 2 alpha-collagen protein; 7) protein kinase B; 8) mitogen-activated protein (MAP) kinase-1 and -2; and 9) growth receptor-bound protein-2. Compared with controls, insulin enhanced the intestinal activity of MAP kinase-2 and protein kinase B by two- and fivefold, respectively, but did not enhance p70/S6 ribosomal kinase. Administration of an antireceptor antibody or MAP-kinase inhibitor PD-98059 but not a PI 3-kinase inhibitor (wortmannin) to sucklings inhibited the effects of insulin on mucosal mass and enzyme expression. We conclude that normal rat enterocytes express all of the receptor substrates and mediators involved in different insulin signaling pathways and that receptor binding initiates a signal enhancing brush-border membrane hydrolase, which appears to be regulated by the cascade of MAP kinases but not by PI 3-kinase. PMID:11208545

Marandi, S; De Keyser, N; Saliez, A; Maernoudt, A S; Sokal, E M; Stilmant, C; Rider, M H; Buts, J P

2001-02-01

191

Myocardial Ischaemia-reperfusion Injury is Attenuated by Intact Glucagon Like Peptide1 (GLP-1) in the In Vitro Rat Heart and may Involve the p70s6K Pathway  

Microsoft Academic Search

Background and methods  Glucagon Like Peptide-1 (GLP-1), one of the most potent incretin hormones, has potential beneficial actions on the ischaemic\\u000a and failing heart. This study sought to further identify the mechanisms of action of GLP-1 on the ischaemic heart using an\\u000a in vitro isolated perfused rat heart model of ischaemic-reperfusion injury (measuring infarct size to area of risk (%)) subjected

Amal K. Bose; Mihaela M. Mocanu; Richard D. Carr; Derek M. Yellon

2007-01-01

192

Acetaldehyde promotes rapamycin-dependent activation of p70 S6K and glucose uptake despite inhibition of Akt and mTOR in dopaminergic SH-SY5Y human neuroblastoma cells  

Microsoft Academic Search

Alcohol intake is one of the important lifestyle factors for the risk of insulin resistance and type 2 diabetes. Acetaldehyde, the major ethanol metabolite which is far more reactive than ethanol, has been postulated to participate in alcohol-induced tissue injury although its direct impact on insulin signaling is unclear. This study was designed to examine the effect of acetaldehyde on

Cindy X. Fang; Xiaoping Yang; Nair Sreejayan; Jun Ren

2007-01-01

193

Macrophage-produced IL-12p70 mediates hemorrhage-induced damage in a complement-dependent manner.  

PubMed

Hemorrhage and hemorrhagic shock instigate intestinal damage and inflammation. Multiple components of the innate immune response, including complement and neutrophil infiltration, are implicated in this pathology. To investigate the interaction of complement activation and other components of the innate immune response during hemorrhage, we treated mice after hemorrhage with CR2-fH, a targeted inhibitor of the alternative complement pathway and assessed intestinal damage and inflammation 2 h after hemorrhage. In wild-type mice, CR2-fH attenuated hemorrhage-induced, midjejunal damage and inflammation as determined by decreased mucosal damage, macrophage infiltration, leukotriene B4, IL-12p40, and TNF-[alpha] production. The critical nature of intestinal macrophage infiltration and activation in the response to hemorrhage was further determined using mice pretreated with clodronate-containing liposomes. The absence of either macrophages or IL-12p70 attenuated intestinal damage. These data suggest that complement activation and macrophage infiltration with IL-12p70 production are critical to hemorrhage-induced midjejunal damage and inflammation. PMID:20577145

Hylton, Diana J; Hoffman, Sara M; Van Rooijen, N; Tomlinson, Stephen; Fleming, Sherry D

2011-02-01

194

The S6KII (rsk) gene of Drosophila melanogaster differentially affects an operant and a classical learning task.  

PubMed

In an attempt to dissect classical and operant conditioning in Drosophila melanogaster, we have isolated the gene for ribosomal S6 kinase II (S6KII). This enzyme is part of a family of serine-threonine kinases that in mammals have been implicated in the MAPK (mitogen-activated protein kinase) signaling cascade controlling (among other processes) synaptic plasticity (long-term potentiation/long-term depression) and memory formation. The human homolog rsk2 has been linked to mental retardation (Coffin-Lowry syndrome). Mutant analysis in Drosophila shows that S6KII serves different functions in operant place learning and classical (pavlovian) olfactory conditioning. Whereas in the null mutant only pavlovian olfactory learning is affected, a P-element insertion mutant reducing the amount of S6KII only affects operant place learning. A mutant lacking part of the N-terminal kinase domain and performing poorly in both learning tasks is dominant in the operant paradigm and recessive in the pavlovian paradigm. The behavioral defects in the pavlovian task can be rescued by the genomic S6KII transgene. Overexpression of S6KII in wild type has a dominant-negative effect on the operant task that is rescued by the null mutant, whereas in the pavlovian task overexpression may even enhance learning performance. PMID:15525759

Putz, Gabriele; Bertolucci, Franco; Raabe, Thomas; Zars, Troy; Heisenberg, Martin

2004-11-01

195

Effect of IRS4 Levels on PI 3-Kinase Signalling.  

PubMed

Insulin receptor substrate 1 (IRS1) and IRS2 are well-characterized adapter proteins that relay signals from receptor tyrosine kinases to downstream components of signalling pathways. In contrast, the function of IRS4 is not well understood. IRS4 overexpression has been associated with acute lymphoblastic leukaemia and subungual exostosis, while point mutations of IRS4 have been found in melanomas. Here, we show that while IRS4 expression is low in most cancer cell lines, IRS4 mRNA and protein levels are markedly elevated in certain cells including the NCI-H720, DMS114, HEK293T and HEK293AAV lines. Surprisingly, IRS4 expression was also strongly induced when HEK293 cells were infected with retroviral particles and selected under puromycin, making IRS4 expression a potential off-target effect of retroviral expression vectors. Cells with high expression of IRS4 displayed high phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels, as well as elevated Akt and p70 S6 kinase activities, even in the absence of growth factors. PI 3-kinase (PI3K) signalling in these cells depends on IRS4, even though these cells also express IRS1/2. Knockdown of IRS4 also inhibited cell proliferation in cells with high levels of IRS4. Together, these findings suggest IRS4 as a potential therapeutic target for cancers with high expression of this protein. PMID:24039912

Hoxhaj, Gerta; Dissanayake, Kumara; Mackintosh, Carol

2013-09-10

196

Inhibition of PI-3 kinase/Akt/mTOR, but not calcineurin signaling, reverses insulin-like growth factor I-induced protection against glucose toxicity in cardiomyocyte contractile function.  

PubMed

Insulin-like growth factor-I (IGF-1) ameliorates cardiac dysfunction in diabetes although the mechanism of action remains poorly understood. This study examined the role of PI-3 kinase/Akt/mammalian target of rapamycin (mTOR) and calcineurin pathways in cardiac effects of IGF-1 against glucose toxicity. Adult rat ventricular myocytes were cultured for 8 h with either normal (NG, 5.5 mM) or high (HG, 25.5 mM) glucose, in the presence or absence of IGF-1 (10-500 nM), the PI-3 kinase/Akt inhibitor LY294002 (10 microM), the mTOR inhibitor rapamycin (20 microM) or the calcineurin inhibitors cyclosporin A (5 microM) or FK506 (10 mg/l). Mechanical properties were evaluated using an IonOptix MyoCam system. HG depressed peak shortening (PS), reduced maximal velocity of shortening/relengthening (+/- dl/dt) and prolongs time-to-90% relengthening (TR90), which were abolished by IGF-1 (100 and 500 nM). Interestingly, the IGF-1-elicited protective effect against HG was nullified by either LY294002 or rapamycin, but not by cyclosporine A or FK506. None of the inhibitors affected cell mechanics. Western blot analysis indicated that HG and IGF-1 stimulated phosphorylation of Akt and mTOR. HG also activated p70s6k and suppressed GSK-3beta phosphorylation. However, the HG-induced alterations in phosphorylation of Akt, mTOR, p70s6k and GSK-3beta were significantly reversed by IGF-1. Protein expression of Akt, mTOR, p70s6k, GSK-3beta, SERCA2a and phospholamban was unaffected by HG, IGF-1 or rapamycin. Rapamycin significantly enhanced Akt phosphorylation whereas it inhibited mTOR phosphorylation. Collectively, our data suggest that IGF-1 may provide cardiac protection against glucose in part through a PI-3 kinase/Akt/mTOR/ p70s6k-dependent and calcineurin-independent pathway. PMID:16135669

Li, Shi-Yan; Fang, Cindy X; Aberle, Nicholas S; Ren, Bonnie H; Ceylan-Isik, Asli F; Ren, Jun

2005-09-01

197

THE ROLE OF MTOR\\/S6K IN DEVELOPMENT AND HUMAN DISEASE  

Microsoft Academic Search

INTRODUCTION. The mTOR\\/S6K signaling pathway is an ancient nutrient effector pathway. With the rise of metazoans this pathway has become integrated with the insulin-controlled class 1 phosphatidyl-inositide-3OH-kinase, PI3K, pathway to regulate growth and development of the organism. The importance of this pathway in the pathogenesis of human diseases has been underscored by the wide use of the rapamycins in a

George Thomas

198

Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity  

Microsoft Academic Search

Elucidating the signalling mechanisms by which obesity leads to impaired insulin action is critical in the development of therapeutic strategies for the treatment of diabetes. Recently, mice deficient for S6 Kinase 1 (S6K1), an effector of the mammalian target of rapamycin (mTOR) that acts to integrate nutrient and insulin signals, were shown to be hypoinsulinaemic, glucose intolerant and have reduced

Sung Hee Um; Francesca Frigerio; Mitsuhiro Watanabe; Frédéric Picard; Manel Joaquin; Melanie Sticker; Stefano Fumagalli; Peter R. Allegrini; Sara C. Kozma; Johan Auwerx; George Thomas

2004-01-01

199

Macrophage IL-12p70 Signaling Prevents HSV-1-Induced CNS Autoimmunity Triggered by Autoaggressive CD4+ Tregs  

PubMed Central

Purpose. CD4+CD25+FoxP3+ naturally occurring regulatory T cells (Tregs) maintain self-tolerance and function to suppress overly exuberant immune responses. However, it is unclear whether innate immune cells modulate Treg function. Here the authors examined the role of innate immunity in lymphomyeloid homeostasis. Methods. The involvement of B cells, dendritic cells (DCs), macrophages, natural killer (NK) cells, and T cells in central nervous system (CNS) demyelination in different strains of mice infected ocularly with herpes simplex virus type 1 (HSV-1) was investigated. Results. The authors found that depletion of macrophages, but not DCs, B cells, NK cells, CD4+ T cells, or CD8+ T cells, induced CNS demyelination irrespective of virus or mouse strain. As with macrophage depletion, mice deficient in interleukin (IL)-12p35 or IL-12p40 showed CNS demyelination after HSV-1 infection, whereas demyelination was undetectable in HSV-1–infected, IL-23p19–deficient, or Epstein-Barr virus–induced gene 3-deficient mice. Demyelination could be rescued in macrophage-depleted mice after the injection of IL-12p70 DNA and in IL-12p35?/? or IL-12p40?/? mice after injection with IL-12p35 or IL-12p40 DNA or with recombinant viruses expressing IL-12p35 or IL-12p40. Using FoxP3-, CD4-, CD8-, or CD25-depletion and gene-deficient mouse approaches, the authors demonstrated that HSV-1–induced demyelination was blocked in the absence of CD4, CD25, or FoxP3 in macrophage-depleted mice. Flow cytometry showed an elevation of CD4+CD25+FoxP3+ T cells in the spleens of infected macrophage-depleted mice, and adoptive transfer of CD4+CD25+ T cells to infected macrophage-depleted severe combined immunodeficient mice induced CNS demyelination. Conclusions. The authors demonstrated that macrophage IL-12p70 signaling plays an important role in maintaining immune homeostasis in the CNS by preventing the development of autoaggressive CD4+ Tregs.

Mott, Kevin R.; Gate, David; Zandian, Mandana; Allen, Sariah J.; Rajasagi, Naveen Kumar; van Rooijen, Nico; Chen, Shuang; Arditi, Moshe; Rouse, Barry T.; Flavell, Richard A.; Town, Terrence; Ghiasi, Homayon

2011-01-01

200

Effect of growth conditions of Lactobacillus gasseri OLL2809 on the immunostimulatory activity for production of interleukin-12 (p70) by murine splenocytes.  

PubMed

Lactobacillus gasseri OLL2809, a probiotic lactic acid bacterium, strongly stimulates interleukin (IL)-12 (p70) production by murine splenocytes; therefore, it is expected to ameliorate allergic diseases. Although many studies have investigated characteristics of the immunostimulatory activity of probiotics, little is known about how bacterial growth conditions affect the activity. In this study, we investigated the effects of the growth conditions of L. gasseri OLL2809 on the stimulation of IL-12 (p70) production. L. gasseri OLL2809 was grown under various culture conditions including different cultivation periods, media, and culture pH, and IL-12 (p70) production by murine splenocytes stimulated with these bacterial cells was determined. The results revealed that IL-12 (p70) production (i) increased with the growth of the bacterial cells and was higher in stationary-phase cells than in logarithmic-phase cells; (ii) it was higher in the cells grown in acidic pH; and (iii) it decreased when the cells were incubated in a buffer at neutral pH prior to heat treatment. These observations indicated that stimulation of IL-12 (p70) production is affected by culture medium pH. In addition, the observations of a difference in the stimulation of IL-12 (p70) production by L. gasseri OLL2809 grown under various conditions are consistent with the characteristics of autolysis. Therefore, it was deduced that the integrity of the bacterial cells is necessary for the stimulatory effect on IL-12 (p70) production and that acidic pH and heat treatment contributed to the stimulation by inhibiting the activity of autolysins indigenous to the bacteria. Our result suggests that cultivation until the stationary phase under acidic pH is required for the effective production of probiotics with immunostimulatory activity. PMID:17936392

Sashihara, Toshihiro; Sueki, Natsuko; Furuichi, Keisuke; Ikegami, Shuji

2007-09-15

201

Nocardia farcinica activates human dendritic cells and induces secretion of interleukin-23 (IL-23) rather than IL-12p70.  

PubMed

Studying the interaction of dendritic cells (DCs) with bacteria controlled by T-cell-mediated immune responses may reveal novel adjuvants for the induction of cellular immunity. Murine studies and the observation that nocardias infect predominantly immunosuppressed patients have suggested that these bacteria may possess an adjuvant potential. Moreover, adjuvants on the basis of the nocardial cell wall have been applied in clinical studies. Since the handling of adjuvants by DCs may determine the type of immune responses induced by a vaccine, the present study aimed at investigating the interaction of immature human monocyte-derived DCs with live or inactivated Nocardia farcinica in vitro and determining the cellular phenotypic changes as well as alterations in characteristic functions, such as phagocytosis, induction of T-cell proliferation, and cytokine secretion. Human DCs ingested N. farcinica and eradicated the bacterium intracellularly. DCs exposed to inactivated N. farcinica were activated, i.e., they developed a mature phenotype, downregulated their phagocytic capacity, and stimulated allogeneic T cells in mixed leukocyte reactions. Soluble factors were not involved in this process. To elucidate the potential adjuvant effect of N. farcinica on the induction of T-cell-mediated immune responses, we characterized the cytokines produced by nocardia-exposed DCs and detected substantial amounts of tumor necrosis factor alpha (TNF-?) and interleukin-12 p40 (IL-12p40). However, nocardia-treated DCs secreted only small amounts of IL-12p70, which were significantly smaller than the amounts of IL-23. Thus, N. farcinica activates DCs, but adjuvants based on this bacterium may have only a limited capacity to induce Th1 immune responses. PMID:22988018

Eisenblätter, Martin; Buchal, Ariane; Gayum, Hermine; Jasny, Edith; Renner Viveros, Pablo; Ulrichs, Timo; Schneider, Thomas; Schumann, Ralf R; Zweigner, Janine; Ignatius, Ralf

2012-09-17

202

Nocardia farcinica Activates Human Dendritic Cells and Induces Secretion of Interleukin-23 (IL-23) Rather than IL-12p70  

PubMed Central

Studying the interaction of dendritic cells (DCs) with bacteria controlled by T-cell-mediated immune responses may reveal novel adjuvants for the induction of cellular immunity. Murine studies and the observation that nocardias infect predominantly immunosuppressed patients have suggested that these bacteria may possess an adjuvant potential. Moreover, adjuvants on the basis of the nocardial cell wall have been applied in clinical studies. Since the handling of adjuvants by DCs may determine the type of immune responses induced by a vaccine, the present study aimed at investigating the interaction of immature human monocyte-derived DCs with live or inactivated Nocardia farcinica in vitro and determining the cellular phenotypic changes as well as alterations in characteristic functions, such as phagocytosis, induction of T-cell proliferation, and cytokine secretion. Human DCs ingested N. farcinica and eradicated the bacterium intracellularly. DCs exposed to inactivated N. farcinica were activated, i.e., they developed a mature phenotype, downregulated their phagocytic capacity, and stimulated allogeneic T cells in mixed leukocyte reactions. Soluble factors were not involved in this process. To elucidate the potential adjuvant effect of N. farcinica on the induction of T-cell-mediated immune responses, we characterized the cytokines produced by nocardia-exposed DCs and detected substantial amounts of tumor necrosis factor alpha (TNF-?) and interleukin-12 p40 (IL-12p40). However, nocardia-treated DCs secreted only small amounts of IL-12p70, which were significantly smaller than the amounts of IL-23. Thus, N. farcinica activates DCs, but adjuvants based on this bacterium may have only a limited capacity to induce Th1 immune responses.

Eisenblatter, Martin; Buchal, Ariane; Gayum, Hermine; Jasny, Edith; Renner Viveros, Pablo; Ulrichs, Timo; Schneider, Thomas; Schumann, Ralf R.; Zweigner, Janine

2012-01-01

203

Ghrelin-induced food intake and adiposity depend on central mTORC1/S6K1 signaling.  

PubMed

Signaling through the mammalian target of rapamycin complex 1 (mTORC1) and its effectors the S6-kinases (S6K) in the hypothalamus is thought to be involved in nutrient sensing and control of food intake. Given the anatomical proximity of this pathway to circuits for the hormone ghrelin, we investigated the potential role of the mTORC1/S6K pathway in mediating the metabolic effects of ghrelin. We found that ghrelin promoted phosphorylation of S6K1 in the mouse hypothalamic cell line N-41 and in the rat hypothalamus after intracerebroventricular administration. Rapamycin, an inhibitor of mTORC1, suppressed ghrelin-induced phosphorylation of hypothalamic S6K1 and increased food intake and insulin in rats. Chronic peripheral administration of ghrelin induced a significant increase in body weight, fat mass and food efficiency in wild-type and S6K2-knockout but not in S6K1-knockout mice. We therefore propose that ghrelin-induced hyperphagia, adiposity and insulin secretion are controlled by a central nervous system involving the mTORC1/S6K1 pathway. PMID:23994018

Stevanovic, Darko; Trajkovic, Vladimir; Müller-Lühlhoff, Sabrina; Brandt, Elisabeth; Abplanalp, William; Bumke-Vogt, Christiane; Liehl, Beate; Wiedmer, Petra; Janjetovic, Kristina; Starcevic, Vesna; Pfeiffer, Andreas F H; Al-Hasani, Hadi; Tschöp, Matthias H; Castañeda, Tamara R

2013-08-29

204

(S)-[6]-Gingerol enhances glucose uptake in L6 myotubes by activation of AMPK in response to [Ca2+]i.  

PubMed

Purpose. The aim of this study was to investigate the mechanism of (S)-[6]-gingerol in promoting glucose uptake in L6 skeletal muscle cells. METHODS. The effect of (S)-[6]-gingerol on glucose uptake in L6 myotubes was examined using 2-[1,2-3H]-deoxy-D-glucose. Intracellular Ca2+ concentration was measured using Fluo-4. Phosphorylation of AMPK? was determined by Western blotting analysis. RESULTS. (S)-[6]-Gingerol time-dependently enhanced glucose uptake in L6 myotubes. (S)-[6]-Gingerol elevated intracellular Ca2+ concentration and subsequently induced a dose- and time-dependent enhancement of threonine172 phosphorylated AMPK? in L6 myotubes via modulation by Ca2+/calmodulin-dependent protein kinase kinase. CONCLUSION. The results indicated that (S)-[6]-gingerol increased glucose uptake in L6 skeletal muscle cells by activating AMPK. (S)-[6]-gingerol, a major component of Zingiber officinale, may have potential for development as an antidiabetic agent. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page. PMID:23958199

Li, Yiming; Tran, Van H; Koolaji, Nooshin; Duke, Colin; Roufogalis, Basil D

2013-01-01

205

Flow cytometric analysis of expression of interleukin-2 receptor beta chain (p70-75) on various leukemic cells  

SciTech Connect

We analyzed the expression of the interleukin-2 receptor (IL-2R) beta chain (p70-75) on various leukemic cells from 44 patients by flow cytometric analysis using the IL-2R beta chain-specific monoclonal antibody, designated Mik-beta 1. Flow cytometric analysis demonstrated the expression of the IL-2R beta chain on granular lymphocytes (GLs) from all eight patients with granular lymphocyte proliferative disorders (GLPDs), on adult T-cell leukemia (ATL) cells from all three patients with ATL, and on T-cell acute lymphoblastic leukemia (T-ALL) cells from one of three patients with T-ALL. Although GLs from all the GLPD patients expressed the IL-2R beta chain alone and not the IL-2R alpha chain (Tac-antigen: p55), ATL and T-ALL cells expressing the beta chain coexpressed the alpha chain. In two of seven patients with common ALL (cALL) and in both patients with B-cell chronic lymphocytic leukemia, the leukemic cells expressed the alpha chain alone. Neither the alpha chain nor the beta chain was expressed on leukemic cells from the remaining 28 patients, including all 18 patients with acute nonlymphocytic leukemia, five of seven patients with cALL, all three patients with multiple myeloma, and two of three patients with T-ALL. These results indicate that three different forms of IL-2R chain expression exist on leukemic cells: the alpha chain alone; the beta chain alone; and both the alpha and beta chains. To examine whether the results obtained by flow cytometric analysis actually reflect functional aspects of the expressed IL-2Rs, we studied the specific binding of 125I-labeled IL-2 (125I-IL-2) to leukemic cells in 18 of the 44 patients. In addition, we performed 125I-IL-2 crosslinking studies in seven patients. The results of IL-2R expression of both 125I-IL-2 binding assay and crosslinking studies were in agreement with those obtained by flow cytometric analysis.

Hoshino, S.; Oshimi, K.; Tsudo, M.; Miyasaka, M.; Teramura, M.; Masuda, M.; Motoji, T.; Mizoguchi, H. (Tokyo Women's Medical College (Japan))

1990-08-15

206

Saporin-s6: a useful tool in cancer therapy.  

PubMed

Thirty years ago, the type 1 ribosome-inactivating protein (RIP) saporin-S6 (also known as saporin) was isolated from Saponaria officinalis L. seeds. Since then, the properties and mechanisms of action of saporin-S6 have been well characterized, and it has been widely employed in the construction of conjugates and immunotoxins for different purposes. These immunotoxins have shown many interesting results when used in cancer therapy, particularly in hematological tumors. The high enzymatic activity, stability and resistance to conjugation procedures and blood proteases make saporin-S6 a very useful tool in cancer therapy. High efficacy has been reported in clinical trials with saporin-S6-containing immunotoxins, at dosages that induced only mild and transient side effects, which were mainly fever, myalgias, hepatotoxicity, thrombocytopenia and vascular leak syndrome. Moreover, saporin-S6 triggers multiple cell death pathways, rendering impossible the selection of RIP-resistant mutants. In this review, some aspects of saporin-S6, such as the chemico-physical characteristics, the structural properties, its endocytosis, its intracellular routing and the pathogenetic mechanisms of the cell damage, are reported. In addition, the recent progress and developments of saporin-S6-containing immunotoxins in cancer immunotherapy are summarized, including in vitro and in vivo pre-clinical studies and clinical trials. PMID:24105401

Polito, Letizia; Bortolotti, Massimo; Mercatelli, Daniele; Battelli, Maria Giulia; Bolognesi, Andrea

2013-10-07

207

Saporin-S6: A Useful Tool in Cancer Therapy  

PubMed Central

Thirty years ago, the type 1 ribosome-inactivating protein (RIP) saporin-S6 (also known as saporin) was isolated from Saponaria officinalis L. seeds. Since then, the properties and mechanisms of action of saporin-S6 have been well characterized, and it has been widely employed in the construction of conjugates and immunotoxins for different purposes. These immunotoxins have shown many interesting results when used in cancer therapy, particularly in hematological tumors. The high enzymatic activity, stability and resistance to conjugation procedures and blood proteases make saporin-S6 a very useful tool in cancer therapy. High efficacy has been reported in clinical trials with saporin-S6-containing immunotoxins, at dosages that induced only mild and transient side effects, which were mainly fever, myalgias, hepatotoxicity, thrombocytopenia and vascular leak syndrome. Moreover, saporin-S6 triggers multiple cell death pathways, rendering impossible the selection of RIP-resistant mutants. In this review, some aspects of saporin-S6, such as the chemico-physical characteristics, the structural properties, its endocytosis, its intracellular routing and the pathogenetic mechanisms of the cell damage, are reported. In addition, the recent progress and developments of saporin-S6-containing immunotoxins in cancer immunotherapy are summarized, including in vitro and in vivo pre-clinical studies and clinical trials.

Polito, Letizia; Bortolotti, Massimo; Mercatelli, Daniele; Battelli, Maria Giulia; Bolognesi, Andrea

2013-01-01

208

Effect of growth conditions of Lactobacillus gasseri OLL2809 on the immunostimulatory activity for production of interleukin-12 (p70) by murine splenocytes  

Microsoft Academic Search

Lactobacillus gasseri OLL2809, a probiotic lactic acid bacterium, strongly stimulates interleukin (IL)-12 (p70) production by murine splenocytes; therefore, it is expected to ameliorate allergic diseases. Although many studies have investigated characteristics of the immunostimulatory activity of probiotics, little is known about how bacterial growth conditions affect the activity. In this study, we investigated the effects of the growth conditions of

Toshihiro Sashihara; Natsuko Sueki; Keisuke Furuichi; Shuji Ikegami

2007-01-01

209

Shock Metamorphism in L Chondrites Above Shock Stage S6  

NASA Astrophysics Data System (ADS)

We investigated several L6 chondrites shocked to between stage S6 and whole rock melting. The study presents the effects of high post-shock temperature and the annealing of high-pressure evidence in highly shocked chondrites.

Hu, J.; Sharp, T. G.; De Carli, P. S.

2013-09-01

210

Supersymmetric AdS6 via T Duality  

NASA Astrophysics Data System (ADS)

We present a new supersymmetric AdS6 solution of type IIB supergravity with SU(2) isometry. Through the AdS/CFT correspondence, this has potentially very interesting implications for 5D fixed point theories. This solution is the result of a non-Abelian T duality on the known supersymmetric AdS6 solution of massive IIA. The SU(2) R symmetry is untouched, leading to sixteen supercharges and preserved supersymmetry.

Lozano, Y.; Ó Colgáin, E.; Rodríguez-Gómez, D.; Sfetsos, K.

2013-06-01

211

mTOR signaling is activated by FLT3 kinase and promotes survival of FLT3-mutated acute myeloid leukemia cells  

PubMed Central

Activating mutations of the FLT3 gene mediate leukemogenesis, at least in part, through activation of PI3K/AKT. The mammalian target of rapamycin (mTOR)-Raptor signaling pathway is known to act downstream of AKT. Here we show that the mTOR effectors, 4EBP1, p70S6K and rpS6, are highly activated in cultured and primary FLT3-mutated acute myeloid leukemia (AML) cells. Introduction of FLT3-ITD expressing constitutively activated FLT3 kinase further activates mTOR and its downstream effectors in BaF3 cells. We also found that mTOR signaling contributes to tumor cell survival, as demonstrated by pharmacologic inhibition of PI3K/AKT/mTOR, or total silencing of the mTOR gene. Furthermore, inhibition of FLT3 kinase results in downregulation of mTOR signaling associated with decreased survival of FLT3-mutated AML cells. These findings suggest that mTOR signaling operates downstream of activated FLT3 kinase thus contributing to tumor cell survival, and may represent a promising therapeutic target for AML patients with mutated-FLT3.

2010-01-01

212

Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) regulates TOR signaling and cell growth during Drosophila development  

PubMed Central

During development, Drosophila larvae undergo a dramatic increase in body mass wherein nutritional and developmental cues are transduced into growth through the activity of complex signaling pathways. Class I phosphoinositide 3-kinases have an established role in this process. In this study we identify Drosophila phosphatidylinositol 5-phosphate 4-kinase (dPIP4K) as a phosphoinositide kinase that regulates growth during larval development. Loss-of-function mutants in dPIP4K show reduced body weight and prolonged larval development, whereas overexpression of dPIP4K results both in an increase in body weight and shortening of larval development. The growth defect associated with dPIP4K loss of function is accompanied by a reduction in the average cell size of larval endoreplicative tissues. Our findings reveal that these phenotypes are underpinned by changes in the signaling input into the target of rapamycin (TOR) signaling complex and changes in the activity of its direct downstream target p70 S6 kinase. Together, these results define dPIP4K activity as a regulator of cell growth and TOR signaling during larval development.

Gupta, Amit; Toscano, Sarah; Trivedi, Deepti; Jones, David R.; Mathre, Swarna; Clarke, Jonathan H.; Divecha, Nullin; Raghu, Padinjat

2013-01-01

213

A Drosophila protein homologous to the human p70 Ku autoimmune antigen interacts with the P transposable element inverted repeats.  

PubMed Central

P transposable elements in Drosophila are mobilized via a cut-and-paste mechanism. This mode of transposition requires repair of both a double-strand break at the donor DNA site and gapped DNA at the target site. Biochemical studies have identified a cellular non-P element-encoded DNA binding protein, termed the inverted repeat binding protein (IRBP), that specifically interacts with the outer half of the 31-bp terminal inverted repeats. Protein sequence information was used to isolate cDNA clones encoding IRBP. Sequence analysis shows that IRBP is related to the 70-kDa subunit of the human Ku autoimmune antigen. The mammalian Ku antigen binds free DNA termini and has been implicated in immunoglobulin VDJ recombination, DNA repair, and transcription. In addition, Ku is the DNA binding subunit of the double-strand DNA-dependent protein kinase. Cytogenetic mapping indicates that the IRBP gene maps to chromosomal position 86E on the right arm of the third chromosome. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5

Beall, E L; Admon, A; Rio, D C

1994-01-01

214

Contribution of S6K1/MAPK Signaling Pathways in the Response to Oxidative Stress: Activation of RSK and MSK by Hydrogen Peroxide  

PubMed Central

Cells respond to different kind of stress through the coordinated activation of signaling pathways such as MAPK or p53. To find which molecular mechanisms are involved, we need to understand their cell adaptation. The ribosomal protein, S6 kinase 1 (S6K1), is a common downstream target of signaling by hormonal or nutritional stress. Here, we investigated the initial contribution of S6K1/MAPK signaling pathways in the cell response to oxidative stress produced by hydrogen peroxide (H2O2). To analyze S6K1 activation, we used the commercial anti-phospho-Thr389-S6K1 antibody most frequently mentioned in the bibliography. We found that this antibody detected an 80-90 kDa protein that was rapidly phosphorylated in response to H2O2 in several human cells. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI3K inhibitors, and knock-down experiments showed that this protein was not S6K1. RSK and MSK proteins were candidate targets of this phosphorylation. We demonstrated that H2O2 stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. This phosphorylation required the activity of either p38 or ERK MAP kinases. Kinase assays showed activation of RSK and MSK by H2O2. Experiments with mouse embryonic fibroblasts from p38 animals’ knockout confirmed these observations. Altogether, these findings show that the S6K1 signaling pathway is not activated under these conditions, clarify previous observations probably misinterpreted by non-specific detection of proteins RSK and MSK by the anti-phospho-Thr389-S6K1 antibody, and demonstrate the specific activation of MAPK signaling pathways through ERK/p38/RSK/MSK by H2O2.

Schneider, Taiane; Bonan, Carla Denise; Bartrons, Ramon; Ventura, Francesc; Rodrigues de Oliveira, Jarbas; Rosa, Jose Luis

2013-01-01

215

Contribution of S6K1/MAPK Signaling Pathways in the Response to Oxidative Stress: Activation of RSK and MSK by Hydrogen Peroxide.  

PubMed

Cells respond to different kind of stress through the coordinated activation of signaling pathways such as MAPK or p53. To find which molecular mechanisms are involved, we need to understand their cell adaptation. The ribosomal protein, S6 kinase 1 (S6K1), is a common downstream target of signaling by hormonal or nutritional stress. Here, we investigated the initial contribution of S6K1/MAPK signaling pathways in the cell response to oxidative stress produced by hydrogen peroxide (H2O2). To analyze S6K1 activation, we used the commercial anti-phospho-Thr389-S6K1 antibody most frequently mentioned in the bibliography. We found that this antibody detected an 80-90 kDa protein that was rapidly phosphorylated in response to H2O2 in several human cells. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI3K inhibitors, and knock-down experiments showed that this protein was not S6K1. RSK and MSK proteins were candidate targets of this phosphorylation. We demonstrated that H2O2 stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. This phosphorylation required the activity of either p38 or ERK MAP kinases. Kinase assays showed activation of RSK and MSK by H2O2. Experiments with mouse embryonic fibroblasts from p38 animals' knockout confirmed these observations. Altogether, these findings show that the S6K1 signaling pathway is not activated under these conditions, clarify previous observations probably misinterpreted by non-specific detection of proteins RSK and MSK by the anti-phospho-Thr389-S6K1 antibody, and demonstrate the specific activation of MAPK signaling pathways through ERK/p38/RSK/MSK by H2O2. PMID:24058693

Siebel, Anna; Cubillos-Rojas, Monica; Santos, Roberto Christ; Schneider, Taiane; Bonan, Carla Denise; Bartrons, Ramon; Ventura, Francesc; Rodrigues de Oliveira, Jarbas; Rosa, Jose Luis

2013-09-18

216

Involvement of phosphorylation of adenosine 5'-monophosphate-activated protein kinase in PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori.  

PubMed

In this study, we investigated inhibition of the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) by prothoracicotropic hormone (PTTH) in prothoracic glands of the silkworm, Bombyx mori. We found that treatment with PTTH in vitro inhibited AMPK phosphorylation in time- and dose-dependent manners, as seen on Western blots of glandular lysates probed with antibody directed against AMPK? phosphorylated at Thr172. Moreover, in vitro inhibition of AMPK phosphorylation by PTTH was also verified by in vivo experiments: injection of PTTH into day 7 last instar larvae greatly inhibited glandular AMPK phosphorylation. PTTH-inhibited AMPK phosphorylation appeared to be partially reversed by treatment with LY294002, indicating involvement of phosphatidylinositol 3-kinase (PI3K) signaling. A chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-?-d-ribofuranoside, AICAR) increased both basal and PTTH-inhibited AMPK phosphorylation. Treatment with AICAR also inhibited PTTH-stimulated ecdysteroidogenesis of prothoracic glands. The mechanism underlying inhibition of PTTH-stimulated ecdysteroidogenesis by AICAR was further investigated by determining the phosphorylation of eIF4E-binding protein (4E-BP) and p70 ribosomal protein S6 kinase (S6K), two known downstream signaling targets of the target of rapamycin complex 1 (TORC1). Upon treatment with AICAR, decreases in PTTH-stimulated phosphorylation of 4E-BP and S6K were detected. In addition, treatment with AICAR did not affect PTTH-stimulated extracellular signal-regulated kinase (ERK) phosphorylation, indicating that AMPK phosphorylation is not upstream signaling for ERK phosphorylation. Examination of gene expression levels of AMPK?, ?, and ? by quantitative real-time PCR (qRT-PCR) showed that PTTH did not affect AMPK transcription. From these results, it is assumed that inhibition of AMPK phosphorylation, which lies upstream of PTTH-stimulated TOR signaling, may play a role in PTTH stimulation of ecdysteroidogenesis. PMID:23671658

Gu, Shi-Hong; Hsieh, Yun-Chin; Young, Shun-Chieh; Lin, Pei-Ling

2013-05-09

217

Faraday effect in Sn2P2S6 crystals.  

PubMed

We have revealed a large Faraday rotation in tin thiohypodiphosphate (Sn(2)P(2)S(6)) crystals, which makes this material promising for magneto-optics. The effective Faraday tensor component and the Verdet constant for the direction of the optic axis have been determined by measuring the pure Faraday rotation in Sn(2)P(2)S(6) crystals with both the single-ray and small-angular polarimetric methods at the normal conditions and a wavelength of 632.8 nm. The effective Verdet constant is found to be equal to 115 rad/T x m. PMID:19002228

Krupych, Oleh; Adamenko, Dmytro; Mys, Oksana; Grabar, Aleksandr; Vlokh, Rostyslav

2008-11-10

218

Serum Adiponectin, TNF-?, IL12p70, and IL13 Levels in Multiple Sclerosis and the Effects of Different Therapy Regimens  

Microsoft Academic Search

Objectives: Multiple sclerosis (MS) is a chronic inflammatory disease of the human central nervous system. In the present study, we aimed to determine adiponectin, tumor necrosis factor-?, interleukin (IL)-12p70, and IL-13 levels in the sera of patients with MS and to investigate the effects of interferon (IFN), glatiramer acetate (GA), and immunosuppressive treatment regimens on these parameters. Methods: Fifty-seven patients

Ugur Musabak; Seref Demirkaya; Gencer Genç; Rahsan Sagkan Ilikci; Zeki Odabasi

2011-01-01

219

Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1  

PubMed Central

Cellular growth signals stimulate anabolic processes. The mechanistic target of rapamycin complex 1 (mTORC1) is a protein kinase that senses growth signals to regulate anabolic growth and proliferation. Activation of mTORC1 led to the acute stimulation of metabolic flux through the de novo pyrimidine synthesis pathway. mTORC1 signaling post-translationally regulated this metabolic pathway via its downstream target ribosomal protein S6 kinase 1 (S6K1), which directly phosphorylates S1859 on CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotatase), the enzyme that catalyzes the first three steps of de novo pyrimidine synthesis. Growth signaling through mTORC1 thus stimulates the production of new nucleotides to accommodate an increase in RNA and DNA synthesis needed for ribosome biogenesis and anabolic growth.

Ben-Sahra, Issam; Howell, Jessica J.; Asara, John M.; Manning, Brendan D.

2013-01-01

220

Hydrogen Sulfide Inhibits High Glucose-induced Matrix Protein Synthesis by Activating AMP-activated Protein Kinase in Renal Epithelial Cells*?  

PubMed Central

Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H2S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase ?, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase ? inhibitor, had the same effect. Renal cortical content of cystathionine ?-synthase and cystathionine ?-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.

Lee, Hak Joo; Mariappan, Meenalakshmi M.; Feliers, Denis; Cavaglieri, Rita C.; Sataranatarajan, Kavithalakshmi; Abboud, Hanna E.; Choudhury, Goutam Ghosh; Kasinath, Balakuntalam S.

2012-01-01

221

S6K1- and betaTRCP-Mediated Degradation of PDCD4 Promotes Protein Translation and Cell Growth  

Microsoft Academic Search

The tumor suppressor programmed cell death protein 4 (PDCD4) inhibits the translation initiation factor eIF4A, an RNA helicase that catalyzes the unwinding of secondary structure at the 5' untranslated region (5'UTR) of messenger RNAs (mRNAs). In response to mitogens, PDCD4 was rapidly phosphorylated on Ser67 by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase SCFbetaTRCP. Expression in

N. Valerio Dorrello; Angelo Peschiaroli; Daniele Guardavaccaro; Nancy H. Colburn; Nicholas E. Sherman; Michele Pagano

2006-01-01

222

Mutagenic and cytotoxic properties of 6-thioguanine, S6-methylthioguanine, and guanine-S6-sulfonic acid.  

PubMed

Thiopurine drugs, including 6-thioguanine ((S)G), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of (S)G nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. (S)G in DNA can be methylated by S-adenosyl-l-methionine to give S(6)-methylthioguanine (S(6)mG) and oxidized by UVA light to render guanine-S(6)-sulfonic acid ((SO3H)G). Here, we constructed single-stranded M13 shuttle vectors carrying a (S)G, S(6)mG, or (SO3H)G at a unique site and allowed the vectors to propagate in wild-type and bypass polymerase-deficient Escherichia coli cells. Analysis of the replication products by using the competitive replication and adduct bypass and a slightly modified restriction enzyme digestion and post-labeling assays revealed that, although none of the three thionucleosides considerably blocked DNA replication in all transfected E. coli cells, both S(6)mG and (SO3H)G were highly mutagenic, which resulted in G-->A mutation at frequencies of 94 and 77%, respectively, in wild-type E. coli cells. Deficiency in bypass polymerases does not result in alteration of mutation frequencies of these two lesions. In contrast to what was found from previous steady-state kinetic analysis, our data demonstrated that 6-thioguanine is mutagenic, with G-->A transition occurring at a frequency of approximately 10%. The mutagenic properties of 6-thioguanine and its derivatives revealed in the present study offered important knowledge about the biological implications of these thionucleosides. PMID:18591241

Yuan, Bifeng; Wang, Yinsheng

2008-06-30

223

A small molecule inhibitor of Pim protein kinases blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma  

PubMed Central

The serine/threonine Pim kinases are up-regulated in specific hematologic neoplasms, and play an important role in key signal transduction pathways, including those regulated by MYC, MYCN, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. We demonstrate that SMI-4a, a novel benzylidene-thiazolidine-2, 4-dione small molecule inhibitor of the Pim kinases, kills a wide range of both myeloid and lymphoid cell lines with precursor T-cell lymphoblastic leukemia/lymphoma (pre–T-LBL/T-ALL) being highly sensitive. Incubation of pre–T-LBL cells with SMI-4a induced G1 phase cell-cycle arrest secondary to a dose-dependent induction of p27Kip1, apoptosis through the mitochondrial pathway, and inhibition of the mammalian target of rapamycin C1 (mTORC1) pathway based on decreases in phospho-p70 S6K and phospho-4E-BP1, 2 substrates of this enzyme. In addition, treatment of these cells with SMI-4a was found to induce phosphorylation of extracellular signal-related kinase1/2 (ERK1/2), and the combination of SMI-4a and a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor was highly synergistic in killing pre–T-LBL cells. In immunodeficient mice carrying subcutaneous pre–T-LBL tumors, treatment twice daily with SMI-4a caused a significant delay in the tumor growth without any change in the weight, blood counts, or chemistries. Our data suggest that inhibition of the Pim protein kinases may be developed as a therapeutic strategy for the treatment of pre–T-LBL.

Lin, Ying-Wei; Beharry, Zanna M.; Hill, Elizabeth G.; Song, Jin H.; Wang, Wenxue; Xia, Zuping; Zhang, Zhenhua; Aplan, Peter D.; Aster, Jon C.; Smith, Charles D.

2010-01-01

224

FRAP DNA-dependent protein kinase mediates a late signal transduced from ultraviolet-induced DNA damage.  

PubMed

Ultraviolet radiation induces signal transduction at both early (<6 h) and late (>6 h) times after exposure. The inflammatory and immunosuppressive cytokine tumor necrosis factor alpha is induced at late times, and is induced by ultraviolet-induced DNA damage, as defects in DNA repair increase, and enhanced photoproduct repair reduces, tumor necrosis factor alpha expression. Here we show that late tumor necrosis factor alpha gene expression is sensitive to rapamycin, implicating FKBP12-rapamycin-associated protein, a member of the DNA protein kinase family, as a signal transducer of ultraviolet-induced DNA damage. FKBP12-rapamycin-associated protein was localized in the nucleus of keratinocytes and its level was increased following ultraviolet irradiation. Immuno- precipitated FKBP12-rapamycin-associated protein was stimulated by ultraviolet-irradiated DNA to phosphorylate p53 in vitro, and in vivo rapamycin reduced ultraviolet induction of p53 by 20%. Rapamycin further inhibited the ultraviolet-induced phosphorylation of the FKBP12-rapamycin-associated protein downstream target kinase p70S6K. In mice, topical application of rapamycin before ultraviolet exposure protected against suppression of the contact hypersensitivity that is a hallmark of ultraviolet-induced cytokine gene expression. These results demonstrate that the FKBP12-rapamycin-associated DNA protein kinase transduces the signal of ultraviolet-induced DNA damage into production of immunosuppressive cytokines at late times after ultraviolet irradiation. PMID:10771484

Yarosh, D B; Cruz, P D; Dougherty, I; Bizios, N; Kibitel, J; Goodtzova, K; Both, D; Goldfarb, S; Green, B; Brown, D

2000-05-01

225

Interleukin-12p70 Expression by Dendritic Cells of HIV-1-Infected Patients Fails to Stimulate gag-Specific Immune Responses  

PubMed Central

A variety of immune-based therapies has been developed in order to boost or induce protective CD8+ T cell responses in order to control HIV replication. Since dendritic cells (DCs) are professional antigen-presenting cells (APCs) with the unique capability to stimulate naïve T cells into effector T cells, their use for the induction of HIV-specific immune responses has been studied intensively. In the present study we investigated whether modulation of the activation state of DCs electroporated with consensus codon-optimized HxB2 gag mRNA enhances their capacity to induce HIV gag-specific T cell responses. To this end, mature DCs were (i) co-electroporated with mRNA encoding interleukin (IL)-12p70 mRNA, or (ii) activated with a cytokine cocktail consisting of R848 and interferon (IFN)-?. Our results confirm the ability of HxB2 gag-expressing DCs to expand functional HIV-specific CD8+ T cells. However, although most of the patients had detectable gag-specific CD8+ T cell responses, no significant differences in the level of expansion of functional CD8+ T cells could be demonstrated when comparing conventional or immune-modulated DCs expressing IL-12p70. This result which goes against expectation may lead to a re-evaluation of the need for IL-12 expression by DCs in order to improve T-cell responses in HIV-1-infected individuals.

Van Gulck, Ellen; Cools, Nathalie; Atkinson, Derek; Bracke, Lotte; Vereecken, Katleen; Vekemans, Marc; Van Tendeloo, Viggo F. I.; Berneman, Zwi N.; Vanham, Guido

2012-01-01

226

TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h.  

PubMed

Mammalian target-of-rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF-mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin-1. Torin-1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1-eIF3h-is phosphorylated and detected in polysomes in response to auxin. In TOR-deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF-mRNAs and eIF3h was impaired. Transient expression of eIF3h-S178D in plant protoplasts specifically upregulates uORF-mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation. PMID:23524850

Schepetilnikov, Mikhail; Dimitrova, Maria; Mancera-Martínez, Eder; Geldreich, Angèle; Keller, Mario; Ryabova, Lyubov A

2013-03-22

227

Antiferromagnetic resonance in Mn2P2S6  

NASA Astrophysics Data System (ADS)

The antiferromagnetic resonance (AFMR) spectra along the principal magnetic axes x, y, z of the compound Mn2P2S6 have been investigated in detail in a wide range of frequencies (8-142 GHz) and magnetic fields (up to 75 kOe) at T=4.2 K. It is shown that this compound is a biaxial magnetic substance. At H=0, there are two gaps in the spin-wave spectrum: (101.46+/-0.1) and (115.52+/-0.1) GHz. The AFMR data were used to determine the effective magnetic-anisotropy fields: Ha1=0.619 kOe and Ha2=0.803 kOe. An anomalous interaction of the AFMR branches in Mn2P2S6 has been detected, along with the appearance of coupled spin-spin oscillations of the same symmetry. An additional absorption in the AFMR spectrum is a local mode associated with breakdown of translational order in a low-dimension magnet. It is shown for the first time that the appearance of additional absorption peaks depends on the sample-cooling rate. It is proven that the observed peaks are associated with the internal modes of the domain boundaries.

Kobets, M. I.; Dergachev, K. G.; Gnatchenko, S. L.; Khats'ko, E. N.; Vysochanskii, Yu. M.; Gurzan, M. I.

2009-12-01

228

IL-2 Suppression of IL-12p70 by a Recombinant HSV-1 Expressing IL-2 Induces T-Cell Auto-Reactivity and CNS Demyelination  

PubMed Central

To evaluate the role of cellular infiltrates in CNS demyelination in immunocompetent mice, we have used a model of multiple sclerosis (MS) in which different strains of mice are infected with a recombinant HSV-1 expressing IL-2. Histologic examination of the mice infected with HSV-IL-2 demonstrates that natural killer cells, dendritic cells, B cells, and CD25 (IL-2r?) do not play any role in the HSV-IL-2-induced demyelination. T cell depletion, T cell knockout and T cell adoptive transfer experiments suggest that both CD8+ and CD4+ T cells contribute to HSV-IL-2-induced CNS demyelination with CD8+ T cells being the primary inducers. In the adoptive transfer studies, all of the transferred T cells irrespective of their CD25 status at the time of transfer were positive for expression of FoxP3 and depletion of FoxP3 blocked CNS demyelination by HSV-IL-2. The expression levels of IL-12p35 relative to IL-12p40 differed in BM-derived macrophages infected with HSV-IL-2 from those infected with wild-type HSV-1. HSV-IL-2-induced demyelination was blocked by injecting HSV-IL-2-infected mice with IL-12p70 DNA. This study demonstrates that suppression of the IL-12p70 function of macrophages by IL-2 causes T cells to become auto-aggressive. Interruption of this immunoregulatory axis results in demyelination of the optic nerve, the spinal cord and the brain by autoreactive T cells in the HSV-IL-2 mouse model of MS.

Zandian, Mandana; Mott, Kevin R.; Allen, Sariah J.; Chen, Shuang; Arditi, Moshe; Ghiasi, Homayon

2011-01-01

229

Role of ERK/mTOR signaling in TGFbeta-modulated focal adhesion kinase mRNA stability and protein synthesis in cultured rat IEC-6 intestinal epithelial cells.  

PubMed

Increasing evidence is available showing the importance of the FAK (focal adhesion kinase) protein level in the migration and homeostasis of intestinal cells. TGFbeta (transforming growth factor beta) modulates FAK protein expression in a complex fashion not only by inducing the activation of p38 and Smad signaling resulting in increased fak promoter activity and increased FAK protein levels, but also by activating ERK (extracellular signal regulated kinases), p38, and the Smad pathway. We show that the blockade of ERK signaling by a specific MEK (MAPK kinase) inhibitor attenuates TGFbeta-induced FAK mRNA stability and reduces FAK protein levels in rat IEC-6 intestinal epithelial cells. The mTOR (mammalian target of rapamycin)-specific inhibitor rapamycin and small interfering RNAs for mTOR and p70(S6) kinase also block TGFbeta-induced FAK protein synthesis. Furthermore, we have found that a TGFbeta-induced increase in wound closures in monolayers of these cells is abolished in the presence ERK or mTOR inhibition. Thus, TGFbeta also modulates FAK protein levels in cultured rat IEC-6 intestinal epithelial cells via ERK activation, acting at the transcriptional level to complement Smad signaling and at on the translational level via the mTOR pathway downstream of ERK, which in turn promotes intestinal epithelial cell migration. PMID:19340459

Suer, Silke; Ampasala, Dinakar; Walsh, Mary F; Basson, Marc D

2009-04-02

230

S6K1 and 4E-BP1 Are Independent Regulated and Control Cellular Growth in Bladder Cancer  

PubMed Central

Aberrant activation and mutation status of proteins in the phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and the mitogen activated protein kinase (MAPK) signaling pathways have been linked to tumorigenesis in various tumors including urothelial carcinoma (UC). However, anti-tumor therapy with small molecule inhibitors against mTOR turned out to be less successful than expected. We characterized the molecular mechanism of this pathway in urothelial carcinoma by interfering with different molecular components using small chemical inhibitors and siRNA technology and analyzed effects on the molecular activation status, cell growth, proliferation and apoptosis. In a majority of tested cell lines constitutive activation of the PI3K was observed. Manipulation of mTOR or Akt expression or activity only regulated phosphorylation of S6K1 but not 4E-BP1. Instead, we provide evidence for an alternative mTOR independent but PI3K dependent regulation of 4E-BP1. Only the simultaneous inhibition of both S6K1 and 4E-BP1 suppressed cell growth efficiently. Crosstalk between PI3K and the MAPK signaling pathway is mediated via PI3K and indirect by S6K1 activity. Inhibition of MEK1/2 results in activation of Akt but not mTOR/S6K1 or 4E-BP1. Our data suggest that 4E-BP1 is a potential new target molecule and stratification marker for anti cancer therapy in UC and support the consideration of a multi-targeting approach against PI3K, mTORC1/2 and MAPK.

Nawroth, Roman; Stellwagen, Florian; Schulz, Wolfgang A.; Stoehr, Robert; Hartmann, Arndt; Krause, Bernd J.; Gschwend, Juergen E.; Retz, Margitta

2011-01-01

231

Networking with mitogen-activated protein kinases.  

PubMed

Mitogen activated protein (MAP) kinases and their target ribosomal protein S6 (RSK) kinases have been recognized as shared components in the intracellular signaling pathways of many diverse cytokines. Recent studies have extended this protein kinase cascade by identifying the major activator of vertebrate MAP kinases as a serine/threonine/tyrosine-protein kinase called MEK, which is related to yeast mating factor-regulated protein kinases encoded by the STE7 and byr1 genes. MEK, in turn, may be activated following its phosphorylation on serine by either of the kinases encoded by proto-oncogenes raf1 or mos, as well as by p78mekk, which is related to the yeast STE11 and byr2 gene products. Isoforms of all of these protein kinases may specifically combine to assemble distinct modules for intracellular signal transmission. However, the fundamental architecture of these protein kinase cascades has been highly conserved during eukaryotic evolution. PMID:7935348

Pelech, S L; Charest, D L; Mordret, G P; Siow, Y L; Palaty, C; Campbell, D; Charlton, L; Samiei, M; Sanghera, J S

1993-11-01

232

Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells  

PubMed Central

Aims Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells. Methods We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry. Results In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC50 concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels. Conclusion Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.

Fei, Shi-Jiang; Zhang, Xu-Chao; Dong, Song; Cheng, Hua; Zhang, Yi-Fang; Huang, Ling; Zhou, Hai-Yu; Xie, Zhi; Chen, Zhi-Hong; Wu, Yi-Long

2013-01-01

233

Protein Kinase RSK Family - Roles in Prostate Cancer.  

National Technical Information Service (NTIS)

The Ser/Thr protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of mitogen-activated protein kinase but its roles in prostate cancer have not been previously examined. We have found that RSK regulates the growth of the hum...

D. Lannigan

2006-01-01

234

AVP inhibits EGF-stimulated MAP kinase cascade in Madin-Darby canine kidney cells  

Microsoft Academic Search

AVP inhibits EGF-stimulated MAP kinase cascade in Madin-Darby canine kidney cells. We investigated the effects of epidermal growth factor (EGF) and arginine vasopressin (AVP) on Raf-1-MAP kinase cascade, including Raf-1-kinase (Raf-1-K), MAP kinase kinase (MAPKK), MAP kinase (MAPK) and S6 kinase (S6K) in Madin-Darby canine kidney (MDCK) cells. In a dose-dependent manner (10?10M to 10?6M), EGF increased autophosphorylation of Raf-1-K

Takehisa Yamada; Yoshio Terada; Miwako K Homma; Hiroshi Nonoguchi; Sei Sasaki; Yasuhito Yuasa; Kimio Tomita; Fumiaki Marumo

1995-01-01

235

Monitoring of TNFR1, IL-2R?, HGF, CCL8, IL-8 and IL-12p70 following HSCT and their role as GVHD biomarkers in paediatric patients.  

PubMed

No predictive factors are currently available to establish patient-specific GVHD risk. A panel of six serum cytokines (TNF receptor 1, IL-2 receptor alfa (IL-2R?), hepatocyte growth factor (HGF), monocyte chemo-attractant protein-2, IL-8, IL-12p70) were monitored at established time points (days -1, +1, +7, +14, +21, +28 and +60) in 170 paediatric hematopoietic SCT (HSCT) recipients. We found that higher concentrations of IL-2R? on days +14 and +21 together with HGF on days +14 and +21 were significantly associated at a higher probability of both grade II-IV GVHD (on day +14 it was: 60% vs 28%, P=0.007) and grade III-IV (on day +14 it was: 40% vs 15%, P=0.001). The higher IL-8 serum concentration on day +28 was associated with a lower probability of chronic GVHD being 4% vs 29% (P=0.01) for patients with higher vs lower IL-8 serum concentration. These findings were confirmed when the analysis was restricted to the the matched unrelated donor group. In conclusion, even if the serum cytokine levels were related to several variables associated with HSCT, we identified two cytokines as predictors of GVHD II-IV and III-IV, translating into a higher TRM risk (17% vs 3%, P=0.004). PMID:23584443

Berger, M; Signorino, E; Muraro, M; Quarello, P; Biasin, E; Nesi, F; Vassallo, E; Fagioli, F

2013-04-15

236

ErbB receptor tyrosine kinase network inhibition radiosensitizes carcinoma cells  

SciTech Connect

Purpose The expression of epidermal growth factor receptor (EGFR)-CD533, a truncation mutant of the wild-type EGFR, radiosensitizes carcinoma and malignant glioma cell lines. This deletion mutant disrupts EGFR activation and downstream signaling through the formation of inhibitory dimerizations. In this study, the effects of EGFR-CD533 on other ErbB receptor tyrosine kinase (RTK) family members were quantified to better understand the mechanism of EGFR-CD533-mediated radiosensitization. Methods and Materials Breast carcinoma cell lines with different ErbB RTK expression profiles were transduced with EGFR or ErbB2 deletion mutants (EGFR-CD533 and ErbB2-CD572) using an adenoviral vector. ErbB RTK activation, mitogen activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/p70S6K signaling, and clonogenic survival were determined for expression of each deletion mutant. Results EGFR-CD533 radiosensitizes carcinoma cells with either high EGFR expression (MDA-MB231) or low EGFR expression (T47D) through significant blockade of the ErbB RTK network. Analysis of clonogenic survival demonstrate significant enhancement of the {alpha}/{beta} ratios, as determined by the linear-quadratic model. Split-dose survival experiments confirm that EGFR-CD533 reduces the repair of cellular damage after ionizing radiation. Conclusion Expression of EGFR-CD533 inhibits the ErbB RTK network and radiosensitizes carcinoma cells irrespective of the ErbB RTK expression patterns, and ErbB2-CD572 does not radiosensitize cells with low EGFR expression. These studies demonstrate that the mechanism of action for EGFR-CD533-mediated radiosensitization is inhibition of the ErbB RTK network, and is an advantage for radiosensitizing multiple malignant cell types.

Contessa, Joseph N. [Department of Radiation Oncology, Medical College of Virginia/Virginia Commonwealth University, Richmond VA (United States)]. E-mail: jcontess@med.umich.edu; Abell, Angela [Department of Radiation Oncology, Medical College of Virginia/Virginia Commonwealth University, Richmond VA (United States); Valerie, Kristoffer [Department of Radiation Oncology, Medical College of Virginia/Virginia Commonwealth University, Richmond VA (United States); Lin, Peck-Sun [Department of Radiation Oncology, Medical College of Virginia/Virginia Commonwealth University, Richmond VA (United States); Schmidt-Ullrich, Rupert K. [Department of Radiation Oncology, Medical College of Virginia/Virginia Commonwealth University, Richmond VA (United States)

2006-07-01

237

Simultaneous inhibition of pan-phosphatidylinositol-3-kinases and MEK as a potential therapeutic strategy in peripheral T-cell lymphomas  

PubMed Central

Peripheral T-cell lymphomas are very aggressive hematologic malignancies for which there is no targeted therapy. New, rational approaches are necessary to improve the very poor outcome in these patients. Phosphatidylinositol-3-kinase is one of the most important pathways in cell survival and proliferation. We hypothesized that phosphatidylinositol-3-kinase inhibitors could be rationally selected drugs for treating peripheral T-cell lymphomas. Several phosphatidylinositol-3-kinase isoforms were inhibited genetically (using small interfering RNA) and pharmacologically (with CAL-101 and GDC-0941 compounds) in a panel of six peripheral and cutaneous T-cell lymphoma cell lines. Cell viability was measured by intracellular ATP content; apoptosis and cell cycle changes were checked by flow cytometry. Pharmacodynamic biomarkers were assessed by western blot. The PIK3CD gene, which encodes the ? isoform of phosphatidylinositol-3-kinase, was overexpressed in cell lines and primary samples, and correlated with survival pathways. However, neither genetic nor specific pharmacological inhibition of phosphatidylinositol-3-kinase ? affected cell survival. In contrast, the pan-phosphatidylinositol-3-kinase inhibitor GDC-0941 arrested all T-cell lymphoma cell lines in the G1 phase and induced apoptosis in a subset of them. We identified phospho-GSK3? and phospho-p70S6K as potential biomarkers of phosphatidylinositol-3-kinase inhibitors. Interestingly, an increase in ERK phosphorylation was observed in some GDC -0941-treated T-cell lymphoma cell lines, suggesting the presence of a combination of phosphatidylinositol-3-kinase and MEK inhibitors. A highly synergistic effect was found between the two inhibitors, with the combination enhancing cell cycle arrest at G0/G1 in all T-cell lymphoma cell lines, and reducing cell viability in primary tumor T cells ex vivo. These results suggest that the combined treatment of pan-phosphatidylinositol-3-kinase + MEK inhibitors could be more effective than single phosphatidylinositol-3-kinase inhibitor treatment, and therefore, that this combination could be of therapeutic value for treating peripheral and cutaneous T-cell lymphomas.

Martin-Sanchez, Esperanza; Rodriguez-Pinilla, Socorro M.; Sanchez-Beato, Margarita; Lombardia, Luis; Dominguez-Gonzalez, Beatriz; Romero, Diana; Odqvist, Lina; Garcia-Sanz, Pablo; Wozniak, Magdalena B.; Kurz, Guido; Blanco-Aparicio, Carmen; Mollejo, Manuela; Alves, F. Javier; Menarguez, Javier; Gonzalez-Palacios, Fernando; Rodriguez-Peralto, Jose Luis; Ortiz-Romero, Pablo L.; Garcia, Juan F.; Bischoff, James R.; Piris, Miguel A.

2013-01-01

238

The endocannabinoid anandamide downregulates IL-23 and IL-12 subunits in a viral model of multiple sclerosis: evidence for a cross-talk between IL-12p70/IL-23 axis and IL-10 in microglial cells.  

PubMed

Theiler's virus (TMEV) infection of the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). The endocannabinoid system represents a novel therapeutic target for autoimmune and chronic inflammatory diseases due to its anti-inflammatory properties by regulating cytokine network. IL-12p70 and IL-23 are functionally related heterodimeric cytokines that play a crucial role in the pathogenesis of MS. In the present study we showed that the endocannabinoid anandamide (AEA) downregulated the gene expression of IL-12p70 and IL-23 forming subunits mRNAs in the spinal cord of TMEV-infected mice and ameliorated motor disturbances. This was accompanied by significant decreases on the serological levels of IL-12p70/IL-23 and more interestingly, of IL-17A. In contrast, serum levels of IL-10 resulted elevated. In addition, we studied the signalling pathways involved in the regulation of IL-12p70/IL-23 and IL-10 expression in TMEV-infected microglia and addressed the possible interactions of AEA with these pathways. AEA acted through the ERK1/2 and JNK pathways to downregulate IL-12p70 and IL-23 while upregulating IL-10. These effects were partially mediated by CB2 receptor activation. We also described an autocrine circuit of cross-talk between IL-12p70/IL-23 and IL-10, since endogenously produced IL-10 negatively regulates IL-12p70 and IL-23 cytokines in TMEV-infected microglia. This suggests that by altering the cytokine network, AEA could indirectly modify the type of immune responses within the CNS. Accordingly, pharmacological modulation of endocannabinoids might be a useful tool for treating neuroinflammatory diseases. PMID:21310228

Correa, Fernando; Hernangómez-Herrero, Miriam; Mestre, Leyre; Loría, Frida; Docagne, Fabian; Guaza, Carmen

2011-02-16

239

Viral factor TAV recruits TOR/S6K1 signalling to activate reinitiation after long ORF translation  

PubMed Central

The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral reinitiation factor transactivator–viroplasmin (TAV) exceptionally promotes reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor reinitiation-supporting protein (RISP) to regenerate reinitiation-competent ribosomal complexes. Here, we show that TAV function in reinitiation depends on physical association with TOR, with TAV–TOR binding being critical for both translation reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP—a novel and specific target of TOR/S6K1—in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and reinitiation. Thus, activation of reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of reinitiation-competent ribosomal complexes.

Schepetilnikov, Mikhail; Kobayashi, Kappei; Geldreich, Angele; Caranta, Carole; Robaglia, Christophe; Keller, Mario; Ryabova, Lyubov A

2011-01-01

240

Association of IL-12p70 and IL-6:IL-10 ratio with autism-related behaviors in 22q11.2 deletion syndrome: a preliminary report.  

PubMed

22q11.2 deletion syndrome (22q11DS) is a genetic disorder that conveys a significant risk for the development of social behavior disorders, including autism and schizophrenia. Also known as DiGeorge syndrome, 22q11DS is the second most common genetic disorder and is characterized by an elevated risk for immune dysfunction, up to 77% of individuals have an identifiable immune deficiency. We hypothesize that this immune dysfunction could contribute to the elevated risk of impaired social behavior seen in 22q11DS. The current study begins to elucidate these immune deficits and link them with the behavioral alterations associated with the disorder. Serum concentrations of a series of cytokines were examined, using a multiplex immunoassay, in sixteen individuals with 22q11DS and screened for autism-related behavior using the Autism Diagnostic Interview-Revised (ADI-R). This preliminary study examined correlations between specific immune proteins and each of the ADI-R algorithm scores (social, communication, and repetitive behavior). The inflammatory cytokine IL-1?, as well as the ratio between the inflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10, were correlated with social scores (r=0.851, p=0.004; r=0.580, p=0.018). In addition, the inflammatory cytokines interferon gamma and IL-12p70 were correlated with repetitive behaviors (r=0.795, p=0.033; r=0.774, p=0.002). Interestingly, IL-12 has been reported to be increased in autistic children. These data show a positive association between severity of autism-related behaviors and level of serum concentrations of inflammatory cytokines in individuals with 22q11DS, providing a basis for further inquiry. PMID:23353117

Ross, Heather E; Guo, Ying; Coleman, Karlene; Ousley, Opal; Miller, Andrew H

2013-01-24

241

Black raspberry extracts inhibit benzo(a)pyrene diol-epoxide-induced activator protein 1 activation and VEGF transcription by targeting the phosphotidylinositol 3-kinase/Akt pathway.  

PubMed

Previous studies have shown that freeze-dried black raspberry extract fractions inhibit benzo(a)pyrene [B(a)P]-induced transformation of Syrian hamster embryo cells and benzo(a)pyrene diol-epoxide [B(a)PDE]-induced activator protein-1 (AP-1) activity in mouse epidermal Cl 41 cells. The phosphotidylinositol 3-kinase (PI-3K)/Akt pathway is critical for B(a)PDE-induced AP-1 activation in mouse epidermal Cl 41 cells. In the present study, we determined the potential involvement of PI-3K and its downstream kinases on the inhibition of AP-1 activation by black raspberry fractions, RO-FOO3, RO-FOO4, RO-ME, and RO-DM. In addition, we investigated the effects of these fractions on the expression of the AP-1 target genes, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Pretreatment of Cl 41 cells with fractions RO-F003 and RO-ME reduced activation of AP-1 and the expression of VEGF, but not iNOS. In contrast, fractions RO-F004 and RO-DM had no effect on AP-1 activation or the expression of either VEGF or iNOS. Consistent with inhibition of AP-1 activation, the RO-ME fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase (p70(S6k)). In addition, overexpression of the dominant negative PI-3K mutant delta p85 reduced the induction of VEGF by B(a)PDE. It is likely that the inhibitory effects of fractions RO-FOO3 and RO-ME on B(a)PDE-induced AP-1 activation and VEGF expression are mediated by inhibition of the PI-3K/Akt pathway. In view of the important roles of AP-1 and VEGF in tumor development, one mechanism for the chemopreventive activity of black raspberries may be inhibition of the PI-3K/Akt/AP-1/VEGF pathway. PMID:16397275

Huang, Chuanshu; Li, Jingxia; Song, Lun; Zhang, Dongyun; Tong, Qiangsong; Ding, Min; Bowman, Linda; Aziz, Robeena; Stoner, Gary D

2006-01-01

242

The role of c-Myc on granulocyte colony-stimulating factor-dependent neutrophilic proliferation and differentiation of HL60 cells  

Microsoft Academic Search

We have previously suggested that phosphatidylinositol 3-kinase (PI3K)\\/p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells on the basis of analysis of transferrin receptor (Trf-R)-positive (Trf-R+) and -negative (Trf-R?) cells that appear after treatment with dimethyl sulfoxide (DMSO). In the present study, we analyzed the downstream events of p70 S6K in

Toshie Kanayasu-Toyoda; Teruhide Yamaguchi; Tadashi Oshizawa; Eriko Uchida; Takao Hayakawa

2003-01-01

243

Expression of the SNT-1/FRS2 phosphotyrosine binding domain inhibits activation of MAP kinase and PI3-kinase pathways and antiestrogen resistant growth induced by FGF-1 in human breast carcinoma cells.  

PubMed

Fibroblast growth factor (FGF) signaling can bypass the requirement for estrogen receptor (ER) activation in the growth of ER-positive (ER+) breast cancer cells. Fibroblast growth factor-1 stimulation leads to phosphorylation of the adaptor protein Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT-1) on C-terminal tyrosine residues, whereas it is constitutively bound through its N-terminal phosphotyrosine-binding domain (PTB) to FGF receptors (FGFRs). By expressing the PTB domain of SNT-1 (SNT-1 PTB) in an inducible manner in an ER+ breast carcinoma line, ML20, we asked whether we could uncouple FGFR activation from its downstream signaling components and abrogate FGF-1-induced antiestrogen-resistant growth. Induction of SNT-1 PTB resulted in a significant decrease of FGF-1-dependent tyrosine phosphorylation of endogenous SNT-1, strong inhibition of complex formation between SNT-1, Gab-1 and Sos-1, and reduced activation of Ras, mitogen-activated protein kinase (MAP kinase), and Akt. SNT-1 PTB also inhibited the phosphorylation of p70S6K on Thr421/Ser424 and Ser411, which may result from the abrogation of MAP kinase activity. Moreover, we also observed a decreased phosphorylation of the MAP kinase-independent site Thr389. This may reflect both inhibition of PI-3 kinase pathways and mammalian target of rapamycin (mTOR)-dependent signaling, as the phosphorylation of Thr389 site was sensitive to treatment with the PI3-K and mTOR inhibitors, LY294002 and rapamycin, respectively. Collectively these results suggest that SNT-1 plays a pivotal role in FGF-dependent activation of the Ras-MAP kinase, PI-3 kinase, and mTOR pathways in these cells. Fibroblast growth factor-1 dependent colony formation of ML20 cells in media containing the pure antiestrogen ICI 182,780 was also markedly inhibited upon induction of SNT-1 PTB, suggesting that blockade of FGFR-SNT-1 interactions might abrogate FGF-mediated antiestrogen resistance in breast cancers. PMID:16682955

Manuvakhova, M; Thottassery, J V; Hays, S; Qu, Z; Rentz, S S; Westbrook, L; Kern, F G

2006-05-08

244

Comparative Immunohistochemical Analysis of Ochratoxin A Tumourigenesis in Rats and Urinary Tract Carcinoma in Humans; Mechanistic Significance of p-S6 Ribosomal Protein Expression  

PubMed Central

Ochratoxin A (OTA) is considered to be a possible human urinary tract carcinogen, based largely on a rat model, but no molecular genetic changes in the rat carcinomas have yet been defined. The phosphorylated-S6 ribosomal protein is a marker indicating activity of the mammalian target of rapamycin, which is a serine/threonine kinase with a key role in protein biosynthesis, cell proliferation, transcription, cellular metabolism and apoptosis, while being functionally deregulated in cancer. To assess p-S6 expression we performed immunohistochemistry on formalin-fixed and paraffin-embedded tumours and normal tissues. Marked intensity of p-S6 expression was observed in highly proliferative regions of rat renal carcinomas and a rare angiosarcoma, all of which were attributed to prolonged exposure to dietary OTA. Only very small OTA-generated renal adenomas were negative for p-S6. Examples of rat subcutaneous fibrosarcoma and testicular seminoma, as well as of normal renal tissue, showed no or very weak positive staining. In contrast to the animal model, human renal cell carcinoma, upper urinary tract transitional cell carcinoma from cases of Balkan endemic nephropathy, and a human angiosarcoma were negative for p-S6. The combined findings are reminiscent of constitutive changes in the rat tuberous sclerosis gene complex in the Eker strain correlated with renal neoplasms, Therefore rat renal carcinogenesis caused by OTA does not obviously mimic human urinary tract tumourigenesis.

Gazinska, Patrycja; Herman, Diana; Gillett, Cheryl; Pinder, Sarah; Mantle, Peter

2012-01-01

245

S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth.  

PubMed

The tumor suppressor programmed cell death protein 4 (PDCD4) inhibits the translation initiation factor eIF4A, an RNA helicase that catalyzes the unwinding of secondary structure at the 5' untranslated region (5'UTR) of messenger RNAs (mRNAs). In response to mitogens, PDCD4 was rapidly phosphorylated on Ser67 by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase SCF(betaTRCP). Expression in cultured cells of a stable PDCD4 mutant that is unable to bind betaTRCP inhibited translation of an mRNA with a structured 5'UTR, resulted in smaller cell size, and slowed down cell cycle progression. We propose that regulated degradation of PDCD4 in response to mitogens allows efficient protein synthesis and consequently cell growth. PMID:17053147

Dorrello, N Valerio; Peschiaroli, Angelo; Guardavaccaro, Daniele; Colburn, Nancy H; Sherman, Nicholas E; Pagano, Michele

2006-10-20

246

Effect of S6 Tail Mutations on Charge Movement in Shaker Potassium Channels  

Microsoft Academic Search

The cytoplasmic ends of the four S6 transmembrane segments of voltage-gated potassium channels converge in a bundle crossing that acts as the activation gate that opens in response to a depolarization. To explore whether the cytoplasmic extension of the S6 segment (the S6 tail) plays a role in coupling voltage sensor and activation gate movements, we examined the effect of

Shinghua Ding; Richard Horn

2003-01-01

247

Phosphorylation of translation factors in response to anoxia in turtles, Trachemys scripta elegans: role of the AMP-activated protein kinase and target of rapamycin signalling pathways.  

PubMed

Long-term survival of oxygen deprivation by animals with well-developed anoxia tolerance depends on multiple biochemical adaptations including strong metabolic rate depression. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in the suppression of protein synthesis that occurs when turtles experience anoxic conditions. AMPK activity and the phosphorylation state of ribosomal translation factors were measured in liver, heart, red muscle and white muscle of red-eared slider turtles (Trachemys scripta elegans) subjected to 20 h of anoxic submergence. AMPK activity increased twofold in white muscle of anoxic turtles compared with aerobic controls but remained unchanged in liver and red muscle, whereas in heart AMPK activity decreased by 40%. Immunoblotting with phospho-specific antibodies revealed that eukaryotic elongation factor-2 phosphorylation at the inactivating Thr56 site increased six- and eightfold in red and white muscles from anoxic animals, respectively, but was unchanged in liver and heart. The phosphorylation state of the activating Thr389 site of p70 ribosomal protein S6 kinase was reduced under anoxia in red muscle and heart but was unaffected in liver and white muscle. Exposure to anoxia decreased 40S ribosomal protein S6 phosphorylation in heart and promoted eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) dephosphorylation in red muscle, but surprisingly increased 4E-BP1 phosphorylation in white muscle. The changes in phosphorylation state of translation factors suggest that organ-specific patterns of signalling and response are involved in achieving the anoxia-induced suppression of protein synthesis in turtles. PMID:19579060

Rider, Mark H; Hussain, Nusrat; Dilworth, Stephen M; Storey, Kenneth B

2009-07-05

248

Protein Kinases  

NSDL National Science Digital Library

This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein kinases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the genomics and evolutionary relationships among kinases and then proceeds to describe the structure-function relationships of specific kinases, the molecular mechanisms underlying substrate specificity, and selected issues in regulation of kinase activity.

Avrom Caplan (Mount Sinai School of Medicine;Department of Pharmacology and Biological Chemistry REV)

2005-02-22

249

Protein Kinase RSK Family - Roles in Prostate Cancer.  

National Technical Information Service (NTIS)

The Ser/Thr protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of MAPK but its roles in prostate cancer have not previously been examined. We have now discovered that RSK2 regulates the expression of PSA, an important dia...

D. A. Lannigan

2005-01-01

250

The endocannabinoid anandamide downregulates IL23 and IL12 subunits in a viral model of multiple sclerosis: Evidence for a cross-talk between IL12p70\\/IL23 axis and IL10 in microglial cells  

Microsoft Academic Search

Theiler’s virus (TMEV) infection of the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). The endocannabinoid system represents a novel therapeutic target for autoimmune and chronic inflammatory diseases due to its anti-inflammatory properties by regulating cytokine network. IL-12p70 and IL-23 are functionally related

Fernando Correa; Miriam Hernangómez-Herrero; Leyre Mestre; Frida Loría; Fabian Docagne; Carmen Guaza

2011-01-01

251

The nuts and bolts of AGC protein kinases  

Microsoft Academic Search

The AGC kinase subfamily of protein kinases contains 60 members, including PKA, PKG and PKC. The family comprises some intensely examined protein kinases (such as Akt, S6K, RSK, MSK, PDK1 and GRK) as well as many less well-studied enzymes (such as SGK, NDR, LATS, CRIK, SGK494, PRKX, PRKY and MAST). Research has shed new light onto the architecture and regulatory

Laura R. Pearce; David Komander; Dario R. Alessi

2010-01-01

252

Hierarchical Cd4SiS6/SiO2 Heterostructure Nanowire Arrays  

PubMed Central

Novel hierarchical Cd4SiS6/SiO2 based heterostructure nanowire arrays were fabricated on silicon substrates by a one-step thermal evaporation of CdS powder. The as-grown products were characterized using scanning electron microscopy, X-ray diffraction, and transmission electron microscopy. Studies reveal that a typical hierarchical Cd4SiS6/SiO2 heterostructure nanowire is composed of a single crystalline Cd4SiS6 nanowire core sheathed with amorphous SiO2 sheath. Furthermore, secondary nanostructures of SiO2 nanowires are highly dense grown on the primary Cd4SiS6 core-SiO2 sheath nanowires and formed hierarchical Cd4SiS6/SiO2 based heterostructure nanowire arrays which stand vertically on silicon substrates. The possible growth mechanism of hierarchical Cd4SiS6/SiO2 heterostructure nanowire arrays is proposed. The optical properties of hierarchical Cd4SiS6/SiO2 heterostructure nanowire arrays are investigated using Raman and Photoluminescence spectroscopy.

2010-01-01

253

Evolutionary conservation of nuclear and nucleolar targeting sequences in yeast ribosomal protein S6A  

SciTech Connect

Over 1 billion years ago, the animal kingdom diverged from the fungi. Nevertheless, a high sequence homology of 62% exists between human ribosomal protein S6 and S6A of Saccharomyces cerevisiae. To investigate whether this similarity in primary structure is mirrored in corresponding functional protein domains, the nuclear and nucleolar targeting signals were delineated in yeast S6A and compared to the known human S6 signals. The complete sequence of S6A and cDNA fragments was fused to the 5'-end of the LacZ gene, the constructs were transiently expressed in COS cells, and the subcellular localization of the fusion proteins was detected by indirect immunofluorescence. One bipartite and two monopartite nuclear localization signals as well as two nucleolar binding domains were identified in yeast S6A, which are located at homologous regions in human S6 protein. Remarkably, the number, nature, and position of these targeting signals have been conserved, albeit their amino acid sequences have presumably undergone a process of co-evolution with their corresponding rRNAs.

Lipsius, Edgar [Zentrum fuer Experimentelle Medizin, Institut fuer Molekulare Zellbiologie, Universitaet Hamburg, Hamburg (Germany); Walter, Korden [Zentrum fuer Experimentelle Medizin, Institut fuer Molekulare Zellbiologie, Universitaet Hamburg, Hamburg (Germany); Leicher, Torsten [Zentrum fuer Experimentelle Medizin, Institut fuer Molekulare Zellbiologie, Universitaet Hamburg, Hamburg (Germany); Phlippen, Wolfgang [Zentrum fuer Experimentelle Medizin, Institut fuer Molekulare Zellbiologie, Universitaet Hamburg, Hamburg (Germany); Bisotti, Marc-Angelo [Zentrum fuer Experimentelle Medizin, Institut fuer Molekulare Zellbiologie, Universitaet Hamburg, Hamburg (Germany); Kruppa, Joachim [Zentrum fuer Experimentelle Medizin, Institut fuer Molekulare Zellbiologie, Universitaet Hamburg, Hamburg (Germany)]. E-mail: kruppa@uke.uni-hamburg.de

2005-08-12

254

CCL2 Protects Prostate Cancer PC3 Cells from Autophagic Death via Phosphatidylinositol 3-Kinase/AKT-dependent Survivin Up-regulation*  

PubMed Central

Resistance to cell death is a hallmark of cancer. Autophagy is a survival mechanism activated in response to nutrient deprivation; however, excessive autophagy will ultimately induce cell death in a nonapoptotic manner. The present study demonstrates that CCL2 protects prostate cancer PC3 cells from autophagic death, allowing prolonged survival in serum-free conditions. Upon serum starvation, CCL2 induced survivin up-regulation in PC3, DU 145, and C4-2B prostate cancer cells. Both cell survival and survivin expression were stunted in CCL2-stimulated PC3 cells when treated either with the phosphatidylinositol 3-kinase inhibitor LY294002 (2 ?m) or the Akt-specific inhibitor-X (Akti-X; 2.5 ?m). Furthermore, CCL2 significantly reduced light chain 3-II (LC3-II) in serum-starved PC3; in contrast, treatment with LY294002 or Akti-X reversed the effect of CCL2 on LC3-II levels, suggesting that CCL2 signaling limits autophagy in these cells. Upon serum deprivation, the analysis of LC3 localization by immunofluorescence revealed a remarkable reduction in LC3 punctate after CCL2 stimulation. CCL2 treatment also resulted in a higher sustained mTORC1 activity as measured by an increase in phospho-p70S6 kinase (Thr389). Rapamycin, an inducer of autophagy, both down-regulated survivin and decreased PC3 cell viability in serum-deprived conditions. Treatment with CCL2, however, allowed cells to partially resist rapamycin-induced death, which correlated with survivin protein levels. In two stable transfectants expressing survivin-specific short hairpin RNA, generated from PC3, survivin protein levels controlled both cell viability and LC3 localization in response to CCL2 treatment. Altogether, these findings indicate that CCL2 protects prostate cancer PC3 cells from autophagic death via the phosphatidylinositol 3-kinase/Akt/survivin pathway and reveal survivin as a critical molecule in this survival mechanism.

Roca, Hernan; Varsos, Zachary; Pienta, Kenneth J.

2008-01-01

255

Perspectives of Targeting mTORC1-S6K1 in Cardiovascular Aging  

PubMed Central

The global population aging is accelerating and age-associated diseases including cardiovascular diseases become more challenging. The underlying mechanisms of aging and age-associated cardiovascular dysfunction remain elusive. There are substantial evidences demonstrating a pivotal role of the mammalian target of rapamycin complex 1 (mTORC1) and its down-stream effector S6K1 signaling in mammalian lifespan regulation and age-related diseases such as type II diabetes mellitus and cancer. The role of mTORC1–S6K1 in age-related cardiovascular diseases is, however, largely unknown and the available experimental results are controversial. This review article primarily summarizes the most recent advances toward understanding the role of mTORC1–S6K1 in cardiovascular aging and discusses the future perspectives of targeting mTORC1–S6K1 signaling as a healthy lifespan extension modality in anti-aging and anti-cardiovascular aging.

Ming, Xiu-Fen; Montani, Jean-Pierre; Yang, Zhihong

2011-01-01

256

Structure and function of MK5/PRAK: the loner among the mitogen-activated protein kinase-activated protein kinases.  

PubMed

Mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways that control pivotal cellular processes including proliferation, differentiation, survival, apoptosis, gene regulation, and motility. MAPK pathways consist of a relay of consecutive phosphorylation events exerted by MAPK kinase kinases, MAPK kinases, and MAPKs. Conventional MAPKs are characterized by a conserved Thr-X-Tyr motif in the activation loop of the kinase domain, while atypical MAPKs lack this motif and do not seem to be organized into the classical three-tiered kinase cascade. One functional group of conventional and atypical MAPK substrates consists of protein kinases known as MAPK-activated protein kinases. Eleven mammalian MAPK-activated protein kinases have been identified, and they are divided into five subgroups: the ribosomal-S6-kinases RSK1-4, the MAPK-interacting kinases MNK1 and 2, the mitogen- and stress-activated kinases MSK1 and 2, the MAPK-activated protein kinases MK2 and 3, and the MAPK-activated protein kinase MK5 (also referred to as PRAK). MK5/PRAK is the only MAPK-activated protein kinase that is a substrate for both conventional and atypical MAPK, while all other MAPKAPKs are exclusively phosphorylated by conventional MAPKs. This review focuses on the structure, activation, substrates, functions, and possible implications of MK5/PRAK in malignant and nonmalignant diseases. PMID:23729623

Moens, Ugo; Kostenko, Sergiy

2013-09-01

257

Role of GalNAc4S-6ST in astrocytic tumor progression.  

PubMed

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) is the sulfotransferase responsible for biosynthesis of highly sulfated chondroitin sulfate CS-E. Although involvements of CS-E in neuronal cell functions have been extensively analyzed, the role of GalNAc4S-6ST in astrocytic tumor progression remains unknown. Here, we reveal that GalNAc4S-6ST transcripts were detected in astrocytic tumors derived from all 30 patients examined using quantitative reverse transcription-PCR analysis. Patients with high GalNAc4S-6ST mRNA expression had significantly worse outcome compared with patients with low expression, and multivariate survival analysis disclosed that GalNAc4S-6ST is an independent poor prognostic factor for astrocytic tumors. We then tested whether CS-E enhanced haptotaxic migration of glioblastoma U251-MG cells that endogenously express both the CS-E's scaffold tyrosine phosphatase ? (PTP?) and GalNAc4S-6ST, in the presence of CS-E's preferred ligands, pleiotrophin (PTN) or midkine (MK), using a modified Boyden chamber method. Haptotaxic stimulation of cell migration by PTN was most robust on control siRNA-transfected U251-MG cells, while that enhancing effect was cancelled following transduction of GalNAc4S-6ST siRNA. Similar results were obtained using MK, suggesting that both PTN and MK enhance migration of U251-MG cells by binding to CS-E. We also found that PTP? as well as PTN and MK were frequently expressed in astrocytic tumor cells. Thus, our findings indicate that GalNAc4S-6ST mRNA expressed by astrocytic tumor cells is associated with poor patient prognosis likely by enhancing CS-E-mediated tumor cell motility in the presence of PTN and/or MK. PMID:23349846

Kobayashi, Tatsuya; Yan, Huimin; Kurahashi, Yasuhiro; Ito, Yuki; Maeda, Hiroshi; Tada, Tsuyoshi; Hongo, Kazuhiro; Nakayama, Jun

2013-01-17

258

Safety assessment of biopharmaceuticals: Japanese perspective on ICH S6 guideline maintenance.  

PubMed

Safety assessment of biopharmaceuticals in preclinical studies is guided by the ICH S6 guideline issued in 1997. Along with enormous experiences and knowledge on safety assessment of some classes of biopharmaceuticals over the last decade, the necessity and feasibility of updating the guideline has been discussed. According to a recommendation by safety experts at the ICH meeting in Chicago in 2006, regional discussions of ICH S6 were held in the USA, EU and Japan. The meeting to clarify the values, challenges and recommendations for ICH S6 from Japanese perspective was held as a part of the first Drug Evaluation Forum in Tokyo on August 10, 2007. Of utmost importance, the "case-by-case" approach must be preserved as the basic principle of the ICH S6 guideline. It is our opinion that oligonucleotides, siRNA, aptamers and related molecules should be excluded from ICH S6 and may be more appropriate for separate guidance. However, based on experiences and accumulated knowledge, there are a number of issues that can be updated including new types of biopharmaceuticals such as bioconjugates, use of homologous proteins and transgenic animals, reproductive/developmental toxicity studies in non-human primates, in vitro cardiac ion channel assay and alternative approaches for carcinogenicity assessment. Preliminary recommendations for some of these topics were outlined at the meeting. The overall Japanese recommendation is that the ICH S6 guideline should be updated to address these topics. PMID:18670158

Nakazawa, Takahiro; Kurokawa, Misao; Kimura, Kazuya; Wakata, Akihiro; Hisada, Shigeru; Inoue, Tadashi; Sagami, Fumio; Heidel, Shawn M; Kawakami, Koji; Shinoda, Kazutoshi; Onodera, Hiroshi; Kumagai, Yuji; Ohno, Yasuo; Kawamura, Nobuyuki; Yamazaki, Tsuneyoshi; Inoue, Tohru

2008-08-01

259

Involvement of PI3K-AKT-mTOR pathway in protein kinase CKII inhibition-mediated senescence in human colon cancer cells.  

PubMed

Cellular senescence is a tumor suppression mechanism. We previously reported that CKII downregulation induces senescence in human lung fibroblast IMR-90 and colon cancer HCT116 cells. In this study, potential longevity drugs, including rapamycin, vitamin C, and vitamin E, blocked CKII downregulation-mediated senescence through reduction of reactive oxygen species (ROS) production in HCT116 cells. Since rapamycin is a mammalian target of rapamycin (mTOR) inhibitor, we examined the roles of mTOR and its upstream regulators phosphatidylinositol 3-kinase (PI3K) and AKT in CKII inhibition-mediated senescence. CKII? knock-down or CKII inhibitor treatment strikingly increased phosphorylation of mTOR, p70S6K, an mTOR substrate, and AKT, whereas CKII? overexpression reduced this phosphorylation event. This result indicated that CKII inhibition activated the PI3K-AKT-mTOR pathway. Further, pharmacological inhibition of PI3K and AKT attenuated ROS production and senescence in CKII-downregulated cells. Taken together, these results demonstrate, for the first time, that the PI3K-AKT-mTOR-ROS pathway is necessary for CKII inhibition-mediated cellular senescence. PMID:23523798

Park, Ji Hye; Kim, Jin Joo; Bae, Young-Seuk

2013-03-21

260

PI3 kinase regulation of skeletal muscle hypertrophy and atrophy.  

PubMed

Activation of the PI3 kinase pathway can induce skeletal muscle hypertrophy, defined as an increase in skeletal muscle mass. In mammals, skeletal muscle hypertrophy occurs as a result of an increase in the size, as opposed to the number, of pre-existing skeletal muscle fibers. This pathway's effects on skeletal muscle have been implicated most prominently downstream of Insulin-like growth factor 1 signaling. IGF-1's pro-hypertrophy activity comes predominantly through its ability to activate the Phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Akt is a serine-threonine protein kinase that can induce protein synthesis and block the transcriptional upregulation of key mediators of skeletal muscle atrophy, the E3 ubiquitin ligases MuRF1 and MAFbx (also called Atrogin-1), by phosphorylating and thereby inhibiting the nuclear translocation of the FOXO (also called "forkhead") family of transcription factors. Once phosphorylated by Akt, the FOXOs are excluded from the nucleus, and upregulation of MuRF1 and MAFbx is blocked. MuRF1 and MAFbx mediate atrophy by ubiquitinating particular protein substrates, causing them to undergo degradation by the proteasome. MuRF1's substrates include several components of the sarcomeric thick filament, including Myosin Heavy Chain (MyHC). Thus, by blocking MuRF1 activation, IGF-1 helps prevent the breakdown of the thick filament under atrophy conditions.IGF1/PI3K/Akt signaling also can dominantly inhibit the effects of a secreted protein called "myostatin," which is a member of the TGF? family of proteins. Deletion or inhibition of myostatin causes an increase in skeletal muscle size, because myostatin acts both to inhibit myoblast differentiation and to block the Akt pathway. Thus by blocking myostatin, PI3K/Akt activation stimulates differentiation and protein synthesis by this distinct mechanism. Myostatin induces the phosphorylation and activation of the transcription factors of Smad2 and Smad3, downstream of the ActRII (Activin Receptor type II)/Alk (Activin Receptor-like kinase) receptor complex. Other TGF?-like molecules can also block differentiation, including TGF-b1, GDF-11, activinA, BMP-2 and BMP-7. As mentioned, myostatin also downregulates the Akt/mTOR/p70S6 protein synthesis pathway, which mediates both differentiation in myoblasts and hypertrophy in myotubes. Blockade of the Akt/mTOR pathway, using siRNA to RAPTOR, a component of "TORC1" (TOR signaling Complex 1), increases myostatin-induced phosphorylation of Smad2; this establishes a "feed-forward mechanism," because myostatin can downregulates TORC1, and this downregulation in turn amplifies myostatin signaling. Blockade of RAPTOR also facilitates myostatin's inhibition of muscle differentiation. When added to post-differentiated myotubes, myostatin causes a decrease in their diameter - however, this does not happen through the normal "atrophy pathway." Rather than causing upregulation of the E3 ubiquitin ligases MuRF1 and MAFbx, previously shown to mediate skeletal muscle atrophy, myostatin decreases expression of these atrophy markers in differentiated myotubes, as well as other genes normally upregulated during differentiation, such as MyoD and myogenin. These findings show that myostatin signaling acts by blocking genes induced during differentiation, even in a myotube, as opposed to activating the distinct "atrophy program." PMID:20593312

Glass, David J

2010-01-01

261

HemoHIM ameliorates the persistent down-regulation of Th1-like immune responses in fractionated ?-irradiated mice by modulating the IL-12p70-STAT4 signaling pathway.  

PubMed

Whole body irradiated mice appear to experience a down-regulation of the helper T (Th)1-like immune response, and maintain a persistent immunological imbalance. In the current study, we evaluated the effect of HemoHIM (an herbal product made from Angelica Radix, Cnidium officinale , and Paeonia japonica cultivated in Korea) to ameliorate the immunological imbalance induce in fractionated ?-irradiated mice. The mice were exposed to ? rays twice a week (0.5 Gy fractions) for a total dose of 5 Gy, and HemoHIM was administrated orally from 1 week before the first irradiation to 1 week before the final analysis. All experiments were performed 4 and 6 months after their first exposure. HemoHIM ameliorated the Th1- and Th2-related immune responses normally occur in irradiated mice with or without dinitrophenylated keyhole limpet hemocyanin immunization. HemoHIM also restored the natural killer cell activities without changing the percentage of natural killer cells in irradiated mice. Furthermore, the administration of HemoHIM prevented the reduction in levels of interleukin-12p70 in irradiated mice. Finally, we found that HemoHIM enhanced the phosphorylation of signal transducer and activator of transcription (STAT) 4 that was reduced in irradiated mice. Our findings suggest that HemoHIM ameliorates the persistent down-regulation of Th1-like immune responses by modulating the IL-12p70/pSTAT4 signaling pathway. PMID:22439601

Park, Hae-Ran; Jo, Sung-Kee; Choi, Nam-Hee; Jung, Uhee

2012-04-02

262

PDK1 regulates growth through Akt and S6K in Drosophila  

Microsoft Academic Search

The insulin\\/insulin-like growth factor-1 signaling pathway promotes growth in invertebrates and vertebrates by increasing the levels of phosphatidylinositol 3,4,5-triphosphate through the activation of p110 phosphatidylinositol 3-kinase. Two key effectors of this pathway are the phosphoinositide-dependent protein kinase 1 (PDK1) and Akt\\/PKB. Although genetic analysis in Caenorhabditis elegans has implicated Akt as the only relevant PDK1 substrate, cell culture studies have

Felix Rintelen; Hugo Stocker; George Thomas; Ernst Hafen

2001-01-01

263

Kinase dysfunction and kinase inhibitors.  

PubMed

With recent advances in molecular biology, abnormalities in cancer cells that contribute to dysregulation of cell survival and proliferation are being characterized with greater precision. Through this process, key abnormalities in cancer cells involving proteins that regulate signal transduction, migration, mitosis and other critical processes have been identified. Such abnormalities often involve a class of proteins called kinases that act to phosphorylate other proteins in the cell, resulting in activation of these proteins in the absence of appropriate stimulation/regulation. Given their role in tumour biology, substantial effort has been directed at blocking the function of these proteins. Several approaches have been used, including monoclonal antibodies and small molecule inhibitors. While antibodies are primarily directed at cell surface proteins, small molecule inhibitors, also known as kinase inhibitors, target proteins throughout the cell. A variety of kinase inhibitors have been approved for the treatment of human cancers. In some instances, these inhibitors have exhibited significant clinical efficacy, and it is likely that their biological activity will be further enhanced as combination regimens with standard treatment modalities are explored. The use of kinase inhibitors in dogs and cats is relatively recent, although two inhibitors, toceranib (Palladia; Pfizer Animal Health, Madison, NJ, USA) and masitinib (Kinavet; Catalent Pharma Solutions, Somerset, NJ, USA) have been approved by the Federal Drug Administration (USA) for use in dogs. This article reviews the biology of protein kinase dysfunction in human and animal cancers, and the application of specific kinase inhibitors to veterinary cancer patients. PMID:23331696

London, Cheryl A

2013-02-01

264

Downregulation of protein kinase CK2 induces autophagic cell death through modulation of the mTOR and MAPK signaling pathways in human glioblastoma cells  

PubMed Central

Glioblastoma multiforme is the most common primary brain tumor and one of the most aggressive types of cancer in adults. Survival signaling and apoptosis resistance are hallmarks of malignant glioma cells. However, recent studies have shown that other types of cell death such as autophagy can be induced in malignant glioma cells. This suggests that stimulation of this process may be explored in new therapeutic strategies against glioblastoma multiforme. Protein kinase CK2 is a highly conserved and constitutively active enzyme that promotes numerous cellular processes such as survival, proliferation and differentiation. CK2 has been found elevated in several malignancies including brain tumors, and to confer resistance against chemotherapeutic agents and apoptotic stimuli. Recently, we have shown that the siRNA-mediated downregulation of CK2 leads to cell death in DNA-PK-proficient human glioblastoma cells. We show, here, that lack of CK2 results in significant induction of autophagic cell death in two human glioblastoma cell lines, M059K and T98G, as indicated by the positive staining of cells with the acidotropic dye acridine orange, and the specific recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosome membranes. Induction of autophagy is accompanied by CK2-dependent decreased phosphorylation of p70 ribosomal S6 and AKT kinases and significantly reduced expression levels of Raptor. In contrast, phosphorylation and activity levels of ERK1/2 are enhanced suggesting an inhibition of the PI3K/AKT/mTORC1 and activation of the ERK1/2 pathways. Furthermore, siRNA-mediated silencing of CK2 results in increased mitochondrial superoxide production in both glioblastoma cell lines. However, mitochondrial reactive oxygen species release correlates with induction of autophagy only in T98G cells. Taken together, our findings identify CK2 as a novel component of the autophagic machinery and underline the potential of its downregulation to kill glioblastoma cells by overcoming the resistance to multiple anticancer agents.

OLSEN, BIRGITTE B.; SVENSTRUP, TINA H.; GUERRA, BARBARA

2012-01-01

265

Augmentation of antitumor immunity by fusions of ethanol-treated tumor cells and dendritic cells stimulated via dual TLRs through TGF-?1 blockade and IL-12p70 production.  

PubMed

The therapeutic efficacy of fusion cell (FC)-based cancer vaccine generated with whole tumor cells and dendritic cells (DCs) requires the improved immunogenicity of both cells. Treatment of whole tumor cells with ethanol resulted in blockade of immune-suppressive soluble factors such as transforming growth factor (TGF)-?1, vascular endothelial growth factor, and IL-10 without decreased expression of major histocompatibility complex (MHC) class I and the MUC1 tumor-associated antigen. Moreover, the ethanol-treated tumor cells expressed "eat-me" signals such as calreticulin (CRT) on the cell surface and released immunostimulatory factors such as heat shock protein (HSP)90? and high-mobility group box 1 (HMGB1). A dual stimulation of protein-bound polysaccharides isolated from Coriolus versicolor (TLR2 agonist) and penicillin-inactivated Streptococcus pyogenes (TLR4 agonist) led human monocyte-derived DCs to produce HSP90? and multiple cytokines such as IL-12p70 and IL-10. Interestingly, incorporating ethanol-treated tumor cells and TLRs-stimulated DCs during the fusion process promoted fusion efficiency and up-regulated MHC class II molecules on a per fusion basis. Moreover, fusions of ethanol-treated tumor cells and dual TLRs-stimulated DCs (E-tumor/FCs) inhibited the production of multiple immune-suppressive soluble factors including TGF-?1 and up-regulated the production of IL-12p70 and HSP90?. Most importantly, E-tumor/FCs activated T cells capable of producing high levels of IFN-?, resulting in augmented MUC1-specific CTL induction. Collectively, our results illustrate the synergy between ethanol-treated whole tumor cells and dual TLRs-stimulated DCs in inducing augmented CTL responses in vitro by FC preparations. The alternative system is simple and may provide a platform for adoptive immunotherapy. PMID:23717436

Koido, Shigeo; Homma, Sadamu; Okamoto, Masato; Namiki, Yoshihisa; Takakura, Kazuki; Takahara, Akitaka; Odahara, Shunichi; Tsukinaga, Shintaro; Yukawa, Toyokazu; Mitobe, Jimi; Matsudaira, Hiroshi; Nagatsuma, Keisuke; Kajihara, Mikio; Uchiyama, Kan; Arihiro, Seiji; Imazu, Hiroo; Arakawa, Hiroshi; Kan, Shin; Hayashi, Kazumi; Komita, Hideo; Kamata, Yuko; Ito, Masaki; Hara, Eiichi; Ohkusa, Toshifumi; Gong, Jianlin; Tajiri, Hisao

2013-05-24

266

Augmentation of Antitumor Immunity by Fusions of Ethanol-Treated Tumor Cells and Dendritic Cells Stimulated via Dual TLRs through TGF-?1 Blockade and IL-12p70 Production  

PubMed Central

The therapeutic efficacy of fusion cell (FC)-based cancer vaccine generated with whole tumor cells and dendritic cells (DCs) requires the improved immunogenicity of both cells. Treatment of whole tumor cells with ethanol resulted in blockade of immune-suppressive soluble factors such as transforming growth factor (TGF)-?1, vascular endothelial growth factor, and IL-10 without decreased expression of major histocompatibility complex (MHC) class I and the MUC1 tumor-associated antigen. Moreover, the ethanol-treated tumor cells expressed “eat-me” signals such as calreticulin (CRT) on the cell surface and released immunostimulatory factors such as heat shock protein (HSP)90? and high-mobility group box 1 (HMGB1). A dual stimulation of protein-bound polysaccharides isolated from Coriolus versicolor (TLR2 agonist) and penicillin-inactivated Streptococcus pyogenes (TLR4 agonist) led human monocyte-derived DCs to produce HSP90? and multiple cytokines such as IL-12p70 and IL-10. Interestingly, incorporating ethanol-treated tumor cells and TLRs-stimulated DCs during the fusion process promoted fusion efficiency and up-regulated MHC class II molecules on a per fusion basis. Moreover, fusions of ethanol-treated tumor cells and dual TLRs-stimulated DCs (E-tumor/FCs) inhibited the production of multiple immune-suppressive soluble factors including TGF-?1 and up-regulated the production of IL-12p70 and HSP90?. Most importantly, E-tumor/FCs activated T cells capable of producing high levels of IFN-?, resulting in augmented MUC1-specific CTL induction. Collectively, our results illustrate the synergy between ethanol-treated whole tumor cells and dual TLRs-stimulated DCs in inducing augmented CTL responses in vitro by FC preparations. The alternative system is simple and may provide a platform for adoptive immunotherapy.

Koido, Shigeo; Homma, Sadamu; Okamoto, Masato; Namiki, Yoshihisa; Takakura, Kazuki; Takahara, Akitaka; Odahara, Shunichi; Tsukinaga, Shintaro; Yukawa, Toyokazu; Mitobe, Jimi; Matsudaira, Hiroshi; Nagatsuma, Keisuke; Kajihara, Mikio; Uchiyama, Kan; Arihiro, Seiji; Imazu, Hiroo; Arakawa, Hiroshi; Kan, Shin; Hayashi, Kazumi; Komita, Hideo; Kamata, Yuko; Ito, Masaki; Hara, Eiichi; Ohkusa, Toshifumi; Gong, Jianlin; Tajiri, Hisao

2013-01-01

267

Crystal structure of the ribosomal protein S6 from Thermus thermophilus.  

PubMed

The amino acid sequence and crystal structure of the ribosomal protein S6 from the small ribosomal subunit of Thermus thermophilus have been determined. S6 is a small protein with 101 amino acid residues. The 3D structure, which was determined to 2.0 A resolution, consists of a four-stranded anti-parallel beta-sheet with two alpha-helices packed on one side. Similar folding patterns have been observed for other ribosomal proteins and may suggest an original RNA-interacting motif. Related topologies are also found in several other nucleic acid-interacting proteins and based on the assumption that the structure of the ribosome was established early in the molecular evolution, the possibility that an ancestral RNA-interacting motif in ribosomal proteins is the evolutionary origin for the nucleic acid-interacting domain in large classes of ribonucleic acid binding proteins should be considered. PMID:8137808

Lindahl, M; Svensson, L A; Liljas, A; Sedelnikova, S E; Eliseikina, I A; Fomenkova, N P; Nevskaya, N; Nikonov, S V; Garber, M B; Muranova, T A

1994-03-15

268

Superplasticity of low-workability nickel—Base alloy ZhS6KP  

Microsoft Academic Search

1.In tension at 1100° at a strain rate of 1.66·10-3 sec-1 alloy ZhS6KP with an original fine-grained structure prepared by rolling, extrusion, and hydrostatic extrusion exhibits superplasticity.2.For practical utilization of the superplasticity it is necessary that the alloy be heat treated after deformation, which substantially increases the strength characteristics.3.The sensitivity coefficient of resistance to deformation to the strain rate m

V. F. Kalugin; E. I. Razuvaev; L. R. Medvedovskaya

1975-01-01

269

STEREOSELECTIVE SYNTHESIS OF CHIRALLY-DEUTERATED D-(S)-[6-**2H1]GLUCOSE  

Technology Transfer Automated Retrieval System (TEKTRAN)

Chirally-deuterated D-(S)-[6-**2H1]glucose has been prepared in good overall yield from D-[6,6'-**2H2]glucose by a short step synthesis utilizing (R)-(+)-Alpine-Borane. Suitably protected methyl [6,6-**2H2]-2,3,4-tri-O-benzyl-D-glucopyranoside was prepared and the deuterated O-6 primary alcohol was...

270

Cysteine Mutagenesis and Computer Modeling of the S6 Region of an Intermediate Conductance IKCa Channel  

Microsoft Academic Search

Cysteine-scanning mutagenesis (SCAM) and computer-based modeling were used to investigate key structural features of the S6 transmembrane segment of the calcium-activated Kchannel of intermediate con- ductance IKCa. Our SCAM results show that the interaction of (2-(trimethylammonium)ethyl) methanethiosul- fonate bromide (MTSET) with cysteines engineered at positions 275, 278, and 282 leads to current inhibition. This effect was state dependent as MTSET

Manuel Simoes; Line Garneau; Hélène Klein; Umberto Banderali; Fadi Hobeila; Benoit Roux; Lucie Parent; Rémy Sauvé

2002-01-01

271

Distinct subdomains of the KCNQ1 S6 segment determine channel modulation by different KCNE subunits.  

PubMed

Modulation of voltage-gated potassium (KV) channels by the KCNE family of single transmembrane proteins has physiological and pathophysiological importance. All five KCNE proteins (KCNE1-KCNE5) have been demonstrated to modulate heterologously expressed KCNQ1 (KV7.1) with diverse effects, making this channel a valuable experimental platform for elucidating structure-function relationships and mechanistic differences among members of this intriguing group of accessory subunits. Here, we specifically investigated the determinants of KCNQ1 inhibition by KCNE4, the least well-studied KCNE protein. In CHO-K1 cells, KCNQ1, but not KCNQ4, is strongly inhibited by coexpression with KCNE4. By studying KCNQ1-KCNQ4 chimeras, we identified two adjacent residues (K326 and T327) within the extracellular end of the KCNQ1 S6 segment that determine inhibition of KCNQ1 by KCNE4. This dipeptide motif is distinct from neighboring S6 sequences that enable modulation by KCNE1 and KCNE3. Conversely, S6 mutations (S338C and F340C) that alter KCNE1 and KCNE3 effects on KCNQ1 do not abrogate KCNE4 inhibition. Further, KCNQ1-KCNQ4 chimeras that exhibited resistance to the inhibitory effects of KCNE4 still interact biochemically with this protein, implying that accessory subunit binding alone is not sufficient for channel modulation. These observations indicate that the diverse functional effects observed for KCNE proteins depend, in part, on structures intrinsic to the pore-forming subunit, and that distinct S6 subdomains determine KCNQ1 responses to KCNE1, KCNE3, and KCNE4. PMID:19687231

Vanoye, Carlos G; Welch, Richard C; Daniels, Melissa A; Manderfield, Lauren J; Tapper, Andrew R; Sanders, Charles R; George, Alfred L

2009-08-17

272

Simulation, experiment, and evolution: Understanding nucleation in protein S6 folding  

PubMed Central

In this study, we explore nucleation and the transition state ensemble of the ribosomal protein S6 using a Monte Carlo (MC) Go model in conjunction with restraints from experiment. The results are analyzed in the context of extensive experimental and evolutionary data. The roles of individual residues in the folding nucleus are identified, and the order of events in the S6 folding mechanism is explored in detail. Interpretation of our results agrees with, and extends the utility of, experiments that shift ?-values by modulating denaturant concentration and presents strong evidence for the realism of the mechanistic details in our MC Go model and the structural interpretation of experimental ?-values. We also observe plasticity in the contacts of the hydrophobic core that support the specific nucleus. For S6, which binds to RNA and protein after folding, this plasticity may result from the conformational flexibility required to achieve biological function. These results present a theoretical and conceptual picture that is relevant in understanding the mechanism of nucleation in protein folding.

Hubner, Isaac A.; Oliveberg, Mikael; Shakhnovich, Eugene I.

2004-01-01

273

Attenuation of ribosomal protein S6 phosphatase activity in chicken embryo fibroblasts transformed by Rous sarcoma virus.  

PubMed Central

In chicken embryo fibroblasts, phosphorylation of the 40S ribosomal protein S6 increases during G1 but returns to basal level by mitosis. In contrast, in Rous sarcoma virus (RSV)-transformed fibroblasts, S6 remains highly phosphorylated throughout mitosis. This study investigated the mechanism by which RSV alters the pattern of S6 phosphorylation. Pulse-chase experiments demonstrate that phosphate turnover in S6 is rapid in normal cells and in cells infected with an RSV transformation-defective virus. In contrast, phosphate turnover in S6 is severely reduced in cells infected with temperature-sensitive RSV at a temperature permissive for transformation, indicating a diminished S6 phosphatase activity. Fractionation of cell lysates by DEAE chromatography showed an almost threefold lower S6 phosphatase activity in RSV-transformed versus normal cells. The S6 phosphatase was sensitive to inhibitor 2 and specifically recognized by an antibody to type 1 phosphatase (PP1). The S6 phosphatase activity recovered by immunoprecipitation of PP1 was threefold lower in transformed cells, but the steady-state level of expression and the rate of synthesis of PP1 were not altered by oncogenic transformation. Together, the results show that transformation by RSV reduced the S6-PP1 activity. Images

Belandia, B; Brautigan, D; Martin-Perez, J

1994-01-01

274

Combination of pharmacophore hypothesis, genetic function approximation model, and molecular docking to identify novel inhibitors of S6K1.  

PubMed

S6K1 has emerged as a potential target for the treatment for obesity, type II diabetes and cancer diseases. Discovery of S6K1 inhibitors has thus attracted much attention in recent years. In this investigation, a hybrid virtual screening method that involves pharmacophore hypothesis, genetic function approximation (GFA) model, and molecular docking technology has been used to discover S6K1 inhibitors especially with novel scaffolds. The common feature pharmacophore hypothesis and GFA regression model of S6K1 inhibitors were first developed and applied in a virtual screen of the Specs database for retrieving S6K1 inhibitors. Then, the molecular docking method was carried out to re-filter these screened compounds. Finally, 60 compounds with promising S6K1 inhibitory activity were carefully selected and have been handed over to the other group to complete the follow-up compound synthesis (or purchase) and activity test. PMID:23982212

Zhang, Hui; Xiang, Ming-Li; Liang, Jun-Yu; Zeng, Tao; Zhang, Xiao-Nuo; Zhang, Ji; Yang, Sheng-Yong

2013-08-28

275

Cross-talk between protein kinase A and the MAPK-activated protein kinases RSK1 and MK5.  

PubMed

Typical mammalian mitogen-activated protein kinase (MAPK) pathways consist of a cascade of three consecutive phosphorylation events exerted by a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK), and finally a MAPK. MAPKs not only target non-protein kinase substrates, they can also phosphorylate other protein kinases designated as MAPK-activated protein kinases (MAPKAPK). The MAPKAPK family includes the ribosomal-S6-kinases (RSK1-4), the MAPK-interacting kinases (MNK1 and 2), the mitogen-and stress-activated kinases (MSK1 and 2), and the MAPKAPK (MK2, 3, and 5) subfamilies. Although several reports indicate extensive cross-talk between the MAPK and protein kinase A (PKA) pathways, evidence of a direct interaction at the level of the MAPKAPK only appeared recently. The MAPKAPKs RSK1 and MK5 can bind to PKA, but the features of these interactions are distinct. This review discusses the different characteristics of regulating the activity and subcellular localization of MK5 and RSK1 by PKA and the functional implications of these interactions. PMID:20849292

Kostenko, Sergiy; Shiryaev, Alexey; Dumitriu, Gianina; Gerits, Nancy; Moens, Ugo

2010-09-18

276

Cysteine Mutagenesis and Computer Modeling of the S6 Region of an Intermediate Conductance IKCa Channel  

PubMed Central

Cysteine-scanning mutagenesis (SCAM) and computer-based modeling were used to investigate key structural features of the S6 transmembrane segment of the calcium-activated K+ channel of intermediate conductance IKCa. Our SCAM results show that the interaction of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) with cysteines engineered at positions 275, 278, and 282 leads to current inhibition. This effect was state dependent as MTSET appeared less effective at inhibiting IKCa in the closed (zero Ca2+ conditions) than open state configuration. Our results also indicate that the last four residues in S6, from A283 to A286, are entirely exposed to water in open IKCa channels, whereas MTSET can still reach the 283C and 286C residues with IKCa maintained in a closed state configuration. Notably, the internal application of MTSET or sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) caused a strong Ca2+-dependent stimulation of the A283C, V285C, and A286C currents. However, in contrast to the wild-type IKCa, the MTSET-stimulated A283C and A286C currents appeared to be TEA insensitive, indicating that the MTSET binding at positions 283 and 286 impaired the access of TEA to the channel pore. Three-dimensional structural data were next generated through homology modeling using the KcsA structure as template. In accordance with the SCAM results, the three-dimensional models predict that the V275, T278, and V282 residues should be lining the channel pore. However, the pore dimensions derived for the A283–A286 region cannot account for the MTSET effect on the closed A283C and A286 mutants. Our results suggest that the S6 domain extending from V275 to V282 possesses features corresponding to the inner cavity region of KcsA, and that the COOH terminus end of S6, from A283 to A286, is more flexible than predicted on the basis of the closed KcsA crystallographic structure alone. According to this model, closure by the gate should occur at a point located between the T278 and V282 residues.

Simoes, Manuel; Garneau, Line; Klein, Helene; Banderali, Umberto; Hobeila, Fadi; Roux, Benoit; Parent, Lucie; Sauve, Remy

2002-01-01

277

Immunological evidence for structural homology between Drosophila melanogaster (S14), rabbit liver (S12), Saccharomyces cerevisiae (S25), Bacillus subtilis (S6), and Escherichia coli (S6) ribosomal proteins.  

PubMed Central

Specific antibodies directed against Drosophila melanogaster acidic ribosomal protein S14 were used in a comparative study of eucaryotic and procaryotic ribosomes by immunoblotting and enzyme-linked immunosorbent assays. Common antigenic determinants and, thus, structural homology were found between D. melanogaster, Saccharomyces cerevisiae (S25), rabbit liver (S12), Bacillus subtilis (S6), and Escherichia coli (S6) ribosomes. Images

Chooi, W Y; Otaka, E

1984-01-01

278

Platelet-derived growth factor-induced Akt phosphorylation requires mTOR/Rictor and phospholipase C-?1, whereas S6 phosphorylation depends on mTOR/Raptor and phospholipase D  

PubMed Central

Mammalian target of rapamycin (mTOR) can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor) and mTORC2 (containing Rictor). Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF)-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictor-null cells the phosphorylation of phospholipase C?1 (PLC?1) and protein kinase C (PKC) was impaired, and the PKC? protein levels strongly reduced. Furthermore, interfering with the PLC?/Ca2+/PKC pathway inhibited PDGF-BB-induced Akt phosphorylation. In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD). It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner and promotes phosphorylation of the S6 protein, whereas mTORC2, in concert with PLC? signaling, promotes Akt phosphorylation.

2013-01-01

279

Differential roles of S6 domain hinges in the gating of KCNQ potassium channels.  

PubMed

Voltage-gated K+ channel activation is proposed to result from simultaneous bending of all S6 segments away from the central axis, enlarging the aperture of the pore sufficiently to permit diffusion of K+ into the water-filled central cavity. The hinge position for the bending motion of each S6 segment is proposed to be a Gly residue and/or a Pro-Val-Pro motif in Kv1-Kv4 channels. The KCNQ1 (Kv7.1) channel has Ala-336 in the Gly-hinge position and Pro-Ala-Gly. Here we show that mutation of Ala-336 to Gly in KCNQ1 increased current amplitude and shifted the voltage dependence of activation to more negative potentials, consistent with facilitation of hinge activity that favors the open state. In contrast, mutation of Ala-336 to Cys or Thr shifted the voltage dependence of activation to more positive potentials and reduced current amplitude. Mutation of the putative Gly hinge to Ala in KCNQ2 (Kv7.2) abolished channel function. Mutation-dependent changes in current amplitude, but not kinetics, were found in heteromeric KCNQ1/KCNE1 channels. Mutation of the Pro or Gly of the Pro-Ala-Gly motif to Ala abolished KCNQ1 function and introduction of Gly in front of the Ala-mutations partially recovered channel function, suggesting that flexibility at the PAG is important for channel activation. PMID:16326905

Seebohm, Guiscard; Strutz-Seebohm, Nathalie; Ureche, Oana N; Baltaev, Ravshan; Lampert, Angelika; Kornichuk, Ganna; Kamiya, Kaichiro; Wuttke, Thomas V; Lerche, Holger; Sanguinetti, Michael C; Lang, Florian

2005-12-02

280

The PP242 Mammalian Target of Rapamycin (mTOR) Inhibitor Activates Extracellular Signal-regulated Kinase (ERK) in Multiple Myeloma Cells via a Target of Rapamycin Complex 1 (TORC1)/ Eukaryotic Translation Initiation Factor 4E (eIF-4E)/RAF Pathway and Activation Is a Mechanism of Resistance*  

PubMed Central

Activation of PI3-K-AKT and ERK pathways is a complication of mTOR inhibitor therapy. Newer mTOR inhibitors (like pp242) can overcome feedback activation of AKT in multiple myeloma (MM) cells. We, thus, studied if feedback activation of ERK is still a complication of therapy with such drugs in this tumor model. PP242 induced ERK activation in MM cell lines as well as primary cells. Surprisingly, equimolar concentrations of rapamycin were relatively ineffective at ERK activation. Activation was not correlated with P70S6kinase inhibition nor was it prevented by PI3-kinase inhibition. ERK activation was prevented by MEK inhibitors and was associated with concurrent stimulation of RAF kinase activity but not RAS activation. RAF activation correlated with decreased phosphorylation of RAF at Ser-289, Ser-296, and Ser-301 inhibitory residues. Knockdown studies confirmed TORC1 inhibition was the key proximal event that resulted in ERK activation. Furthermore, ectopic expression of eIF-4E blunted pp242-induced ERK phosphorylation. Since pp242 was more potent than rapamycin in causing sequestering of eIF-4E, a TORC1/4E-BP1/eIF-4E-mediated mechanism of ERK activation could explain the greater effectiveness of pp242. Use of MEK inhibitors confirmed ERK activation served as a mechanism of resistance to the lethal effects of pp242. Thus, although active site mTOR inhibitors overcome AKT activation often seen with rapalog therapy, feedback ERK activation is still a problem of resistance, is more severe than that seen with use of first generation rapalogs and is mediated by a TORC1- and eIF-4E-dependent mechanism ultimately signaling to RAF.

Hoang, Bao; Benavides, Angelica; Shi, Yijiang; Yang, Yonghui; Frost, Patrick; Gera, Joseph; Lichtenstein, Alan

2012-01-01

281

Inhibition of mTOR Kinase by AZD8055 Can Antagonize Chemotherapy-induced Cell Death through Autophagy Induction and Down-regulation of p62/Sequestosome 1*  

PubMed Central

AZD8055 is an ATP-competitive inhibitor of mammalian target of rapamycin (mTOR) that forms two multiprotein complexes, mTORC1 and mTORC2, and negatively regulates autophagy. We demonstrate that AZD8055 stimulates and potentiates chemotherapy-mediated autophagy, as shown by LC3I-II conversion and down-regulation of the ubiquitin-binding protein p62/sequestosome 1. AZD8055-induced autophagy was pro-survival as shown by its ability to attenuate cell death and DNA damage (p-H2AX), and to enhance clonogenic survival by cytotoxic chemotherapy. Autophagy inhibition by siRNA against Beclin 1 or LC3B, or by chloroquine, partially reversed the cytoprotective effect of AZD8055 that was independent of cell cycle inhibition. The pro-survival role of autophagy was confirmed using ectopic expression of Beclin 1 that conferred cytoprotection. To determine whether autophagy-mediated down-regulation of p62/sequestosome 1 contributes to its pro-survival role, we generated p62 knockdown cells using shRNA that showed protection from chemotherapy-induced cell death and DNA damage. We also overexpressed wild-type (wt) p62 that promoted chemotherapy-induced cell death, whereas mutated p62 at functional domains (PB1, UBA) failed to do so. The ability of ectopic wt p62 to promote cell death was blocked by AZD8055. AZD8055 was shown to inhibit phosphorylation of the autophagy-initiating kinase ULK1 at Ser757 and inhibited known targets of mTORC1 (p-mTOR Ser2448, p70S6K, p-S6, p4EBP1) and mTORC2 (p-mTOR Ser2481, p-AKT Ser473). Knockdown of mTOR, but not Raptor or Rictor, reduced p-ULK1 at Ser757 and enhanced chemotherapy-induced autophagy that resulted in a similar cytoprotective effect as shown for AZD8055. In conclusion, AZD8055 inhibits mTOR kinase and ULK1 phosphorylation to induce autophagy whose pro-survival effect is due, in part, to down-regulation of p62.

Huang, Shengbing; Yang, Zhineng J.; Yu, Chunrong; Sinicrope, Frank A.

2011-01-01

282

S6:S18 ribosomal protein complex interacts with a structural motif present in its own mRNA.  

PubMed

Prokaryotic ribosomal protein genes are typically grouped within highly conserved operons. In many cases, one or more of the encoded proteins not only bind to a specific site in the ribosomal RNA, but also to a motif localized within their own mRNA, and thereby regulate expression of the operon. In this study, we computationally predicted an RNA motif present in many bacterial phyla within the 5' untranslated region of operons encoding ribosomal proteins S6 and S18. We demonstrated that the S6:S18 complex binds to this motif, which we hereafter refer to as the S6:S18 complex-binding motif (S6S18CBM). This motif is a conserved CCG sequence presented in a bulge flanked by a stem and a hairpin structure. A similar structure containing a CCG trinucleotide forms the S6:S18 complex binding site in 16S ribosomal RNA. We have constructed a 3D structural model of a S6:S18 complex with S6S18CBM, which suggests that the CCG trinucleotide in a specific structural context may be specifically recognized by the S18 protein. This prediction was supported by site-directed mutagenesis of both RNA and protein components. These results provide a molecular basis for understanding protein-RNA recognition and suggest that the S6S18CBM is involved in an auto-regulatory mechanism. PMID:23980204

Matelska, Dorota; Purta, Elzbieta; Panek, Sylwia; Boniecki, Michal J; Bujnicki, Janusz M; Dunin-Horkawicz, Stanislaw

2013-08-26

283

ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions  

PubMed Central

Conserved signaling pathways that activate the mitogen-activated protein kinases (MAPKs) are involved in relaying extracellular stimulations to intracellular responses. The MAPKs coordinately regulate cell proliferation, differentiation, motility, and survival, which are functions also known to be mediated by members of a growing family of MAPK-activated protein kinases (MKs; formerly known as MAPKAP kinases). The MKs are related serine/threonine kinases that respond to mitogenic and stress stimuli through proline-directed phosphorylation and activation of the kinase domain by extracellular signal-regulated kinases 1 and 2 and p38 MAPKs. There are currently 11 vertebrate MKs in five subfamilies based on primary sequence homology: the ribosomal S6 kinases, the mitogen- and stress-activated kinases, the MAPK-interacting kinases, MAPK-activated protein kinases 2 and 3, and MK5. In the last 5 years, several MK substrates have been identified, which has helped tremendously to identify the biological role of the members of this family. Together with data from the study of MK-knockout mice, the identities of the MK substrates indicate that they play important roles in diverse biological processes, including mRNA translation, cell proliferation and survival, and the nuclear genomic response to mitogens and cellular stresses. In this article, we review the existing data on the MKs and discuss their physiological functions based on recent discoveries.

Roux, Philippe P.; Blenis, John

2004-01-01

284

ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions.  

PubMed

Conserved signaling pathways that activate the mitogen-activated protein kinases (MAPKs) are involved in relaying extracellular stimulations to intracellular responses. The MAPKs coordinately regulate cell proliferation, differentiation, motility, and survival, which are functions also known to be mediated by members of a growing family of MAPK-activated protein kinases (MKs; formerly known as MAPKAP kinases). The MKs are related serine/threonine kinases that respond to mitogenic and stress stimuli through proline-directed phosphorylation and activation of the kinase domain by extracellular signal-regulated kinases 1 and 2 and p38 MAPKs. There are currently 11 vertebrate MKs in five subfamilies based on primary sequence homology: the ribosomal S6 kinases, the mitogen- and stress-activated kinases, the MAPK-interacting kinases, MAPK-activated protein kinases 2 and 3, and MK5. In the last 5 years, several MK substrates have been identified, which has helped tremendously to identify the biological role of the members of this family. Together with data from the study of MK-knockout mice, the identities of the MK substrates indicate that they play important roles in diverse biological processes, including mRNA translation, cell proliferation and survival, and the nuclear genomic response to mitogens and cellular stresses. In this article, we review the existing data on the MKs and discuss their physiological functions based on recent discoveries. PMID:15187187

Roux, Philippe P; Blenis, John

2004-06-01

285

Lifetime of the 7s6d D12 atomic state of radium  

NASA Astrophysics Data System (ADS)

The lifetime of the 7s6d D12 state of atomic radium is determined to be 385(45)?s using cold R226a atoms prepared in a magneto-optical trap. The D12 state is populated from the decay of the P11 state which is excited by a pulse of 483 nm light. The decay of the D12 state is observed by detecting delayed fluorescence at 714 nm from the last step in the decay sequence P11-D12-P31-S10 . The measured lifetime is compared to a number of theoretical calculations. An improved value of the 7s7p P11 level of 20715.598(6)cm-1 is obtained.

Trimble, W. L.; Sulai, I. A.; Ahmad, I.; Bailey, K.; Graner, B.; Greene, J. P.; Holt, R. J.; Korsch, W.; Lu, Z.-T.; Mueller, P.; O'Connor, T. P.

2009-11-01

286

TOR signalling and control of cell growth  

Microsoft Academic Search

TOR, phosphatidylinositol 3-kinase, p70s6k, and 4E-BP1 have recently emerged as components of a major signalling pathway that is dedicated to protein translation and thus to cell growth. This pathway appears to be conserved, at least in part, in yeast, slime molds, plants, flies, and mammals. TOR and phosphatidylinositol 3-kinase control p70s6k and 4E-BP1, which, in turn, directly control the translation

George Thomas; Michael N Hall

1997-01-01

287

Stress value in 2003 Dayao M S6.1 earthquake source region  

NASA Astrophysics Data System (ADS)

The M S6.2 Dayao, Yunnan, earthquake occurred on July 21, 2003, followed by a major M S6.1 earthquake about 88 days later in the same region. Hypocenters of the two earthquakes are almost in the same place. Based on the P wave first motion polarities of the two aftershock sequences recorded by temporary stations, we have studied the stress field in the aftershock zone and obtained the two stress field directions in Dayao region using the new version of PKU Grid Test Software provided by Chunquan Yu. Assuming that the rotation of the stress field is caused by the second main shock, we estimated the crustal stress value in the focal region by using the stress value calculation method proposed by Yongge Wan. The estimated maximum, intermediate and minimum principal stresses are 166.3 MPa, 158.7 MPa and 151 MPa, respectively, before the second main shock. The normal and shear stresses projected on the fault plane of the second main shock before it occurred are 157.3 MPa, 7.4 MPa, and are 158.8 MPa, 0.2 MPa after it occurred, respectively. The perturbed input parameters experiments attest the stability of the solution. The result shows that the preseismic shear stress is larger than the post-seismic one, and their difference corresponds to the stress drop approximately. The estimated compressive stress level is very high, but the differential stress is low. The result is helpful for friction coefficient estimation, plate motion simulation and related studies.

Xu, Xiaofeng; Wan, Yongge; Wang, Huilin

2011-08-01

288

A Possible Mechanism for Hyperthermic Radiosensitization Mediated through Hyperthermic Lability of Ku Subunits in DNA-Dependent Protein Kinase  

Microsoft Academic Search

DNA-dependent protein kinase (DNA-PK), composed of p470 catalytic subunit and p85\\/p70 heterodimer of Ku autoantigen, is considered a critical enzyme in DNA double-strand break repair. We purified DNA-PK from human leukaemic MOLT-4 cells by successive column chromatography and separated into p470 and Ku subunits by ultracentrifugation in glycerol gradient. We studied hyperthermic stability of DNA-PK holoenzyme and its separated subunits

Yoshihisa Matsumoto; Norio Suzuki; Kazuo Sakai; Akeshi Morimatsu; Kazuya Hirano; Hiromu Murofushi

1997-01-01

289

Elastic and acoustooptic properties of Sn2P2S6 crystals: Effect of ferroelectric phase transition  

NASA Astrophysics Data System (ADS)

We report on the studies for temperature dependences of elastic stiffness coefficients in Sn2P2S6 crystals. Basing on the construction of acoustic velocity surfaces, we have determined the parameters of the slowest acoustic wave that propagates in Sn2P2S6 crystals. The acoustooptic figure of merit for the case of acoustooptic interaction with this wave is estimated as ˜0.8 × 10-12 s3/kg. We have shown that Sn2P2S6 is very close to the conditions of tricritical point on the (x, T)- and (p, T)-phase diagrams of the solid solutions Sn2P2(SexS1-x)6. The critical exponent ? of the heat capacity for the Sn2P2S6 crystals is equal to 0.42 ± 0.03.

Mys, O.; Zapeka, B.; Martynyuk-Lototska, I.; Vlokh, R.

2012-12-01

290

Copper ordering in lamellar CuMP2S6 (M= Cr, In): Transition to an antiferroelectric or ferroelectric phase  

Microsoft Academic Search

The transition to an antiferroelectric or ferroelectric phase in the lamellar compound CuMP2S6 (M= Cr or In) is shown to be triggered by the appearance of an ordered copper sublattice. Cu(I) ions in CuCrP2S6, which are distributed over several sites at room temperature, freeze into an intermediate quasi-antipolar state between 190 and 175K, and then form an array of alternating

V. B. Cajipea; J. Ravez; V. Maisonneuve; A. Simon; C. Payen; R. Von Der Muhll; J. E. Fischer

1996-01-01

291

Mice Deficient in Ribosomal Protein S6 Phosphorylation Suffer from Muscle Weakness that Reflects a Growth Defect and Energy Deficit  

Microsoft Academic Search

BackgroundMice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all five phosphorylatable serine residues by alanines (rpS6P?\\/?), are viable and fertile. However, phenotypic characterization of these mice and embryo fibroblasts derived from them, has established the role of these modifications in the regulation of the size of several cell types, as well as pancreatic ?-cell function and

Igor Ruvinsky; Maximiliano Katz; Avigail Dreazen; Yuval Gielchinsky; Ann Saada; Nanette Freedman; Eyal Mishani; Gabriel Zimmerman; Judith Kasir; Oded Meyuhas; Rafael Linden

2009-01-01

292

The Putative RNA Helicase HELZ Promotes Cell Proliferation, Translation Initiation and Ribosomal Protein S6 Phosphorylation  

PubMed Central

The hypoxia–inducible transcription factor (HIF) is a key component of the cellular adaptation mechanisms to hypoxic conditions. HIF? subunits are degraded by prolyl-4-hydroxylase domain (PHD) enzyme-dependent prolyl-4-hydroxylation of LxxLAP motifs that confer oxygen-dependent proteolytic degradation. Interestingly, only three non-HIF? proteins contain two conserved LxxLAP motifs, including the putative RNA helicase with a zinc finger domain HELZ. However, HELZ proteolytic regulation was found to be oxygen-independent, supporting the notion that a LxxLAP sequence motif alone is not sufficient for oxygen-dependent protein destruction. Since biochemical pathways involving RNA often require RNA helicases to modulate RNA structure and activity, we used luciferase reporter gene constructs and metabolic labeling to demonstrate that HELZ overexpression activates global protein translation whereas RNA-interference mediated HELZ suppression had the opposite effect. Although HELZ interacted with the poly(A)-binding protein (PABP) via its PAM2 motif, PABP was dispensable for HELZ function in protein translation. Importantly, downregulation of HELZ reduced translational initiation, resulting in the disassembly of polysomes, in a reduction of cell proliferation and hypophosphorylation of ribosomal protein S6.

Hasgall, Philippe A.; Hoogewijs, David; Faza, Marius B.; Panse, Vikram G.; Wenger, Roland H.; Camenisch, Gieri

2011-01-01

293

Role of Elongation and Secondary Pathways in S6 Amyloid Fibril Growth  

PubMed Central

The concerted action of a large number of individual molecular level events in the formation and growth of fibrillar protein structures creates a significant challenge for differentiating between the relative contributions of different self-assembly steps to the overall kinetics of this process. The characterization of the individual steps is, however, an important requirement for achieving a quantitative understanding of this general phenomenon which underlies many crucial functional and pathological pathways in living systems. In this study, we have applied a kinetic modeling approach to interpret experimental data obtained for the aggregation of a selection of site-directed mutants of the protein S6 from Thermus thermophilus. By studying a range of concentrations of both the seed structures, used to initiate the reaction, and of the soluble monomer, which is consumed during the growth reaction, we are able to separate unambiguously secondary pathways from primary nucleation and fibril elongation. In particular, our results show that the characteristic autocatalytic nature of the growth process originates from secondary processes rather than primary nucleation events, and enables us to derive a scaling law which relates the initial seed concentration to the onset of the growth phase.

Lorenzen, Nikolai; Cohen, Samuel I.A.; Nielsen, S?ren B.; Herling, Therese W.; Christiansen, Gunna; Dobson, Christopher M.; Knowles, Tuomas P.J.; Otzen, Daniel

2012-01-01

294

Protein Kinase C-mediated Phosphorylation and Activation of PDE3A Regulate cAMP Levels in Human Platelets*S?  

PubMed Central

The elevation of [cAMP]i is an important mechanism of platelet inhibition and is regulated by the opposing activity of adenylyl cyclase and phosphodiesterase (PDE). In this study, we demonstrate that a variety of platelet agonists, including thrombin, significantly enhance the activity of PDE3A in a phosphorylation-dependent manner. Stimulation of platelets with the PAR-1 agonist SFLLRN resulted in rapid and transient phosphorylation of PDE3A on Ser312, Ser428, Ser438, Ser465, and Ser492, in parallel with the PKC (protein kinase C) substrate, pleckstrin. Furthermore, phosphorylation and activation of PDE3A required the activation of PKC, but not of PI3K/PKB, mTOR/p70S6K, or ERK/RSK. Activation of PKC by phorbol esters also resulted in phosphorylation of the same PDE3A sites in a PKC-dependent, PKB-independent manner. This was further supported by the finding that IGF-1, which strongly activates PI3K/PKB, but not PKC, did not regulate PDE3A. Platelet activation also led to a PKC-dependent association between PDE3A and 14-3-3 proteins. In contrast, cAMP-elevating agents such as PGE1 and forskolin-induced phosphorylation of Ser312 and increased PDE3A activity, but did not stimulate 14-3-3 binding. Finally, complete antagonism of PGE1-evoked cAMP accumulation by thrombin required both Gi and PKC activation. Together, these results demonstrate that platelet activation stimulates PKC-dependent phosphorylation of PDE3A on Ser312, Ser428, Ser438, Ser465, and Ser492 leading to a subsequent increase in cAMP hydrolysis and 14-3-3 binding.

Hunter, Roger W.; MacKintosh, Carol; Hers, Ingeborg

2009-01-01

295

Decreased IRS Signaling Impairs ?-Cell Cycle Progression and Survival in Transgenic Mice Overexpressing S6K in ?-Cells  

PubMed Central

OBJECTIVE The purpose of this study was to evaluate the role of the S6K arm of mammalian target of rapamycin complex 1 (mTORC1) signaling in regulation of ?-cell mass and function. Additionally, we aimed to delineate the importance of in vivo S6K activation in the regulation of insulin signaling and the extent to which alteration of insulin receptor substrate (IRS) signaling modulates ?-cell mass and function. RESEARCH DESIGN AND METHODS The current experiments describe the phenotype of transgenic mice overexpressing a constitutively active form of S6K under the control of the rat insulin promoter. RESULTS Activation of S6K signaling in these mice improved insulin secretion in the absence of changes in ?-cell mass. The lack of ?-cell mass expansion resulted from decreased G1-S progression and increased apoptosis. This phenotype was associated with increased p16 and p27 and decreased Cdk2 levels. The changes in cell cycle were accompanied by diminished survival signals because of impaired IRS/Akt signaling. CONCLUSIONS This work defines the importance of S6K in regulation of ?-cell cycle, cell size, function, and survival. These experiments also demonstrate that in vivo downregulation of IRS signaling by TORC1/S6K induces ?-cell insulin resistance, and that this mechanism could explain some of the abnormalities that ultimately result in ?-cell failure and diabetes in conditions of nutrient overload.

Elghazi, Lynda; Balcazar, Norman; Blandino-Rosano, Manuel; Cras-Meneur, Corentin; Fatrai, Szabolcs; Gould, Aaron P.; Chi, Maggie M.; Moley, Kelle H.; Bernal-Mizrachi, Ernesto

2010-01-01

296

SUPPLEMENTARY COMPARISON: APMP.EM-S6: Bilateral supplementary comparison of resistance  

NASA Astrophysics Data System (ADS)

A bilateral supplementary comparison of resistance, APMP.EM-S6, was conducted between the National Institute of Metrology Thailand (NIMT) and the CSIRO National Measurement Laboratory of Australia (NML). The comparison covered seven values of resistance, 0.1 ?, 1 ?, 100 ?, 10 k?, 100 k?, 1 M? and 100 M?. The 0.1 ? resistor was a YEW type 2792, the 100 M? resistor was an IET type SRC and the other five resistors were Fluke type 742A. The resistors were supplied by NIMT, and NML was the pilot laboratory for the comparison. The measurements for the comparison were made between December 2003 and April 2004. The resistors were measured on three separate occasions by NIMT and between each of these occasions the resistors were sent to NML for measurement. The resistors of nominal values 0.1 ?, 1 ?, 100 ? and 10 k? were measured as four-terminal resistors while the 100 k?, 1 M? and 100 M? resistors were measured as two-terminal resistors. The quantity En (the absolute value of the difference between the values obtained at the two laboratories divided by the root sum square of the expanded uncertainties of the two values) was calculated for each resistance value used in the comparison. In all cases En was less than 0.5. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the APMP, according to the provisions of the Mutual Recognition Arrangement (MRA).

Charoensook, Ajchara; Jassadajin, Chaiwat; Chen, Henry; Ricketts, Brian

2004-01-01

297

A Genome-wide RNAi Screen for Polypeptides that Alter rpS6 Phosphorylation  

PubMed Central

Mammalian target of rapamycin (mTOR) is a giant protein kinase that controls cell proliferation, growth, and metabolism. mTOR is regulated by nutrient availability, by mitogens, and by stress, and operates through two independently regulated hetero-oligomeric complexes. We have attempted to identify the cellular components necessary to maintain the activity of mTOR complex 1 (mTORC1), the amino acid-dependent, rapamycin-inhibitable complex, using a whole genome approach involving RNAi-induced depletion of cellular polypeptides. We have used a pancreatic ductal adenocarcinoma (PDAC) cell line, Mia-PaCa for this screen; as with many pancreatic cancers, these cells exhibit constitutive activation of mTORC1. PDAC is the most common form of pancreatic cancer and the 5-year survival rate remains 3–5% despite current nonspecific and targeted therapies. Although rapamycin-related mTOR inhibitors have yet to demonstrate encouraging clinical responses, it is now evident that this class of compounds is capable of only partial mTORC1 inhibition. Identifying previously unappreciated proteins needed for maintenance of mTORC1 activity may provide new targets and lead to the development of beneficial therapies for pancreatic cancer.

Papageorgiou, Angela; Avruch, Joseph

2012-01-01

298

Silver ionic and electronic conductivity in Ag9GaS6, Ag9AlS6, AgGaS2, AgAlS2, and AgAl5S8  

NASA Astrophysics Data System (ADS)

Silver ionic and electronic conductivity in phases in the Ag2S-M2S3, M = Al, Ga, systems have been investigated using dc methods with ionically reversible electrodes and ac methods. Measurements on the mixed conductors Ag9GaS6 and the new phase Ag9AlS6, both with high silver ionic conductivity, the chalcopyrites AgGaS2 and AgAlS2, both with predominate silver ionic conductivity, and the mixed conducting spinel, AgAl5S8, are reported. In addition, a schematic version of the Ag2S-Al2S3 phase diagram is presented.

Hellstrom, E. E.; Huggins, R. A.

1980-11-01

299

The substrate specificity and structure of mitogen-activated protein (MAP) kinase-activated protein kinase-2.  

PubMed Central

The substrate specificity of mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MAPKAP kinase-2) was investigated by using synthetic peptides related to the N-terminus of glycogen synthase. The minimum sequence required for efficient phosphorylation was found to be Xaa-Xaa-Hyd-Xaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa, where Hyd is a bulky hydrophobic residue (Phe > Leu > Val >> Ala), and the peptide Lys-Lys-Phe-Asn-Arg-Thr-Leu-Ser-Val-Ala was phosphorylated with a Km of 9.3 microM and Vmax. of 10 mumol/min per mg. MAPKAP kinase-1 (a homologue of ribosomal protein S6 kinase) also requires an arginine three residues N-terminal to the serine (position n-3), but not a hydrophobic residue at position n-5. Neither MAPKAP kinase-1 nor MAPKAP kinase-2 could tolerate a proline residue at position n + 1, indicating that their specificities do not overlap with that of MAP kinase. The specificity of calmodulin-dependent protein kinase-II resembled that of MAPKAP kinase-2, except that it could tolerate replacement of the arginine by a lysine and the phosphorylation-site serine by a threonine residue. Partial cDNAs encoding MAPKAP kinase-2 were isolated from rabbit and human skeletal muscle and human teratocarcinoma libraries, and Northern-blotting experiments revealed a single 3.3 kb mRNA transcript present at similar levels in six human tissues examined. The catalytic domain was most similar (35-40% identity) to calmodulin-dependent protein kinases II and IV, phosphorylase kinase, putative serine kinase H1 and the C-terminal domain of MAPKAP kinase-1, which form one branch of the protein kinase phylogenetic tree. The sequence N-terminal to the catalytic domain is proline-rich and contains two putative SH3-binding sites. The threonine residue phosphorylated by MAP kinase lies immediately C-terminal to the catalytic domain and is followed by a nuclear localization signal, Lys-Lys-(Xaa)10-Lys-Arg-Arg-Lys-Lys, near the C-terminus. Images Figure 3

Stokoe, D; Caudwell, B; Cohen, P T; Cohen, P

1993-01-01

300

Effect of spin ordering on structure and structural transitions in the (MnS)6 magic cluster  

NASA Astrophysics Data System (ADS)

Many manganese-based magnetic materials exhibit exciting technologically important properties (e.g. colossal magnetoresistance, giant magnetocaloric effect) but, as yet, relatively little is known at the nanoscale. Recently, manganese sulfide clusters were produced in a laser ablation experiment, revealing sizes with special stability, i.e. magic clusters. Here, we use density functional calculations to investigate the (MnS)6 magic cluster. Specifically, the interplay between magnetism, structure and energetic stability in nine low lying minima (MnS)6 structures is investigated and rationalized. We further calculate the energetic barriers for interconversion between the two lowest lying (MnS)6 isomers and discuss their possible relevance to premelting cluster dynamics.

Lewoczko, April D.; BelBruno, Joseph J.; Bromley, Stefan T.

2013-01-01

301

KEA: kinase enrichment analysis  

PubMed Central

Motivation: Multivariate experiments applied to mammalian cells often produce lists of proteins/genes altered under treatment versus control conditions. Such lists can be projected onto prior knowledge of kinase–substrate interactions to infer the list of kinases associated with a specific protein list. By computing how the proportion of kinases, associated with a specific list of proteins/genes, deviates from an expected distribution, we can rank kinases and kinase families based on the likelihood that these kinases are functionally associated with regulating the cell under specific experimental conditions. Such analysis can assist in producing hypotheses that can explain how the kinome is involved in the maintenance of different cellular states and can be manipulated to modulate cells towards a desired phenotype. Summary: Kinase enrichment analysis (KEA) is a web-based tool with an underlying database providing users with the ability to link lists of mammalian proteins/genes with the kinases that phosphorylate them. The system draws from several available kinase–substrate databases to compute kinase enrichment probability based on the distribution of kinase–substrate proportions in the background kinase–substrate database compared with kinases found to be associated with an input list of genes/proteins. Availability: The KEA system is freely available at http://amp.pharm.mssm.edu/lib/kea.jsp Contact: avi.maayan@mssm.edu

Lachmann, Alexander; Ma'ayan, Avi

2009-01-01

302

Probing the Higgs branch of 5d fixed point theories with dual giant gravitons in AdS6  

NASA Astrophysics Data System (ADS)

We consider the warped Ad{S_6}× {S^4}/{{{Z}}n} backgrounds dual to certain 5d quiver gauge theories. By studying dual giant gravitons in the AdS 6 geometry we are able to partially probe the Higgs branch of these theories. We show how the quantization of the phase space of such dual giants coincides with the counting of holomorphic functions on {{{C}}^2}/{{{Z}}n} , which is the geometric part of the Higgs branch for these theories.

Bergman, Oren; Rodríguez-Gómez, Diego

2012-12-01

303

Syntheses and characterizations of compounds Ba4F4XGa2S6 (X = Cr, Mn, Fe) and Ba4F4MnIn2S6 with 2D layered structures.  

PubMed

Four new 2D layered quinary sulfides, Ba4F4CrGa2S6 (1), Ba4F4MnGa2S6 (2), Ba4F4FeGa2S6 (3) and Ba4F4MnIn2S6 (4), have been synthesized by the traditional solid state reaction method. Single crystal X-ray diffraction analyses show that the isostructural complexes 1, 3 and 4 belong to the Ba2F2Fe1.5S3 structure type and crystallize in the space group Pnma of the orthorhombic system, whereas the complex 2 crystallizes in the space group Cmca. The crystal structures of the four compounds can be viewed as the alternated stacking of the fluorite type [Ba2F2](2+) blocks and the newly discovered [X0.5GaS3](2-) or [X0.5InS3](2-) blocks. First-principles electronic structure calculations performed with DFT indicate that the title compounds are semiconductors with the band gaps of 1.83, 3.21, 1.16 and 2.93 eV for 1, 2, 3 and 4, respectively. PMID:23698170

Luo, Zhong-Zhen; Lin, Chen-Sheng; Cheng, Wen-Dan; Li, Yuan-Bing; Zhang, Hao; Zhang, Wei-Long; He, Zhang-Zhen

2013-05-22

304

Protein Kinase Inhibitors.  

National Technical Information Service (NTIS)

Protein kinase inhibitors are disclosed having utility in the treatment of protein kinase-mediated diseases and conditions, such as cancer. The compounds of this invention have the following structure: 1 including steroisomers, prodrugs and pharmaceutical...

L. H. Hurley D. Mahadevan H. Han D. J. Bearss H. Vankayalapati

2004-01-01

305

Protein Kinase Inhibitors.  

National Technical Information Service (NTIS)

Protein kinase inhibitors are disclosed having utility in the treatment of protein kinase-mediated diseases and conditions, such as cancer. The compounds of this invention have the following structure: 1 including steroisomers, prodrugs and pharmaceutical...

L. H. Hurley D. Mahadevan H. Han D. J. Bearss H. Vankayalapati S. Bashyam

2005-01-01

306

Evolution of Creatine Kinase.  

National Technical Information Service (NTIS)

A comparison was made of the electrophoretic patterns of creatine kinase from different organs and from different species of frogs, fish and birds. A particularly interesting correlation was found between the brain creatine kinase patterns of the 26 bird ...

M. E. Eppenberger H. M. Eppenberger N. O. Kaplan

1967-01-01

307

Hypoxia-Induced Invadopodia Formation Involves Activation of NHE-1 by the p90 Ribosomal S6 Kinase (p90RSK)  

Microsoft Academic Search

The hypoxic and acidic microenvironments in tumors are strongly associated with malignant progression and metastasis, and have thus become a central issue in tumor physiology and cancer treatment. Despite this, the molecular links between acidic pH- and hypoxia-mediated cell invasion\\/metastasis remain mostly unresolved. One of the mechanisms that tumor cells use for tissue invasion is the generation of invadopodia, which

Fabrice Lucien; Karine Brochu-Gaudreau; Dominique Arsenault; Kelly Harper; Claire M. Dubois

2011-01-01

308

90-kDa ribosomal S6 kinase 1 is inhibited by S-glutathionylation of its active-site cysteine residue during oxidative stress.  

PubMed

Previously, we reported that p90-RSK1 phosphorylates neuronal nitric oxide synthase (nNOS) at Ser847 in cells treated with mitogens, leading to the inhibition of NOS activity. Here, we show RSK1 Cys223 glutathionylation limits the activity of the enzyme following an oxidative stimulus and attenuates the nNOS phosphorylation. Treatment of RSK1 with diamide/glutathione results in inactivation of the enzyme in vitro. Mutagenesis studies confirmed that S-glutathionylation of Cys223 is both necessary and sufficient for this inhibition of RSK1. In transfected cells expressing RSK1 and nNOS, treatment with diamide caused a decrease in EGF-induced phosphorylation of nNOS at Ser847. Cells expressing mutant RSK1 (C223S) proved resistant in this regard. Thus, RSK1 Cys223 glutathionylation may contribute to regulate the levels of NO in the brain. PMID:23624076

Takata, Tsuyoshi; Tsuchiya, Yukihiro; Watanabe, Yasuo

2013-04-25

309

The fruit juice of Morinda citrifolia (noni) downregulates HIF-1? protein expression through inhibition of PKB, ERK-1/2, JNK-1 and S6 in manganese-stimulated A549 human lung cancer cells.  

PubMed

High exposure of manganese is suggested to be a risk factor for many lung diseases. Evidence suggests anticancerous and antiangiogenic effects by products derived from Morinda citrifolia (noni) fruit. In this study, we investigated the effect of noni fruit juice (NFJ) on the expression of HIF-1?, a tumor angiogenic transcription factor in manganese-chloride (manganese)-stimulated A549 human lung carcinoma cells. Treatment with manganese largely induced expression of HIF-1? protein but did not affect HIF-1? mRNA expression in A549 cells, suggesting the metal-mediated co- and/or post-translational HIF-1? upregulation. Manganese treatment also led to increased phosphorylation of extracellular-regulated protein kinase-1/2 (ERK-1/2), c-Jun N-terminal kinase-1 (JNK-1), protein kinase B (PKB), S6 and eukaryotic translation initiation factor-2? (eIF-2?) in A549 cells. Of note, the exposure of NFJ inhibited the manganese-induced HIF-1? protein upregulation in a concentration-dependent manner. Importantly, as assessed by results of pharmacological inhibition and siRNA transfection studies, the effect of NFJ on HIF-1? protein downregulation seemed to be largely associated with the ability of NFJ to interfere with the metal's signaling to activate PKB, ERK-1/2, JNK-1 and S6 in A549 cells. It was further shown that NFJ could repress the induction of HIF-1? protein by desferoxamine or interleukin-1? (IL-1?), another HIF-1? inducer in A549 cells. Thus, the present study provides the first evidence that NFJ has the ability to strongly downregulate manganese-induced HIF-1? protein expression in A549 human lung cancer cells, which may suggest the NFJ-mediated beneficial effects on lung pathologies in which manganese and HIF-1? overexpression play pathogenic roles. PMID:22179285

Jang, Byeong-Churl

2011-12-14

310

Conserved herpesvirus protein kinases  

Microsoft Academic Search

Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine\\/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however, along with a conserved role, individual kinases may have unique functions in the context of

Edward Gershburg; Joseph S. Pagano

2008-01-01

311

THE THERMAL NEUTRON TEMPERATURE IN NRX SELF-SERVE POSITIONS S-3-5 AND S-6-3  

Microsoft Academic Search

A measurement of relative thermal neutron temperatures based on the ; determination of relative fission cross sections from measurements of fission ; product gamma activities is described. The results for two irradiation positions ; in the NRX reflector were 120 plus or minus 10 deg C for S-3-5 and 138 plus or ; minus 1O deg C for S-6-3. The

Bigham

1958-01-01

312

Role for the Plasmodium sporozoite-specific transmembrane protein S6 in parasite motility and efficient malaria transmission  

PubMed Central

Malaria transmission occurs by intradermal deposition of Plasmodium sporozoites during the infectious bite of a female Anopheles mosquito. After formation in midgut-associated oocysts sporozoites actively enter mosquito salivary glands and subsequently invade host hepatocytes where they transform into clinically silent liver stages. To date, two sporozoite-specific transmembrane proteins have been identified that perform vital functions in natural malaria transmission. The sporozoite invasin TRAP drives sporozoite motility and target cell entry whereas the adhesin MAEBL mediates sporozoite recognition of and attachment to salivary glands. Here, we demonstrate that the sporozoite-specific transmembrane protein S6 is required for efficient malaria transmission to the vertebrate host. Targeted deletion of S6 results in severe impairment of sporozoite gliding motility and invasion of mosquito salivary glands. During sporozoite maturation S6 expression is tightly regulated by transcriptional and translational control. We propose that S6 functions together with TRAP/MIC2 family invasins to direct fast, efficient and specific cell entry and, ultimately, life cycle progression of the malaria sporozoite.

Steinbuechel, Marion; Matuschewski, Kai

2009-01-01

313

Repression of GCN5 Histone Acetyltransferase Activity via Bromodomain-Mediated Binding and Phosphorylation by the Ku-DNA-Dependent Protein Kinase Complex  

Microsoft Academic Search

GCN5, a putative transcriptional adapter in humans and yeast, possesses histone acetyltransferase (HAT) activity which has been linked to GCN5's role in transcriptional activation in yeast. In this report, we dem- onstrate a functional interaction between human GCN5 (hGCN5) and the DNA-dependent protein kinase (DNA-PK) holoenzyme. Yeast two-hybrid screening detected an interaction between the bromodomain of hGCN5 and the p70

NICKOLAI A. BARLEV; VLADIMIR POLTORATSKY; TOM OWEN-HUGHES; CAROL YING; LIN LIU; JERRY L. WORKMAN; SHELLEY L. BERGER

314

Trpc1 ion channel modulates phosphatidylinositol 3-kinase/Akt pathway during myoblast differentiation and muscle regeneration.  

PubMed

We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1(-/-) and Trpc1(+/+) murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1(-/-) muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1(-/-) mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1(-/-) muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1(-/-) primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca(2+) or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca(2+) through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca(2+)-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration. PMID:22399301

Zanou, Nadège; Schakman, Olivier; Louis, Pierre; Ruegg, Urs T; Dietrich, Alexander; Birnbaumer, Lutz; Gailly, Philippe

2012-03-06

315

Trpc1 Ion Channel Modulates Phosphatidylinositol 3-Kinase/Akt Pathway during Myoblast Differentiation and Muscle Regeneration*  

PubMed Central

We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1?/? and Trpc1+/+ murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1?/? muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1?/? mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1?/? muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1?/? primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca2+ or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca2+ through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca2+-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.

Zanou, Nadege; Schakman, Olivier; Louis, Pierre; Ruegg, Urs T.; Dietrich, Alexander; Birnbaumer, Lutz; Gailly, Philippe

2012-01-01

316

Structural basis for activation of the autoinhibitory C-terminal kinase domain of p90 RSK2  

SciTech Connect

The X-ray structure at 2.0-{angstrom} resolution of the p90 ribosomal S6 kinase 2 C-terminal kinase domain revealed a C-terminal autoinhibitory {alpha}L-helix that was embedded in the kinase scaffold and determines the inactive kinase conformation. We suggest a mechanism of activation through displacement of the {alpha}L-helix and rearrangement of the conserved residue Glu500, as well as the reorganization of the T-loop into the active conformation.

Malakhova, Margarita; Tereshko, Valentina; Lee, Sung-Young; Yao, Ke; Cho, Yong-Yeon; Bode, Ann; Dong, Zigang (UMM); (UC)

2010-02-04

317

Metabolic Regulation by Leucine of Translation Initiation Through the mTOR-Signaling Pathway by Pancreatic  Cells  

Microsoft Academic Search

Recent findings have demonstrated that the branched- chain amino acid leucine can activate the translational regulators, phosphorylated heat- and acid-stable pro- tein regulated by insulin (PHAS-I) and p70 S6 kinase (p70 s6k ), in an insulin-independent and rapamycin-sen- sitive manner through mammalian target of rapamycin (mTOR), although the mechanism for this activation is undefined. It has been previously established that

Guang Xu; Guim Kwon; Wilhelm S. Cruz; Connie A. Marshall; Michael L. McDaniel

2001-01-01

318

Protein Kinases and Addiction  

PubMed Central

Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction.

Lee, Anna M.; Messing, Robert O.

2011-01-01

319

A new family of protein kinases— The mitochondrial protein kinases  

Microsoft Academic Search

Molecular cloning has provided evidence for a new family of protein kinases in eukaryotic cells. These kinases show no sequence similarity with other eukaryotic protein kinases, but are related by sequence to the histidine protein kinases found in prokaryotes. These protein kinases, responsible for phosphorylation and inactivation of the branched-chain ?-ketoacid dehydrogenase and pyruvate dehydrogenase complexes, are located exclusively in

Robert A. Harris; Kirill M. Popov; Yu Zhao; Natalia Y. Kedishvili; Yoshiharu Shimomura; David W. Crabb

1995-01-01

320

Rapid stimulation of Ser/Thr protein kinases following treatment of Swiss 3T3 cells with bombesin. Involvement of casein kinase-2 in the signaling pathway of bombesin.  

PubMed

Treatment of quiescent Swiss 3T3 mouse fibroblasts with bombesin resulted in a rapid 6-8-fold stimulation of cytosolic Ser/Thr kinase activities toward the S6 peptide (RRLSSLR), myelin basic protein (MBP), and the G peptide (SPQPSRRGSESSEE). Anion exchange Mono Q chromatography resolved multiple S6 peptide- and G peptide kinase activities and two MBP kinase peaks. Both MBP- and several S6 peptide kinase peaks could be inactivated by PCSL (PP2A2) phosphatase action. This indicates that the bombesin-induced activation of these enzymes is mediated by a Ser/Thr phosphorylation event. The S6 peptide kinases as well as the two MBP kinases stimulated in response to bombesin are similar to those activated by epidermal growth factor in Swiss 3T3 fibroblasts which suggests that the early events of the signal transduction pathway mediated by these growth factors in Swiss 3T3 cells may converge in the activation of common Ser/Thr kinases. Bombesin, which acts as a sole mitogen for Swiss 3T3 fibroblasts, also produced a several-fold increase in the kinase activity toward the RRREEESEEE peptide, a specific substrate for CK-2. This kinase activity was heparin-sensitive and also measurable with the G peptide (SPQPSRRGSESSEE) and GS-1 peptide (YRRAAVPPSPSPSLSRHSSPHQSEDEE), which contain consensus sequences for phosphorylation by CK-2. The bombesin-stimulated CK-2 activity could not be measured in whole cytosols but was revealed by the anion exchange chromatography step. The activation of CK-2 was not reversed by PCSL phosphatase action. The implication of CK-2 in the signal transduction pathway of bombesin is discussed. PMID:1577810

Agostinis, P; Van Lint, J; Sarno, S; De Witte, P; Vandenheede, J R; Merlevede, W

1992-05-15

321

Spatial Productivity Variations During Formation of Sapropels S5 and S6 in the Mediterranean Sea: Evidence from Ba Concentrations  

NASA Astrophysics Data System (ADS)

We investigated five time equivalent core sections from the Balearic Sea, the easternmost Levantine Basin and Southwest, South, and Southeast of Crete to reconstruct spatial patterns of productivity during deposition of sapropels S5 and S6 in the Mediterranean Sea (MS). We used accumulation rate of biogenic Ba to compute paleoproductivity. Maximum surface water productivity was found during deposition of S5 and pronounced spatial variability is evident. In the Balearic Sea, coeval sediment intervals show very little productivity change, suggesting that chemical and biological environments in the eastern and western MS were decoupled in the Pleistocene. We interpret the spatial variability as the result of two different modes of nutrient delivery to the photic zone: riverine derived nutrient input and shoaling of the pycnocline/nutricline to the photic zone. The productivity increase during the formation of S6 was moderate compared to S5 and had less marked spatial variability within the study area. Given that S6 has formed during a glacial interval, glacial boundary conditions such as high wind stress and/or cooler surface water temperatures apparently favoured lateral and vertical mixing and prevented the development of spatial gradients within the Eastern MS. A non-sapropel sediment interval with elevated Ba content and depleted oxygen isotope ratios in planktonic foraminifer calcite was detected between S6 and S5 and corresponds to the weak northern hemisphere insolation maximum at 150 kyr. At this time, productivity apparently increased up to 5 times over surrounding intervals, but benthic fauna show that the deep water remained oxic. In our interpretation, the interval denotes a failed sapropel, when an strong monsoon did not manage to push the Eastern MS into permanent stratification. The comparison of interglacial and glacial sapropels stresses the relevance of climatic boundary conditions in the northern catchment in determining the facies and spatial variability of sapropels within the Eastern MS.

Weldeab, S.; Schmiedl, G.; Hemleben, C.; Emeis, K.

2001-12-01

322

Characterization of Methanosarcina barkeri MST and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-76IT  

Microsoft Academic Search

Members of the genus Methanosarcina are recognized as major aceticlastic methanogens, and several species which thrive in low-salt, pH-neutral culture medium at mesophilic temperatures have been described. However, the environmental conditions which support the fastest growth of these species (Methanosarcina barkeri MST (T = type strain) and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-761T) have not been reported previously.

GLORIA M. MAESTROJUAN; DAVID R. BOONE

323

Moment tensor inversion of focal mechanism for the aftershock sequence of 1982 Lulong M S =6.1 earthquake  

Microsoft Academic Search

Using the moment tensor inversion method, we calculate the focal mechanisms of the aftershock sequence of the M\\u000a S=6.1 Lulong earthquake occurred on October 19, 1982 in Hebei Province. We found that the pressure axis in Lulong basin is\\u000a nearly in the east-west direction with an azimuth of N74E. However, in the north of the basin the stress axis changes

Wen-Jun Li; Pei-De Wang; Qi-Fu Chen

2006-01-01

324

MAP kinase and pain  

Microsoft Academic Search

Mitogen-activated protein kinases (MAPKs) are important for intracellular signal transduction and play critical roles in regulating neural plasticity and inflammatory responses. The MAPK family consists of three major members: extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK), which represent three separate signaling pathways. Accumulating evidence shows that all three MAPK pathways contribute to pain sensitization after tissue and

Ru-Rong Ji; Robert W. Gereau IV; Marzia Malcangio; Gary R. Strichartz

2009-01-01

325

mGluR-Dependent Long-Term Depression Is Associated with Increased Phosphorylation of S6 and Synthesis of Elongation Factor 1A but Remains Expressed in S6K-Deficient Mice  

Microsoft Academic Search

Metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) in the hippocampus re- quires rapid protein synthesis, which suggests that mGluR activation is coupled to signaling pathways that regulate translation. Herein, we have investigated the signaling pathways that couple group I mGluRs to ribosomal S6 protein phosphorylation and 5oligopyrimidine tract (5TOP)-encoded protein synthesis during mGluR-LTD. We found that mGluR-LTD was associated with increased

Marcia D. Antion; Lingfei Hou; Helen Wong; Charles A. Hoeffer; Eric Klann

2008-01-01

326

Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR  

Microsoft Academic Search

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) can activate multiple signaling pathways in parallel, including those involving Janus kinase (JAK)–signal transducers and activators of transcription (STATs) [1], mitogen-activated protein kinase (MAPK) [2], phosphatidylinositol 3-kinase (PI 3-kinase) and mammalian target of rapamycin (mTOR)–p70 S6 kinase [3]. Crosstalk occurs between these pathways, because studies have shown that STAT3 requires phosphorylation on

Kiyotaka Yokogami; Shinichiro Wakisaka; Joseph Avruch; Steven A. Reeves

2000-01-01

327

?-Caryophyllene oxide inhibits growth and induces apoptosis through the suppression of PI3K/AKT/mTOR/S6K1 pathways and ROS-mediated MAPKs activation.  

PubMed

Both PI3K/AKT/mTOR/S6K1 and mitogen activated protein kinase (MAPK) signaling cascades play an important role in cell proliferation, survival, angiogenesis, and metastasis of tumor cells. In the present report, we investigated the effects of ?-caryophyllene oxide (CPO), a sesquiterpene isolated from essential oils of medicinal plants such as guava (Psidium guajava), oregano (Origanum vulgare L.), cinnamon (Cinnamomum spp.) clove (Eugenia caryophyllata), and black pepper (Piper nigrum L.) on the PI3K/AKT/mTOR/S6K1 and MAPK activation pathways in human prostate and breast cancer cells. We found that CPO not only inhibited the constitutive activation of PI3K/AKT/mTOR/S6K1 signaling cascade; but also caused the activation of ERK, JNK, and p38 MAPK in tumor cells. CPO induced increased reactive oxygen species (ROS) generation from mitochondria, which is associated with the induction of apoptosis as characterized by positive Annexin V binding and TUNEL staining, loss of mitochondrial membrane potential, release of cytochrome c, activation of caspase-3, and cleavage of PARP. Inhibition of ROS generation by N-acetylcysteine (NAC) significantly prevented CPO-induced apoptosis. Subsequently, CPO also down-regulated the expression of various downstream gene products that mediate cell proliferation (cyclin D1), survival (bcl-2, bcl-xL, survivin, IAP-1, and IAP-2), metastasis (COX-2), angiogenesis (VEGF), and increased the expression of p53 and p21. Interestingly, we also observed that CPO can significantly potentiate the apoptotic effects of various pharmacological PI3K/AKT inhibitors when employed in combination in tumor cells. Overall, these findings suggest that CPO can interfere with multiple signaling cascades involved in tumorigenesis and used as a potential therapeutic candidate for both the prevention and treatment of cancer. PMID:21924548

Park, Kyung-Ran; Nam, Dongwoo; Yun, Hyung-Mun; Lee, Seok-Geun; Jang, Hyeung-Jin; Sethi, Gautam; Cho, Somi K; Ahn, Kwang Seok

2011-08-26

328

Phosphorylated Ndr kinase  

US Patent & Trademark Office Database

The present invention relates to the phosphorylated form of nuclear serine/threonine protein kinase, designated nuclear, Dbf2-related kinase (Ndr), and provides assays and materials for identifying modulators thereof. The invention relates to the fields of molecular biology, chemistry, pharmacology, and screening technology.

2011-08-30

329

Tec family kinases.  

PubMed

Tec family kinases (TFKs) are ancient kinases, vital for the development of both mammals and fruit flies. The first TFKs were described 20 years ago, and one of them, BTK, was found to be vital for B-lymphocyte development. However, during the last five years several seminal discoveries have been made, significantly advancing our understanding of these signal transducers. PMID:21518256

Yu, Liang; Smith, C I Edvard

2011-05-18

330

A 500-MSample\\/s, 6-bit Nyquist-rate ADC for disk-drive read-channel applications  

Microsoft Academic Search

The analog-to-digital conversion required in most disk-drive read-channel applications is designed for good dynamic and noise performance over a wide-input frequency range. This paper presents a 500-MSample\\/s, 6-bit analog to-digital converter (ADC) and its embedded implementation inside a disk-drive read channel, using a 0.35-?m CMOS double-poly (only one poly layer was used in the ADC), triple-metal process. The converter achieves

Iuri Mehr; Declan Dalton

1999-01-01

331

The natural killer cell receptor specific for HLA-A allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140-kD disulphide-linked dimer  

PubMed Central

Human natural killer (NK) cells express inhibitory receptors that are specific for different groups of HLA-C or HLA-B alleles. The majority of these receptors belong to the immunoglobulin (Ig) superfamily and are characterized by two or three extracellular Ig-like domains. Here we describe a novel inhibitory NK receptor that is specific for a group of HLA-A alleles. The HLA-A3-specific NK cell clone DP7 has been used for mice immunization. Two mAbs, termed Q66 and Q241, bound to the immunizing clone and stained only a subset of NK cell populations or clones. Among Q66 mAb-reactive clones, we further selected those that did not express any of the previously identified HLA-class I-specific NK receptors. These clones did not lyse HLA-A3+ (or -A11+) target cells, but lysis of these targets could be detected in the presence of Q66 or Q241 mAbs. On the other hand, target cells expressing other HLA- A alleles, including -A1, -A2, and -A24, were efficiently lysed. Moreover, none of the HLA-C or HLA-B alleles that were tested exerted a protective effect. Q66+, but not Q66- NK cell clones, expressed messenger RNA coding for a novel 3 Ig domain protein homologous to the HLA-C (p58) and HLA-B (p70) receptors. The corresponding cDNA (cl.1.1) was used to generate transient and stable transfectants in COS7 and NIH3T3 cell lines, respectively. Both types of transfectants were specifically stained by Q66 and Q241 mAbs. Since the cytoplasmic tail of Q66-reactive molecules was at least 11 amino acid longer than the other known p58/p70 molecules, we could generate an antiserum specific for the COOH-terminus of Q66-reactive molecules, termed PGP-3. PGP-3 immunoprecipitated, only from Q66+ NK cells, molecules displaying a molecular mass of 140 kD, under nonreducing conditions, which resolved, under reducing conditions, in a 70-kD band. Thus, differently from the other p58/p70 receptors, Q66-reactive molecules appear to be expressed as disulphide-linked dimers and were thus termed p140. The comparative analysis of the amino acid sequences of p58, p70, and p140 molecules revealed the existence of two cysteins proximal to the transmembrane region, only in the amino acid sequence of p140 molecules.

1996-01-01

332

Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma12  

PubMed Central

The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR) and MET are activated in subsets of mesothelioma, suggesting that these kinases might represent novel therapeutic targets in this notoriously chemotherapy-resistant cancer. However, clinical trials have shown little activity for EGFR inhibitors in mesothelioma. Despite the evidence for RTK activation in mesothelioma pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. Using phospho-RTK and immunoblot assays, we herein demonstrate activation of multiple RTKs (EGFR, MET, AXL, and ERBB3) in individual mesothelioma cell lines but not in normal mesothelioma cells. Inhibition of mesothelioma multi-RTK signaling was accomplished using combinations of RTK direct inhibitors or by inhibition of the RTK chaperone, heat shock protein 90 (HSP90). Multi-RTK inhibition by the HSP90 inhibitor 17-allyloamino-17-demethoxygeldanamycin (17-AAG) had a substantially greater effect on mesothelioma proliferation and survival compared with inhibition of individual activated RTKs. HSP90 inhibition also suppressed phosphorylation of downstream signaling intermediates (AKT, mitogen-activated protein kinase, and S6); upregulated the p53, p21, and p27 cell cycle checkpoints; induced G2 phase arrest; induced caspase 3/7 activity; and led to an increase in the sub-G1 apoptotic population. These compelling proapoptotic and antiproliferative responses indicate that HSP90 inhibition warrants clinical evaluation as a novel therapeutic strategy in mesothelioma.

Ou, Wen-Bin; Hubert, Christopher; Corson, Joseph M; Bueno, Raphael; Flynn, Daniel L; Sugarbaker, David J; Fletcher, Jonathan A

2011-01-01

333

Kinase Suppressor of Ras Is Ceramide-Activated Protein Kinase  

Microsoft Academic Search

A proline-directed serine\\/threonine ceramide-activated protein (CAP) kinase mediates transmembrane signaling through the sphingomyelin pathway. CAP kinase reportedly initiates proinflammatory TNF? action by phosphorylating and activating Raf-1. The present studies delineate kinase suppressor of Ras (KSR), identified genetically in Caenorhabditis elegans and Drosophila, as CAP kinase. Mouse KSR, like CAP kinase, renatures and autophosphorylates as a 100-kDa membrane-bound polypeptide. KSR overexpression

Yuhua Zhang; Bei Yao; Sylvie Delikat; Shariff Bayoumy; Xin-Hua Lin; Subham Basu; Michael McGinley; Po-Ying Chan-Hui; Henri Lichenstein; Richard Kolesnick

1997-01-01

334

Explanation of the EPR g factors for Co2+ impurities in trigonal Cd2P2S6 crystal  

NASA Astrophysics Data System (ADS)

By extending the theory of Abragam and Pryce, the formulas of EPR parameters g? and g? for 3d7 ion in trigonal octahedral crystals are established from a cluster approach. In these formulas, not only the configuration interaction effect, but also the covalency effect are considered and the parameters related to both effects and the trigonal distortion are determined from the optical spectra and the structural data of the crystal under study. Based on these formulas, the g? and g? of Cd2P2S6:Co2+ crystal are reasonably explained from its structural data. The relationship between the sign of /?g (=g?-g?) and the trigonal distortion (elongated or compressed) of ligand octahedron, which is opposite to that obtained in the previous paper, is given and the results are discussed.

Zheng, Wen-Chen; Wu, Shao-Yi

2001-12-01

335

Isotope Shifts of the ^1So (6s)^2 ? ^3P_1(6s6p) Transition in Ytterbium  

NASA Astrophysics Data System (ADS)

A laser beam modulated by either an acousto-optic or electro-optic modulator was used to excite the Yb ^1So (6s)^2 ? ^3P1 (6s6p) transition. Fluorescence was recorded as the laser frequency was scanned across the resonance. Each isotope generated multiple peaks in the spectrum separated by the modulation frequency which permitted the frequency scan to be calibrated. The resulting isotope shifts agree well with the very best data obtained using an interferometer to monitor the change in laser frequency. Optical modulators have the advantage of being relatively inexpensive and simpler to operate than interferometers whose length must be stabilized using a laser which is locked to an atomic transition, for accurate results.

Li, Jian; van Wijngaarden, William

1997-04-01

336

Heat capacity and electrical resistance of (PbySn1-y)2P2S6 chalcogenides  

NASA Astrophysics Data System (ADS)

We present the results of study of heat capacity and electrical resistance of the (PbySn1-y)2P2S6 chalcogenides where Pb content varies from 0 till 0.8. We have studied the temperature behaviour of heat capacity from the point of Pb influence on change of transition temperature at the temperature region 50-400 K Moreover, we present the results of study of temperature dependence of electrical resistance and influence of an applied magnetic field up to 3 T The electrical resistance was studied in the temperature range 50-400 K due to low temperature semiconducting behaviour. We studied the influence of Pb content on phase transition and relaxation processes.

Il'kovi?, S.; Reiffers, M.; Šebe?, V.; Šterbáková, K.; Burger, V.; Parma, L.; Chobal, O.; Rizak, I.

2012-12-01

337

Degradation of Si-Al aluminide coating after service of turbine blades made of ZhS6K superalloy  

NASA Astrophysics Data System (ADS)

Aero engine turbine blades made of nickel-based superalloys are characterized by very good mechanical properties, but their hot corrosion resistance is insufficient. Therefore, various protective coatings must be applied. These coatings are typically made of diffusive aluminide coatings based on the ?-NiAl intermetallic phase. Although the oxidation resistance and hot corrosion resistance of these coatings are very good, their thermal resistance is relatively poor. As a result, turbine blades with aluminide coatings are prone to degradation in case of overheating. In this paper we study the degradation of the Si-Al aluminide coating on turbine blades made of ZhS6K superalloy during overheating in the DV2 jet engine.

Chmiela, B.; Kianicová, M.; Soza?ska, M.; Swad?ba, L.

2012-05-01

338

Ab initio quantum chemical investigation of arsenic sulfide molecular diversity from As4S6 and As4  

NASA Astrophysics Data System (ADS)

The structural diversity of arsenic sulfide molecules in compositions between As4S6 and As4 was investigated using ab initio quantum chemical calculations. The As4S6 molecule consists of four trigonal pyramid coordinations of As atoms bonding to three S atoms. In the As4S5 composition, only one type of molecular configuration corresponds to an uzonite-type molecule. In the As4S4 composition, two molecular configurations exist with realgar-type and pararealgar-type molecules. Three molecular configurations are in the As4S3 composition. The first configuration comprises trigonal pyramidal As atom coordinations of two types: bonding to two S atoms and one As atom, and bonding to one S atom and two As atoms. The second is the molecular configuration of dimorphite. The third comprises trigonal pyramidal As atom coordinations of two types: bonding to three As atoms, and bonding to one As atom and two S atoms. The As4S2 composition allows molecular configurations of two types. One is comprised of trigonal pyramidal As atom configurations of one type bonding to two As atoms and one S atom. The other comprises trigonal pyramidal As atom coordinations of three types: bonding to two S atoms and one As atoms, bonding to one S atom and two As atoms, and bonding to three As atoms. The As4S molecule has trigonal pyramidal As atom coordinations of two types: bonding to one S atom and two As atoms, and bonding to three As atoms. The As4S composition permits only one molecular configuration, which suggests that the mineral duranusite comprises the As4S molecular geometry. In all, ten molecular configurations are predicted in the molecular hierarchy of the arsenic sulfide binary system. The simulated Raman spectral profiles are helpful in searching for undiscovered arsenic sulfide minerals.

Kyono, Atsushi

2013-10-01

339

[New kinase inhibitors].  

PubMed

Treatment of rheumatoid arthritis (RA) has dramatically changed during the last 15 years. A limited number of conventional disease-modifying antirheumatic drugs (DMARD) in combination with non-steroid anti-inflammatory drugs (NSAID) and corticosteroids are facing a variety of biologics that are increasingly being used. Because of the high costs of biologics as well as the necessity for subcutaneous or intravenous administration, there is currently a growing interest in new and potent oral compounds such as the small molecules. Inflammatory pathways and mechanisms in signal transduction have been characterized in detail. Instead of neutralizing the action of a proinflammatory cytokine by antagonizing its biologic effect by an antibody, these small molecules interfere with the intracellular pathways of the inflammatory cascade. Intracellular kinases are among these enzymes which are crucially involved in intracellular signal transduction. Kinase inhibitors have been successfully used within the last few years in the treatment of various hematological malignancies, such as imatinib in patients with chronic myeloid leukemia. More recently, the Janus kinase (JAK) inhibitor tofacitinib has been evaluated as a potential new treatment option in RA and is awaiting approval. While an overview about JAK inhibition will be given elsewhere, other inhibitors such as spleen tyrosine kinase (Syk) inhibitor, mitogen-activated protein kinase (MAPK) inhibitor and Bruton's tyrosine kinase (Btk) inhibitor are currently in preclinical and clinical development. PMID:22777068

Rubbert-Roth, A

2012-08-01

340

Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases.  

PubMed

The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (?, ?, ?, and ?), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries. PMID:21372320

Cargnello, Marie; Roux, Philippe P

2011-03-01

341

Identification of IRS-1 Ser-1101 as a target of S6K1 in nutrient- and obesity-induced insulin resistance  

PubMed Central

S6K1 has emerged as a critical signaling component in the development of insulin resistance through phosphorylation and inhibition of IRS-1 function. This effect can be triggered directly by nutrients such as amino acids or by insulin through a homeostatic negative-feedback loop. However, the role of S6K1 in mediating IRS-1 phosphorylation in a physiological setting of nutrient overload is unresolved. Here we show that S6K1 directly phosphorylates IRS-1 Ser-1101 in vitro in the C-terminal domain of the protein and that mutation of this site largely blocks the ability of amino acids to suppress IRS-1 tyrosine and Akt phosphorylation. Consistent with this finding, phosphorylation of IRS-1 Ser-1101 is increased in the liver of obese db/db and wild-type, but not S6K1?/?, mice maintained on a high-fat diet and is blocked by siRNA knockdown of S6K1 protein. Finally, infusion of amino acids in humans leads to the concomitant activation of S6K1, phosphorylation of IRS-1 Ser-1101, a reduction in IRS-1 function, and insulin resistance in skeletal muscle. These findings indicate that nutrient- and hormonal-dependent activation of S6K1 causes insulin resistance in mice and humans, in part, by mediating IRS-1 Ser-1101 phosphorylation.

Tremblay, Frederic; Brule, Sophie; Hee Um, Sung; Li, Yu; Masuda, Kohei; Roden, Michael; Sun, Xiao Jian; Krebs, Michael; Polakiewicz, Roberto D.; Thomas, George; Marette, Andre

2007-01-01

342

Identification of IRS-1 Ser-1101 as a target of S6K1 in nutrient- and obesity-induced insulin resistance.  

PubMed

S6K1 has emerged as a critical signaling component in the development of insulin resistance through phosphorylation and inhibition of IRS-1 function. This effect can be triggered directly by nutrients such as amino acids or by insulin through a homeostatic negative-feedback loop. However, the role of S6K1 in mediating IRS-1 phosphorylation in a physiological setting of nutrient overload is unresolved. Here we show that S6K1 directly phosphorylates IRS-1 Ser-1101 in vitro in the C-terminal domain of the protein and that mutation of this site largely blocks the ability of amino acids to suppress IRS-1 tyrosine and Akt phosphorylation. Consistent with this finding, phosphorylation of IRS-1 Ser-1101 is increased in the liver of obese db/db and wild-type, but not S6K1(-/-), mice maintained on a high-fat diet and is blocked by siRNA knockdown of S6K1 protein. Finally, infusion of amino acids in humans leads to the concomitant activation of S6K1, phosphorylation of IRS-1 Ser-1101, a reduction in IRS-1 function, and insulin resistance in skeletal muscle. These findings indicate that nutrient- and hormonal-dependent activation of S6K1 causes insulin resistance in mice and humans, in part, by mediating IRS-1 Ser-1101 phosphorylation. PMID:17709744

Tremblay, Frédéric; Brûlé, Sophie; Hee Um, Sung; Li, Yu; Masuda, Kohei; Roden, Michael; Sun, Xiao Jian; Krebs, Michael; Polakiewicz, Roberto D; Thomas, George; Marette, André

2007-08-20

343

Effects of an S6 strain of Mycoplasma gallisepticum challenge at onset of lay on digestive and reproductive tract characteristics in commercial layers.  

PubMed

Mycoplasma gallisepticum (MG), a reproductive/respiratory pathogen in poultry, has been implicated in suboptimum egg production and decreased hatchability. Commercial layer hens raised in a controlled environment were inoculated with the S6 strain of MG at 20 wk of age. The S6 inoculation had no effect on bird weight, egg production, digestive tract weight and length, or histopathologic lesion scores, although significant differences were noted in the lengths and weights of various portions of the reproductive tract. This study shows that S6MG inoculation does not detrimentally affect layer hen performance when in the absence of environmental stressors customary to a caged layer facility. PMID:12713163

Parker, T A; Branton, S L; Jones, M S; Peebles, E D; Gerard, P D; Willeford, K O; Pharr, G T; Maslin, W R

344

PI(3) kinase is associated with a mechanism of immunoresistance in breast and prostate cancer  

PubMed Central

Immune escape describes a critical event whereby tumor cells adopt an immunoresistant phenotype to escape adaptive surveillance. We show that expression of a pivotal negative regulator of T-cell function, B7-H1, correlates with PI(3) kinase activation in breast and prostate cancer patients. B7-H1-mediated immunoresistance can be attenuated by inhibitors of the PI(3) kinase pathway, and is dependent on S6K1-mediated translational regulation of B7-H1 protein. Breast and prostate carcinoma cells with activated PI(3) kinase lose the immunoresistant phenotype after treatment with B7-H1 siRNA. Conversely, breast and prostate carcinoma cells with minimal PI(3) kinase activation adopt an immunoresistant phenotype when engineered to overexpress B7-H1 protein. These observations describe a mechanism for immune escape from tumor dormancy in humans that relates to oncogenesis.

Crane, CA; Panner, A; Murray, JC; Wilson, SP; Xu, H; Chen, L; Simko, JP; Waldman, FM; Pieper, RO; Parsa, AT

2013-01-01

345

Orphan kinases turn eccentric  

PubMed Central

PCTAIRE kinases (PCTK) are a highly conserved, but poorly characterized, subgroup of cyclin-dependent kinases (CDK). They are characterized by a conserved catalytic domain flanked by N- and C-terminal extensions that are involved in cyclin binding. Vertebrate genomes contain three highly similar PCTAIRE kinases (PCTK1,2,3, a.k.a., CDK16,17,18), which are most abundant in post-mitotic cells in brain and testis. Consistent with this restricted expression pattern, PCTK1 (CDK16) has recently been shown to be essential for spermatogenesis. PCTAIREs are activated by cyclin Y (CCNY), a highly conserved single cyclin fold protein. By binding to N-myristoylated CCNY, CDK16 is targeted to the plasma membrane. Unlike conventional cyclin-CDK interactions, binding of CCNY to CDK16 not only requires the catalytic domain, but also domains within the N-terminal extension. Interestingly, phosphorylation within this domain blocks CCNY binding, providing a novel means of cyclin-CDK regulation. By using these functional characteristics, we analyzed “PCTAIRE” sequence containing protein kinase genes in genomes of various organisms and found that CCNY and CCNY-dependent kinases are restricted to eumetazoa and possibly evolved along with development of a central nervous system. Here, we focus on the structure and regulation of PCTAIREs and discuss their established functions.

Mikolcevic, Petra; Rainer, Johannes; Geley, Stephan

2012-01-01

346

Conserved herpesvirus protein kinases  

PubMed Central

Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task.

Gershburg, Edward; Pagano, Joseph S.

2008-01-01

347

The Janus kinases (Jaks).  

PubMed

The Janus kinase (Jak) family is one of ten recognized families of non-receptor tyrosine kinases. Mammals have four members of this family, Jak1, Jak2, Jak3 and Tyrosine kinase 2 (Tyk2). Birds, fish and insects also have Jaks. Each protein has a kinase domain and a catalytically inactive pseudo-kinase domain, and they each bind cytokine receptors through amino-terminal FERM (Band-4.1, ezrin, radixin, moesin) domains. Upon binding of cytokines to their receptors, Jaks are activated and phosphorylate the receptors, creating docking sites for signaling molecules, especially members of the signal transducer and activator of transcription (Stat) family. Mutations of the Drosophila Jak (Hopscotch) have revealed developmental defects, and constitutive activation of Jaks in flies and humans is associated with leukemia-like syndromes. Through the generation of Jak-deficient cell lines and gene-targeted mice, the essential, nonredundant functions of Jaks in cytokine signaling have been established. Importantly, deficiency of Jak3 is the basis of human autosomal recessive severe combined immunodeficiency (SCID); accordingly, a selective Jak3 inhibitor has been developed, forming a new class of immunosuppressive drugs. PMID:15575979

Yamaoka, Kunihiro; Saharinen, Pipsa; Pesu, Marko; Holt, Vance E T; Silvennoinen, Olli; O'Shea, John J

2004-11-30

348

The Janus kinases (Jaks)  

PubMed Central

The Janus kinase (Jak) family is one of ten recognized families of non-receptor tyrosine kinases. Mammals have four members of this family, Jak1, Jak2, Jak3 and Tyrosine kinase 2 (Tyk2). Birds, fish and insects also have Jaks. Each protein has a kinase domain and a catalytically inactive pseudo-kinase domain, and they each bind cytokine receptors through amino-terminal FERM (Band-4.1, ezrin, radixin, moesin) domains. Upon binding of cytokines to their receptors, Jaks are activated and phosphorylate the receptors, creating docking sites for signaling molecules, especially members of the signal transducer and activator of transcription (Stat) family. Mutations of the Drosophila Jak (Hopscotch) have revealed developmental defects, and constitutive activation of Jaks in flies and humans is associated with leukemia-like syndromes. Through the generation of Jak-deficient cell lines and gene-targeted mice, the essential, nonredundant functions of Jaks in cytokine signaling have been established. Importantly, deficiency of Jak3 is the basis of human autosomal recessive severe combined immunodeficiency (SCID); accordingly, a selective Jak3 inhibitor has been developed, forming a new class of immunosuppressive drugs.

Yamaoka, Kunihiro; Saharinen, Pipsa; Pesu, Marko; Holt, Vance ET; Silvennoinen, Olli; O'Shea, John J

2004-01-01

349

Turnover of the Active Fraction of IRS1 Involves Raptor-mTOR- and S6K1Dependent Serine Phosphorylation in Cell Culture Models of Tuberous Sclerosis  

Microsoft Academic Search

The TSC1-TSC2\\/Rheb\\/Raptor-mTOR\\/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for

O. Jameel Shah; Tony Hunter

2006-01-01

350

Behavioural differences between C57BL/6 and 129S6/SvEv strains are reinforced by environmental enrichment.  

PubMed

Housing in enriched environment has been advocated as a means for controlled variation of environmental conditions in transgenic studies to explore interactions between genes and surroundings. In the present study, behavioural phenotypes of C57Bl/6 (B6) and 129S6/SvEv (129) mice, housed in either standard laboratory conditions or environmentally enriched conditions, were explored. Housing in enriched conditions increased exploratory activity in the plus-maze and reduced habituation in the locomotor activity test in B6 mice, whereas in 129 mice increased hot plate latencies and reduced aggression were observed. Compared to B6, 129 strain displayed lower exploratory activity in the plus-maze and locomotor activity test, longer hot plate latencies, spent more time immobile in the forced swim test and engaged more in social interaction. These behavioural differences between the two strains were reproducible independent of pre-experimental housing conditions. Moreover, environmental enrichment accentuated dissimilarities between the strains in the plus-maze, locomotor activity, hot plate and forced swim test. By contrast, strain differences in anxiety-like behaviours in the plus-maze test and in aggressive encounters in the resident-intruder test were not reproducible in mice housed in alternative environmental conditions, suggesting a strong contribution of environmental factors to the development of these phenotypes. It is concluded that the application of environmental enrichment in addition to standard housing conditions is a meaningful approach for testing reproducibility of behavioural findings within one laboratory. PMID:18687379

Abramov, Urho; Puussaar, Triinu; Raud, Sirli; Kurrikoff, Kaido; Vasar, Eero

2008-08-05

351

Dissociative stereoisomerization of persulfuranes via 10-S-5 persulfonium salts. Geometrical isomers of octahedral 12-S-6 species  

SciTech Connect

The reaction of bromine trifluoride with sulfurane gives rise to the first reported isolation of geometrical isomers of 12-S-6 sulfur, the trans- and cis-difluorodialkoxydiarylpersulfuranes. The cis isomer is 1.6 +/- 0.1 kcal/mol more favored in free energy than the all-trans isomer in methylene chloride solution at 25/sup 0/C. Calorimetry showed trans-to-cis isomerization to be exothermic by 2.0 +/- 0.5 kcal/mol. The rapid, acid-catalyzed isomerization of trans isomer to give cis isomer occurs by a dissociative mechanism through a pentacoordinate cationic sulfur intermediate, a 10-S-5 persulfonium ion, which was isolated at its hexafluorophosphate salt. A lower limit for the activation barrier of the unobserved nondissociative isomerization of trans to cis is 50 kcal/mol in quinoline solution. Polarization and other effects on the relative bond lengths and energies of the isomers are discussed. The results of complete X-ray structure determinations for trans and cis are described together with a PMO argument rationalizing the observed order of bond lengths.

Michalak, R.S.; Martin, J.C.

1982-01-01

352

PAK family kinases  

PubMed Central

The p21-activated kinases (PAKs) are a family of Ser/Thr protein kinases that are represented by six genes in humans (PAK 1–6), and are found in all eukaryotes sequenced to date. Genetic and knockdown experiments in frogs, fish and mice indicate group I PAKs are widely expressed, required for multiple tissue development, and particularly important for immune and nervous system function in the adult. The group II PAKs (human PAKs 4–6) are more enigmatic, but their restriction to metazoans and presence at cell-cell junctions suggests these kinases emerged to regulate junctional signaling. Studies of protozoa and fungal PAKs show that they regulate cell shape and polarity through phosphorylation of multiple cytoskeletal proteins, including microtubule binding proteins, myosins and septins. This chapter discusses what we know about the regulation of PAKs and their physiological role in different model organisms, based primarily on gene knockout studies.

Zhao, Zhuo-shen; Manser, Ed

2012-01-01

353

Phosphatidylinositol kinase from rabbit reticulocytes  

SciTech Connect

Phosphatidylinositol (PI) kinase was isolated from the postribosomal supernatant of rabbit reticulocytes. This activity was identified by the formation of a product that comigrated with phosphatidylinositol-4-phosphate (PIP) when purified PI was phosphorylated in the presence of (/sup 32/P)ATP and Mg/sup 2 +/. Three major peaks of PI kinase activity were resolved by chromatography on DEAE-cellulose. The first peak eluted at 50-100 mM NaCl together with several serine protein kinases, casein kinase (CK) I and protease activated kinase (PAK) I and II. The PI kinase was subsequently separated from the protein kinases by chromatography on phosphocellulose. The second peak eluted at 125-160 mM NaCl and contained another lipid kinase activity that produced a product which comigrated with phosphatidic acid on thin layer chromatography. The third peak, which eluted at 165-200 mM NaCl, partly comigrated with casein kinase (CK) II and an active protein kinase(s) which phosphorylated mixed histone and histone I. CK II and the histone kinase activities were also separated by chromatography on phosphocelluslose. The different forms of PI kinase were characterized and compared with respect to substrate and salt requirements.

Tuazon, P.T.; Heng, A.B.W.; Traugh, J.A.

1986-05-01

354

Evolution of herpesvirus thymidine kinases from cellular deoxycytidine kinase  

Microsoft Academic Search

The thymidine kinases encoded by herpesviruses of higher vertebrates form a distinct group and are unrelated to the thymidine kinases (TKs) of other organisms. Their evolutionary source has not been identified, but our analysis has revealed a clear relationship with a sequence of human deoxycytidine kinase (dCK) published recently. We report the sequence of the putative TK of channel catfish

Patrick T. Harrison; Russell Thompson; Andrew J. Davison

1991-01-01

355

The Exquisite Regulation of PLD2 by a Wealth of Interacting Proteins: S6K, Grb2, Sos, WASp and Rac2 (and a Surprise Discovery: PLD2 Is a GEF)  

PubMed Central

PLD catalyzes the conversion of the membrane phospholipid phosphatidylcholine to choline and phosphatidic acid (PA). PLD's mission in the cell is two-fold: phospholipid turnover with maintenance of the structural integrity of cellular/intracellular membranes and cell signaling through PA and its metabolites. Precisely, through its product of the reaction, PA, PLD has been implicated in a variety of physiological cellular functions, such as intracellular protein trafficking, cytoskeletal dynamics, chemotaxis of leukocytes and cell proliferation. The catalytic (HKD) and regulatory (PH and PX) domains were studied in detail in the PLD1 isoform, but PLD2 was traditionally studied in lesser detail and much less was known about its regulation. Our laboratory has been focusing on the study of PLD2 regulation in mammalian cells. Over the past few years, we have reported, in regards to the catalytic action of PLD, that PA is a chemoattractant agent that binds to and signals inside the cell through the ribosomal S6 kinases (S6K). Regarding the regulatory domains of PLD2, we have reported the discovery of the PLD2 interaction with Grb2 via Y169 in the PX domain, and further association to Sos, which results in an increase of de novo DNA synthesis and an interaction (also with Grb2) via the adjacent residue Y179, leading to the regulation of cell ruffling, chemotaxis and phagocytosis of leukocytes. We also review the complex regulation by tyrosine phosphorylation by epidermal growth factor receptor (EGF-R), Janus Kinase 3 (JAK3) and Src and the role of phosphatases. Recently, there is evidence supporting a new level of regulation of PLD2 at the PH domain, by the discovery of CRIB domains and a Rac2-PLD2 interaction that leads to a dual (positive and negative) effect on its enzymatic activity. Lastly, we review the surprising finding of PLD2 acting as a GEF. A phospholipase such as PLD that exists already in the cell membrane that acts directly on Rac allows a quick response of the cell without intermediary signaling molecules. This provides only the latest level of PLD2 regulation in a field that promises newer and exciting advances in the next few years.

Gomez-Cambronero, Julian

2011-01-01

356

Cyclophilin represents a novel class of protein kinases  

SciTech Connect

Cyclophilin (CyP, Mr 17,737, pI 9.6), a highly specific cytosolic receptor for cyclosporin A (CsA) has ser/thr protein kinase activity. Incorporation of /sup 32/P into bovine histone H/sub 3/ (BH/sub 3/) was catalyzed by major and minor CyP isozymes at the same rate. Salt effects were biphasic with optimal kinase activity between 50-100 mM Na/sup +/ or K/sup +/. Kinase activity was maximal at 37/sup 0/C, stable for 5 min at 45/sup 0/, labile at 56/sup 0/, optimal between pH 6.8 and 8.0 and had an apparent Km of 20 uM ATP with both isozymes. The specific activity of CyP was 1.0 nmole P/mg protein/min with chicken histone H/sub 1/ (CH/sub 1/), 0.2 nmoles P/mg prot/min with BH/sub 3/ and less than 0.01 nmoles P/mg prot/min with synapsin, casein, phosvitin, and ribosomal protein S6. Cofactors including Mn/sup + +/, Zn/sup + +/, Ca/sup + +/, phosphatidyl serine, diolein and phorbol ester, cAMP, cGMP and Ca/sup + +/ did not affect basal CyP kinase activity. CsA (<200 ng/ml) inhibited phosphorylation of CH/sub 3/ by 50% but did not inhibit phosphorylation of other histones; 2ug CsA/ml was required to cause 50% inhibition of cAMP and Ca/sup + +//CaM dependent kinases. A non-immunosuppressive analog (Me-leu-11-CsA) that does not bind to CyP did not inhibit CH/sub 3/ phosphorylation. Thus, CyP is a novel protein kinase that mediates immunosuppression by CsA.

Harding, M.W.; Gorelick, F.S.; Handschumacher, R.E.

1987-05-01

357

Molecular pharmacology of phosphatidylinositol 3-kinase inhibition in human glioma.  

PubMed

Gliomas are primary brain tumors with poor prognosis that exhibit frequent abnormalities in phosphatidylinositol 3-kinase (PI3 kinase) signaling. We investigated the molecular mechanism of action of the isoform-selective class I PI3 kinase and mTOR inhibitor PI-103 in human glioma cells. The potent inhibitory effects of PI-103 on the PI3 kinase pathway were quantified. PI-103 and the mTOR inhibitor rapamycin both inhibited ribosomal protein S6 phosphorylation but there were clear differences in the response of upstream components of the PI3 kinase pathway, such as phosphorylation of Thr(308)-AKT, that were inhibited by PI-103 but not rapamycin. Gene expression profiling identified altered expression of genes encoding regulators of the cell cycle and cholesterol metabolism, and genes modulated by insulin or IGF1 signaling, rapamycin treatment or nutrient starvation. PI-103 decreased expression of positive regulators of G(1)/S phase progression and increased expression of the negative cell cycle regulator p27(kip1). A reversible PI-103-mediated G(1) cell cycle arrest occurred without significant apoptosis, consistent with the altered gene expression detected. PI-103 induced vacuolation and processing of LC-3i to LC-3ii, which are features of an autophagic response. In contrast to PI-103, LY294002 and PI-387 induced apoptosis, indicative of likely off-target effects. PI-103 interacted synergistically or additively with cytotoxic agents used in the treatment of glioma, namely vincristine, BCNU and temozolomide. Compared to individual treatments, the combination of PI-103 with temozolomide significantly improved the response of U87MG human glioma xenografts. Our results support the therapeutic potential for PI3 kinase inhibitors with a PI-103-like profile as therapeutic agents for the treatment of glioma. PMID:19177002

Guillard, Sandrine; Clarke, Paul A; Te Poele, Robert; Mohri, Zahra; Bjerke, Lynn; Valenti, Melanie; Raynaud, Florence; Eccles, Suzanne A; Workman, Paul

2009-02-16

358

A Novel Function of eIF2? Kinases as Inducers of the Phosphoinositide-3 Kinase Signaling Pathway  

PubMed Central

Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the ? subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2? kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR?/? or PERK?/? mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2? kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2? phosphorylation as demonstrated by the inactivation of endogenous eIF2? by small interfering RNA or utilization of MEFs bearing the eIF2? Ser51Ala mutation. Our data reveal a novel property of eIF2? kinases as activators of PI3K signaling and cell survival.

Kazemi, Shirin; Mounir, Zineb; Baltzis, Dionissios; Raven, Jennifer F.; Wang, Shuo; Krishnamoorthy, Jothi-Latha; Pluquet, Olivier; Pelletier, Jerry

2007-01-01

359

Expression studies on four members of the pMGA multigene family in Mycoplasma gallisepticum S6.  

PubMed

A large family of related genes known as pMGA exists in the avian pathogen Mycoplasma gallisepticum but only a single member of this family was previously found to be expressed in one strain of this bacterium. In this work two unrelated strains of M. gallisepticum were also shown by amino-terminal sequencing to express a unique pMGA polypeptide in both cases. To investigate pMGA gene selection in M. gallisepticum, mRNA expression was analysed in M. gallisepticum strain 56 using reverse transcription-PCR (RT-PCR) and Northern blot techniques with probes for several members of the pMGA multigene family. It was shown that the pMGA message is 2.2 kb in size and is monocistronic. RT-PCR detected four different pMGA mRNA molecules but their relative yields were significantly affected by magnesium concentration. By quantitative Northern analysis, the relative abundances of the four pMGA mRNAs in M. gallisepticum S6 total RNA was determined: the pMGA1.1 mRNA predominated [1.88 ng (micrograms total RNA)-1] but at least three other pMGA genes were found to be transcribed but at much lower levels (20 to 40-fold lower). The pMGA1.1 mRNA is expressed at a level five times higher than the tuf gene, known to be one of the most abundantly expressed proteins in the prokaryotic cell. The start point of transcription for pMGA1.1 was determined and probable promoter assigned. From these data it appears likely that transcriptional control plays a major role in the selection of pMGA gene expression in the M. gallisepticum cell. PMID:8535528

Glew, M D; Markham, P F; Browning, G F; Walker, I D

1995-11-01

360

The S4-S5 Linker of KCNQ1 Channels Forms a Structural Scaffold with the S6 Segment Controlling Gate Closure*  

PubMed Central

In vivo, KCNQ1 ?-subunits associate with the ?-subunit KCNE1 to generate the slowly activating cardiac potassium current (IKs). Structurally, they share their topology with other Kv channels and consist out of six transmembrane helices (S1–S6) with the S1–S4 segments forming the voltage-sensing domain (VSD). The opening or closure of the intracellular channel gate, which localizes at the bottom of the S6 segment, is directly controlled by the movement of the VSD via an electromechanical coupling. In other Kv channels, this electromechanical coupling is realized by an interaction between the S4-S5 linker (S4S5L) and the C-terminal end of S6 (S6T). Previously we reported that substitutions for Leu353 in S6T resulted in channels that failed to close completely. Closure could be incomplete because Leu353 itself is the pore-occluding residue of the channel gate or because of a distorted electromechanical coupling. To resolve this and to address the role of S4S5L in KCNQ1 channel gating, we performed an alanine/tryptophan substitution scan of S4S5L. The residues with a “high impact” on channel gating (when mutated) clustered on one side of the S4S5L ?-helix. Hence, this side of S4S5L most likely contributes to the electromechanical coupling and finds its residue counterparts in S6T. Accordingly, substitutions for Val254 resulted in channels that were partially constitutively open and the ability to close completely was rescued by combination with substitutions for Leu353 in S6T. Double mutant cycle analysis supported this cross-talk indicating that both residues come in close contact and stabilize the closed state of the channel.

Labro, Alain J.; Boulet, Inge R.; Choveau, Frank S.; Mayeur, Evy; Bruyns, Tine; Loussouarn, Gildas; Raes, Adam L.; Snyders, Dirk J.

2011-01-01

361

Sarafotoxin 6c (S6c) reduces infarct size and preserves mRNA for the ETB receptor in the ischemic/reperfused myocardium of anesthetized rats.  

PubMed

The aims of this study were to determine if the ETB receptor agonist, sarafotoxin 6c (S6c) reduces myocardial infarct size following myocardial ischemia and reperfusion and to investigate whether any changes in mRNA for endothelin receptors in the injured myocardium were modified by S6c pretreatment. Hypnorm/Hypnovel anesthetized rats were subjected to occlusion of the left main coronary artery for 30 minutes, followed by 120 minutes reperfusion. Animals were administered a bolus dose of S6c (0.24 nmol kg-1 i.v., n = 10) or saline (n = 15) 5 minutes prior to occlusion. At the end of reperfusion, hearts were stained with Evan's Blue dye to delineate area at risk. A 1.5- to 2.0-mm thick slice was cut transmurally 1 mm below the site of ligation for assessment of infarct size by triphenyltetrazolium chloride. A further transmural slice (2.5-3-mm thick) was cut for assessment of receptor mRNA levels by RTPCR. Administration of S6c caused a transient fall in mean arterial blood pressure (MABP) prior to occlusion and attenuated the fall in MABP induced by coronary occlusion. S6c significantly reduced infarct size (13 +/- 4% of area of slice at risk) compared with control hearts (35 +/- 5%; P < 0.05). In control hearts, there was a marked reduction in mRNA content for both ETA (50% reduction) and ETB (70% reduction) receptors in the ischemic zone, compared with non-ischemic tissue. In hearts pre-treated with S6c there was a reduction in ETA, but not ETB receptor mRNA in the ischemic zone. This study has shown that S6c reduces myocardial infarct size and results in preservation of ETB receptor mRNA in ischemic/reperfused tissue. PMID:15243294

Crockett, Thomas R; Gray, Gillian A; Kane, Kathleen A; Wainwright, Cherry L

2004-08-01

362

Pyruvate kinase isozymes in man  

Microsoft Academic Search

Anti human M2 type and anti human L type pyruvate kinase sera allowed us to distinguish two groups of pyruvate kinase in man. Erythrocyte and liver (L type) enzymes on the one hand were inhibited by anti L and not at all by anti M2 serum; pyruvate kinase from all the other tissues on the other hand were inhibited by

Joelle Marie; Axel Kahn; Pierre Boivin

1976-01-01

363

The Ca 2 \\/Calmodulin-dependent Protein Kinase Kinases Are AMP-activated Protein Kinase Kinases* ? S  

Microsoft Academic Search

The AMP-activated protein kinase (AMPK) is an im- portant regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells,

Rebecca L. Hurley; Kristin A. Anderson; Jeanne M. Franzone; Bruce E. Kemp; Anthony R. Means; Lee A. Witters

2005-01-01

364

Cyclin-Dependent Kinases  

Microsoft Academic Search

Cyclin-dependent kinases (CDKs) have been recognized as key regulators of cell cycle progression. Alteration and deregulation of CDK activity are pathogenic hallmarks of neoplasia. Therefore, inhibitors or modulators would be of interest to explore as novel therapeutic agents in cancer, as well as other hyperproliferative disorders. Flavopiridol is a semisynthetic flavonoid that emerged from an empirical screening program as a

Edward A. Sausville; Daniel Zaharevitz; Robert Gussio; Laurent Meijer; Maryse Louarn-Leost; Conrad Kunick; Robert Schultz; Tyler Lahusen; Donna Headlee; Sherman Stinson; Susan G. Arbuckand; Adrian Senderowicz

1999-01-01

365

Plant 5-Methylthioribose Kinase  

PubMed Central

Activity of 5-methylthioribose kinase, the enzyme which catalyzes the ATP-dependent formation of 1-phospho-5-methylthioribose, has been revealed in the extracts from various higher plant species. Almost 2,000-fold-purified enzyme has been obtained from yellow lupin (Lupinus luteus L. cv Topaz) seed extract. Molecular weight of the native enzyme is 70,000 as judged by gel filtration. The lupin 5-methylthioribose kinase exhibits a strict requirement for divalent metal ions. Among the ions tested, only Mg2+ and Mn2+ acted as cofactors. The curve of kinase initial velocity versus pH reaches plateau at pH 10 to 10.5. The Km values calculated for 5-methylthioribose and ATP are 4.3 and 8.3 micromolar, respectively. Among nucleoside triphosphates tested as potential phosphate donors, only dATP could substitute in the reaction for ATP. 5-Isobutylthioribose, an analog of 5-methylthioribose, proved to be the ?-ATP-phosphate acceptor, too. The compound inhibits competitively synthesis of 1-phospho-5-methylthioribose (Ki = 1.4 micromolar). Lupin 5-methylthioribose kinase is completely and irreversibly inhibited by the antisulfhydryl reagent, p-hydroxymercuribenzoate. As in bacteria (Ferro, Barrett, Shapiro 1978 J Biol Chem 253: 6021-6025), the enzyme may be involved in a new, alternative pathway of methionine synthesis in plant tissues.

Guranowski, Andrzej

1983-01-01

366

Oncoprotein protein kinase  

DOEpatents

An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK.

Karin, Michael (San Diego, CA); Hibi, Masahiko (San Diego, CA); Linn, Anning (La Jolla, CA)

1996-01-01

367

Phosphatidylinositol 3-kinase-gamma Activates Bruton's Tyrosine Kinase in Concert with Src Family Kinases  

Microsoft Academic Search

Bruton's tyrosine kinase (Btk) is essential for normal B lymphocyte development and function. The activity of Btk is partially regulated by transphosphorylation within its kinase domain by Src family kinases at residue Tyr-551 and subsequent autophosphorylation at Tyr-223. Activation correlates with Btk association with cellular membranes. Based on specific loss of function mutations, the Btk pleckstrin homology (PH) domain plays

Zuomei Li; Matthew I. Wahl; Alicia Eguinoa; Leona