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1

Phosphoinositide 3-Kinase Dependent Inhibition as a Broad Basis for Opponent Coding in Mammalian Olfactory Receptor Neurons  

PubMed Central

Phosphoinositide 3-kinase (PI3K) signaling has been implicated in mediating inhibitory odorant input to mammalian olfactory receptor neurons (ORNs). To better understand the breadth of such inhibition in odor coding, we screened a panel of odorants representing different chemical classes, as well as odorants known to occur in a natural odor object (tomato), for their ability to rapidly activate PI3K-dependent inhibitory signaling. Odorants were screened on dissociated native rat ORNs before and after pre-incubation with the PI3K-isoform specific blockers AS252424 and TGX221. Many different odorants increased their excitatory strength for particular ORNs following PI3K blockade in a manner consistent with activating PI3K-dependent inhibitory signaling in those cells. The PI3K-dependent inhibitory odorants overlapped with conventional excitatory odorants, but did not share the same bias, indicating partial partitioning of the odor space. Finding that PI3K-dependent inhibition can be activated by a wide range of otherwise conventional excitatory odorants strongly implies PI3K-dependent inhibition provides a broad basis for opponent coding in mammalian ORNs. PMID:23585911

Ukhanov, Kirill; Corey, Elizabeth A.; Ache, Barry W.

2013-01-01

2

Phosphoinositide 3-kinase-dependent phosphorylation of the dual adaptor for phosphotyrosine and 3-phosphoinositides by the Src family of tyrosine kinase.  

PubMed

We recently identified a novel adaptor protein, termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), that possesses a Src homology (SH2) domain and a pleckstrin homology (PH) domain. DAPP1 exhibits a high-affinity interaction with PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2), which bind to the PH domain. In the present study we show that when DAPP1 is expressed in HEK-293 cells, the agonists insulin, insulin-like growth factor-1 and epidermal growth factor induce the phosphorylation of DAPP1 at Tyr(139). Treatment of cells with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or expression of a dominant-negative PI 3-kinase prevent phosphorylation of DAPP1 at Tyr(139), and a PH-domain mutant of DAPP1, which does not interact with PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2), is not phosphorylated at Tyr(139) following agonist stimulation of cells. Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPP1 in unstimulated cells. We demonstrated that Tyr(139) of DAPP1 is likely to be phosphorylated in vivo by a Src-family tyrosine kinase, since the specific Src-family inhibitor, PP2, but not an inactive variant of this drug, PP3, prevented the agonist-induced tyrosine phosphorylation of DAPP1. Src, Lyn and Lck tyrosine kinases phosphorylate DAPP1 at Tyr(139) in vitro at similar rates in the presence or absence of PtdIns(3,4,5)P(3), and overexpression of these kinases in HEK-293 cells induces the phosphorylation of Tyr(139). These findings indicate that, following activation of PI 3-kinases, PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2) bind to DAPP1, recruiting it to the plasma membrane where it becomes phosphorylated at Tyr(139) by a Src-family tyrosine kinase. PMID:10880360

Dowler, S; Montalvo, L; Cantrell, D; Morrice, N; Alessi, D R

2000-07-15

3

Exendin-4 increases insulin sensitivity via a PI-3-kinase-dependent mechanism: contrasting effects of GLP-1.  

PubMed

The insulinotropic agent, exendin-4, is a long-acting analogue of glucagon-like peptide-1 (GLP-1) which improves glucose tolerance in humans and animals with diabetes, but the underlying mechanisms and the effects of exendin-4 on peripheral (muscle/fat) insulin action are unclear. Previous in vivo and clinical studies have been difficult to interpret because of complex, simultaneous changes in insulin and glucagon levels and possible effects on hepatic metabolism. Thus, the comparative effects of exendin-4 and GLP-1 on insulin-stimulated 2-[3H]deoxyglucose (2-DOG) uptake were measured in fully differentiated L6 myotubes and 3T3-adipocytes, including co-incubation with inhibitors of the PI-3-kinase (wortmannin) and mitogen-activated protein (MAP) kinase (PD098059) pathways. In L6 myotubes, there was a concentration-dependent and PI-3-kinase-dependent increase in insulin-stimulated 2-DOG uptake with exendin-4 and GLP-1, e.g. for exendin-4 the C(I-200) value (concentration of insulin required to increase 2-DOG uptake 2-fold) decreased from 1.3 +/- 1.4 x 10(-7)M (insulin alone, n=16) to 5.9 +/- 1.3 x 10(-8)M (insulin+exendin-4 0.1nM, n=18, P<0.03). A similar insulin-sensitizing effect was observed with exendin-4 in 3T3-adipocytes, but GLP-1 had no effect on adipocyte insulin sensitivity. In conclusion, this is the first direct evidence showing that exendin-4 increases insulin-stimulated glucose uptake in muscle and fat derived cells via a pathway that involves PI-3-kinase activation. Furthermore, the contrasting responses of exendin and GLP-1 in 3T3-adipocytes suggest that the peripheral insulin-sensitizing effect of exendin-4 (in contrast to the insulinotropic effect) does not involve the GLP-1 receptor pathway. PMID:11911852

Idris, Iskandar; Patiag, Divina; Gray, Samuel; Donnelly, Richard

2002-03-01

4

Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.  

PubMed Central

Insulin plays a key role in regulating a wide range of cellular processes. However, until recently little was known about the signalling pathways that are involved in linking the insulin receptor with downstream responses. It is now apparent that the activation of class 1a phosphoinositide 3-kinase (PI 3-kinase) is necessary and in some cases sufficient to elicit many of insulin's effects on glucose and lipid metabolism. The lipid products of PI 3-kinase act as both membrane anchors and allosteric regulators, serving to localize and activate downstream enzymes and their protein substrates. One of the major ways these lipid products of PI 3-kinase act in insulin signalling is by binding to pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB) and in the process regulating the phosphorylation of PKB by PDK. Using mechanisms such as this, PI 3-kinase is able to act as a molecular switch to regulate the activity of serine/threonine-specific kinase cascades important in mediating insulin's effects on endpoint responses. PMID:9677303

Shepherd, P R; Withers, D J; Siddle, K

1998-01-01

5

The distinct roles of Ras and Rac in PI 3-kinase-dependent protrusion during EGF-stimulated cell migration  

PubMed Central

Summary Cell migration involves the localized extension of actin-rich protrusions, a process that requires Class I phosphoinositide 3-kinases (PI 3-kinases). Both Rac and Ras have been shown to regulate actin polymerization and activate PI 3-kinase. However, the coordination of Rac, Ras and PI 3-kinase activation during epidermal growth factor (EGF)-stimulated protrusion has not been analyzed. We examined PI 3-kinase-dependent protrusion in MTLn3 rat adenocarcinoma cells. EGF-stimulated phosphatidyl-inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] levels showed a rapid and persistent response, as PI 3-kinase activity remained elevated up to 3 minutes. The activation kinetics of Ras, but not Rac, coincided with those of leading-edge PtdIns(3,4,5)P3 production. Small interfering RNA (siRNA) knockdown of K-Ras but not Rac1 abolished PtdIns(3,4,5)P3 production at the leading edge and inhibited EGF-stimulated protrusion. However, Rac1 knockdown did inhibit cell migration, because of the inhibition of focal adhesion formation in Rac1 siRNA-treated cells. Our data show that in EGF-stimulated MTLn3 carcinoma cells, Ras is required for both PtdIns(3,4,5)P3 production and lamellipod extension, whereas Rac1 is required for formation of adhesive structures. These data suggest an unappreciated role for Ras during protrusion, and a crucial role for Rac in the stabilization of protrusions required for cell motility. PMID:17698922

Yip, Shu-Chin; El-Sibai, Mirvat; Coniglio, Salvatore J.; Mouneimne, Ghassan; Eddy, Robert J.; Drees, Beth E.; Neilsen, Paul O.; Goswami, Sumanta; Symons, Marc; Condeelis, John S.; Backer, Jonathan M.

2014-01-01

6

Phosphoinositide and Inositolpolyphosphate Signalling in Defense Responses of Arabidopsis thaliana Challenged by Mechanical Wounding  

Microsoft Academic Search

Various biochemical signals are implicated in Arabidopsis wound signalling, including jasmonic acid (JA), sal- icylic acid, auxin, and Ca21. Here, we report on cross-talk of phytohormones with phosphoinositide signals not previously implicated in plant wound responses. Within 30 min of mechanical wounding of Arabidopsis rosette-leaves, the levels of the lipid-derived soluble inositolpolyphosphate, inositol 1,4,5-trisphosphate (InsP3), increased four to five-fold. Concom-

Alina Mosblech; Sabine Konig; Irene Stenzel; Peter Grzeganek; Ivo Feussner; Ingo Heilmann

2008-01-01

7

Phosphoinositide 3-Kinase C2? Regulates Cytoskeletal Organization and Cell Migration via Rac-dependent Mechanisms  

PubMed Central

Receptor-linked class I phosphoinositide 3-kinases (PI3Ks) induce assembly of signal transduction complexes through protein–protein and protein–lipid interactions that mediate cell proliferation, survival, and migration. Although class II PI3Ks have the potential to make the same phosphoinositides as class I PI3Ks, their precise cellular role is currently unclear. In this report, we demonstrate that class II phosphoinositide 3-kinase C2? (PI3KC2?) associates with the Eps8/Abi1/Sos1 complex and is recruited to the EGF receptor as part of a multiprotein signaling complex also involving Shc and Grb2. Increased expression of PI3KC2? stimulated Rac activity in A-431 epidermoid carcinoma cells, resulting in enhanced membrane ruffling and migration speed of the cells. Conversely, expression of dominant negative PI3KC2? reduced Rac activity, membrane ruffling, and cell migration. Moreover, PI3KC2?-overexpressing cells were protected from anoikis and displayed enhanced proliferation, independently of Rac function. Taken together, these findings suggest that PI3KC2? regulates the migration and survival of human tumor cells by distinct molecular mechanisms. PMID:16775008

Katso, Roy M.; Pardo, Olivier E.; Palamidessi, Andrea; Franz, Clemens M.; Marinov, Marin; De Laurentiis, Angela; Downward, Julian; Scita, Giorgio; Ridley, Anne J.; Waterfield, Michael D.

2006-01-01

8

Cholesterol stabilizes fluid phosphoinositide domains.  

PubMed

Local accumulation of phosphoinositides (PIPs) is an important factor for a broad range of cellular events including membrane trafficking and cell signaling. The negatively charged phosphoinositide headgroups can interact with cations or cationic proteins and this electrostatic interaction has been identified as the main phosphoinositide clustering mechanism. However, an increasing number of reports show that phosphoinositide-mediated signaling events are at least in some cases cholesterol dependent, suggesting other possible contributors to the segregation of phosphoinositides. Using fluorescence microscopy on giant unilamellar vesicles and monolayers at the air/water interface, we present data showing that cholesterol stabilizes fluid phosphoinositide-enriched phases. The interaction with cholesterol is observed for all investigated phosphoinositides (PI(4)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3) as well as phosphatidylinositol. We find that cholesterol is present in the phosphoinositide-enriched phase and that the resulting phase is fluid. Cholesterol derivatives modified at the hydroxyl group (cholestenone, cholesteryl ethyl ether) do not promote formation of phosphoinositide domains, suggesting an instrumental role of the cholesterol hydroxyl group in the observed cholesterol/phosphoinositide interaction. This leads to the hypothesis that cholesterol participates in an intermolecular hydrogen bond network formed among the phosphoinositide lipids. We had previously reported that the intra- and intermolecular hydrogen bond network between the phosphoinositide lipids leads to a reduction of the charge density at the phosphoinositide phosphomonoester groups (Kooijman et al., 2009). We believe that cholesterol acts as a spacer between the phosphoinositide lipids, thereby reducing the electrostatic repulsion, while participating in the hydrogen bond network, leading to its further stabilization. To illustrate the effect of phosphoinositide segregation on protein binding, we show that binding of the tumor suppressor protein PTEN to PI(5)P and PI(4,5)P2 is enhanced in the presence of cholesterol. These results provide new insights into how phosphoinositides mediate important cellular events. PMID:24556334

Jiang, Zhiping; Redfern, Roberta E; Isler, Yasmin; Ross, Alonzo H; Gericke, Arne

2014-09-01

9

Phosphoinositide phosphatases in cell biology and disease.  

PubMed

Phosphoinositides are essential signaling molecules linked to a diverse array of cellular processes in eukaryotic cells. The metabolic interconversions of these phospholipids are subject to exquisite spatial and temporal regulation executed by arrays of phosphatidylinositol (PtdIns) and phosphoinositide-metabolizing enzymes. These include PtdIns- and phosphoinositide-kinases that drive phosphoinositide synthesis, and phospholipases and phosphatases that regulate phosphoinositide degradation. In the past decade, phosphoinositide phosphatases have emerged as topics of particular interest. This interest is driven by the recent appreciation that these enzymes represent primary mechanisms for phosphoinositide degradation, and because of their ever-increasing connections with human diseases. Herein, we review the biochemical properties of six major phosphoinositide phosphatases, the functional involvements of these enzymes in regulating phosphoinositide metabolism, the pathologies that arise from functional derangements of individual phosphatases, and recent ideas concerning the involvements of phosphoinositide phosphatases in membrane traffic control. PMID:20043944

Liu, Yang; Bankaitis, Vytas A

2010-07-01

10

TLR-induced activation of neutrophils promotes histamine production via a PI3 kinase dependent mechanism  

PubMed Central

Histamine is a bioactive amine that exerts immunomodulatory functions, including many allergic symptoms. It is preformed and stored in mast cells and basophils but recent evidence suggests that other cell types produce histamine in an inducible fashion. During infection, it has been suggested that neutrophils may produce histamine. We also observed that histamine is released in a neutrophil-mediated LPS-induced model of acute lung injury. Therefore, we sought to examine whether innate signals promote histamine production by neutrophils. Bone marrow-derived neutrophils stimulated with a range of TLR agonists secreted histamine in response to LPS or R837, suggesting TLR4 or TLR7 are important. LPS-driven histamine was enhanced by coculture with GM-CSF and led to a transient release of histamine that peaked at 8 hours post stimulation. This was dependent upon de novo synthesis of histamine, since cells derived from histidine decarboxylase (HDC) deficient mice were unable to produce histamine but did generate reactive oxygen species upon stimulation. Using pharmacological inhibitors, we show that histamine production requires PI3 kinase, which has been shown to regulate other neutrophil functions, including activation and selective granule release. However, unlike mast cells, HDC deficiency did not alter the granule structure of neutrophils, suggesting that histamine does not participate in granule integrity in these cells. Consequently, our findings establish that neutrophils generate histamine in response to a select panel of innate immune triggers and that this might contribute to acute lung injury responses. PMID:21903133

Smuda, Craig; Wechsler, Joshua B; Bryce, Paul J

2011-01-01

11

Small GTPases and phosphoinositides in the regulatory mechanisms of macropinosome formation and maturation  

PubMed Central

Macropinosome formation requires the sequential activation of numerous signaling pathways that coordinate the actin-driven formation of plasma membrane protrusions (ruffles) and circular ruffles (macropinocytic cups), followed by the closure of these macropinocytic cups into macropinosomes. In the process of macropinosome formation, localized productions of phosphoinositides such as PI(4,5)P2 and PI(3,4,5)P3 spatiotemporally orchestrate actin polymerization and rearrangement through recruiting and activating a variety of actin-associated proteins. In addition, the sequential activation of small GTPases, which are known to be master regulators of the actin cytoskeleton, plays a pivotal role in parallel with phosphoinositides. To complete macropinosome formation, phosphoinositide breakdown and Rho GTPase deactivation must occur in appropriate timings. After the nascent macropinosomes are formed, phosphoinositides and several Rab GTPases control macropinosome maturation by regulating vesicle trafficking and membrane fusion. In this review, we summarize recent advances in our understanding of the critical functions of phosphoinositide metabolism and small GTPases in association with their downstream effectors in macropinocytosis. PMID:25324782

Egami, Youhei; Taguchi, Tomohiko; Maekawa, Masashi; Arai, Hiroyuki; Araki, Nobukazu

2014-01-01

12

Phosphoinositides as Regulators of Protein-Chromatin Interactions  

NSDL National Science Digital Library

The molecular function of phospholipids in the nucleus has been only partially elucidated. The upsurge of epigenetic research has contributed to increased interest in nuclear phospholipids, such as phosphoinositides, and their involvement in gene transcription. However, the mechanisms by which phosphoinositides regulate transcription is still unknown at the molecular level. Certain phosphoinositide species can regulate protein-chromatin and protein–nucleic acid interactions, and specific nuclear target proteins link nuclear signaling lipids to gene expression. We propose that a phosphoinositide-mediated detachment of proteins from chromatin is a general biological mechanism that partly underlies the signaling effects of nuclear phosphoinositides.

Keijo Viiri (School of Medicine and Tampere University Hospital;University of Tampere REV); Markku Maki (School of Medicine and Tampere University Hospital;University of Tampere REV); Olli Lohi (School of Medicine and Tampere University Hospital;University of Tampere REV)

2012-05-01

13

Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism.  

PubMed Central

In response to growth factors, mTOR (mammalian target of rapamycin) has been identified as a central component of the signalling pathways that control the translational machinery and cell growth. Signalling through mTOR has also been shown to be necessary for the mechanical load-induced growth of cardiac and skeletal muscles. Although the mechanisms involved for mechanically induced activation of mTOR are not known, it has been suggested that activation of PI3K (phosphoinositide 3-kinase) and protein kinase B (Akt), via the release of locally acting growth factors, underlies this process. In the present study, we show that mechanically stimulating (passive stretch) the skeletal muscle ex vivo results in the activation of mTOR-dependent signalling events. The activation of mTOR-dependent signalling events was necessary for an increase in translational efficiency, demonstrating the physiological significance of this pathway. Using pharmacological inhibitors, we show that activation of mTOR-dependent signalling occurs through a PI3K-independent pathway. Consistent with these results, mechanically induced signalling through mTOR was not disrupted in muscles from Akt1-/- mice. In addition, ex vivo co-incubation experiments, along with in vitro conditioned-media experiments, demonstrate that a mechanically induced release of locally acting autocrine/paracrine growth factors was not sufficient for the activation of the mTOR pathway. Taken together, our results demonstrate that mechanical stimuli can activate the mTOR pathway independent of PI3K/Akt1 and locally acting growth factors. Thus mechanical stimuli and growth factors provide distinct inputs through which mTOR co-ordinates an increase in the translational efficiency. PMID:15030312

Hornberger, Troy A; Stuppard, Rudy; Conley, Kevin E; Fedele, Mark J; Fiorotto, Marta L; Chin, Eva R; Esser, Karyn A

2004-01-01

14

Targeting the phosphoinositide 3-kinase pathway in hematologic malignancies  

PubMed Central

The phosphoinositide 3-kinase pathway represents an important anticancer target because it has been implicated in cancer cell growth, survival, and motility. Recent studies show that PI3K may also play a role in the development of resistance to currently available therapies. In a broad range of cancers, various components of the phosphoinositide 3-kinase signaling axis are genetically modified, and the pathway can be activated through many different mechanisms. The frequency of genetic alterations in the phosphoinositide 3-kinase pathway, coupled with the impact in oncogenesis and disease progression, make this signaling axis an attractive target in anticancer therapy. A better understanding of the critical function of the phosphoinositide 3-kinase pathway in leukemias and lymphomas has led to the clinical evaluation of novel rationally designed inhibitors in this setting. Three main categories of phosphoinositide 3-kinase inhibitors have been developed so far: agents that target phosphoinositide 3-kinase and mammalian target of rapamycin (dual inhibitors), pan-phosphoinositide 3-kinase inhibitors that target all class I isoforms, and isoform-specific inhibitors that selectively target the ?, -?, -?, or -? isoforms. Emerging data highlight the promise of phosphoinositide 3-kinase inhibitors in combination with other therapies for the treatment of patients with hematologic malignancies. Further evaluation of phosphoinositide 3-kinase inhibitors in first-line or subsequent regimens may improve clinical outcomes. This article reviews the role of phosphoinositide 3-kinase signaling in hematologic malignancies and the potential clinical utility of inhibitors that target this pathway. PMID:24425689

Jabbour, Elias; Ottmann, Oliver G.; Deininger, Michael; Hochhaus, Andreas

2014-01-01

15

Channelopathies linked to plasma membrane phosphoinositides  

PubMed Central

The plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) controls the activity of most ion channels tested thus far through direct electrostatic interactions. Mutations in channel proteins that change their apparent affinity to PIP2 can lead to channelopathies. Given the fundamental role that membrane phosphoinositides play in regulating channel activity, it is surprising that only a small number of channelopathies have been linked to phosphoinositides. This review proposes that for channels whose activity is PIP2-dependent and for which mutations can lead to channelopathies, the possibility that the mutations alter channel-PIP2 interactions ought to be tested. Similarly, diseases that are linked to disorders of the phosphoinositide pathway result in altered PIP2 levels. In such cases, it is proposed that the possibility for a concomitant dysregulation of channel activity also ought to be tested. The ever-growing list of ion channels whose activity depends on interactions with PIP2 promises to provide a mechanism by which defects on either the channel protein or the phosphoinositide levels can lead to disease. PMID:20396900

Logothetis, Diomedes E.; Petrou, Vasileios I.; Adney, Scott K.; Mahajan, Rahul

2014-01-01

16

Early activation of mTORC1 signalling in response to mechanical overload is independent of phosphoinositide 3-kinase/Akt signalling  

PubMed Central

Abstract The mammalian target of rapamycin complex 1 (mTORC1) functions as a central integrator of a wide range of signals that modulate protein metabolism and cell growth. However, the contributions of individual pathways regulating mTORC1 activity in skeletal muscle are poorly defined. The purpose of this study was to determine the regulatory mechanisms that contribute to mTORC1 activation during mechanical overload-induced skeletal muscle hypertrophy. Consistent with previous studies, mechanical overload induced progressive hypertrophy of the plantaris muscle which was associated with significant increases in total RNA content and protein metabolism. mTORC1 was activated after a single day of overload as indicated by a significant increase in S6K1 phosphorylation at T389 and T421/S424. In contrast, Akt activity, as assessed by Akt phosphorylation status (T308 and S473), phosphorylation of direct downstream targets (glycogen synthase kinase 3 ?, proline-rich Akt substrate 40 kDa and tuberous sclerosis 2 (TSC2)) and a kinase assay, was not significantly increased until 2–3 days of overload. Inhibition of phosphoinositide 3-kinase (PI3K) activity by wortmannin was sufficient to block insulin-dependent signalling but did not prevent the early activation of mTORC1 in response to overload. We identified that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent pathway was activated at day 1 after overload. In addition, a target of MEK/ERK signalling, phosphorylation of TSC2 at S664, was also increased at this early time point. These observations demonstrate that in vivo, mTORC1 activation at the early phase of mechanical overload in skeletal muscle occurs independently of PI3K/Akt signalling and provide evidence that the MEK/ERK pathway may contribute to mTORC1 activation through phosphorylation of TSC2. PMID:21300751

Miyazaki, Mitsunori; McCarthy, John J; Fedele, Mark J; Esser, Karyn A

2011-01-01

17

TGF?-Induced PI 3 Kinase-Dependent Mnk-1 Activation is Necessary for Ser-209 Phosphorylation of eIF4E and Mesangial Cell Hypertrophy  

PubMed Central

Transforming growth factor? (TGF?)-induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGF? signaling remains largely unexplored. TGF? time-dependently stimulated eIF4E phosphorylation at Ser-209 concomitant with enhanced phosphorylation of Erk1/2 (extracellular signal regulated kinase1/2) and MEK (mitogen-activated and extracellular signal-regulated kinase kinase) in mesangial cells. Inhibition of Erk1/2 by MEK inhibitor or by expression of dominant negative Erk2 blocked eIF4E phosphorylation, resulting in attenuation of TGF?-induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active (CA) MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGF?. Pharmacological or dominant negative inhibition of phosphatidylinositol (PI) 3 kinase decreased MEK/Erk1/2 phosphorylation leading to suppression of eIF4E phosphorylation. Inducible phosphorylation of eIF4E at Ser-209 is mediated by Mnk-1 (mitogen-activated protein kinase signal-integrating kinase-1). Both PI 3 kinase and Erk1/2 promoted phosphorylation of Mnk-1 in response to TGF?. Dominant negative Mnk-1 significantly inhibited TGF?-stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E-4EBP-1 complex, decreased TGF?-stimulated phosphorylation of eIF4E without any effect on Mnk-1 phosphorylation. Furthermore, mutant eIF4E S209D, which mimics phosphorylated eIF4E, promoted protein synthesis and hypertrophy similar to TGF?. These results were confirmed using phosphorylation deficient mutant of eIF4E. Together our results highlight a significant role of dissociation of 4EBP-1-eIF4E complex for Mnk-1-mediated phosphorylation of eIF4E. Moreover, we conclude that TGF?-induced noncanonical signaling circuit involving PI 3 kinase-dependent Mnk-1-mediated phosphorylation of eIF4E at Ser-209 is required to facilitate mesangial cell hypertrophy. PMID:23359369

DAS, FALGUNI; GHOSH-CHOUDHURY, NANDINI; BERA, AMIT; KASINATH, BALAKUNTALAM S.; CHOUDHURY, GOUTAM GHOSH

2013-01-01

18

Phosphoinositides alter lipid bilayer properties.  

PubMed

Phosphatidylinositol-4,5-bisphosphate (PIP2), which constitutes ?1% of the plasma membrane phospholipid, plays a key role in membrane-delimited signaling. PIP2 regulates structurally and functionally diverse membrane proteins, including voltage- and ligand-gated ion channels, inwardly rectifying ion channels, transporters, and receptors. In some cases, the regulation is known to involve specific lipid-protein interactions, but the mechanisms by which PIP2 regulates many of its various targets remain to be fully elucidated. Because many PIP2 targets are membrane-spanning proteins, we explored whether the phosphoinositides might alter bilayer physical properties such as curvature and elasticity, which would alter the equilibrium between membrane protein conformational states-and thereby protein function. Taking advantage of the gramicidin A (gA) channels' sensitivity to changes in lipid bilayer properties, we used gA-based fluorescence quenching and single-channel assays to examine the effects of long-chain PIP2s (brain PIP2, which is predominantly 1-stearyl-2-arachidonyl-PIP2, and dioleoyl-PIP2) on bilayer properties. When premixed with dioleoyl-phosphocholine at 2 mol %, both long-chain PIP2s produced similar changes in gA channel function (bilayer properties); when applied through the aqueous solution, however, brain PIP2 was a more potent modifier than dioleoyl-PIP2. Given the widespread use of short-chain dioctanoyl-phosphoinositides, we also examined the effects of diC8-phosphoinositol (PI), PI(4,5)P2, PI(3,5)P2, PI(3,4)P2, and PI(3,4,5)P3. The diC8 phosphoinositides, except for PI(3,5)P2, altered bilayer properties with potencies that decreased with increasing head group charge. Nonphosphoinositide diC8 phospholipids generally were more potent bilayer modifiers than the polyphosphoinositides. These results show that physiological increases or decreases in plasma membrane PIP2 levels, as a result of activation of PI kinases or phosphatases, are likely to alter lipid bilayer properties, in addition to any other effects they may have. The results further show that exogenous PIP2, as well as structural analogues that differ in acyl chain length or phosphorylation state, alters lipid bilayer properties at the concentrations used in many cell physiological experiments. PMID:23712549

Rusinova, Radda; Hobart, E Ashley; Koeppe, Roger E; Andersen, Olaf S

2013-06-01

19

Phosphoinositides alter lipid bilayer properties  

PubMed Central

Phosphatidylinositol-4,5-bisphosphate (PIP2), which constitutes ?1% of the plasma membrane phospholipid, plays a key role in membrane-delimited signaling. PIP2 regulates structurally and functionally diverse membrane proteins, including voltage- and ligand-gated ion channels, inwardly rectifying ion channels, transporters, and receptors. In some cases, the regulation is known to involve specific lipid–protein interactions, but the mechanisms by which PIP2 regulates many of its various targets remain to be fully elucidated. Because many PIP2 targets are membrane-spanning proteins, we explored whether the phosphoinositides might alter bilayer physical properties such as curvature and elasticity, which would alter the equilibrium between membrane protein conformational states—and thereby protein function. Taking advantage of the gramicidin A (gA) channels’ sensitivity to changes in lipid bilayer properties, we used gA-based fluorescence quenching and single-channel assays to examine the effects of long-chain PIP2s (brain PIP2, which is predominantly 1-stearyl-2-arachidonyl-PIP2, and dioleoyl-PIP2) on bilayer properties. When premixed with dioleoyl-phosphocholine at 2 mol %, both long-chain PIP2s produced similar changes in gA channel function (bilayer properties); when applied through the aqueous solution, however, brain PIP2 was a more potent modifier than dioleoyl-PIP2. Given the widespread use of short-chain dioctanoyl-phosphoinositides, we also examined the effects of diC8-phosphoinositol (PI), PI(4,5)P2, PI(3,5)P2, PI(3,4)P2, and PI(3,4,5)P3. The diC8 phosphoinositides, except for PI(3,5)P2, altered bilayer properties with potencies that decreased with increasing head group charge. Nonphosphoinositide diC8 phospholipids generally were more potent bilayer modifiers than the polyphosphoinositides. These results show that physiological increases or decreases in plasma membrane PIP2 levels, as a result of activation of PI kinases or phosphatases, are likely to alter lipid bilayer properties, in addition to any other effects they may have. The results further show that exogenous PIP2, as well as structural analogues that differ in acyl chain length or phosphorylation state, alters lipid bilayer properties at the concentrations used in many cell physiological experiments. PMID:23712549

Hobart, E. Ashley; Koeppe, Roger E.; Andersen, Olaf S.

2013-01-01

20

Phosphoinositides and vesicular membrane traffic  

PubMed Central

Phosphoinositide lipids were initially discovered as precursors for specific second messengers involved in signal transduction, but have now taken the center stage in controlling many essential processes at virtually every cellular membrane. In particular, phosphoinositides play a critical role in regulating membrane dynamics and vesicular transport. The unique distribution of certain phosphoinositides at specific intracellular membranes makes these molecules uniquely suited to direct organelle-specific trafficking reactions. In this regulatory role, phosphoinositides cooperate specifically with small GTPases from the Arf and Rab families. This review will summarize recent progress in the study of phosphoinositides in membrane trafficking and organellar organization and highlight the particular relevance of these signaling pathways in disease. PMID:22281700

Mayinger, Peter

2012-01-01

21

Phosphoinositide signalling in Drosophila.  

PubMed

Phosphoinositides (PtdInsPs) are lipids that mediate a range of conserved cellular processes in eukaryotes. These include the transduction of ligand binding to cell surface receptors, vesicular transport and cytoskeletal function. The nature and functions of PtdInsPs were initially elucidated through biochemical experiments in mammalian cells. However, over the years, genetic and cell biological analysis in a range of model organisms including S. cerevisiae, D. melanogaster and C. elegans have contributed to an understanding of the involvement of PtdInsPs in these cellular events. The fruit fly Drosophila is an excellent genetic model for the analysis of cell and developmental biology as well as physiological processes, particularly analysis of the complex relationship between the cell types of a metazoan in mediating animal physiology. PtdInsP signalling pathways are underpinned by enzymes that synthesise and degrade these molecules and also by proteins that bind to these lipids in cells. In this review we provide an overview of the current understanding of PtdInsP signalling in Drosophila. We provide a comparative genomic analysis of the PtdInsP signalling toolkit between Drosophila and mammalian systems. We also review some areas of cell and developmental biology where analysis in Drosophila might provide insights into the role of this lipid-signalling pathway in metazoan biology. This article is part of a Special Issue entitled Phosphoinositides. PMID:25449646

Balakrishnan, Sruthi S; Basu, Urbashi; Raghu, Padinjat

2014-10-30

22

Class III phosphoinositide 3-kinase--Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes.  

PubMed

Increased proteolysis contributes to muscle atrophy that prevails in many diseases. Elucidating the signalling pathways responsible for this activation is of obvious clinical importance. Autophagy is a ubiquitous degradation process, induced by amino acid starvation, that delivers cytoplasmic components to lysosomes. Starvation markedly stimulates autophagy in myotubes, and the present studies investigate the mechanisms of this regulation. In C(2)C(12) myotubes incubated with serum growth factors, amino acid starvation stimulated autophagic proteolysis independently of p38 and p42/p44 mitogen-activated protein kinases, but in a PI3K (phosphoinositide 3-kinase)-dependent manner. Starvation, however, did not alter activities of class I and class II PI3Ks, and was not sufficient to affect major signalling proteins downstream from class I PI3K (glycogen synthase kinase, Akt/protein kinase B and protein S6). In contrast, starvation increased class III PI3K activity in whole-myotube extracts. In fact, this increase was most pronounced for a population of class III PI3K that coimmunoprecipitated with Beclin1/Apg6 protein, a major determinant in the initiation of autophagy. Stimulation of proteolysis was reproduced by feeding myotubes with synthetic dipalmitoyl-PtdIns3 P, the class III PI3K product. Conversely, protein transfection of anti-class III PI3K inhibitory antibody into starved myotubes inverted the induction of proteolysis. Therefore, independently of class I PI3K/Akt, protein S6 and mitogen-activated protein kinase pathways, amino acid starvation stimulates proteolysis in myotubes by regulating class III PI3K-Beclin1 autophagic complexes. PMID:12967324

Tassa, Amina; Roux, Marie Paule; Attaix, Didier; Bechet, Daniel M

2003-12-15

23

Class III phosphoinositide 3-kinase--Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes.  

PubMed Central

Increased proteolysis contributes to muscle atrophy that prevails in many diseases. Elucidating the signalling pathways responsible for this activation is of obvious clinical importance. Autophagy is a ubiquitous degradation process, induced by amino acid starvation, that delivers cytoplasmic components to lysosomes. Starvation markedly stimulates autophagy in myotubes, and the present studies investigate the mechanisms of this regulation. In C(2)C(12) myotubes incubated with serum growth factors, amino acid starvation stimulated autophagic proteolysis independently of p38 and p42/p44 mitogen-activated protein kinases, but in a PI3K (phosphoinositide 3-kinase)-dependent manner. Starvation, however, did not alter activities of class I and class II PI3Ks, and was not sufficient to affect major signalling proteins downstream from class I PI3K (glycogen synthase kinase, Akt/protein kinase B and protein S6). In contrast, starvation increased class III PI3K activity in whole-myotube extracts. In fact, this increase was most pronounced for a population of class III PI3K that coimmunoprecipitated with Beclin1/Apg6 protein, a major determinant in the initiation of autophagy. Stimulation of proteolysis was reproduced by feeding myotubes with synthetic dipalmitoyl-PtdIns3 P, the class III PI3K product. Conversely, protein transfection of anti-class III PI3K inhibitory antibody into starved myotubes inverted the induction of proteolysis. Therefore, independently of class I PI3K/Akt, protein S6 and mitogen-activated protein kinase pathways, amino acid starvation stimulates proteolysis in myotubes by regulating class III PI3K-Beclin1 autophagic complexes. PMID:12967324

Tassa, Amina; Roux, Marie Paule; Attaix, Didier; Bechet, Daniel M

2003-01-01

24

Short-Chain Phosphoinositide Partitioning into Plasma Membrane Models  

PubMed Central

Phosphoinositides are vital for many cellular signaling processes, and therefore a number of approaches to manipulating phosphoinositide levels in cells or excised patches of cell membranes have been developed. Among the most common is the use of “short-chain” phosphoinositides, usually dioctanoyl phosphoinositol phosphates. We use isothermal titration calorimetry to determine partitioning of the most abundant phosphoinositol phosphates, PI(4)P and PI(4,5)P2 into models of the intracellular and extracellular facing leaflets of neuronal plasma membranes. We show that phosphoinositide mole fractions in the lipid membrane reach physiological levels at equilibrium with reasonable solution concentrations. Finally we explore the consequences of our results for cellular electrophysiology. In particular, we find that TRPV1 is more selective for PI(4,5)P2 than PI(4)P and activated by extremely low membrane mole fractions of PIPs. We conclude by discussing how the logic of our work extends to other experiments with short-chain phosphoinositides. For delayed rectifier K+ channels, consideration of the membrane mole fraction of PI(4,5)P2 lipids with different acyl chain lengths suggests a different mechanism for PI(4,5)P2 regulation than previously proposed. Inward rectifier K+ channels apparent lack of selectivity for certain short-chain PIPs may require reinterpretation in view of the PIPs different membrane partitioning. PMID:24314079

Collins, Marcus D.; Gordon, Sharona E.

2013-01-01

25

Gold nanoparticle supported phospholipid membranes as a biomimetic biosensor platform for phosphoinositide signaling detection.  

PubMed

Enzyme mediated phosphoinositide signaling plays important regulatory roles in diverse cellular processes and has close implication in human diseases. However, detection of phosphoinositide enzymes remains a challenge because of the difficulty in discriminating the phosphorylation patterns of phosphoinositide. Here we develop a novel enzyme-activated gold nanoparticles (AuNPs) assembly strategy as a homogeneous colorimetric biosensor for activity detection of phosphoinositide kinases and phosphatases. This strategy utilizes a biomimetic mechanism of phosphoinositide signaling, in which AuNP supported phospholipid membranes are constructed to mimic the cellular membrane substrate, and AuNPs modified with the pleckstrin homology (PH) domain of cytosolic proteins are designed for specific, multivalent recognition of phosphorylated phosphoinositides. This biomimetic strategy enables efficient enzymatic reactions of the substrate and highly selective detection of target enzyme. The biosensor is demonstrated for the detection of phosphoinositide 3-kinase (PI3K) and phosphatase with tensin homology (PTEN). The results revealed that it allows sensitive, rapid visual detection of the enzymes with pM detection limits and four-decade wide dynamic ranges, and is capable of detecting enzyme activities in complex cell lysate samples. This biosensor might provide a general biosensor platform for high-throughput detection of phosphoinositide enzymes with high sensitivity and selectivity in biomedical research and clinical diagnostics. PMID:24994507

Wen, Qian; Liu, Si-Jia; Tang, Li-Juan; Tang, Ying; Jiang, Jian-Hui

2014-12-15

26

Plant phosphoinositide-specific phospholipase C  

PubMed Central

Phosphoinositide-specific phospholipase C (PI-PLC) belongs to an important class of enzymes involved in signaling related to lipids. They hydrolyze a membrane-associated phospholipid, phosphatidylinositol-4,5-bisphosphate, to produce inositol-1,4,5-trisphosphate and diacylglycerol. The role of PI-PLC and the mechanism behind its functioning is well studied in animal system; however, mechanism of plant PI-PLC functioning remains largely obscure. Here, we attempted to summarize the understanding regarding plant PI-PLC mechanism of regulation, localization, and domain association. Using sedimentation based phospholipid binding assay and surface plasmon resonance spectroscopy, it was demonstrated that C2 domain of plant PI-PLC alone is capable of targeting membranes. Moreover, change in surface hydrophobicity upon calcium stimulus is the key element in targeting plant PI-PLC from soluble fractions to membranes. This property of altering surface hydrophobicity plays a pivot role in regulation of PI-PLC activity. PMID:22902702

Rupwate, Sunny D.; Rajasekharan, Ram

2012-01-01

27

Phosphoinositide Signaling: New Tools and Insights  

NSDL National Science Digital Library

Phosphoinositides constitute only a small fraction of cellular phospholipids, yet their importance in the regulation of cellular functions can hardly be overstated. The rapid metabolic response of phosphoinositides after stimulation of certain cell surface receptors was the first indication that these lipids could serve as regulatory molecules. These early observations opened research areas that ultimately clarified the plasma membrane role of phosphoinositides in Ca2+ signaling. However, research of the last 10 years has revealed a much broader range of processes dependent on phosphoinositides. These lipids control organelle biology by regulating vesicular trafficking, and they modulate lipid distribution and metabolism more generally via their close relationship with lipid transfer proteins. Phosphoinositides also regulate ion channels, pumps, and transporters as well as both endocytic and exocytic processes. The significance of phosphoinositides found within the nucleus is still poorly understood, and a whole new research concerns the highly phosphorylated inositols that also appear to control multiple nuclear processes. The expansion of research and interest in phosphoinositides naturally created a demand for new approaches to determine where, within the cell, these lipids exert their effects. Imaging of phosphoinositide dynamics within live cells has become a standard cell biological method. These new tools not only helped us localize phosphoinositides within the cell but also taught us how tightly phosphoinositide control can be linked with distinct effector protein complexes. The recent progress allows us to understand the underlying causes of certain human diseases and design new strategies for therapeutic interventions.

2009-08-01

28

Photoreceptor phagocytosis is mediated by phosphoinositide signaling  

PubMed Central

Circadian oscillations in peripheral tissues, such as the retinal compartment of the eye, are critical to anticipating changing metabolic demands. Circadian shedding of retinal photoreceptor cell discs with subsequent phagocytosis by the neighboring retinal pigmented epithelium (RPE) is essential for removal of toxic metabolites and lifelong survival of these postmitotic neurons. Defects in photoreceptor phagocytosis can lead to severe retinal pathology, but the biochemical mechanisms remain poorly defined. By first documenting a 2.8-fold burst of photoreceptor phagocytosis events in the mouse eye in the morning compared with the afternoon by serial block face imaging, we established time points to assess transcriptional readouts by RNA sequencing (RNA-Seq). We identified 365 oscillating protein-coding transcripts that implicated the phosphoinositide lipid signaling network mediating the discrete steps of photoreceptor phagocytosis. Moreover, examination of overlapping cistromic sites by core clock transcription factors and promoter elements of these effector genes provided a functional basis for the circadian cycling of these transcripts. RNA-Seq also revealed oscillating expression of 16 long intergenic noncoding RNAs and key histone modifying enzymes critical for circadian gene expression. Our phenotypic and genotypic characterization reveals a complex global landscape of overlapping and temporally controlled networks driving the essential circadian process in the eye.—Mustafi, D., Kevany, B. M., Genoud, C., Bai, X., Palczewski, K. Photoreceptor phagocytosis is mediated by phosphoinositide signaling. PMID:23913857

Mustafi, Debarshi; Kevany, Brian M.; Genoud, Christel; Bai, Xiaodong; Palczewski, Krzysztof

2013-01-01

29

The Phosphoinositide 3-Kinase Pathway  

NSDL National Science Digital Library

Phosphorylated lipids are produced at cellular membranes during signaling events and contribute to the recruitment and activation of various signaling components. The role of phosphoinositide 3-kinase (PI3K), which catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate, in cell survival pathways; the regulation of gene expression and cell metabolism; and cytoskeletal rearrangements are highlighted. The PI3K pathway is implicated in human diseases including diabetes and cancer, and understanding the intricacies of this pathway may provide new avenues for therapuetic intervention.

Lewis Cantley (Beth Israel Deaconess Medical Center;Department of Cell Biology, Harvard Medical School and Division of Signal Transduction)

2002-05-31

30

Stress-ING Out: Phosphoinositides Mediate the Cellular Stress Response  

NSDL National Science Digital Library

Phosphoinositides regulate numerous cellular processes required for growth, proliferation, and motility. Whereas phosphoinositide signal transduction pathways within the cytosol have been well characterized, nuclear signaling pathways remain poorly understood. Accumulating experimental data have now started to uncover critical functions for nuclear phosphoinositides. In particular, phosphoinositides modulate the activity of the tumor suppressor protein ING2 in response to extracellular stress. These findings highlight a previously uncharacterized function for phosphoinositides and implicate their metabolism in signaling pathways critical for cell survival.

Matthew W. Bunce (University of Wisconsin;Department of Pharmacology REV); Michael L. Gonzales (University of Wisconsin;Department of Pharmacology REV); Richard A. Anderson (University of Wisconsin;Department of Pharmacology REV)

2006-11-07

31

Phosphoinositides: Key modulators of energy metabolism.  

PubMed

Phosphoinositides are key players in many trafficking and signaling pathways. Recent advances regarding the synthesis, location and functions of these lipids have dramatically improved our understanding of how and when these lipids are generated and what their roles are in animal physiology. In particular, phosphoinositides play a central role in insulin signaling, and manipulation of PtdIns(3,4,5)P3 levels in particular, may be an important potential therapeutic target for the alleviation of insulin resistance associated with obesity and the metabolic syndrome. In this article we review the metabolism, regulation and functional roles of phosphoinositides in insulin signaling and the regulation of energy metabolism. This article is part of a Special Issue entitled Phosphoinositides. PMID:25463477

Bridges, Dave; Saltiel, Alan R

2014-11-20

32

Mutations in Phosphoinositide Metabolizing Enzymes and Human Disease  

NSDL National Science Digital Library

Phosphoinositides are implicated in the regulation of a wide variety of cellular functions. Their importance in cellular and organismal physiology is underscored by the growing number of human diseases linked to perturbation of kinases and phosphatases that catalyze interconversion from one phosphoinositide to another. Many such enzymes are attractive targets for therapeutic interventions. Here, we review diseases linked to inheritable or somatic mutations of these enzymes. Phosphatidylinositol (PtdIns), a membrane phospholipid, can be reversibly phosphorylated at the 3, 4, and 5 positions of the inositol ring to generate seven phosphoinositides [PI3P, PI4P, PI5P, PI(3,4)P2, PI(4,5)P2, PI(3,5)P2, and PI(3,4,5)P3] (FIGURE 1A). The importance of this metabolism in cell regulation was first established in the context of studies on stimulus-secretion coupling. It was found that many stimuli that trigger secretion also trigger enhanced turnover of PtdIns and phosphoinositides (42). Subsequently, it became clear that phospholipase C-dependent hydrolysis of PI(4,5)P2 to generate the second messenger molecules diacyl glycerol and Ins(1,4,5)P3 (IP3) is a mechanism through which many cell surface receptors, including many receptors that stimulate secretion, transduce their signals (10). Diacyl glycerol binds and regulates protein kinase C and a variety of other effectors, whereas IP3 triggers calcium release from the endoplasmic reticulum (10, 42). In another signal transduction pathway, PI(4,5)P2 is cleaved by phospholipase A2 to generate arachidonic acid, a precursor of many signaling molecules.

Heather J. McCrea (Yale University); Pietro De Camilli (Yale University School of Medicine)

2009-02-01

33

BIN1/M-Amphiphysin2 induces clustering of phosphoinositides to recruit its downstream partner dynamin  

NASA Astrophysics Data System (ADS)

Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

Picas, Laura; Viaud, Julien; Schauer, Kristine; Vanni, Stefano; Hnia, Karim; Fraisier, Vincent; Roux, Aurélien; Bassereau, Patricia; Gaits-Iacovoni, Frédérique; Payrastre, Bernard; Laporte, Jocelyn; Manneville, Jean-Baptiste; Goud, Bruno

2014-12-01

34

BIN1/M-Amphiphysin2 induces clustering of phosphoinositides to recruit its downstream partner dynamin.  

PubMed

Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins. PMID:25487648

Picas, Laura; Viaud, Julien; Schauer, Kristine; Vanni, Stefano; Hnia, Karim; Fraisier, Vincent; Roux, Aurélien; Bassereau, Patricia; Gaits-Iacovoni, Frédérique; Payrastre, Bernard; Laporte, Jocelyn; Manneville, Jean-Baptiste; Goud, Bruno

2014-01-01

35

Physical Foundations of PTEN/Phosphoinositide Interaction  

NASA Astrophysics Data System (ADS)

Phosphoinositides act as signaling molecules by recruiting critical effectors to specific subcellular membranes to regulate cell proliferation, apoptosis and cytoskeletal reorganization, which requires a tight regulation of phosphoinositide generation and turnover as well as a high degree of compartmentalization. PTEN is a phosphatase specific for the 3 position of the phosophoinositide ring that is deleted or mutated in many different disease states. PTEN association with membranes requires the interaction of its C2 domain with phosphatidylserine and the interaction of its N-terminal end with phosphatidylinositol-4,5-bisphophate (PI(4,5)P2). We have investigated PTEN/PI(4,5)P2 interaction and found that Lys13 is crucial for the observed binding. We also found that the presence of cholesterol enhances PTEN binding to mixed PI(4,5)P2/POPC vesicles. Fluorescence microscopy experiments utilizing GUVs yielded results consistent with enhanced phosphoinositide domain formation in the presence of cholesterol. These experiments were accompanied by zeta potential measurements and solid state MAS ^31P-NMR experiments aimed at investigating the ionization behavior of phosphoinositides.

Gericke, Arne; Jiang, Zhiping; Redfern, Roberta E.; Kooijman, Edgar E.; Ross, Alonzo H.

2009-03-01

36

FERM Domain Phosphoinositide Binding Targets Merlin to the Membrane and Is Essential for Its Growth-Suppressive Function ?  

PubMed Central

The neurofibromatosis type 2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of plasma membrane-actin cytoskeleton linkers. For ezrin, phosphatidylinositol 4,5-bisphosphate (PIP2) binding to the amino-terminal FERM domain is required for its conformational activation, proper subcellular localization, and function, but less is known about the role of phosphoinositide binding for merlin. Current evidence indicates that association with the membrane is important for merlin to function as a growth regulator; however, the mechanisms by which merlin localizes to the membrane are less clear. Here, we report that merlin binds phosphoinositides, including PIP2, via a conserved binding motif in its FERM domain. Abolition of FERM domain-mediated phosphoinositide binding of merlin displaces merlin from the membrane and releases it into the cytosol without altering the folding of merlin. Importantly, a merlin protein whose FERM domain cannot bind phosphoinositide is defective in growth suppression. Retargeting the mutant merlin into the membrane using a dual-acylated amino-terminal decapeptide from Fyn is sufficient to restore the growth-suppressive properties to the mutant merlin. Thus, FERM domain-mediated phosphoinositide binding and membrane association are critical for the growth-regulatory function of merlin. PMID:21402777

Mani, Timmy; Hennigan, Robert F.; Foster, Lauren A.; Conrady, Deborah G.; Herr, Andrew B.; Ip, Wallace

2011-01-01

37

Ceramide dissociates 3'-phosphoinositide production from pleckstrin homology domain translocation.  

PubMed Central

Numerous hormones, cytokines and transforming oncogenes activate phosphoinositide 3-kinase (PI-3K), a lipid kinase that initiates signal transduction cascades regulating cellular proliferation, survival, protein synthesis and glucose metabolism. PI-3K catalyses the production of the 3'-phosphoinositides PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3), which recruit downstream effector enzymes to the membrane via their pleckstrin homology (PH) domains. Recent studies have indicated that another signalling lipid, the sphingolipid ceramide, inhibits several PI-3K-dependent events, including insulin-stimulated glucose uptake and growth-factor-stimulated cell survival. Here we show that ceramide analogues specifically prevent the recruitment of the PtdIns(3,4,5)P(3)-binding proteins Akt/protein kinase B (PKB) or the general receptor for phosphoinositides-1 (GRP1). Specifically, the short-chain ceramide derivative C2-ceramide inhibited the platelet-derived growth factor (PDGF)-stimulated translocation of full-length Akt/PKB, as well as truncated proteins encoding only the PH domains of Akt/PKB or GRP1. C2-ceramide did not alter the membrane localization of the PH domain for phospholipase Cdelta, which preferentially binds PtdIns(4,5)P(2), nor did it affect the PDGF-stimulated production of PtdIns(3,4)P(2) or PtdIns(3,4,5)P(3). Interestingly, a glucosylceramide synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol (PDMP), shown previously to increase intracellular ceramide concentrations without affecting PI-3K [Rani, Abe, Chang, Rosenzweig, Saltiel, Radin and Shayman (1995) J. Biol. Chem. 270, 2859-2867], recapitulated the inhibitory effects of C2-ceramide on PDGF-stimulated Akt/PKB phosphorylation. These studies indicate that ceramide prevents the translocation of certain PtdIns(3,4,5)P(3)-binding proteins, despite the presence of a full complement of PtdIns(3,4)P(2) or PtdIns(3,4,5)P(3). Furthermore, these findings suggest a mechanism by which stimuli that induce ceramide synthesis could negate the fundamental signalling pathways initiated by PI-3K. PMID:11171115

Stratford, S; DeWald, D B; Summers, S A

2001-01-01

38

Phosphoinositide 3-kinase p110? in immunity.  

PubMed

The rapid and accurate response of leukocytes to environmental cues is critical for a proper inflammatory reaction to foreign particles or invading microbes. In the last decade, the signal transduction enzyme phosphoinositide 3-kinase ? (PI3K?) has emerged as a critical modulator of leukocyte responses, with its effects spanning from recruitment to the site of inflammation to the production of reactive oxygen species. These findings initially obtained from genetically modified mice have led to the development of experimental anti-inflammatory inhibitors with reasonable selectivity and specificity. While such molecules have not yet reached clinical use, preclinical studies combining genetics and pharmacology continue to provide new therapeutic indications for targeting PI3K?. Thus, this review focuses on the latest discoveries regarding PI3K? function in leukocytes and on the most recent findings in disease models related to immunity. PMID:21800408

Costa, Carlotta; Martin-Conte, Erica L; Hirsch, Emilio

2011-09-01

39

Phosphoinositides in the mammalian endo-lysosomal network  

PubMed Central

The endo-lysosomal system is an interconnected tubulo-vesicular network that acts as a sorting station to process and distribute internalised cargo. This network accepts cargoes from both the plasma membrane and the biosynthetic pathway, and directs these cargos either towards the lysosome for degradation, the peri-nuclear recycling endosome for return to the cell surface, or to the trans-Golgi network. These intracellular membranes are variously enriched in different phosphoinositides that help to shape compartmental identity. These lipids act to localise a number of phosphoinositide-binding proteins that function as sorting machineries to regulate endosomal cargo sorting. Herein we discuss regulation of these machineries by phosphoinositides and explore how phosphoinositide-switching contributes toward sorting decisions made at this platform. PMID:22374088

Cullen, Peter J.; Carlton, Jeremy G.

2014-01-01

40

PITPs as Targets for Selectively Interfering With Phosphoinositide Signaling in Cells  

PubMed Central

Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production, and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs, nor PITPs in general, have been exploited as targets for chemical inhibition for such purposes. Herein, we validate the first small molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and are effective inhibitors in vitro and in vivo. We further establish Sec14 is the sole essential NPPM target in yeast, that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects, and demonstrate NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof-of-concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies. PMID:24292071

Nile, Aaron H.; Tripathi, Ashutosh; Yuan, Peihua; Mousley, Carl J.; Suresh, Sundari; Wallace, Iain Michael; Shah, Sweety D.; Pohlhaus, Denise Teotico; Temple, Brenda; Nislow, Corey; Giaever, Guri; Tropsha, Alexander; Davis, Ronald W.; St Onge, Robert P.; Bankaitis, Vytas A.

2013-01-01

41

Global approaches for the elucidation of phosphoinositide-binding proteins.  

PubMed

Phosphoinositide lipids (PIPns) control numerous critical biological pathways, typically through the regulation of protein function driven by non-covalent protein-lipid binding interactions. Despite the importance of these systems, the unraveling of the full scope of protein-PIPn interactions has represented a significant challenge due to the massive complexity associated with these events, including the large number of diverse proteins that bind to these lipids, variations in the mechanisms by which proteins bind to lipids, and the presence of multiple distinct PIPn isomers. As a result of this complexity, global methods in which numerous proteins that bind PIPns can be identified and characterized simultaneously from complex samples, which have been enabled by key technological advancements, have become popular as an efficient means for tackling this challenge. This review article provides an overview of advancements in large-scale methods for profiling protein-PIPn binding, including experimental methods, such as affinity enrichment, microarray analysis and activity-based protein profiling, as well as computational methods, and combined computational/experimental efforts. PMID:24220499

Best, Michael D

2014-09-01

42

Targeting phosphoinositide 3-kinase ? for allergic asthma.  

PubMed

Chronic inflammation in the lung has long been linked to the pathogenesis of asthma. Central to this airway inflammation is a T-cell response to allergens, with Th2 cytokines driving the differentiation, survival and function of the major inflammatory cells involved in the allergic cascade. PI3K? (phosphoinositide 3-kinase ?) is a lipid kinase, expressed predominantly in leucocytes, where it plays a critical role in immune receptor signalling. A selective PI3K? inhibitor is predicted to block T-cell activation in the lung, reducing the production of pro-inflammatory Th2 cytokines. PI3K? is also involved in B-cell and mast cell activation. Therefore the inhibition of PI3K? should dampen down the inflammatory cascade involved in the asthmatic response through a wide breadth of pharmacology. Current anti-inflammatory therapies, which are based on corticosteroids, are effective in controlling inflammation in mild asthmatics, but moderate/severe asthmatic patients remain poorly controlled, experiencing recurrent exacerbations. Corticosteroids have no effect on mast cell degranulation and do not act directly on B-cells, so, overall, a PI3K? inhibitor has the potential to deliver improvements in onset of action, efficacy and reduced exacerbations in moderate/severe asthmatics. Additionally, PI3K? inhibition is expected to block effects of Th17 cells, which are increasingly implicated in steroid-insensitive asthma. PMID:22260698

Rowan, Wendy C; Smith, Janet L; Affleck, Karen; Amour, Augustin

2012-02-01

43

4-Methylhomoibotenic Acid Activates a Novel Metabotropic Glutamate Receptor Coupled to Phosphoinositide Hydrolysis1  

E-print Network

to Phosphoinositide Hydrolysis1 DOROTHY S. CHUNG, STEPHEN F. TRAYNELIS, T. J. MURPHY and P. JEFFREY CONN DepartmentGluR1 and mGluR5 are coupled to phosphoinositide hydrolysis in expression systems, and both the phosphoinositide hydrolysis response to DHPG in rat cortical slices. In contrast, LY341495 did not block

Traynelis, Stephen F.

44

Synthesis and Function of Membrane Phosphoinositides in Budding Yeast, Saccharomyces cerevisiae  

PubMed Central

It is now well appreciated that derivatives of phosphatidylinositol (PtdIns) are key regulators of many cellular processes in eukaryotes. Of particular interest are phosphoinositides (mono- and polyphosphorylated adducts to the inositol ring in PtdIns), which are located at the cytoplasmic face of cellular membranes. Phosphoinositides serve both a structural and a signaling role via their recruitment of proteins that contain phosphoinositide-binding domains. Phosphoinositides also have a role as precursors of several types of second messengers for certain intracellular signaling pathways. Realization of the importance of phosphoinositides has brought increased attention to characterization of the enzymes that regulate their synthesis, interconversion, and turnover. Here we review the current state of our knowledge about the properties and regulation of the ATP-dependent lipid kinases responsible for synthesis of phosphoinositides and also the additional temporal and spatial controls exerted by the phosphatases and a phospholipase that act on phosphoinositides in yeast. PMID:17382260

Strahl, Thomas; Thorner, Jeremy

2007-01-01

45

Regulation of ion transport proteins by membrane phosphoinositides  

Microsoft Academic Search

Over the past decade, there has been an explosion in the number of membrane transport proteins that have been shown to be sensitive to the abundance of phosphoinositides in the plasma membrane. These proteins include voltage-gated potassium and calcium channels, ion channels that mediate sensory and nociceptive responses, epithelial transport proteins and ionic exchangers. Each of the regulatory lipids is

Nikita Gamper; Mark S. Shapiro

2007-01-01

46

INTRODUCTION Increasing evidence points to the phosphoinositide signalling  

E-print Network

of the phosphoinositide pathway and is teratogenic. We report the effects of lithium on the first cell cycles of sea, 1990). These effects can be reversed by the addition of myo-inositol (Berridge et al., 1989; Busa, 1988; Busa and Gimlich, 1989). However, the effects of lithium on early embryonic cell cycles have not been

Newcastle upon Tyne, University of

47

Nephrin mediates actin reorganization via phosphoinositide 3-kinase in podocytes  

Microsoft Academic Search

Nephrin is a slit diaphragm protein critical for structural and functional integrity of visceral glomerular epithelial cells (podocytes) and is known to be tyrosine phosphorylated by Src family kinases. We studied the role of phosphoinositide 3-kinase (PI3K), activated via the phosphorylation of nephrin, in actin cytoskeletal reorganization of cultured rat podocytes. Phosphorylation of rat nephrin by the Fyn kinase markedly

J Zhu; N Sun; L Aoudjit; H Li; H Kawachi; S Lemay; T Takano

2008-01-01

48

Activation of TRPV1 channels inhibits mechanosensitive Piezo channel activity by depleting membrane phosphoinositides.  

PubMed

Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor phosphatidylinositol 4-phosphate [PI(4)P] from the plasma membrane through Ca(2+)-induced phospholipase C? (PLC?) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLC? indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 and PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin. PMID:25670203

Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

2015-01-01

49

An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway  

PubMed Central

Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3K?, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinase? (PI3K?), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis. PMID:16103228

Shin, Hye-Won; Hayashi, Mitsuko; Christoforidis, Savvas; Lacas-Gervais, Sandra; Hoepfner, Sebastian; Wenk, Markus R.; Modregger, Jan; Uttenweiler-Joseph, Sandrine; Wilm, Matthias; Nystuen, Arne; Frankel, Wayne N.; Solimena, Michele; De Camilli, Pietro; Zerial, Marino

2005-01-01

50

Phosphoinositide binding by the SNX27 FERM domain regulates localisation at the immune synapse of activated T-cells.  

PubMed

Sorting nexin 27 (SNX27) controls the endosomal to cell-surface recycling of diverse transmembrane protein cargos. Critical to this function is the recruitment of SNX27 to endosomes through the binding of phosphatidylinositol-3-phosphate (PtdIns3P) by the phox-homology (PX) domain. In T cells, SNX27 is polarized to the immunological synapse (IS) in an activation-dependent manner, but the molecular mechanisms underlying SNX27 translocation remain to be clarified. Here, we examined the phosphoinositide lipid-binding capabilities of full-length SNX27, and discovered a novel PtdInsP binding site within the C-terminal 4.1/ezrin/radixin/moesin (FERM) domain. This binding site showed a clear preference for di and tri-phosphorylated phophoinositides, and the interaction was confirmed through biophysical, mutagenesis and modeling approaches. At the IS of activated T-cells cell signaling regulates phosphoinositide dynamics, and we find that perturbing phosphoinositide binding by the SNX27 FERM domain alters its distribution in both endosomal recycling compartments and PtdIns(3,4,5)P3-enriched domains of the plasma membrane during synapse formation. Our results suggest that SNX27 undergoes dynamic partitioning between different membrane domains during IS assembly, and underscore the contribution of unique lipid interactions for SNX27 orchestration of cargo trafficking. PMID:25472716

Ghai, Rajesh; Tello-Lafoz, Maria; Norwood, Suzanne J; Yang, Zhe; Clairfeuille, Thomas; Teasdale, Rohan D; Mérida, Isabel; Collins, Brett M

2014-12-01

51

Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis  

PubMed Central

Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH2PO4 and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH2PO4 concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×105 in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10?18 to 10?20 mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications. PMID:23000742

Wang, Kelong; Jiang, Dechen; Sims, Christopher E.; Allbritton, Nancy L.

2012-01-01

52

Myotubularin phosphoinositide phosphatases: cellular functions and disease pathophysiology.  

PubMed

The myotubularin family of phosphoinositide phosphatases includes several members mutated in neuromuscular diseases or associated with metabolic syndrome, obesity, and cancer. Catalytically dead phosphatases regulate their active homologs by heterodimerization and potentially represent key players in the phosphatase-kinase balance. Although the enzymatic specificity for phosphoinositides indicates a role for myotubularins in endocytosis and membrane trafficking, recent findings in cellular and animal models suggest that myotubularins regulate additional processes including cell proliferation and differentiation, autophagy, cytokinesis, and cytoskeletal and cell junction dynamics. In this review, we discuss how myotubularins regulate such diverse processes, emphasizing newly identified functions in a physiological and pathological context. A better understanding of myotubularin pathophysiology will pave the way towards therapeutic strategies. PMID:22578719

Hnia, Karim; Vaccari, Ilaria; Bolino, Alessandra; Laporte, Jocelyn

2012-06-01

53

Phosphoinositides Direct Equine Infectious Anemia Virus Gag Trafficking and Release  

PubMed Central

Phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), the predominant phosphoinositide on the plasma membrane, binds the matrix (MA) protein of Human Immunodeficiency Virus type 1 (HIV-1) and Equine Infectious Anemia Virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P2 is critical for HIV-1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments hence the significance of its interaction with PI(4,5)P2 is unclear. We therefore investigated the binding in vitro of other phosphoinositides to EIAV MA and whether intracellular association with compartments bearing these phosphoinositides was important for assembly and release of virus-like particles (VLPs) formed by Gag. In vitro, EIAV MA bound PI(3)P with higher affinity than PI(4,5)P2 as revealed by NMR spectra upon lipid titration. Gag was detected on the plasma membrane and in compartments enriched in PI(3,5)P2. Treatment of cells with YM201636, a kinase inhibitor that blocks production of PI(3,5)P2 from PI(3)P, caused Gag to co-localize with aberrant compartments and inhibited VLP release. In contrast to HIV-1, release of EIAV VLPs was not significantly diminished by co-expression with 5-phosphatase IV, an enzyme that specifically depletes PI(4,5)P2 from the plasma membrane. However, co-expression with synaptojanin 2, a phosphatase with broader specificity, diminished VLP production. PI-binding pocket mutations caused striking budding defects, as revealed by electron microscopy. One of the mutations also modified Gag-Gag interaction, as suggested by altered bimolecular fluorescence complementation. We conclude that phosphoinositide-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release. PMID:21176037

Fernandes, Fiona; Chen, Kang; Ehrlich, Lorna S.; Jin, Jing; Chen, Min H.; Medina, Gisselle N.; Symons, Marc; Montelaro, Ronald; Donaldson, Julie; Tjandra, Nico; Carter, Carol A.

2011-01-01

54

Analysis of the murine phosphoinositide 3-kinase ? gene  

Microsoft Academic Search

Phosphoinositide 3-kinase ? is preferentially expressed in leukocytes. PI3K? is activated by ?? subunits of heterotrimeric G-proteins, which thus link seven transmembrane helix receptor activation to phosphatidylinositol (3,4,5)-trisphosphate production. Here we describe the molecular cloning of the murine PI3K? cDNA, the PI3K? gene structure, its chromosomal assignment and the analysis of promoter activity. The mouse cDNA shares 86% identity to

Emilio Hirsch; Matthias P. Wymann; Enrico Patrucco; Emanuela Tolosano; Ginette Bulgarelli-Leva; Stefano Marengo; Mariano Rocchi; Fiorella Altruda

2000-01-01

55

Phosphoinositide phosphatases: just as important as the kinases.  

PubMed

Phosphoinositide phosphatases comprise several large enzyme families with over 35 mammalian enzymes identified to date that degrade many phosphoinositide signals. Growth factor or insulin stimulation activates the phosphoinositide 3-kinase that phosphorylates phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] to form phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)], which is rapidly dephosphorylated either by PTEN (phosphatase and tensin homologue deleted on chromosome 10) to PtdIns(4,5)P(2), or by the 5-phosphatases (inositol polyphosphate 5-phosphatases), generating PtdIns(3,4)P(2). 5-phosphatases also hydrolyze PtdIns(4,5)P(2) forming PtdIns(4)P. Ten mammalian 5-phosphatases have been identified, which regulate hematopoietic cell proliferation, synaptic vesicle recycling, insulin signaling, and embryonic development. Two 5-phosphatase genes, OCRL and INPP5E are mutated in Lowe and Joubert syndrome respectively. SHIP [SH2 (Src homology 2)-domain inositol phosphatase] 2, and SKIP (skeletal muscle- and kidney-enriched inositol phosphatase) negatively regulate insulin signaling and glucose homeostasis. SHIP2 polymorphisms are associated with a predisposition to insulin resistance. SHIP1 controls hematopoietic cell proliferation and is mutated in some leukemias. The inositol polyphosphate 4-phosphatases, INPP4A and INPP4B degrade PtdIns(3,4)P(2) to PtdIns(3)P and regulate neuroexcitatory cell death, or act as a tumor suppressor in breast cancer respectively. The Sac phosphatases degrade multiple phosphoinositides, such as PtdIns(3)P, PtdIns(4)P, PtdIns(5)P and PtdIns(3,5)P(2) to form PtdIns. Mutation in the Sac phosphatase gene, FIG4, leads to a degenerative neuropathy. Therefore the phosphatases, like the lipid kinases, play major roles in regulating cellular functions and their mutation or altered expression leads to many human diseases. PMID:22403078

Dyson, Jennifer M; Fedele, Clare G; Davies, Elizabeth M; Becanovic, Jelena; Mitchell, Christina A

2012-01-01

56

Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation  

PubMed Central

Phosphoinositides (PIs) make up only a small fraction of cellular phospholipids, yet they control almost all aspects of a cell's life and death. These lipids gained tremendous research interest as plasma membrane signaling molecules when discovered in the 1970s and 1980s. Research in the last 15 years has added a wide range of biological processes regulated by PIs, turning these lipids into one of the most universal signaling entities in eukaryotic cells. PIs control organelle biology by regulating vesicular trafficking, but they also modulate lipid distribution and metabolism via their close relationship with lipid transfer proteins. PIs regulate ion channels, pumps, and transporters and control both endocytic and exocytic processes. The nuclear phosphoinositides have grown from being an epiphenomenon to a research area of its own. As expected from such pleiotropic regulators, derangements of phosphoinositide metabolism are responsible for a number of human diseases ranging from rare genetic disorders to the most common ones such as cancer, obesity, and diabetes. Moreover, it is increasingly evident that a number of infectious agents hijack the PI regulatory systems of host cells for their intracellular movements, replication, and assembly. As a result, PI converting enzymes began to be noticed by pharmaceutical companies as potential therapeutic targets. This review is an attempt to give an overview of this enormous research field focusing on major developments in diverse areas of basic science linked to cellular physiology and disease. PMID:23899561

2013-01-01

57

Allosteric Activation of the Phosphoinositide Phosphatase Sac1 by Anionic Phospholipids  

PubMed Central

Sac family phosphoinositide phosphatases comprise an evolutionarily conserved family of enzymes in eukaryotes. Our recently determined crystal structure of the Sac phosphatase domain of yeast Sac1, the founding member of the Sac family proteins, revealed a unique conformation of the catalytic P-loop and a large positively charged groove at the catalytic site. We now report a unique mechanism for the regulation of its phosphatase activity. Sac1 is an allosteric enzyme that can be activated by its product phosphatidylinositol or anionic phospholipid phosphatidylserine. The activation of Sac1 may involve conformational changes of the catalytic P-loop induced by direct binding with the regulatory anionic phospholipids in the large cationic catalytic groove. These findings highlight the fact that lipid composition of the substrate membrane plays an important role in the control of Sac1 function. PMID:22452743

2012-01-01

58

Two structural components in CNGA3 support regulation of cone CNG channels by phosphoinositides.  

PubMed

Cyclic nucleotide-gated (CNG) channels in retinal photoreceptors play a crucial role in vertebrate phototransduction. The ligand sensitivity of photoreceptor CNG channels is adjusted during adaptation and in response to paracrine signals, but the mechanisms involved in channel regulation are only partly understood. Heteromeric cone CNGA3 (A3) + CNGB3 (B3) channels are inhibited by membrane phosphoinositides (PIP(n)), including phosphatidylinositol 3,4,5-triphosphate (PIP(3)) and phosphatidylinositol 4,5-bisphosphate (PIP(2)), demonstrating a decrease in apparent affinity for cyclic guanosine monophosphate (cGMP). Unlike homomeric A1 or A2 channels, A3-only channels paradoxically did not show a decrease in apparent affinity for cGMP after PIP(n) application. However, PIP(n) induced an ?2.5-fold increase in cAMP efficacy for A3 channels. The PIP(n)-dependent change in cAMP efficacy was abolished by mutations in the C-terminal region (R643Q/R646Q) or by truncation distal to the cyclic nucleotide-binding domain (613X). In addition, A3-613X unmasked a threefold decrease in apparent cGMP affinity with PIP(n) application to homomeric channels, and this effect was dependent on conserved arginines within the N-terminal region of A3. Together, these results indicate that regulation of A3 subunits by phosphoinositides exhibits two separable components, which depend on structural elements within the N- and C-terminal regions, respectively. Furthermore, both N and C regulatory modules in A3 supported PIP(n) regulation of heteromeric A3+B3 channels. B3 subunits were not sufficient to confer PIP(n) sensitivity to heteromeric channels formed with PIP(n)-insensitive A subunits. Finally, channels formed by mixtures of PIP(n)-insensitive A3 subunits, having complementary mutations in N- and/or C-terminal regions, restored PIP(n) regulation, implying that intersubunit N-C interactions help control the phosphoinositide sensitivity of cone CNG channels. PMID:23530136

Dai, Gucan; Peng, Changhong; Liu, Chunming; Varnum, Michael D

2013-04-01

59

Phosphoinositide synthesis and degradation in isolated rat liver peroxisomes  

Microsoft Academic Search

Analyzing peroxisomal phosphoinositide (PId#) synthesis in highly purified rat liver peroxisomes we found synthesis of phosphatidylinositol 4-phosphate (PtdIns4P), PtdIns(4,5)P2 and PtdIns(3,5)P2. PtdIns3P was hardly detected in vitro, however, was observed in vivo after [32P]-phosphate labeling of primary rat hepatocytes. In comparison with other subcellular organelles peroxisomes revealed a unique PId pattern suggesting peroxisomal specificity of the observed synthesis. Use of

Boyan Jeynov; Dorothee Lay; Frank Schmidt; Sabina Tahirovic; Wilhelm W. Just

2006-01-01

60

Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities.  

PubMed Central

The second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is generated by the action of phosphoinositide 3-kinase (PI 3-kinase), and regulates a plethora of cellular processes. An approach for dissecting the mechanisms by which these processes are regulated is to identify proteins that interact specifically with PtdIns(3,4,5)P(3). The pleckstrin homology (PH) domain has become recognized as the specialized module used by many proteins to interact with PtdIns(3,4,5)P(3). Recent work has led to the identification of a putative phosphatidylinositol 3,4,5-trisphosphate-binding motif (PPBM) at the N-terminal regions of PH domains that interact with this lipid. We have searched expressed sequence tag databases for novel proteins containing PH domains possessing a PPBM. Surprisingly, many of the PH domains that we identified do not bind PtdIns(3,4,5)P(3), but instead possess unexpected and novel phosphoinositide-binding specificities in vitro. These include proteins possessing PH domains that interact specifically with PtdIns(3,4)P(2) [TAPP1 (tandem PH-domain-containing protein-1) and TAPP2], PtdIns4P [FAPP1 (phosphatidylinositol-four-phosphate adaptor protein-1)], PtdIns3P [PEPP1 (phosphatidylinositol-three-phosphate-binding PH-domain protein-1) and AtPH1] and PtdIns(3,5)P(2) (centaurin-beta2). We have also identified two related homologues of PEPP1, termed PEPP2 and PEPP3, that may also interact with PtdIns3P. This study lays the foundation for future work to establish the phospholipid-binding specificities of these proteins in vivo, and their physiological role(s). PMID:11001876

Dowler, S; Currie , R A; Campbell , D G; Deak, M; Kular, G; Downes, C P; Alessi, D R

2000-01-01

61

Phosphoinositide 3-kinase ? regulates chromosome segregation in mitosis.  

PubMed

Class I(A) phosphoinositide 3-kinases (PI3K) are enzymes composed of a p85 regulatory and a p110 catalytic subunit that control formation of 3-poly-phosphoinositides (PIP(3)). The PI3K pathway regulates cell survival, migration, and division, and is mutated in approximately half of human tumors. For this reason, it is important to define the function of the ubiquitous PI3K subunits, p110? and p110?. Whereas p110? is activated at G1-phase entry and promotes protein synthesis and gene expression, p110? activity peaks in S phase and regulates DNA synthesis. PI3K activity also increases at the onset of mitosis, but the isoform activated is unknown; we have examined p110? and p110? function in mitosis. p110? was activated at mitosis entry and regulated early mitotic events, such as PIP(3) generation, prometaphase progression, and spindle orientation. In contrast, p110? was activated near metaphase and controlled dynein/dynactin and Aurora B activities in kinetochores, chromosome segregation, and optimal function of the spindle checkpoint. These results reveal a p110? function in preserving genomic stability during mitosis. PMID:23051731

Silió, Virginia; Redondo-Muñoz, Javier; Carrera, Ana C

2012-12-01

62

Odorant-stimulated phosphoinositide signaling in mammalian olfactory receptor neurons  

PubMed Central

Recent evidence has revived interest in the idea that phosphoinositides (PIs) may play a role in signal transduction in mammalian olfactory receptor neurons (ORNs). To provide direct evidence that odorants indeed activate PI signaling in ORNs, we used adenoviral vectors carrying two different fluorescently tagged probes, the pleckstrin homology (PH) domains of phospholipase C?1 (PLC?1) and the general receptor of phosphoinositides (GRP1), to monitor PI activity in the dendritic knobs of ORNs in vivo. Odorants mobilized PI(4,5)P2/IP3 and PI(3,4,5)P3, the substrates and products of PLC and PI3K. We then measured odorant activation of PLC and PI3K in olfactory ciliary-enriched membranes in vitro using a phospholipid overlay assay and ELISAs. Odorants activated both PLC and PI3K in the olfactory cilia within 2 sec of odorant stimulation. Odorant-dependent activation of PLC and PI3K in the olfactory epithelium could be blocked by enzyme-specific inhibitors. Odorants activated PLC and PI3K with partially overlapping specificity. These results provide direct evidence that odorants indeed activate PI signaling in mammalian ORNs in a manner that is consistent with the idea that PI signaling plays a role in olfactory transduction. PMID:19781634

Klasen, K.; Corey, E.A.; Kuck, F.; Wetzel, C.H.; Hatt, H.; Ache, B.W.

2009-01-01

63

Alcohol induced changes in phosphoinositide signaling system in rat brain  

SciTech Connect

Agonist-induced phosphoinositide break down functions as a signal generating system in a manner similar to the C-AMP system. In order to examine if the changes produced by chronic ethanol treatment on membrane lipid composition and metabolism effect the cellular functions of the neuron, the authors have examined the effect of chronic ethanol exposure on norepinephrine (NE) serotonin (5HT) and calcium ionophore (CI) stimulated phosphoinositide (PI) hydrolysis in rat cortical slices. Rats were maintained on liber-decarli diet alcohol and control liquid diet containing isocaloric sucrose substitute for two months. They were then sacrificed and brain was removed for determination of PI turnover. 5HT stimulated {sup 3}H- inositol monophosphate ({sup 3}H-IPI) formation was significantly lower in the cortex of alcohol treated rats as compared to control rats. However, neither CI nor NE stimulated IP1 formation was significantly different from control rats. The results thus indicate that chronic exposure to ethanol decreases 5HT induced PI breakdown in rat cortex. In order to examine if this decrease is related to a decrease in 5HT2 receptors, or decreased in coupling of receptor to the effector pathway, the authors are currently determining the number and affinity of 5HT2 receptors in alcohol treated rats.

Pandey, S.; Piano, M.; Schwertz, D.; Davis, J.; Pandey, G. (Univ. of Illinois, Chicago (United States))

1991-03-11

64

Species-related differences in inotropic effects of angiotensin II in mammalian ventricular muscle: receptors, subtypes and phosphoinositide hydrolysis.  

PubMed Central

1. Experiments were carried out to clarify the mechanisms responsible for variations in the positive inotropic effect (PIE) of angiotensin II (AII) on ventricular muscles from various mammals. We examined the density of AII receptors, the relative proportions of receptor subtypes and the acceleration of the hydrolysis of phosphoinositide that was induced by AII, as well as the PIE of AII in ventricular muscles from the rabbit, dog, rat and ferret. 2. In the rabbit, AII (1 microM) in the presence of bupranolol (0.3 microM) and prazosin (0.1 microM) elicited a concentration-dependent PIE, which was antagonized by a selective AT1 subtype antagonist, losartan, but not by an AT2 antagonist, PD123319. AII did not have any inotropic effects in ventricular muscles from the dog, rat and ferret. 3. Specific high-affinity binding of [125I]-AII, with a similar Kd value in each case (1-2 nM), was observed with membrane fractions derived from ventricular muscle of all four species tested. 4. In the rabbit, losartan and PD123319 each displaced approximately 50% of [125I]-AII specific binding having high affinity for the receptors, and indicating that AT1 and AT2 subtypes were present in equal numbers. In the other species the AT1 subtype of receptors was predominant. 5. In all four species AII caused a concentration-dependent acceleration of the hydrolysis of phosphoinositide in ventricular slices that had been prelabelled with myo-[3H]-inositol. 6. The results indicate that the signal-transduction process distal to acceleration of the hydrolysis of phosphoinositide may be responsible for the wide range of species variations in the inotropic action of AII on mammalian ventricular myocardium. PMID:7881743

Ishihata, A; Endoh, M

1995-01-01

65

Phosphoinositide synthesis and degradation in isolated rat liver peroxisomes.  

PubMed

Analyzing peroxisomal phosphoinositide (PId(#)) synthesis in highly purified rat liver peroxisomes we found synthesis of phosphatidylinositol 4-phosphate (PtdIns4P), PtdIns(4,5)P(2) and PtdIns(3,5)P(2). PtdIns3P was hardly detected in vitro, however, was observed in vivo after [(32)P]-phosphate labeling of primary rat hepatocytes. In comparison with other subcellular organelles peroxisomes revealed a unique PId pattern suggesting peroxisomal specificity of the observed synthesis. Use of phosphatase inhibitors enhanced the amount of PtdIns4P. The results obtained provide evidence that isolated rat liver peroxisomes synthesize PIds and suggest the association of PId 4-kinase and PId 5-kinase and PId 4-phosphatase activities with the peroxisomal membrane. PMID:17045591

Jeynov, Boyan; Lay, Dorothee; Schmidt, Frank; Tahirovic, Sabina; Just, Wilhelm W

2006-10-30

66

Cardiac regulation by phosphoinositide 3-kinases and PTEN.  

PubMed

The diverse effects mediated by PI3K/PTEN (phosphoinositide 3-kinase/phosphatase and tensin homologue deleted on chromosome 10) signalling in the heart clearly support an important biological and pathophysiological role for this signalling cascade. PI3Ks are a family of evolutionarily conserved lipid kinases that mediate many cellular responses to physiological and pathophysiological stimuli. Class I PI3K can be activated by either receptor tyrosine kinase/cytokine receptor activation (class IA) or G-protein-coupled receptors (class IB), leading to the generation of phosphatidyl inositol (3,4,5)P3 and recruitment and activation of Akt/protein kinase B, 3'-phosphoinositide-dependent kinase-1 (PDK1), or monomeric G-proteins, and phosphorylation of a wide range of downstream targets including glycogen synthase kinase 3beta (GSK3beta), mTOR (mammalian target of rapamycin), p70S6 kinase, endothelial nitric oxide synthase, and several anti-apoptotic effectors. Class IA (PI3Kalpha, beta, and delta) and class IB (PI3Kgamma) PI3Ks mediate distinct phenotypes in the heart under negative control by the 3'-lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4,5)P3 to generate PtdIns(4,5)P2. PI3Kalpha, PI3Kgamma, and PTEN are expressed in cardiomyocytes, fibroblasts, endothelial cells, and vascular smooth muscle cells, where they modulate cell survival, hypertrophy, contractility, metabolism, and mechanotransduction. The PI3K/PTEN signalling pathways are involved in a wide variety of diseases including myocardial hypertrophy and contractility, heart failure, and preconditioning. In this review, we discuss the signalling pathways mediated by PI3K class I isoforms and PTEN and their roles in cardiac structure and function. PMID:19147653

Oudit, Gavin Y; Penninger, Josef M

2009-05-01

67

Functional Anatomy of Phospholipid Binding And Regulation of Phosphoinositide Homeostasis By Proteins of the Sec14 Superfamily  

SciTech Connect

Sec14, the major yeast phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein, regulates essential interfaces between lipid metabolism and membrane trafficking from the trans-Golgi network (TGN). How Sec14 does so remains unclear. We report that Sec14 binds PtdIns and PtdCho at distinct (but overlapping) sites, and both PtdIns- and PtdCho-binding activities are essential Sec14 activities. We further show both activities must reside within the same molecule to reconstitute a functional Sec14 and for effective Sec14-mediated regulation of phosphoinositide homeostasis in vivo. This regulation is uncoupled from PtdIns-transfer activity and argues for an interfacial presentation mode for Sec14-mediated potentiation of PtdIns kinases. Such a regulatory role for Sec14 is a primary counter to action of the Kes1 sterol-binding protein that antagonizes PtdIns 4-OH kinase activity in vivo. Collectively, these findings outline functional mechanisms for the Sec14 superfamily and reveal additional layers of complexity for regulating phosphoinositide homeostasis in eukaryotes.

Schaaf, G.; Ortlund, E.A.; Tyeryar, K.R.; Mousley, C.J.; Ile, K.E.; Garrett, T.A.; Ren, J.; Woolls, M.J.; Raetz, C.R.H.; Redinbo, M.R.; Bankaitis, V.A.

2009-05-27

68

Expression and evolution of the phosphoinositide-specific phospholipase C gene family in Arabidopsis thaliana  

Microsoft Academic Search

Phosphoinositide-specific phospholipase C cleaves the substrate phosphatidylinositol 4,5-bisphosphate and generates inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, both of which are second messengers in the phosphoinositide signal transduction pathways operative in animal cells. Five PI-PLC isoforms, ?, ?, ?, ? and ?, have been identified in mammals. Plant PI-PLCs are structurally close to the mammalian PI-PLC-? isoform. The Arabidopsis genome contains nine AtPLC

I. Made Tasma; Volker Brendel; Steven A. Whitham; Madan K. Bhattacharyya

2008-01-01

69

Phosphoinositide kinases and the synthesis of polyphosphoinositides in higher plant cells  

NASA Technical Reports Server (NTRS)

Phosphoinositides are a family of inositol-containing phospholipids which are present in all eukaryotic cells. Although in most cells these lipids, with the exception of phosphatidylinositol, constitute only a very minor proportion of total cellular lipids, they have received immense attention by researchers in the past 15-20 years. This is due to the discovery that these lipids, rather than just having structural functions, play key roles in a wide range of important cellular processes. Much less is known about the plant phosphoinositides than about their mammalian counterparts. However, it has been established that a functional phosphoinositide system exists in plant cells and it is becoming increasingly clear that inositol-containing lipids are likely to play many important roles throughout the life of a plant. It is not our intention to give an exhaustive overview of all aspects of the field, but rather we focus on the phosphoinositide kinases responsible for the synthesis of all phosphorylated forms of phosphatidylinositol. Also, we mention some of the aspects of current phosphoinositide research which, in our opinion, are most likely to provide a suitable starting point for further research into the role of phosphoinositides in plants.

Drobak, B. K.; Dewey, R. E.; Boss, W. F.; Davies, E. (Principal Investigator)

1999-01-01

70

Genetic enhancement of behavioral itch responses in mice lacking phosphoinositide 3-kinase-? (PI3K?).  

PubMed

Phosphoinositide 3-kinases (PI3Ks) are important for synaptic plasticity and various brain functions. The only class IB isoform of PI3K, PI3K?, has received the most attention due to its unique roles in synaptic plasticity and cognition. However, the potential role of PI3K? in sensory transmission, such as pain and itch has not been examined. In this study, we present the evidence for the first time, that genetic deletion of PI3K? enhanced scratching behaviours in histamine-dependent and protease-activated receptor 2 (PAR-2)-dependent itch. In contrast, PI3K?-deficient mice did not exhibit enhanced scratching in chloroquine-induced itch, suggesting that PI3K? selectively contributes to certain types of behavioal itch response. Furthermore, PI3K?-deficient mice exhibited normal acute nociceptive responses to thermal and mechanical noxious stimuli. Behavioral licking responses to intraplantar injections of formalin and mechanical allodynia in a chronic inflammatory pain model (CFA) were also not affected by PI3K? gene deletion. Our findings indicate that PI3K? selectively contributes to behavioral itching induced by histamine and PAR-2 agonist, but not chloroquine agonist. PMID:22168443

Lee, Bolam; Descalzi, Giannina; Baek, Jinhee; Kim, Jae-Ick; Lee, Hye-Ryeon; Lee, Kyungmin; Kaang, Bong-Kiun; Zhuo, Min

2011-01-01

71

Inhibition of phosphoinositide 3-kinase ? attenuates inflammation, obesity, and cardiovascular risk factors.  

PubMed

Phosphoinositide 3-kinase ? (PI3K?) plays a central role in inflammation, allergy, cardiovascular, and metabolic disease. Obesity is accompanied by chronic, low-grade inflammation. As PI3K? plays a major role in leukocyte recruitment, targeting of PI3K? has been considered to be a strategy for attenuating progression of obesity to insulin resistance and type 2 diabetes. Indeed, PI3K? null mice are protected from high fat diet-induced obesity, metabolic inflammation, fatty liver, and insulin resistance. The lean phenotype of the PI3K?-null mice has been linked to increased thermogenesis and energy expenditure. Surprisingly, the increase in fat mass and metabolic aberrations were not linked to PI3K? activity in the hematopoietic compartment. Thermogenesis and oxygen consumption are modulated by PI3K? lipid kinase-dependent and -independent signaling mechanisms. PI3K? signaling controls metabolic and inflammatory stress, and may provide an entry point for therapeutic strategies in metabolic disease, inflammation, and cardiovascular disease. PMID:23551103

Wymann, Matthias P; Solinas, Giovanni

2013-03-01

72

Phosphoinositide 3-kinase ?/? inhibition does not prevent concanavalin A-induced hepatitis.  

PubMed

An increasing number of studies have suggested that phosphoinositide 3-kinase-? (PI3K?) and PI3K? are involved in the pathogenesis of autoimmune and inflammatory diseases, such as asthma and atherosclerosis. However, the underlying mechanism of acute hepatitis remains unknown. The present study aimed to determine the effect of PI3K?/? inhibition on hepatic injury in a murine model of hepatitis induced by concanavalin A (ConA). It was demonstrated that the pharmacological inhibition of PI3K?/? by TG100-115 did not prevent liver damage following ConA challenge. Furthermore, the PI3K?/? inhibition resulted in elevated transaminase activity in the serum, aggravated hepatic lesions characterized by hepatic necrosis, increased inflammatory cell infiltration and apoptosis of hepatocytes. Survival tests demonstrated that TG100-115 significantly increased the death rate of mice following ConA challenge. In addition, TG100-115 increased the serum levels of the proinflammatory cytokine IL-2 following ConA injection. These results may oppose the development of PI3K?/? inhibitors as therapeutic agents, particularly for the treatment of human hepatitis. PMID:23969545

Liu, Yuanyuan; Xiong, Li; Chang, Ying; Tang, Jianying; Ang, Wei; Yang, Tao; Pi, Weiyi; Yang, Xiaoyan; Ye, Weiwei; Luo, Youfu; Wang, Zhenling

2013-11-01

73

Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.  

PubMed

Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degradation. Here we show that O-GlcNAc transferase (OGT) harbours a previously unrecognized type of phosphoinositide-binding domain. After induction with insulin, phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma membrane, where the enzyme catalyses dynamic modification of the insulin signalling pathway by O-GlcNAc. This results in the alteration in phosphorylation of key signalling molecules and the attenuation of insulin signal transduction. Hepatic overexpression of OGT impairs the expression of insulin-responsive genes and causes insulin resistance and dyslipidaemia. These findings identify a molecular mechanism by which nutritional cues regulate insulin signalling through O-GlcNAc, and underscore the contribution of this modification to the aetiology of insulin resistance and type 2 diabetes. PMID:18288188

Yang, Xiaoyong; Ongusaha, Pat P; Miles, Philip D; Havstad, Joyce C; Zhang, Fengxue; So, W Venus; Kudlow, Jeffrey E; Michell, Robert H; Olefsky, Jerrold M; Field, Seth J; Evans, Ronald M

2008-02-21

74

Phosphoinositide regulation of inward rectifier potassium (Kir) channels  

PubMed Central

Inward rectifier potassium (Kir) channels are integral membrane proteins charged with a key role in establishing the resting membrane potential of excitable cells through selective control of the permeation of K+ ions across cell membranes. In conjunction with secondary anionic phospholipids, members of this family are directly regulated by phosphoinositides (PIPs) in the absence of other proteins or downstream signaling pathways. Different Kir isoforms display distinct specificities for the activating PIPs but all eukaryotic Kir channels are activated by PI(4,5)P2. On the other hand, the bacterial KirBac1.1 channel is inhibited by PIPs. Recent crystal structures of eukaryotic Kir channels in apo and lipid bound forms reveal one specific binding site per subunit, formed at the interface of N- and C-terminal domains, just beyond the transmembrane segments and clearly involving some of the key residues previously identified as controlling PI(4,5)P2 sensitivity. Computational, biochemical, and biophysical approaches have attempted to address the energetic determinants of PIP binding and selectivity among Kir channel isoforms, as well as the conformational changes that trigger channel gating. Here we review our current understanding of the molecular determinants of PIP regulation of Kir channel activity, including in context with other lipid modulators, and provide further discussion on the key questions that remain to be answered. PMID:24409153

Fürst, Oliver; Mondou, Benoit; D'Avanzo, Nazzareno

2014-01-01

75

Targeting phosphoinositide 3-kinase ? for the treatment of respiratory diseases.  

PubMed

Asthma and chronic obstructive pulmonary disease (COPD) are characterized in their pathogenesis by chronic inflammation in the airways. Phosphoinositide 3-kinase ? (PI3K?), a lipid kinase expressed predominantly in leukocytes, is thought to hold much promise as a therapeutic target for such inflammatory conditions. Of particular interest for the treatment of severe respiratory disease is the observation that inhibition of PI3K? may restore steroid effectiveness under conditions of oxidative stress. PI3K? inhibition may also prevent recruitment of inflammatory cells, including T lymphocytes and neutrophils, as well as the release of proinflammatory mediators, such as cytokines, chemokines, reactive oxygen species, and proteolytic enzymes. In addition, targeting the PI3K? pathway could reduce the incidence of pathogen-induced exacerbations by improving macrophage-mediated bacterial clearance. In this review, we discuss the potential and highlight the unknowns of targeting PI3K? for the treatment of respiratory disease, focusing on recent developments in the role of the PI3K? pathway in inflammatory cell types believed to be critical to the pathogenesis of COPD. PMID:23551101

Sriskantharajah, Srividya; Hamblin, Nicole; Worsley, Sally; Calver, Andrew R; Hessel, Edith M; Amour, Augustin

2013-03-01

76

CD28 co-stimulates TCR/CD3-induced phosphoinositide turnover in human T lymphocytes.  

PubMed

Upon engagement of TCR with peptide-MHC complexes displayed on the surface of antigen-presenting cells, T lymphocytes undergo a sustained elevation of intracellular Ca(2+) concentration([Ca(2+)](i)), which is required for cytokine production. In the present work, we investigate how inositol lipid metabolism can be activated for a prolonged time to ensure a sustained link between receptor triggering and downstream signaling effectors. Four lines of evidence indicate that an extensive phosphoinositide turnover induced by TCR and CD28 engagement allows this task to be accomplished: (i) continuous phosphoinositide breakdown is required for a sustained [Ca(2+)](i )increase in antigen-stimulated T cells; (ii) TCR triggering results in a continuous release of inositol phosphates from the cell membrane paralleled by a massive and sustained phosphoinositide re-synthesis due to free inositol re-incorporation; (iii) TCR-induced phosphoinositide turnover is strongly increased by CD28 ligation; and (iv) CD28 engagement augments and sustains the TCR-induced [Ca(2+)](i )increase. Our results show that the T cell pool of phosphoinositides is continuously re-formed during T cell-APC cognate interaction, thereby explaining how sustained receptor triggering can transduce an equally sustained [Ca(2+)](i) increase. Importantly, our data identify a novel step in the signaling cascade where co-stimulation converges with TCR-generated signals to sustain and amplify the activation process. PMID:11500828

Zaru, R; Berrie, C P; Iurisci, C; Corda, D; Valitutti, S

2001-08-01

77

Probing the regulation of TASK potassium channels by PI4,5P? with switchable phosphoinositide phosphatases.  

PubMed

TASK channels are background K+ channels that contribute to the resting conductance in many neurons. A key feature of TASK channels is the reversible inhibition by Gq-coupled receptors, thereby mediating the dynamic regulation of neuronal activity by modulatory transmitters. The mechanism that mediates channel inhibition is not fully understood. While it is clear that activation of G?q is required, the immediate signal for channel closure remains controversial. Experimental evidence pointed to either phospholipase C (PLC)-mediated depletion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) as the cause for channel closure or to a direct inhibitory interaction of active G?q with the channel. Here, we address the role of PI(4,5)P2 for G-protein-coupled receptor (GPCR)-mediated TASK inhibition by using recently developed genetically encoded tools to alter phosphoinositide (PI) concentrations in the living cell.When expressed in CHO cells, TASK-1- and TASK-3-mediated currents were not affected by depletion of plasma membrane PI(4,5)P2 either via the voltage-activated phosphatase Ci-VSP or via chemically triggered recruitment of a PI(4,5)P2-5'-phosphatase. Depletion of both PI(4,5)P2 and PI(4)P via membrane recruitment of a novel engineered dual-specificity phosphatase also did not inhibit TASK currents. In contrast, each of these methods produced robust inhibition of the bona fide PI(4,5)P2-dependent channel KCNQ4. Efficient depletion of PI(4,5)P2 and PI(4)P was further confirmed with a fluorescent phosphoinositide sensor. Moreover, TASK channels recovered normally from inhibition by co-expressed muscarinic M1 receptors when resynthesis of PI(4,5)P2 was prevented by depletion of cellular ATP. These results demonstrate that TASK channel activity is independent of phosphoinositide concentrations within the physiological range. Consequently, Gq-mediated inhibition of TASK channels is not mediated by depletion of PI(4,5)P2. PMID:21540350

Lindner, Moritz; Leitner, Michael G; Halaszovich, Christian R; Hammond, Gerald R V; Oliver, Dominik

2011-07-01

78

Phosphoinositides direct equine infectious anemia virus gag trafficking and release.  

PubMed

Phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2) ], the predominant phosphoinositide (PI) on the plasma membrane, binds the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P(2) is critical for HIV-1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments; hence, the significance of its interaction with PI(4,5)P(2) is unclear. We therefore investigated the binding in vitro of other PIs to EIAV MA and whether intracellular association with compartments bearing these PIs was important for assembly and release of virus-like particles (VLPs) formed by Gag. In vitro, EIAV MA bound phosphatidylinositol 3-phosphate [PI(3)P] with higher affinity than PI(4,5)P(2) as revealed by nuclear magnetic resonance (NMR) spectra upon lipid titration. Gag was detected on the plasma membrane and in compartments enriched in phosphatidylinositol 3,5-biphosphate [PI(3,5)P(2) ]. Treatment of cells with YM201636, a kinase inhibitor that blocks production of PI(3,5)P(2) from PI(3)P, caused Gag to colocalize with aberrant compartments and inhibited VLP release. In contrast to HIV-1, release of EIAV VLPs was not significantly diminished by coexpression with 5-phosphatase IV, an enzyme that specifically depletes PI(4,5)P(2) from the plasma membrane. However, coexpression with synaptojanin 2, a phosphatase with broader specificity, diminished VLP production. PI-binding pocket mutations caused striking budding defects, as revealed by electron microscopy. One of the mutations also modified Gag-Gag interaction, as suggested by altered bimolecular fluorescence complementation. We conclude that PI-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release. PMID:21176037

Fernandes, Fiona; Chen, Kang; Ehrlich, Lorna S; Jin, Jing; Chen, Min H; Medina, Gisselle N; Symons, Marc; Montelaro, Ronald; Donaldson, Julie; Tjandra, Nico; Carter, Carol A

2011-04-01

79

Phosphoinositide 3-kinase ? inhibits cardiac GSK-3 independently of Akt.  

PubMed

Activation of cardiac phosphoinositide 3-kinase ? (PI3K?) by growth factors, such as insulin, or activation of PI3K? downstream of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors stimulates the activity of the kinase Akt, which phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). We found that PI3K? inhibited GSK-3 independently of the insulin-PI3K?-Akt axis. Although insulin treatment activated Akt in PI3K? knockout mice, phosphorylation of GSK-3 was decreased compared to control mice. GSK-3 is activated when dephosphorylated by the protein phosphatase 2A (PP2A), which is activated when methylated by the PP2A methyltransferase PPMT-1. PI3K? knockout mice showed increased activity of PPMT-1 and PP2A and enhanced nuclear export of the GSK-3 substrate NFATc3. GSK-3 inhibits cardiac hypertrophy, and the hearts of PI3K? knockout mice were smaller compared to those of wild-type mice. Cardiac overexpression of a catalytically inactive PI3K? (PI3K?(inact)) transgene in PI3K? knockout mice reduced the activities of PPMT-1 and PP2A and increased phosphorylation of GSK-3. Furthermore, PI3K? knockout mice expressing the PI3K?(inact) transgene had larger hearts than wild-type or PI3K? knockout mice. Our studies show that a kinase-independent function of PI3K? could directly inhibit GSK-3 function by preventing the PP2A-PPMT-1 interaction and that this inhibition of GSK-3 was independent of Akt. PMID:23354687

Mohan, Maradumane L; Jha, Babal K; Gupta, Manveen K; Vasudevan, Neelakantan T; Martelli, Elizabeth E; Mosinski, John David; Naga Prasad, Sathyamangla V

2013-01-01

80

Insulin activates the alpha isoform of class II phosphoinositide 3-kinase.  

PubMed

The novel class II phosphoinositide (PI) 3-kinases are characterized by the presence of a C-terminal C2 domain, but little is known about their regulation. We find insulin causes a rapid 2-3-fold increase in the activity of PI 3-kinase C2alpha (PI3K-C2alpha) in CHO-IR cells, 3T3-L1 adipocytes, and fully differentiated L5L6 myotubes. No insulin-induced activation of PI3K-C2alpha was observed in cell types known to have low responsiveness to insulin including HEK 293 cells, 3T3-L1 preadipocytes, and undifferentiated L5L6 myoblasts. The mechanism of activation of PI3K-C2alpha by insulin differs from that of class Ia PI 3-kinases in that insulin stimulation did not cause PI3K-C2alpha to associate with IRS-1 or insulin receptor. PI3K-C2alpha existed as a doublet, and insulin stimulation caused a redistribution from the lower molecular weight band to the higher molecular weight band, suggesting phosphorylation-induced bandshift. Consistent with this, in vitro phosphatase treatment reduced the intensity of the upper band back to that seen in unstimulated cells. This suggests that insulin-induced phosphorylation could play a role in regulation of the activity of PI3K-C2alpha. The finding that insulin activates PI3K-C2alpha in cell types known to possess a wide range of responses to insulin suggests that PI3K-C2alpha is a novel component of insulin-stimulated signaling cascades. PMID:10329640

Brown, R A; Domin, J; Arcaro, A; Waterfield, M D; Shepherd, P R

1999-05-21

81

Reduced Phosphoinositide 3-Kinase (p110?) Activation Increases the Susceptibility to Atrial Fibrillation  

PubMed Central

Atrial fibrillation (AF) is the most common sustained arrhythmia presenting at cardiology departments. A limited understanding of the molecular mechanisms responsible for the development of AF has hindered treatment strategies. The purpose of this study was to assess whether reduced activation of phosphoinositide 3-kinase (PI3K, p110?) makes the compromised heart susceptible to AF. Risk factors for AF, including aging, obesity, and diabetes, have been associated with insulin resistance that leads to depressed/defective PI3K signaling. However, to date, there has been no link between PI3K(p110?) and AF. To address this question, we crossed a cardiac-specific transgenic mouse model of dilated cardiomyopathy (DCM) with a cardiac-specific transgenic mouse expressing a dominant negative mutant of PI3K (dnPI3K; reduces PI3K activity). Adult (?4.5 months) double-transgenic (dnPI3K-DCM), single-transgenic (DCM-Tg, dnPI3K-Tg), and nontransgenic mice were subjected to morphological, functional/ECG, microarray, and biochemical analyses. dnPI3K-DCM mice developed AF and had depressed cardiac function as well as greater atrial enlargement and fibrosis than DCM-Tg mice. AF was not detected in other groups. Aged DCM-Tg mice (?15 months) with a similar phenotype to dnPI3K-DCM mice (4.5 months) did not develop AF, suggesting loss of PI3K activity directly contributed to the AF phenotype. Furthermore, increasing PI3K activity reduced atrial fibrosis and improved cardiac conduction in DCM-Tg mice. Finally, in atrial appendages from patients with AF, PI3K activation was lower compared with tissue from patients in sinus rhythm. These results suggest a link between PI3K(p110?) and AF. PMID:19679877

Pretorius, Lynette; Du, Xiao-Jun; Woodcock, Elizabeth A.; Kiriazis, Helen; Lin, Ruby C.Y.; Marasco, Silvana; Medcalf, Robert L.; Ming, Ziqiu; Head, Geoffrey A.; Tan, Joon Win; Cemerlang, Nelly; Sadoshima, Junichi; Shioi, Tetsuo; Izumo, Seigo; Lukoshkova, Elena V.; Dart, Anthony M.; Jennings, Garry L.; McMullen, Julie R.

2009-01-01

82

Phosphoinositide 3-kinase at the crossroad between endocytosis and signaling of cytokine receptors  

PubMed Central

Class I phosphoinositide 3-kinase (PI3K) is a lipid kinase playing key roles in many signaling pathways regulating cell survival and growth. Besides its important role in signal transduction, PI3K is also involved in actin and membrane reorganization such as protrusion, adhesion, phagocytosis and macropinocytosis. Receptor-mediated endocytosis is initiated by plasma membrane reorganization creating buds that then mature to small vesicles. Whereas most of endocytic mechanisms involve actin polymerization, PI3K requirement has not been clearly investigated. Our study identifies class I PI3K as a key player in clathrin-independent endocytosis of the interleukin 2 receptor (IL-2R) in contrast to the clathrin-dependent entry of transferrin (Tf). IL-2R is a cytokine receptor, inducing several signaling cascades such as PI3K, that are essential for the immune response. We have shown previously that IL-2R can be internalized with or without IL-2 and this process requires dynamin, actin and their regulators cortactin, N-WASP, Rac1 and the kinases Pak. Our recent work reveals that PI3K regulates Rac1 during IL-2R uptake in two ways: via its catalytic activity (p110) and via its regulatory factor (p85). Indeed, the catalytic activity of PI3K is required for both constitutive and IL-2 induced uptake of cytokine receptors, in lymphocytes as well as in epithelial cells. Interestingly, Vav2, a Rac1 GTPase exchange factor (GEF) induced upon PI3K activation, is specifically involved and recruited during IL-2R uptake. The second action of PI3K is via its regulatory subunit, p85, which binds activated Rac1 and IL-2R; this interaction being enhanced upon IL-2 treatment. Thus, PI3K regulates both the activation of Rac1 and its recruitment during IL-2R endocytosis. Finally, our results identify a link between cytokine receptors signaling and clathrin-independent endocytosis. PMID:23986799

Basquin, Cyril; Sauvonnet, Nathalie

2013-01-01

83

Phosphoinositide 3-kinase at the crossroad between endocytosis and signaling of cytokine receptors.  

PubMed

Class I phosphoinositide 3-kinase (PI3K) is a lipid kinase playing key roles in many signaling pathways regulating cell survival and growth. Besides its important role in signal transduction, PI3K is also involved in actin and membrane reorganization such as protrusion, adhesion, phagocytosis and macropinocytosis. Receptor-mediated endocytosis is initiated by plasma membrane reorganization creating buds that then mature to small vesicles. Whereas most of endocytic mechanisms involve actin polymerization, PI3K requirement has not been clearly investigated. Our study identifies class I PI3K as a key player in clathrin-independent endocytosis of the interleukin 2 receptor (IL-2R) in contrast to the clathrin-dependent entry of transferrin (Tf). IL-2R is a cytokine receptor, inducing several signaling cascades such as PI3K, that are essential for the immune response. We have shown previously that IL-2R can be internalized with or without IL-2 and this process requires dynamin, actin and their regulators cortactin, N-WASP, Rac1 and the kinases Pak. Our recent work reveals that PI3K regulates Rac1 during IL-2R uptake in two ways: via its catalytic activity (p110) and via its regulatory factor (p85). Indeed, the catalytic activity of PI3K is required for both constitutive and IL-2 induced uptake of cytokine receptors, in lymphocytes as well as in epithelial cells. Interestingly, Vav2, a Rac1 GTPase exchange factor (GEF) induced upon PI3K activation, is specifically involved and recruited during IL-2R uptake. The second action of PI3K is via its regulatory subunit, p85, which binds activated Rac1 and IL-2R; this interaction being enhanced upon IL-2 treatment. Thus, PI3K regulates both the activation of Rac1 and its recruitment during IL-2R endocytosis. Finally, our results identify a link between cytokine receptors signaling and clathrin-independent endocytosis. PMID:23986799

Basquin, Cyril; Sauvonnet, Nathalie

2013-07-01

84

The basal level of intracellular calcium gates the activation of phosphoinositide 3-kinase-Akt signaling by brain-derived neurotrophic factor in cortical neurons.  

PubMed

Brain-derived neurotrophic factor (BDNF) mediates survival and neuroplasticity through the activation of phosphoinositide 3-kinase-Akt pathway. Although previous studies suggested the roles of mitogen-activated protein kinase, phospholipase C-gamma-mediated intracellular calcium ([Ca2+]i) increase, and extracellular calcium influx in regulating Akt activation, the cellular mechanisms are largely unknown. We demonstrated that sub-nanomolar BDNF significantly induced Akt activation in developing cortical neurons. The TrkB-dependent Akt phosphorylation at S473 and T308 required only phosphoinositide 3-kinase, but not phospholipase C and mitogen-activated protein kinase activity. Blocking NMDA receptors, L-type voltage-gated calcium channels, and chelating extracellular calcium by EGTA failed to block BDNF-induced Akt phosphorylation. In contrast, chelating [Ca2+]i by 1,2-bis(o-aminophenoxy)ethane-N,N,N ',N '-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) abolished Akt phosphorylation. Interestingly, sub-nanomolar BDNF did not stimulate [Ca2+]i increase under our culture conditions. Together with that NMDA- and membrane depolarization-induced [Ca2+]i increase did not activate Akt, we conclude that the basal level of [Ca2+]i gates BDNF function. Furthermore, inhibiting calmodulin by W13 suppressed Akt phosphorylation. On the other hand, inhibition of protein phosphatase 1 by okadaic acid and tautomycin rescued Akt phosphorylation in BAPTA-AM and W13-treated neurons. We further demonstrated that the phosphorylation of phosphoinositide-dependent kinase-1 did not correlate with Akt phosphorylation at T308. Our results suggested novel roles of basal [Ca2+]i, rather than activity-induced calcium elevation, in BDNF-Akt signaling. PMID:18485103

Zheng, Fei; Soellner, Deborah; Nunez, Joseph; Wang, Hongbing

2008-08-01

85

Triggering Actin Comets Versus Membrane Ruffles: Distinctive Effects of Phosphoinositides on Actin Reorganization  

PubMed Central

A limited set of phosphoinositide membrane lipids regulate diverse cellular functions including proliferation, differentiation, and migration. We developed two techniques based on rapamycin-induced protein dimerization to rapidly change the concentration of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. First, we increased PI(4,5)P2 synthesis from phosphatidylinositol 4-phosphate [PI(4)P] using a membrane recruitable form of PI(4)P 5-kinase, and found that COS-7, HeLa, and HEK293 cells formed bundles of motile actin filaments known as actin comets. In contrast, a second technique that increased the concentration of PI(4,5)P2 without consuming PI(4)P induced membrane ruffles. These distinct phenotypes were mediated by dynamin-mediated vesicular trafficking and mutually inhibitory crosstalk between the small guanosine triphosphatases Rac and RhoA. Our results indicate that the effect of PI(4,5)P2 on actin reorganization depends on the abundance of other phosphoinositides, such as PI(4)P. Thus, combinatorial regulation of phosphoinositide concentrations may contribute to the diversity of phosphoinositide functions. PMID:22169478

Ueno, Tasuku; Falkenburger, Björn H.; Pohlmeyer, Christopher; Inoue, Takanari

2012-01-01

86

Oncogenic mutations mimic and enhance dynamic events in the natural activation of phosphoinositide  

E-print Network

Oncogenic mutations mimic and enhance dynamic events in the natural activation of phosphoinositide110/p85 and a spectrum of oncogenic mutants using hydrogen/deuterium exchange mass spectrometry (HDX to an activated form on membranes entails four distinct events. Analysis of oncogenic mutations shows that all up

Williams, Roger L.

87

Original Paper Abstract: Phosphoinositide-specific phospholipase C (PI-PLC)  

E-print Network

Original Paper Abstract: Phosphoinositide-specific phospholipase C (PI-PLC) hydrolyzes the membrane. DNA synthesis, a measure of cell growth, was significantly suppressed by the PI-PLC-specific inhibitor,5-pyrrolidinedione did not cause any suppression. Acti- vation of PI-PLC by MS salts increased DNA synthesis and abol

Bhattacharyya, Madan Kumar

88

Initiation and maintenance of NGF-stimulated neurite outgrowth requires activation of a phosphoinositide 3-kinase  

PubMed Central

Summary Application of nerve growth factor (NGF) to PC12 cells stimulates a programme of physiological changes leading to the development of a sympathetic neuron like phenotype, one aspect of which is the development of a neuronal morphology characterised by the outgrowth of neuritic processes. We have investigated the role of phosphoinositide 3-kinase in NGF-stimulated morphological differentiation through two approaches: firstly, preincubation with wortmannin, a reputedly specific inhibitor of phosphoinositide kinases, completely inhibited initial morphological responses to NGF, the formation of actin filament rich microspikes and subsequent neurite outgrowth. This correlated with wortmannin inhibition of NGF-stimulated phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) and phosphatidylinositol(3,4)bisphosphate (PtdIns(3,4)P2) production and with inhibition of NGF-stimulated phosphoinositide 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Secondly, the overexpression of a mutant p85 regulatory subunit of the phosphoinositide 3-kinase, which cannot interact with the catalytic p110 subunit, also substantially inhibited the initiation of NGF-stimulated neurite outgrowth. In addition, we found that wortmannin caused a rapid collapse of more mature neurites formed following several days exposure of PC12 cells to NGF. These results indicate that NGF-stimulated neurite outgrowth requires the activity of a tyrosine kinase regulated PI3-kinase and suggest that the primary product of this enzyme, PtdInsP3, is a necessary second messenger for the cytoskeletal and membrane reorganization events which occur during neuronal differentiation. PMID:8838652

Jackson, Trevor R.; Blader, Ira J.; Hammonds-Odie, Latanya P.; Burga, Christina R.; Cooke, Frank; Hawkins, Phillip T.; Wolf, Andrea G.; Heldman, Katherine A.; Theibert, Anne B.

2015-01-01

89

Phosphoinositide 3-kinase gamma in T cell biology and disease therapy.  

PubMed

Phosphoinositide 3-kinase gamma (PI3K?) kinase activity is important for its signaling functions in T cell development, activation, differentiation, and trafficking. Protection of PI3K? knockout mice from disease in multiple autoimmune models suggests that targeting PI3K? alone, or in combination with PI3K?, could be a promising approach to disease therapy. PMID:23551102

Fung-Leung, Wai-Ping

2013-03-01

90

Competitive fluorescence polarization assays for the detection of phosphoinositide kinase and phosphatase activity.  

PubMed

We describe the development and implementation of competitive fluorescence polarization (FP) based assays for determining activity of phosphoinositide 3-kinase (PI 3-K) and the type-II SH2-domain-containing inositol 5-phosphatase (SHIP2). These assays are based on the interaction of specific phosphoinositide binding proteins with fluorophore-labeled phosphoinositide and inositol phosphate tracers. Enzyme reaction products are detected by their ability to compete with the fluorescent tracers for protein binding, leading to an increase in the amount of free tracer and a decrease in polarization (mP) values. A variety of fluorophore-labeled tracers were evaluated, and assay sensitivity and specificity for products of PI 3-K and SHIP2 activity was determined. Assay performance was evaluated using recombinant PI 3-Kalpha and SHIP2 with diC(8)-PI(4,5)P(2) and diC(8)-PI(3,4,5)P(3) as respective substrates. IC(50) values for previously characterized PI 3-K inhibitors were within expected ranges. These assays are homogeneous, sensitive, and rapid, and suitable for HTS applications, and will facilitate screening for novel inhibitors of phosphoinositide kinases and phosphatases in drug development. PMID:12769675

Drees, Beth E; Weipert, Amber; Hudson, Heather; Ferguson, Colin G; Chakravarty, Leena; Prestwich, Glenn D

2003-06-01

91

AGE-RELATED CHANGES IN RECEPTOR-MEDIATED PHOSPHOINOSITIDE HYDROLYSIS IN VARIOUS REGIONS OF RAT BRAIN  

EPA Science Inventory

The effects of age on cholinergic markers and receptor-stimulated phosphoinositide hydrolysis was dined in the frontal cortex and striatum of male Fischer-344 rats. holine acetyltransferase activity was decreased 27% in the striatum of aged (24 month) rats cared to young (3 month...

92

ERBB-2 overexpression confers PI 3 ? kinase-dependent invasion capacity on human mammary epithelial cells  

PubMed Central

Amplification and overexpression of ERBB-2 in human breast cancer is thought to play a significant role in the progression of the disease; however, its precise role in the aetiology of altered phenotypes associated with human breast cancer is unknown. We have previously shown that exogenous overexpression of ERBB-2 conferred growth factor independence on human mammary epithelial cells. In this study, we show that ERBB-2 overexpression also causes the cells to acquire other characteristics exhibited by human breast cancer cells, such as anchorage-independent growth and invasion capabilities. ERBB-2-induced invasion is dependent on fibronectin and correlates with the down-regulation of cell surface ?4 integrin. In addition ERBB-2 co-immunoprecipitates with focal adhesion kinase (FAK) in these cells. We have also shown, by use of exogenously expressed PTEN and by treatment with the PI3?-kinase inhibitor LY294002, that ERBB-2-induced invasion is dependent on the PI3?-kinase pathway; however, PTEN does not dephosphorylate FAK in these cells. © 2000 Cancer Research Campaign PMID:10682681

Ignatoski, K M Woods; Maehama, T; Markwart, S M; Dixon, J E; Livant, D L; Ethier, S P

2000-01-01

93

Regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) by Src involves tyrosine phosphorylation of PDK1 and Src homology 2 domain binding.  

PubMed

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr(9) and Tyr(373/376)) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr(9) antibodies showed that the level of Tyr(9) phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr(9), distinct from Tyr(373/376), is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr(9) phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation. PMID:18024423

Yang, Keum-Jin; Shin, Sanghee; Piao, Longzhen; Shin, Eulsoon; Li, Yuwen; Park, Kyeong Ah; Byun, Hee Sun; Won, Minho; Hong, Janghee; Kweon, Gi Ryang; Hur, Gang Min; Seok, Jeong Ho; Chun, Taehoon; Brazil, Derek P; Hemmings, Brian A; Park, Jongsun

2008-01-18

94

Research article Expression and evolution of the phosphoinositide-specific phospholipase C gene family in Arabidopsis thaliana  

Microsoft Academic Search

Phosphoinositide-specific phospholipase C cleaves the substrate phosphatidylinositol 4,5-bisphosphate and generates inositol 1,4,5- trisphosphate and 1,2-diacylglycerol, both of which are second messengers in the phosphoinositide signal transduction pathways operative in animal cells. Five PI-PLC isoforms, b, g, d, 3 and z, have been identified in mammals. Plant PI-PLCs are structurally close to the mammalian PI-PLC-z isoform. The Arabidopsis genome contains nine

I. Made Tasma; Volker Brendel; Steven A. Whitham; Madan K. Bhattacharyya

95

Diverse Kir modulators act in close proximity to residues implicated in phosphoinositide binding  

PubMed Central

Inwardly rectifying potassium (Kir) channels were the first shown to be directly activated by phosphoinositides in general and phosphatidylinositol bisphosphate (PIP2) in particular. Atomic resolution structures have been determined for several mammalian and bacterial Kir channels. Basic residues, identified through mutagenesis studies to contribute to the sensitivity of the channel to PIP2, have been mapped onto the three dimensional channel structure and their localization has given rise to a plausible model that can explain channel activation by PIP2. Moreover, mapping onto the three-dimensional channel structure sites involved in the modulation of Kir channel activity by a diverse group of regulatory molecules, revealed a striking proximity to residues implicated in phosphoinositide binding. These observations support the hypothesis that the observed dependence of diverse modulators on channel–PIP2 interactions stems from their localization within distances that can affect PIP2-interacting residues. PMID:17495041

Logothetis, Diomedes E; Lupyan, Dmitry; Rosenhouse-Dantsker, Avia

2007-01-01

96

Atrial natriuretic peptide inhibits the phosphoinositide hydrolysis in murine Leydig tumor cells  

Microsoft Academic Search

The ability of ANP to inhibit the hydrolysis of phosphoinositides was examined in [3H] myoinositol-labeled intact murine Leydig tumor (MA-10) cells. Arginine vasopressin (AVP) stimulated the formation of inositol monophosphate (IP1), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) both in a time- and dose- dependent manner in MA-10 cells. ANP inhibited the AVP-induced formation of IP1, IP2, and IP3 in

Madan L. Khurana; Kailash N. Pandey

1996-01-01

97

Thrombin-induced phosphoinositide hydrolysis in platelets. Receptor occupancy and desensitization.  

PubMed Central

The relationship between occupancy of thrombin receptors on platelets and enhanced phosphoinositide hydrolysis was analysed by examination of the dose-response relationship, the effects of thrombin inhibitors and the contribution of secondary effects. Washed human platelets were labelled with [3H]inositol, and agonist-induced accumulation of labelled inositol phosphates was measured. The dose-response curves and the time courses for alpha-thrombin- or gamma-thrombin-induced accumulation of inositol phosphates were similar to those for dense-granule secretion. Addition of the thrombin inhibitor hirudin to thrombin-activated platelets revealed that the continuous presence of active thrombin was required to maintain the accumulation of labelled inositol phosphates; the total production of inositol phosphates increased with longer periods of exposure to thrombin, reaching a maximum between 5 and 10 min. After activation with thrombin, the ability of a second, greater, addition of thrombin to induce additional phosphoinositide hydrolysis decreased with time; it was absent within 10 min after the first addition. The failure to sustain accumulation of labelled inositol phosphates or to respond to a second addition of thrombin beyond 10 min was not due to depletion of the pool of labelled precursors, because the platelets retained their ability to respond to collagen. Addition of ADP-consuming enzymes decreased sensitivity to thrombin, but inhibition of cyclo-oxygenase with indomethacin did not impair the thrombin-induced hydrolysis of phosphoinositides. It was concluded that thrombin-induced hydrolysis of phosphoinositides has characteristics consistent with mediation by a receptor that is similar to that that triggers dense-granule secretion, requires continuous presence of active thrombin to be maintained, is mediated by a receptor that displays thrombin-induced desensitization, and is only partially enhanced by secondary agents. PMID:3036080

Huang, E M; Detwiler, T C

1987-01-01

98

Fusion, Leakage and Surface Hydrophobicity of Vesicles Containing Phosphoinositides: Influence of Steric and Electrostatic Effects  

Microsoft Academic Search

Calcium and lanthanum ion-induced fusion of lipid vesicles containing phosphatidylinositol (PI), phosphatidylinositol-4-monophosphate (PIP), phosphatidylinositol-4,5-bisphosphate (PIP2) or phosphatidylinositol-3,4,5-trisphosphate (PIP3) and its associated membrane properties, e.g., surface dielectric constant and vesicle leakage, were studied by fluorescence methods. The presence of poly-phosphorylated phosphoinositides (PPI) in lipid vesicles enhanced fusion, depending on the PPI phosphorylation level and the PPI concentration, as determined by the

M. Müller; O. Zschörnig; S. Ohki; K. Arnold

2003-01-01

99

Alterations in phosphoinositide signaling and G-protein levels in depressed suicide brain  

Microsoft Academic Search

The function of the phosphoinositide signal transduction system and the levels of heterotrimeric G-protein ?-subunits were examined in postmortem prefrontal cortex regions (8\\/9) and region (10) from suicide victims with major depression and matched control subjects without psychiatric illness. The hydrolysis of [3H]phosphatidylinositol (PI) stimulated by phospholipase C, GTP-?-S, NaF, and neurotransmitter receptor agonists was measured in membrane preparations from

Mary A. Pacheco; Craig Stockmeier; Herbert Y. Meltzer; James C. Overholser; Ginny E. Dilley; Richard S. Jope

1996-01-01

100

Phosphoinositide3 Kinase-Akt Pathway Controls Cellular Entry of Ebola Virus  

Microsoft Academic Search

The phosphoinositide-3 kinase (PI3K) pathway regulates diverse cellular activities related to cell growth, migration, survival, and vesicular trafficking. It is known that Ebola virus requires endocytosis to establish an infection. However, the cellular signals that mediate this uptake were unknown for Ebola virus as well as many other viruses. Here, the involvement of PI3K in Ebola virus entry was studied.

Mohammad F. Saeed; Andrey A. Kolokoltsov; Alexander N. Freiberg; Michael R. Holbrook; Robert A. Davey

2008-01-01

101

TRPV1 channels are intrinsically heat sensitive and negatively regulated by phosphoinositide lipids  

PubMed Central

Summary The capsaicin receptor, TRPV1, is regulated by phosphatidylinositol-4,5-bisphosphate (PIP2), although the precise nature of this effect (i.e., positive or negative) remains controversial. Here, we reconstitute purified TRPV1 into artificial liposomes, where it is gated robustly by capsaicin, protons, spider toxins and, notably, heat, demonstrating intrinsic sensitivity of the channel to both chemical and thermal stimuli. TRPV1 is fully functional in the absence of phosphoinositides, arguing against their proposed obligatory role in channel activation. Rather, introduction of various phosphoinositides, including PIP2, PI4P and PI, inhibits TRPV1, supporting a model whereby phosphoinositide turnover contributes to thermal hyperalgesia by disinhibiting the channel. Using an orthogonal chemical strategy, we show that association of the TRPV1 C-terminus with the bilayer modulates channel gating, consistent with phylogenetic data implicating this domain as a key regulatory site for tuning stimulus sensitivity. Beyond TRPV1, these findings are relevant to understanding how membrane lipids modulate other “receptor-operated” TRP channels. PMID:23439120

Cao, Erhu; Cordero-Morales, Julio F.; Liu, Beiying; Qin, Feng; Julius, David

2013-01-01

102

Fetal bovine serum induces changes in fatty acid composition of Trypanosoma cruzi phosphoinositides.  

PubMed

Fetal bovine serum (FBS) is a necessary constituent of the culture media employed to foster the growth of Trypanosoma cruzi epimastigote forms. In different laboratories, the serum is used at final concentrations of 5 or 10%. We have normally supplemented the complex medium with 10% FBS. Under this condition we have described the fatty acid composition of the total lipids and of the phosphoinositide fractions. Additionally, we have reported the increase of polyphosphoinositides and phosphatidic acid after cholinergic stimulation. Since further attempts to reproduce these results with 5% FBS in the culture medium were not successful, the effect of the FBS concentration on the fatty acid composition of phospholipids from the T. cruzi epimastigote forms was thoroughly examined. This work showed that when the FBS concentration supplementing the culture medium was reduced from 10 to 5%, the fatty acid composition of the phosphoinositides was altered while the other major phospholipids were not significantly affected. The most relevant result was the decrease in the content of linoleic acid (18:2) and the increase of palmitoleic acid (16:1) in phosphatidylinositol 4,5-bisphosphate. Phosphatidylinositol (PI) and phosphatidylinositol phosphate also exhibited similar changes in the same fatty acids. The C2 fatty acid composition of the phosphoinositides, under the same conditions, is also reported here for the first time. PMID:8590410

Racagni, G; de Lema, M G; Hernández, G; Machado-Domenech, E E

1995-10-01

103

Ankyrin-G palmitoylation and ?II-spectrin binding to phosphoinositide lipids drive lateral membrane assembly  

PubMed Central

Ankyrin-G and ?II-spectrin colocalize at sites of cell–cell contact in columnar epithelial cells and promote lateral membrane assembly. This study identifies two critical inputs from lipids that together provide a rationale for how ankyrin-G and ?II-spectrin selectively localize to Madin-Darby canine kidney (MDCK) cell lateral membranes. We identify aspartate-histidine-histidine-cysteine 5/8 (DHHC5/8) as ankyrin-G palmitoyltransferases required for ankyrin-G lateral membrane localization and for assembly of lateral membranes. We also find that ?II-spectrin functions as a coincidence detector that requires recognition of both ankyrin-G and phosphoinositide lipids for its lateral membrane localization. DHHC5/8 and ?II-spectrin colocalize with ankyrin-G in micrometer-scale subdomains within the lateral membrane that are likely sites for palmitoylation of ankyrin-G. Loss of either DHHC5/8 or ankyrin-G–?II-spectrin interaction or ?II-spectrin–phosphoinositide recognition through its pleckstrin homology domain all result in failure to build the lateral membrane. In summary, we identify a functional network connecting palmitoyltransferases DHHC5/8 with ankyrin-G, ankyrin-G with ?II-spectrin, and ?II-spectrin with phosphoinositides that is required for the columnar morphology of MDCK epithelial cells. PMID:25049274

He, Meng; Abdi, Khadar M.

2014-01-01

104

ERK and phosphoinositide 3-kinase temporally coordinate different modes of actin-based motility during embryonic wound healing  

PubMed Central

Summary Embryonic wound healing provides a perfect example of efficient recovery of tissue integrity and homeostasis, which is vital for survival. Tissue movement in embryonic wound healing requires two functionally distinct actin structures: a contractile actomyosin cable and actin protrusions at the leading edge. Here, we report that the discrete formation and function of these two structures is achieved by the temporal segregation of two intracellular upstream signals and distinct downstream targets. The sequential activation of ERK and phosphoinositide 3-kinase (PI3K) signalling divides Xenopus embryonic wound healing into two phases. In the first phase, activated ERK suppresses PI3K activity, and is responsible for the activation of Rho and myosin-2, which drives actomyosin cable formation and constriction. The second phase is dominated by restored PI3K signalling, which enhances Rac and Cdc42 activity, leading to the formation of actin protrusions that drive migration and zippering. These findings reveal a new mechanism for coordinating different modes of actin-based motility in a complex tissue setting, namely embryonic wound healing. PMID:23986484

Li, Jingjing; Zhang, Siwei; Soto, Ximena; Woolner, Sarah; Amaya, Enrique

2013-01-01

105

Phosphoinositide Kinase-3 Status Associated With Presence or Absence of Human Papillomavirus in Head and Neck Squamous Cell Carcinomas  

SciTech Connect

Purpose: To investigate phosphoinositide kinase-3 (PI3K) activation in relation to human papillomavirus (HPV) status in head and neck squamous cell carcinoma (HNSCC). Methods and Materials: Gene expression microarray data were analyzed to determine differentially expressed genes between HPV(+) and HPV(-) HNSCC. PIK3CA gene expression was confirmed by quantitative reverse transcriptase-polymerase chain reaction in seven HPV(+) and seven HPV(-) primary HNSCCs. PIK3CA mutation status in three HPV(+) and nine HPV(-) cell lines was determined by polymerase chain reaction amplification of hot spot exons (1, 9, 20) followed by direct sequencing. Results: PIK3CA was overexpressed in HPV(+)-associated HNSCC compared with the expression in HPV(-) HNSCC. Activation of PIK3CA by mutation was found in 1 of the 12 tested HNSCC cell lines. Conclusion: Activation of PI3K by mutation of PIK3CA is rare in HNSCC cell lines and was not found in three HPV(+) cell lines. One mechanism by which HPV-associated HNSCC might activate PI3K is increased expression of PIK3CA.

Yarbrough, Wendell G. [Department of Otolaryngology, Vanderbilt University, Nashville, TN (United States); Department of Cancer Biology, Vanderbilt University, Nashville, TN (United States); Ingram Cancer Center, Vanderbilt University, Nashville, TN (United States)], E-mail: Wendell.yarbrough@vanderbilt.edu; Whigham, Amy; Brown, Brandee [Department of Otolaryngology, Vanderbilt University, Nashville, TN (United States); Roach, Michael [Department of Cancer Biology, Vanderbilt University, Nashville, TN (United States); Slebos, Robbert [Department of Otolaryngology, Vanderbilt University, Nashville, TN (United States); Department of Cancer Biology, Vanderbilt University, Nashville, TN (United States)

2007-10-01

106

C5a controls TLR-induced IL-10 and IL-12 production independent of phosphoinositide 3-kinase.  

PubMed

The complement system is a classic central player in innate immunity. Most pathogens activate both complement and the toll-like receptor (TLR) pathway. Therefore, to provide a more comprehensive understanding of innate immunity, it is important to understand the crosstalk between these two systems. Mouse macrophages produce IL-12 and IL-10 in response to TLR ligands such as LPS, CpG, Poly I:C and Malp2. The TLR-induced IL-12 production was decreased, while that of IL-10 was increased by concurrent stimulation with a complement fragment C5a. Pharmacological studies have suggested that C5a regulates TLR4-induced IL-12 production in a phosphoinositide 3-kinase (PI3K)-dependent mechanism. In the present study, however, we found that the C5a-mediated changes can be observed in macrophages from mice lacking PI3K p85? or PI3K p110?. The result indicates that the C5a action is PI3K-independent; neither class IA nor class IB PI3K subtype is involved in this regulation. The actions of C5a were sensitive to pertussis toxin and PD98059, suggesting a role of G protein-mediated activation of the Erk1/2 pathway. PMID:21106540

Okazaki, Natsumi; Hazeki, Kaoru; Izumi, Tatsuhiro; Nigorikawa, Kiyomi; Hazeki, Osamu

2011-03-01

107

Phosphoinositide 3-kinase C2alpha is activated upon smooth muscle cell migration and regulated by alpha(v)beta(3) integrin engagement.  

PubMed

The involvement of phosphoinositide 3-kinase C2alpha in vascular smooth muscle cell migration was investigated. Products of phosphoinositide 3-kinase, phosphatidylinositol-3-phosphate, and phosphatidylinositol-3,4-bis-phosphate were increased upon smooth muscle cell migration but their synthesis was affected only partially by phosphoinositide 3-kinase inhibitors, wortmannin and LY-294002. Using specific antibody, we showed that the wortmannin/LY-294002 poorly sensitive phosphoinositide 3-kinase C2alpha is expressed in smooth muscle cells. Measurement of phosphoinositide 3-kinase C2alpha activity in vitro, after immunoprecipitation, clearly demonstrated its activation upon smooth muscle cell migration. Moreover, for the first time, phosphoinositide 3-kinase C2alpha was found to be differentially regulated by alpha(v)beta(3) and alpha(v)beta(5) integrin engagement. Finally, we have identified two new potential phosphoinositide 3-kinase C2alpha-binding proteins, p70 and p110, which both may be tyrosine phosphorylated. Thus, phosphoinositide 3-kinase C2alpha might represent a new regulatory pathway of cell migration downstream of integrin engagement. PMID:12237112

Paulhe, Frédérique; Perret, Bertrand; Chap, Hugues; Iberg, Niggi; Morand, Olivier; Racaud-Sultan, Claire

2002-09-20

108

Phosphoinositide 5Phosphatase Fig4p Is Required for both Acute Rise and Subsequent Fall in Stress-Induced Phosphatidylinositol 3,5-Bisphosphate Levels  

Microsoft Academic Search

Phosphoinositide lipids regulate complex events via the recruitment of proteins to a specialized region of the membrane at a specific time. Precise control of both the synthesis and turnover of phosphoinositide lipids is integral to membrane trafficking, signal transduction, and cytoskeletal rearrangements. Little is known about the acute regulation of the levels of these signaling lipids. When Saccharomyces cerevisiae cells

Jason E. Duex; Johnathan J. Nau; Emily J. Kauffman; Lois S. Weisman

2006-01-01

109

Characterization of a G protein-activated phosphoinositide 3-kinase in vascular smooth muscle cell nuclei.  

PubMed

Recent studies highlight the existence of an autonomous nuclear polyphosphoinositide metabolism related to cellular proliferation and differentiation. However, only few data document the nuclear production of the putative second messengers, the 3-phosphorylated phosphoinositides, by the phosphoinositide 3-kinase (PI3K). In the present paper, we examine whether GTP-binding proteins can directly modulate 3-phosphorylated phosphoinositide metabolism in membrane-free nuclei isolated from pig aorta smooth muscle cells (VSMCs). In vitro PI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), whereas guanosine 5'-(beta-thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTPgammaS-induced PtdIns(3,4,5)P(3) synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P(3) accumulation, suggesting that nuclear G(i)/G(0) proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of Galpha(0) proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85alpha subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3Kgamma. These results indicate the existence of a G protein-activated PI3K inside VSMC nucleus that might be involved in the control of VSMC proliferation and in the pathogenesis of vascular proliferative disorders. PMID:11303021

Bacqueville, D; Déléris, P; Mendre, C; Pieraggi, M T; Chap, H; Guillon, G; Perret, B; Breton-Douillon, M

2001-06-22

110

Characterization of cholinergic muscarinic receptor-stimulated phosphoinositide metabolism in brain from immature rats  

SciTech Connect

Hydrolysis of phosphoinositides elicited by stimulation of cholinergic muscarinic receptors has been studied in brain from neonatal (7-day-old) rats in order to determine: (1) whether the neonatal rat could provide a good model system to study this signal-transduction pathway; and (2) whether potential differences with adult nerve tissue would explain the differential, age-related effects of cholinergic agonists. Accumulation of (3H) inositol phosphates in (3H)inositol prelabeled slices from neonatal and adult rats was measured as an index of phosphoinositide metabolism. Full (acetylcholine, methacholine, carbachol) and partial (oxotremorine, bethanechol) agonists had qualitatively similar, albeit quantitatively different, effects in neonatal and adult rats. Atropine and pirenzepine effectively blocked the carbachol-induced response with inhibition constants of 1.2 and 20.7 nM, respectively. In all brain areas, response to all agonists was higher in neonatal than adult rats, and in hippocampus and cerebral cortex the response was higher than in cerebellum or brainstem. The relative intrinsic activity of partial agonists was higher in the latter two areas (0.6-0.7) than in the former two (0.3-0.4). Carbachol-stimulated phosphoinositide metabolism in brain areas correlated well with the binding of (3H)QNB (r2 = 0.627) and, particularly, with (3H)pirenzepine (r2 = 0.911). In cerebral cortex the effect of carbachol was additive to that of norepinephrine and glutamate. The presence of calcium (250-500 microM) was necessary for maximal response to carbachol to be elicited; the EC50 value for Ca2+ was 65.4 microM. Addition of EDTA completely abolished the response. Removal of sodium ions from the incubation medium reduced the response to carbachol by 50%.

Balduini, W.; Murphy, S.D.; Costa, L.G. (Univ. of Washington, Seattle (USA))

1990-05-01

111

Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells  

PubMed Central

Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3K? (phosphoinositide 3-kinase ?) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3K? in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3K? is pre-formed as a 1:1 p110?:p85? complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3K? was recruited to the PM, but oncogenic forms of PI3K? were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3K?. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity. PMID:24597785

Layton, Meredith J.; Rynkiewicz, Natalie K.; Ivetac, Ivan; Horan, Kristy A.; Mitchell, Christina A.; Phillips, Wayne A.

2014-01-01

112

Phosphoinositide-mediated oligomerization of a defensin induces cell lysis  

PubMed Central

Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique ‘cationic grip’ configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. DOI: http://dx.doi.org/10.7554/eLife.01808.001 PMID:24692446

Poon, Ivan KH; Baxter, Amy A; Lay, Fung T; Mills, Grant D; Adda, Christopher G; Payne, Jennifer AE; Phan, Thanh Kha; Ryan, Gemma F; White, Julie A; Veneer, Prem K; van der Weerden, Nicole L; Anderson, Marilyn A; Kvansakul, Marc; Hulett, Mark D

2014-01-01

113

Phosphoinositide-mediated oligomerization of a defensin induces cell lysis.  

PubMed

Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique 'cationic grip' configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. DOI: http://dx.doi.org/10.7554/eLife.01808.001. PMID:24692446

Poon, Ivan Kh; Baxter, Amy A; Lay, Fung T; Mills, Grant D; Adda, Christopher G; Payne, Jennifer Ae; Phan, Thanh Kha; Ryan, Gemma F; White, Julie A; Veneer, Prem K; van der Weerden, Nicole L; Anderson, Marilyn A; Kvansakul, Marc; Hulett, Mark D

2014-01-01

114

Phosphoinositide-metabolizing enzymes at the interface between membrane traffic and cell signalling  

PubMed Central

Phosphoinositides (PIs) have long been known to have important roles in cell signalling. During the past decade, it has become clear that these lipids also act as constitutive signals that aid in defining organelle identity, and are short-lived recruiters and regulators of cytoskeletal and membrane dynamics. Recent studies have provided important clues as to how regulated activation of PI-metabolizing enzymes and recruitment of their binding proteins might cooperate in targeting distinct pools of PIs to different cell physiological functions. PMID:17330069

Krauß, Michael; Haucke, Volker

2007-01-01

115

Inositol pyrophosphates regulate cell death and telomere length through phosphoinositide 3-kinase-related protein kinases.  

PubMed

Inositol pyrophosphates physiologically regulate vesicular endocytosis, ribosomal disposition, and directly phosphorylate proteins. Here we demonstrate roles in cell death and regulation of telomere length. Lethal actions of wortmannin and caffeine are selectively abolished in yeast mutants that cannot synthesize inositol pyrophosphates. Wortmannin and caffeine appear to act through the phosphoinositide 3-kinase-related protein kinases Tel1 and Mec1, known regulators of telomere length. Inositol pyrophosphates physiologically antagonize the actions of these kinases, which is demonstrated by the fact that yeast mutants with reduced or elevated levels of inositol pyrophosphates, respectively, display longer and shorter telomeres. PMID:15665079

Saiardi, Adolfo; Resnick, Adam C; Snowman, Adele M; Wendland, Beverly; Snyder, Solomon H

2005-02-01

116

The PDZ2 domain of zonula occludens-1 and -2 is a phosphoinositide binding domain  

Microsoft Academic Search

Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins\\u000a that play a fundamental role in the assembly of tight junctions and establishment of cell polarity. Here, we show that the\\u000a second PDZ domain of ZO-1 and ZO-2 binds phosphoinositides (PtdInsP) and we identified critical residues involved in the interaction.\\u000a Furthermore, peptide and PtdInsP binding of

Kris Meerschaert; Moe Phyu Tun; Eline Remue; Ariane De Ganck; Ciska Boucherie; Berlinda Vanloo; Gisèle Degeest; Joël Vandekerckhove; Pascale Zimmermann; Nitin Bhardwaj; Hui Lu; Wonhwa Cho; Jan Gettemans

2009-01-01

117

Inositol pyrophosphates regulate cell death and telomere length through phosphoinositide 3-kinase-related protein kinases  

PubMed Central

Inositol pyrophosphates physiologically regulate vesicular endocytosis, ribosomal disposition, and directly phosphorylate proteins. Here we demonstrate roles in cell death and regulation of telomere length. Lethal actions of wortmannin and caffeine are selectively abolished in yeast mutants that cannot synthesize inositol pyrophosphates. Wortmannin and caffeine appear to act through the phosphoinositide 3-kinase-related protein kinases Tel1 and Mec1, known regulators of telomere length. Inositol pyrophosphates physiologically antagonize the actions of these kinases, which is demonstrated by the fact that yeast mutants with reduced or elevated levels of inositol pyrophosphates, respectively, display longer and shorter telomeres. PMID:15665079

Saiardi, Adolfo; Resnick, Adam C.; Snowman, Adele M.; Wendland, Beverly; Snyder, Solomon H.

2005-01-01

118

Suppression of Phosphoinositide 3-Kinase Signaling and Alteration of Multiple Ion Currents in Drug-Induced Long QT Syndrome  

PubMed Central

Many drugs, including some commonly used medications, can cause abnormal heart rhythms and sudden death, as manifest by a prolonged QT interval in the electrocardiogram. Cardiac arrhythmias caused by drug-induced long QT syndrome are thought to result mainly from reductions in the delayed rectifier potassium ion (K+) current IKr. Here, we report a mechanism for drug-induced QT prolongation that involves changes in multiple ion currents caused by a decrease in phosphoinositide 3-kinase (PI3K) signaling. Treatment of canine cardiac myocytes with inhibitors of tyrosine kinases or PI3Ks caused an increase in action potential duration that was reversed by intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate. The inhibitors decreased the delayed rectifier K+ currents IKr and IKs, the L-type calcium ion (Ca2+) current ICa,L, and the peak sodium ion (Na+) current INa and increased the persistent Na+ current INaP. Computer modeling of the canine ventricular action potential showed that the drug-induced change in any one current accounted for less than 50% of the increase in action potential duration. Mouse hearts lacking the PI3K p110? catalytic subunit exhibited a prolonged action potential and QT interval that were at least partly a result of an increase in INaP. These results indicate that down-regulation of PI3K signaling directly or indirectly via tyrosine kinase inhibition prolongs the QT interval by affecting multiple ion channels. This mechanism may explain why some tyrosine kinase inhibitors in clinical use are associated with increased risk of life-threatening arrhythmias. PMID:22539774

Lu, Zhongju; Wu, Chia-Yen C.; Jiang, Ya-Ping; Ballou, Lisa M.; Clausen, Chris; Cohen, Ira S.; Lin, Richard Z.

2012-01-01

119

Epigallocatechin gallate (EGCG), a major component of green tea, is a dual phosphoinositide-3-kinase/mTOR inhibitor  

SciTech Connect

Research highlights: {yields} Epigallocatechin-3-gallate (EGCG) is an ATP-competitive inhibitor of PI3K and mTOR with Ki values around 300 nM. {yields} EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231and A549 cells. {yields} Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site. {yields} These results suggest another important molecular mechanism for the anticancer activities of EGCG. -- Abstract: The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The key nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) with K{sub i} values of 380 and 320 nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.

Van Aller, Glenn S., E-mail: glenn.s.van.aller@gsk.com [Department of Cancer Research, GlaxoSmithKline, Collegeville, PA 19426 (United States); Carson, Jeff D. [Department of Cancer Research, GlaxoSmithKline, Collegeville, PA 19426 (United States)] [Department of Cancer Research, GlaxoSmithKline, Collegeville, PA 19426 (United States); Tang, Wei; Peng, Hao; Zhao, Lin [Discovery Biology, BioDuro, No. 29 Life Science Park Road, Changping, Beijing (China)] [Discovery Biology, BioDuro, No. 29 Life Science Park Road, Changping, Beijing (China); Copeland, Robert A.; Tummino, Peter J. [Department of Cancer Research, GlaxoSmithKline, Collegeville, PA 19426 (United States)] [Department of Cancer Research, GlaxoSmithKline, Collegeville, PA 19426 (United States); Luo, Lusong [Discovery Biology, BioDuro, No. 29 Life Science Park Road, Changping, Beijing (China)] [Discovery Biology, BioDuro, No. 29 Life Science Park Road, Changping, Beijing (China)

2011-03-11

120

Spatial Regulation of Membrane Fusion Controlled by Modification of Phosphoinositides  

PubMed Central

Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG) facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE) assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLC?), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P2) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism. PMID:20808914

Dumas, Fabrice; Byrne, Richard D.; Vincent, Ben; Hobday, Tina M. C.; Poccia, Dominic L.; Larijani, Banafshé

2010-01-01

121

Electrostatics of phosphoinositide bilayer membranes. Theoretical and experimental results.  

PubMed Central

We made fluorescence, electron paramagnetic resonance (EPR), electrophoretic mobility, and ionizing electrode measurements to study the effect of the monovalent lipid phosphatidylinositol (PI) and the trivalent lipid phosphatidylinositol 4,5-bisphosphate (PIP2) on the electrostatic potential adjacent to bilayer membranes. When the membranes were formed from mixtures of PI and the zwitterionic lipid phosphatidylcholine (PC), the Gouy-Chapman-Stern (GCS) theory described adequately the dependence of potential on distance (0, 1, 2 nm) from the membrane, mole % negative lipid, and [KCI]. Furthermore, all EPR and fluorescence probes reported identical surface potentials with a PC/PI membrane. With PC/PIP2 membranes, however, the anionic (coion) probes reported less negative potentials than the cationic (counterion) probes; the deviations from the GCS theory were greater for the coions than the counterions. Discreteness-of-charge theories based on the Poisson-Boltzmann equation incorrectly predict that deviations from the GCS theory should be greater for counterions than for coions. We discuss a consistent statistical mechanical theory that takes into account three effects ignored in the GCS theory: the finite size of the ions in the double layer, the electrical interaction between pairs of ions (correlation effects), and the mobile discrete nature of the surface charges. This theory correctly predicts that deviations from GCS theory should be negligible for monovalent lipids, significant for trivalent lipids, and greater for coions than for counterions. PMID:2156577

Langner, M; Cafiso, D; Marcelja, S; McLaughlin, S

1990-01-01

122

Phosphoinositide regulation of integrin trafficking required for muscle attachment and maintenance.  

PubMed

Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. We show that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila melanogaster, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease. PMID:21347281

Ribeiro, Inês; Yuan, Lin; Tanentzapf, Guy; Dowling, James J; Kiger, Amy

2011-01-01

123

Participation of the phosphoinositide metabolism in the hypersensitive response of Citrus limon against Alternaria alternata.  

PubMed

Lemon seedlings inoculated with Alternaria alternata develop a hypersensitive response (HR) that includes the induction of Phenylalanine ammonia-lyase (PAL, E. C. 4.3.1.5) and the synthesis of scoparone. The signal transduction pathway involved in the development of this response is unknown. We used several inhibitors of the Phosphoinositide (PI) animal system to study a possible role of Inositol-1,4,5-triphosphate (IP3) in the transduction of the fungal conidia signal in Citrus limon. The HR was only partially inhibited by EGTA, suggesting that not only external but internal calcium as well are necessary for a complete development of the HR. In this plant system, Alternaria alternata induced an early accumulation of the second messenger IP3. When lemon seedlings were watered long term with LiCl, an inhibitor of the phosphoinositide cycle, the IP3 production was reduced, and the LiCl-watered plants could neither induce PAL nor synthesize scoparone in response to fungal conidia. Furthermore, neomycin, a Phospholipase C (PLC, E. C. 3.1.4.3) inhibitor, also inhibited PAL induction and scoparone synthesis in response to A. alternata. These results suggest that IP3 could be involved in the signal transduction pathway for the development of the HR of Citrus limon against A. alternata. PMID:11471522

Ortega, X; Pérez, L M

2001-01-01

124

Yunis-Varón syndrome is caused by mutations in FIG4, encoding a phosphoinositide phosphatase.  

PubMed

Yunis-Varón syndrome (YVS) is an autosomal-recessive disorder with cleidocranial dysplasia, digital anomalies, and severe neurological involvement. Enlarged vacuoles are found in neurons, muscle, and cartilage. By whole-exome sequencing, we identified frameshift and missense mutations of FIG4 in affected individuals from three unrelated families. FIG4 encodes a phosphoinositide phosphatase required for regulation of PI(3,5)P(2) levels, and thus endosomal trafficking and autophagy. In a functional assay, both missense substitutions failed to correct the vacuolar phenotype of Fig4-null mouse fibroblasts. Homozygous Fig4-null mice exhibit features of YVS, including neurodegeneration and enlarged vacuoles in neurons. We demonstrate that Fig4-null mice also have small skeletons with reduced trabecular bone volume and cortical thickness and that cultured osteoblasts accumulate large vacuoles. Our findings demonstrate that homozygosity or compound heterozygosity for null mutations of FIG4 is responsible for YVS, the most severe known human phenotype caused by defective phosphoinositide metabolism. In contrast, in Charcot-Marie-Tooth disease type 4J (also caused by FIG4 mutations), one of the FIG4 alleles is hypomorphic and disease is limited to the peripheral nervous system. This genotype-phenotype correlation demonstrates that absence of FIG4 activity leads to central nervous system dysfunction and extensive skeletal anomalies. Our results describe a role for PI(3,5)P(2) signaling in skeletal development and maintenance. PMID:23623387

Campeau, Philippe M; Lenk, Guy M; Lu, James T; Bae, Yangjin; Burrage, Lindsay; Turnpenny, Peter; Román Corona-Rivera, Jorge; Morandi, Lucia; Mora, Marina; Reutter, Heiko; Vulto-van Silfhout, Anneke T; Faivre, Laurence; Haan, Eric; Gibbs, Richard A; Meisler, Miriam H; Lee, Brendan H

2013-05-01

125

Structural Basis for Activation and Inhibition of Class I Phosphoinositide 3-Kinases  

NSDL National Science Digital Library

Phosphoinositide 3-kinases (PI3Ks) phosphorylate a hydroxyl group on phosphoinositide lipids. The 3-phosphorylated inositol lipids act as membrane-resident second messengers, recruiting downstream signaling components that control cell growth, proliferation, differentiation, survival, and motility. The best studied of the PI3Ks, the class I enzymes, are heterodimers with a catalytic and a regulatory subunit and have been implicated in many human diseases. Class I PI3Ks can be stimulated downstream of receptor tyrosine kinases and heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors as well as small G proteins of the Ras superfamily. Both the catalytic and regulatory subunits have a multidomain organization. Crystal structures, biochemical analysis, and oncogenic mutations in PI3Ks have shown that interdomain interactions are not static but undergo regulated conformational cycles, resulting in enzyme activation or inhibition. This Review, which contains 7 figures and 104 references, highlights the molecular details of how their regulatory partners selectively inhibit and activate PI3K isoforms.

Oscar Vadas (Cambridge;Laboratory of Molecular Biology REV); John E. Burke (Singapore;Nanyang Technological University REV); Xuxiao Zhang (Singapore;Nanyang Technological University REV); Alex Berndt (Cambridge;Laboratory of Molecular Biology REV); Roger L. Williams (Cambridge;Laboratory of Molecular Biology REV)

2011-10-18

126

Phosphoinositide Regulation of Integrin Trafficking Required for Muscle Attachment and Maintenance  

PubMed Central

Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. We show that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila melanogaster, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease. PMID:21347281

Ribeiro, Inês; Yuan, Lin; Tanentzapf, Guy; Dowling, James J.; Kiger, Amy

2011-01-01

127

Expression of Phosphoinositide-specific phospholipase C enzymes in human osteosarcoma cell lines.  

PubMed

The definition of the number and nature of signal transduction pathways networking in the pathogenesis of osteosarcoma raised great interest. Intracellular calcium ions are important second messengers implicated in the control of cell death. The calcium concentration is regulated by signal transduction pathways, including the Phosphoinositides (PI) signaling. Phosphatydil inositol (4,5) bisphosphate (PIP2) is critical for many cellular activities. The levels of PIP2 are regulated by means of Phosphoinositide-specific Phospholipase C (PI-PLC) family of enzymes. We delineated the panel of expression of PI-PLC enzymes in four human osteosarcoma cell lines. In MG-63 cell line, PI-PLC ?1, ?2, ?3, ?4, ?1, ?2, ?1, ?3 and ? resulted expressed. In 143B cell line, PI-PLC ?1, ?2, ?3, ?4, ?1, ?2, ?1, ?3 and ? were expressed. In SaOS-2 cell line, PI-PLC ?1, ?3, ?4, ?1, ?2, ?1, ?3, ? and ?1. In Hs888 cell line, PI-PLC ?1, ?3, ?4, ?1, ?1, ?3, ?4, ? and ?1 the administration of U-73122 to cultures briefly modifies the levels of PI-PLC transcripts. The obtained complete expression panel of PI-PLC isoforms will be a useful tool for further functional studies about the role of the PI signal transduction pathway in osteosarcoma. PMID:23430511

Lo Vasco, Vincenza Rita; Leopizzi, Martina; Chiappetta, Caterina; Puggioni, Chiara; Di Cristofano, Claudio; Della Rocca, Carlo

2013-06-01

128

Changes in phosphoinositide-specific phospholipase C and phospholipase A 2 activity in ischemic and reperfused rat heart  

Microsoft Academic Search

Phospholipid metabolism is altered during ischemia and post-ischemic reperfusion. Past studies demonstrating elevated myocardial free fatty acid and lysophospholipid content infer accelerated phospholipid degradation involving phospholipase A activity. Recently, ischemic and postischemic reperfusion (reperfusion) have been shown to affect levels of phosphoinositide (PPI) degradation products. Considering the role of PPI turnover in regulation of cellular calcium homeostasis, our laboratory and

D. W. Schwertz; J. Halverson

1992-01-01

129

Discovery and optimization of a series of 2-aminothiazole-oxazoles as potent phosphoinositide 3-kinase ? inhibitors.  

PubMed

A novel series of 2-aminothiazole-oxazoles was designed and synthesized as part of efforts to develop potent phosphoinositide 3-kinase ? (PI3K?) inhibitors. The modification of a high-throughput screening hit, compound 1, resulted in the identification of compounds 10 and 15, which displayed potent inhibitory activities in enzyme-based and cell-based assays. PMID:23122859

Oka, Yusuke; Yabuuchi, Tetsuya; Fujii, Yasuyuki; Ohtake, Hidenori; Wakahara, Shunichi; Matsumoto, Kayo; Endo, Mayumi; Tamura, Yunoshin; Sekiguchi, Yoshinori

2012-12-15

130

The Phosphoinositide 3-Kinase Regulatory Subunit p85  Can Exert Tumor Suppressor Properties through Negative Regulation of Growth Factor Signaling  

E-print Network

Phosphoinositide 3-kinase (PI3K) plays a critical role in tumorigenesis, and the PI3K p85 regulatory subunit exerts both positive and negative effects on signaling. Expression of Pik3r1, the gene encoding p85, is decreased ...

Taniguchi, Cullen M.

131

Phosphoinositide-3 Kinase Inhibition Modulates Responses to Rhinovirus by Mechanisms that Are Predominantly Independent of Autophagy  

PubMed Central

Human rhinoviruses (HRV) are a major cause of exacerbations of airways disease. Aspects of cell signalling responses to HRV infection remain unclear, particularly with regard to signalling via PI3K, and the PI3K-dependent pathway, autophagy. We investigated the roles of PI3K and autophagy in the responses of epithelial cells to major and minor group HRV infection. The PI3K inhibitor 3-MA, commonly used to inhibit autophagy, markedly reduced HRV-induced cytokine induction. Further investigation of potential targets of 3-MA and comparison of results using this inhibitor to a panel of general and class I-selective PI3K inhibitors showed that several PI3Ks cooperatively regulate responses to HRV. Targeting by siRNA of the autophagy proteins Beclin-1, Atg7, LC3, alone or in combination, or targeting of the autophagy-specific class III PI3K had at most only modest effects on HRV-induced cell signalling as judged by induction of proinflammatory cytokine production. Our data indicate that PI3K and mTOR are involved in induction of proinflammatory cytokines after HRV infection, and that autophagy has little role in the cytokine response to HRV or control of HRV replication. PMID:25541728

Ismail, Saila; Stokes, Clare A.; Prestwich, Elizabeth C.; Roberts, Rebecca L.; Juss, Jatinder K.; Sabroe, Ian; Parker, Lisa C.

2014-01-01

132

Salicylic acid modulates levels of phosphoinositide dependent-phospholipase C substrates and products to remodel the Arabidopsis suspension cell transcriptome  

PubMed Central

Basal phosphoinositide-dependent phospholipase C (PI-PLC) activity controls gene expression in Arabidopsis suspension cells and seedlings. PI-PLC catalyzes the production of phosphorylated inositol and diacylglycerol (DAG) from phosphoinositides. It is not known how PI-PLC regulates the transcriptome although the action of DAG-kinase (DGK) on DAG immediately downstream from PI-PLC is responsible for some of the regulation. We previously established a list of genes whose expression is affected in the presence of PI-PLC inhibitors. Here this list of genes was used as a signature in similarity searches of curated plant hormone response transcriptome data. The strongest correlations obtained with the inhibited PI-PLC signature were with salicylic acid (SA) treatments. We confirm here that in Arabidopsis suspension cells SA treatment leads to an increase in phosphoinositides, then demonstrate that SA leads to a significant 20% decrease in phosphatidic acid, indicative of a decrease in PI-PLC products. Previous sets of microarray data were re-assessed. The SA response of one set of genes was dependent on phosphoinositides. Alterations in the levels of a second set of genes, mostly SA-repressed genes, could be related to decreases in PI-PLC products that occur in response to SA action. Together, the two groups of genes comprise at least 40% of all SA-responsive genes. Overall these two groups of genes are distinct in the functional categories of the proteins they encode, their promoter cis-elements and their regulation by DGK or phospholipase D. SA-regulated genes dependent on phosphoinositides are typical SA response genes while those with an SA response that is possibly dependent on PI-PLC products are less SA-specific. We propose a model in which SA inhibits PI-PLC activity and alters levels of PI-PLC products and substrates, thereby regulating gene expression divergently. PMID:25426125

Ruelland, Eric; Pokotylo, Igor; Djafi, Nabila; Cantrel, Catherine; Repellin, Anne; Zachowski, Alain

2014-01-01

133

Targeting the phosphoinositide 3-kinase (PI3K) pathway in cancer  

PubMed Central

The phosphoinositide 3-kinase (PI3K) pathway, a critical signal transduction system linking oncogenes and multiple receptor classes to many essential cellular functions, is perhaps the most commonly activated signaling pathway in human cancer. This pathway thus presents both an opportunity and a challenge for cancer therapy. Even as inhibitors that target PI3K isoforms and other major nodes in the pathway including AKT and mTOR reach clinical trials, major issues remain. Here we highlight recent progress made in our understanding of the PI3K pathway and discuss both the promises and challenges for the therapeutic development of agents targeting the PI3K pathway in cancer. PMID:19644473

Liu, Pixu; Cheng, Hailing; Roberts, Thomas M.; Zhao, Jean J.

2011-01-01

134

Actin and phosphoinositide recruitment to fully formed Candida albicans phagosomes in mouse macrophages.  

PubMed

Candida albicans is a dimorphic yeast that enters macrophages (Mphi) via the beta-glucan receptor dectin-1. Phagocytosis of C. albicans is characterized by actin polymerization, Syk kinase activation and rapid acquisition of phagolysosomal markers. In mice, C. albicans are able to resist the harsh environment of the phagosome and form pseudohyphae inside the phagolysosomal compartment, eventually extending from the Mphi. In this study, we investigated these unique C. albicans phagosomes and found that actin localized dynamically around the phagosomes, before disintegrating. Membrane phosphoinositides, PI(4,5)P(2), PI(3,4,5)P(3), PI(3,4)P(2), and PI(3)P also localized to the phagosomes. Localization was not related to actin polymerization, and inhibitor studies showed that polymerization of actin on the C. albicans phagosome was independent of PI3K. The ability of mature C. albicans phagosomes to stimulate actin polymerization could facilitate the escape of the growing yeast from the Mphi. PMID:20375582

Heinsbroek, Sigrid E M; Kamen, Lynn A; Taylor, Philip R; Brown, Gordon D; Swanson, Joel; Gordon, Siamon

2009-01-01

135

Phosphoinositide breakdown in isolated myelin is stimulated by GTP analogues and calcium.  

PubMed

Purified myelin from rat brainstem, prelabeled in vivo by intracerebral injection of [3H]myoinositol, showed enhanced breakdown of phosphoinositides on treatment with 5'-guanylylimidodiphosphate [Gpp-(NH)p] and Ca2+. Concentration variation of the former in the presence of Ca2+ showed a dose-dependent release of inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3), while inositol 1-phosphate (IP) release was erratic. Concentration-dependent release of IP2 and IP3 was also observed with Ca2+ as the variable in the presence of Gpp(NH)p. Carbachol, when present, did not enhance the stimulatory effect of Gpp(NH)p alone. Addition of diphosphoglycerate during incubation enhanced IP3 at the expense of IP2, suggesting the presence of IP3 phosphatase in myelin. PMID:1965838

Golly, F; Larocca, J N; Ledeen, R W

1990-11-01

136

Aluminium perturbs oscillatory phosphoinositide-mediated calcium signalling in hormone-stimulated hepatocytes.  

PubMed Central

Aluminium is known to be toxic to cells from bone, brain and bone marrow but the molecular target(s) affected by Al3+ are not known. We show here that Al3+ disrupts the oscillatory free Ca2+ responses of hepatocytes exposed to the Ca2(+)-mobilizing agonist phenylephrine. Al3+ initially increases the frequency of the oscillations and later induces broad Ca2+ spikes lasting several minutes. These broad spikes persist after removal of both agonist and Al3+ from the medium. In the absence of agonist, Al3+ has no effect on free Ca2+. The data suggest that some component(s) of the receptor-phosphoinositide-Ca2+ signalling pathway might be the site at which Al3+ exerts toxic effects. PMID:2167073

Schöfl, C; Sanchez-Bueno, A; Dixon, C J; Woods, N M; Lee, J A; Cuthbertson, K S; Cobbold, P H; Birchall, J D

1990-01-01

137

Discovery of a novel series of potent and orally bioavailable phosphoinositide 3-kinase ? inhibitors.  

PubMed

The phosphoinositide 3-kinases (PI3Ks) have been linked to an extraordinarily diversified group of cellular functions making these enzymes compelling targets for the treatment of disease. A large body of evidence has linked PI3K? to the modulation of autoimmune and inflammatory processes making it an intriguing target for drug discovery. Our high-throughput screening (HTS) campaign revealed two hits that were nominated for further optimization studies. The in vitro activity of the first HTS hit, designated as the sulfonylpiperazine scaffold, was optimized utilizing structure-based design. However, nonoptimal pharmacokinetic properties precluded this series from further studies. An overlay of the X-ray structures of the sulfonylpiperazine scaffold and the second HTS hit within their complexes with PI3K? revealed a high degree of overlap. This feature was utilized to design a series of hybrid analogues including advanced leads such as 31 with desirable potency, selectivity, and oral bioavailability. PMID:22548342

Leahy, James W; Buhr, Chris A; Johnson, Henry W B; Kim, Byung Gyu; Baik, Taegon; Cannoy, Jonah; Forsyth, Timothy P; Jeong, Joon Won; Lee, Matthew S; Ma, Sunghoon; Noson, Kevin; Wang, Longcheng; Williams, Matthew; Nuss, John M; Brooks, Eric; Foster, Paul; Goon, Leanne; Heald, Nathan; Holst, Charles; Jaeger, Christopher; Lam, Scott; Lougheed, Julie; Nguyen, Lam; Plonowski, Arthur; Song, Joanne; Stout, Thomas; Wu, Xiang; Yakes, Michael F; Yu, Peiwen; Zhang, Wentao; Lamb, Peter; Raeber, Olivia

2012-06-14

138

Phosphoinositide 3-kinase ? mediates microglial phagocytosis via lipid kinase-independent control of cAMP.  

PubMed

Microglial phagocytosis plays a key role in neuroprotective and neurodegenerative responses of the innate immune system in the brain. Here we investigated the regulatory function of phosphoinositide 3-kinase ? (PI3K?) in phagocytosis of bacteria and Zymosan particles by mouse brain microglia in vitro and in vivo. Using genetic and pharmacological approaches our data revealed PI3K? as an essential mediator of microglial phagocytosis. Unexpectedly, microglia expressing lipid kinase deficient mutant PI3K? exhibited similar phagocytosis as wild-type cells. These data suggest kinase-independent stimulation of cAMP phosphodiesterase activity by PI3K? as a crucial mediator of phagocytosis. In sum our findings indicate PI3K?-dependent suppression of cAMP signaling as a critical regulatory element of microglial phagocytosis. PMID:23276671

Schmidt, C; Schneble, N; Müller, J P; Bauer, R; Perino, A; Marone, R; Rybalkin, S D; Wymann, M P; Hirsch, E; Wetzker, R

2013-03-13

139

Axon-myelin transfer of phospholipids and phospholipid precursors. Labeling of myelin phosphoinositides through axonal transport.  

PubMed

Previous studies have provided evidence for axon-to-myelin transfer of intact lipids and lipid precursors for reutilization by myelin enzymes. Several of the lipid constituents of myelin showed significant contralateral/ipsilateral ratios of incorporated radioactivity, indicative of axonal origin, whereas proteins and certain other lipids did not participate in this transfer-reutilization process. The present study will examine the labeling of myelin phosphoinositides by this pathway. Both 32PO4 and [3H]inositol were injected monocularly into 7-9-wk-old rabbits and myelin was isolated 7 or 21 days later from pooled optic tracts and superior colliculi. In total lipids 32P counts of the isolated myelin samples showed significant contralateral/ipsilateral ratios as well as increasing magnitude of contralateral-ipsilateral differences during the time interval. Thin-layer chromatographic isolation of the myelin phosphoinositides revealed significant 32P-labeling of these species, with PIP and PIP2 showing time-related increases. This resembled the labeling pattern of the major phospholipids from rabbit optic system myelin in a previous study and suggested incorporation of axon-derived phosphate by myelin-associated enzymes. The 32P label in PI, on the other hand, remained constant between 7 and 21 days, suggesting transfer of intact lipid. This was supported by the labeling pattern with [3H]inositol, which also showed no increase over time for PI. These results suggest axon-myelin transfer of intact PI followed by myelin-localized incorporation of axon-derived phosphate groups into PIP and PIP2. The general topic of axon-myelin transfer of phospholipids and phospholipid precursors is reviewed. PMID:1282330

Ledeen, R W; Golly, F; Haley, J E

1992-01-01

140

Phosphoinositide-signaling is one component of a robust plant defense response.  

PubMed

The phosphoinositide pathway and inositol-1,4,5-triphosphate (InsP3) have been implicated in plant responses to many abiotic stresses; however, their role in response to biotic stress is not well characterized. In the current study, we show that both basal defense and systemic acquired resistance responses are affected in transgenic plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase) which have greatly reduced InsP3 levels. Flagellin induced Ca(2+)-release as well as the expressions of some flg22 responsive genes were attenuated in the InsP 5-ptase plants. Furthermore, the InsP 5-ptase plants were more susceptible to virulent and avirulent strains of Pseudomonas syringae pv. tomato (Pst) DC3000. The InsP 5-ptase plants had lower basal salicylic acid (SA) levels and the induction of SAR in systemic leaves was reduced and delayed. Reciprocal exudate experiments showed that although the InsP 5-ptase plants produced equally effective molecules that could trigger PR-1 gene expression in wild type plants, exudates collected from either wild type or InsP 5-ptase plants triggered less PR-1 gene expression in InsP 5-ptase plants. Additionally, expression profiles indicated that several defense genes including PR-1, PR-2, PR-5, and AIG1 were basally down regulated in the InsP 5-ptase plants compared with wild type. Upon pathogen attack, expression of these genes was either not induced or showed delayed induction in systemic leaves. Our study shows that phosphoinositide signaling is one component of the plant defense network and is involved in both basal and systemic responses. The dampening of InsP3-mediated signaling affects Ca(2+) release, modulates defense gene expression and compromises plant defense responses. PMID:24966862

Hung, Chiu-Yueh; Aspesi, Peter; Hunter, Melissa R; Lomax, Aaron W; Perera, Imara Y

2014-01-01

141

Phosphoinositide Binding Differentially Regulates NHE1 Na+/H+ Exchanger-dependent Proximal Tubule Cell Survival*  

PubMed Central

Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na+/H+ exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P2 binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP3 > PIP2 > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P2-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na+/H+ exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P3, which localized to basolateral membranes. Divergent PI(4,5)P2 and PI(3,4,5)P3 effects on NHE1-dependent Na+/H+ exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase C? and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P2 and PI(3,4,5)P3. In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P2, which leads to PI 3-kinase activation, and PI(4,5)P2 phosphorylation. The resulting PI(3,4,5)P3 dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P2. PMID:22020933

Abu Jawdeh, Bassam G.; Khan, Shenaz; Deschênes, Isabelle; Hoshi, Malcolm; Goel, Monu; Lock, Jeffrey T.; Shinlapawittayatorn, Krekwit; Babcock, Gerald; Lakhe-Reddy, Sujata; DeCaro, Garren; Yadav, Satya P.; Mohan, Maradumane L.; Naga Prasad, Sathyamangla V.; Schilling, William P.; Ficker, Eckhard; Schelling, Jeffrey R.

2011-01-01

142

Alternative splicing governs cone cyclic nucleotide-gated (CNG) channel sensitivity to regulation by phosphoinositides.  

PubMed

Precursor mRNA encoding CNGA3 subunits of cone photoreceptor cyclic nucleotide-gated (CNG) channels undergoes alternative splicing, generating isoforms differing in the N-terminal cytoplasmic region of the protein. In humans, four variants arise from alternative splicing, but the functional significance of these changes has been a persistent mystery. Heterologous expression of the four possible CNGA3 isoforms alone or with CNGB3 subunits did not reveal significant differences in basic channel properties. However, inclusion of optional exon 3, with or without optional exon 5, produced heteromeric CNGA3 + CNGB3 channels exhibiting an ?2-fold greater shift in K1/2,cGMP after phosphatidylinositol 4,5-biphosphate or phosphatidylinositol 3,4,5-trisphosphate application compared with channels lacking the sequence encoded by exon 3. We have previously identified two structural features within CNGA3 that support phosphoinositides (PIPn) regulation of cone CNG channels: N- and C-terminal regulatory modules. Specific mutations within these regions eliminated PIPn sensitivity of CNGA3 + CNGB3 channels. The exon 3 variant enhanced the component of PIPn regulation that depends on the C-terminal region rather than the nearby N-terminal region, consistent with an allosteric effect on PIPn sensitivity because of altered N-C coupling. Alternative splicing of CNGA3 occurs in multiple species, although the exact variants are not conserved across CNGA3 orthologs. Optional exon 3 appears to be unique to humans, even compared with other primates. In parallel, we found that a specific splice variant of canine CNGA3 removes a region of the protein that is necessary for high sensitivity to PIPn. CNGA3 alternative splicing may have evolved, in part, to tune the interactions between cone CNG channels and membrane-bound phosphoinositides. PMID:24675082

Dai, Gucan; Sherpa, Tshering; Varnum, Michael D

2014-05-01

143

Signaling via Class IA Phosphoinositide 3-Kinases (PI3K) in Human, Breast-Derived Cell Lines  

PubMed Central

We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (?, ?, ? and ?) and p50–101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110?, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110?>>?>? with undetectable p110?. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110?-, but not ?- or ?- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110?, but not ?- or ?- activity. In the presence of single, endogenous alleles of onco-mutant p110? (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110? inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN?/? cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110?, but not ?- or ?- activity; in PTEN?/? MCF10a it remained, like the parental cells, p110?-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110? is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110? augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110? function in the absence of PTEN. PMID:24124465

Juvin, Veronique; Malek, Mouhannad; Anderson, Karen E.; Dion, Carine; Chessa, Tamara; Lecureuil, Charlotte; Ferguson, G. John; Cosulich, Sabina

2013-01-01

144

Signaling via class IA Phosphoinositide 3-kinases (PI3K) in human, breast-derived cell lines.  

PubMed

We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (?, ?, ? and ?) and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110?, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110?>?>? with undetectable p110?. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110?-, but not ?- or ?- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110?, but not ?- or ?- activity. In the presence of single, endogenous alleles of onco-mutant p110? (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110? inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/-) cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110?, but not ?- or ?- activity; in PTEN(-/-) MCF10a it remained, like the parental cells, p110?-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110? is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110? augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110? function in the absence of PTEN. PMID:24124465

Juvin, Veronique; Malek, Mouhannad; Anderson, Karen E; Dion, Carine; Chessa, Tamara; Lecureuil, Charlotte; Ferguson, G John; Cosulich, Sabina; Hawkins, Phillip T; Stephens, Len R

2013-01-01

145

Differential regulation of phosphoinositide metabolism by alphaVbeta3 and alphaVbeta5 integrins upon smooth muscle cell migration.  

PubMed

Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins. PMID:11551924

Paulhe, F; Racaud-Sultan, C; Ragab, A; Albiges-Rizo, C; Chap, H; Iberg, N; Morand, O; Perret, B

2001-11-01

146

Phosphoinositide 3-kinase p110? promotes lumen formation through the enhancement of apico-basal polarity and basal membrane organization.  

PubMed

Signalling triggered by adhesion to the extracellular matrix plays a key role in the spatial orientation of epithelial polarity and formation of lumens in glandular tissues. Phosphoinositide 3-kinase signalling in particular is known to influence the polarization process during epithelial cell morphogenesis. Here, using Madin-Darby canine kidney epithelial cells grown in 3D culture, we show that the p110? isoform of phosphoinositide 3-kinase co-localizes with focal adhesion proteins at the basal surface of polarized cells. Pharmacological, siRNA- or kinase-dead-mediated inhibition of p110? impair the early stages of lumen formation, resulting in inverted polarized cysts, with no laminin or type IV collagen assembly at cell/extracellular matrix contacts. p110? also regulates the organization of focal adhesions and membrane localization of dystroglycan. Thus, we uncover a previously unrecognized role for p110? in epithelial cells in the orientation of the apico-basal axis and lumen formation. PMID:25583025

Peng, Juan; Awad, Aline; Sar, Sokhavuth; Hamze Komaiha, Ola; Moyano, Romina; Rayal, Amel; Samuel, Didier; Shewan, Annette; Vanhaesebroeck, Bart; Mostov, Keith; Gassama-Diagne, Ama

2015-01-01

147

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

148

Wortmannin Inactivates Phosphoinositide 3Kinase by Covalent Modification of Lys802, a Residue Involved in the Phosphate Transfer Reaction  

Microsoft Academic Search

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA- dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortman- nin on

MATTHIAS P. WYMANN; GINETTE BULGARELLI-LEVA; MARKETA J. ZVELEBIL; LUCIANO PIROLA; BART VANHAESEBROECK; MICHAEL D. WATERFIELD

149

Constitutive Macropinocytosis in Oncogene transformed Fibroblasts Depends on Sequential Permanent Activation of Phosphoinositide 3Kinase and Phospholipase C  

Microsoft Academic Search

Macropinocytosis results from the closure of lamellipodia generated by membrane ruffling, thereby reflecting cortical actin dynamics. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection for expression of dominant-positive, wild-type phosphoinositide 3-kinase (PI3K) regulatory subunit p85a constitutively led to stress fiber disruption, cortical actin recruitment, extensive ruffling, and macropinosome formation, as measured by a selective accel- eration

Mustapha Amyere; Bernard Payrastre; Ulrike Krause; Patrick Van Der Smissen; Alex Veithen; Pierre J. Courtoy

150

Alterations of phosphoinositide-specific phospholipase C and protein kinase C in the myocardium of spontaneously hypertensive rats  

Microsoft Academic Search

Summary In order to determine whether phosphoinositide metabolism is altered in hypertensive cardiac hypertrophy, phospholipase C (PLC) and protein kinase C activities were measured in hearts from 4- and 20-week-old spontaneously hypertensive rats (SHR) and age-matched, normotensive Wister-Kyoto rats (WKY). PLC activities were assayed using phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP2) as substrates to assess the substrate specificity. PI-hydrolyzing PLC activity

N. Makita; H. Yasuda

1990-01-01

151

Direct Association of Heat Shock Protein 20 (HSPB6) with Phosphoinositide 3-kinase (PI3K) in Human Hepatocellular Carcinoma: Regulation of the PI3K Activity  

PubMed Central

HSP20 (HSPB6), one of small heat shock proteins (HSPs), is constitutively expressed in various tissues and has several functions. We previously reported that the expression levels of HSP20 in human hepatocellular carcinoma (HCC) cells inversely correlated with the progression of HCC, and that HSP20 suppresses the growth of HCC cells via the AKT and mitogen-activated protein kinase signaling pathways. However, the exact mechanism underlying the effect of HSP20 on the regulation of these signaling pathways remains to be elucidated. To clarify the details of this effect in HCC, we explored the direct targets of HSP20 in HCC using human HCC-derived HuH7 cells with HSP20 overexpression. HSP20 proteins in the HuH7 cells were coimmunoprecipitated with the p85 regulatory subunit and p110 catalytic subunit of phosphoinositide 3-kinase (PI3K), an upstream kinase of AKT. Although HSP20 overexpression in HCC cells failed to affect the expression levels of PI3K, the activity of PI3K in the unstimulated cells and even in the transforming growth factor-? stimulated cells were downregulated by HSP20 overexpression. The association of HSP20 with PI3K was also observed in human HCC tissues in vivo. These findings strongly suggest that HSP20 directly associates with PI3K and suppresses its activity in HCC, resulting in the inhibition of the AKT pathway, and subsequently decreasing the growth of HCC. PMID:24223153

Matsushima-Nishiwaki, Rie; Kumada, Takashi; Nagasawa, Tomoaki; Suzuki, Mariko; Yasuda, Eisuke; Okuda, Seiji; Maeda, Atsuyuki; Kaneoka, Yuji; Toyoda, Hidenori; Kozawa, Osamu

2013-01-01

152

Direct association of heat shock protein 20 (HSPB6) with phosphoinositide 3-kinase (PI3K) in human hepatocellular carcinoma: regulation of the PI3K activity.  

PubMed

HSP20 (HSPB6), one of small heat shock proteins (HSPs), is constitutively expressed in various tissues and has several functions. We previously reported that the expression levels of HSP20 in human hepatocellular carcinoma (HCC) cells inversely correlated with the progression of HCC, and that HSP20 suppresses the growth of HCC cells via the AKT and mitogen-activated protein kinase signaling pathways. However, the exact mechanism underlying the effect of HSP20 on the regulation of these signaling pathways remains to be elucidated. To clarify the details of this effect in HCC, we explored the direct targets of HSP20 in HCC using human HCC-derived HuH7 cells with HSP20 overexpression. HSP20 proteins in the HuH7 cells were coimmunoprecipitated with the p85 regulatory subunit and p110 catalytic subunit of phosphoinositide 3-kinase (PI3K), an upstream kinase of AKT. Although HSP20 overexpression in HCC cells failed to affect the expression levels of PI3K, the activity of PI3K in the unstimulated cells and even in the transforming growth factor-? stimulated cells were downregulated by HSP20 overexpression. The association of HSP20 with PI3K was also observed in human HCC tissues in vivo. These findings strongly suggest that HSP20 directly associates with PI3K and suppresses its activity in HCC, resulting in the inhibition of the AKT pathway, and subsequently decreasing the growth of HCC. PMID:24223153

Matsushima-Nishiwaki, Rie; Kumada, Takashi; Nagasawa, Tomoaki; Suzuki, Mariko; Yasuda, Eisuke; Okuda, Seiji; Maeda, Atsuyuki; Kaneoka, Yuji; Toyoda, Hidenori; Kozawa, Osamu

2013-01-01

153

The p85 subunit of phosphoinositide 3-kinase is associated with beta-catenin in the cadherin-based adhesion complex.  

PubMed Central

Cell adhesion is fundamental to establishing and maintaining the discrete tissues in multicellular organisms. Adhesion must be sufficiently strong to preserve tissue architecture, whilst having the capacity to readily dissociate to permit fundamental processes, such as wound repair, to occur. However, very little is known about the signalling mechanisms involved in temporary down-regulation of cell adhesion to facilitate such processes. Cadherins are the principal mediators of cell-cell adhesion in a wide variety of tissues and species and form multi-protein complexes with cytosolic and cytoskeletal proteins to express their full adhesive capacity. In the present study we report that the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) is associated with the cadherin-based adhesion complex in human epithelial cells. The interaction of p85 with the complex is via beta-catenin. We also show that the interaction of p85 and beta-catenin is direct, involves the N-terminal Src homology domain 2 of p85 and is regulated by tyrosine phosphorylation. These data suggest that PI 3-kinase may play a role in the functional regulation of the cadherin-based adhesion complex. PMID:11716761

Woodfield, R J; Hodgkin, M N; Akhtar, N; Morse, M A; Fuller, K J; Saqib, K; Thompson, N T; Wakelam, M J

2001-01-01

154

Activation of phosphoinositide 3-kinase by D2 receptor prevents apoptosis in dopaminergic cell lines.  

PubMed Central

Whereas dopamine agonists are known to provide symptomatic benefits for Parkinson's disease, recent clinical trials suggest that they might also be neuroprotective. Laboratory studies demonstrate that dopamine agonists can provide neuroprotective effects in a number of model systems, but the role of receptor-mediated signalling in these effects is controversial. We find that dopamine agonists have robust, concentration-dependent anti-apoptotic activity in PC12 cells that stably express human D(2L) receptors from cell death due to H(2)O(2) or trophic withdrawal and that the protective effects are abolished in the presence of D(2)-receptor antagonists. D(2) agonists are also neuroprotective in the nigral dopamine cell line SN4741, which express endogenous D(2) receptors, whereas no anti-apoptotic activity is observed in native PC12 cells, which do not express detectable D(2) receptors. Notably, the agonists studied differ in their relative efficacy to mediate anti-apoptotic effects and in their capacity to stimulate [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding, an indicator of G-protein activation. Studies with inhibitors of phosphoinositide 3-kinase (PI 3-kinase), extracellular-signal-regulated kinase or p38 mitogen-activated protein kinase indicate that the PI 3-kinase pathway is required for D(2) receptor-mediated cell survival. These studies indicate that certain dopamine agonists can complex with D(2) receptors to preferentially transactivate neuroprotective signalling pathways and to mediate increased cell survival. PMID:12683952

Nair, Venugopalan D; Olanow, C Warren; Sealfon, Stuart C

2003-01-01

155

Activation of phosphoinositide 3-kinase by D2 receptor prevents apoptosis in dopaminergic cell lines.  

PubMed

Whereas dopamine agonists are known to provide symptomatic benefits for Parkinson's disease, recent clinical trials suggest that they might also be neuroprotective. Laboratory studies demonstrate that dopamine agonists can provide neuroprotective effects in a number of model systems, but the role of receptor-mediated signalling in these effects is controversial. We find that dopamine agonists have robust, concentration-dependent anti-apoptotic activity in PC12 cells that stably express human D(2L) receptors from cell death due to H(2)O(2) or trophic withdrawal and that the protective effects are abolished in the presence of D(2)-receptor antagonists. D(2) agonists are also neuroprotective in the nigral dopamine cell line SN4741, which express endogenous D(2) receptors, whereas no anti-apoptotic activity is observed in native PC12 cells, which do not express detectable D(2) receptors. Notably, the agonists studied differ in their relative efficacy to mediate anti-apoptotic effects and in their capacity to stimulate [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding, an indicator of G-protein activation. Studies with inhibitors of phosphoinositide 3-kinase (PI 3-kinase), extracellular-signal-regulated kinase or p38 mitogen-activated protein kinase indicate that the PI 3-kinase pathway is required for D(2) receptor-mediated cell survival. These studies indicate that certain dopamine agonists can complex with D(2) receptors to preferentially transactivate neuroprotective signalling pathways and to mediate increased cell survival. PMID:12683952

Nair, Venugopalan D; Olanow, C Warren; Sealfon, Stuart C

2003-07-01

156

TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration  

PubMed Central

Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration. PMID:24311986

Rousseau, Adrien; McEwen, Alastair G.; Poussin-Courmontagne, Pierre; Rognan, Didier; Nominé, Yves; Rio, Marie-Christine; Tomasetto, Catherine; Alpy, Fabien

2013-01-01

157

Effects of Novel Isoform-Selective Phosphoinositide 3-Kinase Inhibitors on Natural Killer Cell Function  

PubMed Central

Phosphoinositide 3-kinases (PI3Ks) are promising targets for therapeutic development in cancer. The class I PI3K isoform p110? has received considerable attention in oncology because the gene encoding p110? (PIK3CA) is frequently mutated in human cancer. However, little is known about the function of p110? in lymphocyte populations that modulate tumorigenesis. We used recently developed investigational inhibitors to compare the function of p110? and other isoforms in natural killer (NK) cells, a key cell type for immunosurveillance and tumor immunotherapy. Inhibitors of all class I isoforms (pan-PI3K) significantly impaired NK cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity against tumor cells, whereas p110?-selective inhibitors had no effect. In NK cells stimulated through NKG2D, p110? inhibition modestly reduced PI3K signaling output as measured by AKT phosphorylation. Production of IFN-? and NK cell-derived chemokines was blocked by a pan-PI3K inhibitor and partially reduced by a p110?inhibitor, with lesser effects of p110? inhibitors. Oral administration of mice with MLN1117, a p110? inhibitor in oncology clinical trials, had negligible effects on NK subset maturation or terminal subset commitment. Collectively, these results support the targeting of PIK3CA mutant tumors with selective p110? inhibitors to preserve NK cell function. PMID:24915189

Yea, Sung Su; So, Lomon; Mallya, Sharmila; Lee, Jongdae; Rajasekaran, Kamalakannan; Malarkannan, Subramaniam; Fruman, David A.

2014-01-01

158

Spatiotemporal model of a key step in endocytosis: SNX9 recruitment via phosphoinositides  

E-print Network

Clathrin mediated endocytosis (CME) is an ubiquitous cellular pathway that regulates central aspects of cell physiology such as nutrient uptake, modulation of signal transduction, synaptic transmission and membrane turn-over. Endocytic vesicle formation depends on the timed production of specific phosphoinositides and their interactions with various endocytic proteins. Recently, it has been found that phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) produced by the class II phosphatidylinositol 3-kinase C2alpha plays a key role in the recruitment of the PX-BAR domain protein SNX9, which is proposed to play a role in the constriction of the endocytic vesicle neck [Posor et al, Nature 499, p233 (2013)]. Interestingly, SNX9 and its close paralog SNX18 are not fully specific to PI(3,4)P2 but can also bind other phospholipids, in particular to PI(4,5)P2, an abundant plasma membrane lipid required for the recruitment of many endocytic proteins. In order to understand the dynamical interplay between phospholipids a...

Schöneberg, Johannes; Posor, York; Haucke, Volker; Noe, Frank

2013-01-01

159

Ablation of phosphoinositide-3-kinase class II alpha suppresses hepatoma cell proliferation  

SciTech Connect

Cancer such as hepatocellular carcinoma (HCC) is characterized by complex perturbations in multiple signaling pathways, including the phosphoinositide-3-kinase (PI3K/AKT) pathways. Herein we investigated the role of PI3K catalytic isoforms, particularly class II isoforms in HCC proliferation. Among the siRNAs tested against the eight known catalytic PI3K isoforms, specific ablation of class II PI3K alpha (PIK3C2{alpha}) was the most effective in impairing cell growth and this was accompanied by concomitant decrease in PIK3C2{alpha} mRNA and protein levels. Colony formation ability of cells deficient for PIK3C2{alpha} was markedly reduced and growth arrest was associated with increased caspase 3 levels. A small but significant difference in gene dosage and expression levels was detected between tumor and non-tumor tissues in a cohort of 19 HCC patients. Taken together, these data suggest for the first time that in addition to class I PI3Ks in cancer, class II PIK3C2{alpha} can modulate HCC cell growth.

Ng, Stanley K.L. [Singapore Immunology Network A-STAR (Singapore)] [Singapore Immunology Network A-STAR (Singapore); Neo, Soek-Ying, E-mail: neo_soek_ying@sics.a-star.edu.sg [Singapore Immunology Network A-STAR (Singapore)] [Singapore Immunology Network A-STAR (Singapore); Yap, Yann-Wan [Singapore Immunology Network A-STAR (Singapore)] [Singapore Immunology Network A-STAR (Singapore); Karuturi, R. Krishna Murthy; Loh, Evelyn S.L. [Genome Institute of Singapore A-STAR (Singapore)] [Genome Institute of Singapore A-STAR (Singapore); Liau, Kui-Hin [Department of General Surgery, Tan Tock Seng Hospital (Singapore)] [Department of General Surgery, Tan Tock Seng Hospital (Singapore); Ren, Ee-Chee, E-mail: ren_ee_chee@immunol.a-star.edu.sg [Singapore Immunology Network A-STAR (Singapore) [Singapore Immunology Network A-STAR (Singapore); Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore (Singapore)

2009-09-18

160

Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers  

PubMed Central

Class IA phosphoinositide 3-kinases (PI3Ks) signal downstream of tyrosine kinases and Ras and control a wide variety of biological responses. In mammals, these heterodimeric PI3Ks consist of a p110 catalytic subunit (p110?, p110?, or p110?) bound to any of five distinct regulatory subunits (p85?, p85?, p55?, p55?, and p50?, collectively referred to as “p85s”). The relative expression levels of p85 and p110 have been invoked to explain key features of PI3K signaling. For example, free (i.e., non-p110-bound) p85? has been proposed to negatively regulate PI3K signaling by competition with p85/p110 for recruitment to phosphotyrosine docking sites. Using affinity and ion exchange chromatography and quantitative mass spectrometry, we demonstrate that the p85 and p110 subunits are present in equimolar amounts in mammalian cell lines and tissues. No evidence for free p85 or p110 subunits could be obtained. Cell lines contain 10,000–15,000 p85/p110 complexes per cell, with p110? and p110? being the most prevalent catalytic subunits in nonleukocytes and leukocytes, respectively. These results argue against a role of free p85 in PI3K signaling and provide insights into the nonredundant functions of the different class IA PI3K isoforms. PMID:17470792

Geering, Barbara; Cutillas, Pedro R.; Nock, Gemma; Gharbi, Severine I.; Vanhaesebroeck, Bart

2007-01-01

161

Notch-induced T cell development requires phosphoinositide-dependent kinase 1.  

PubMed

Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T lymphocyte development. A key finding is that PDK1 is required for the expression of key nutrient receptors in T cell progenitors: CD71 the transferrin receptor and CD98 a subunit of L-amino acid transporters. PDK1 is also essential for Notch-mediated trophic and proliferative responses in thymocytes. A PDK1 mutant PDK1 L155E, which supports activation of PKB but no other AGC kinases, can restore CD71 and CD98 expression in pre-T cells and restore thymocyte differentiation. However, PDK1 L155E is insufficient for thymocyte proliferation. The role of PDK1 in thymus development thus extends beyond its ability to regulate PKB. In addition, PDK1 phosphorylation of AGC kinases such as S6K and RSK is also necessary for thymocyte development. PMID:17599070

Kelly, April P; Finlay, David K; Hinton, Heather J; Clarke, Rosie G; Fiorini, Emma; Radtke, Freddy; Cantrell, Doreen A

2007-07-25

162

Evaluation of variation in the phosphoinositide-3-kinase catalytic subunit alpha oncogene and breast cancer risk  

PubMed Central

Background: Somatic mutations in phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA) are frequent in breast tumours and have been associated with oestrogen receptor (ER) expression, human epidermal growth factor receptor-2 overexpression, lymph node metastasis and poor survival. The goal of this study was to evaluate the association between inherited variation in this oncogene and risk of breast cancer. Methods: A single-nucleotide polymorphism from the PIK3CA locus that was associated with breast cancer in a study of Caucasian breast cancer cases and controls from the Mayo Clinic (MCBCS) was genotyped in 5436 cases and 5280 controls from the Cancer Genetic Markers of Susceptibility (CGEMS) study and in 30?949 cases and 29?788 controls from the Breast Cancer Association Consortium (BCAC). Results: Rs1607237 was significantly associated with a decreased risk of breast cancer in MCBCS, CGEMS and all studies of white Europeans combined (odds ratio (OR)=0.97, 95% confidence interval (CI) 0.95–0.99, P=4.6 × 10?3), but did not reach significance in the BCAC replication study alone (OR=0.98, 95% CI 0.96–1.01, P=0.139). Conclusion: Common germline variation in PIK3CA does not have a strong influence on the risk of breast cancer PMID:22033276

Stevens, K N; Garcia-Closas, M; Fredericksen, Z; Kosel, M; Pankratz, V S; Hopper, J L; Dite, G S; Apicella, C; Southey, M C; Schmidt, M K; Broeks, A; Van ‘t Veer, L J; Tollenaar, R A E M; Fasching, P A; Beckmann, M W; Hein, A; Ekici, A B; Johnson, N; Peto, J; dos Santos Silva, I; Gibson, L; Sawyer, E; Tomlinson, I; Kerin, M J; Chanock, S; Lissowska, J; Hunter, D J; Hoover, R N; Thomas, G D; Milne, R L; Pérez, JI Arias; González-Neira, A; Benítez, J; Burwinkel, B; Meindl, A; Schmutzler, R K; Bartrar, C R; Hamann, U; Ko, Y D; Brüning, T; Chang-Claude, J; Hein, R; Wang-Gohrke, S; Dörk, T; Schürmann, P; Bremer, M; Hillemanns, P; Bogdanova, N; Zalutsky, J V; Rogov, Y I; Antonenkova, N; Lindblom, A; Margolin, S; Mannermaa, A; Kataja, V; Kosma, V-M; Hartikainen, J; Chenevix-Trench, G; Chen, X; Peterlongo, P; Bonanni, B; Bernard, L; Manoukian, S; Wang, X; Cerhan, J; Vachon, C M; Olson, J; Giles, G G; Baglietto, L; McLean, C A; Severi, G; John, E M; Miron, A; Winqvist, R; Pylkäs, K; Jukkola-Vuorinen, A; Grip, M; Andrulis, I; Knight, J A; Glendon, G; Mulligan, A M; Cox, A; Brock, I W; Elliott, G; Cross, S S; Pharoah, P P; Dunning, A M; Pooley, K A; Humphreys, M K; Wang, J; Kang, D; Yoo, K-Y; Noh, D-Y; Sangrajrang, S; Gabrieau, V; Brennan, P; McKay, J; Anton-Culver, H; Ziogas, A; Couch, F J; Easton, D F

2011-01-01

163

Critical role for phosphoinositide 3-kinase gamma in parasite invasion and disease progression of cutaneous leishmaniasis.  

PubMed

Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase ? (PI3K?), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3K?, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana. Using the isoform-selective PI3K? inhibitor, AS-605240 and PI3K? gene-deficient mice, we show that selective blockade or deficiency of PI3K? significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial drug sodium stibogluconate in treatment of cutaneous leishmaniasis caused by L. mexicana. These findings reveal a unique role for PI3K? in Leishmania invasion and establishment of chronic infection, and demonstrate that therapeutic targeting of host pathways involved in establishment of infection may be a viable strategy for treating infections caused by obligate intracellular pathogens such as Leishmania. PMID:22232690

Cummings, Hannah E; Barbi, Joseph; Reville, Patrick; Oghumu, Steve; Zorko, Nicholas; Sarkar, Anasuya; Keiser, Tracy L; Lu, Bao; Rückle, Thomas; Varikuti, Sanjay; Lezama-Davila, Claudio; Wewers, Mark D; Whitacre, Caroline; Radzioch, Danuta; Rommel, Christian; Seveau, Stéphanie; Satoskar, Abhay R

2012-01-24

164

Blockade of class IB phosphoinositide-3 kinase ameliorates obesity-induced inflammation and insulin resistance.  

PubMed

Obesity and insulin resistance, the key features of metabolic syndrome, are closely associated with a state of chronic, low-grade inflammation characterized by abnormal macrophage infiltration into adipose tissues. Although it has been reported that chemokines promote leukocyte migration by activating class IB phosphoinositide-3 kinase (PI3K?) in inflammatory states, little is known about the role of PI3K? in obesity-induced macrophage infiltration into tissues, systemic inflammation, and the development of insulin resistance. In the present study, we used murine models of both diet-induced and genetically induced obesity to examine the role of PI3K? in the accumulation of tissue macrophages and the development of obesity-induced insulin resistance. Mice lacking p110? (Pik3cg(-/-)), the catalytic subunit of PI3K?, exhibited improved systemic insulin sensitivity with enhanced insulin signaling in the tissues of obese animals. In adipose tissues and livers of obese Pik3cg(-/-) mice, the numbers of infiltrated proinflammatory macrophages were markedly reduced, leading to suppression of inflammatory reactions in these tissues. Furthermore, bone marrow-specific deletion and pharmacological blockade of PI3K? also ameliorated obesity-induced macrophage infiltration and insulin resistance. These data suggest that PI3K? plays a crucial role in the development of both obesity-induced inflammation and systemic insulin resistance and that PI3K? can be a therapeutic target for type 2 diabetes. PMID:21436039

Kobayashi, Naoki; Ueki, Kohjiro; Okazaki, Yukiko; Iwane, Aya; Kubota, Naoto; Ohsugi, Mitsuru; Awazawa, Motoharu; Kobayashi, Masatoshi; Sasako, Takayoshi; Kaneko, Kazuma; Suzuki, Miho; Nishikawa, Yoshitaka; Hara, Kazuo; Yoshimura, Kotaro; Koshima, Isao; Goyama, Susumu; Murakami, Koji; Sasaki, Junko; Nagai, Ryozo; Kurokawa, Mineo; Sasaki, Takehiko; Kadowaki, Takashi

2011-04-01

165

Phosphoinositide-containing polymerized liposomes: stable membrane-mimetic vesicles for protein-lipid binding analysis.  

PubMed

Stable phosphoinositide (PIP(n))-containing liposomes were prepared using polydiacetylene photochemistry. Tethered pentacosadiynyl inositol polyphosphate (InsP(n)) analogues of Ins(1,3,4)P(3), Ins(1,4,5)P(3), and Ins(1,3,4,5)P(4) were synthesized, incorporated into vesicles made up of diyne-phosphatidylcholine and -phosphatidylethanolamine, and polymerized by UV irradiation. The polymerized liposome nanoparticles showed markedly increased stability over conventional PIP(n)-containing vesicles as a result of the covalent conjugated ene-yne network in the acyl chains. The polymerized liposomes were specifically recognized by PIP(n) binding PH domains in liposome overlay assays and amplified luminescent proximity homogeneous assays. Moreover, the biotin moiety allowed attachment of the nanoparticles to a streptavidin-coated sensor chips in surface plasmon resonance (SPR) sensor. The PIP(n) headgroups displayed on SPR sensors showed higher affinities for PH domains and PIP(n) monoclonal antibodies than did monomeric PIP(n)-analogues with biotinylated acyl chains. PMID:16287244

Ferguson, Colin G; James, Robyn D; Bigman, Cleve S; Shepard, Donnie A; Abdiche, Yasmina; Katsamba, Phinikoula S; Myszka, David G; Prestwich, Glenn D

2005-01-01

166

The Phosphoinositide-3-Kinase-Akt-mTOR Pathway as a Therapeutic Target in Breast Cancer  

PubMed Central

The phosphoinositide-3-kinase (PI3-kinase)-Akt-mTOR pathway is a central signal transduction pathway that regulates many critical aspects of normal and cancer physiology, including cell proliferation, apoptosis, cell morphology and migration, protein synthesis, and integration of metabolism. In breast cancer, somatic mutations that activate the pathway occur in more than 50% of tumors, underscoring the potentially broad impact of targeting the pathway for therapy. A vast body of preclinical data demonstrates the efficacy of pathway inhibition on tumor growth, and evidence also shows that activation of the pathway occurs in models of acquired resistance to hormonal therapy. This preclinical work led to the investigation of allosteric mTOR inhibitors, everolimus and temsirolimus, in metastatic hormone receptor–positive breast cancer. The recent BOLERO-2 trial comparing everolimus plus exemestane versus placebo plus exemestane in women with resistance to nonsteroidal aromatase inhibitors demonstrated a 6-month improvement in progression-free survival and led to FDA approval of everolimus for this indication in the United States. This landmark trial is the first demonstration of significant clinical benefit using drugs targeting this pathway in breast cancer. Many questions remain about the role of everolimus and other pathway-targeting drugs in clinical development in breast cancer treatment. This article reviews the role of the PI3-kinase-Akt-mTOR pathway in breast cancer biology and the clinical trial evidence available to date. PMID:23744866

Lauring, Josh; Park, Ben Ho; Wolff, Antonio C.

2014-01-01

167

The phosphoinositide-3-kinase-Akt-mTOR pathway as a therapeutic target in breast cancer.  

PubMed

The phosphoinositide-3-kinase (PI3-kinase)-Akt-mTOR pathway is a central signal transduction pathway that regulates many critical aspects of normal and cancer physiology, including cell proliferation, apoptosis, cell morphology and migration, protein synthesis, and integration of metabolism. In breast cancer, somatic mutations that activate the pathway occur in more than 50% of tumors, underscoring the potentially broad impact of targeting the pathway for therapy. A vast body of preclinical data demonstrates the efficacy of pathway inhibition on tumor growth, and evidence also shows that activation of the pathway occurs in models of acquired resistance to hormonal therapy. This preclinical work led to the investigation of allosteric mTOR inhibitors, everolimus and temsirolimus, in metastatic hormone receptor-positive breast cancer. The recent BOLERO-2 trial comparing everolimus plus exemestane versus placebo plus exemestane in women with resistance to nonsteroidal aromatase inhibitors demonstrated a 6-month improvement in progression-free survival and led to FDA approval of everolimus for this indication in the United States. This landmark trial is the first demonstration of significant clinical benefit using drugs targeting this pathway in breast cancer. Many questions remain about the role of everolimus and other pathway-targeting drugs in clinical development in breast cancer treatment. This article reviews the role of the PI3-kinase-Akt-mTOR pathway in breast cancer biology and the clinical trial evidence available to date. PMID:23744866

Lauring, Josh; Park, Ben Ho; Wolff, Antonio C

2013-06-01

168

Metastasis of human gastric adenocarcinoma partly depends on phosphoinositide-specific phospholipase ?1 expression.  

PubMed

It is known that phosphoinositide-specific phospholipases ?1(PLC?1) can trigger several signalling pathways to regulate cell proliferation, differentiation, and metastasis. However, whether this kinase is highly expressive and active in human gastric adenocarcinomas, and whether it can play an important role in the development of the cancer, have not yet been investigated. The aim of the study was to investigate the expression of PLC?1 in human gastric adenocarcinoma, while the question of whether PLC?1 can be activated through protein kinase B (Akt) signalling pathways to regulate cell migration was further explored using human gastric adenocarcinoma BGC-823 cell line. The expression of PLC?1 in human adenocarcinoma was detected using immunohistochemical staining. The BGC-823 cells were cultured and treated with inhibitors or transfected with plasmid construction. The cell migration of BGC-823 cells was measured with wound healing assay, cell migration assay, and the ruffling assay. The expression levels of PLC?1 and its related signal molecules in BGC-823 cells were assessed using Western blot analysis or gelatine zymography assay. PLC?1 was highly expressed in humangastric adenocarcinomas, especially in the region with lymph node metastasis. It was shown that migration of BGC-823 cells in vitro depends on PLC?1 activation. This activation is mediated through Akt, an upstream of PLC?1 that triggers the PLC?1/extracellular signal-regulated kinase (ERK)/matrix metalloproteinase (MMP) pathway in BGC-823 cells. PLC?1 activities play an important role in the metastasis of gastric adenocarcinoma, and may serve as a potential therapeutic target in this type of cancer. PMID:25308733

Zhuang, Luhua; Zhang, Bingchang; Zeng, Guoqing; Dai, Lianzhi; Qian, Hongliu; Hu, Tianhui; Song, Gang; Zhang, Bing; Xia, Chun

2014-01-01

169

Role of phosphoinositide 3-kinase in the pathogenesis of acute pancreatitis  

PubMed Central

A large body of experimental and clinical data supports the notion that inflammation in acute pancreatitis has a crucial role in the pathogenesis of local and systemic damage and is a major determinant of clinical severity. Thus, research has recently focused on molecules that can regulate the inflammatory processes, such as phosphoinositide 3-kinases (PI3Ks), a family of lipid and protein kinases involved in intracellular signal transduction. Studies using genetic ablation or pharmacologic inhibitors of different PI3K isoforms, in particular the class I PI3K? and PI3K?, have contributed to a greater understanding of the roles of these kinases in the modulation of inflammatory and immune responses. Recent data suggest that PI3Ks are also involved in the pathogenesis of acute pancreatitis. Activation of the PI3K signaling pathway, and in particular of the class IB PI3K? isoform, has a significant role in those events which are necessary for the initiation of acute pancreatic injury, namely calcium signaling alteration, trypsinogen activation, and nuclear factor-?B transcription. Moreover, PI3K? is instrumental in modulating acinar cell apoptosis, and regulating local neutrophil infiltration and systemic inflammatory responses during the course of experimental acute pancreatitis. The availability of PI3K inhibitors selective for specific isoforms may provide new valuable therapeutic strategies to improve the clinical course of this disease. This article presents a brief summary of PI3K structure and function, and highlights recent advances that implicate PI3Ks in the pathogenesis of acute pancreatitis. PMID:25386068

Lupia, Enrico; Pigozzi, Luca; Goffi, Alberto; Hirsch, Emilio; Montrucchio, Giuseppe

2014-01-01

170

PPAR? Activation Acts Cooperatively with 3-Phosphoinositide-Dependent Protein Kinase-1 to Enhance Mammary Tumorigenesis  

PubMed Central

Peroxisome proliferator-activated receptor? (PPAR?) is a transcription factor that is associated with metabolic gene regulation and inflammation. It has been implicated in tumor promotion and in the regulation of 3-phosphoinositide-dependent kinase-1 (PDK1). PDK1 is a key regulator of the AGC protein kinase family, which includes the proto-oncogene AKT/PKB implicated in several malignancies, including breast cancer. To assess the role of PDK1 in mammary tumorigenesis and its interaction with PPAR?, transgenic mice were generated in which PDK1 was expressed in mammary epithelium under the control of the MMTV enhancer/promoter region. Transgene expression increased pT308AKT and pS9GSK3?, but did not alter phosphorylation of mTOR, 4EBP1, ribosomal protein S6 and PKC?. The transgenic mammary gland also expressed higher levels of PPAR? and a gene expression profile resembling wild-type mice maintained on a diet containing the PPAR? agonist, GW501516. Both wild-type and transgenic mice treated with GW501516 exhibited accelerated rates of tumor formation that were more pronounced in transgenic animals. GW501516 treatment was accompanied by a distinct metabolic gene expression and metabolomic signature that was not present in untreated animals. GW501516-treated transgenic mice expressed higher levels of fatty acid and phospholipid metabolites than treated wild-type mice, suggesting the involvement of PDK1 in enhancing PPAR?-driven energy metabolism. These results reveal that PPAR? activation elicits a distinct metabolic and metabolomic profile in tumors that is in part related to PDK1 and AKT signaling. PMID:21297860

Pollock, Claire B.; Yin, Yuzhi; Yuan, Hongyan; Zeng, Xiao; King, Sruthi; Li, Xin; Kopelovich, Levy; Albanese, Chris; Glazer, Robert I.

2011-01-01

171

Endosomal Maturation, Rab7 GTPase and Phosphoinositides in African Swine Fever Virus Entry  

PubMed Central

Here we analyzed the dependence of African swine fever virus (ASFV) infection on the integrity of the endosomal pathway. Using confocal immunofluorescence with antibodies against viral capsid proteins, we found colocalization of incoming viral particles with early endosomes (EE) during the first minutes of infection. Conversely, viral capsid protein was not detected in acidic late endosomal compartments, multivesicular bodies (MVBs), late endosomes (LEs) or lysosomes (LY). Using an antibody against a viral inner core protein, we found colocalization of viral cores with late compartments from 30 to 60 minutes postinfection. The absence of capsid protein staining in LEs and LYs suggested that virus desencapsidation would take place at the acid pH of these organelles. In fact, inhibitors of intraluminal acidification of endosomes caused retention of viral capsid staining virions in Rab7 expressing endosomes and more importantly, severely impaired subsequent viral protein production. Endosomal acidification in the first hour after virus entry was essential for successful infection but not thereafter. In addition, altering the balance of phosphoinositides (PIs) which are responsible of the maintenance of the endocytic pathway impaired ASFV infection. Early infection steps were dependent on the production of phosphatidylinositol 3-phosphate (PtdIns3P) which is involved in EE maturation and multivesicular body (MVB) biogenesis and on the interconversion of PtdIns3P to phosphatidylinositol 3, 5-biphosphate (PtdIns(3,5)P2). Likewise, GTPase Rab7 activity should remain intact, as well as processes related to LE compartment physiology, which are crucial during early infection. Our data demonstrate that the EE and LE compartments and the integrity of the endosomal maturation pathway orchestrated by Rab proteins and PIs play a central role during early stages of ASFV infection. PMID:23133661

Cuesta-Geijo, Miguel A.; Galindo, Inmaculada; Hernáez, Bruno; Quetglas, Jose Ignacio; Dalmau-Mena, Inmaculada; Alonso, Covadonga

2012-01-01

172

Role of phosphoinositide 3-kinase in the pathogenesis of acute pancreatitis.  

PubMed

A large body of experimental and clinical data supports the notion that inflammation in acute pancreatitis has a crucial role in the pathogenesis of local and systemic damage and is a major determinant of clinical severity. Thus, research has recently focused on molecules that can regulate the inflammatory processes, such as phosphoinositide 3-kinases (PI3Ks), a family of lipid and protein kinases involved in intracellular signal transduction. Studies using genetic ablation or pharmacologic inhibitors of different PI3K isoforms, in particular the class I PI3K? and PI3K?, have contributed to a greater understanding of the roles of these kinases in the modulation of inflammatory and immune responses. Recent data suggest that PI3Ks are also involved in the pathogenesis of acute pancreatitis. Activation of the PI3K signaling pathway, and in particular of the class IB PI3K? isoform, has a significant role in those events which are necessary for the initiation of acute pancreatic injury, namely calcium signaling alteration, trypsinogen activation, and nuclear factor-?B transcription. Moreover, PI3K? is instrumental in modulating acinar cell apoptosis, and regulating local neutrophil infiltration and systemic inflammatory responses during the course of experimental acute pancreatitis. The availability of PI3K inhibitors selective for specific isoforms may provide new valuable therapeutic strategies to improve the clinical course of this disease. This article presents a brief summary of PI3K structure and function, and highlights recent advances that implicate PI3Ks in the pathogenesis of acute pancreatitis. PMID:25386068

Lupia, Enrico; Pigozzi, Luca; Goffi, Alberto; Hirsch, Emilio; Montrucchio, Giuseppe

2014-11-01

173

Phosphoinositides Are Involved in Control of the Glucose-Dependent Growth Resumption That Follows the Transition Phase in Streptomyces lividans?  

PubMed Central

The interruption of the sblA gene of Streptomyces lividans was previously shown to lead to relief of glucose repression of the normally strongly glucose-repressed ?-amylase gene. In addition to this relief, an early entry into stationary phase was observed when cells were grown in a minimal medium containing glucose as the main carbon source. In this study, we established that this mutant does not resume growth after the transition phase when cultured in the complex glucose-rich liquid medium R2YE and sporulates much earlier than the wild-type strain when plated on solid R2YE. These phenotypic differences, which were abolished when glucose was omitted from the R2YE medium, correlated with a reduced glucose uptake ability of the sblA mutant strain. sblA was shown to encode a bifunctional enzyme possessing phospholipase C-like and phosphoinositide phosphatase activities. The cleavage of phosphoinositides by SblA seems necessary to trigger the glucose-dependent renewed growth that follows the transition phase. The transient expression of sblA that takes place just before the transition phase is consistent with a regulatory role for this gene during the late stages of growth. The tight temporal control of sblA expression was shown to depend on two operator sites. One, located just upstream of the ?35 promoter region, likely constitutes a repressor binding site. The other, located 170 bp downstream of the GTG sblA translational start codon, may be involved in the regulation of the degradation of the sblA transcript. This study suggests that phosphoinositides constitute important regulatory molecules in Streptomyces, as they do in eukaryotes. PMID:17122350

Chouayekh, H.; Nothaft, H.; Delaunay, S.; Linder, M.; Payrastre, B.; Seghezzi, N.; Titgemeyer, F.; Virolle, M. J.

2007-01-01

174

Role of the phosphoinositide pathway in the light-dependent C4 phosphoenolpyruvate carboxylase phosphorylation cascade in Digitaria sanguinalis protoplasts.  

PubMed

Stimulus-response coupling in animal cells frequently involves the hydrolysis of PtdIns(4,5)P(2) which is catalysed by phosphoinositide-specific phospholipase C (PI-PLC). There is an increasing body of evidence for PI-PLC-based signalling in plant cells; however, the physiological role of this system remains poorly documented in plants. Our data provide the first evidence that a PI-PLC-based signalling system is a committed step in the transduction chain controlling the phosphorylation state of C(4) phosphoenolpyruvate carboxylase (PEPC), the regulation of which is central to the assimilation of atmospheric CO(2) in C(4) plants. PMID:11171220

Coursol, S; Pierre, J N; Vidal, J

2000-12-01

175

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region*  

PubMed Central

The small intestinal BB Na+/H+ antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P3) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His6 proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475–589), F2 (amino acids 590–667), F3 (amino acids 668–747), and F4 (amino acids 748–832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P2 and PI(3,4,5)P3 bound only to the NHE3 F1 fusion protein (amino acids 475–589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na+/H+ exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P2 and PI(3,4,5)P3 binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P2 and PI(3,4,5)P3 binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P2 or PI(3,4,5)P3 binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr501–Arg512 and Arg520–Arg552) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression. PMID:20736165

Mohan, Sachin; Tse, Chung Ming; Gabelli, Sandra B.; Sarker, Rafiquel; Cha, Boyoung; Fahie, Kamau; Nadella, Mythili; Zachos, Nicholas C.; Tu-Sekine, Becky; Raben, Daniel; Amzel, L. Mario; Donowitz, Mark

2010-01-01

176

NHE3 activity is dependent on direct phosphoinositide binding at the N terminus of its intracellular cytosolic region.  

PubMed

The small intestinal BB Na(+)/H(+) antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P(3)) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His(6) proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475-589), F2 (amino acids 590-667), F3 (amino acids 668-747), and F4 (amino acids 748-832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P(2) and PI(3,4,5)P(3) bound only to the NHE3 F1 fusion protein (amino acids 475-589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na(+)/H(+) exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P(2) and PI(3,4,5)P(3) binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P(2) and PI(3,4,5)P(3) binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P(2) or PI(3,4,5)P(3) binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr(501)-Arg(512) and Arg(520)-Arg(552)) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression. PMID:20736165

Mohan, Sachin; Tse, Chung Ming; Gabelli, Sandra B; Sarker, Rafiquel; Cha, Boyoung; Fahie, Kamau; Nadella, Mythili; Zachos, Nicholas C; Tu-Sekine, Becky; Raben, Daniel; Amzel, L Mario; Donowitz, Mark

2010-11-01

177

Phylogenomics of phosphoinositide lipid kinases: perspectives on the evolution of second messenger signaling and drug discovery  

PubMed Central

Background Phosphoinositide lipid kinases (PIKs) generate specific phosphorylated variants of phosatidylinositols (PtdIns) that are critical for second messenger signaling and cellular membrane remodeling. Mammals have 19 PIK isoforms spread across three major families: the PtIns 3-kinases (PI3Ks), PtdIns 4-kinases (PI4Ks), and PtdIns-P (PIP) kinases (PIPKs). Other eukaryotes have fewer yet varying PIK complements. PIKs are also an important, emerging class of drug targets for many therapeutic areas including cancer, inflammatory and metabolic diseases and host-pathogen interactions. Here, we report the genomic occurrences and evolutionary relationships or phylogenomics of all three PIK families across major eukaryotic groups and suggest potential ramifications for drug discovery. Results Our analyses reveal four core eukaryotic PIKs which are type III PIK4A and PIK4B, and at least one homolog each from PI3K (possibly PIK3C3 as the ancestor) and PIP5K families. We also applied evolutionary analyses to PIK disease ontology and drug discovery. Mutated PIK3CA are known to be oncogenic and several inhibitors are in anti-cancer clinical trials. We found conservation of activating mutations of PIK3CA in paralogous isoforms suggesting specific functional constraints on these residues. By mapping published compound inhibition data (IC50s) onto a phylogeny of PI3Ks, type II PI4Ks and distantly related, MTOR, ATM, ATR and PRKDC kinases, we also show that compound polypharmacology corresponds to kinase evolutionary relationships. Finally, we extended the rationale for drugs targeting PIKs of malarial Plasmodium falciparum, and the parasites, Leishmania sp. and Trypanosoma sp. by identifying those PIKs highly divergent from human homologs. Conclusion Our phylogenomic analysis of PIKs provides new insights into the evolution of second messenger signaling. We postulate two waves of PIK diversification, the first in metazoans with a subsequent expansion in cold-blooded vertebrates that was post-emergence of Deutrostomia\\Chordata but prior to the appearance of mammals. Reconstruction of the evolutionary relationships among these lipid kinases also adds to our understanding of their roles in various diseases and assists in their development as potential drug targets. PMID:21208444

2011-01-01

178

Bradykinin-stimulated phosphoinositide metabolism in cultured canine tracheal smooth muscle cells.  

PubMed Central

1. Stimulation of bradykinin (BK) receptors coupled to phosphoinositide (PI) hydrolysis was investigated in canine cultured tracheal smooth muscle cells (TSMCs). BK, kallidin, and des-Arg9-BK, stimulated [3H]-inositol phosphates (IPs) accumulation in a dose-dependent manner with half-maximal responses (EC50) at 20 +/- 5, 13 +/- 4, and 2.3 +/- 0.7 nM, (n = 5), respectively. 2. D-Arg[Hyp3, D-Phe7]-BK and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK, B2 receptor antagonists, were equipotent in blocking the BK-induced IPs accumulation with pKB = 7.1 and 7.3, respectively. 3. Short-term exposure of TSMCs to phorbol 12-myristate 13-acetate (PMA, 1 microM) attenuated BK-stimulated IPs accumulation. The concentrations of PMA that gave half-maximal and maximal inhibition of BK-induced IPs accumulation were 15 +/- 4 nM and 1 microM, n = 3, respectively. The inhibitory effect of PMA on BK-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. 4. Prolonged incubation of TSMCs with PMA for 24 h, resulted in a recovery of receptor responsiveness which may be due to down-regulation of PKC. The inactive phorbol ester, 4 alpha-phorbol 12, 13-didecanoate at 1 microM, did not inhibit this response. 5. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the G protein(s) can be directly activated by AlF4-, which is uncoupled from phospholipase C by PMA treatment. 6. Incubation of TSMCs in the absence of external Ca2+ or upon removal of Ca2+ by addition of EGTA, caused a decrease in IPs accumulation without changing the basal levels. Addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs stimulated IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or BK.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 5 Figure 6 PMID:8012698

Yang, C. M.; Hsia, H. C.; Chou, S. P.; Ong, R.; Hsieh, J. T.; Luo, S. F.

1994-01-01

179

The basal level of intracellular calcium gates the activation of phosphoinositide 3-kinase - Akt signaling by brain-derived neurotrophic factor in cortical neurons  

PubMed Central

Brain derived neurotrophic factor (BDNF) mediates survival and neuroplasticity through the activation of phosphoinositide 3-kinase (PI3K)-Akt pathway. Although previous studies suggested the roles of MAPK, PLC-?-mediated intra-cellular calcium ([Ca2+]i) increase, and extra-cellular calcium influx in regulating Akt activation, the cellular mechanisms are largely unknown. We demonstrated that sub-nanomolar BDNF significantly induced Akt activation in developing cortical neurons. The TrkB-dependent Akt phosphorylation at S473 and T308 required only PI3K, but not PLC and MAPK activity. Blocking NMDA receptors, L-type voltage-gated calcium channels, and chelating extra-cellular calcium by EGTA failed to block BDNF-induced Akt phosphorylation. In contrast, chelating [Ca2+]i by BAPTA-AM abolished Akt phosphorylation. Interestingly, sub-nanomolar BDNF did not stimulate [Ca2+]i increase under our culture conditions. Together with that NMDA- and membrane depolarization-induced [Ca2+]i increase did not activate Akt, we conclude that the basal level of [Ca2+]i gates BDNF function. Furthermore, inhibiting calmodulin by W13 suppressed Akt phosphorylation. On the other hand, inhibition of protein phosphatase 1 by okadaic acid and tautomycin rescued Akt phosphorylation in BAPTA- and W13-treated neurons. We further demonstrated that the phosphorylation of PDK1 did not correlate with Akt phosphorylation at T308. Our results suggested novel roles of basal [Ca2+]i, rather than activity-induced calcium elevation, in BDNF-Akt signaling. PMID:18485103

Zheng, Fei; Soellner, Deborah; Nunez, Joseph; Wang, Hongbing

2008-01-01

180

Cellular transformation and activation of the phosphoinositide-3-kinase-Akt cascade by the ETV6-NTRK3 chimeric tyrosine kinase requires c-Src.  

PubMed

The ETV6-NTRK3 (EN) chimeric tyrosine kinase, a potent oncoprotein expressed in tumors derived from multiple cell lineages, functions as a constitutively active protein-tyrosine kinase. ETV6-NTRK expression leads to the constitutive activation of two major effector pathways of wild-type NTRK3, namely, the Ras-mitogen-activated protein kinase (MAPK) mitogenic pathway and the phosphoinositide-3-kinase (PI3K)-Akt pathway mediating cell survival, and both are required for EN transformation. However, it remains unclear how ETV6-NTRK3 activates Ras-Erk1/2 and/or PI3K-Akt cascades. Here, we define some aspects of the molecular mechanisms regulating ETV6-NTRK-dependent Ras-Erk1/2 and PI3K-Akt activation. We show that ETV6-NTRK3 associates with c-Src, and that treatment with SU6656, a c-Src inhibitor, completely blocks ETV6-NTRK-transforming activity. Treatment of NIH3T3 cells expressing ETV6-NTRK3 with SU6656 attenuated the activation of Ras-Erk1/2 and PI3K-Akt. Suppression of c-Src by RNA interference in NIH3T3-ETV6-NTRK3 cells resulted in markedly decreased expression of cyclin D1 and suppression of activation of Ras-Erk1/2 and PI3K-Akt. However, in Src-deficient cells, the ETV6-NTRK3 failed to activate the PI3K-Atk pathway, but not the Ras-Erk1/2 pathway. Therefore, these data indicate that ETV6-NTRK3 induces the PI3K-Akt cascade through the activation of c-Src. PMID:17409427

Jin, Wook; Yun, Chohee; Hobbie, Amy; Martin, Matthew J; Sorensen, Poul H B; Kim, Seong-Jin

2007-04-01

181

Activation and Membrane Binding of Retinal Protein Kinase B?/Akt1 is Regulated through Light-Dependent Generation of Phosphoinositides  

PubMed Central

Akt is a phospholipid-binding protein and the downstream effector of the phosphoinositide 3-kinase (PI3K) pathway. Akt has three isoforms: Akt1, Akt2, and Akt3. All of these isoforms are expressed in rod photoreceptor cells, but the individual functions of each isoform are not known. In this study we found that light induces the activation of Akt1. The membrane binding of Akt1 to rod outer segments (ROS) is insulin receptor (IR)/PI3K-dependent as demonstrated by reduced binding of Akt1 to ROS membranes of photoreceptor-specific IR knockout mice. Membrane binding of Akt1 is mediated through its Pleckstrin homology (PH) domain. To determine whether binding of the PH domain of Akt1 to photoreceptor membranes is regulated by light, various green fluorescent protein (GFP)/Akt1-PH domain fusion proteins were expressed in rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The R25C mutant PH domain of Akt1, which does not bind phosphoinositides, failed to associate with plasma membranes in a light-dependent manner. This study suggests that light-dependent generation of phosphoinositides regulates the activation and membrane binding of Akt1 in vivo. Our results also suggest that actin cytoskeletal organization may be regulated through light-dependent generation of phosphoinositides. PMID:18823366

Li, Guiyuan; Rajala, Ammaji; Wiechmann, Allan F.; Anderson, Robert E.; Rajala, Raju V.S.

2008-01-01

182

Response to platelet-activating factor in human platelets stored and aged in plasma. Decrease in aggregation, phosphoinositide turnover, and receptor affinity  

SciTech Connect

Human platelet concentrates were stored in polyolefin bags at 22 to 24 degrees C on a horizontal shaker for up to 8 days. At different intervals, aliquots of platelet-rich plasma (PRP) were removed aseptically and five variables, i.e., platelet counts, morphology, platelet-activating factor (PAF)-stimulated aggregation, phosphoinositide turnover, and (3H)PAF binding to platelet receptors, were studied. The number of platelets did not change during the 8 days of storage. Scanning electron microscopy of the platelets revealed a gradual morphologic change from biconcave flat discs to irregular, crenated forms. The PAF-induced aggregation of platelets declined with time of storage. A decrease to 50 percent of the Day 1 aggregatory response to PAF was evident on Day 2, and there was a further decline to about 20 percent by Day 6. Similarly, PAF receptor-coupled phosphoinositide turnover, as monitored by 32P incorporation into individual phosphoinositides, decreased dramatically with storage. After 2 to 3 days of storage, the phosphoinositide turnover was reduced to 50 percent of the original response, and it continued to decline to about 25 percent of original response by Day 5 or 6. The binding of (3H)PAF to washed human platelets indicated subtle changes between Days 2 and 4, which became more noticeable by Day 6. These results have raised the possibility of changes in the number of the receptors and/or their affinity for the ligand during storage. We conclude that although the number of platelets was maintained during storage for 8 days, a general deterioration of their responses to PAF occurred at the levels of cell surface receptor, transmembrane signaling (phosphoinositide turnover), and response (aggregation).

Shukla, S.D.; Morrison, W.J.; Klachko, D.M.

1989-07-01

183

Dominant-Activating, Germline Mutations in Phosphoinositide 3-Kinase p110? Cause T Cell Senescence and Human Immunodeficiency  

PubMed Central

The p110? subunit of phosphoinositide 3-kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. Here we report three different germline, heterozygous, gain-of-function mutations in the PIK3CD gene encoding p110? in fourteen patients from seven families. These patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and CMV and/or EBV viremia. Strikingly, naïve and central memory T cells were severely deficient, while senescent effector T cells were over-represented. In vitro, patient T cells exhibited increased phosphorylation of Akt and hyperactivation of mTOR, enhanced glucose uptake and terminal effector differentiation. Importantly, treatment with rapamycin to inhibit mTOR activity in vivo partially restored naïve T cells, largely rescued the in vitro T cell defects, and improved clinical course. PMID:24165795

Lucas, Carrie L.; Kuehn, Hye Sun; Zhao, Fang; Niemela, Julie E.; Deenick, Elissa K.; Palendira, Umaimainthan; Avery, Danielle T.; Moens, Leen; Cannons, Jennifer L.; Biancalana, Matthew; Stoddard, Jennifer; Ouyang, Weiming; Frucht, David L.; Rao, V. Koneti; Atkinson, T. Prescott; Agharahimi, Anahita; Hussey, Ashleigh A.; Folio, Les R.; Olivier, Kenneth N.; Fleisher, Thomas A.; Pittaluga, Stefania; Holland, Steven M.; Cohen, Jeffrey I.; Oliviera, Joao B.; Tangye, Stuart G.; Schwartzberg, Pamela L.; Lenardo, Michael J.; Uzel, Gulbu

2014-01-01

184

Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors  

SciTech Connect

Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

Medina, Jesus R.; Becker, Christopher J.; Blackledge, Charles W.; Duquenne, Celine; Feng, Yanhong; Grant, Seth W.; Heerding, Dirk; Li, William H.; Miller, William H.; Romeril, Stuart P.; Scherzer, Daryl; Shu, Arthur; Bobko, Mark A.; Chadderton, Antony R.; Dumble, Melissa; Gardiner, Christine M.; Gilbert, Seth; Liu, Qi; Rabindran, Sridhar K.; Sudakin, Valery; Xiang, Hong; Brady, Pat G.; Campobasso, Nino; Ward, Paris; Axten, Jeffrey M. (GSKPA)

2014-10-02

185

The putative phosphoinositide-specific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth.  

PubMed Central

Using the polymerase chain reaction technique, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) in the yeast Saccharomyces cerevisiae. The nucleotide sequence indicates that the gene encodes a polypeptide of 869 amino acid residues with a calculated molecular mass of 101 kDa. This polypeptide has both the X and Y regions conserved among mammalian PLC-beta, -gamma, and -delta, and the structure is most similar to that of mammalian PLC-delta. This putative yeast PLC gene has been designated PLC1. Disruption of PLC1 results in slow growth or lethality for cells, depending on their genetic background and the medium, indicating that PLC1 is important for cell growth. Expression of rat PLC-delta 1 cDNA suppressed the growth defect of plc1 disruptants, strongly suggesting that PLC1 encodes PLC. Images Fig. 4 Fig. 5 PMID:8383328

Yoko-o, T; Matsui, Y; Yagisawa, H; Nojima, H; Uno, I; Toh-e, A

1993-01-01

186

CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions.  

PubMed

Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositides (PIPn) such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIPn regulation. We determined the functional consequences of this mutation using electrophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIPn inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIPn sensitivity depended on an intact amino (NH2) terminal PIPn regulation module. These observations led us to hypothesize that L633P enhances PIPn inhibition by altering the coupling between NH2- and COOH-terminal regions of CNGA3. A recombinant COOH-terminal fragment partially restored normal PIPn sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the L633P mutation. PMID:23552282

Dai, Gucan; Varnum, Michael D

2013-07-15

187

Phosphoinositide 3-kinase targeting by the ? galactoside binding protein cytokine negates akt gene expression and leads aggressive breast cancer cells to apoptotic death  

Microsoft Academic Search

INTRODUCTION: Phosphoinositide 3-kinase (PI3K)-activated signalling has a critical role in the evolution of aggressive tumourigenesis and is therefore a prime target for anticancer therapy. Previously we have shown that the ? galactoside binding protein (?GBP) cytokine, an antiproliferative molecule, induces functional inhibition of class 1A and class 1B PI3K. Here, we have investigated whether, by targeting PI3K, ?GBP has therapeutic

Valerie Wells; Livio Mallucci

2009-01-01

188

Modulation of phosphoinositide 3-kinase activation by cholesterol level suggests a novel positive role for lipid rafts in lysophosphatidic acid signalling  

Microsoft Academic Search

Methyl-?-cyclodextrin (M?CD) was used to explore a role for cholesterol-enriched plasma membrane microdomains in coupling lysophosphatidic acid (LPA) stimulation to phosphoinositide 3-kinase (PI3K) activation. Cholesterol depletion strongly inhibited the production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in Vero cells stimulated with LPA. In agreement, the phosphorylation of Akt\\/protein kinase B, but not of Erk kinases, was suppressed by M?CD. M?CD

Christine Peres; Armelle Yart; Bertrand Perret; Jean-Pierre Salles; Patrick Raynal

2003-01-01

189

Aggregated Beta Amyloid Peptide 1–40 Decreases Ca2+- and Cholinergic Receptor-Mediated Phosphoinositide Degradation by Alteration of Membrane and Cytosolic Phospholipase C in Brain Cortex  

Microsoft Academic Search

The effects of full-length amyloid ß protein, Aß (1–40), on phosphoinositide-specific phospholipase C (PLC) were investigated in synaptic plasma membranes (SPM) and cytosol prepared from the cerebral cortex of adult rats. Moreover, the role of Aß (1–40) on the activation of lipid peroxidation was evaluated. The activity of phospholipase C (PLC) acting on phosphatidylinositol (PI) and phosphatidylinositol-4, 5-bisphosphate (PIP2) was

Agata Zambrzycka; Robert P. Strosznajder; Joanna B. Strosznajder

2000-01-01

190

Targeted Expression of the Class II Phosphoinositide 3Kinase in Drosophila melanogaster Reveals Lipid Kinase-Dependent Effects on Patterning and Interactions with Receptor Signaling Pathways  

Microsoft Academic Search

Phosphoinositide 3-kinases (PI3Ks) can be divided into three distinct classes (I, II, and III) on the basis of their domain structures and the lipid signals that they generate. Functions have been assigned to the class I and class III enzymes but have not been established for the class II PI3Ks. We have obtained the first evidence for a biological function

Lindsay K. MacDougall; Mary Elizabeth Gagou; Sally J. Leevers; Ernst Hafen; Michael D. Waterfield

2004-01-01

191

A Direct Linkage between the Phosphoinositide 3-Kinase-AKT Signaling Pathway and the Mammalian Target of Rapamycin in Mitogen-stimulated and Transformed Cells1  

Microsoft Academic Search

The microbially derived antiproliferative agent rapamycin inhibits cell growth by interfering with the signaling functions of the mammalian target of rapamycin (mTOR). In this study, we demonstrate that inter- leukin-3 stimulation induces a wortmannin-sensitive increase in mTOR kinase activity in a myeloid progenitor cell line. The involvement of phosphoinositide 3*-kinase (PI3K) in the regulation of mTOR activity was further suggested

Aleksandar Sekulic; Christine C. Hudson; James L. Homme; Peng Yin; Diane M. Otterness; Larry M. Karnitz; Robert T. Abraham

2000-01-01

192

Acylated and unacylated ghrelin attenuate isoproterenol-induced lipolysis in isolated rat visceral adipocytes through activation of phosphoinositide 3-kinase ? and phosphodiesterase 3B.  

PubMed

The acylated peptide ghrelin (AG) and its endogenous non-acylated isoform (UAG) protect cardiomyocytes, pancreatic ?-cells, and preadipocytes from apoptosis, and induce preadipocytes differentiation into adipocytes. These events are mediated by AG and UAG binding to a still unidentified receptor, which determines the activation of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) ERK1/2. AG and UAG also possess antilipolytic activity in vitro, but the underlying mechanism remains unknown. Thus, the objective of the current study was to characterize the molecular events involved in AG/UAG receptor signaling cascade. We treated rat primary visceral adipocytes with isoproterenol (ISO) and forskolin (FSK) to stimulate lipolysis, simultaneously incubating them with or without AG or UAG. Both peptides blocked ISO- and FSK-induced lipolysis. By direct measurement of cAMP intracellular content, we demonstrated that AG/UAG effect was associated to a reduction of ISO-induced cAMP accumulation. Moreover, the cAMP analog 8Br-cAMP abolished AG/UAG effect. As AG and UAG were ineffective against lipolysis induced by db-cAMP, another poorly hydrolyzable cAMP analog, phosphodiesterase (PDE) involvement was hypothesized. Indeed, cilostamide, a specific PDE3B inhibitor, blocked AG/UAG effect on ISO-induced lipolysis. Furthermore, the PI3K inhibitor wortmannin and AKT inhibitor 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4piperidinyl)-2H-benzimidazol-2-one trifluoroacetate also blocked AG/UAG action, suggesting a role in PDE3B activation. In particular, PI3K isoenzyme gamma (PI3K?) selective inhibition through the compound AS605240 prevented AG/UAG effect on ISO-stimulated lipolysis, hampering AKT phosphorylation on Ser(473). Taken together, these data demonstrate for the first time that AG/UAG attenuation of ISO-induced lipolysis involves PI3K?/AKT and PDE3B. PMID:21435395

Baragli, Alessandra; Ghè, Corrado; Arnoletti, Elisa; Granata, Riccarda; Ghigo, Ezio; Muccioli, Giampiero

2011-06-01

193

Activated PTHLH Coupling Feedback Phosphoinositide to G-Protein Receptor Signal-Induced Cell Adhesion Network in Human Hepatocellular Carcinoma by Systems-Theoretic Analysis  

PubMed Central

Studies were done on analysis of biological processes in the same high expression (fold change ?2) activated PTHLH feedback-mediated cell adhesion gene ontology (GO) network of human hepatocellular carcinoma (HCC) compared with the corresponding low expression activated GO network of no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection). Activated PTHLH feedback-mediated cell adhesion network consisted of anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolism, cell adhesion, cell differentiation, cell-cell signaling, G-protein-coupled receptor protein signaling pathway, intracellular transport, metabolism, phosphoinositide-mediated signaling, positive regulation of transcription, regulation of cyclin-dependent protein kinase activity, regulation of transcription, signal transduction, transcription, and transport in HCC. We proposed activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network. Our hypothesis was verified by the different activated PTHLH feedback-mediated cell adhesion GO network of HCC compared with the corresponding inhibited GO network of no-tumor hepatitis/cirrhotic tissues, or the same compared with the corresponding inhibited GO network of HCC. Activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network included BUB1B, GNG10, PTHR2, GNAZ, RFC4, UBE2C, NRXN3, BAP1, PVRL2, TROAP, and VCAN in HCC from GEO dataset using gene regulatory network inference method and our programming. PMID:22997493

Wang, Lin; Huang, Juxiang; Jiang, Minghu; Lin, Hong; Qi, Lianxiu; Diao, Haizhen

2012-01-01

194

CAMK1 phosphoinositide signal-mediated protein sorting and transport network in human hepatocellular carcinoma (HCC) by biocomputation.  

PubMed

We data-analyzed and constructed the high-expression CAMK1 phosphoinositide signal-mediated protein sorting and transport network in human hepatocellular carcinoma (HCC) compared with low-expression (fold change ? 2) no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection) in GEO data set, using integration of gene regulatory network inference method with gene ontology (GO). Our result showed that CAMK1 transport subnetwork upstream KCNQ3, LCN2, NKX2_5, NUP62, SORT1, STX1A activated CAMK1, and downstream CAMK1-activated AFP, ENAH, KPNA2, SLC4A3; CAMK1 signal subnetwork upstream BRCA1, DKK1, GPSM2, LEF1, NR5A1, NUP62, SORT1, SSTR5, TBL3 activated CAMK1, and downstream CAMK1-activated MAP2K6, SFRP4, SSTR5, TSHB, UBE2C in HCC. We proposed that CAMK1 activated network enhanced endosome to lysosome transport, endosome transport via multivesicular body sorting pathway, Golgi to endosome transport, intracellular protein transmembrane transport, intracellular protein transport, ion transport, mRNA transport, plasma membrane to endosome transport, potassium ion transport, protein transport, vesicle-mediated transport, anion transport, intracellular transport, androgen receptor signaling pathway, cell surface receptor-linked signal transduction, hormone-mediated signaling, induction of apoptosis by extracellular signals, signal transduction by p53 class mediator resulting in transcription of p21 class mediator, signal transduction resulting in induction of apoptosis, phosphoinositide-mediated signaling, Wnt receptor signaling pathway, as a result of inducing phosphoinositide signal-mediated protein sorting, and transport in HCC. Our hypothesis was verified by CAMK1 functional regulation subnetwork containing positive regulation of calcium ion transport via voltage gated calcium channel, cell proliferation, DNA repair, exocytosis, I-kappaB kinase/NF-kappaB cascade, immunoglobulin-mediated immune response, mast cell activation, natural killer cell-mediated cytotoxicity directed against tumor cell target, protein ubiquitination, sodium ion transport, survival gene product activity, T cell-mediated cytotoxicity, transcription, transcription from RNA polymerase II promoter, transcription initiation from RNA polymerase II promoter, transcription via serum response element binding, exit from mitosis, ubiquitin ligase activity during mitotic cell cycle, regulation of angiogenesis, apoptosis, cell growth, cell proliferation, cyclin-dependent protein kinase activity, gene expression, insulin secretion, steroid biosynthesis, transcription from RNA polymerase II promoter, transcription from RNA polymerase III promoter, cell cycle, cell migration, DNA recombination, and protein metabolism; also by CAMK1 negative functional regulation subnetwork including negative regulation of apoptosis, cell proliferation, centriole replication, fatty acid biosynthesis, lipoprotein lipase activity, MAPK activity, progression through cell cycle, transcription, transcription from RNA polymerase II promoter, cell growth, phosphorylation, and ubiquitin ligase activity during mitotic cell cycle in HCC. PMID:24825433

Wang, Lin; Huang, Juxiang; Jiang, Minghu; Chen, Qingchun; Jiang, Zhenfu; Feng, Haitao

2014-11-01

195

Endogenous mono-ADP-ribosylation of the free Gbetagamma prevents stimulation of phosphoinositide 3-kinase-gamma and phospholipase C-beta2 and is activated by G-protein-coupled receptors.  

PubMed Central

We have recently demonstrated that the beta subunit of the heterotrimeric G-proteins is endogenously mono-ADP-ribosylated in intact cells. The modified betagamma heterodimer loses its ability to inhibit calmodulin-stimulated type 1 adenylate cyclase and, remarkably, is de-ADP-ribosylated by a cytosolic hydrolase that completes an ADP-/de-ADP-ribosylation cycle of potential physiological relevance. In the present study, we show that this ADP-ribosylation might indeed be a general mechanism for termination of betagamma signalling, since the ADP-ribosylated betagamma subunit is also unable to activate both phosphoinositide 3-kinase-gamma and phospholipase C-beta2. Moreover, we show that beta subunit ADP-ribosylation is induced by G-protein-coupled receptor activation, since hormone stimulation of Chinese-hamster ovary plasma membranes leads to increases in beta subunit labelling. This occurs when betagamma is in its active heterodimeric conformation, since full inhibition of this modification can be achieved by binding of GDP-alphai3 to the betagamma heterodimer. Taken together, these findings delineate a pathway that arises from the activation of a G-protein-coupled receptor and leads to the inhibition of betagamma activity through its reversible mono-ADP-ribosylation. PMID:12149126

Lupi, Rosita; Dani, Nadia; Dietrich, Alexander; Marchegiani, Adriano; Turacchio, Sabrina; Berrie, Christopher P; Moss, Joel; Gierschik, Peter; Corda, Daniela; Di Girolamo, Maria

2002-01-01

196

Sodium antimony gluconate induces generation of reactive oxygen species and nitric oxide via phosphoinositide 3-kinase and mitogen-activated protein kinase activation in Leishmania donovani-infected macrophages.  

PubMed

Pentavalent antimony complexes, such as sodium stibogluconate and sodium antimony gluconate (SAG), are still the first choice for chemotherapy against various forms of leishmaniasis, including visceral leishmaniasis, or kala-azar. Although the requirement of a somewhat functional immune system for the antileishmanial action of antimony was reported previously, the cellular and molecular mechanism of action of SAG was not clear. Herein, we show that SAG induces extracellular signal-regulated kinase 1 (ERK-1) and ERK-2 phosphorylation through phosphoinositide 3-kinase (PI3K), protein kinase C, and Ras activation and p38 mitogen-activated protein kinase (MAPK) phosphorylation through PI3K and Akt activation. ERK-1 and ERK-2 activation results in an increase in the production of reactive oxygen species (ROS) 3 to 6 h after SAG treatment, while p38 MAPK activation and subsequent tumor necrosis factor alpha release result in the production of nitric oxide (NO) 24 h after SAG treatment. Thus, this study has provided the first evidence that SAG treatment induces activation of some important components of the intracellular signaling pathway, which results in an early wave of ROS-dependent parasite killing and a stronger late wave of NO-dependent parasite killing. This opens up the possibility of this metalloid chelate being used in the treatment of various diseases either alone or in combination with other drugs and vaccines. PMID:16641451

Mookerjee Basu, Jayati; Mookerjee, Ananda; Sen, Prosenjit; Bhaumik, Suniti; Sen, Pradip; Banerjee, Subha; Naskar, Ksudiram; Choudhuri, Soumitra K; Saha, Bhaskar; Raha, Sanghamitra; Roy, Syamal

2006-05-01

197

Phospholipase C ? and Phosphoinositide 3-Kinase Link Cytokines to ERK Activation in Hematopoietic Cells With Normal and Oncogenic Kras  

PubMed Central

Oncogenic K-Ras proteins, such as K-RasG12D, accumulate in the active, guanosine triphosphate (GTP)–bound conformation and stimulate signaling through effector kinases. The presence of the K-RasG12D oncoprotein at a similar abundance to that of endogenous wild-type K-Ras results in only minimal phosphorylation and activation of the canonical Raf–mitogen-activated or extracellular signal–regulated protein kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) and phosphoinositide-3 kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR) signaling cascades in primary hematopoietic cells, and these pathways remain dependent on growth factors for efficient activation. Here, we show that phospholipase C ? (PLC-?), PI3K, and their generated second messengers link activated cytokine receptors to Ras and ERK signaling in differentiated bone marrow cells and in a cell population highly enriched for leukemia stem cells. Cells expressing endogenous oncogenic K-RasG12D remained dependent on the second messenger diacylglycerol for the efficient activation of Ras-ERK signaling. These data raise the unexpected possibility of therapeutically targeting proteins that function upstream of oncogenic Ras in cancer. PMID:24300897

Diaz-Flores, Ernesto; Goldschmidt, Hana; Depeille, Philippe; Ng, Victor; Akutagawa, Jon; Krisman, Kimberly; Crone, Michael; Burgess, Michael R.; Williams, Olusegun; Houseman, Benjamin; Shokat, Kevan; Sampath, Deepak; Bollag, Gideon; Roose, Jeroen P.; Braun, Benjamin S.; Shannon, Kevin

2014-01-01

198

Phosphoinositide-Dependent Kinase 1 and mTORC2 Synergistically Maintain Postnatal Heart Growth and Heart Function in Mice  

PubMed Central

The protein kinase Akt plays a critical role in heart function and is activated by phosphorylation of threonine 308 (T308) and serine 473 (S473). While phosphoinositide-dependent kinase 1 (PDK1) is responsible for Akt T308 phosphorylation, the identities of the kinases for Akt S473 phosphorylation in the heart remain controversial. Here, we disrupted mTOR complex 2 (mTORC2) through deletion of Rictor in the heart and found normal heart growth and function. Rictor deletion caused significant reduction of Akt S473 phosphorylation but enhanced Akt T308 phosphorylation, suggesting that a high level of Akt T308 phosphorylation maintains Akt activity and heart function. Deletion of Pdk1 in the heart caused significantly enhanced Akt S473 phosphorylation that was suppressed by removal of Rictor, leading to worsened dilated cardiomyopathy (DCM) and accelerated heart failure in Pdk1-deficient mice. In addition, we found that increasing Akt S473 phosphorylation through deletion of Pten or chemical inhibition of PTEN reversed DCM and heart failure in Pdk1-deficient mice. Investigation of heart samples from human DCM patients revealed changes similar to those in the mouse models. These results demonstrated that PDK1 and mTORC2 synergistically promote postnatal heart growth and maintain heart function in postnatal mice. PMID:24662050

Zhao, Xia; Lu, Shuangshuang; Nie, Junwei; Hu, Xiaoshan; Luo, Wen; Wu, Xiangqi; Liu, Hailang; Feng, Qiuting; Chang, Zai; Liu, Yaoqiu; Cao, Yunshan; Sun, Haixiang; Li, Xinli; Hu, Yali

2014-01-01

199

Disruption of Phosphoinositide-Specific Phospholipases C?1 Contributes to Extracellular Matrix Synthesis of Human Osteoarthritis Chondrocytes  

PubMed Central

Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation including extracellular matrix (ECM) degradation and cell loss. It is known that phosphoinositide-specific phospholipase ?1 (PLC?1) can trigger several signaling pathways to regulate cell metabolism. However, whether this kinase is expressive and active in human OA chondrocytes and its role in the pathological progression of OA have not been investigated. The current study was designed to investigate the PLC?1 expression in human OA cartilage, and whether PLC?1 was involved in the ECM synthesis had been further explored using cultured human OA chondrocytes. Our results indicated that PLC?1 was highly expressed in human OA chondrocytes. In our further study using the cultured human OA chondrocytes, the results demonstrated that the disruption of PLC?1 by its inhibitor, U73122, and siRNA contributed to the ECM synthesis of human OA chondrocytes through regulating the expression of ECM-related signaling molecules, including MMP-13, Col II, TIMP1, Sox-9, and AGG. Furthermore, PLC?1/IP3/Ca(2+)/CaMK II signaling axis regulated the ECM synthesis of human chondrocytes through triggering mTOR/P70S6K/S6 pathway. In summary, our results suggested that PLC-?1 activities played an important role in the ECM synthesis of human OA chondrocytes, and may serve as a therapeutic target for treating OA. PMID:25073093

Zeng, Guoqing; Cui, Xu; Liu, Zejun; Zhao, Honghai; Zheng, Xinpeng; Zhang, Bing; Xia, Chun

2014-01-01

200

Leukotriene D4 receptor-mediated hydrolysis of phosphoinositide and mobilization of calcium in sheep tracheal smooth muscle cells  

SciTech Connect

A sheep tracheal smooth muscle primary culture cell system was developed to characterize leukotriene D4 (LTD4) receptor-mediated biochemical and pharmacological effects. (/sup 3/H)LTD4 binding to the enriched plasma membrane receptor was specific, stereoselective and saturable. LTE4 and high affinity receptor antagonists bound to the receptors with a rank-order potency that was expected from previous smooth muscle contraction studies. In the (/sup 3/H)myoinositol labeled cells, LTD4 and LTE4 induced phosphoinositide hydrolysis. The biosynthesis of (/sup 3/H)inositol-trisphosphate was rapid and the induction of biosynthesis of (/sup 3/H)inositol-monophosphate by LTs was stereoselective and specific and was inhibited specifically by a receptor antagonist, SKF 104353. In the fura-2 loaded smooth muscle cells, LTD4 and LTE4 induced transient intracellular Ca++ mobilization. The fura-2/Ca++ transient was stereoselective and specific and was inhibited by receptor antagonist, SKF 104353. These results suggest that the cultured sheep tracheal smooth muscle cells have plasma membrane receptors for LTD4. These receptors were coupled to a phospholipase C that, when activated by agonists, induced hydrolysis of inositol containing phospholipids. The hydrolysis products, e.g. diacylglycerol and inositol-trisphosphate, may serve as intracellular messengers that trigger or contribute to the contractile effect in sheep tracheal smooth muscle.

Mong, S.; Miller, J.; Wu, H.L.; Crooke, S.T.

1988-02-01

201

Rab25 is responsible for phosphoinositide 3-kinase/AKT?mediated cisplatin resistance in human epithelial ovarian cancer cells.  

PubMed

Rab25, a member of the Rab family of small guanosine triphosphatase, was reported to have an essential role in the development of human epithelial ovarian cancer. The present study demonstrated that Rab25 mediated the sensitivity of ovarian cancer to cisplatin, a first?line chemotherapeutic agent for the treatment of ovarian cancer in the clinic. Overexpression of Rab25 and increased phosphoinositide 3?kinase (PI3K)/AKT signaling were detected in cisplatin?resistant SKOV?3 cells compared with those in cisplatin?sensitive ES?2 cells. The results of the present study indicated that cisplatin resistance was primarily due to reduced G1 cell cycle arrest following cisplatin treatment in SKOV?3 cells. By contrast, the corresponding phenomenon was not observed following treatment with a Rab25?specific small interfering RNA or treatment with the PI3K/AKT inhibitor LY294002. Of note, inhibition of the PI3K/AKT pathway reduced Rab25 gene expression and sensitized SKOV?3 cells to cisplatin. Furthermore, knockdown of Rab25 showed an effect comparable with blocking the PI3K/AKT pathway. In conclusion, the results of the present study demonstrated that PI3K/AKT and Rab25 significantly contributed to cisplatin resistance in human epithelial ovarian cancer; in addition, silencing Rab25 or inhibiting the PI3K/AKT pathway markedly increased the sensitivity of these cells to cisplatin. PMID:25405658

Fan, Yang; Wang, Long; Han, Xuechuan; Liu, Xueqin; Ma, Hongyun

2015-03-01

202

Class-IA phosphoinositide 3-kinase p110? Triggers GPCR-induced superoxide production in p110?-deficient murine neutrophils.  

PubMed

Studies with knockout mice have indicated that the only isoform of phosphoinositide 3-kinase (PI3K) functioning in the oxidative burst of mouse neutrophils in response to heterotrimeric guanine nucleotide-binding protein-coupled receptor (GPCR) agonists is a class-IB PI3K, p110?. In the present study, we observed that the cells from p110?(-/-) mice gain a response to N-formyl-Met-Leu-Phe (fMLP) after priming with cytochalasin E. Even the unprimed cells, which show no response to fMLP, produce a significant amount of superoxide, when an effective agonist of the mouse-type fMLP receptors, Trp-Lys-Tyr-Met-Val-D-Met, is used to stimulate the cells. These results suggested that the class-IA isoforms (p110?, p110?, and p110?) of PI3K are sufficient to trigger and maintain superoxide production. Examination of the effects of isoform-specific inhibitors suggested that the p110? isoform is the primary PI3K triggering the response to GPCR agonists when p110? is absent. PMID:23149576

Nigorikawa, Kiyomi; Hazeki, Kaoru; Kumazawa, Takashi; Itoh, Yuhta; Hoshi, Megumi; Hazeki, Osamu

2012-01-01

203

p87 and p101 subunits are distinct regulators determining class IB phosphoinositide 3-kinase (PI3K) specificity.  

PubMed

Class IB phosphoinositide 3-kinase ? (PI3K?) comprises a single catalytic p110? subunit, which binds to two non-catalytic subunits, p87 or p101, and controls a plethora of fundamental cellular responses. The non-catalytic subunits are assumed to be redundant adaptors for G?? enabling G-protein-coupled receptor-mediated regulation of PI3K?. Growing experimental data provide contradictory evidence. To elucidate the roles of the non-catalytic subunits in determining the specificity of PI3K?, we tested the impact of p87 and p101 in heterodimeric p87-p110? and p101-p110? complexes on the modulation of PI3K? activity in vitro and in living cells. RT-PCR, biochemical, and imaging data provide four lines of evidence: (i) specific expression patterns of p87 and p101, (ii) up-regulation of p101, providing the basis to consider p87 as a protein forming a constitutively and p101 as a protein forming an inducibly expressed PI3K?, (iii) differences in basal and stimulated enzymatic activities, and (iv) differences in complex stability, all indicating apparent diversity within class IB PI3K?. In conclusion, expression and activities of PI3K? are modified differently by p87 and p101 in vitro and in living cells, arguing for specific regulatory roles of the non-catalytic subunits in the differentiation of PI3K? signaling pathways. PMID:24014027

Shymanets, Aliaksei; Prajwal; Bucher, Kirsten; Beer-Hammer, Sandra; Harteneck, Christian; Nürnberg, Bernd

2013-10-25

204

Elevation of cyclic AMP decreases phosphoinositide turnover and inhibits thrombin-induced secretion in human platelets  

Microsoft Academic Search

Elevation of cyclic AMP (cAMP) in platelets inhibits agonist-induced, G protein-mediated responses and activation of polyphosphoinositide-specific phospholipase C (PLC) by ill-defined mechanism(s). Signal transduction steps downstream of PLC are inhibited by elevated cAMP, suggesting an inhibitory effect of cAMP, via protein kinase A, on PLC. In [32P]i-prelabeled platelets, forskolin increased intracellular cAMP (104 nmol\\/1011 cells at 10?5 M forskolin) and

Anita Ryningen; Baard Olav Jensen; Holm Holmsen

1998-01-01

205

The Sec14 superfamily and mechanisms for crosstalk between lipid metabolism and lipid signaling.  

PubMed

Lipid signaling pathways define central mechanisms for cellular regulation. Productive lipid signaling requires an orchestrated coupling between lipid metabolism, lipid organization and the action of protein machines that execute appropriate downstream reactions. Using membrane trafficking control as primary context, we explore the idea that the Sec14-protein superfamily defines a set of modules engineered for the sensing of specific aspects of lipid metabolism and subsequent transduction of 'sensing' information to a phosphoinositide-driven 'execution phase'. In this manner, the Sec14 superfamily connects diverse territories of the lipid metabolome with phosphoinositide signaling in a productive 'crosstalk' between these two systems. Mechanisms of crosstalk, by which non-enzymatic proteins integrate metabolic cues with the action of interfacial enzymes, represent unappreciated regulatory themes in lipid signaling. PMID:19926291

Bankaitis, Vytas A; Mousley, Carl J; Schaaf, Gabriel

2010-03-01

206

Spatial and Temporal Regulation of 3-Phosphoinositides by PI 3Kinase and PTEN Mediates Chemotaxis  

Microsoft Academic Search

We have investigated the mechanisms of leading edge formation in chemotaxing Dictyostelium cells. We demonstrate that while phosphatidylinositol 3-kinase (PI3K) transiently translocates to the plasma membrane in response to chemoattractant stimulation and to the leading edge in chemotaxing cells, PTEN, a negative regulator of PI3K pathways, exhibits a reciprocal pattern of localization. By uniformly localizing PI3K along the plasma membrane,

Satoru Funamoto; Ruedi Meili; Susan Lee; Lisa Parry; Richard A. Firtel

2002-01-01

207

A novel macrolide/fluoroketolide, solithromycin (CEM-101), reverses corticosteroid insensitivity via phosphoinositide 3-kinase pathway inhibition  

PubMed Central

Background and Purpose Corticosteroid insensitivity is a major therapeutic problem for some inflammatory diseases including chronic obstructive pulmonary disease (COPD), and it is known to be induced by reduced histone deacetylase (HDAC)-2 activities via activation of the phosphoinositide 3-kinase (PI3K) pathway. The aim of this study is to evaluate effects of a novel macrolide/fluoroketolide, solithromycin (SOL, CEM-101), on corticosteroid sensitivity induced by oxidative stress. Experimental Approach Corticosteroid sensitivity was determined by IC50/EC50 of dexamethasone (Dex) on TNF-?-induced CXCL8 production in U937 monocytic cell line and peripheral blood mononuclear cells (PBMC) from COPD patients. Activities of HDAC and protein phosphatase 2A (PP2A) were measured by fluorescence-based assay in cells exposed to hydrogen peroxide (H2O2). We also investigated steroid insensitive airway neutrophilia in cigarette smoke exposed mice in vivo. Key Results SOL (10 ?M) restored Dex sensitivity in PBMC from COPD patients, H2O2-treated U937 cells and phorbol 12-myristate 13-acetate-differentiated U937 cells. In addition, SOL restored HDAC activity with concomitant inhibition of Akt phosphorylation as surrogate marker of PI3K activation. The inhibition of Akt phosphorylation by SOL was due to increased PP2A phosphatase activity, which was reduced in COPD and oxidative stress model. Other known macrolides, such as eryhthromycin, clarithromycin and azithromycin, were significantly less effective in these responses. In cigarette smoke-exposed mice, SOL (100 mg kg?1, po) showed significant but weak inhibition of neutrophilia, whereas Dex (10 mg kg?1, p.o.) showed no such effect. However, a combination of SOL and Dex inhibited neutrophilia by over 50%. Conclusions and Implications SOL has potential as novel therapy for corticosteroid-insensitive diseases such as COPD. PMID:23758162

Kobayashi, Y; Wada, H; Rossios, C; Takagi, D; Charron, C; Barnes, P J; Ito, K

2013-01-01

208

Propofol mediates signal transducer and activator of transcription 3 activation and crosstalk with phosphoinositide 3-kinase/AKT  

PubMed Central

We previously demonstrated that propofol, an intravenous anesthetic with anti-oxidative properties, activated the phosphoinositide 3-kinase (PI3K)/AKT pathway to increase the expression of B cell lymphoma (Bcl)-2 and, therefore the anti-apoptotic potential on cardiomyocytes. Here, we wanted to determine if propofol can also activate the Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 pathway, another branch of cardioprotective signaling. The cellular response of nuclear factor kappa B (NF?B) and STAT3 was also evaluated. Cardiac H9c2 cells were treated by propofol alone or in combination with pretreatment by inhibitors for JAK2/STAT3 or PI3K/AKT pathway. STAT3 and AKT phosphorylation, and STAT3 translocation were measured by western blotting and immunofluorescence staining, respectively. Propofol treatment significantly increased STAT3 phosphorylation at both tyrosine 705 and serine 727 residues. Sustained early phosphorylation of STAT3 was observed with 25~75 ?M propofol at 10 and 30 min. Nuclear translocation of STAT3 was seen at 4 h after treatment with 50 ?M propofol. In cultured H9c2 cells, we further demonstrated that propofol-induced STAT3 phosphorylation was reduced by pretreatment with PI3K/AKT pathway inhibitors wortmannin or API-2. Conversely, pretreatment with JAK2/STAT3 pathway inhibitor AG490 or stattic inhibited propofol-induced AKT phosphorylation. In addition, propofol induced NF?B p65 subunit perinuclear translocation. Inhibition or knockdown of STAT3 was associated with increased levels of the NF?B p65 subunit. Our results suggest that propofol induces an adaptive response by dual activation and crosstalk of cytoprotective PI3K/AKT and JAK2/STAT3 pathways. Rationale to apply propofol clinically as a preemptive cardioprotectant during cardiac surgery is supported by our findings. PMID:25105067

Shravah, Jayant; Wang, Baohua; Pavlovic, Marijana; Kumar, Ujendra; Chen, David DY; Luo, Honglin; Ansley, David M

2014-01-01

209

The Phosphoinositide 3-Kinase Isoform PI3K? Regulates Osteoclast-Mediated Bone Resorption in Humans and Mice  

PubMed Central

Objective While phosphoinositide 3-kinases (PI3Ks) are involved in various intracellular signal transduction processes, the specific functions of the different PI3K isoforms are poorly understood. We have previously shown that the PI3K? isoform is required for arthritis development in the K/BxN serum–transfer model. Since osteoclasts play a critical role in pathologic bone loss during inflammatory arthritis and other diseases, we undertook this study to test the role of PI3K? in osteoclast development and function using a combined genetic and pharmacologic approach. Methods The role of PI3K? in primary human and murine osteoclast cultures was tested with the PI3K?-selective inhibitor TGX221 and by using PI3K??/? mice. The trabecular bone architecture of PI3K??/? mice was evaluated using micro–computed tomography and histomorphometric analyses. Results The expression of PI3K? was strongly and specifically up-regulated during in vitro osteoclast differentiation. In vitro development of large multinucleated osteoclasts from human or murine progenitors and their resorption capacity were strongly reduced by the PI3K? inhibitor TGX221 or by the genetic deficiency of PI3K?. This was likely due to defective cytoskeletal reorganization and vesicular trafficking, since PI3K??/? mouse multinucleated cells failed to form actin rings and retained intracellular acidic vesicles and cathepsin K. In contrast, osteoclast-specific gene expression and the survival and apoptosis of osteoclasts were not affected. PI3K??/? mice had significantly increased trabecular bone volume and showed abnormal osteoclast morphology with defective resorption pit formation. Conclusion PI3K? plays an important role in osteoclast development and function and is required for in vivo bone homeostasis. PMID:24719382

Gy?ri, Dávid; Csete, Dániel; Benk?, Szilvia; Kulkarni, Suhasini; Mandl, Péter; Dobó-Nagy, Csaba; Vanhaesebroeck, Bart; Stephens, Len; Hawkins, Phillip T; Mócsai, Attila

2014-01-01

210

Differential effects of polychlorinated biphenyl congeners on phosphoinositide hydrolysis and protein kinase C translocation in rat cerebellar granule cells.  

PubMed

Previous reports from our laboratory have suggested that the neuroactivity of some polychlorinated biphenyl (PCB) congeners is associated with perturbations in cellular Ca(2+)-homeostasis. We have characterized further the neurochemical effects of PCBs on signal transduction in primary cultures of cerebellar granule cells. The present experiments found that neither 2,2'-dichlorobiphenyl (DCBP), an ortho-substituted congener, nor 3,3',4,4',5-pentachlorobiphenyl (PCBP), a non-ortho-substituted congener, affected basal phosphoinositide (PI) hydrolysis in cerebellar granule cells. However, at concentrations up to 50 microM, DCBP potentiated carbachol-stimulated PI hydrolysis, while decreasing it at 100 microM. PCBP, on the other hand, had no effect on carbachol-stimulated PI hydrolysis in concentrations up to 100 microM. [3H]Phorbol ester ([3H]PDBu) binding was used to determine protein kinase C (PKC) translocation. DCBP increased [3H]PDBu binding in a concentration-dependent manner and a twofold increase was observed at 100 microM in cerebellar granule cells. PCBP had no effect on [3H]PDBu binding at concentrations up to 100 microM. The effect of DCBP on [3H]PDBu binding was time-dependent and was also dependent on the presence of external Ca2+ in the medium. To test the hypothesis that DCBP increases [3H]PDBu binding by acting on receptor-activated calcium channels, the effects of DCBP were compared to those of L-glutamate. The effects of DCBP (50 microM) and glutamate (20 microM) were additive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7859093

Kodavanti, P R; Shafer, T J; Ward, T R; Mundy, W R; Freudenrich, T; Harry, G J; Tilson, H A

1994-10-31

211

How Cells Integrate Complex Stimuli: The Effect of Feedback from Phosphoinositides and Cell Shape on Cell Polarization and Motility  

PubMed Central

To regulate shape changes, motility and chemotaxis in eukaryotic cells, signal transduction pathways channel extracellular stimuli to the reorganization of the actin cytoskeleton. The complexity of such networks makes it difficult to understand the roles of individual components, let alone their interactions and multiple feedbacks within a given layer and between layers of signalling. Even more challenging is the question of if and how the shape of the cell affects and is affected by this internal spatiotemporal reorganization. Here we build on our previous 2D cell motility model where signalling from the Rho family GTPases (Cdc42, Rac, and Rho) was shown to organize the cell polarization, actin reorganization, shape change, and motility in simple gradients. We extend this work in two ways: First, we investigate the effects of the feedback between the phosphoinositides (PIs) , and Rho family GTPases. We show how that feedback increases heights and breadths of zones of Cdc42 activity, facilitating global communication between competing cell “fronts”. This hastens the commitment to a single lamellipodium initiated in response to multiple, complex, or rapidly changing stimuli. Second, we show how cell shape feeds back on internal distribution of GTPases. Constraints on chemical isocline curvature imposed by boundary conditions results in the fact that dynamic cell shape leads to faster biochemical redistribution when the cell is repolarized. Cells with frozen cytoskeleton, and static shapes, consequently respond more slowly to reorienting stimuli than cells with dynamic shape changes, the degree of the shape-induced effects being proportional to the extent of cell deformation. We explain these concepts in the context of several in silico experiments using our 2D computational cell model. PMID:22396633

Edelstein-Keshet, Leah

2012-01-01

212

An integrin-targeted, pan-isoform, phosphoinositide-3 kinase inhibitor, SF1126, has activity against multiple myeloma in vivo  

PubMed Central

Purpose Multiple reports point to an important role for the phosphoinositide-3 kinase (PI3K) and AKT signaling pathways in tumor survival and chemoresistance in multiple myeloma (MM). The goals of our study were: (1) to generate the preclinical results necessary to justify a Phase I clinical trial of SF1126 in hematopoietic malignancies including multiple myeloma, and (2) to begin combining pan PI-3 kinase inhibitors with other agents to augment antitumor activity of this class of agent in preparation for combination therapy in Phase I/II trials. Methods We determined the in vitro activity of SF1126 with16 human MM cell lines. In vivo tumor growth suppression was determined with human myeloma (MM.1R) xenografts in athymic mice. In addition, we provide evidence that SF1126 has pharmacodynamic activity in the treatment of patients with MM. Results SF1126 was cytotoxic to all tested MM lines and potency was augmented by the addition of bortezomib. SF1126 affected MM.1R cell line signaling in vitro, inhibiting phospho-AKT, phospho-ERK, and the hypoxic stabilization of HIF1?. Tumor growth was 94% inhibited, with a marked decrease in both cellular proliferation (PCNA immunostaining) and angiogenesis (tumor microvessel density via CD31 immunostaining). Our clinical results demonstrate pharmacodynamic knockdown of p-AKT in primary patient derived MM tumor cells in vivo. Conclusions Our results establish three important points: (1) SF1126, a pan PI-3 kinase inhibitor has potent antitumor activity against multiple myeloma in vitro and in vivo, (2) SF1126 displays augmented antimyeloma activity when combined with proteasome inhibitor, bortezomib/Velcade®, and (3) SF1126 blocks the IGF-1 induced activation of AKT in primary MM tumor cells isolated from SF1126 treated patients The results support the ongoing early Phase I clinical trial in MM and suggest a future Phase I trial in combination with bortezomib in hematopoietic malignancies. PMID:23355037

De, Pradip; Dey, Nandini; Terakedis, Breanne; Bersagel, Leif; Li, Zhi Hua; Mahadevan, Daruka; Garlich, Joseph R.; Trudel, Suzanne; Makale, Milan T.; Durden, Donald L.

2013-01-01

213

Targeting phosphoinositide 3-kinase ? in airway smooth muscle cells to suppress interleukin-13-induced mouse airway hyperresponsiveness.  

PubMed

We recently reported that phosphoinositide 3-kinase ? (PI3K?) directly regulates airway smooth muscle (ASM) contraction by modulating Ca(2+) oscillations. Because ASM contraction plays a critical role in airway hyperresponsiveness (AHR) of asthma, the aim of the present study was to determine whether targeting PI3K? in ASM cells could suppress AHR in vitro and in vivo. Intranasal administration into mice of interleukin-13 (IL-13; 10 ?g per mouse), a key pathophysiologic cytokine in asthma, induced AHR after 48 h, as assessed by invasive tracheostomy. Intranasal administration of a broad-spectrum PI3K inhibitor or a PI3K?-specific inhibitor 1 h before AHR assessment attenuated IL-13 effects. Airway responsiveness to bronchoconstrictor agonists was also examined in precision-cut mouse lung slices pretreated without or with IL-13 for 24 h. Acetylcholine and serotonin dose-response curves indicated that IL-13-treated lung slices had a 40 to 50% larger maximal airway constriction compared with controls. Furthermore, acetylcholine induced a larger initial Ca(2+) transient and increased Ca(2+) oscillations in IL-13-treated primary mouse ASM cells compared with control cells, correlating with increased cell contraction. As expected, PI3K? inhibitor treatment attenuated IL-13-augmented airway contractility of lung slices and ASM cell contraction. In both control and IL-13-treated ASM cells, small interfering RNA-mediated knockdown of PI3K? by 70% only reduced the initial Ca(2+) transient by 20 to 30% but markedly attenuated Ca(2+) oscillations and contractility of ASM cells by 50 to 60%. This report is the first to demonstrate that PI3K? in ASM cells is important for IL-13-induced AHR and that acute treatment with a PI3K? inhibitor can ameliorate AHR in a murine model of asthma. PMID:22543031

Jiang, Haihong; Xie, Yan; Abel, Peter W; Toews, Myron L; Townley, Robert G; Casale, Thomas B; Tu, Yaping

2012-08-01

214

Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane  

PubMed Central

Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85? subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85? coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane. PMID:17074976

Stein, Alexander T.; Ufret-Vincenty, Carmen A.; Hua, Li; Santana, Luis F.; Gordon, Sharona E.

2006-01-01

215

The Arabidopsis DREB2 genetic pathway is constitutively repressed by basal phosphoinositide-dependent phospholipase C coupled to diacylglycerol kinase  

PubMed Central

Phosphoinositide-dependent phospholipases C (PI-PLCs) are activated in response to various stimuli. They utilize substrates provided by type III-Phosphatidylinositol-4 kinases (PI4KIII) to produce inositol triphosphate and diacylglycerol (DAG) that is phosphorylated into phosphatidic acid (PA) by DAG-kinases (DGKs). The roles of PI4KIIIs, PI-PLCs, and DGKs in basal signaling are poorly understood. We investigated the control of gene expression by basal PI-PLC pathway in Arabidopsis thaliana suspension cells. A transcriptome-wide analysis allowed the identification of genes whose expression was altered by edelfosine, 30 ?M wortmannin, or R59022, inhibitors of PI-PLCs, PI4KIIIs, and DGKs, respectively. We found that a gene responsive to one of these molecules is more likely to be similarly regulated by the other two inhibitors. The common action of these agents is to inhibit PA formation, showing that basal PI-PLCs act, in part, on gene expression through their coupling to DGKs. Amongst the genes up-regulated in presence of the inhibitors, were some DREB2 genes, in suspension cells and in seedlings. The DREB2 genes encode transcription factors with major roles in responses to environmental stresses, including dehydration. They bind to C-repeat motifs, known as Drought-Responsive Elements that are indeed enriched in the promoters of genes up-regulated by PI-PLC pathway inhibitors. PA can also be produced by phospholipases D (PLDs). We show that the DREB2 genes that are up-regulated by PI-PLC inhibitors are positively or negatively regulated, or indifferent, to PLD basal activity. Our data show that the DREB2 genetic pathway is constitutively repressed in resting conditions and that DGK coupled to PI-PLC is active in this process, in suspension cells and seedlings. We discuss how this basal negative regulation of DREB2 genes is compatible with their stress-triggered positive regulation. PMID:23964284

Djafi, Nabila; Vergnolle, Chantal; Cantrel, Catherine; Wietrzyñski, Wojciech; Delage, Elise; Cochet, Françoise; Puyaubert, Juliette; Soubigou-Taconnat, Ludivine; Gey, Delphine; Collin, Sylvie; Balzergue, Sandrine; Zachowski, Alain; Ruelland, Eric

2013-01-01

216

Increasing Plasma Membrane Phosphatidylinositol(4,5)Bisphosphate Biosynthesis Increases Phosphoinositide Metabolism in Nicotiana tabacum[W][OA  

PubMed Central

A genetic approach was used to increase phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] biosynthesis and test the hypothesis that PtdInsP kinase (PIPK) is flux limiting in the plant phosphoinositide (PI) pathway. Expressing human PIPKI? in tobacco (Nicotiana tabacum) cells increased plasma membrane PtdIns(4,5)P2 100-fold. In vivo studies revealed that the rate of 32Pi incorporation into whole-cell PtdIns(4,5)P2 increased >12-fold, and the ratio of [3H]PtdInsP2 to [3H]PtdInsP increased 6-fold, but PtdInsP levels did not decrease, indicating that PtdInsP biosynthesis was not limiting. Both [3H]inositol trisphosphate and [3H]inositol hexakisphosphate increased 3-and 1.5-fold, respectively, in the transgenic lines after 18 h of labeling. The inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] binding assay showed that total cellular Ins(1,4,5)P3/g fresh weight was >40-fold higher in transgenic tobacco lines; however, even with this high steady state level of Ins(1,4,5)P3, the pathway was not saturated. Stimulating transgenic cells with hyperosmotic stress led to another 2-fold increase, suggesting that the transgenic cells were in a constant state of PI stimulation. Furthermore, expressing Hs PIPKI? increased sugar use and oxygen uptake. Our results demonstrate that PIPK is flux limiting and that this high rate of PI metabolism increased the energy demands in these cells. PMID:17496116

Im, Yang Ju; Perera, Imara Y.; Brglez, Irena; Davis, Amanda J.; Stevenson-Paulik, Jill; Phillippy, Brian Q.; Johannes, Eva; Allen, Nina S.; Boss, Wendy F.

2007-01-01

217

Piperine inhibits type II phosphatidylinositol 4-kinases: a key component in phosphoinositides turnover.  

PubMed

Piperine has been shown to have anti-inflammatory properties. The molecular mechanisms by which it mediates anti-inflammatory activities remain elusive. Type II phosphatidylinositol 4-kinase(s) are key components in Fc?RI receptor-mediated signaling leading to inflammatory mediators release in RBL-2H3 cells. The effects of piperine on IgE-mediated signaling and mast cell degranulation were investigated. Pretreatment of RBL-2H3 cells with piperine inhibited IgE-induced activation of type II PtdIns 4-kinase(s). In vitro lipid kinase assays showed piperine-inhibited type II PtdIns 4-kinase activity in a dose-dependent fashion with no effect on PtdIns 3-kinase activity. Concomitantly, pretreatment of RBL-2H3 cells with piperine also inhibited IgE-induced ?-hexosaminidase release in RBL-2H3 cells. These results suggest that type II PtdIns 4-kinases are part of piperine-mediated anti-inflammatory signaling mechanisms. PMID:24671493

Bojjireddy, Naveen; Sinha, Ranjeet Kumar; Subrahmanyam, Gosukonda

2014-08-01

218

Alterations in the MA and NC Domains Modulate Phosphoinositide-Dependent Plasma Membrane Localization of the Rous Sarcoma Virus Gag Protein  

PubMed Central

Retroviral Gag proteins direct virus particle assembly from the plasma membrane (PM). Phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] plays a role in PM targeting of several retroviral Gag proteins. Here we report that depletion of intracellular PI(4,5)P2 and phosphatidylinositol-(3,4,5)-triphosphate [PI(3,4,5)P3] levels impaired Rous sarcoma virus (RSV) Gag PM localization. Gag mutants deficient in nuclear trafficking were less sensitive to reduction of intracellular PI(4,5)P2 and PI(3,4,5)P3, suggesting a possible connection between Gag nuclear trafficking and phosphoinositide-dependent PM targeting. PMID:23325682

Nadaraia-Hoke, Shorena; Bann, Darrin V.; Lochmann, Timothy L.; Gudleski-O'Regan, Nicole

2013-01-01

219

Hesperidin induces apoptosis and triggers autophagic markers through inhibition of Aurora-A mediated phosphoinositide-3-kinase/Akt/mammalian target of rapamycin and glycogen synthase kinase-3 beta signalling cascades in experimental colon carcinogenesis.  

PubMed

Abnormalities in the homeostasis mechanisms involved in cell survival and apoptosis are contributing factors for colon carcinogenesis. Interventions of these mechanisms by pharmacologically safer agents gain predominance in colon cancer prevention. We previously reported the chemopreventive efficacy of hesperidin against colon carcinogenesis. In the present study, we aimed at investigating the potential of hesperidin over the abrogated Aurora-A coupled pro-survival phosphoinositide-3-kinase (PI3K)/Akt signalling cascades. Further, the role of hesperidin over apoptosis and mammalian target of rapamycin (mTOR) mediated autophagic responses were studied. Azoxymethane (AOM) induced mouse model of colon carcinogenesis was involved in this study. Hesperidin treatment was provided either in initiation/post-initiation mode respectively. Hesperidin significantly altered AOM mediated anti-apoptotic scenario by modulating Bax/Bcl-2 ratio together with enhanced cytochrome-c release and caspase-3, 9 activations. In addition, hesperidin enhanced p53-p21 axis with concomitant decrease in cell cycle regulator. Hesperidin treatment caused significant up-regulation of tumour suppressor phosphatase and tensin homologue (PTEN) with a reduction in the expression of AOM mediated p-PI3K and p-Akt. Additionally, hesperidin administration exhibited inhibition against p-mTOR expression which in turn led to stimulation of autophagic markers Beclin-1 and LC3-II. Aurora-A an upstream regulator of PI3K/Akt pathway was significantly inhibited by hesperidin. Furthermore, hesperidin administration restored glycogen synthase kinase-3 beta (GSK-3?) activity which in turn prevented the accumulation of oncoproteins ?-catenin, c-jun and c-myc. Taken together, hesperidin supplementation initiated apoptosis via targeted inhibition of constitutively activated Aurora-A mediated PI3K/Akt/GSK-3? and mTOR pathways coupled with autophagic stimulation against AOM induced colon carcinogenesis. PMID:25047426

Saiprasad, Gowrikumar; Chitra, Palanivel; Manikandan, Ramar; Sudhandiran, Ganapasam

2014-09-01

220

Integrin ?v?3 mediates the synergetic regulation of core-binding factor ?1 transcriptional activity by gravity and insulin-like growth factor-1 through phosphoinositide 3-kinase signaling.  

PubMed

Mechanical stimulation and biological factors coordinately regulate bone development and regeneration; however, the underlying mechanisms are poorly understood. Microgravity induces bone loss, which may be partly related to the development of resistance to local cytokines, including insulin-like growth factor 1 (IGF-1). Here, we report the involvement of integrin ?v?3 in microgravity-associated bone loss. An established OSE-3T3 cell model was stably transfected with a 6OSE2 (Osteoblast-Specific Element 2)-luciferase reporter and cultured under simulated microgravity (SMG) and hypergravity (HG) conditions in the presence or absence of IGF-1, the disintegrin echistatin, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, or combinations of these agents. Activity of core-binding factor ?1 (Cbfa1), an essential transcription factor for osteoblastic differentiation and osteogenesis, was reflected by luciferase activity. Different gravity conditions affected the induction of IGF-1 and subsequent effects on Cbfa1 transcription activity. SMG and HG influenced the expression and activity of integrin ?v?3 and phosphorylation level of p85. LY294002 inhibited the effects of HG or IGF-1 on Cbfa1 activity, indicating that HG and IGF-1 could increase Cbfa1 activity via PI3K signaling. Inhibition of integrin ?v?3 by echistatin attenuated the induction of IGF-1 and thus its effect on Cbfa1 activity under normal and HG conditions. Co-immunoprecipitation demonstrated that integrin ?3 interacted with insulin receptor substrate 1, and that this interaction was decreased under SMG and increased under HG conditions. These results suggest that integrin ?v?3 mediates the synergetic regulation of Cbfa1 transcription activity by gravity and IGF-1 via PI3K signaling. PMID:25263523

Dai, Zhongquan; Guo, Feima; Wu, Feng; Xu, Hongjie; Yang, Chao; Li, Jinqiao; Liang, Peilong; Zhang, Hongyu; Qu, Lina; Tan, Yingjun; Wan, Yumin; Li, Yinghui

2014-12-01

221

Phosphoinositide 3-kinase ? regulates migration and invasion of synoviocytes in rheumatoid arthritis.  

PubMed

Cartilage destruction mediated by invasive fibroblast-like synoviocytes (FLS) plays a central role in pathogenesis of rheumatoid arthritis (RA). Increased cell migration and degradation of extracellular matrix are fundamental to these processes. The class I PI3Ks control cell survival, proliferation, and migration, which might be involved in cartilage damage in RA. PI3K? isoform was recently identified as a key regulator of FLS growth and survival, suggesting that it could contribute to synoviocyte aggressive behavior. Therefore, we assessed the role of PI3K? in RA synoviocyte migration and invasion. We observed that PI3K? inhibition or small interfering RNA knockdown decreased platelet-derived growth factor (PDGF)-mediated migration and invasion of FLS. We then showed that PI3K? regulates the organization of actin cytoskeleton and lamellipodium formation during PDGF stimulation. To gain insight into molecular mechanisms, we examined the effect of PI3K? inhibition on Rac1/PAK, FAK, and JNK activation. Our studies suggest that Rac1/PAK is key target of PDGF-mediated PI3K? signaling, whereas FAK and JNK are not involved. Thus, PI3K? contributes to multiple aspects of the pathogenic FLS behavior in RA. These observations, together with previous findings that PI3K? regulates FLS growth and survival, suggest that PI3K? inhibition could be chondroprotective in RA by modulating synoviocyte growth, migration, and invasion. PMID:24470496

Bartok, Beatrix; Hammaker, Deepa; Firestein, Gary S

2014-03-01

222

PTEN Regulation, a Novel Function for the p85 Subunit of Phosphoinositide 3-Kinase  

NSDL National Science Digital Library

Timely regulation of phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] abundance in cells is essential for the control of cellular homeostasis. The concentrations of these lipids are low in quiescent cells but rapidly and transiently increase following growth factor receptor (GFR) stimulation, which triggers cellular metabolic changes, proliferation, survival, and motility. Class IA phosphatidylinositol 3-kinase (PI3K), which is composed of a p85 (regulatory) and p110 (catalytic) subunits, is the enzyme generating PI(3,4)P2 and PI(3,4,5)P3 following GFR stimulation. Although the steps in GFR-induced activation of PI3K , are relatively well known, the mechanisms for subsequent 3-polyphospho-PI down-regulation are less understood. Examination of frequent genetic alterations in human cancer showed that PTEN (phosphatase with tensin homology on chromosome 10) is the major enzyme that decreases PI(3,4)P2 and PI(3,4,5)P3 cell content. Nonetheless, interpretation of the complexity of PTEN regulation remains a matter of debate. The recent description of diminished PTEN activity in liver-conditional knockout mice lacking the p85α PI3K regulatory subunit reveals a previously unknown p85α-dependent negative-feedback pathway that controls PI(3,4)P2 and PI(3,4,5)P3 half-life by regulating PTEN.

Domingo F. Barber (Universidad Autonoma de Madrid;Centro Nacional de Biotecnologia/Consejo Superiod de Investigaciones Cientificas REV); Maria Alvarado-Kristensson (Universidad Autonoma de Madrid;Centro Nacional de Biotecnologia/Consejo Superiod de Investigaciones Cientificas REV); Ana Gonzalez-Garcia (Universidad Autonoma de Madrid;Centro Nacional de Biotecnologia/Consejo Superiod de Investigaciones Cientificas REV); Rafael Pulido (Centro de Investigacion Principe Felipe; REV); Ana C. Carrera (Universidad Autonoma de Madrid;Centro Nacional de Biotecnologia/Consejo Superiod de Investigaciones Cientificas REV)

2006-11-21

223

Cholecystokinin receptors: disparity between phosphoinositide breakdown and amylase releasing activity of CCK analogues in pancreas  

SciTech Connect

Cholecystokinin (CCK) peptides are a family of hormones which also occur in brain. In pancreas CCK stimulates the release of amylase, a process that is dependent on the mobilization of intracellular Ca/sup 2 +/. Recent evidence suggests that inositol 1,4,5-trisphosphate, the breakdown product of phosphatidylinositol 4,5-bisphosphate, is responsible for the rise in intracellular Ca/sup 2 +/. Their laboratory has developed assays to study synthetic CCK analogues using radioligand binding, PI breakdown and amylase release. They have shown that there are good correlations among these three assay systems for the carboxy terminal fragments of CCK/sub 8/. Recently, they have discovered synthetic analogues of CCK/sub 4/ that are full agonists in amylase release but are ineffective in causing PI breakdown. In particular, A-61576, Boc-5-amino-2-indolemethylene-pent-2-ene-1-oyl-Leu-Asp-Phe-NH/sub 2/, is a full agonist in the amylase releasing assay, but is devoid of PI stimulating activity. A-61576 completely reverses the stimulation of PI response induced by CCK/sub 8/, indicative of an antagonist. Since a mechanism other than the PI breakdown is responsible for amylase release by A-61576, they suggest that separate receptors are responsible for PI breakdown and amylase release.

Lin, C.W.; Grant, D.; Bianchi, B.; Miller, T.; Witte, D.; Shue, Y.K.; Nadzan, A.

1986-03-05

224

p110? phosphoinositide 3-kinase represses IgE switch by potentiating BCL6 expression.  

PubMed

PI3Ks are key signaling enzymes required for triggering many immunological functions. In B lymphocytes, PI3K signaling is required for Ag-induced proliferation and robust production of most Ab isotypes. Paradoxically, PI3K was found to have a negatively regulatory function regarding Ab class switch recombination, and blockade of PI3K can strongly potentiate IgE switch. In this article, we explore the mechanisms of this unexpected negative regulatory function of PI3K regarding IgE. We demonstrate that p110? PI3K selectively regulates IgE switch in a B cell-intrinsic manner by controlling germline transcription of the IgE promoter (?GLT). Although p110? can regulate transcription of activation-induced cytidine deaminase via Akt, repression of ?GLT and IgE switch is not dependent on Akt signaling. Inhibition of p110?, but not Akt, leads to reduced expression of transcriptional repressor B cell lymphoma 6 (BCL6) and concomitant upregulation of ?GLT and other BCL6-target genes. p110? inhibitor treatment strikingly alters the balance between BCL6 and IRF4 (a transcription factor that antagonizes BCL6), leading to increased IRF4 and decreased BCL6 expression levels in germinal center B cells. Ectopic expression of BCL6 can partially overcome the elevated ?GLTs and potentiated IgE switching in p110?-inhibited B cells. To our knowledge, these results provide the first evidence that p110? PI3K signaling regulates BCL6 expression and indicate that PI3K promotes the germinal center B cell program and selectively represses IgE switch by maintaining sufficient levels of BCL6. PMID:22422878

Zhang, Ting-ting; Makondo, Kennedy J; Marshall, Aaron J

2012-04-15

225

Glucagon-like peptide-2-stimulated protein synthesis through the PI 3-kinase-dependent Akt-mTOR signaling pathway  

Technology Transfer Automated Retrieval System (TEKTRAN)

Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive neuropeptide that exerts diverse actions in the gastrointestinal tract, including enhancing mucosal cell survival and proliferation. GLP-2 stimulates mucosal growth in vivo with an increased rate of protein synthesis. However, it was unclear w...

226

PI 3-kinase-dependent phosphorylation of Plk1–Ser99 promotes association with 14-3-3? and is required for metaphase–anaphase transition  

PubMed Central

Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1–Thr210 phosphorylation. Plk1–Ser99 phosphorylation creates a docking site for 14-3-3?, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3? or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1–Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1–Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1–Thr210 phosphorylation, but also by Plk1 binding to 14-3-3? following Plk1–Ser99 phosphorylation downstream of the PI3K–Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase. PMID:23695676

Kasahara, Kousuke; Goto, Hidemasa; Izawa, Ichiro; Kiyono, Tohru; Watanabe, Nobumoto; Elowe, Sabine; Nigg, Erich A; Inagaki, Masaki

2013-01-01

227

Interaction between phosphoinositide turnover system and cyclic AMP pathway for the secretion of pancreastatin and somatostatin from QGP-1N cells.  

PubMed

It is found that secretion of pancreastatin and somatostatin from QGP-1N cells is regulated through muscarinic receptor-mediated activation of phosphatidylinositide hydrolysis system. In this report, whether the cAMP pathway interacts with the phosphoinositide turnover system for the secretion of pancreastatin and somatostatin from QGP-1N cells through muscarinic receptors was studied. Stimulation of QGP-1N cells with carbachol increased intracellular cAMP levels. The carbachol-induced increase in cAMP levels was inhibited by atropine. Calcium ionophore (A23187) and phorbol 12-myristate 13-acetate increased cAMP synthesis. Dibutyryl cAMP, forskolin and theophylline stimulated secretion of pancreastatin and somatostatin. When either dibutyryl cAMP, forskolin or theophylline was added in culture medium with A23187, phorbol ester or carbachol, a synergistic effect was found on pancreastatin and somatostatin secretion. These results suggest that interaction between the phosphoinositide turnover system and the cAMP pathway occurs in QGP-1N cells through muscarinic receptor stimulation for the secretion of pancreastatin and somatostatin. PMID:1352680

Tateishi, K; Funakoshi, A; Kitayama, N; Matsuoka, Y

1992-06-30

228

A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress–induced membrane biogenesis  

PubMed Central

Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches. PMID:24403601

Ren, Jihui; Pei-Chen Lin, Coney; Pathak, Manish C.; Temple, Brenda R. S.; Nile, Aaron H.; Mousley, Carl J.; Duncan, Mara C.; Eckert, Debra M.; Leiker, Thomas J.; Ivanova, Pavlina T.; Myers, David S.; Murphy, Robert C.; Brown, H. Alex; Verdaasdonk, Jolien; Bloom, Kerry S.; Ortlund, Eric A.; Neiman, Aaron M.; Bankaitis, Vytas A.

2014-01-01

229

A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis.  

PubMed

Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches. PMID:24403601

Ren, Jihui; Pei-Chen Lin, Coney; Pathak, Manish C; Temple, Brenda R S; Nile, Aaron H; Mousley, Carl J; Duncan, Mara C; Eckert, Debra M; Leiker, Thomas J; Ivanova, Pavlina T; Myers, David S; Murphy, Robert C; Brown, H Alex; Verdaasdonk, Jolien; Bloom, Kerry S; Ortlund, Eric A; Neiman, Aaron M; Bankaitis, Vytas A

2014-03-01

230

Synaptojanin 1 Mutation in Parkinson's Disease Brings Further Insight into the Neuropathological Mechanisms  

PubMed Central

Synaptojanin 1 (SYNJ1) is a phosphoinositide phosphatase highly expressed in nerve terminals. Its two phosphatase domains dephosphorylate phosphoinositides present in membranes, while its proline-rich domain directs protein-protein interactions with synaptic components, leading to efficient recycling of synaptic vesicles in neurons. Triplication of SYNJ1 in Down's syndrome is responsible for higher level of phosphoinositides, enlarged endosomes, and learning deficits. SYNJ1 downregulation in Alzheimer's disease models is protective towards amyloid-beta peptide (A?) toxicity. One missense mutation in one of SYNJ1 functional domains was recently incriminated in an autosomal recessive form of early-onset Parkinson's disease (PD). In the third decade of life, these patients develop progressive Parkinsonism with bradykinesia, dystonia, and variable atypical symptoms such as cognitive decline, seizures, and eyelid apraxia. The identification of this new gene, together with the fact that most of the known PD proteins play a role in synaptic vesicle recycling and lipid metabolism, points out that synaptic maintenance is a key player in PD pathological mechanisms. Studying PD genes as a network regulating synaptic activity could bring insight into understanding the neuropathological processes of PD and help identify new genes at fault in this devastating disorder. PMID:25302295

Drouet, Valérie; Lesage, Suzanne

2014-01-01

231

Mineral fiber-mediated activation of phosphoinositide-specific phospholipase c in human bronchoalveolar carcinoma-derived alveolar epithelial A549 cells.  

PubMed

Given the role of phosphoinositide-specific phospholipase C (PLC) isozymes in the control of cell growth and differentiation we were prompted to analyze the expression of some of these PLC in human bronchoalveolar carcinoma-derived alveolar epithelial A549 cells. The effects of several fluoro-edenite fibers were compared with those of tremolite, a member of the calcic amphibole group of asbestos that originates from Calabria (Italy), and crocidolite, that, due to its high toxicity, is one of the most studied asbestos amphiboles. Our data show an increased expression of both PLC beta1 and PLC gamma1 in A549 cells treated with asbestos-like fibers, hinting at a role of PLC signalling in those cancerous cells. PMID:19148471

Loreto, Carla; Carnazza, Maria Luisa; Cardile, Venera; Libra, Massimo; Lombardo, Laura; Malaponte, Grazia; Martinez, Giuseppina; Musumeci, Giuseppe; Papa, Veronica; Cocco, Lucio

2009-02-01

232

The Presence of a Constitutively Active Phosphoinositide 3Kinase in Small Cell Lung Cancer Cells Mediates Anchorage-independent Proliferation via a Protein Kinase B and p70s6k-dependentPathway1  

Microsoft Academic Search

Small cell lung cancer (SCLC) is characterized by early and widespread métastases.Anchorage-independent growth is pivotal to the ability of tumor cells to survive and metastasize in vivo and, under I'M vitro condi tions, allows transformed cells to form colonies in semisolid medium. Here, we report that of five SCLC cell lines tested, all exhibited high basal constitutive phosphoinositide 3-kinase (PI

Sarah M. Moore; Robert C. Rintoul; Trevor R. Walker; Edwin R. Chilvers; Christopher Haslett; Tariq Sethi

233

Amplification of Chromosome 1q Genes Encoding the Phosphoinositide Signalling Enzymes PI4KB, AKT3, PIP5K1A and PI3KC2B in Breast Cancer  

PubMed Central

Little is known about the possible oncogenic roles of genes encoding for the phosphatidylinositol 4-kinases, a family of enzymes that regulate an early step in phosphoinositide signalling. To address this issue, the mutational status of all four human phosphatidylinositol 4-kinases genes was analyzed across 852 breast cancer samples using the COSMIC data resource. Point mutations in the phosphatidylinositol 4-kinase genes were uncommon and appeared in less than 1% of the patient samples however, 62% of the tumours had increases in gene copy number for PI4KB which encodes the phosphatidylinositol 4-kinase IIIbeta isozyme. Extending this analysis to subsequent enzymes in the phosphoinositide signalling cascades revealed that the only PIP5K1A, PI3KC2B and AKT3 genes exhibited similar patterns of gene copy number variation. By comparison, gene copy number increases for established oncogenes such as EGFR and HER2/Neu were only evident in 20% of the samples. The PI4KB, PIP5K1A, PI3KC2B and AKT3 genes are related in that they all localize to chromosome 1q which is often structurally and numerically abnormal in breast cancer. These results demonstrate that a gene quartet encoding a potential phosphoinositide signalling pathway is amplified in a subset of breast cancers. PMID:25368680

Waugh, Mark G.

2014-01-01

234

Ceramide Disables 3Phosphoinositide Binding to the Pleckstrin Homology Domain of Protein Kinase B (PKB)\\/Akt by a PKC Dependent Mechanism  

Microsoft Academic Search

Ceramide is generated in response to numerous stress-inducing stimuli and has been implicated in the regulation of diverse cellular responses, including cell death, differentiation, and insulin sensitivity. Recent evidence indicates that ceramide may regulate these responses by inhibiting the stimulus-mediated activation of protein kinase B (PKB), a key determinant of cell fate and insulin action. Here we show that inhibition

Darren J. Powell; Eric Hajduch; Gursant Kular; Harinder S. Hundal

2003-01-01

235

Ephrin type-A receptor 2 regulates sensitivity to paclitaxel in nasopharyngeal carcinoma via the phosphoinositide 3-kinase/Akt signalling pathway  

PubMed Central

Ephrin type-A receptor 2 (EphA2) is a receptor tyrosine kinase that is associated with cancer cell metastasis. There has been little investigation into its impact on the regulation of sensitivity to paclitaxel in nasopharyngeal carcinoma (NPC). In the present study, upregulation of EphA2 expression enhanced the survival of NPC 5-8F cells, compared with control cells exposed to the same concentrations of paclitaxel. Flow cytometry and western blot analysis demonstrated that over-expression of EphA2 decreased NPC cancer cell sensitivity to paclitaxel by regulating paclitaxel-mediated cell cycle progression but not apoptosis in vitro. This was accompanied by alterations in the expression of cyclin-dependent kinase inhibitors, p21 and p27, and of inactive phosphorylated-retinoblastoma protein. Furthermore, paclitaxel stimulation and EphA2 over-expression resulted in activation of the phosphoinositide 3-kinase (PI3K)/Akt signalling pathway in NPC cells. Inhibition of the PI3K/Akt signalling pathway restored sensitivity to paclitaxel in 5-8F cells over-expressing EphA2, which indicated that the PI3K/Akt pathway is involved in EphA2-mediated paclitaxel sensitivity. The current study demonstrated that EphA2 mediates sensitivity to paclitaxel via the regulation of the PI3K/Akt signalling pathway in NPC. PMID:25351620

WANG, YUNYUN; LIU, YONG; LI, GUO; SU, ZHONGWU; REN, SHULING; TAN, PINGQING; ZHANG, XIN; QIU, YUANZHENG; TIAN, YONGQUAN

2015-01-01

236

Melittin stimulates phosphoinositide hydrolysis and placental lactogen release: Arachidonic acid as a link between phospholipase A sub 2 and phospholipase C signal-transduction pathways  

SciTech Connect

Previous investigations from this laboratory have implicated both phospholipase A{sub 2} and phospholipase C in the regulation of human placental lactogen release from human trophoblast. To study further the role of endogenous phospholipase A{sub 2} and the relationship between phospholipase A{sub 2} activation and phosphoinositide metabolism, the authors examined hPL and ({sup 3}H)-inositol release from trophoblast cells in response to agents that stimulate or inhibit the endogenous enzyme. Melittin stimulated rapid, dose-dependent, and reversible increases in the release of hPL, prostaglandin E, and ({sup 3}H)-inositol. Mepacrine inhibited this stimulation. However, mepacrine had no effect on the stimulation of hPL and ({sup 3}H)-inositol release by exogenous arachidonic acid (AA). These results indicate that the stimulation by melittin of phosphoionsitide metabolism and hPL release is mediated by initial activation of phospholipase A{sub 2}. Furthermore, the results support the possibility that AA, released as a consequence of phospholipase A{sub 2} activation, can act as a second messenger linking the two phospholipase pathways.

Zeitler, P.; Handwerger, S. (Univ. of Cincinnati College of Medicine, OH (USA)); Wu, Y.Q. (Duke Univ. Medical Center, Durham, NC (USA))

1991-01-01

237

Loss of phosphoinositide 3-kinase ? decreases migration and activation of phagocytes but not T cell activation in antigen-induced arthritis  

PubMed Central

Background Phosphoinositide 3-kinase ? (PI3K?) has been depicted as a major regulator of inflammatory processes, including leukocyte activation and migration towards several chemokines. This study aims to explore the role of PI3K? in the murine model of antigen-induced arthritis (AIA). Methods Development of AIA was investigated in wildtype and PI3K?-deficient mice as well as in mice treated with a specific inhibitor of PI3K? (AS-605240) in comparison to untreated animals. Inflammatory reactions of leukocytes, including macrophage and T cell activation, and macrophage migration, were studied in vivo and in vitro. Results Genetic deletion or pharmacological inhibition of PI3K? induced a marked decrease of clinical symptoms in early AIA, together with a considerably diminished macrophage migration and activation (lower production of NO, IL-1?, IL-6). Also, macrophage and neutrophil infiltration into the knee joint were impaired in vivo. However, T cell functions, measured by cytokine production (TNF?, IFN?, IL-2, IL-4, IL-5, IL-17) in vitro and DTH reaction in vivo were not altered, and accordingly, disease developed normally at later timepoints Conclusion PI3K? specifically affects phagocyte function in the AIA model but has no impact on T cell activation. PMID:20374644

2010-01-01

238

Functional redundancy of class I phosphoinositide 3-kinase (PI3K) isoforms in signaling growth factor-mediated human neutrophil survival.  

PubMed

We have investigated the contribution of individual phosphoinositide 3-kinase (PI3K) Class I isoforms to the regulation of neutrophil survival using (i) a panel of commercially available small molecule isoform-selective PI3K Class I inhibitors, (ii) novel inhibitors, which target single or multiple Class I isoforms (PI3K?, PI3K?, PI3K?, and PI3K?), and (iii) transgenic mice lacking functional PI3K isoforms (p110?(KO)?(KO) or p110?(KO)). Our data suggest that there is considerable functional redundancy amongst Class I PI3Ks (both Class IA and Class IB) with regard to GM-CSF-mediated suppression of neutrophil apoptosis. Hence pharmacological inhibition of any 3 or more PI3K isoforms was required to block the GM-CSF survival response in human neutrophils, with inhibition of individual or any two isoforms having little or no effect. Likewise, isolated blood neutrophils derived from double knockout PI3K p110?(KO)?(KO) mice underwent normal time-dependent constitutive apoptosis and displayed identical GM-CSF mediated survival to wild type cells, but were sensitized to pharmacological inhibition of the remaining PI3K isoforms. Surprisingly, the pro-survival neutrophil phenotype observed in patients with an acute exacerbation of chronic obstructive pulmonary disease (COPD) was resilient to inactivation of the PI3K pathway. PMID:23029326

Juss, Jatinder K; Hayhoe, Richard P; Owen, Charles E; Bruce, Ian; Walmsley, Sarah R; Cowburn, Andrew S; Kulkarni, Suhasini; Boyle, Keith B; Stephens, Len; Hawkins, Phillip T; Chilvers, Edwin R; Condliffe, Alison M

2012-01-01

239

Activity of the pan-class I phosphoinositide 3-kinase inhibitor NVP-BKM120 in T-cell acute lymphoblastic leukemia.  

PubMed

Constitutively active phosphoinositide 3-kinase (PI3K) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival and drug resistance. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the pan-PI3K inhibitor NVP-BKM120 (BKM120), an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in G2/M phase cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T lymphoblasts, and promoting a dose- and time-dependent dephosphorylation of Akt and S6RP. BKM120 maintained its pro-apoptotic activity against Jurkat cells even when cocultured with MS-5 stromal cells, which mimic the bone marrow microenvironment. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. Moreover, in vivo administration of BKM120 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth, thus prolonging survival time. Taken together, our findings indicate that BKM120, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment for T-ALLs that have aberrant upregulation of the PI3K signaling pathway. PMID:24310736

Lonetti, A; Antunes, I L; Chiarini, F; Orsini, E; Buontempo, F; Ricci, F; Tazzari, P L; Pagliaro, P; Melchionda, F; Pession, A; Bertaina, A; Locatelli, F; McCubrey, J A; Barata, J T; Martelli, A M

2014-06-01

240

Selective inhibitors of phosphoinositide 3-kinase delta: modulators of B-cell function with potential for treating autoimmune inflammatory diseases and B-cell malignancies  

PubMed Central

The delta isoform of the p110 catalytic subunit (p110?) of phosphoinositide 3-kinase is expressed primarily in hematopoietic cells and plays an essential role in B-cell development and function. Studies employing mice lacking a functional p110? protein, as well as the use of highly-selective chemical inhibitors of p110?, have revealed that signaling via p110?-containing PI3K complexes (PI3K?) is critical for B-cell survival, migration, and activation, functioning downstream of key receptors on B cells including the B-cell antigen receptor, chemokine receptors, pro-survival receptors such as BAFF-R and the IL-4 receptor, and co-stimulatory receptors such as CD40 and Toll-like receptors (TLRs). Similarly, this PI3K isoform plays a key role in the survival, proliferation, and dissemination of B-cell lymphomas. Herein we summarize studies showing that these processes can be inhibited in vitro and in vivo by small molecule inhibitors of p110? enzymatic activity, and that these p110? inhibitors have shown efficacy in clinical trials for the treatment of several types of B-cell malignancies including chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). PI3K? also plays a critical role in the activation, proliferation, and tissue homing of self-reactive B cells that contribute to autoimmune diseases, in particular innate-like B-cell populations such as marginal zone (MZ) B cells and B-1 cells that have been strongly linked to autoimmunity. We discuss the potential utility of p110? inhibitors, either alone or in combination with B-cell depletion, for treating autoimmune diseases such as lupus, rheumatoid arthritis, and type 1 diabetes. Because PI3K? plays a major role in both B-cell-mediated autoimmune inflammation and B-cell malignancies, PI3K? inhibitors may represent a promising therapeutic approach for treating these diseases. PMID:22936933

Puri, Kamal D.; Gold, Michael R.

2012-01-01

241

Inhibitory Effects of Isoquinoline Alkaloid Berberine on Ischemia-Induced Apoptosis via Activation of Phosphoinositide 3-Kinase/Protein Kinase B Signaling Pathway  

PubMed Central

Purpose Berberine is a type of isoquinoline alkaloid that has been used to treat various diseases. A neuroprotective effect of berberine against cerebral ischemia has been reported; however, the effects of berberine on apoptosis in relation to reactive astrogliosis and microglia activation under ischemic conditions have not yet been fully evaluated. In the present study, we investigated the effects of berberine on global ischemia-induced apoptosis, and focused on the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in the hippocampus using gerbils. Methods Gerbils received berberine orally once a day for 14 consecutive days, starting one day after surgery. In this study, a step-down avoidance task was used to assess short-term memory. Furthermore, we employed the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to evaluate DNA fragmentation, immunohistochemistry to investigate glial fibriallary acidic protein, CD11b, and caspase-3, and western blot to assess PI3K, Akt, Bax, Bcl-2, and cytochrome c. Results Our results revealed that berberine treatment alleviated ischemia-induced short-term memory impairment. Treatment with berbeine also attenuated ischemia-induced apoptosis and inhibited reactive astrogliosis and microglia activation. Furthermore, berberine enhanced phospho-PI3K and phospho-Akt expression in the hippocampus of ischemic gerbils. Conclusions Berberine exerted a neuroprotective effect against ischemic insult by inhibiting neuronal apoptosis via activation of the PI3K/Akt signaling pathway. The antiapoptotic effect of berberine was achieved through inhibition of reactive astrogliosis and microglia activation. Berberine may therefore serve as a therapeutic agent for stroke-induced neurourological problems. PMID:25279238

Kim, Mia; Shin, Mal Soon; Lee, Jae Min; Cho, Han Sam; Kim, Chang Ju; Kim, Young Joon; Choi, Hey Ran

2014-01-01

242

Fc?R-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome  

PubMed Central

The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b558 and cytosolic p67phox, p47phox, and p40phox subunits that undergo membrane translocation upon cellular activation. The function of p40phox, which binds p67phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40phox in Fc?R-induced oxidase activation, we used immunofluorescence and real-time imaging of Fc?R-induced phagocytosis. YFP-tagged p67phox and p40phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67phox and p47phox accumulation on nascent and internalized phagosomes did not require p40phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40phox PI3P-binding domain or wortmannin. Translocation of p40phox to nascent phagosomes required binding to p67phox but not PI3P, although the loss of PI3P binding reduced p40phox retention after phagosome internalization. We conclude that p40phox functions primarily to regulate Fc?R-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization. PMID:18711001

Tian, Wei; Li, Xing Jun; Stull, Natalie D.; Ming, Wenyu; Suh, Chang-Il; Bissonnette, Sarah A.; Yaffe, Michael B.; Grinstein, Sergio; Atkinson, Simon J.

2008-01-01

243

Eicosapentaenoic acid-enriched phosphatidylcholine isolated from Cucumaria frondosa exhibits anti-hyperglycemic effects via activating phosphoinositide 3-kinase/protein kinase B signal pathway.  

PubMed

Eicosapentaenoic acid-enriched phosphatidylcholine was isolated from the sea cucumber Cucumaria frondosa (Cucumaria-PC) and its effects on streptozotocin (STZ)-induced hyperglycemic rats were investigated. Male Sprague-Dawley rats were randomly divided into normal control, model control (STZ), low- and high-dose Cucumaria-PC groups (STZ + Cucumaria-PC at 25 and 75 mg/Kg·b·wt, intragastrically, respectively). Blood glucose, insulin, glycogen in liver and gastrocnemius were determined over 60 days. Insulin signaling in the rats' gastrocnemius was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The results showed that Cucumaria-PC significantly decreased blood glucose level, increased insulin secretion and glycogen synthesis in diabetic rats. RT-PCR analysis revealed that Cucumaria-PC significantly promoted the expressions of glycometabolism-related genes of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K), protein kinase B (PKB), and glucose transporter 4 (GLUT4) in gastrocnemius. Western blotting assay demonstrated that Cucumaria-PC remarkably enhanced the proteins abundance of IR-?, PI3K, PKB, GLUT4, as well as phosphorylation of Tyr-IR-?, p85-PI3K, Ser473-PKB (P < 0.05 and P < 0.01). These findings suggested that Cucumaria-PC exhibited significant anti-hyperglycemic activities through up-regulating PI3K/PKB signal pathway mediated by insulin. Nutritional supplementation with Cucumaria-PC, if validated for human studies, may offer an adjunctive therapy for diabetes mellitus. PMID:24168893

Hu, Shiwei; Xu, Leilei; Shi, Di; Wang, Jingfeng; Wang, Yuming; Lou, Qiaoming; Xue, Changhu

2014-04-01

244

Hydrogen sulfide regulates Na+/H+ exchanger activity via stimulation of phosphoinositide 3-kinase/Akt and protein kinase G pathways.  

PubMed

Intracellular pH (pH(i)) is an important endogenous modulator of cardiac function. Inhibition of Na(+)/H(+) exchanger-1 (NHE-1) protects the heart by preventing Ca(2+) overload during ischemia/reperfusion. Hydrogen sulfide (H(2)S) has been reported to produce cardioprotection. The present study was designed to investigate the pH regulatory effect of H(2)S in rat cardiac myocytes and evaluate its contribution to cardioprotection. It was found that sodium hydrosulfide (NaHS), at a concentration range of 10 to 1000 ?M, produced sustained decreases in pH(i) in the rat myocytes in a concentration-dependent manner. NaHS also abolished the intracellular alkalinization caused by trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate (U50,488H), which activates NHEs. Moreover, when measured with an NHCl(4) prepulse method, NaHS was found to significantly suppress NHE-1 activity. Both NaHS and cariporide or [5-(2-methyl-5-fluorophenyl)furan-2-ylcarbonyl]guanidine (KR-32568), two NHE inhibitors, protected the myocytes against ischemia/reperfusion injury. However, coadministration of NaHS with KR-32568 did not produce any synergistic effect. Functional study showed that perfusion with NaHS significantly improved postischemic contractile function in isolated rat hearts subjected to ischemia/reperfusion. Blockade of phosphoinositide 3-kinase (PI3K) with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), Akt with Akt VIII, or protein kinase G (PKG) with (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6

Hu, Li-Fang; Li, Yu; Neo, Kay Li; Yong, Qian Chen; Lee, Shiau Wei; Tan, Benny Kwong Huat; Bian, Jin-Song

2011-11-01

245

Platelet activation by bacterial phospholipase C involves phosphoinositide turnover and phosphorylation of 47,000 dalton but not 20,000 dalton protein  

SciTech Connect

This study was conducted to examine the role of phosphoinositides (PIns) and phosphorylation of 47,000 dalton (P47) and 20,000 dalton (P20) proteins in platelet activation by bacterial phospholipase C (PLC). PLC induced serotonin secretion (SS) and platelet aggregation (PA) in a concentration dependent manner. PLC (0.02 U/ml) caused phosphorylation of P47 in a time dependent manner (27% at 0.5 min to 378% at 7 min). PLC did not induce more than 15% phosphorylation of P20 by 7 min. Aspirin (500 ..mu..M) blocked phosphorylation of P20 but did not inhibit SS, PA or phosphorylation of P47. PLC (0.04 U/ml) decreased radioactivity (cpm) in /sup 32/P labeled phosphatidylinositol (PI), PI-4,5-bis-PO4 (PIP2) and PI-4-PO4 (PIP) by 20%, 12% and 7.5% respectively at 15 sec. The level of PI but not that of PIP2 returned to base line in 3 min. PIP level increased above control values within one min. PLC increased phosphatidic acid level (75% at 0.5 min. to 1545% at 3 min). In other experiments PLC produced diacylglycerol (DAG) in a time and concentration dependent manner. However, no DAG was detectable in the first 60 sec. These data suggest that: (a) PIns turnover and phosphorylation of P47 but not that of P20 is involved in platelet activation by PLC; and (b) DAG production from outer membrane phospholipids is not a prerequisite for platelet activation by PLC.

Huzoor-Akbar; Anwer, K.

1986-05-01

246

Class I Phosphoinositide-3-Kinases and Src Kinases Play a Nonredundant Role in Regulation of Adhesion-Independent and -Dependent Neutrophil Reactive Oxygen Species Generation  

PubMed Central

Chemoattractant-induced reactive oxygen species (ROS) generation by adherent neutrophils occurs in two phases: the first is very rapid and transient, and the second one is delayed and lasts up to 30–40 min. We examined the role of phosphoinositide 3-kinases (PI3Ks) and Src-family kinases (SFKs) in these responses using human neutrophils treated with inhibitory compounds or murine neutrophils deficient of PI3K? or Hck, Fgr, and Lyn. Our studies show that PI3K? is indispensable for the early, fMLF-induced ROS generation and AKT and ERK phosphorylation, but is dispensable for the late response to fMLF. Additionally, the response to TNF, an agonist triggering only the delayed phase of ROS generation, was also unaffected in PI3K?-deficient neutrophils. In contrast, inhibition of SFKs by a selective inhibitor in human, or SFK deficiency in murine, neutrophils resulted in the inhibition of both the early and late phase of ROS generation, without affecting the early phase of AKT phosphorylation, but inhibiting the late one. Selective inhibitors of PI3K? and PI3K? markedly reduced both the early and late response to fMLF and TNF in human neutrophils. These findings suggest that class IA PI3Ks may be activated by PI3K? via Ras in the early phase of the response and by SFKs in the late phase. The evidence that inhibition of SFKs in human, or SFK deficiency in murine, neutrophils results in suppression of Vav phosphorylation at all time points of the response to fMLF or TNF suggests that SFKs are indispensable for Vav phosphorylation. PMID:23447687

Fumagalli, Laura; Campa, Carlo C.; Germena, Giulia; Lowell, Clifford A.; Hirsch, Emilio; Berton, Giorgio

2014-01-01

247

The Vr-PLC3 gene encodes a putative plasma membrane-localized phosphoinositide-speci¢c phospholipase C whose expression is induced by abiotic stress in mung bean (Vigna radiata L.)1  

Microsoft Academic Search

Phosphoinositide-speci¢c phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate and diacylglycerol, both of which act as secondary messengers in animal cells. In this report, we identi¢ed in Vigna radiata L. (mung bean) three distinct partial cDNAs (pVr-PLC1, pVr-PLC2, and pVr- PLC3), which encode forms of putative PI-PLC. All three Vr-PLC genes were transcriptionally active and

Yun Ju Kim; Jee Eun Kim; Jae-Hoon Lee; Myoung Hui Lee; Ho Won Jung; Byung Kook Hwang; Inhwan Hwang; Woo Taek Kim

248

Two sites of action for PLD2 inhibitors: The enzyme catalytic center and an allosteric, phosphoinositide biding pocket.  

PubMed

Phospholipase D (PLD) has been implicated in many physiological functions, such as chemotaxis and phagocytosis, as well as pathological functions, such as cancer cell invasion and metastasis. New inhibitors have been described that hamper the role of PLD in those pathologies but their site of action is not known. We have characterized the biochemical and biological behavior of the PLD1/2 dual inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), and the specific PLD2 inhibitor, N-[2-[1-(3-Fluorophenyl)-4-oxo-1,3,-8-triazaspiro[4.5]dec-8-yl]ethyl]-2-naphthalenecarboxamide (NFOT), and found that both FIPI and NFOT are mixed-kinetics inhibitors. Mutagenesis studies indicate that FIPI binds at S757 of PLD2, which is within the HKD2 catalytic site of the enzyme, whereas NFOT binds to PLD2 at two different sites, one being at S757/S648 and another to an allosteric site that is a natural site occupied by PIP2 (R210/R212). This latter site, along with F244/L245/L246, forms a hydrophobic pocket in the PH domain. The mechanism of action of FIPI is a direct effect on the catalytic site (and as such inhibits both PLD1 and PLD2 isoforms), whereas PLD2 affects both the catalytic site (orthosteric) and blocks PIP2 binding to PLD2 (allosteric), which negates the natural enhancing role of PIP2. Moreover, NFOT prevents cell invasion of cancer cells, which does not occur in cells overexpressing PLD2-F244A/L245A/L246A, or PLD2-R210A/R212A, or PLD2-S757/S648 mutants. This study provides new specific knowledge of enzyme regulation and mechanisms of activation and inhibition of PLD2 that are necessary to understand its role in cell signaling and to develop new inhibitors for cancer cell invasion and metastasis. PMID:25532944

Ganesan, Ramya; Mahankali, Madhu; Alter, Gerald; Gomez-Cambronero, Julian

2015-03-01

249

Discovery of N-{5-[3-(3-hydroxypiperidin-1-yl)-1,2,4-oxadiazol-5-yl]-4-methyl-1,3-thiazol-2-yl}acetamide (TASP0415914) as an orally potent phosphoinositide 3-kinase ? inhibitor for the treatment of inflammatory diseases.  

PubMed

Class I phosphoinositide 3-kinases (PI3Ks), particularly PI3K?, have become attractive drug targets for inflammatory and autoimmune disorders such as rheumatoid arthritis. Herein, we describe the synthesis and the structure-activity relationships (SAR) of a series of 2-amino-5-oxadiazolyl thiazoles, culminating in the identification of 8j (TASP0415914), an orally potent inhibitor of phosphoinositide 3-kinase ? (PI3K?). TASP0415914 demonstrated good potency in a cell-based assay and, furthermore, exhibited in vivo efficacy in a collagen induced arthritis (CIA) model in mice after oral administration. PMID:24262886

Oka, Yusuke; Yabuuchi, Tetsuya; Oi, Takahiro; Kuroda, Shoichi; Fujii, Yasuyuki; Ohtake, Hidenori; Inoue, Tomoyuki; Wakahara, Shunichi; Kimura, Kayo; Fujita, Kiyoko; Endo, Mayumi; Taguchi, Kyoko; Sekiguchi, Yoshinori

2013-12-15

250

Phosphoinositide [PI(3,5)P2] lipid-dependent regulation of the general transcriptional regulator Tup1  

PubMed Central

Transcriptional activity of a gene is governed by transcriptional regulatory complexes that assemble/disassemble on the gene and control the chromatin architecture. How cytoplasmic components influence the assembly/disassembly of transcriptional regulatory complexes is poorly understood. Here we report that the budding yeast Saccharomyces cerevisiae has a chromatin architecture-modulating mechanism that is dependent on the endosomal lipid PI(3,5)P2. We identified Tup1 and Cti6 as new, highly specific PI(3,5)P2 interactors. Tup1—which associates with multiple transcriptional regulators, including the HDAC (histone deacetylase) and SAGA complexes—plays a crucial role in determining an activated or repressed chromatin state of numerous genes, including GAL1. We show that, in the context that the Gal4 activation pathway is compromised, PI(3,5)P2 plays an essential role in converting the Tup1-driven repressed chromatin structure into a SAGA-containing activated chromatin structure at the GAL1 promoter. Biochemical and cell biological experiments suggest that PI(3,5)P2 recruits Cti6 and the Cyc8–Tup1 corepressor complex to the late endosomal/vacuolar membrane and mediates the assembly of a Cti6–Cyc8–Tup1 coactivator complex that functions to recruit the SAGA complex to the GAL1 promoter. Our findings provide important insights toward understanding how the chromatin architecture and epigenetic status of a gene are regulated by cytoplasmic components. PMID:21536737

Han, Bong-Kwan; Emr, Scott D.

2011-01-01

251

Phosphoinositide 3-kinase targeting by the ? galactoside binding protein cytokine negates akt gene expression and leads aggressive breast cancer cells to apoptotic death  

PubMed Central

Introduction Phosphoinositide 3-kinase (PI3K)-activated signalling has a critical role in the evolution of aggressive tumourigenesis and is therefore a prime target for anticancer therapy. Previously we have shown that the ? galactoside binding protein (?GBP) cytokine, an antiproliferative molecule, induces functional inhibition of class 1A and class 1B PI3K. Here, we have investigated whether, by targeting PI3K, ?GBP has therapeutic efficacy in aggressive breast cancer cells where strong mitogenic input is fuelled by overexpression of the ErbB2 (also known as HER/neu, for human epidermal growth factor receptor 2) oncoprotein receptor and have used immortalised ductal cells and non-aggressive mammary cancer cells, which express ErbB2 at low levels, as controls. Methods Aggressive BT474 and SKBR3 cancer cells where ErbB2 is overexpressed, MCF10A immortalised ductal cells and non-invasive MCF-7 cancer cells which express low levels of ErbB2, both in their naive state and when forced to mimic aggressive behaviour, were used. Class IA PI3K was immunoprecipitated and the conversion of phosphatidylinositol (4,5)-biphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) assessed by ELISA. The consequences of PI3K inhibition by ?GBP were analysed at proliferation level, by extracellular signal-regulated kinase (ERK) activation, by akt gene expression and by apoptosis. Apoptosis was documented by changes in mitochondrial membrane potential, alteration of the plasma membrane, caspase 3 activation and DNA fragmentation. Phosphorylated and total ERK were measured by Western blot analysis and akt mRNA levels by Northern blot analysis. The results obtained with the BT474 and SKBR3 cells were validated in the MCF10A ductal cells and in non-invasive MCF-7 breast cancer cells forced into mimicking the in vitro behaviour of the BT474 and SKBR3 cells. Results In aggressive breast cancer cells, where mitogenic signalling is enforced by the ErbB2 oncoprotein receptor, functional inhibition of the catalytic activity of PI3K by the ?GBP cytokine and loss of akt mRNA results in apoptotic death. A functional correlation between ERK and the kt gene was also found. The relationship between ERK, akt mRNA, PI3K and cell vulnerability to ?GBP challenge was sustained both in mammary ductal cells forced to mimic an aggressive behaviour and in non-aggressive breast cancer cells undergoing an enforced shift into an aggressive phenotype. Conclusions ?GBP, a newly discovered physiological inhibitor of PI3K, is a selective and potent inducer of apoptosis in aggressive breast cancer cells. Due to its physiological nature, which carries no chemotherapeutic disadvantages, ?GBP has the potential to be safely tested in clinical trials. PMID:19133120

Wells, Valerie; Mallucci, Livio

2009-01-01

252

Effects of 5?fluorouracil and class III phosphoinositide 3?kinase small interfering RNA combination therapy on SGC7901 human gastric cancer cells.  

PubMed

The aim of the present study was to investigate the effects of small interfering RNA?mediated inhibition of Class III phosphoinositide 3?kinase (PI3K) signal transduction on the proliferation, apoptosis and autophagy of SGC7901 gastric cancer cells. The present study also aimed to examine the contribution of autophagic inhibition to the antitumor effects of 5?fluorouracil (5?FU). A PI3K(III)?RNA interference (i)?green fluorescent protein (GFP) recombinant replication adenovirus (AD) and the negative control (NC)?RNAi?GFP control AD were constructed and infected into SGC7901 cells. A methyl thiazolyl tetrazolium assay was used to determine the growth rate of the SGC7901 cells. Immunofluorescent staining was used to detect microtubule?associated protein 1 light chain 3 expression. The mitochondrial membrane potential was measured using the JC?1 fluorescent probe. Autophagic expression was monitored with MDC staining and transmission electron microscopy. The results revealed that following combination treatment of the SGC7901 gastric cancer cells with 5?FU + PI3K(III)?RNAi?AD, the optical density absorbance values at 24, 48 and 72 h were 0.17±1.64, 0.13±4.64 and 0.11±3.56%, respectively, with cell viability inhibition ratios of 45.89±6.67, 72.57±9.48 and 87.51±4.65%, respectively. As compared with the other treatment groups, the inhibition rate in the combined treatment group was significantly higher (P<0.05). The percentages of the cells with green fluorescence in the combined treatment group were 74.4±3.86 (24 h), 82.3±1.84 (48 h) and 92.5±1.1% (72 h), which were larger than those of the other groups. The percentage of cells with green fluorescence became larger, which indicated that the mitochondrion membrane potential had been reduced to a greater extent. MDC staining revealed that the number of autophagic vacuoles in the cells (measured at 24, 48 and 72 h) decreased gradually with time, with more autophagic vacuoles observed in the cells in the control group at 24 h than those in the other treatment groups. Fewest autophagic vacuoles were identified in the combined treatment group. Using a fluorescence microscope, the immune fluorescence expression of microtubule?associated proteins 1A/1B light chain 3A, which is the specific protein of autophagy, in the combined treatment group was observed to be significantly downregulated, as compared with the other groups. As determined by transmission electron microscopic observation of the SGC7901 gastric cancer cells, the degree of autophagy in the combined treatment group was significantly reduced, as compared with that of the other treatment groups. In conclusion, following combined treatment with 5?FU and an inhibitor of class III PI3K signal transduction, the proliferation of SGC7901 cells was significantly suppressed, the mitochondrion membrane potentials were significantly reduced and the expression levels of autophagic markers were significantly downregulated. PMID:25385552

Zhu, Bao-Song; Sun, Jia-Lei; Gong, Wei; Zhang, Xing-Ding; Wu, Yong-You; Xing, Chun-Gen

2015-03-01

253

Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism  

NASA Technical Reports Server (NTRS)

Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser(1179) in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser(116), Thr(497), and Ser(635). Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser(635), which was consistently slower than that at Ser(1179). Thr(497) was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser(116). Whereas shear-stimulated Ser(635) phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser(635) may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.

Boo, Yong Chool; Hwang, Jinah; Sykes, Michelle; Michell, Belinda J.; Kemp, Bruce E.; Lum, Hazel; Jo, Hanjoong

2002-01-01

254

Recent advances in understanding the molecular mechanisms of the development and function of Th17 cells  

PubMed Central

IL-17-producing T helper (Th17) cells comprise a distinct Th subset involved in epithelial cell- and neutrophil-mediated immune responses against extracellular microbes. At the same time, Th17 cells play significant roles in the development of autoimmune diseases including rheumatoid arthritis and multiple sclerosis. Since the identification of Th17 cells approximately a decade ago, the molecular mechanisms of their differentiation have been intensively studied and a number of signaling cascades and transcription factors have been shown to be involved. Here, we review the current knowledge regarding the function of Th17 cells in vivo as well as several key concepts for the molecular mechanisms of Th17 differentiation. We also discuss the emerging roles of phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin complex 1 (mTORC1) and hypoxia-inducible factor 1 (HIF-1) in the differentiation of Th17 cells. PMID:23383714

Kurebayashi, Yutaka; Nagai, Shigenori; Ikejiri, Ai; Koyasu, Shigeo

2013-01-01

255

Activation of mitogen-activated protein kinase by membrane-targeted Raf chimeras is independent of raft localization.  

PubMed

Binding of proteins to the plasma membrane can be achieved with various membrane targeting motifs, including combinations of fatty acids, isoprenoids, and basic domains. In this study, we investigate whether attachment of different membrane targeting motifs influences the signaling capacity of membrane-bound signal transduction proteins by directing the proteins to different membrane microdomains. We used c-Raf-1 as a model for a signaling protein that is activated when membrane-bound. Three different membrane targeting motifs from K-Ras, Fyn, and Src proteins were fused to the N or C terminus of Raf-1. The ability of the modified Rafs to initiate MAPK signaling was then investigated. All three modified Raf-1 constructs activated MAPK to nearly equivalent levels. The extent of localization of the Raf-1 constructs to membrane microdomains known as rafts did not correlate with the level of MAPK activation. Moreover, treatment of cells with the raft disrupting drug methyl-beta-cyclodextrin (MbetaCD) caused activation of MAPK to levels equivalent to those achieved with membrane-targeted Raf constructs. The use of pharmacological agents as well as dominant negative mutants revealed that MAPK activation by MbetaCD proceeds via a phosphoinositide 3-kinase-dependent mechanism that is Ras/Raf-independent. We conclude that cholesterol depletion from the plasma membrane by MbetaCD constitutes an alternative pathway for activating MAPK. PMID:11457834

Chen, X; Resh, M D

2001-09-14

256

Insulin-sensitizing and beneficial lipid-metabolic effects of the water-soluble melanin complex extracted from Inonotus obliquus.  

PubMed

Inonotus obliquus has been traditionally used for treatment of metabolic diseases; however, the mechanism remains to be elucidated. In this study, we found that the water-soluble melanin complex extracted from I. obliquus improved insulin sensitivity and reduced adiposity in high fat (HF)-fed obese mice. When the melanin complex was treated to 3T3-L1 adipocytes, insulin-stimulated glucose uptake was increased significantly, and its phosphoinositide 3-kinase-dependent action was proven with wortmannin treatment. Additionally, dose-dependent increases in Akt phosphorylation and glucose transporter 4 translocation into the plasma membrane were observed in melanin complex-treated cells. Adiponectin gene expression in 3T3-L1 cells incubated with melanin complex increased which was corroborated by increased AMP-activated protein kinase phosphorylation in HepG2 and C2C12 cells treated with conditioned media from the 3T3-L1 culture. Melanin complex-treated 3T3-L1 cells showed no significant change in expression of several lipogenic genes, whereas enhanced expressions of fatty acid oxidative genes were observed. Similarly, the epididymal adipose tissue of melanin complex-treated HF-fed mice had higher expression of fatty acid oxidative genes without significant change in lipogenic gene expression. Together, these results suggest that the water-soluble melanin complex of I. obliquus exerts antihyperglycemic and beneficial lipid-metabolic effects, making it a candidate for promising antidiabetic agent. PMID:24615848

Lee, Jung-Han; Hyun, Chang-Kee

2014-09-01

257

Oleanolic acid induces relaxation and calcium-independent release of endothelium-derived nitric oxide  

PubMed Central

Background and purpose: The present study investigated the mechanisms by which oleanolic acid, a component of olive oil, increases release of nitric oxide (NO). Experimental approach: Measurements of isometric tension, NO concentration, or endothelial cell calcium were made in rat isolated mesenteric arteries. Immunoblotting for endothelial NOS (eNOS) and Akt kinase were performed in primary cultures of human umbilical vein endothelial cells (HUVECs). Key results: Oleanolic acid (3–30??M) evoked endothelium-dependent relaxations in noradrenaline-contracted rat superior and small mesenteric arteries. In rat superior mesenteric arteries, oleanolic acid induced simultaneous increases in NO concentration and relaxation, and these responses were inhibited by an inhibitor of NOS, asymmetric dimethyl-L-arginine (300??M) and by the NO scavenger, oxyhaemoglobin (10??M). Oleanolic acid-evoked NO increases were not reduced in Ca2+-free solution and in the presence of an inhibitor of endoplasmic reticulum calcium-ATPase, thapsigargin (1??M). Oleanolic acid evoked relaxation without changes in endothelial cell calcium, but decreased smooth muscle calcium in arterial segments. Oleanolic acid failed to increase calcium in HUVECs, but increased time-dependently phosphorylation of Akt kinase at Serine473 (Akt-Ser473) and eNOS at Serine1177 (eNOS-Ser1177), which was attenuated by inhibitors of phosphoinositide-3-kinase. Conclusions and implications: This study provides direct evidence that a component of olive oil, oleanolic acid, activated endothelium-dependent release of NO and decreased smooth muscle cell calcium followed by relaxation. The oleanolic acid-evoked endothelium-derived NO release was independent of endothelial cell calcium and involved phosphoinositide-3-kinase-dependent phosphorylation of Akt-Ser473 followed by phosphorylation of eNOS-Ser1177. PMID:18622409

Rodriguez-Rodriguez, R; Stankevicius, E; Herrera, M D; Østergaard, L; Andersen, M R; Ruiz-Gutierrez, V; Simonsen, U

2008-01-01

258

Role of phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways in granulocyte macrophage-colony-stimulating factor failure to delay fas-induced neutrophil apoptosis in elderly humans.  

PubMed

Fas-stimulated neutrophils from elderly individuals show impaired granulocyte macrophage-colony-stimulating factor (GM-CSF)-induced apoptosis cell rescue. Herein, this defect was found to be associated with a significant reduction in GM-CSF-mediated Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Using Akt and ERK1/2 inhibitors, we demonstrated that both kinases were critical for GM-CSF antiapoptotic effects. Whereas Akt inhibition also affected GM-CSF-dependent ERK1/2 phosphorylation, ERK1/2 inhibition did not affect GM-CSF-induced Akt phosphorylation, suggesting that phosphoinositide 3-kinase (PI3-K)/Akt and ERK1/2 are activated in series and that PI3-K is located upstream of ERK1/2 along the GM-CSF-dependent signaling pathway. No age-associated changes in GM-CSF receptor expression were observed. Interestingly, both suppressors of cytokine signaling (SOCS)1 and SOCS3 proteins were significantly higher in unstimulated neutrophils from elderly individuals and, unlike in young individuals, did not further increase following GM-CSF cell triggering. These results indicate that defective PI3-K/Akt/ERK1/2 activation, likely dependent on elevated SOCS1 and SOCS3 levels, may affect the GM-CSF capacity to delay neutrophil apoptosis in elderly persons. PMID:17167151

Tortorella, Cosimo; Simone, Olivia; Piazzolla, Giuseppina; Stella, Isabella; Cappiello, Valentina; Antonaci, Salvatore

2006-11-01

259

Yeast 3-phosphoinositide-dependent protein kinase-1 (PDK1) orthologs Pkh1-3 differentially regulate phosphorylation of protein kinase A (PKA) and the protein kinase B (PKB)/S6K ortholog Sch9.  

PubMed

Pkh1, -2, and -3 are the yeast orthologs of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1). Although essential for viability, their functioning remains poorly understood. Sch9, the yeast protein kinase B and/or S6K ortholog, has been identified as one of their targets. We now have shown that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1-interacting fragment pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrated that Pkh1 phosphorylates Sch9 both in vitro and in vivo on its PDK1 site and that this phosphorylation is essential for a wild type cell size. In vivo phosphorylation on this site disappeared during nitrogen deprivation and rapidly increased again upon nitrogen resupplementation. In addition, we have shown here for the first time that the PDK1 site in protein kinase A is phosphorylated by Pkh1 in vitro, that this phosphorylation is Pkh-dependent in vivo and occurs during or shortly after synthesis of the protein kinase A catalytic subunits. Mutagenesis of the PDK1 site in Tpk1 abolished binding of the regulatory subunit and cAMP dependence. As opposed to PDK1 site phosphorylation of Sch9, phosphorylation of the PDK1 site in Tpk1 was not regulated by nitrogen availability. These results bring new insight into the control and prevalence of PDK1 site phosphorylation in yeast by Pkh protein kinases. PMID:21531713

Voordeckers, Karin; Kimpe, Marlies; Haesendonckx, Steven; Louwet, Wendy; Versele, Matthias; Thevelein, Johan M

2011-06-24

260

An increase in phosphoinositide-specific phospholipase C activity precedes induction of C4 phosphoenolpyruvate carboxylase phosphorylation in illuminated and NH4Cl-treated protoplasts from Digitaria sanguinalis.  

PubMed

A Ca2+-dependent phosphoinositide-specific phospholipase C (PI-PLC) activity has been characterized in the microsomal fraction of Digitaria sanguinalis mesophyll cell protoplasts. Microsomal PI-PLC was found to be inhibited in vitro by a mammalian anti-PLC-delta1 antibody and by the aminosteroide U-73122, an inhibitor of PI-PLC activity in animal cells. In Western blot experiments, the antibody recognized an 85 kDa protein in both microsomal protein extracts from mesophyll protoplasts and rat brain protein extracts containing the authentic enzyme. The involvement of the microsomal PI-PLC in the light-dependent transduction pathway leading to the phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) was investigated in D. sanguinalis protoplasts. A transient increase in the PI-PLC reaction product inositol-1,4,5-trisphosphate (Ins(1,4, 5)P3) was observed in situ during early induction of the C4 PEPC phosphorylation cascade. U-73122, but not the inactive analogue U-73343, efficiently blocked the transient accumulation of Ins(1,4, 5)P3, and both the increase in C4 PEPC kinase activity and C4 PEPC phosphorylation in illuminated and weak base-treated protoplasts. Taken together, these data suggest that PI-PLC-based signalling is a committed step in the cascade controlling the regulation of C4 PEPC phosphorylation in C4 leaves. PMID:10972876

Coursol, S; Giglioli-Guivarc'h, N; Vidal, J; Pierre, J N

2000-08-01

261

MicroRNA-7-regulated TLR9 signaling-enhanced growth and metastatic potential of human lung cancer cells by altering the phosphoinositide-3-kinase, regulatory subunit 3/Akt pathway.  

PubMed

Recent evidence shows that microRNAs (miRNAs) contribute to the biological effects of Toll-like receptor (TLR) signaling on various cells. Our previous data showed that TLR9 signaling could enhance the growth and metastatic potential of human lung cancer cells. However, the potential role of miRNAs in the effects of TLR9 signaling on tumor biology remains unknown. In this paper, we first report that TLR9 signaling could reduce intrinsic miR-7 expression in human lung cancer cells. Furthermore, overexpression of miR-7 can significantly inhibit TLR9 signaling-enhanced growth and metastatic potential of lung cancer cells in vitro and in vivo. Notably, we identify phosphoinositide-3-kinase, regulatory subunit 3 (PIK3R3) as a novel target molecule of miR-7 in lung cancer cells by Western blotting and luciferase report assay. Further study shows that miR-7 inhibits the effects of TLR9 signaling on lung cancer cells through regulation of the PIK3R3/Akt pathway. These data suggest that miR-7 could act as a fine-tuner in regulating the biological effects of TLR9 signaling on human lung cancer cells, which might be helpful to the understanding of the potential role of miRNAs in TLR signaling effects on tumor biology. PMID:23135998

Xu, Lin; Wen, Zhenke; Zhou, Ya; Liu, Zhongmin; Li, Qinchuan; Fei, Guangru; Luo, Junmin; Ren, Tao

2013-01-01

262

CD28 Costimulation: A Source of Vav-1 for TCR Signaling with the Help of SLP-76?  

NSDL National Science Digital Library

T cells require dual stimulation to become activated. When T cells encounter antigen-presenting cells, both the T cell receptor (TCR) and the CD28 coreceptor are ligated and activated. Michel and Acuto discuss how the adaptor SLP-76, which is recruited to the activated TCR complex, and the Rho family guanosine triphosphatase exchanger Vav-1, which is recruited by the CD28 receptor and TCR, may form a macromolecular complex that results in T cells activation. Vav-1 may serve as a central integrator between CD28 signaling and TCR signaling through its indirect effects on phosphoinositide 3-kinase-dependent signaling.

Frederique Michel (Institut Pasteur;Molecular Immunology Unit, Department of Immunology REV); Oreste Acuto (Institut Pasteur;Molecular Immunology Unit, Department of Immunology REV)

2002-08-06

263

Inference on biological mechanisms using an integrated phenotype prediction model.  

PubMed

We propose a methodology for constructing an integrated phenotype prediction model that accounts for multiple pathways regulating a targeted phenotype. The method uses multiple prediction models, each expressing a particular pattern of gene-to-gene interrelationship, such as epistasis. We also propose a methodology using Gene Ontology annotations to infer a biological mechanism from the integrated phenotype prediction model. To construct the integrated models, we employed multiple logistic regression models using a two-step learning approach to examine a number of patterns of gene-to-gene interrelationships. We first selected individual prediction models with acceptable goodness of fit, and then combined the models. The resulting integrated model predicts phenotype as a logical sum of predicted results from the individual models. We used published microarray data on neuroblastoma from Ohira et al (2005) for illustration, constructing an integrated model to predict prognosis and infer the biological mechanisms controlling prognosis. Although the resulting integrated model comprised a small number of genes compared to a previously reported analysis of these data, the model demonstrated excellent performance, with an error rate of 0.12 in a validation analysis. Gene Ontology analysis suggested that prognosis of patients with neuroblastoma may be influenced by biological processes such as cell growth, G-protein signaling, phosphoinositide-mediated signaling, alcohol metabolism, glycolysis, neurophysiological processes, and catecholamine catabolism. PMID:18578362

Enomoto, Yumi; Ushijima, Masaru; Miyata, Satoshi; Matsuura, Masaaki; Ohtaki, Megu

2008-03-01

264

Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase  

PubMed Central

The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3)P-binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25 nM IC50 in vitro, but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3)P-binding probe from endosome membranes, within 1 min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specifically with PtdIns(3)P via its N-terminal PX domain, might be controlled by Vps34. Mutations disrupting PtdIns(3)P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid ~50–60% loss of SGK3 phosphorylation within 1 min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a PtdIns(3)P-binding PX domain. Furthermore, class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~80–90%. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of PtdIns(3)P. The first is produced through phosphorylation of PtdIns by Vps34 at the endosome. The second is due to the conversion of class I PI3K product, PtdIns(3,4,5)P3 into PtdIns(3)P, via the sequential actions of the PtdIns 5-phosphatases [SHIP1/2 (Src homology 2-domain-containing inositol phosphatase 1/2)] and PtdIns 4-phosphatase [INPP4B (inositol polyphosphate 4-phosphatase type II)]. VPS34-IN1 will be a useful probe to delineate physiological roles of the Vps34. Monitoring SGK3 phosphorylation and activity could be employed as a biomarker of Vps34 activity, in an analogous manner by which Akt is used to probe cellular class I PI3K activity. Combining class I (GDC-0941) and class III (VPS34-IN1) PI3K inhibitors could be used as a strategy to better analyse the roles and regulation of the elusive class II PI3K. PMID:25177796

Bago, Ruzica; Malik, Nazma; Munson, Michael J.; Prescott, Alan R.; Davies, Paul; Sommer, Eeva; Shpiro, Natalia; Ward, Richard; Cross, Darren; Ganley, Ian G.; Alessi, Dario R.

2014-01-01

265

Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase.  

PubMed

The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3)P-binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25 nM IC50 in vitro, but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3)P-binding probe from endosome membranes, within 1 min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specifically with PtdIns(3)P via its N-terminal PX domain, might be controlled by Vps34. Mutations disrupting PtdIns(3)P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid ~50-60% loss of SGK3 phosphorylation within 1 min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a PtdIns(3)P-binding PX domain. Furthermore, class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~80-90%. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of PtdIns(3)P. The first is produced through phosphorylation of PtdIns by Vps34 at the endosome. The second is due to the conversion of class I PI3K product, PtdIns(3,4,5)P3 into PtdIns(3)P, via the sequential actions of the PtdIns 5-phosphatases [SHIP1/2 (Src homology 2-domain-containing inositol phosphatase 1/2)] and PtdIns 4-phosphatase [INPP4B (inositol polyphosphate 4-phosphatase type II)]. VPS34-IN1 will be a useful probe to delineate physiological roles of the Vps34. Monitoring SGK3 phosphorylation and activity could be employed as a biomarker of Vps34 activity, in an analogous manner by which Akt is used to probe cellular class I PI3K activity. Combining class I (GDC-0941) and class III (VPS34-IN1) PI3K inhibitors could be used as a strategy to better analyse the roles and regulation of the elusive class II PI3K. PMID:25177796

Bago, Ruzica; Malik, Nazma; Munson, Michael J; Prescott, Alan R; Davies, Paul; Sommer, Eeva; Shpiro, Natalia; Ward, Richard; Cross, Darren; Ganley, Ian G; Alessi, Dario R

2014-11-01

266

Phosphoinositide-specific Phospholipase C ? 1b (PI-PLC?1b) Interactome: Affinity Purification-Mass Spectrometry Analysis of PI-PLC?1b with Nuclear Protein*  

PubMed Central

Two isoforms of inositide-dependent phospholipase C ?1 (PI-PLC?1) are generated by alternative splicing (PLC?1a and PLC?1b). Both isoforms are present within the nucleus, but in contrast to PLC?1a, the vast majority of PLC?1b is nuclear. In mouse erythroid leukemia cells, PI-PLC?1 is involved in the regulation of cell division and the balance between cell proliferation and differentiation. It has been demonstrated that nuclear localization is crucial for the enzymatic function of PI-PLC?1, although the mechanism by which this nuclear import occurs has never been fully characterized. The aim of this study was to characterize both the mechanism of nuclear localization and the molecular function of nuclear PI-PLC?1 by identifying its interactome in Friend's erythroleukemia isolated nuclei, utilizing a procedure that coupled immuno-affinity purification with tandem mass spectrometry analysis. Using this procedure, 160 proteins were demonstrated to be in association with PI-PLC?1b, some of which have been previously characterized, such as the splicing factor SRp20 (Srsf3) and Lamin B (Lmnb1). Co-immunoprecipitation analysis of selected proteins confirmed the data obtained via mass spectrometry. Of particular interest was the identification of the nuclear import proteins Kpna2, Kpna4, Kpnb1, Ran, and Rangap1, as well as factors involved in hematological malignancies and several anti-apoptotic proteins. These data give new insight into possible mechanisms of nuclear trafficking and functioning of this critical signaling molecule. PMID:23665500

Piazzi, Manuela; Blalock, William L.; Bavelloni, Alberto; Faenza, Irene; D'Angelo, Antonietta; Maraldi, Nadir M.; Cocco, Lucio

2013-01-01

267

Mutations in the Arabidopsis Phosphoinositide Phosphatase Gene SAC9 Lead to Overaccumulation of PtdIns(4,5)P2 and Constitutive Expression of the Stress-Response Pathway1  

PubMed Central

Phosphoinositides (PIs) are signaling molecules that regulate cellular events including vesicle targeting and interactions between membrane and cytoskeleton. Phosphatidylinositol (PtdIns)(4,5)P2 is one of the best characterized PIs; studies in which PtdIns(4,5)P2 localization or concentration is altered lead to defects in the actin cytoskeleton and exocytosis. PtdIns(4,5)P2 and its derivative Ins(1,4,5)P3 accumulate in salt, cold, and osmotically stressed plants. PtdIns(4,5)P2 signaling is terminated through the action of inositol polyphosphate phosphatases and PI phosphatases including supressor of actin mutation (SAC) domain phosphatases. In some cases, these phosphatases also act on Ins(1,4,5)P3. We have characterized the Arabidopsis (Arabidopsis thaliana) sac9 mutants. The SAC9 protein is different from other SAC domain proteins in several ways including the presence of a WW protein interaction domain within the SAC domain. The rice (Oryza sativa) and Arabidopsis SAC9 protein sequences are similar, but no apparent homologs are found in nonplant genomes. High-performance liquid chromatography studies show that unstressed sac9 mutants accumulate elevated levels of PtdIns(4,5)P2 and Ins(1,4,5)P3 as compared to wild-type plants. The sac9 mutants have characteristics of a constitutive stress response, including dwarfism, closed stomata, and anthocyanin accumulation, and they overexpress stress-induced genes and overaccumulate reactive-oxygen species. These results suggest that the SAC9 phosphatase is involved in modulating phosphoinsitide signals during the stress response. PMID:15923324

Williams, Mary Elizabeth; Torabinejad, Javad; Cohick, Evan; Parker, Katherine; Drake, Elizabeth J.; Thompson, James E.; Hortter, Michelle; DeWald, Daryll B.

2005-01-01

268

The Vr-PLC3 gene encodes a putative plasma membrane-localized phosphoinositide-specific phospholipase C whose expression is induced by abiotic stress in mung bean ( Vigna radiata L.) 1 1 EMBL accession numbers: AY394079 ( Vr-PLC1), AY461431 ( Vr-PLC2) and AY394078 ( Vr-PLC3)  

Microsoft Academic Search

Phosphoinositide-specific phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate and diacylglycerol, both of which act as secondary messengers in animal cells. In this report, we identified in Vigna radiata L. (mung bean) three distinct partial cDNAs (pVr-PLC1, pVr-PLC2, and pVr-PLC3), which encode forms of putative PI-PLC. All three Vr-PLC genes were transcriptionally active and displayed

Yun Ju Kim; Jee Eun Kim; Jae-Hoon Lee; Myoung Hui Lee; Ho Won Jung; Young Yil Bahk; Byung Kook Hwang; Inhwan Hwang; Woo Taek Kim

2004-01-01

269

Puzzling Mechanisms  

ERIC Educational Resources Information Center

The basis of a good mechanical puzzle is often a puzzling mechanism. This article will introduce some new puzzling mechanisms, like two knots that engage like gears, a chain whose links can be interchanged, and flat gears that do not come apart. It illustrates how puzzling mechanisms can be transformed into real mechanical puzzles, e.g., by…

van Deventer, M. Oskar

2009-01-01

270

Guanylyl cyclase/natriuretic peptide receptor-A signaling antagonizes phosphoinositide hydrolysis, Ca2+ release, and activation of protein kinase C  

PubMed Central

Thus far, three related natriuretic peptides (NPs) and three distinct sub-types of cognate NP receptors have been identified and characterized based on the specific ligand binding affinities, guanylyl cyclase activity, and generation of intracellular cGMP. Atrial and brain natriuretic peptides (ANP and BNP) specifically bind and activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), and C-type natriuretic peptide (CNP) shows specificity to activate guanylyl cyclase/natriuretic peptide receptor-B (GC-B/NPRB). All three NPs bind to natriuretic peptide receptor-C (NPRC), which is also known as clearance or silent receptor. The NPRA is considered the principal biologically active receptor of NP family; however, the molecular signaling mechanisms of NP receptors are not well understood. The activation of NPRA and NPRB produces the intracellular second messenger cGMP, which serves as the major signaling molecule of all three NPs. The activation of NPRB in response to CNP also produces the intracellular cGMP; however, at lower magnitude than that of NPRA, which is activated by ANP and BNP. In addition to enhanced accumulation of intracellular cGMP in response to all three NPs, the levels of cAMP, Ca2+ and inositol triphosphate (IP3) have also been reported to be altered in different cells and tissue types. Interestingly, ANP has been found to lower the concentrations of cAMP, Ca2+, and IP3; however, NPRC has been proposed to increase the levels of these metabolic signaling molecules. The mechanistic studies of decreased and/or increased levels of cAMP, Ca2+, and IP3 in response to NPs and their receptors have not yet been clearly established. This review focuses on the signaling mechanisms of ANP/NPRA and their biological effects involving an increased level of intracellular accumulation of cGMP and a decreased level of cAMP, Ca2+, and IP3 in different cells and tissue systems. PMID:25202235

Pandey, Kailash N.

2014-01-01

271

Moving Forward: Mechanisms of Chemoattractant Gradient Sensing  

NSDL National Science Digital Library

Cells use an internal compass to sense the direction of chemoattractant gradients. This is used to bias pseudopod extension at the front of the cell and to orient cell polarization. Recent studies have highlighted the important roles played by phosphoinositide-3,4,5-triphosphate and small G proteins, but many questions remain.

PhD Jonathan Franca-Koh (Johns Hopkins University School of Medicine Department of Cell Biology)

2004-10-01

272

Lactate Engages Receptor Tyrosine Kinases Axl, Tie2, and Vascular Endothelial Growth Factor Receptor 2 to Activate Phosphoinositide 3-Kinase/Akt and Promote Angiogenesis*  

PubMed Central

Although a high level of lactate is quintessential to both tumors and wound healing, the manner by which lactate impacts endothelial cells to promote angiogenesis and thereby create or restore vascular perfusion to growing tissues has not been fully elucidated. Here we report that lactate activated the PI3K/Akt pathway in primary human endothelial cells. Furthermore, activating this signaling pathway was required for lactate-stimulated organization of endothelial cells into tubes and for sprouting of vessels from mouse aortic explants. Lactate engaged the PI3K/Akt pathway via ligand-mediated activation of the three receptor tyrosine kinases Axl, Tie2, and VEGF receptor 2. Neutralizing the ligands for these receptor tyrosine kinases, pharmacologically inhibiting their kinase activity or suppressing their expression largely eliminated the ability of cells and explants to respond to lactate. Elucidating the mechanism by which lactate communicates with endothelial cells presents a previously unappreciated opportunity to improve our understanding of the angiogenic program and to govern it. PMID:23754286

Ruan, Guo-Xiang; Kazlauskas, Andrius

2013-01-01

273

Plasma membrane nanoporation as a possible mechanism behind infrared excitation of cells  

NASA Astrophysics Data System (ADS)

Objective. Short infrared (IR) laser pulses have been used to stimulate action potentials in neurons both in vivo and in vitro. However, the mechanism(s) underlying this phenomenon has remained elusive. In vitro studies have found that pulsed IR exposure generates a nearly instant change in capacitance in the plasma membrane, characterized by inward rectification, a common feature in pore-forming exposures, such as electrical pulses and acoustic shock waves. Based on this similarity, we hypothesize that the mechanism of IR stimulation is the formation of short-lived nanopores in the plasma membrane. These transient, small-diameter pores allow the influx of extracellular ions that lead to action potential generation, possibly through activation of secondary messenger pathways or depolarization of the cell membrane resulting in activation of voltage-gated ion channels. Approach. A variety of fluorescent markers are used to observe the cell response to IR stimulation to monitor for effects indicative of nanoporation in other modalities. Main results. We observe rapid, transient rises in intracellular Ca2+, influx of YO-PRO-1 and propidium iodide into the cell signifying membrane permeabilization, cellular blebbing and swelling, and activation of the intracellular phosphoinositides lipid signaling pathway. Significance. This conclusion better explains the experimental observations and limitations of IR-induced neurological stimulation and represents a distinct theoretical shift in the understanding of the mechanism of IR-induced stimulation.

Beier, Hope T.; Tolstykh, Gleb P.; Musick, Joshua D.; Thomas, Robert J.; Ibey, Bennett L.

2014-12-01

274

Plasma membrane nanoporation as a possible mechanism behind infrared excitation of cells.  

PubMed

Objective. Short infrared (IR) laser pulses have been used to stimulate action potentials in neurons both in vivo and in vitro. However, the mechanism(s) underlying this phenomenon has remained elusive. In vitro studies have found that pulsed IR exposure generates a nearly instant change in capacitance in the plasma membrane, characterized by inward rectification, a common feature in pore-forming exposures, such as electrical pulses and acoustic shock waves. Based on this similarity, we hypothesize that the mechanism of IR stimulation is the formation of short-lived nanopores in the plasma membrane. These transient, small-diameter pores allow the influx of extracellular ions that lead to action potential generation, possibly through activation of secondary messenger pathways or depolarization of the cell membrane resulting in activation of voltage-gated ion channels. Approach. A variety of fluorescent markers are used to observe the cell response to IR stimulation to monitor for effects indicative of nanoporation in other modalities. Main results. We observe rapid, transient rises in intracellular Ca(2+), influx of YO-PRO-1 and propidium iodide into the cell signifying membrane permeabilization, cellular blebbing and swelling, and activation of the intracellular phosphoinositides lipid signaling pathway. Significance. This conclusion better explains the experimental observations and limitations of IR-induced neurological stimulation and represents a distinct theoretical shift in the understanding of the mechanism of IR-induced stimulation. PMID:25340253

Beier, Hope T; Tolstykh, Gleb P; Musick, Joshua D; Thomas, Robert J; Ibey, Bennett L

2014-12-01

275

Gi-mediated translocation of GLUT4 is independent of p85/p110alpha and p110gamma phosphoinositide 3-kinases but might involve the activation of Akt kinase.  

PubMed Central

Activation of phosphoinositide 3-kinase (PI-3K) is essential for insulin-stimulated translocation of GLUT4 and glucose transport in insulin target tissues. A novel p110gamma PI-3K was reported to be activated by G(i)-coupled receptors via Gbetagamma subunits. We asked whether the stimulation of G(i)-coupled receptors would trigger GLUT4 translocation and glucose uptake by the activation of Gbetagamma-dependent p110gamma PI-3K. We find that this translocation and glucose uptake can be induced by the ligand stimulation of G(i)-coupled alpha(2A) adrenergic receptor and fMet-Leu-Phe receptor in cells stably expressing these receptors. The noradrenaline ('noradrenaline')- and fMet-Leu-Phe-stimulated GLUT4 translocations were abolished by pretreatment with pertussis toxin. Pretreatment with wortmannin or genistein also inhibited the G(i)-mediated GLUT4 translocation. On ligand stimulation of these two kinds of G(i)-coupled receptor, although there was a slight increase in PtdIns(3,4,5)P(3) production, activation of either the p85/p110alpha PI-3K or Gbetagamma-dependent p110gamma PI-3K was not observed even in Chinese hamster ovary cells stably overexpressing exogenous p101/p110gamma. The G(i)-mediated GLUT4 translocation was accompanied by activation of the serine-threonine kinase Akt; the inhibitory effects of pertussis toxin, wortmannin and genistein on G(i)-mediated GLUT4 translocation paralleled their inhibitory effects on Akt activation. In contrast, the activation of some other G(i)-coupled receptors, such as prostaglandin EP3alpha receptor and platelet-activating factor receptor, did not cause either pertussis-toxin-sensitive translocation of GLUT4myc or activation of Akt kinase. These results indicate that the ligand stimulation of some G(i)-coupled receptors triggers GLUT4 translocation that occurs independently of p85/p110alpha-type and p110gamma-type PI-3Ks but might involve the activation of Akt kinase. PMID:10642513

Wang, L; Hayashi, H; Kishi, K; Huang, L; Hagi, A; Tamaoka, K; Hawkins, P T; Ebina, Y

2000-01-01

276

Quantum Mechanics  

NASA Astrophysics Data System (ADS)

Introduction; Part I. Basic Features of Quantum Mechanics: 1. From classical mechanics to quantum mechanics; 2. Quantum observable and states; 3. Quantum dynamics; 4. Examples of quantum dynamics; 5. Density matrix; Part II. More Advanced Topics: 6. Angular momentum and spin; 7. Identical particles; 8. Symmetries and conservation laws; 9. The measurement problem; Part III. Matter and Light: 10. Perturbations and approximation methods; 11. Hydrogen and helium atoms; 12. Hydrogen molecular ion; 13. Quantum optics; Part IV. Quantum Information: State and Correlations: 14. Quantum theory of open systems; 15. State measurement in quantum mechanics; 16. Entanglement: non-separability; 17. Entanglement: quantum information; References; Index.

Auletta, Gennaro; Fortunato, Mauro; Parisi, Giorgio

2014-01-01

277

Modern mechanics  

NSDL National Science Digital Library

We consider the goals of the introductory course in classical mechanics taken by physics majors and argue both that these goals are not well met in actual courses and that the goals themselves should be rethought. We propose alternative goals and describe an introductory "modern mechanics" course that addresses these alternative goals. Included in the description are several genres of homework problems that are nearly absent from traditional mechanics courses at both the introductory and intermediate levels. The intermediate mechanics course could be restructured to exploit a broader foundation laid by the introductory course.

Chabay, Ruth; Sherwood, Bruce

2005-10-11

278

Lysophosphatidylcholine and 7-oxocholesterol modulate Ca2+ signals and inhibit the phosphorylation of endothelial NO synthase and cytosolic phospholipase A2.  

PubMed Central

The oxidation of plasma LDLs (low-density lipoproteins) is a key event in the pathogenesis of atherosclerosis. LPC (lysophosphatidylcholine) and oxysterols are major lipid constitutents of oxidized LDLs. In particular, 7-oxocholesterol has been found in plasma from cardiac patients and atherosclerotic plaque. In the present study, we investigated the ability of 7-oxocholesterol and LPC to regulate the activation of eNOS (endothelial nitric oxide synthase) and cPLA2 (cytosolic phospholipase A2) that synthesize two essential factors for vascular wall integrity, NO (nitric oxide) and arachidonic acid. In endothelial cells from human umbilical vein cords, both 7-oxocholesterol (150 microM) and LPC (20 microM) decreased histamine-induced NO release, but not the release activated by thapsigargin. The two lipids decreased NO release through a PI3K (phosphoinositide 3-kinase)-dependent pathway, and decreased eNOS phosphorylation. Their mechanisms of action were, however, different. The NO release reduction was dependent on superoxide anions in LPC-treated cells and not in 7-oxocholesterol-treated ones. The Ca2+ signals induced by histamine were abolished by LPC, but not by 7-oxocholesterol. The oxysterol also inhibited (i) the histamine- and thapsigargin-induced arachidonic acid release, and (ii) the phosphorylation of both cPLA2 and ERK1/2 (extracellular-signal-regulated kinases 1/2). The results show that 7-oxocholesterol inhibits eNOS and cPLA2 activation by altering a Ca2+-independent upstream step of PI3K and ERK1/2 cascades, whereas LPC desensitizes eNOS by interfering with receptor-activated signalling pathways. This suggests that 7-oxocholesterol and LPC generate signals which cross-talk with heterologous receptors, effects which could appear at early stage of atherosclerosis. PMID:14992685

Millanvoye-Van Brussel, Elisabeth; Topal, Gökce; Brunet, Annie; Do Pham, Thuc; Deckert, Valérie; Rendu, Francine; David-Dufilho, Monique

2004-01-01

279

Fluid Mechanics  

Microsoft Academic Search

This text is intended for the study of fluid mechanics at an intermediate level. The presentation starts with basic concepts, in order to form a sound conceptual structure that can support engineering applications and encourage further learning. The presentation is exact, incorporating both the mathematics involved and the physics needed to understand the various phenomena in fluid mechanics. Where a

David Pnueli; Chaim Gutfinger

1997-01-01

280

Quantum Mechanics  

NASA Astrophysics Data System (ADS)

Preface; 1. Introduction; 2. Mathematical preliminaries; 3. The rules of quantum mechanics; 4. The connection between the fundamental rules and wave mechanics; 5. Further illustrations of the rules of quantum mechanics; 6. Further developments in one-dimensional wave mechanics; 7. The theory of angular momentum; 8. Wave mechanics in three dimensions: hydrogenic atoms; 9. Time-independent approximations for bound state problems; 10. Applications of static perturbation theory; 11. Identical particles; 12. Atomic structure; 13. Molecules; 14. The stability of matter; 15. Photons; 16. Interaction of non-relativistic charged particles and radiation; 17. Further topics in perturbation theory; 18. Scattering; 19. Special relativity and quantum mechanics: the Klein–Gordon equation; 20. The Dirac equation; 21. Interaction of a relativistic spin 1/2 particle with an external electromagnetic field; 22. The Dirac field; 23. Interaction between relativistic electrons, positrons, and photons; 24. The quantum mechanics of weak interactions; 25. The quantum measurement problem; Appendix A: useful inequalities for quantum mechanics; Appendix B: Bell's inequality; Appendix C: spin of the photon: vector spherical waves; Works cited; Bibliography; Index.

Commins, Eugene D.

2014-10-01

281

& Mechanical Engineering  

E-print Network

, thermodynamics, uid mechanics, heat transfer, materials and design. Our graduates are frequently at the leading control, emerging fuel cell technologies, computational uid mechanics, combustion, heat transfer engage in research that is shaping the future of air and ground transportation and improving our

Zhou, Chongwu

282

Grasping Mechanism  

NASA Technical Reports Server (NTRS)

Grasping mechanism captures and locks onto rodlike handle without being precisely aligned with it initially. Mechanism includes two faceplates and three rotatable fingers meshing odd finger between two members of opposite finger pair. Power for fingers supplied by motor powered via harmonic drive reduces speed and increases torque by factor of 160.

Myers, W. Neil; Forbes, John C.

1987-01-01

283

Cytoskeletal Mechanics  

NASA Astrophysics Data System (ADS)

1. Introduction and the biological basis for cell mechanics Mohammad R. K. Mofrad and Roger Kamm; 2. Experimental measurements of intracellular mechanics Paul Janmey and Christoph Schmidt; 3. The cytoskeleton as a soft glassy material Jeffrey Fredberg and Ben Fabry; 4. Continuum elastic or viscoelastic models for the cell Mohammad R. K. Mofrad, Helene Karcher and Roger Kamm; 5. Multiphasic models of cell mechanics Farshid Guuilak, Mansoor A. Haider, Lori A. Setton, Tod A. Laursen and Frank P. T. Baaijens; 6. Models of cytoskeletal mechanics based on tensegrity Dimitrije Stamenovic; 7. Cells, gels and mechanics Gerald H. Pollack; 8. Polymer-based models of cytoskeletal networks F. C. MacKintosh; 9. Cell dynamics and the actin cytoskeleton James L. McGrath and C. Forbes Dewey, Jr; 10. Active cellular motion: continuum theories and models Marc Herant and Micah Dembo; 11. Summary Mohammad R. K. Mofrad and Roger Kamm.

Mofrad, Mohammad R. K.; Kamm, Roger D.

2006-10-01

284

Cytoskeletal Mechanics  

NASA Astrophysics Data System (ADS)

1. Introduction and the biological basis for cell mechanics Mohammad R. K. Mofrad and Roger Kamm; 2. Experimental measurements of intracellular mechanics Paul Janmey and Christoph Schmidt; 3. The cytoskeleton as a soft glassy material Jeffrey Fredberg and Ben Fabry; 4. Continuum elastic or viscoelastic models for the cell Mohammad R. K. Mofrad, Helene Karcher and Roger Kamm; 5. Multiphasic models of cell mechanics Farshid Guuilak, Mansoor A. Haider, Lori A. Setton, Tod A. Laursen and Frank P. T. Baaijens; 6. Models of cytoskeletal mechanics based on tensegrity Dimitrije Stamenovic; 7. Cells, gels and mechanics Gerald H. Pollack; 8. Polymer-based models of cytoskeletal networks F. C. MacKintosh; 9. Cell dynamics and the actin cytoskeleton James L. McGrath and C. Forbes Dewey, Jr; 10. Active cellular motion: continuum theories and models Marc Herant and Micah Dembo; 11. Summary Mohammad R. K. Mofrad and Roger Kamm.

Mofrad, Mohammad R. K.; Kamm, Roger D.

2011-08-01

285

Molecular Mechanisms of Ca2+ Signaling in Neurons Induced by the S100A4 Protein  

PubMed Central

The S100A4 protein belongs to the S100 family of vertebrate-specific proteins possessing both intra- and extracellular functions. In the nervous system, high levels of S100A4 expression are observed at sites of neurogenesis and lesions, suggesting a role of the protein in neuronal plasticity. Extracellular oligomeric S100A4 is a potent promoter of neurite outgrowth and survival from cultured primary neurons; however, the molecular mechanism of this effect has not been established. Here we demonstrate that oligomeric S100A4 increases the intracellular calcium concentration in primary neurons. We present evidence that both S100A4-induced Ca2+ signaling and neurite extension require activation of a cascade including a heterotrimeric G protein(s), phosphoinositide-specific phospholipase C, and diacylglycerol-lipase, resulting in Ca2+ entry via nonselective cation channels and via T- and L-type voltage-gated Ca2+ channels. We demonstrate that S100A4-induced neurite outgrowth is not mediated by the receptor for advanced glycation end products, a known target for other extracellular S100 proteins. However, S100A4-induced signaling depends on interactions with heparan sulfate proteoglycans at the cell surface. Thus, glycosaminoglycans may act as coreceptors of S100 proteins in neurons. This may provide a mechanism by which S100 proteins could locally regulate neuronal plasticity in connection with brain lesions and neurological disorders. PMID:16612001

Kiryushko, Darya; Novitskaya, Vera; Soroka, Vladislav; Klingelhofer, Jorg; Lukanidin, Eugene; Berezin, Vladimir; Bock, Elisabeth

2006-01-01

286

Ca2+ transport in plant cells and mechanisms of transformation of phytochrome-induced photosignals  

NASA Astrophysics Data System (ADS)

The recent data on the influence of phytochrome on the efficiency of Ca2+ translocation across the membranes of oat protoplasts are given. Ca2+ uptake in the protoplasts was shown to be influenced by the red light (R) illumination. This effect was reverted by the following far-red light (FR) illumination. To elucidate the sensitivity to phytochrome-controlling action the screening between the mechanisms of Ca2+ transport across the plasma membranes of oat protoplasts, Na+/Ca2+ and Ca2+/H+ exchangers, Ca2+-pump and Ca2+-channel was done. It was established that phytochrome modulated the activity of Na+/Ca2+-exchanger and Ca2+-pump. The light-mediated oscillations of cytoplasmic Ca2+ concentration in the oat protoplasts were demonstrated using fluorescence probe quin2 loaded into the cells and laser monitoring of fluorescence signal. The evidences were obtained that the oscillations were not the result of the elevation of cytoplasmic Ca2+ concentration and had no connection with Ca2+ pool of mitochondria. The possibility of the relation between the Ca2+ oscillations and phosphoinositide metabolism in plant cell membranes is analyzed. The mechanisms of transformation of primary phytochrome signal into biological effects were discussed.

Volotovski, Igor D.

1995-01-01

287

Tissue Mechanics  

NSDL National Science Digital Library

Students reflect on their experiences making silly putty (the previous hands-on activity in the unit), especially why changing the borax concentration alters the mechanical properties of silly putty and how this pertains to tissue mechanics. Students learn why engineers must understand tissue mechanics in order to design devices that will be implanted or used inside bodies, to study pathologies of tissues and how this alters tissue function, and to design prosthetics. Finally, students learn about collagen, elastin and proteoglycans and their roles in giving body tissues their unique functions. This prepares them for the culminating design-build-test activity of the unit.

Integrated Teaching And Learning Program

288

Mechanical memory  

DOEpatents

A first-in-first-out (FIFO) microelectromechanical memory apparatus (also termed a mechanical memory) is disclosed. The mechanical memory utilizes a plurality of memory cells, with each memory cell having a beam which can be bowed in either of two directions of curvature to indicate two different logic states for that memory cell. The memory cells can be arranged around a wheel which operates as a clocking actuator to serially shift data from one memory cell to the next. The mechanical memory can be formed using conventional surface micromachining, and can be formed as either a nonvolatile memory or as a volatile memory.

Gilkey, Jeffrey C. (Albuquerque, NM); Duesterhaus, Michelle A. (Albuquerque, NM); Peter, Frank J. (Albuquerque, NM); Renn, Rosemarie A. (Albuquerque, NM); Baker, Michael S. (Albuquerque, NM)

2006-05-16

289

Mechanical memory  

DOEpatents

A first-in-first-out (FIFO) microelectromechanical memory apparatus (also termed a mechanical memory) is disclosed. The mechanical memory utilizes a plurality of memory cells, with each memory cell having a beam which can be bowed in either of two directions of curvature to indicate two different logic states for that memory cell. The memory cells can be arranged around a wheel which operates as a clocking actuator to serially shift data from one memory cell to the next. The mechanical memory can be formed using conventional surface micromachining, and can be formed as either a nonvolatile memory or as a volatile memory.

Gilkey, Jeffrey C. (Albuquerque, NM); Duesterhaus, Michelle A. (Albuquerque, NM); Peter, Frank J. (Albuquerque, NM); Renn, Rosemarie A. (Alburquerque, NM); Baker, Michael S. (Albuquerque, NM)

2006-08-15

290

Statistical Mechanics  

Microsoft Academic Search

We review and further develop a mathematical framework for non-equilibrium quantum statistical mechanics recently proposed in (JP4, JP5, JP6, Ru3, Ru4, Ru5, Ru6). In the alge- braic formalism of quantum statistical mechanics we introduce notions of non-equilibrium steady states, entropy production and heat fluxes, and study their properties. Our basic paradigm is a model of a small (finite) quantum system

V. Jaksi ´; C.-A. Pillet

1937-01-01

291

Fault mechanics  

SciTech Connect

Recent observational, experimental, and theoretical modeling studies of fault mechanics are discussed in a critical review of U.S. research from the period 1987-1990. Topics examined include interseismic strain accumulation, coseismic deformation, postseismic deformation, and the earthquake cycle; long-term deformation; fault friction and the instability mechanism; pore pressure and normal stress effects; instability models; strain measurements prior to earthquakes; stochastic modeling of earthquakes; and deep-focus earthquakes. Maps, graphs, and a comprehensive bibliography are provided. 220 refs.

Segall, P. (USAF, Geophysics Laboratory, Hanscom AFB, MA (United States))

1991-01-01

292

Computational mechanics  

SciTech Connect

The Computational Mechanics thrust area sponsors research into the underlying solid, structural and fluid mechanics and heat transfer necessary for the development of state-of-the-art general purpose computational software. The scale of computational capability spans office workstations, departmental computer servers, and Cray-class supercomputers. The DYNA, NIKE, and TOPAZ codes have achieved world fame through our broad collaborators program, in addition to their strong support of on-going Lawrence Livermore National Laboratory (LLNL) programs. Several technology transfer initiatives have been based on these established codes, teaming LLNL analysts and researchers with counterparts in industry, extending code capability to specific industrial interests of casting, metalforming, and automobile crash dynamics. The next-generation solid/structural mechanics code, ParaDyn, is targeted toward massively parallel computers, which will extend performance from gigaflop to teraflop power. Our work for FY-92 is described in the following eight articles: (1) Solution Strategies: New Approaches for Strongly Nonlinear Quasistatic Problems Using DYNA3D; (2) Enhanced Enforcement of Mechanical Contact: The Method of Augmented Lagrangians; (3) ParaDyn: New Generation Solid/Structural Mechanics Codes for Massively Parallel Processors; (4) Composite Damage Modeling; (5) HYDRA: A Parallel/Vector Flow Solver for Three-Dimensional, Transient, Incompressible Viscous How; (6) Development and Testing of the TRIM3D Radiation Heat Transfer Code; (7) A Methodology for Calculating the Seismic Response of Critical Structures; and (8) Reinforced Concrete Damage Modeling.

Goudreau, G.L.

1993-03-01

293

Fluid Mechanics  

NASA Astrophysics Data System (ADS)

This text is intended for the study of fluid mechanics at an intermediate level. The presentation starts with basic concepts, in order to form a sound conceptual structure that can support engineering applications and encourage further learning. The presentation is exact, incorporating both the mathematics involved and the physics needed to understand the various phenomena in fluid mechanics. Where a didactical choice must be made between the two, the physics prevails. Throughout the book the authors have tried to reach a balance between exact presentation, intuitive grasp of new ideas, and creative applications of concepts. This approach is reflected in the examples presented in the text and in the exercises given at the end of each chapter. Subjects treated are hydrostatics, viscous flow, similitude and order of magnitude, creeping flow, potential flow, boundary layer flow, turbulent flow, compressible flow, and non-Newtonian flows. This book is ideal for advanced undergraduate students in mechanical, chemical, aerospace, and civil engineering. Solutions manual available.

Pnueli, David; Gutfinger, Chaim

1997-01-01

294

Turntable mechanism  

NASA Technical Reports Server (NTRS)

In vacuum plasma spraying a turntable must be provided which not only makes it possible to rotate and tilt a heavy workpiece, but to operate at vacuum plasma temperatures to do so. In the vacuum plasma coating of large parts such as combustion chambers of rocket engines, the workpiece must not only be rotated, but it must be tilted. Hence, the turntable must be capable not only of supporting heavy parts, but of angulating such heavy workpieces. And this must be done without drive means failure due to extremely high temperatures under which the turntable mechanism is operated. A turntable mechanism is provided which is capable of operating under such conditions. For cooling the turntable drive mechanism, internal cooling means are included.

Myers, William Neill (inventor)

1993-01-01

295

Turntable mechanism  

NASA Astrophysics Data System (ADS)

In vacuum plasma spraying a turntable must be provided which not only makes it possible to rotate and tilt a heavy workpiece, but to operate at vacuum plasma temperatures to do so. In the vacuum plasma coating of large parts such as combustion chambers of rocket engines, the workpiece must not only be rotated, but it must be tilted. Hence, the turntable must be capable not only of supporting heavy parts, but of angulating such heavy workpieces. And this must be done without drive means failure due to extremely high temperatures under which the turntable mechanism is operated. A turntable mechanism is provided which is capable of operating under such conditions. For cooling the turntable drive mechanism, internal cooling means are included.

Myers, William Neill

1993-10-01

296

Turntable mechanism  

NASA Astrophysics Data System (ADS)

In vacuum plasma spraying a turntable must be provided which not only makes it possible to rotate and tilt a heavy workpiece, but to operate at vacuum plasma temperatures to do so. In the vacuum plasma coating of large parts such as combustion chambers of rocket engines, the workpiece must not only be rotated, but it must be tilted. Hence, the turntable must be capable not only of supporting heavy parts, but of angulating such heavy workpieces. And this must be done without drive means failure due to extremely high temperatures under which the turntable mechanism is operated. A turntable mechanism is provided which is capable of operating under such conditions. For cooling the turntable drive mechanism, internal cooling means are included.

Myers, William Neill

1992-01-01

297

Computational mechanics  

SciTech Connect

The Computational Mechanics thrust area is a vital and growing facet of the Mechanical Engineering Department at Lawrence Livermore National Laboratory (LLNL). This work supports the development of computational analysis tools in the areas of structural mechanics and heat transfer. Over 75 analysts depend on thrust area-supported software running on a variety of computing platforms to meet the demands of LLNL programs. Interactions with the Department of Defense (DOD) High Performance Computing and Modernization Program and the Defense Special Weapons Agency are of special importance as they support our ParaDyn project in its development of new parallel capabilities for DYNA3D. Working with DOD customers has been invaluable to driving this technology in directions mutually beneficial to the Department of Energy. Other projects associated with the Computational Mechanics thrust area include work with the Partnership for a New Generation Vehicle (PNGV) for ''Springback Predictability'' and with the Federal Aviation Administration (FAA) for the ''Development of Methodologies for Evaluating Containment and Mitigation of Uncontained Engine Debris.'' In this report for FY-97, there are five articles detailing three code development activities and two projects that synthesized new code capabilities with new analytic research in damage/failure and biomechanics. The article this year are: (1) Energy- and Momentum-Conserving Rigid-Body Contact for NIKE3D and DYNA3D; (2) Computational Modeling of Prosthetics: A New Approach to Implant Design; (3) Characterization of Laser-Induced Mechanical Failure Damage of Optical Components; (4) Parallel Algorithm Research for Solid Mechanics Applications Using Finite Element Analysis; and (5) An Accurate One-Step Elasto-Plasticity Algorithm for Shell Elements in DYNA3D.

Raboin, P J

1998-01-01

298

Mural propagation of descending vasa recta responses to mechanical stimulation.  

PubMed

To investigate the responses of descending vasa recta (DVR) to deformation of the abluminal surface, we devised an automated method that controls duration and frequency of stimulation by utilizing a stream of buffer from a micropipette. During stimulation at one end of the vessel, fluorescent responses from fluo4 or bis[1,3-dibutylbarbituric acid-(5)] trimethineoxonol [DiBAC?(3)], indicating cytoplasmic calcium ([Ca²?]CYT) or membrane potential, respectively, were recorded from distant cells. Alternately, membrane potential was recorded from DVR pericytes by nystatin whole cell patch-clamp. Mechanical stimulation elicited reversible [Ca²?)]CYT responses that increased with frequency. Individual pericyte responses along the vessel were initiated within a fraction of a second of one another. Those responses were inhibited by gap junction blockade with 18 ?-glycyrrhetinic acid (100 ?M) or phosphoinositide 3 kinase inhibition with 2-morpholin-4-yl-8-phenylchromen-4-one (50 ?M). [Ca²?]CYT responses were blocked by removal of extracellular Ca²? or L-type voltage-gated channel blockade with nifedipine (10 ?M). At concentrations selective for the T-type channel blockade, mibefradil (100 nM) was ineffective. During mechanostimulation, pericytes rapidly depolarized, as documented with either DiBAC4(3) fluorescence or patch-clamp recording. Single stimuli yielded depolarizations of 22.5 ± 2.2 mV while repetitive stimuli at 0.1 Hz depolarized pericytes by 44.2 ± 4.0 mV. We conclude that DVR are mechanosensitive and that rapid transmission of signals along the vessel axis requires participation of gap junctions, L-type Ca²? channels, and pericyte depolarization. PMID:23698119

Zhang, Zhong; Payne, Kristie; Cao, Chunhua; Pallone, Thomas L

2013-08-01

299

Mechanical Madness  

NSDL National Science Digital Library

In this online Flash game, learners test their engineering know-how, moving a collection of mechanical parts onto a board to make complete a system of parts that will move a ball from start to finish. Three levels of play (easy, medium and hard) allow learners to work up to this machine challenge.

Twin Cities Public Television, Inc.

2006-01-01

300

Cratering mechanics  

NASA Technical Reports Server (NTRS)

Main concepts and theoretical models which are used for studying the mechanics of cratering are discussed. Numerical two-dimensional calculations are made of explosions near a surface and high-speed impact. Models are given for the motion of a medium during cratering. Data from laboratory modeling are given. The effect of gravitational force and scales of cratering phenomena is analyzed.

Ivanov, B. A.

1986-01-01

301

Mechanical Technician.  

ERIC Educational Resources Information Center

This document contains 33 units to consider for use in a tech prep competency profile for the occupation of mechanical technician. All the units listed will not necessarily apply to every situation or tech prep consortium, nor will all the competencies within each unit be appropriate. Several units appear within each specific occupation and would…

Ohio State Univ., Columbus. Center on Education and Training for Employment.

302

Quantum Mechanics  

NSDL National Science Digital Library

This website contains a number of descriptions of quantum mechanical phenomena, using 3D animations to illustrate the physics. The goal is to introduce basic concepts and phenomena using simulations rather than complex mathematics. The time-dependence of quantum systems is a focus of this material.

De Raedt, Hans; Michielsen, Kristel

2010-03-25

303

Mechanical allodynia.  

PubMed

Mechanical allodynia (other pain) is a painful sensation caused by innocuous stimuli like light touch. Unlike inflammatory hyperalgesia that has a protective role, allodynia has no obvious biological utility. Allodynia is associated with nerve damage in conditions such as diabetes, and is likely to become an increasing clinical problem. Unfortunately, the mechanistic basis of this enhanced sensitivity is incompletely understood. In this review, we describe evidence for the involvement of candidate mechanosensitive channels such as Piezo2 and their role in allodynia, as well as the peripheral and central nervous system mechanisms that have also been implicated in this form of pain. Specific treatments that block allodynia could be very useful if the cell and molecular basis of the condition could be determined. There are many potential mechanisms underlying this condition ranging from alterations in mechanotransduction and sensory neuron excitability to the actions of inflammatory mediators and wiring changes in the CNS. As with other pain conditions, it is likely that the range of redundant mechanisms that cause allodynia will make therapeutic intervention problematic. PMID:24846747

Lolignier, Stéphane; Eijkelkamp, Niels; Wood, John N

2015-01-01

304

Rhizobium nod factor signaling. Evidence for a g protein-mediated transduction mechanism  

PubMed Central

Rhizobium nodulation (Nod) factors are lipochitooligosaccharide signals that elicit key symbiotic developmental responses in the host legume root. In this study, we have investigated Nod factor signal transduction in the Medicago root epidermis by using a pharmacological approach in conjunction with transgenic plants expressing the Nod factor-responsive reporter construct pMtENOD12-GUS. Evidence for the participation of heterotrimeric G proteins in Nod factor signaling has come from three complementary observations: (1) the amphiphilic peptides mastoparan and Mas7, known G protein agonists, are able to mimic Nod factor-induced epidermal MtENOD12 expression; (2) growth of plants in nodulation-inhibiting conditions (10 mM NH4NO3) leads to a dramatic reduction in both Nod factor- and mastoparan-elicited gene expression; and (3) bacterial pertussis toxin, a well-characterized G protein antagonist, blocks the activities of both the Nod factor and mastoparan. In addition, we have found that antagonists that interfere with phospholipase C activity (neomycin and U73122) and Ca2+ influx/release (EGTA, La3+, and ruthenium red) block Nod factor/mastoparan activity. Taken together, these results are consistent with a Nod factor signal transduction mechanism involving G protein mediation coupled to the activation of both phosphoinositide and Ca2+ second messenger pathways. PMID:9596628

Pingret, JL; Journet, EP; Barker, DG

1998-01-01

305

Structure and mechanism of an intramembrane liponucleotide synthetase central for phospholipid biosynthesis  

PubMed Central

Phospholipids are elemental building-block molecules for biological membranes. Biosynthesis of phosphatidylinositol, phosphatidylglycerol and phosphatidylserine requires a central liponucleotide intermediate named cytidine-diphosphate diacylglycerol (CDP-DAG). The CDP-DAG synthetase (Cds) is an integral membrane enzyme catalysing the formation of CDP-DAG, an essential step for phosphoinositide recycling during signal transduction. Here we report the structure of the Cds from Thermotoga maritima (TmCdsA) at 3.4?Å resolution. TmCdsA forms a homodimer and each monomer contains nine transmembrane helices arranged into a novel fold with three domains. An unusual funnel-shaped cavity penetrates half way into the membrane, allowing the enzyme to simultaneously accept hydrophilic substrate (cytidine 5?-triphosphate (CTP)/deoxy-CTP) from cytoplasm and hydrophobic substrate (phosphatidic acid) from membrane. Located at the bottom of the cavity, a Mg2+-K+ hetero-di-metal centre coordinated by an Asp-Asp dyad serves as the cofactor of TmCdsA. The results suggest a two-metal-ion catalytic mechanism for the Cds-mediated synthesis of CDP-DAG at the membrane–cytoplasm interface. PMID:24968740

Liu, Xiuying; Yin, Yan; Wu, Jinjun; Liu, Zhenfeng

2014-01-01

306

Mechanical Weathering  

NSDL National Science Digital Library

This activity was designed to give students an opportunity to realize that all rocks weather mechanically and each specific rock type has its own particular rate of weathering. Students discover that mechanical weathering is the process of breaking down bedrock into smaller fragments by physical as opposed to chemical means and that rock weathering, although it seems to occur slowly in human terms, is an extremely significant part of the rock cycle. They will learn that weathered rock materials are called sediments and are the structural basis for soils and can also be compacted into sedimentary rock. Students will realize that rock weathering rates vary widely depending on mineral content, texture, rock type, and climate and that differential weathering (varying weathering rates for two or more rock types in physical contact with each other) has given rise to some of the world's most breathtaking scenery.

307

Mechanical clutch  

NASA Technical Reports Server (NTRS)

The present invention is directed to a mechanical clutch which limits transmission of torque to a desired, predetermined maximum torque from a first clutch plate to a second clutch plate. More specifically, the mechanical clutch includes at least one stepper member, preferably three or more evenly spaced stepper members, which transmit the torque from a first clutch plate to a second clutch plate providing a desired maximum torque is not exceeded. However, if the desired maximum torque is exceeded, the stepper member will rotate and move between the clutch plates so that the torque to the second clutch plate does not exceed the desired maximum torque. The desired maximum torque is set by the axial force compressing the stepper member between the clutch plates and when the applied torque to the first clutch plate exceeds the desired torque, the stepper member will rotate between the clutch plates rather than transmit that torque to the second clutch plate.

Withey, Michael M. (Inventor); Lucas-Dean, Rob G. (Inventor)

1995-01-01

308

Mechanical capacitor  

NASA Technical Reports Server (NTRS)

A new energy storage system (the mechanical capacitor), using a spokeless magnetically levitated composite ring rotor, is described and design formulas for sizing the components are presented. This new system is configured around a permanent magnet (flux biased) suspension which has active servo control in the radial direction and passive control in the axial direction. The storage ring is used as a moving rotor and electronic commutation of the stationary armature coils is proposed. There is no mechanical contact with the rotating spokeless ring; therefore, long life and near zero rundown losses are projected. A 7-kW h system is sized to demonstrate feasibility. A literature review of flywheel energy storage systems is also presented and general formulas are developed for comparing rotor geometries.

Kirk, J. A.; Studer, P. A.; Evans, H. E.

1976-01-01

309

Biofluid Mechanics  

Microsoft Academic Search

\\u000a In contrast to the topics discussed in previous chapters, biofluid mechanics is concerned with flows that are influenced by\\u000a flexible biological surfaces. We distinguish between flows past living bodies in air or in water, such as bird flight or the swimming of fish, and internal flows, such as the closed blood circulation of living beings. In the previous millions of

Herbert Oertel

2010-01-01

310

Materials & Mechanics  

NSDL National Science Digital Library

This page presents activities related to materials and mechanics. The lesson plans provided use ideas about building structure to strengthen understanding of larger science topics, the main one being force. Video clips and activities are provided on the topics of bending & torque, tension & compression and shear. Each activity includes a detailed description which will assist teachers in setting up these useful, practical in class lessons.

2013-07-22

311

Mechanical Engineering ME 3720 FLUID MECHANICS  

E-print Network

Mechanical Engineering ME 3720 FLUID MECHANICS Pre-requisite: ME 2330 Co-requisite: ME 3210) to develop an understanding of the physical mechanisms and the mathematical models of fluid mechanics of fluid mechanics problems in engineering practice. The basic principles of fluid mechanics

Panchagnula, Mahesh

312

PI(4,5)P2-Mediated Cell Signaling: Emerging Principles and PTEN as a Paradigm for Regulatory Mechanism  

PubMed Central

PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) is a relatively common anionic lipid that regulates cellular functions by multiple mechanisms. Hydrolysis of PI(4,5)P2 by phospholipase C yields inositol trisphosphate and diacylglycerol. Phosphorylation by phosphoinositide 3-kinase yields PI(3,4,5)P3, which is a potent signal for survival and proliferation. Also, PI(4,5)P2 can bind directly to integral and peripheral membrane proteins. As an example of regulation by PI(4,5)P2, we discuss phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in detail. PTEN is an important tumor suppressor and hydrolyzes PI(3,4,5)P3. PI(4,5)P2 enhances PTEN association with the plasma membrane and activates its phosphatase activity. This is a critical regulatory mechanism, but a detailed description of this process from a structural point of view is lacking. The disordered lipid bilayer environment hinders structural determinations of membrane-bound PTEN. A new method to analyze membrane-bound protein measures neutron reflectivity for proteins bound to tethered phospholipid membranes. These methods allow determination of the orientation and shape of membrane-bound proteins. In combination with molecular dynamics simulations, these studies will provide crucial structural information that can serve as a foundation for our understanding of PTEN regulation in normal and pathological processes. PMID:23775692

Gericke, Arne; Leslie, Nicholas R.; Lösche, Mathias; Ross, Alonzo H.

2013-01-01

313

Molecular Mechanics  

PubMed Central

Molecular Mechanics (MM) force fields are the methods of choice for protein simulations, which are essential in the study of conformational flexibility. Given the importance of protein flexibility in drug binding, MM is involved in most if not all Computational Structure-Based Drug Discovery (CSBDD) projects. This section introduces the reader to the fundamentals of MM, with a special emphasis on how the target data used in the parametrization of force fields determine their strengths and weaknesses. Variations and recent developments such as polarizable force fields are discussed. The section ends with a brief overview of common force fields in CSBDD. PMID:23947650

Vanommeslaeghe, Kenno; Guvench, Olgun; MacKerell, Alexander D.

2014-01-01

314

Information Mechanics  

E-print Network

I hypothesize the unification of action and entropy and suggest an interpretation where this hypothesis is able to reconcile the Bohemian and Copenhagen interpretations of quantum mechanics. I explore the hypothesis implications to the discretization of space; both for a particle and for the vacuum itself. I argue the second law of thermodynamics is the justification for the principle of least action. Similarities with the spin networks of quantum loop gravity are found and the exact simplified area of the network is given. An experiment to test the theory is suggested. I conclude with comments on the non-local interpretation of nature.

John L. Haller Jr

2015-01-29

315

Applied Mechanics  

NSDL National Science Digital Library

This resource, created by National Aerospace Technical Education Center (SpaceTEC), is centered on applied mechanics. The introduction states that it should âÂÂprovide a basis for demonstrating a basic knowledge which will allow you to: interpret a basic drawing, produce a layout/template and fabricate a project.â Topics in this PowerPoint presentation include: machine shop safety, hand tools, drills, measurement, hardware, materials, basic calculators, blueprint reading and the interpretation of technical drawings. The resource features a quiz, bibliography, a design handbook and a virtual machine shop. Overall, this is thorough presentation of this material. It totals nearly one-hundred twenty slides in length. Instructors could use this either as a presentation or simply to enhance existing curriculum.

2009-10-06

316

quantum mechanics  

PubMed Central

-symmetric quantum mechanics (PTQM) has become a hot area of research and investigation. Since its beginnings in 1998, there have been over 1000 published papers and more than 15 international conferences entirely devoted to this research topic. Originally, PTQM was studied at a highly mathematical level and the techniques of complex variables, asymptotics, differential equations and perturbation theory were used to understand the subtleties associated with the analytic continuation of eigenvalue problems. However, as experiments on -symmetric physical systems have been performed, a simple and beautiful physical picture has emerged, and a -symmetric system can be understood as one that has a balanced loss and gain. Furthermore, the phase transition can now be understood intuitively without resorting to sophisticated mathe- matics. Research on PTQM is following two different paths: at a fundamental level, physicists are attempting to understand the underlying mathematical structure of these theories with the long-range objective of applying the techniques of PTQM to understanding some of the outstanding problems in physics today, such as the nature of the Higgs particle, the properties of dark matter, the matter–antimatter asymmetry in the universe, neutrino oscillations and the cosmological constant; at an applied level, new kinds of -synthetic materials are being developed, and the phase transition is being observed in many physical contexts, such as lasers, optical wave guides, microwave cavities, superconducting wires and electronic circuits. The purpose of this Theme Issue is to acquaint the reader with the latest developments in PTQM. The articles in this volume are written in the style of mini-reviews and address diverse areas of the emerging and exciting new area of -symmetric quantum mechanics. PMID:23509390

Bender, Carl M; DeKieviet, Maarten; Klevansky, S. P.

2013-01-01

317

Windmill mechanism  

SciTech Connect

An improved windmill mechanism for adjusting the position of a wind responsive assembly in relation to wind is disclosed. The preferred embodiment comprises a fabric sail mounted on the end of an arm which extends from a power output shaft. A torque sensor is disposed on the arm to sense the torque contribution through that arm to the power output shaft in response to wind acting upon the fabric sail on that arm. The position of the fabric sail is adjusted on the arm by means of a control processor which controls a trim-motor and a magnetic brake. The control processor receives the torque signal provided from the sensor and provides adjustment of the fabric sail in accordance with the torque signal. The control operates to position the sail in a running mode over the semi-circular path segment of rotation of the arm which has a leeward component of motion. It is also effective to position the sail to tacking modes at the beginning and ending of the semi-circular path segment and the flutter mode in the middle of that segment which has a windward component of motion. The control is also effective to automatically adjust for changes in the prevailing wind direction. The sails are supported on flexible mast elements which provide automatic feathering of the sails in response to wind gusts and high wind velocities.

Yang, W. H.

1985-07-23

318

MECHANICAL ENGINEERING The Department of Mechanical Engineer-  

E-print Network

, Solid Mechanics, and Thermal Sciences. Departmental brochures that provide a more detailed description machine, I-DEAS, NX, and AutoCAD. Solid Mechanics The mechanical behavior of advanced materials and applied to solid mechanics problems such as fracture, wave propagation, metal forming, vibration

Ge, Qiaode Jeff

319

S-Adenosylmethionine and Methylthioadenosine Inhibit ?-Catenin Signaling by Multiple Mechanisms in Liver and Colon Cancer.  

PubMed

S-Adenosylmethionine (SAMe), the principal methyl donor that is available as a nutritional supplement, and its metabolite methylthioadenosine (MTA) exert chemopreventive properties against liver and colon cancer in experimental models. Both agents reduced ?-catenin expression on immunohistochemistry in a murine colitis-associated colon cancer model. In this study, we examined the molecular mechanisms involved. SAMe or MTA treatment in the colitis-associated cancer model lowered total ?-catenin protein levels by 47 and 78%, respectively. In an orthotopic liver cancer model, increasing SAMe levels by overexpressing methionine adenosyltransferase 1A also reduced total ?-catenin levels by 68%. In both cases, lower cyclin D1 and c-Myc expression correlated with lower ?-catenin levels. In liver (HepG2) and colon (SW480, HCT116) cancer cells with constitutively active ?-catenin signaling, SAMe and MTA treatment inhibited ?-catenin activity by excluding it from the nuclear compartment. However, in liver (Huh-7) and colon (RKO) cancer cells expressing wild-type Wnt/?-catenin, SAMe and MTA accelerated ?-catenin degradation by a glycogen synthase kinase 3-?-dependent mechanism. Both agents lowered protein kinase B activity, but this was not mediated by inhibiting phosphoinositide 3-kinase. Instead, both agents increased the activity of protein phosphatase 2A, which inactivates protein kinase B. The effect of MTA on lowering ?-catenin is direct and not mediated by its conversion to SAMe, as blocking this conversion had no influence. In conclusion, SAMe and MTA inhibit Wnt/?-catenin signaling in colon and liver cancer cells regardless of whether this pathway is aberrantly induced, making them ideal candidates for chemoprevention and/or chemotherapy in these cancers. PMID:25338671

Li, Tony W H; Peng, Hui; Yang, Heping; Kurniawidjaja, Steven; Panthaki, Parizad; Zheng, Yuhua; Mato, José M; Lu, Shelly C

2015-01-01

320

Department of Mechanical & Aerospace  

E-print Network

Developing Leaders of Innovation Department of Mechanical & Aerospace Engineering #12;Emphasizing, the U.Va. Mechanical and Aerospace Engineering Department prepares students to create the processes of Mechanical and Aerospace Engineering, our mechanical engineering undergraduate students develop skills

Acton, Scott

321

The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo.  

PubMed

We have tested the binding specificities of the pleckstrin homology (PH) domains of protein kinase B (PKB) and GRP1 (general receptor for phosphoinositides-1), expressed as green fluorescent protein (GFP) fusion proteins [PH(PKB)GFP and PH(GRP1)GFP respectively] in HEK 293 cells and Swiss 3T3 cells, using confocal microscopy. Stimulation of HEK 293 cells with insulin caused a small, but sustained, increase in PtdIns(3,4,5)P(3) levels, detected using a radioligand displacement assay, which was mirrored by the translocation of PH(PKB)GFP and PH(GRP1)GFP from the cytosol to the plasma membrane of live, transfected cells. Similar results were obtained using Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) and expressing either PH(PKB)GFP or PH(GRP1)GFP. Biochemical analyses confirmed the accumulation of both PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) in response to PDGF, but only the latter was present at increased levels in Swiss 3T3 cells 30 min after an oxidative stress (1 mM H(2)O(2)). Concomitantly, only PH(PKB)GFP, and not PH(GRP1)GFP, was localized at plasma membranes after 30 min of treatment with H(2)O(2). The fusion proteins appear accurately to report the spatial and temporal distribution of PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) in intact cells. These results establish the lipid selectivity of these PH domains in vivo, and further emphasize the overlapping, but distinct, second messenger roles of PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2). PMID:10585883

Gray, A; Van Der Kaay, J; Downes, C P

1999-12-15

322

The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo.  

PubMed Central

We have tested the binding specificities of the pleckstrin homology (PH) domains of protein kinase B (PKB) and GRP1 (general receptor for phosphoinositides-1), expressed as green fluorescent protein (GFP) fusion proteins [PH(PKB)GFP and PH(GRP1)GFP respectively] in HEK 293 cells and Swiss 3T3 cells, using confocal microscopy. Stimulation of HEK 293 cells with insulin caused a small, but sustained, increase in PtdIns(3,4,5)P(3) levels, detected using a radioligand displacement assay, which was mirrored by the translocation of PH(PKB)GFP and PH(GRP1)GFP from the cytosol to the plasma membrane of live, transfected cells. Similar results were obtained using Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) and expressing either PH(PKB)GFP or PH(GRP1)GFP. Biochemical analyses confirmed the accumulation of both PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) in response to PDGF, but only the latter was present at increased levels in Swiss 3T3 cells 30 min after an oxidative stress (1 mM H(2)O(2)). Concomitantly, only PH(PKB)GFP, and not PH(GRP1)GFP, was localized at plasma membranes after 30 min of treatment with H(2)O(2). The fusion proteins appear accurately to report the spatial and temporal distribution of PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) in intact cells. These results establish the lipid selectivity of these PH domains in vivo, and further emphasize the overlapping, but distinct, second messenger roles of PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2). PMID:10585883

Gray, A; Van Der Kaay, J; Downes, C P

1999-01-01

323

MECHANICAL ENGINEERING AND APPLIED MECHANICS (EG) {MEAM}  

E-print Network

; II. Statics and Structures; and III. Kinematics and Dynamics. Topics include: vector analysis and energy, and dynamics of particles. Case studies will be introduced, and the role of Newtonian mechanicsMECHANICAL ENGINEERING AND APPLIED MECHANICS (EG) {MEAM} 091. Shop Tr: Manual Milling. (C

Fang-Yen, Christopher

324

Phospholipase A{sub 2} is involved in the mechanism of activation of neutrophils by polychlorinated biphenyls  

SciTech Connect

Aroclor 1242, a mixture of polychlorinated biphenyls (PCBs), activates neutrophils to produce superoxide anion (O{sub 2}{sup {minus}}) by a mechanism that involves phospholipase C-dependent hydrolysis of membrane phosphoinositides; however, subsequent signal transduction mechanisms are unknown. This study determines whether phospholipase A{sub 2}-dependent release of arachidonic acid is involved in PCB-induced O{sub 2}{sup {minus}} production. O{sub 2}{sup {minus}} production was measured in vitro in glycogen-elicited, rat neutrophils in the presence and absence of the inhibitors of phospholipase A{sub 2}: quinacrine, 4-bromophenacyl bromide (BPB), and manoalide. All three agents significantly decreased the amount of O{sub 2}{sup {minus}} detected during stimulation of neutrophils with Aroclor 1242. Similar inhibition occurred when neutrophils were activated with the classical stimuli, formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate. The effects of BPB and manoalide were not a result of cytotoxicity or other nonspecific effects. Significant release of {sup 3}H-arachidonic acid preceded O{sub 2}{sup {minus}} production in neutrophils stimulated with Aroclor 1242 or fMLP. Manoalide, at a concentration that abolished O{sub 2}{sup {minus}} production, also inhibited the release of {sup 3}H-arachidonate. Aspirin, zileuton, or WEB 2086 did not affect Aroclor 1242-induced O{sub 2}{sup {minus}} production, suggesting that eicosanoids and platelet-activating factor are not needed for neutrophil activation by PCBs. Activation of phos-pholipase A{sub 2} and O{sub 2}{sup {minus}} production do not appear to involve the Ah receptor. These data suggest that Aroclor 1242 stimulates neutrophils to produce O{sub 2}{sup {minus}} by a mechanism that involves phospholipase A{sub 2}-dependent release of arachiodonic acid. 49 refs., 6 figs., 2 tabs.

Tithof, P.K.; Schiamberg, E.; Ganey, P.E. [Univ. of Michigan, Ann Arbor, MI (United States); Peters-Golden, M. [Michigan State Univ., East Lansing, MI (United States)

1996-01-01

325

Mechanical & Aerospace Engineering  

E-print Network

Mechanical & Aerospace Engineering Strategic Plan 2014-2018 College of Engineering, Architecture & Technology #12;Mechanical and Aerospace Engineering at Oklahoma State University was organized as Mechanical, the School was reorganized as Mechanical and Aerospace Engineering. Since then, the mechanical-aerospace bond

326

Mechanical systems: A compilation  

NASA Technical Reports Server (NTRS)

A compilation of several mechanized systems is presented. The articles are contained in three sections: robotics, industrial mechanical systems, including several on linear and rotary systems and lastly mechanical control systems, such as brakes and clutches.

1975-01-01

327

Mechanical & Industrial Engineering  

E-print Network

Mechanical & Industrial Engineering 1 Welcome MIE Industrial Advisory Board October 15, 2010 #12;Mechanical & Industrial Engineering 2 MIE Dorothy Adams Undergraduate/Graduate Secretary David Schmidt Associate Professor & Graduate Program Director #12;Mechanical & Industrial Engineering 3 MIE James Rinderle

Mountziaris, T. J.

328

Molecular Mechanism of Membrane Binding of the GRP1 PH Domain  

PubMed Central

The pleckstrin homology (PH) domain of the general receptor of phosphoinositides 1 (GRP1) protein selectively binds to a rare signaling phospholipid, phosphatidylinositol (3,4,5)-trisphosphate (PIP3), in the membrane. The specific PIP3 lipid docking of GRP1 PH domain is essential to protein cellular function and is believed to occur in a stepwise process, electrostatic-driven membrane association followed by the specific PIP3 binding. By a combination of all-atom molecular dynamics (MD) simulations, coarse-grained analysis, electron paramagnetic resonance (EPR) membrane docking geometry, and fluorescence resonance energy transfer (FRET) kinetic studies, we have investigated the search and bind process in the GRP1 PH domain at the molecular scale. We simulated the two membrane binding states of the GRP1 PH domain in the PIP3 search process, before and after the GRP1 PH domain docks with the PIP3 lipid. Our results suggest that the background anionic phosphatidylserine lipids, which constitute around one-fifth of the membrane by composition, play a critical role in the initial stages of recruiting protein to the membrane surface through non-specific electrostatic interactions. Our data also reveal a previously unseen transient membrane association mechanism that is proposed to enable a two-dimensional “hopping” search of the membrane surface for the rare PIP3 target lipid. We further modeled the PIP3-bound membrane–protein system using the EPR membrane docking structure for the MD simulations, quantitatively validating the EPR membrane docking structure and augmenting our understanding of the binding interface with atomic-level detail. Several observations and hypotheses reached from our MD simulations are also supported by experimental kinetic studies. PMID:23747485

Lai, Chun-Liang; Srivastava, Anand; Pilling, Carissa; Chase, Anna R.; Falke, Joseph J.; Voth, Gregory A.

2014-01-01

329

Molecular mechanism of apoptosis induction in skin cancer cells by the centipedegrass extract  

PubMed Central

Background Centipedegrass extract (CGE) is mainly composed of maysin and its derivatives, which are recognized internationally as natural compounds. Compared to other flavonoids, maysin has a unique structure in that mannose is bound to the flavonoid backbone. CGE exhibits some biological properties in that it can function as an anti-oxidant, anti-inflammatory, anti-adipogenic, and insecticidal. Whether CGE has other biological functions, such as anti-cancer activity, is unknown. Methods B16F1 (mouse) and SKMEL-5 (human) cells were treated with CGE, and their subsequent survival was determined using MTT assay. We performed a cell cycle analysis using propidium iodide (PI), and detected apoptosis using double staining with annexin V-FITC/PI. In addition, we examined mitochondrial membrane potentials using flow cytometry, as well as signaling mechanisms with an immunoblotting analysis. Results CGE inhibited skin cancer cell growth by arresting the cell cycle in the G2/M phase, and increased both early and late apoptotic cell populations without affecting normal cells. Furthermore, we observed mitochondrial transmembrane depolarization, increased cytochrome-c release, caspase-3 and caspase-7 activation, and increased poly ADP-ribose polymerase degradation. CGE also downregulated activation of p-AKT, p-glycogen synthase kinase-3? (GSK-3?), and p-BAD in a time-dependent manner. LY294002 inhibition of phosphoinositide 3-kinase (PI3K) significantly sensitized skin cancer cells, which led to an increase in CGE-induced apoptosis. Conclusions CGE controlled skin cancer cell growth by inhibiting the PI3K/AKT/GSK-3? signaling pathway and activating the effector caspases. This study is the first to demonstrate anti-cancer properties for CGE, and that CGE may be an effective therapeutic agent for treating skin cancer. PMID:24325618

2013-01-01

330

Defense Mechanisms: A Bibliography.  

ERIC Educational Resources Information Center

This bibliography includes studies of defense mechanisms, in general, and studies of multiple mechanisms. Defense mechanisms, briefly and simply defined, are the unconscious ego defendants against unpleasure, threat, or anxiety. Sigmund Freud deserves the clinical credit for studying many mechanisms and introducing them in professional literature.…

Pedrini, D. T.; Pedrini, Bonnie C.

331

Mechanical & Industrial Engineering  

E-print Network

Mechanical & Industrial Engineering 1 Welcome MIE Industrial Advisory Board May 5th, 2011 #12;Mechanical & Industrial Engineering 2 IAB 2010-2011 · David K. Anderson ­ Alden Research Laboratory, Inc went on for three weeks Mechanical & Industrial Engineering 6 #12;Reza Shahbazian Yassar Mechanical

Mountziaris, T. J.

332

A PtdIns4,5P2-regulated nuclear poly(A) polymerase controls expression of select mRNAs  

Microsoft Academic Search

Phosphoinositides are a family of lipid signalling molecules that regulate many cellular functions in eukaryotes. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2), the central component in the phosphoinositide signalling circuitry, is generated primarily by type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIalpha, PIPKIbeta and PIPKIgamma). In addition to functions in the cytosol, phosphoinositides are present in the nucleus, where they modulate several functions; however, the mechanism by

David L. Mellman; Michael L. Gonzales; Chunhua Song; Christy A. Barlow; Ping Wang; Christina Kendziorski; Richard A. Anderson

2008-01-01

333

Internal pipe attachment mechanism  

DOEpatents

An attachment mechanism is described for repairing or extending fluid carrying pipes, casings, conduits, etc. utilizing one-way motion of spring tempered fingers to provide a mechanical connection between the attachment mechanism and the pipe. The spring tempered fingers flex to permit insertion into a pipe to a desired insertion depth. The mechanical connection is accomplished by reversing the insertion motion and the mechanical leverage in the fingers forces them outwardly against the inner wall of the pipe. A seal is generated by crushing a sealing assembly by the action of setting the mechanical connection. 6 figures.

Bast, R.M.; Chesnut, D.A.; Henning, C.D.; Lennon, J.P.; Pastrnak, J.W.; Smith, J.A.

1994-12-13

334

External Resource: Mechanical Weathering  

NSDL National Science Digital Library

A student activity with teacher's sheet, to give the students an opportunity to realize that all rocks weather mechanically and each specific rock type has its own particular rate of weathering. Mechanical weathering is the process of breaking down bedroc

1900-01-01

335

Mechanical and Aerospace Engineering  

E-print Network

Mechanical and Aerospace Engineering seminar Plasma Actuators for Aerodynamic Flow Control Abstract Plasma actuators have become very popular in aerodynamic flow control research over the last) or thermal (Joule heating) although there is some unavoidable overlap between these mechanisms. Interest

336

Ninteenth Aerospace Mechanisms Symposium  

NASA Technical Reports Server (NTRS)

The proceedings of the 19th Aerospace Mechanisms Symposium are reported. Technological areas covered include space lubrication, bearings, aerodynamic devices, spacecraft/Shuttle latches, deployment, positioning, and pointing. Devices for spacecraft docking and manipulator and teleoperator mechanisms are also described.

1985-01-01

337

Mechanical Properties of Polymers.  

ERIC Educational Resources Information Center

Mechanical properties (stress-strain relationships) of polymers are reviewed, taking into account both time and temperature factors. Topics include modulus-temperature behavior of polymers, time dependence, time-temperature correspondence, and mechanical models. (JN)

Aklonis, J. J.

1981-01-01

338

Programmable mechanical metamaterials.  

PubMed

We create mechanical metamaterials whose response to uniaxial compression can be programmed by lateral confinement, allowing monotonic, nonmonotonic, and hysteretic behavior. These functionalities arise from a broken rotational symmetry which causes highly nonlinear coupling of deformations along the two primary axes of these metamaterials. We introduce a soft mechanism model which captures the programmable mechanics, and outline a general design strategy for confined mechanical metamaterials. Finally, we show how inhomogeneous confinement can be explored to create multistability and giant hysteresis. PMID:25379923

Florijn, Bastiaan; Coulais, Corentin; van Hecke, Martin

2014-10-24

339

Programmable Mechanical Metamaterials  

E-print Network

We create mechanical metamaterials whose response to uniaxial compression can be programmed by lateral confinement, allowing monotonic, non-monotonic and hysteretic behavior. These functionalities arise from a broken rotational symmetry which causes highly nonlinear coupling of deformations along the two primary axes of these metamaterials. We introduce a soft mechanism model which captures the programmable mechanics, and outline a general design strategy for confined mechanical metamaterials. Finally, we show how inhomogeneous confinement can be explored to create multi stability and giant hysteresis.

Bastiaan Florijn; Corentin Coulais; Martin van Hecke

2014-07-17

340

UNSATURATED SOIL MECHANICS IMPLEMENTATION  

E-print Network

UNSATURATED SOIL MECHANICS IMPLEMENTATION DURING PAVEMENT CONSTRUCTION QUALITY ASSURANCE Mn !! Performance Based Construction QA !! Unsaturated Soil Mechanics !! What We've Learned !! Next Steps #12.6-6.0 5 - 7 19 0.8 5 7 - 9 24 1.1 4 9 - 11 28 1.2 4 #12;Unsaturated Soil Mechanics #12;Fundamentals

Minnesota, University of

341

Missing Mechanism Information  

ERIC Educational Resources Information Center

The first recommendation Kazdin made for advancing the psychotherapy research knowledge base, improving patient care, and reducing the gulf between research and practice was to study the mechanisms of therapeutic change. He noted, "The study of mechanisms of change has received the least attention even though understanding mechanisms may well be…

Tryon, Warren W.

2009-01-01

342

Hybrid Mechanical Systems  

E-print Network

We discuss hybrid systems in which a mechanical oscillator is coupled to another (microscopic) quantum system, such as trapped atoms or ions, solid-state spin qubits, or superconducting devices. We summarize and compare different coupling schemes and describe first experimental implementations. Hybrid mechanical systems enable new approaches to quantum control of mechanical objects, precision sensing, and quantum information processing.

Philipp Treutlein; Claudiu Genes; Klemens Hammerer; Martino Poggio; Peter Rabl

2015-01-06

343

Fractional quantum mechanics  

Microsoft Academic Search

A path integral approach to quantum physics has been developed. Fractional path integrals over the paths of the Lévy flights are defined. It is shown that if the fractality of the Brownian trajectories leads to standard quantum and statistical mechanics, then the fractality of the Lévy paths leads to fractional quantum mechanics and fractional statistical mechanics. The fractional quantum and

Nikolai Laskin

2000-01-01

344

Giovanni Gallavotti STATISTICAL MECHANICS  

E-print Network

Giovanni Gallavotti STATISTICAL MECHANICS Short Treatise Roma 1999 #12; . #12; Short treatise of Statistical Mechanics Giovanni Gallavotti Dipartimento di Fisica Universitâ??a di Roma La Sapienza 00185 Roma that started with my involvement as Coordinator of the Statistical Mechanics section of the Italian Encyclo

Roma "La Sapienza", Università di

345

Statistical Mechanics its applications  

E-print Network

Statistical Mechanics and its applications Dr A. Alavi Part II Chemistry 2007-2008 1 #12;I. INTRODUCTION Statistical Mechanics is concerned with how to describe the behaviour of systems containing large, and how this is done, as well as its theoretical justification, is the subject of Statistical Mechanics. A

Alavi, Ali

346

Giovanni Gallavotti STATISTICAL MECHANICS  

E-print Network

Giovanni Gallavotti STATISTICAL MECHANICS Short Treatise Roma 1999 #12;. #12;Short treatise of Statistical Mechanics Giovanni Gallavotti Dipartimento di Fisica Universit`a di Roma La Sapienza 00185 Roma that started with my involvement as Coordinator of the Statistical Mechanics section of the Italian Encyclo

Roma "La Sapienza", Università di

347

Department of Mechanical Engineering  

E-print Network

Department of Mechanical Engineering Undergraduate Program 2006-2007 Northern Illinois University DeKalb, IL #12;DEPARTMENT OF MECHANICAL ENGINEERING NORTHERN ILLINOIS UNIVERSITY Web: http://www.ceet.niu.edu/depts/me/ Phone: 815-753-9979 The mission of the Department of Mechanical Engineering is to provide an up

Karonis, Nicholas T.

348

Department of Mechanical Engineering  

E-print Network

Department of Mechanical Engineering Undergraduate Program 2007-2008 Northern Illinois University DeKalb, IL #12;DEPARTMENT OF MECHANICAL ENGINEERING NORTHERN ILLINOIS UNIVERSITY Web: http://www.ceet.niu.edu/depts/me/ Phone: 815-753-9979 The mission of the Department of Mechanical Engineering is to provide an up

Karonis, Nicholas T.

349

Mechanical & Industrial Engineering  

E-print Network

Mechanical & Industrial Engineering Mario A. Rotea Professor and Department Head #12;2Mechanical & Industrial Engineering Outline · Undergraduate Degree Programs · Graduate Degree Programs · The Faculty · The Research · Summary #12;3Mechanical & Industrial Engineering Undergraduate Programs ­ BSME & BSIE 0 20 40 60

Mountziaris, T. J.

350

Basic Engineer Equipment Mechanic.  

ERIC Educational Resources Information Center

This student guide, one of a series of correspondence training courses designed to improve the job performance of members of the Marine Corps, deals with the skills needed by basic engineer equipment mechanics. Addressed in the four individual units of the course are the following topics: mechanics and their tools (mechanics, hand tools, and power…

Marine Corps Inst., Washington, DC.

351

Inhibition of Rac activation as a mechanism for negative regulation of actin cytoskeletal reorganization and cell motility by cAMP  

PubMed Central

cAMP has been found to play a role in mediating the negative regulation of cell motility, although its underlying molecular mechanism remains poorly understood. By using CHO (Chinese-hamster ovary) cells that express the EP2 subtype of PGE2 (prostaglandin E2) receptors, we provide evidence that an increase in cellular cAMP content leads to inhibition of cellular Rac activity, which serves as a mechanism for this negative regulation. In CHO cells expressing EP2, but not in vector control cells, PGE2 dose-dependently inhibited chemotaxis towards IGF-I (insulin-like growth factor-I), which is a Rac-dependent process, with the maximal 75% inhibition observed at 10?8 M PGE2. EP2 stimulation failed to inhibit tyrosine phosphorylation either of IGF-I receptor or IRS-1 (insulin receptor substrate-1), or activation of phosphoinositide 3-kinase or Akt in response to IGF-I, but potently and dose-dependently inhibited IGF-I-induced activation of cellular Rac activity and membrane ruffling. However, PGE2 failed to inhibit Val12-Rac-induced membrane ruffling. Similar to the case of CHO cells, PGE2 inhibited PDGF (platelet-derived growth factor)-induced Rac activation and chemotaxis in vascular smooth muscle cells endogenously expressing EP2. The inhibitory effects of PGE2 on IGF-I-induced chemotaxis, membrane ruffling and Rac activation were faithfully reproduced by a low concentration of forskolin, which induced a comparable extent of cAMP elevation as with 10?8 M PGE2, and were potentiated by isobutylmethylxanthine. The protein kinase A inhibitor Rp isomer of adenosine 3?,5?-cyclic monophosphorothioate reduced PGE2 inhibition of Rac activation and chemotaxis. These results indicate that EP2 mediates Rac inhibition through a mechanism involving cAMP and protein kinase A, thereby inhibiting membrane ruffling and chemotaxis. PMID:15377280

2004-01-01

352

Mediation and mechanism.  

PubMed

The concepts of mediation and mechanism are contrasted and logical implications holding between theses two concepts are described. The concept of mediation can be formalized using counterfactual definitions of indirect effects; the concept of mechanism can be formalized within the sufficient cause framework. It is shown that both concepts can be illustrated using a single causal diagram. It is also shown that mediation implies mechanism but mechanism need not imply mediation. Discussion is given regarding how the distinction between "statistical causality" and "mechanistic causality" is blurred by recent work in causal inference concerning methods for testing for mediation and mechanism. PMID:19330454

VanderWeele, Tyler J

2009-01-01

353

Catalytic Domain of Phosphoinositide-specific Phospholipase C (PLC)  

E-print Network

Biology, Chester Beatty Laboratories, Fulham Road, London SW3 6JB, the Medical Research Council Laboratory residues (His311 , Asn312 , Glu341 , Asp343 , His356 , Glu390 , Lys438 , Lys440 , Ser522 , Arg549 , and Tyr , and Asp343 resulted in a shift toward higher calcium concentrations required for PIP2 hydrolysis

Williams, Roger L.

354

JournalofCellScience ERK and phosphoinositide 3-kinase temporally  

E-print Network

to draw the epithelium and connective tissue forward together in a `purse-string-like' fashion (Martin recovery of tissue integrity and homeostasis, which is vital for survival. Tissue movement in embryonic in a complex tissue setting, namely embryonic wound healing. Key words: ERK, PI3K, Wound healing, Rho GTPases

Amaya, Enrique

355

Role of phospholipase C? in physiological phosphoinositide signaling networks  

PubMed Central

Receptor-initiated phospholipase C activation and generation of IP3 and DAG are important common triggers for a diversity of signal transduction processes in many cell types. Contributing to this diversity is the existence and differential cellular and subcellular distribution of distinct phospholipase C isoforms with distinct regulatory prop- erties. The recently identified PLC? enzyme is an isoform that is uniquely regulated by multiple upstream signals including ras-family GTP binding proteins as well as heterotrimeric G-proteins. In this review we will consider the well documented biochemical regulation of this isoform in the context of cell and whole animal physiology and in the context of other G protein-regulated PLC isoforms. These studies together reveal a surprisingly wide range of unexpected functions for PLC? in cellular signaling, physiology and disease. PMID:22286105

Smrcka, Alan V.; Brown, Joan Heller; Holz, George G.

2012-01-01

356

Phosphoinositides influence pathogen surfing: EPEC rights the SHIP.  

PubMed

Tir, a translocated effector protein from enteropathogenic E. coli (EPEC), contains two phosphotyrosines that initiate cellular signaling cascades, leading to localized actin polymerization into pedestals. A recent study now shows that two additional tyrosines within Tir recruit the inositol phosphatase SHIP2 to generate a PI(3,4)P2-enriched membrane platform that stabilizes pedestal assembly. PMID:20114020

Campellone, Kenneth G

2010-01-21

357

Mechanical Seal Assembly  

SciTech Connect

An improved mechanical seal assembly is provided for sealing rotating shafts with respect to their shaft housings, wherein the rotating shafts are subject to substantial axial vibrations. The mechanical seal assembly generally includes a rotating sealing ring fixed to the shaft, a non-rotating sealing ring adjacent to and in close contact with the rotating sealing ring for forming an annular seal about the shaft, and a mechanical diode element that applies a biasing force to the non-rotating sealing ring by means of hemispherical joint. The alignment of the mechanical diode with respect to the sealing rings is maintained by a series of linear bearings positioned axially along a desired length of the mechanical diode. Alternative embodiments include mechanical or hydraulic amplification components for amplifying axial displacement of the non-rotating sealing ring and transferring it to the mechanical diode.

Kotlyar, Oleg M.

1999-06-18

358

Mechanical seal assembly  

DOEpatents

An improved mechanical seal assembly is provided for sealing rotating shafts with respect to their shaft housings, wherein the rotating shafts are subject to substantial axial vibrations. The mechanical seal assembly generally includes a rotating sealing ring fixed to the shaft, a non-rotating sealing ring adjacent to and in close contact with the rotating sealing ring for forming an annular seal about the shaft, and a mechanical diode element that applies a biasing force to the non-rotating sealing ring by means of hemispherical joint. The alignment of the mechanical diode with respect to the sealing rings is maintained by a series of linear bearings positioned axially along a desired length of the mechanical diode. Alternative embodiments include mechanical or hydraulic amplification components for amplifying axial displacement of the non-rotating sealing ring and transfering it to the mechanical diode.

Kotlyar, Oleg M. (Salt Lake City, UT)

2002-01-01

359

Mechanical seal assembly  

DOEpatents

An improved mechanical seal assembly is provided for sealing rotating shafts with respect to their shaft housings, wherein the rotating shafts are subject to substantial axial vibrations. The mechanical seal assembly generally includes a rotating sealing ring fixed to the shaft, a non-rotating sealing ring adjacent to and in close contact with the rotating sealing ring for forming an annular seal about the shaft, and a mechanical diode element that applies a biasing force to the non-rotating sealing ring by means of hemispherical joint. The alignment of the mechanical diode with respect to the sealing rings is maintained by a series of linear bearings positioned axially along a desired length of the mechanical diode. Alternative embodiments include mechanical or hydraulic amplification components for amplifying axial displacement of the non-rotating sealing ring and transferring it to the mechanical diode.

Kotlyar, Oleg M. (Salt Lake City, UT)

2001-01-01

360

The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site.  

PubMed

Forkhead in rhabdomyosarcoma (FKHR) is a transcription factor that has been implicated in the control of gene expression by insulin, as well as the regulation of apoptosis by survival factors. These signals trigger the protein kinase B (PKB)-catalysed phosphorylation of FKHR at three residues (Thr(24), Ser(256) and Ser(319)) by a phosphoinositide 3-kinase-dependent pathway that results in the nuclear exit and inactivation of this transcription factor. Here, we have identified a conserved residue (Ser(329)) as a novel in vivo phosphorylation site on FKHR. Ser(329) phosphorylation also decreases the ability of FKHR to stimulate gene transactivation and reduces the proportion of FKHR present in the nucleus. However, unlike the residues targetted by PKB, Ser(329) is phosphorylated in unstimulated HEK-293 cells, and phosphorylation is not increased by stimulation with insulin-like growth factor-1 or by transfection with 3-phosphoinositide-dependent protein kinase-1. We have also purified a protein kinase to near homogeneity from rabbit skeletal muscle that phosphorylates FKHR at Ser(329) specifically and identified it as DYRK1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A). We find that FKHR and DYRK1A co-localize in discrete regions of the nucleus and can be co-immunoprecipitated from cell extracts. These experiments suggest that DYRK1A may phosphorylate FKHR at Ser(329) in vivo. PMID:11311120

Woods, Y L; Rena, G; Morrice, N; Barthel, A; Becker, W; Guo, S; Unterman, T G; Cohen, P

2001-05-01

361

Regulation of osteoclasts by membrane-derived lipid mediators.  

PubMed

Osteoclasts are bone-resorbing cells of monocytic origin. An imbalance between bone formation and resorption can lead to osteoporosis or osteopetrosis. Osteoclastogenesis is triggered by RANKL- and IP3-induced Ca(2+) influx followed by activation of NFATc1, a master transcription factor for osteoclastogenic gene regulation. During differentiation, osteoclasts undergo cytoskeletal remodeling to migrate and attach to the bone surface. Simultaneously, they fuse with each other to form multinucleated cells. These processes require PI3-kinase-dependent cytoskeletal protein activation to initiate cytoskeletal remodeling, resulting in the formation of circumferential podosomes and fusion-competent protrusions. In multinucleated osteoclasts, circumferential podosomes mature into stabilized actin rings, which enables the formation of a ruffled border where intensive membrane trafficking is executed. Membrane lipids, especially phosphoinositides, are key signaling molecules that regulate osteoclast morphology and act as second messengers and docking sites for multiple important effectors. We examine the critical roles of phosphoinositides in the signaling cascades that regulate osteoclast functions. PMID:23296124

Oikawa, Tsukasa; Kuroda, Yukiko; Matsuo, Koichi

2013-09-01

362

The mechanism of fracture  

SciTech Connect

This book presents the papers given at a conference on the fracture mechanics of metals. Topics considered at the conference included microcrack mechanics, pressurized thermal shock behavior of LWR pressure vessels, stress intensity factors, submerged arc welding, weldments in power plants, pipeline weld quality, natural gas tanks, cast iron for spent nuclear fuel shipping casks, pipe ruptures, physical radiation effects, pressure tubes, hydrogen embrittlement, critical flaw size curves, and the fracture mechanics of steels in turbines of power stations.

Goel, V.S.

1985-01-01

363

CRCHD - CPRCHD Mechanisms  

Cancer.gov

The U54 mechanism supports the activities of a full, competitive, comprehensive partnership program known as the CPRCHD. The U54 mechanism is designed for institutions targeting underserved populations and Cancer Centers that have already conducted considerable prior planning and evaluation through the U56 mechanism and are ready to implement or have implemented their partnerships in inter-institutional cancer research projects, cancer training, outreach, and education.

364

CRCHD - PRCHD Mechanisms  

Cancer.gov

The U54 mechanism supports the activities of a full, competitive, comprehensive partnership program known as the PACHE. The U54 mechanism is designed for institutions targeting underserved populations and Cancer Centers that have already conducted considerable prior planning and evaluation through the U56 mechanism and are ready to implement or have implemented their partnerships in inter-institutional cancer research projects, cancer training, outreach, and education.

365

CRCHD - PRCHD Mechanisms  

Cancer.gov

The U54 mechanism supports the activities of a full, competitive, comprehensive partnership program known as the PRCHD. The U54 mechanism is designed for institutions targeting underserved populations and Cancer Centers that have already conducted considerable prior planning and evaluation through the U56 mechanism and are ready to implement or have implemented their partnerships in inter-institutional cancer research projects, cancer training, outreach, and education.

366

Space Mechanisms Technology Workshop  

NASA Technical Reports Server (NTRS)

The Mechanical Components Branch at NASA Glenn Research Center hosted a workshop on Tuesday, May 14, 2002, to discuss space mechanisms technology. The theme for this workshop was 'Working in the Cold,' a focus on space mechanisms that must operate at low temperatures. We define 'cold' as below -60C (210 K), such as would be found near the equator of Mars. However, we are also concerned with much colder temperatures such as in permanently dark craters of the Moon (about 40 K).

Oswald, Fred B. (Editor)

2002-01-01

367

Integrated Mechanical & Electrical Engineering (IMEE)  

E-print Network

mechanics Integrated control system design Mathematics I Circuit theory Solid mechanics I Thermodynamics Design materials and manufacturing I Mathematics II Digital electronics Solid mechanics II Design

Burton, Geoffrey R.

368

More on Atonic Mechanics  

NASA Astrophysics Data System (ADS)

We have shown that crystal based experiments, such as those of Davisson and Germer, do not empirically verify de Broglie's matter-wave hypothesis. We named this theory Atonic Mechanics. This mechanics has also been used to accurately calculate the hundreds of helium atom energy levels tabulated by NIST. We have joined Atonic Mechanics with Einstein's General Relativity. Now we show how fractional values of h-bar arise for the angular momentum of the helium atom in this theory. We now also discuss energy and momentum in Atonic Mechanics.

Phillips, Alfred

2007-03-01

369

Boston University Department of Mechanical Engineering ME 303 Fluid Mechanics  

E-print Network

Boston University Department of Mechanical Engineering ME 303 ­ Fluid Mechanics Fall 2011 Class: EK301 Engineering Mechanics Course Textbook: Fundamentals of Fluid Mechanics, 6th ed., Munson B. Reference Texts: Fluid Mechanics, Landau and Lifshitz, Vol. 6 Fluid Mechanics, Y. Cengel and J. Cimbala

370

Structural Mechanics & Solid Mechanics A finite element toolbox to MATLAB  

E-print Network

Structural Mechanics & Solid Mechanics Department of Mechanics and Materials CALFEM A finite 1999 c Copyright 1992­99 by the Division of Structural Mechanics and the Department of Solid Mechanics The Department of Solid Mechanics Lund University PO Box 118 S­221 00 Lund SWEDEN Phone: +46 46 222 0000 Fax: +46

Ehrhardt, Matthias

371

Cell Mechanics and Mechanosensitivity 1 Cell Mechanics and  

E-print Network

) Review of Fluid mechanics 6) Review of Statistical Mechanics 7) Methods of Cell Mechanics. . Requirements Some background in basic cell and molecular biology and in introductory solid or fluid mechanicsCell Mechanics and Mechanosensitivity 1 Fall 2013 MW 12-2:00 BE535 Cell Mechanics

Vajda, Sandor

372

Hippocampal protein expression is differentially affected by chronic paroxetine treatment in adolescent and adult rats: a possible mechanism of “paradoxical” antidepressant responses in young persons  

PubMed Central

Selective serotonin reuptake inhibitors (SSRIs) are commonly recognized as the pharmacological treatment of choice for patients with depressive disorders, yet their use in adolescent populations has come under scrutiny following reports of minimal efficacy and an increased risk of suicidal ideation and behavior in this age group. The biological mechanisms underlying these effects are largely unknown. Accordingly, the current study examined changes in hippocampal protein expression following chronic administration of paroxetine in drinking water (target dose = 10 mg/kg for 22 days) to adult and adolescent rats. Results indicated age-specific changes in protein expression, with paroxetine significantly altering expression of 8 proteins in adolescents only and 10 proteins solely in adults. A further 12 proteins were significantly altered in both adolescents and adults. In adults, protein changes were generally suggestive of a neurotrophic and neuroprotective effect of paroxetine, with significant downregulation of apoptotic proteins Galectin 7 and Cathepsin B, and upregulation of the neurotrophic factor Neurogenin 1 and the antioxidant proteins Aldose reductase and Carbonyl reductase 3. Phosphodiesterase 10A, a signaling protein associated with major depressive disorder, was also downregulated (-6.5-fold) in adult rats. Adolescent rats failed to show the neurotrophic and neuroprotective effects observed in adults, instead displaying upregulation of the proapoptotic protein BH3-interacting domain death agonist (4.3-fold). Adolescent protein expression profiles also suggested impaired phosphoinositide signaling (Protein kinase C: -3.1-fold) and altered neurotransmitter transport and release (Syntaxin 7: 5.7-fold; Dynamin 1: -6.9-fold). The results of the present study provide clues as to possible mechanisms underlying the atypical response of human adolescents to paroxetine treatment. PMID:23847536

Karanges, Emily A.; Kashem, Mohammed A.; Sarker, Ranjana; Ahmed, Eakhlas U.; Ahmed, Selina; Van Nieuwenhuijzen, Petra S.; Kemp, Andrew H.; McGregor, Iain S.

2013-01-01

373

Bacteriophage resistance mechanisms  

Microsoft Academic Search

Phages are now acknowledged as the most abundant microorganisms on the planet and are also possibly the most diversified. This diversity is mostly driven by their dynamic adaptation when facing selective pressure such as phage resistance mechanisms, which are widespread in bacterial hosts. When infecting bacterial cells, phages face a range of antiviral mechanisms, and they have evolved multiple tactics

Simon J. Labrie; Julie E. Samson; Sylvain Moineau

2010-01-01

374

INJECTION WELL MECHANICAL INTEGRITY  

EPA Science Inventory

EPA's underground injection control regulations require that all injection wells demonstrate mechanical integrity. The regulations state that an injection well has mechanical integrity if (1) there is no significant leak in the casing, tubing or packer and (2) there is no signifi...

375

Engineer Equipment Mechanic.  

ERIC Educational Resources Information Center

Developed as part of the Marine Corps Institute (MCI) correspondence training program, this course on engineer equipment mechanics is designed to advance the professional competence of privates through sergeants as equipment mechanics, Military Occupation Specialty 1341, and is adaptable for nonmilitary instruction. Introductory materials include…

Marine Corps Inst., Washington, DC.

376

Neural Mechanisms of Cardioprotection  

PubMed Central

This review highlights the importance of neural mechanisms capable of protecting the heart against lethal ischemia/reperfusion injury. Increased parasympathetic (vagal) activity limits myocardial infarction, and recent data suggest that activation of autonomic reflex pathways contributes to powerful innate mechanisms of cardioprotection underlying the remote ischemic conditioning phenomena. PMID:24583769

Gourine, Alexander V.

2014-01-01

377

Advanced Visual Quantum Mechanics  

NSDL National Science Digital Library

This page provides links to a range of teaching materials for use in an upper-level undergraduate quantum mechanics course. These are developed from some of the concepts of the Visual Quantum Mechanics course for high school and introductory college classes. Materials inlcude tutorial activities in concepts of energy diagrams, probability, and wavefunctions, and some computer activities.

Axmann, Wally; Group, Kansas S.

2004-04-04

378

Mechanisms (session summary)  

Microsoft Academic Search

Takuya Katayama lead the discussion on Mechanisms for Software Process Description. He classified software process views as reflected in the position papers into functional, behavioral and enactional, described some generally desirable properties of software process mechanisms, and listed those projects that seemed to have obtained non-toy experience.He made a plea for each of these projects to answer a questionnaire that,

Gail E. Kaiser

1990-01-01

379

Cytokinesis Mechanics and Mechanosensing  

PubMed Central

Cytokinesis shape change occurs through the interfacing of three modules, cell mechanics, myosin II-mediated contractile stress generation and sensing, and a control system of regulatory proteins, which together ensure flexibility and robustness. This integrated system then defines the stereotypical shape changes of successful cytokinesis, which occurs under a diversity of mechanical contexts and environmental conditions. PMID:22761196

West-Foyle, Hoku; Robinson, Douglas N.

2012-01-01

380

Safety Critical Mechanisms  

NASA Technical Reports Server (NTRS)

Spaceflight mechanisms have a reputation for being difficult to develop and operate successfully. This reputation is well earned. Many circumstances conspire to make this so: the environments in which the mechanisms are used are extremely severe, there is usually limited or no maintenance opportunity available during operation due to this environment, the environments are difficult to replicate accurately on the ground, the expense of the mechanism development makes it impractical to build and test many units for long periods of time before use, mechanisms tend to be highly specialized and not prone to interchangeability or off-the-shelf use, they can generate and store a lot of energy, and the nature of mechanisms themselves, as a combination of structures, electronics, etc. designed to accomplish specific dynamic performance, makes them very complex and subject to many unpredictable interactions of many types. In addition to their complexities, mechanism are often counted upon to provide critical vehicle functions that can result in catastrophic events should the functions not be performed. It is for this reason that mechanisms are frequently subjected to special scrutiny in safety processes. However, a failure tolerant approach, along with good design and development practices and detailed design reviews, can be developed to allow such notoriously troublesome mechanisms to be utilized confidently in safety-critical applications.

Robertson, Brandan

2008-01-01

381

Graduate Studies Environmental Fluid Mechanics  

E-print Network

Graduate Studies Environmental Fluid Mechanics and Water Resources Engineering ENVIRONMENTAL FLUID MECHANICS AND WATER RESOURCES ENGINEERING (EFMWR) The environmental fluid mechanics and water resources Environmental Fluid Mechanics and Hydraulic Engineering research focuses on turbulent entrainment, transport

Jacobs, Laurence J.

382

Statistical Mechanics with focus on  

E-print Network

Statistical Mechanics with focus on Liquids, Solutions and Colloidal Systems Course contents A. Foundations of statistical mechanics Classical dynamics ­ Hamilton's and Liouville's equations The concept thermodynamics and statistical mechanics. B. Liquid state theory; Equilibrium statistical mechanics for liquids

Johannesson, Henrik

383

Magnetic capture docking mechanism  

NASA Technical Reports Server (NTRS)

A mechanism uses a magnetic field to dock a satellite to a host vehicle. A docking component of the mechanism residing on the host vehicle has a magnet that is used to induce a coupled magnetic field with a docking component of the mechanism residing on the satellite. An alignment guide axially aligns the docking component of the satellite with the docking component of the host device dependent on the coupled magnetic field. Rotational alignment guides are used to rotationally align the docking component of the satellite with the docking component of the host device. A ball-lock mechanism is used to mechanically secure the docking component of the host vehicle and the docking component of the satellite.

Howard, Nathan (Inventor); Nguyen, Hai D. (Inventor)

2010-01-01

384

Membrane quantum mechanics  

NASA Astrophysics Data System (ADS)

We consider the multiple M2-branes wrapped on a compact Riemann surface and study the arising quantum mechanics by taking the limit where the size of the Riemann surface goes to zero. The IR quantum mechanical models resulting from the BLG-model and the ABJM-model compactified on a torus are N = 16 and N = 12 superconformal gauged quantum mechanics. After integrating out the auxiliary gauge fields we find OSp (16 | 2) and SU (1 , 1 | 6) quantum mechanics from the reduced systems. The curved Riemann surface is taken as a holomorphic curve in a Calabi-Yau space to preserve supersymmetry and we present a prescription of the topological twisting. We find the N = 8 superconformal gauged quantum mechanics that may describe the motion of two wrapped M2-branes in a K3 surface.

Okazaki, Tadashi

2015-01-01

385

Membrane Quantum Mechanics  

E-print Network

We consider the multiple M2-branes wrapped on a compact Riemann surface and study the arising quantum mechanics by taking the limit where the size of the Riemann surface goes to zero. The IR quantum mechanical models resulting from the BLG-model and the ABJM-model compactified on a torus are N = 16 and N = 12 superconformal gauged quantum mechanics. After integrating out the auxiliary gauge fields we find OSp(16|2) and SU(1,1|6) quantum mechanics from the reduced systems. The curved Riemann surface is taken as a holomorphic curve in a Calabi-Yau space to preserve supersymmetry and we present a prescription of the topological twisting. We find the N = 8 superconformal gauged quantum mechanics that may describe the motion of two wrapped M2-branes in a K3 surface.

Tadashi Okazaki

2014-11-03

386

Graduate Studies Environmental Fluid Mechanics  

E-print Network

Graduate Studies Environmental Fluid Mechanics and Water Resources Engineering GRADUATE COURSESResourcesManagement · IntermediateFluidMechanics · AdvancedFluidMechanics · EnvironmentalFluidMechanics · AdvancedEnvironmental FluidMechanics · FluidMechanicsofOrganisms · OpenChannelHydraulics · SedimentTransport · ComputationalFluidMechanics

Storici, Francesca

387

The Mechanisms of Involuntary Attention  

ERIC Educational Resources Information Center

We tested 3 mechanisms of involuntary attention: (1) a perceptual enhancement mechanism, (2) a response-decision mechanism, and (3) a serial-search mechanism. Experiment 1 used a response deadline technique to compare the perceptual enhancement and the decision mechanisms and found evidence consistent with the decision mechanism. Experiment 2 used…

Prinzmetal, William; Ha, Ruby; Khani, Aniss

2010-01-01

388

Tethered satellite control mechanism  

NASA Technical Reports Server (NTRS)

The tethered satellite control mechanisms consist of four major subsystems. The reel drive mechanism stores the tether. It is motor driven and includes a level wind to uniformly feed the tether to the reel. The lower boom mechanism serves two primary functions: (1) it measures tether length and velocity as the tether runs through the mechanism, and (2) it reads the tether tension at the reel. It also provides change the direction for the tether from the reel to the upper boom mechanism. The deployment boom positions the upper boom mechanism with satellite out of the cargo bay. The deployment function places the 500-kg satellite 20 m away from the Space Shuttle (producing a small natural gravity gradient force), impacts an initial velocity to the satellite for deployment, and allows for satellite docking at a safe distance from the body of the Space Shuttle. The upper boom mechanism (UBM) services three functions: (1) it provides tether control to the satellite as the satellite swings in and out of plane; (2) it reads tether tension in the low range during the early deployment and final retrieval parts of the mission; and (3) it produces additional tether tension at the reel when tether tension to the satellite is in the low range.

Kyrias, G. M.

1983-01-01

389

Electronic door locking mechanism  

DOEpatents

The invention is a motorized linkage for engaging a thumb piece in a door mechanism. The device has an exterior lock assembly with a small battery cell and combination lock. Proper entry by a user of a security code allows the battery to operate a small motor within the exterior lock assembly. The small motor manipulates a cam-plunger which moves an actuator pin into a thumb piece. The user applies a force on to the thumb piece. This force is transmitted by the thumb piece to a latch engagement mechanism by the actuator pin. The latch engagement mechanism operates the door latch.

Williams, Gary Lin (428 E. Third Ave., Kennewick, WA 99336); Kirby, Patrick Gerald (1010 W. Fifteenth Pl., Kennewick, WA 99337)

1997-01-01

390

Rotary mechanical latch  

DOEpatents

A rotary mechanical latch for positive latching and unlatching of a rotary device with a latchable rotating assembly having a latching gear that can be driven to latched and unlatched states by a drive mechanism such as an electric motor. A cam arm affixed to the latching gear interfaces with leading and trailing latch cams affixed to a flange within the drive mechanism. The interaction of the cam arm with leading and trailing latch cams prevents rotation of the rotating assembly by external forces such as those due to vibration or tampering.

Spletzer, Barry L.; Martinez, Michael A.; Marron, Lisa C.

2012-11-13

391

Is quantum mechanics exact?  

NASA Astrophysics Data System (ADS)

We formulate physically motivated axioms for a physical theory which for systems with a finite number of degrees of freedom uniquely lead to quantum mechanics as the only nontrivial consistent theory. Complex numbers and the existence of the Planck constant common to all systems arise naturally in this approach. The axioms are divided into two groups covering kinematics and basic measurement theory, respectively. We show that even if the second group of axioms is dropped, there are no deformations of quantum mechanics which preserve the kinematic axioms. Thus, any theory going beyond quantum mechanics must represent a radical departure from the usual a priori assumptions about the laws of nature.

Kapustin, Anton

2013-06-01

392

Electronic door locking mechanism  

DOEpatents

The invention is a motorized linkage for engaging a thumb piece in a door mechanism. The device has an exterior lock assembly with a small battery cell and combination lock. Proper entry by a user of a security code allows the battery to operate a small motor within the exterior lock assembly. The small motor manipulates a cam-plunger which moves an actuator pin into a thumb piece. The user applies a force on to the thumb piece. This force is transmitted by the thumb piece to a latch engagement mechanism by the actuator pin. The latch engagement mechanism operates the door latch. 6 figs.

Williams, G.L.; Kirby, P.G.

1997-10-21

393

The orbital mechanics of flight mechanics  

NASA Technical Reports Server (NTRS)

A reference handbook on modern dynamic orbit theory is presented. Starting from the most basic inverse-square law, the law of gravity for a sphere is developed, and the motion of point masses under the influence of a sphere is considered. The reentry theory and the orbital theory are discussed along with the relative motion between two bodies in orbit about the same planet. Relative-motion equations, rectangular coordinates, and the mechanics of simple rigid bodies under the influence of a gravity gradient field are also discussed.

Dunning, R. S.

1973-01-01

394

Mechanical Arm Control  

E-print Network

This paper discusses three main problems associated with the control of the motion of a mechanical arm. 1) Transformation between different coordinate systems associated with the arm. 2) Calculation of detailed ...

Waters, Richard C.

1979-10-01

395

Quantum Mechanics II (Undergraduate)  

E-print Network

, and applications of quantum mechanics to materials science/solid-state physics. Grades: Homework: 15%, Midertm: 40 other selected topics from quantum information (see the QUNET reference) and solid-state physics. All

Nickrent, Daniel L.

396

Mechanisms for Robust Cognition.  

PubMed

To function well in an unpredictable environment using unreliable components, a system must have a high degree of robustness. Robustness is fundamental to biological systems and is an objective in the design of engineered systems such as airplane engines and buildings. Cognitive systems, like biological and engineered systems, exist within variable environments. This raises the question, how do cognitive systems achieve similarly high degrees of robustness? The aim of this study was to identify a set of mechanisms that enhance robustness in cognitive systems. We identify three mechanisms that enhance robustness in biological and engineered systems: system control, redundancy, and adaptability. After surveying the psychological literature for evidence of these mechanisms, we provide simulations illustrating how each contributes to robust cognition in a different psychological domain: psychomotor vigilance, semantic memory, and strategy selection. These simulations highlight features of a mathematical approach for quantifying robustness, and they provide concrete examples of mechanisms for robust cognition. PMID:25352094

Walsh, Matthew M; Gluck, Kevin A

2014-10-29

397

Mechanical ventilator - infants  

MedlinePLUS

Ventilator - infants; Respirator - infants ... WHY IS A MECHANICAL VENTILATOR USED? A ventilator is used to provide breathing support for ill or immature babies. Sick or premature babies are often ...

398

Failure mechanisms in MEMS.  

SciTech Connect

MEMS components by their very nature have different and unique failure mechanisms than their macroscopic counterparts. This paper discusses failure mechanisms observed in various MEMS components and technologies. MEMS devices fabricated using bulk and surface micromachining process technologies are emphasized. MEMS devices offer uniqueness in their application, fabrication, and functionality. Their uniqueness creates various failure mechanisms not typically found in their bulk or IC counterparts. In ICs, electrical precautions are taken to mitigate failure. In MEMS, both electrical and mechanical precautions must be enacted to reduce the risk of failure and increased reliability. Unlike ICs, many MEMS components are designed to interact with their environment, making the fabrication, testing, and packaging processes critical for the success of the device.

Walraven, Jeremy Allen

2003-07-01

399

Ultralight, ultrastiff mechanical metamaterials  

E-print Network

The mechanical properties of ordinary materials degrade substantially with reduced density because their structural elements bend under applied load. We report a class of microarchitected materials that maintain a nearly ...

Zheng, Xiaoyu

400

Applied Mechanics Materials  

NSDL National Science Digital Library

This site from SpaceTEC National Aerospace Technical Education Center presents materials on applied mechanics in the aerospace workplace. Topics include machine shop safety, non-cutting hand tools, cutting hand tools, drilling, measurement, basic calculations, hardware and blueprints.

2010-10-18

401

Playing at Statistical Mechanics  

ERIC Educational Resources Information Center

Discussed are the applications of counting techniques of a sorting game to distributions and concepts in statistical mechanics. Included are the following distributions: Fermi-Dirac, Bose-Einstein, and most probable. (RH)

Clark, Paul M.; And Others

1974-01-01

402

Hasbun Classical Mechanics Package  

NSDL National Science Digital Library

The Hasbun Classical Mechanics Package is a self-contained Java package of OSP programs in support of the textbook: Classical Mechanics with MATLAB Applications. Classical Mechanics with MATLAB Applications is a resource for the advanced undergraduate taking introduction to classical mechanics. Filled with comprehensive examples and thorough descriptions, this text guides students through the complex topics of rigid body motion, moving coordinate systems, Lagrange's equations, small vibrations, the Euler algorithm, and much more. Step-by-step illustrations, examples and computational physics tools further enhance learning and understanding by demonstrating accessible ways of obtaining mathematical solutions. In addition to the numerous examples throughout, each chapter contains a section of MATLAB code to introduce the topic of programming scripts and their modification for the reproduction of graphs and simulations.

Hasbun, Javier

2008-06-23

403

Coordination Mechanisms George Christodoulou  

E-print Network

Coordination Mechanisms George Christodoulou Elias Koutsoupias Akash Nanavati§ Abstract We of Athens. Email: elias@di.uoa.gr § Computer Science Department, University of California Los Angeles. Email

Koutsoupias, Elias

404

Mechanical harvesting of broilers.  

PubMed

At the end of the growout phase of broiler production essentially all broilers are caught and loaded into coops or cages entirely by hand. The catching of broilers remains one of the few aspects of broiler production not yet automated. Vacuum systems, permanent conveyors recessed in the floors of growout facilities, collecting mats, scoops, and self-propelled, combine-type devices have been tried with little success over the past 30 yr. Renewed efforts at developing mechanical broiler harvesters have been encouraging in recent years. Several equipment companies in North America and Europe have developed prototype mechanical harvesters that appear to have promise. The benefits of mechanical harvesting as compared to hand catching include lower costs and improved working conditions. In addition, studies conducted thus far indicate that mechanical harvesting will improve bird welfare both from a stress and injury standpoint. PMID:9872581

Lacy, M P; Czarick, M

1998-12-01

405

Ultrasound—biophysics mechanisms  

PubMed Central

Ultrasonic biophysics is the study of mechanisms responsible for how ultrasound and biological materials interact. Ultrasound-induced bioeffect or risk studies focus on issues related to the effects of ultrasound on biological materials. On the other hand, when biological materials affect the ultrasonic wave, this can be viewed as the basis for diagnostic ultrasound. Thus, an understanding of the interaction of ultrasound with tissue provides the scientific basis for image production and risk assessment. Relative to the bioeffect or risk studies, that is, the biophysical mechanisms by which ultrasound affects biological materials, ultrasound-induced bioeffects are generally separated into thermal and nonthermal mechanisms. Ultrasonic dosimetry is concerned with the quantitative determination of ultrasonic energy interaction with biological materials. Whenever ultrasonic energy is propagated into an attenuating material such as tissue, the amplitude of the wave decreases with distance. This attenuation is due to either absorption or scattering. Absorption is a mechanism that represents that portion of ultrasonic wave that is converted into heat, and scattering can be thought of as that portion of the wave, which changes direction. Because the medium can absorb energy to produce heat, a temperature rise may occur as long as the rate of heat production is greater than the rate of heat removal. Current interest with thermally mediated ultrasound-induced bioeffects has focused on the thermal isoeffect concept. The non-thermal mechanism that has received the most attention is acoustically generated cavitation wherein ultrasonic energy by cavitation bubbles is concentrated. Acoustic cavitation, in a broad sense, refers to ultrasonically induced bubble activity occurring in a biological material that contains pre-existing gaseous inclusions. Cavitation-related mechanisms include radiation force, microstreaming, shock waves, free radicals, microjets and strain. It is more challenging to deduce the causes of mechanical effects in tissues that do not contain gas bodies. These ultrasonic biophysics mechanisms will be discussed in the context of diagnostic ultrasound exposure risk concerns. PMID:16934858

O'Brien, William D.

2007-01-01

406

Comparing finite mechanisms  

Microsoft Academic Search

Summary.   This paper obtains finite analogues to propositions that a previous literature obtained about the informational efficiency\\u000a of mechanisms whose possible messages form a continuum. Upon reaching an equilibrium message, to which all persons “agree”,\\u000a a mechanism obtains an action appropriate to the organization's environment. Each person's privately observed characteristic\\u000a (a part of the organization's environment) enters her agreement rule.

Leonid Hurwicz; Thomas Marschak

2003-01-01

407

Interprocess Communications Mechanisms  

NSDL National Science Digital Library

This website created by David A Rusling explains the various components of Interprocess Communications Mechanisms supported by Linux. Signals, pipes, and sockets are the main features of this website with a section dedicated to each. Each section contains descriptions and explanations, sub-headings, diagrams, and/or charts. This is a great overview to Interprocess Communications Mechanisms and teachers and students alike will find this a worthwhile bookmark in their browsers.

Rusling, David

408

Particle Acceleration Mechanisms  

Microsoft Academic Search

In this paper we review the possible mechanisms for production of non-thermal electrons which are responsible for the observed\\u000a non-thermal radiation in clusters of galaxies. Our primary focus is on non-thermal Bremsstrahlung and inverse Compton scattering,\\u000a that produce hard X-ray emission. We first give a brief review of acceleration mechanisms and point out that in most astrophysical\\u000a situations, and in

V. Petrosian; A. M. Bykov

2008-01-01

409

Mechanisms flown on LDEF  

NASA Technical Reports Server (NTRS)

A wide variety of mechanisms were flown on the Long Duration Exposure Facility (LDEF). These include canisters, valves, gears, drive train assemblies, and motors. This report will provide the status of the Systems SIG effort into documenting, integrating, and developing 'lessons learned' for the variety of mechanisms flown on the LDEF. Results will include both testing data developed by the various experimenters and data acquired by testing of hardware at Boeing.

Dursch, Harry; Spear, Steve

1992-01-01

410

Visual Quantum Mechanics  

NSDL National Science Digital Library

Visual Quantum Mechanics provides illustrations of quantum mechanics using computer-generated animations. Visualizations provide learning experiences for beginners and offer new insights to the advanced student or researcher. This project includes the development of multimedia software for teaching and scientific software for the solution of the Shrodinger equation and the visualization of these solutions in two and three dimensions. The materials presented here are related to two texts by the author.

Thaller, Bernd

2004-07-10

411

Mechanics of Materials  

NSDL National Science Digital Library

MecMovies is an extensive collection of examples, theory, and games designed to complement the entire Mechanics of Materials course. The software features impressive graphics and animation that are highly effective in visually communicating course concepts to students. Special emphasis is placed on developing the learner's understanding and proficiency in basic concepts and skills through interactive exercises and games. Classroom implementation of the software has produced improved student performance and more positive student attitudes regarding the Mechanics of Materials course.

Philpot, Timothy A.

2008-10-24

412

Time in quantum mechanics  

E-print Network

TIME IN QUANTUM MECHANICS A Thesis by KIMBERLY R. CHAPIN Submitted to Texas A8M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Approved as to style and content by: Marian O. Scully (Chair... of Committee) Edward S. Fry (Member) aan Laane (Member) Thomas W. Adair, III (Head of Department) August 1997 Major Subject: Physics TIME IN QIJANTUM MECHANICS A Thesis by KIMBERLY R. CHAPIN Submitted to the Oflice of Graduate Studies of Texas A...

Chapin, Kimberly R.

2012-06-07

413

Ultrasound-biophysics mechanisms.  

PubMed

Ultrasonic biophysics is the study of mechanisms responsible for how ultrasound and biological materials interact. Ultrasound-induced bioeffect or risk studies focus on issues related to the effects of ultrasound on biological materials. On the other hand, when biological materials affect the ultrasonic wave, this can be viewed as the basis for diagnostic ultrasound. Thus, an understanding of the interaction of ultrasound with tissue provides the scientific basis for image production and risk assessment. Relative to the bioeffect or risk studies, that is, the biophysical mechanisms by which ultrasound affects biological materials, ultrasound-induced bioeffects are generally separated into thermal and non-thermal mechanisms. Ultrasonic dosimetry is concerned with the quantitative determination of ultrasonic energy interaction with biological materials. Whenever ultrasonic energy is propagated into an attenuating material such as tissue, the amplitude of the wave decreases with distance. This attenuation is due to either absorption or scattering. Absorption is a mechanism that represents that portion of ultrasonic wave that is converted into heat, and scattering can be thought of as that portion of the wave, which changes direction. Because the medium can absorb energy to produce heat, a temperature rise may occur as long as the rate of heat production is greater than the rate of heat removal. Current interest with thermally mediated ultrasound-induced bioeffects has focused on the thermal isoeffect concept. The non-thermal mechanism that has received the most attention is acoustically generated cavitation wherein ultrasonic energy by cavitation bubbles is concentrated. Acoustic cavitation, in a broad sense, refers to ultrasonically induced bubble activity occurring in a biological material that contains pre-existing gaseous inclusions. Cavitation-related mechanisms include radiation force, microstreaming, shock waves, free radicals, microjets and strain. It is more challenging to deduce the causes of mechanical effects in tissues that do not contain gas bodies. These ultrasonic biophysics mechanisms will be discussed in the context of diagnostic ultrasound exposure risk concerns. PMID:16934858

O'Brien, William D

2007-01-01

414

Mechanics of the Orbita  

PubMed Central

The oculomotor periphery was formerly regarded as a simple mechanism executing complex behaviors explicitly specified by innervation. It is now recognized that several fundamental aspects of ocular motility are properties of the extraocular muscles (EOMs) and their associated connective tissue pulleys. The Active Pulley Hypothesis proposes that rectus and inferior oblique EOMs have connective tissue soft pulleys that are actively controlled by the direction action of the EOMs’ orbital layers. Functional imaging and histology have suggested that the rectus pulley array constitutes an inner mechanism, similar to a gimbal, that is rotated torsionally around the orbital axis by an outer mechanism driven by the oblique EOMs. This arrangement may mechanically account for several commutative aspects of ocular motor control, including Listing’s law, yet permits implementation of noncommutative motility as during the vestibulo-ocular reflex. Recent human behavioral studies, as well neurophysiology in monkeys, are consistent with mechanical rather than central neural implementation of Listing’s law. Pathology of the pulley system is associated with predictable patterns of strabismus that are surgically treatable when the pathologic anatomy is characterized by imaging. This mechanical determination may imply limited possibilities for neural adaptation to some ocular motor pathologies, but indicates greater potential for surgical treatments. PMID:17314483

Demer, Joseph L.

2008-01-01

415

Rituximab: mechanism of action  

PubMed Central

Rituximab is a mainstay in the therapy for a broad variety of B-cell malignancies. Despite its undeniable therapeutic value, we still do not fully understand the mechanisms of action responsible for rituximab's anti-tumor effects. Direct signaling, complement dependent cellular cytotoxicity and antibody dependent cellular cytotoxicity all appear to play a role in rituximab efficacy. In vitro, animal model and clinical data addressing each of these mechanisms of action are reviewed, as are data speaking to the complexity of interactions between these mechanisms. Taken together, these data suggest different mechanisms are likely important in different scenarios. Study of the complex mechanisms of action that contribute to the clinical efficacy of rituximab have led to novel clinical trials including novel combinations, schedules, and generation of additional antibodies designed to have even greater effect. Such studies need to be accompanied by rigorous correlative analysis if we are to understand the importance of various mechanisms of action of rituximab and use that information to improve on what is already an indispensable approach to therapy. PMID:20350658

Weiner, George J.

2010-01-01

416

MECH 386 INDUSTRIAL FLUID MECHANICS INDUSTRIAL FLUID MECHANICS  

E-print Network

MECH 386 ­ INDUSTRIAL FLUID MECHANICS 1 INDUSTRIAL FLUID MECHANICS MECH 386 Contact information Dr This course is an introduction to industrial fluid mechanics. According to J. C. R. Hunt (a famous fluid mechanics specialist): "industrial fluid mechanics broadly covers those aspects of the design, manufacture

417

Nonlinear Solid Mechanics SOLID MECHANICS AND ITS APPLICATIONS  

E-print Network

Nonlinear Solid Mechanics #12;SOLID MECHANICS AND ITS APPLICATIONS Volume 160 Series Editors: G of the series covers the entire spectrum of solid mechanics. Thus it includes the foundation of mechanics.springer.com/series/6557 #12;Adnan Ibrahimbegovic Nonlinear Solid Mechanics Theoretical Formulations and Finite Element

418

Wear and Tear - Mechanical  

NASA Technical Reports Server (NTRS)

The focus of this chapter is on the long term wear and tear, or aging, of the mechanical subsystem of a spacecraft. The mechanical subsystem is herein considered to be the primary support structure (as in a skeleton or exoskeleton) upon which all other spacecraft systems rest, and the associated mechanisms. Mechanisms are devices which have some component that moves at least once, in response to some type of passive or active control system. For the structure, aging may proceed as a gradual degradation of mechanical properties and/or function, possibly leading to complete structural failure over an extended period of time. However, over the 50 years of the Space Age such failures appear to be unusual. In contrast, failures for mechanisms are much more frequent and may have a very serious effect on mission performance. Just as on Earth, all moving devices are subject to normal (and possibly accelerated) degradation from mechanical wear due to loss or breakdown of lubricant, misalignment, temperature cycling effects, improper design/selection of materials, fatigue, and a variety of other effects. In space, such environmental factors as severe temperature swings (possibly 100's of degrees C while going in and out of direct solar exposure), hard vacuum, micrometeoroids, wear from operation in a dusty or contaminated environment, and materials degradation from radiation can be much worse. In addition, there are some ground handling issues such as humidity, long term storage, and ground transport which may be of concern. This chapter addresses the elements of the mechanical subsystem subject to wear, and identifies possible causes. The potential impact of such degradation is addressed, albeit with the recognition that the impact of such wear often depends on when it occurs and on what specific components. Most structural elements of the mechanical system typically are conservatively designed (often to a safety factor of greater than approximately 1.25 on yield for unmanned spacecraft) but do not have backup structure due to the added mass this would impose, and also due to the fact that structural elements can be accurately modeled mathematically and in test. Critical mechanisms or devices may have backups, or alternate work-arounds, since characterization of these systems in a 1g environment is less accurate than structure, and repair in-space is often impossible.

Swanson, Theodore

2008-01-01

419

Department of Mechanical Engineering "Mechanics of Stretchable Electronics and  

E-print Network

Batteries" Professor Yonggang Huang Joseph Cummings Professor Dept. of Civil and Environmental Engineering of Mechanical Engineering and Civil and Environmental Engineering at Northwestern University. He has broadDepartment of Mechanical Engineering presents "Mechanics of Stretchable Electronics and Stretchable

Zakhor, Avideh

420

Mechanical Components Branch Overview  

NASA Technical Reports Server (NTRS)

Mr. James Zakrajsek, chief of the Mechanical Components Branch, presented an overview of research conducted by the branch. Branch members perform basic research on mechanical components and systems, including gears and bearings, turbine seals, structural and thermal barrier seals, and space mechanisms. The research is focused on propulsion systems for present and advanced aerospace vehicles. For rotorcraft and conventional aircraft, we conduct research to develop technology needed to enable the design of low noise, ultra safe geared drive systems. We develop and validate analytical models for gear crack propagation, gear dynamics and noise, gear diagnostics, bearing dynamics, and thermal analyses of gear systems using experimental data from various component test rigs. In seal research we develop and test advanced turbine seal concepts to increase efficiency and durability of turbine engines. We perform experimental and analytical research to develop advanced thermal barrier seals and structural seals for current and next generation space vehicles. In space mechanisms, we conduct fundamental research on lubricants, materials, components and mechanisms subjected to deep space and planetary environments.

2001-01-01

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