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1

The P2Y(12) receptor induces platelet aggregation through weak activation of the alpha(IIb)beta(3) integrin--a phosphoinositide 3-kinase-dependent mechanism.  

PubMed

High concentrations of adenosine-5'-diphosphate ADP are able to induce partial aggregation without shape change of P2Y(1) receptor-deficient mouse platelets through activation of the P2Y(12) receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R-phycoerythrin-conjugated JON/A antibody (JON/A-PE), an antibody which recognizes activated mouse alpha(IIb)beta(3) integrin, revealed a low level activation of alpha(IIb)beta(3) in P2Y(1) receptor-deficient platelets in response to 100 microM ADP or 1 microM 2MeS-ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose-dependent manner. Global phosphorylation of (32)P-labeled platelets showed that P2Y(12)-mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P(20)) or pleckstrin (P(47)) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y(12) receptor is able to trigger a P2Y(1) receptor-independent inside-out signal leading to alpha(IIb)beta(3) integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the alpha(IIb)beta(3) integrin, and (iii) the transduction pathway triggered by the P2Y(12) receptor is independent of PKC but dependent on phosphoinositide 3-kinase. PMID:11566191

Kauffenstein, G; Bergmeier, W; Eckly, A; Ohlmann, P; Léon, C; Cazenave, J P; Nieswandt, B; Gachet, C

2001-09-14

2

RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism  

SciTech Connect

In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85{alpha} and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.

Yamaki, Nao [Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Negishi, Manabu [Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Katoh, Hironori [Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501 (Japan)]. E-mail: hirokato@pharm.kyoto-u.ac.jp

2007-08-01

3

Positive and Negative Regulation of Phosphoinositide 3-Kinase-Dependent Signaling Pathways by Three Different Gene Products of the p85alpha Regulatory Subunit  

Microsoft Academic Search

Phosphoinositide (PI) 3-kinase is a key mediator of insulin-dependent metabolic actions, including stimu- lation of glucose transport and glycogen synthesis. The gene for the p85a regulatory subunit yields three splicing variants, p85a, AS53\\/p55a, and p50a. All three have (i) a C-terminal structure consisting of two Src homology 2 domains flanking the p110 catalytic subunit-binding domain and (ii) a unique N-terminal

KOHJIRO UEKI; PETRA ALGENSTAEDT; FRANCK MAUVAIS-JARVIS; C. RONALD KAHN

2000-01-01

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

t-DARPP regulates phosphatidylinositol-3-kinase-dependent cell growth in breast cancer  

PubMed Central

Background Recent reports have shown that t-DARPP (truncated isoform of DARPP-32) can mediate trastuzumab resistance in breast cancer cell models. In this study, we evaluated expression of t-DARPP in human primary breast tumors, and investigated the role of t-DARPP in regulating growth and proliferation in breast cancer cells. Results Quantitative real time RT-PCR analysis using primers specific for t-DARPP demonstrated overexpression of t-DARPP in 36% of breast cancers (13/36) as opposed to absent to very low t-DARPP expression in normal breast tissue (p < 0.05). The mRNA overexpression of t-DARPP was overwhelmingly observed in ductal carcinomas, including invasive ductal carcinomas and intraductal carcinomas, rather than other types of breast cancers. The immunohistochemistry analysis of DARPP-32/t-DARPP protein(s) expression in breast cancer tissue microarray that contained 59 tumors and matched normal tissues when available indicated overexpression in 35.5% of primary breast tumors that were more frequent in invasive ductal carcinomas (43.7%; 21/48). In vitro studies showed that stable overexpression of t-DARPP in MCF-7 cells positively regulated proliferation and anchorage-dependent and -independent growth. Furthermore, this effect was concomitant with induction of phosphorylation of AKTser473 and its downstream target phosphoser9 GSK3?, and increased Cyclin D1 and C-Myc protein levels. The knockdown of endogenous t-DARPP in HCC1569 cells led to a marked decrease in phosphorylation of AKTsser473 and GSK3?ser9. The use of PI3K inhibitor LY294002 or Akt siRNA abrogated the t-DARPP-mediated phosphorylation of AKTser473 and led to a significant reduction in cell growth. Conclusions Our findings underscore the potential role of t-DARPP in regulating cell growth and proliferation through PI3 kinase-dependent mechanism. PMID:20836878

2010-01-01

6

Deletion of the phosphoinositide 3-Kinase p110(gamma) gene attenuates murine atherosclerosis  

Technology Transfer Automated Retrieval System (TEKTRAN)

Inflammatory cell activation by chemokines requires intracellular signaling through phosphoinositide 3-kinase (PI3-kinase) and the PI3-kinase-dependent protein serine/threonine kinase Akt. Atherosclerosis is a chronic inflammatory process driven by oxidatively modified (atherogenic) lipoproteins, ch...

7

ICAM-1 cytoplasmic tail regulates endothelial glutathione synthesis through a NOX4/PI3-kinase-dependent pathway  

PubMed Central

We previously reported that ICAM-1 expression modulates endothelial intracellular glutathione (GSH) metabolism through unknown mechanisms. Here we report that the cytoplasmic tail of ICAM-1 is critically involved in governing intracellular GSH production. Peptides containing the antennapedia cell-permeative sequence (AP) or an AP peptide linked to the transmembrane and cytosolic tail of ICAM-1 (AP-ICAM) were synthesized and used to measure alterations in redox status in cultured endothelial cells and determine their biological effect. Treatment with AP-ICAM significantly increased GSH concentrations and glutamate–cysteine ligase (GCL) activity over time. Measuring reactive oxygen species (ROS) production with DCF revealed a rapid increase in ROS generation after AP-ICAM treatment. Measurement of superoxide production with hydroethidium revealed biphasic production at 30 min and 6 h after treatment with AP-ICAM. Apocynin, DPI, catalase, or SOD attenuated AP-ICAM-dependent ROS production, GCL activity, and GSH production, implicating superoxide production and dismutation to peroxide. Consistent with these findings, NOX4 siRNA knockdown blocked AP-ICAM peptide increases in GSH or GCL activity, demonstrating the importance of NADPH oxidase. Last, inhibition of PI3-kinase activity with LY 294002 or wortmannin blocked AP-ICAM GSH induction and ROS production. These data reveal that the ICAM-1 cytoplasmic tail regulates production of endothelial GSH through a NOX4/PI3-kinase-dependent redox-sensitive pathway. PMID:20633529

Pattillo, Christopher B.; Pardue, Sibile; Shen, Xinggui; Fang, Kai; Langston, Will; Jourd'heuil, David; Kavanagh, Terrance J.; Patel, Rakesh P.; Kevil, Christopher G.

2014-01-01

8

A central role for phosphoinositide hydrolysis in activating the lytic mechanism of human natural killer cells.  

PubMed Central

Using neomycin, which inhibits phosphoinositide breakdown, cytotoxicity mediated by natural killer (NK) cells was suppressed in a dose-dependent manner, with complete inhibition at 16 mM. Generation of inositol phosphates in effector cells after target cell binding was inhibited in the presence of neomycin. The formation of effector to target cell conjugates was not affected. Neomycin-induced inhibition of NK killing was abolished when TPA was added to the cytotoxic assays. This reconstitution was dependent upon extracellular Ca2+. When the intracellular free Ca2+ level in effector cells was reduced from 73 +/- 11 nM to 43 +/- 3 nM (n = 4) using the Ca2+ indicator dye, Quin 2, NK killing was markedly reduced. Inhibiting the enzyme diacylglycerol (DG) kinase in effector cells with 10 microM R59022 (DG kinase inhibitor) potentiates NK killing, suggesting an increase in protein kinase C (PKC) activity due to accumulation of DG. The PKC inhibitor, H-7, suppressed NK killing in a concentration-dependent manner. These results demonstrate that phosphoinositide metabolism is an early event and its derived second messengers play a central role in activating the lytic mechanism of NK cells. PMID:2162323

Chow, S C; Jondal, M

1990-01-01

9

Acanthamoeba castellanii Induces Host Cell Death via a Phosphatidylinositol 3-Kinase-Dependent Mechanism  

Microsoft Academic Search

Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and trans- migrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate be- longing to the T1 genotype on human brain microvascular endothelial cells, which constitute

James Sissons; Kwang Sik Kim; Monique Stins; Samantha Jayasekera; Selwa Alsam; Naveed Ahmed Khan

2005-01-01

10

Simvastatin increases excitability in the hippocampus via a PI3 kinase-dependent mechanism.  

PubMed

Simvastatin is an HMG-CoA reductase inhibitor commonly used in the clinic to treat hypercholesterolemia. In addition, simvastatin has been shown to cross the blood-brain barrier and pleiotropic effects of simvastatin have been reported including anti-inflammatory properties, enhancement of neurite outgrowth, and memory enhancement properties. However, little has been reported on the effects of simvastatin on basal synaptic transmission and neuronal excitability. Here we report that simvastatin increases the fEPSP, the N-methyl-D-aspartate (NMDA) receptor-mediated fEPSP using extracellular recordings in the dendritic region of the CA1 of hippocampal slices taken from 8-week-old C57Black6J mice. In addition, we found that simvastatin perfusion causes a change in the input/output curve and a decrease of the paired-pulse facilitation ratio, indicating respectively an increase of the neuronal excitability and neurotransmitter release. We have also observed that acute application of simvastatin increased the amplitude of the compound action potential in the CA1 region. Notably, using LY294002, we have demonstrated that this effect was PI3K dependent and was occluded if the animals had previously received a diet supplemented with simvastatin. We have finally shown that the simvastatin-mediated increase of the compound action potential amplitude was also present in hippocampal slices from aged mice. PMID:25701710

Métais, C; Hughes, B; Herron, C E

2015-04-16

11

Acanthamoeba castellanii Induces Host Cell Death via a Phosphatidylinositol 3-Kinase-Dependent Mechanism  

PubMed Central

Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and transmigrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate belonging to the T1 genotype on human brain microvascular endothelial cells, which constitute the blood-brain barrier. Using an apoptosis-specific enzyme-linked immunosorbent assay, we showed that Acanthamoeba induces programmed cell death in brain microvascular endothelial cells. Next, we observed that Acanthamoeba specifically activates phosphatidylinositol 3-kinase. Acanthamoeba-mediated brain endothelial cell death was abolished using LY294002, a phosphatidylinositol 3-kinase inhibitor. These results were further confirmed using brain microvascular endothelial cells expressing dominant negative forms of phosphatidylinositol 3-kinase. This is the first demonstration that Acanthamoeba-mediated brain microvascular endothelial cell death is dependent on phosphatidylinositol 3-kinase. PMID:15845472

Sissons, James; Kim, Kwang Sik; Stins, Monique; Jayasekera, Samantha; Alsam, Selwa; Khan, Naveed Ahmed

2005-01-01

12

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

13

Mechanisms and implications of phosphoinositide 3-kinase in promoting neutrophil trafficking into inflamed tissue  

Microsoft Academic Search

The phosphoinositide 3-kinase (PI3K) catalytic subunit p110 is expressed in neutrophils and is thought to play a role in their accumulation at sites of inflamma- tion by contributing to chemoattractant- directed migration. We report here that p110 is present in endothelial cells and participates in neutrophil trafficking by modulating the proadhesive state of these cells in response to tumor necrosis

Kamal D. Puri; Teresa A. Doggett; Jason Douangpanya; Yonghao Hou; William T. Tino; Tim Wilson; Thomas Graf; Elizabeth Clayton; Martin Turner; Joel S. Hayflick; Thomas G. Diacovo

2004-01-01

14

Phosphoinositide3-Kinase-Dependent, MyD88Independent Induction of CC-Type Chemokines Characterizes the Macrophage Response to Toxoplasma gondii Strains with High Virulence  

Microsoft Academic Search

Chemokines play an important role in inflammation and infection due to their ability to recruit cells of innate and adaptive immunity. Here we examined mouse macrophage chemokine responses during intracel- lular infections with high- and low-virulence Toxoplasma gondii strains. The high-virulence type I strain RH induced a large panel of CC-type chemokines, whereas responses elicited by strains PTG (type II)

Chiang W. Lee; Woraporn Sukhumavasi; Eric Y. Denkers

2007-01-01

15

Phosphoinositide 3-kinase is involved in the tumor-specific activation of human breast cancer cell Na(+)/H(+) exchange, motility, and invasion induced by serum deprivation.  

PubMed

Whereas the tumor acidic extracellular pH plays a crucial role in the invasive process, the mechanism(s) behind this acidification, especially in low nutrient conditions, are unclear. The regulation of the Na(+)/H(+) exchanger (NHE) and invasion by serum deprivation were studied in a series of breast epithelial cell lines representing progression from non-tumor to highly metastatic cells. Whereas serum deprivation reduced lactate production in all three cells lines, it inhibited NHE activity in the non-tumor cells and stimulated it in the tumor cells with a larger stimulation in the metastatic cells. The stimulation of NHE in the tumor cell lines was the result of an increased affinity of the internal H(+) regulatory site of the NHE without changes in sodium kinetics or expression. Serum deprivation conferred increased cell motility and invasive ability that were abrogated by specific inhibition of the NHE. Inhibition of phosphoinositide 3-kinase by overexpression of a dominant-negative mutant or wortmannin incubation inhibited NHE activity and invasion in serum replete conditions while potentiating the serum deprivation-dependent activation of the NHE and invasion. These results indicate that the up-regulation of the NHE by a phosphoinositide 3-kinase-dependent mechanism plays an essential role in increased tumor cell invasion induced by serum deprivation. PMID:10681510

Reshkin, S J; Bellizzi, A; Albarani, V; Guerra, L; Tommasino, M; Paradiso, A; Casavola, V

2000-02-25

16

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)

2012-05-01

17

Phosphoinositides and phagocytosis  

PubMed Central

Phosphoinositide 3 kinases (PI3Ks)* are known as regulators of phagocytosis. Recent results demonstrate that class I and III PI3Ks act consecutively in phagosome formation and maturation, and that their respective products, phosphatidylinositol 3,4,5-trisphosphate (PI[3,4,5]P3) and phosphatidylinositol 3-phosphate (PI[3]P), accumulate transiently at different stages. Phagosomes containing Mycobacterium tuberculosis do not acquire the PI(3)P-binding protein EEA1, which is required for phagosome maturation. This suggests a possible mechanism of how this microorganism evades degradation in phagolysosomes. PMID:11581282

Gillooly, David J.; Simonsen, Anne; Stenmark, Harald

2001-01-01

18

The phosphoinositide 3-kinase signaling pathway in normal and malignant B cells: activation mechanisms, regulation and impact on cellular functions  

PubMed Central

The phosphoinositide 3-kinase (PI3K) pathway is a central signal transduction axis controlling normal B cell homeostasis and activation in humoral immunity. The p110? PI3K catalytic subunit has emerged as a critical mediator of multiple B cell functions. The activity of this pathway is regulated at multiple levels, with inositol phosphatases PTEN and SHIP both playing critical roles. When deregulated, the PI3K pathway can contribute to B cell malignancies and autoantibody production. This review summarizes current knowledge on key mechanisms that activate and regulate the PI3K pathway and influence normal B cell functional responses including the development of B cell subsets, antigen presentation, immunoglobulin isotype switch, germinal center responses, and maintenance of B cell anergy. We also discuss PI3K pathway alterations reported in select B cell malignancies and highlight studies indicating the functional significance of this pathway in malignant B cell survival and growth within tissue microenvironments. Finally, we comment on early clinical trial results, which support PI3K inhibition as a promising treatment of chronic lymphocytic leukemia. PMID:22908014

Pauls, Samantha D.; Lafarge, Sandrine T.; Landego, Ivan; Zhang, Tingting; Marshall, Aaron J.

2012-01-01

19

The phosphoinositide 3-kinase signaling pathway in normal and malignant B cells: activation mechanisms, regulation and impact on cellular functions.  

PubMed

The phosphoinositide 3-kinase (PI3K) pathway is a central signal transduction axis controlling normal B cell homeostasis and activation in humoral immunity. The p110? PI3K catalytic subunit has emerged as a critical mediator of multiple B cell functions. The activity of this pathway is regulated at multiple levels, with inositol phosphatases PTEN and SHIP both playing critical roles. When deregulated, the PI3K pathway can contribute to B cell malignancies and autoantibody production. This review summarizes current knowledge on key mechanisms that activate and regulate the PI3K pathway and influence normal B cell functional responses including the development of B cell subsets, antigen presentation, immunoglobulin isotype switch, germinal center responses, and maintenance of B cell anergy. We also discuss PI3K pathway alterations reported in select B cell malignancies and highlight studies indicating the functional significance of this pathway in malignant B cell survival and growth within tissue microenvironments. Finally, we comment on early clinical trial results, which support PI3K inhibition as a promising treatment of chronic lymphocytic leukemia. PMID:22908014

Pauls, Samantha D; Lafarge, Sandrine T; Landego, Ivan; Zhang, Tingting; Marshall, Aaron J

2012-01-01

20

Insulin stimulates increased catalytic activity of phosphoinositide-dependent kinase-1 by a phosphorylation-dependent mechanism.  

PubMed

Phosphoinositide-dependent kinase-1 (PDK-1) is a serine-threonine kinase downstream from PI 3-kinase that phosphorylates and activates other important kinases such as Akt that are essential for cell survival and metabolism. Previous reports have suggested that PDK-1 has constitutive catalytic activity that is not regulated by stimulation of cells with growth factors. We now show that insulin stimulation of NIH-3T3(IR) cells or rat adipose cells may significantly increase the intrinsic catalytic activity of PDK-1. Insulin treatment of NIH-3T3(IR) fibroblasts overexpressing PDK-1 increased both phosphorylation of recombinant PDK-1 in intact cells and PDK-1 kinase activity in an immune-complex kinase assay. Insulin stimulation of rat adipose cells also increased catalytic activity of endogenous PDK-1 immunoprecipitated from the cells. Both insulin-stimulated phosphorylation and activity of PDK-1 were inhibited by wortmannin and reversed by treatment with the phosphatase PP-2A. A mutant PDK-1 with a disrupted PH domain (W538L) did not undergo phosphorylation or demonstrate increased kinase activity in response to insulin stimulation. Similarly, a PDK-1 phosphorylation site point mutant (S244A) had no increase in kinase activity in response to insulin stimulation. Thus, the insulin-stimulated increase in PDK-1 catalytic activity may involve PI 3-kinase- and phosphorylation-dependent mechanisms. We conclude that the basal constitutive catalytic activity of PDK-1 in NIH-3T3(IR) cells and rat adipose cells can be significantly increased upon insulin stimulation. PMID:11570885

Chen, H; Nystrom, F H; Dong, L Q; Li, Y; Song, S; Liu, F; Quon, M J

2001-10-01

21

Phosphatidylinositol 3-Kinase dependent upregulation of the epidermal growth factor receptor upon Flotillin-1 depletion in breast cancer cells  

PubMed Central

Background Flotillin-1 and flotillin-2 are two homologous and ubiquitously expressed proteins that are involved in signal transduction and membrane trafficking. Recent studies have reported that flotillins promote breast cancer progression, thus making them interesting targets for breast cancer treatment. In the present study, we have investigated the underlying molecular mechanisms of flotillins in breast cancer. Methods Human adenocarcinoma MCF7 breast cancer cells were stably depleted of flotillins by means of lentivirus mediated short hairpin RNAs. Western blotting, immunofluorescence and quantitative real-time PCR were used to analyze the expression of proteins of the epidermal growth factor receptor (EGFR) family. Western blotting was used to investigate the effect of EGFR stimulation or inhibition as well as phosphatidylinositol 3-kinase (PI3K) inhibition on mitogen activated protein kinase (MAPK) signaling. Rescue experiments were performed by stable transfection of RNA intereference resistant flotillin proteins. Results We here show that stable knockdown of flotillin-1 in MCF7 cells resulted in upregulation of EGFR mRNA and protein expression and hyperactivation of MAPK signaling, whereas ErbB2 and ErbB3 expression were not affected. Treatment of the flotillin knockdown cells with an EGFR inhibitor reduced the MAPK signaling, demonstrating that the increased EGFR expression and activity is the cause of the increased signaling. Stable ectopic expression of flotillins in the knockdown cells reduced the increased EGFR expression, demonstrating a direct causal relationship between flotillin-1 expression and EGFR amount. Furthermore, the upregulation of EGFR was dependent on the PI3K signaling pathway which is constitutively active in MCF7 cells, and PI3K inhibition resulted in reduced EGFR expression. Conclusions This study demonstrates that flotillins may not be suitable as cancer therapy targets in cells that carry certain other oncogenic mutations such as PI3K activating mutations, as unexpected effects are prone to emerge upon flotillin knockdown which may even facilitate cancer cell growth and proliferation. PMID:24304721

2013-01-01

22

Aggregation of Phosphoinositides at Phisiological Calcium Concentrations  

NASA Astrophysics Data System (ADS)

Phosphoinositides play a crucial role in many cellular functions such as calcium signaling, endocytosis, exocytosis and the targeting of proteins to specific membrane sites. To maintain functional specificity, it has been suggested that phosphoinositides are spatially organized in ``pools'' in the cellular plasma membrane. A possible mechanism that could induce and regulate such organization of phosphoinositides is their interaction with Ca2+ ions. Understanding the physicochemical mechanism that can regulate membrane structure is a crucial step in the development of adaptive biomimetic membrane systems. Using Langmuir monolayers, we investigated the effect of bivalent calcium and magnesium cations on the surface pressure-area/lipid isotherm of monolayers of phosphatidylinositol (PI), phosphatidylinositol bisphosphate (PIP2) and dioleoylphosphatidylglycerol (DOPG) and dipalmitoylphosphatidylcholine (DPPC). It is found that the decrease of area per lipid, i.e. the increase in aggregation, is dependent on both the lipid's head group charge, the bivalent cation and temperature. However, electrostatics are not sufficient to account for all experimental observations. Thus additional interactions between ions and phosphoinositides need to be considered.

Kazadi Badiambile, Adolphe; Forstner, Martin B.

2012-02-01

23

Adapter protein with a pleckstrin homology (PH) and an Src homology 2 (SH2) domain (APS) and SH2-B enhance insulin-receptor autophosphorylation, extracellular-signal-regulated kinase and phosphoinositide 3-kinase-dependent signalling.  

PubMed Central

Adapter protein with a pleckstrin homology (PH) and an Src homology 2 (SH2) domain (APS) and SH2-B are adapter proteins and substrates that interact with the activation loop of the insulin-receptor (IR) kinase. These proteins are homologous and share substantial sequence similarity. We previously showed [Ahmed, Smith and Pillay, FEBS Lett. 475, 31-34], for the first time, that insulin-stimulated phosphorylation of APS led to interaction with c-Cbl in 3T3-L1 adipocytes and in transfected Chinese-hamster ovary (CHO) cells. In the present study, we find that insulin stimulates the membrane translocation and phosphorylation of APS to a much greater extent than SH2-B, despite the structural similarity of these proteins. Expression of APS or SH2-B delays IR tyrosine and IR substrate (IRS) dephosphorylation. This enhancement of signalling is also observed downsteam of the receptor. In control cells that lack APS, following insulin stimulation, extracellular-signal-regulated kinase (ERK) and Akt kinase reach maximal activation and then decline to basal levels by 60 min. In contrast, in APS- and SH2-B-expressing cells, ERK and Akt kinase activation remains at peak levels at 60 min. These effects may occur because these proteins either stabilize the active conformation or prevent dephosphorylation of the IR. We therefore conclude that, despite the ability to couple to c-Cbl, APS functions as a positive regulator of IR signalling and, although SH2-B is a poor substrate for the IR, its association with the IR allows it to regulate pathways downstream of the receptor independently of its phosphorylation. PMID:12521378

Ahmed, Zamal; Pillay, Tahir S

2003-01-01

24

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

25

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

26

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

27

Analysis of Phosphoinositides in Protein Trafficking  

Microsoft Academic Search

Phosphoinositides are key regulators of vesicle-mediated protein trafficking. Their roles include recruiting vesicle coat and effector proteins to the site of budding and promoting vesicle fusion. The intracellular levels of phosphoinositides and their localization to intracellular membranes are critical to their functions. An analytical procedure was developed that optimizes the recovery of radiolabeled cellular phosphoinositides. Quantitative analyses of yeast cellular

Hiroko Hama; Jon Y. Takemoto; Daryll B. DeWald

2000-01-01

28

MECHANICAL STIMULI REGULATE RAPAMYCIN-SENSITIVE SIGNALLING BY A PHOSPHOINOSITIDE 3-KINASE-, PROTEIN KINASE B- AND GROWTH FACTOR-INDEPENDENT MECHANISM  

Technology Transfer Automated Retrieval System (TEKTRAN)

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 ca...

29

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

30

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

31

Arf6-independent GPI-anchored Protein-enriched Early Endosomal Compartments Fuse with Sorting Endosomes via a Rab5/Phosphatidylinositol-3?-Kinase–dependent Machinery  

PubMed Central

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3?-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes. PMID:16760436

Kalia, Manjula; Kumari, Sudha; Chadda, Rahul; Hill, Michelle M.; Parton, Robert G.

2006-01-01

32

Prospects & Overviews Pairing phosphoinositides with calcium  

E-print Network

-bound factors, including soluble NSF attachment protein receptors (SNAREs), Rab GTPases, and phosphoinositides dinucleotide phosphate; NSF, N-ethylmaleimide-sensitive fusion protein; PIP, phosphoinositide; SNARE, soluble NSF attachment protein receptors; TGN, trans-Golgi network; TPC, two-pore channel; TRP, transient

Xu, Haoxing

33

Ion Induced Changes in Phosphoinositide Monolayers at Phisiological Concentrations  

NASA Astrophysics Data System (ADS)

Phosphoinositides (PIPs) play a crucial role in many cellular process that occur at the plasma membrane such as calcium release, exocytosis or endocytosis. In order to specifically regulate these functions PIPs must segregate in pools at the plasma membrane. A possible mechanism that could induce and regulate such organization of phosphoinositides is their interaction with bivalent cations. Understanding the physicochemical mechanism that can regulate membrane structure is a crucial step in the development of adaptive biomimetic membrane systems. Using Langmuir monolayers, we investigated the effect of calcium and magnesium on the surface pressure-area/lipid isotherm of monolayer of phosphatidylinositol (PI), phosphatidylinositol bisphosphate (PIP2), dioleoylphosphatidylglycerol (DOPG) and palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). It is found that the decrease of area per lipid, i.e. the increase in aggregation, is mostly dependent on the lipid's head group charge but ion specific. In addition, we discuss changes in free energy and compressibility of these monolayer-ion systems.

Kazadi Badiambile, Adolphe; Forstner, Martin B.

2013-03-01

34

Aluminum interaction with phosphoinositide-associated signal transduction  

Microsoft Academic Search

Concerning molecular and cellular mechanisms of aluminum toxicity, recent studies support the hypothesis that interactions\\u000a of aluminum ions with elements of signal transduction pathways are apparently primary events in cells. In the case of the\\u000a phosphoinositide-associated signalling pathway of neuroblastoma cells, guanine nucleotide-binding proteins (G proteins) and\\u000a a phosphatidylinositol-4,5-diphosphate (PIP2)-specific phospholipase C are probable interaction sites for inhibitory actions of

Alfred Haug; Biao Shi; Victor Vitorello

1994-01-01

35

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)

2006-11-07

36

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)

2009-02-01

37

The phosphoinositide 3-kinase pathway and therapy resistance in cancer  

PubMed Central

The phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling network is a master regulator of processes that contribute to tumorigenesis and tumor maintenance. The PI3K pathway also plays a critical role in driving resistance to diverse anti-cancer therapies. This review article focuses on mechanisms by which the PI3K pathway contributes to therapy resistance in cancer, and highlights potential combination therapy strategies to circumvent resistance driven by PI3K signaling. In addition, resistance mechanisms that limit the clinical efficacy of small molecule inhibitors of the PI3K pathway are discussed. PMID:25750731

2015-01-01

38

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

39

CDP-diacylglycerol synthetase-controlled phosphoinositide availability limits VEGFA signaling and vascular morphogenesis  

PubMed Central

Understanding the mechanisms that regulate angiogenesis and translating these into effective therapies are of enormous scientific and clinical interests. In this report, we demonstrate the central role of CDP-diacylglycerol synthetase (CDS) in the regulation of VEGFA signaling and angiogenesis. CDS activity maintains phosphoinositide 4,5 bisphosphate (PIP2) availability through resynthesis of phosphoinositides, whereas VEGFA, mainly through phospholipase C?1, consumes PIP2 for signal transduction. Loss of CDS2, 1 of 2 vertebrate CDS enzymes, results in vascular-specific defects in zebrafish in vivo and failure of VEGFA-induced angiogenesis in endothelial cells in vitro. Absence of CDS2 also results in reduced arterial differentiation and reduced angiogenic signaling. CDS2 deficit-caused phenotypes can be successfully rescued by artificial elevation of PIP2 levels, and excess PIP2 or increased CDS2 activity can promote excess angiogenesis. These results suggest that availability of CDS-controlled resynthesis of phosphoinositides is essential for angiogenesis. PMID:22649102

Pan, Weijun; Pham, Van N.; Stratman, Amber N.; Castranova, Daniel; Kamei, Makoto; Kidd, Kameha R.; Lo, Brigid D.; Shaw, Kenna M.; Torres-Vazquez, Jesus; Mikelis, Constantinos M.; Gutkind, J. Silvio; Davis, George E.

2012-01-01

40

Decoding the role of phosphoinositides in phototropin signaling involved in chloroplast movements  

PubMed Central

In angiosperms, light-dependent chloroplast movements are exclusively mediated by UVA/blue light receptors - phototropins. The two photoreceptors of Arabidopsis thaliana, phot1 and phot2, have overlapping roles in the control of these movements. Experiments performed in different plant species point to the participation of phosphoinositides in blue light-controlled chloroplast relocations. Here, we report a summary of recent findings presenting the involvement of phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 3- and 4-phosphates in weak blue light-mediated (accumulation) and strong blue light-mediated (avoidance) responses of chloroplasts. The blue light-activated alterations in phosphoinositide concentration are partly responsible for cytosolic Ca2+ changes. Ca2+ influx from apoplast does not seem to be involved in the mechanism of movement responses. In summary, interplay between phosphoinositides and intracellular Ca2+ regulates chloroplast redistribution in response to blue light in higher plants. PMID:23733070

Aggarwal, Chhavi; Labuz, Justyna; Gabry?, Halina

2013-01-01

41

Phosphoinositide 3-kinase in immunological systems  

Microsoft Academic Search

Phosphoinositide 3-kinases (PI3Ks) are an evolutionarily conserved family of signal transducing enzymes. A great variety of stimuli activate PI3K, leading to the transient accumulation of its lipid products in cell membranes. These lipids serve as second messengers to regulate the location and activity of an array of downstream effector molecules. In cells of the mammalian immune system, PI3K is activated

David A Fruman; Lewis C Cantley

2002-01-01

42

D-3 phosphoinositide metabolism in cells treated with platelet-derived growth factor.  

PubMed Central

Despite extensive analysis of phosphoinositide 3-hydroxykinases (PI 3-kinases) at the molecular level, comparatively little is known about the mechanisms by which products of these enzymes exert their expected second-messenger functions. This study examines the metabolism of D-3 phosphoinositides in mouse Ph-N2 fibroblasts lacking the platelet-derived growth factor (PDGF) alpha-receptor. Treatment of these cultures with BB PDGF, but not AA PDGF, resulted in transient activation of PI 3-kinase activity measured in vitro. Treatment of myo-[3H]inositol-labelled Ph-N2 cells with BB PDGF resulted in the rapid induction of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 and, to a smaller extent, PtdIns3P. The appearance of PtdIns(3,4,5)P3 preceded that of PtdIns(3,4)P2 and PtdIns3P after the addition of PDGF, suggesting that PtdIns(4,5)P2 is the preferred substrate of the agoniststimulated PI 3-kinase in intact cells. Treatment of both resting and PDGF-stimulated cells with the fungal metabolite wortmannin resulted in pronounced, selective effects on the levels of all D-3 phosphoinositides. Kinetic studies with this PI 3-kinase inhibitor revealed the presence of at least two independent routes for the biosynthesis of D-3 phosphoinositides in PDGF-treated cells. PMID:8920990

Whiteford, C C; Best, C; Kazlauskas, A; Ulug, E T

1996-01-01

43

Decreased platelet phosphoinositide turnover and enhanced platelet activation in IDDM  

SciTech Connect

Individuals with diabetes mellitus may have increased in vivo platelet activity. Abnormal platelet function could contribute to the increased incidence of vascular disease in diabetes mellitus. The biochemical mechanism(s) for platelet hyperactivation is unknown. We examined the hypothesis that platelet phosphoinositide turnover, a key signal-transducing mechanism involved in platelet activation, was abnormal in diabetic subjects. Platelets were harvested from 16 subjects with insulin-dependent diabetes mellitus (IDDM) and 19 healthy, nondiabetic control subjects of comparable age. Plasma beta-thromboglobulin (beta-TBG), a specific marker of platelet activity in vivo, was increased in IDDM (67.1 +/- 7.3 ng/ml) compared with control (41.0 +/- 6.0 ng/ml) subjects (P less than .005). (32P)orthophosphate (32Pi) incorporation into the individual phosphoinositides and phosphatidic acid (PA) reached isotopic equilibrium by 120 min for IDDM and control subjects. Specific activity (dpm 32P/micrograms phosphorus) of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was not different between IDDM and control subjects. Under these conditions, basal 32Pi incorporation into PIP2 and PIP but not phosphatidylinositol (PI) or PA was significantly lower in IDDM subjects. There was significantly decreased (32P)PIP2 and (32P)PIP hydrolysis and decreased (32P)PA formation in IDDM after platelet stimulation with 4 U/ml human thrombin. There were no differences in (32P)PI hydrolysis between the two groups. The mass of PIP2 was reduced (P less than .005) in the platelets from IDDM (0.71 +/- 0.23 nmol/10(9) platelets) compared with control (1.65 +/- 0.53 nmol/10(9) platelets) subjects. Similarly, PIP was lower (P less than .001) in IDDM (0.66 +/- 0.09 nmol/10(9) platelets) than in control (2.92 +/- 0.43 nmol/10(9) platelets) subjects.

Bastyr, E.J. 3d.; Kadrofske, M.M.; Dershimer, R.C.; Vinik, A.I. (Univ. of Michigan, Ann Arbor (USA))

1989-09-01

44

Caspase-activated phosphoinositide binding by CNT-1 promotes apoptosis by inhibiting the AKT pathway.  

PubMed

Inactivation of cell-survival factors is a crucial step in apoptosis. The phosphoinositide 3-kinase (PI3K)-AKT signaling pathway promotes cell growth, proliferation and survival, and its deregulation causes cancer. How this pathway is suppressed to promote apoptosis is poorly understood. Here we report the identification of a CED-3 caspase substrate in Caenorhabditis elegans, CNT-1, that is cleaved during apoptosis to generate an N-terminal phosphoinositide-binding fragment (tCNT-1). tCNT-1 translocates from the cytoplasm to the plasma membrane and blocks AKT binding to phosphatidylinositol (3,4,5)-trisphosphate, thereby disabling AKT activation and its prosurvival activity. Our findings reveal a new mechanism that negatively regulates AKT cell signaling to promote apoptosis and that may restrict cell growth and proliferation in normal cells. PMID:25383666

Nakagawa, Akihisa; Sullivan, Kelly D; Xue, Ding

2014-12-01

45

Urotensin-II Receptor Stimulation of Cardiac L-type Ca2+ Channels Requires the ?? Subunits of Gi/o-protein and Phosphatidylinositol 3-Kinase-dependent Protein Kinase C ?1 Isoform.  

PubMed

Recent studies have demonstrated that urotensin-II (U-II) plays important roles in cardiovascular actions including cardiac positive inotropic effects and increasing cardiac output. However, the mechanisms underlying these effects of U-II in cardiomyocytes still remain unknown. We show by electrophysiological studies that U-II dose-dependently potentiates L-type Ca(2+) currents (ICa,L) in adult rat ventricular myocytes. This effect was U-II receptor (U-IIR)-dependent and was associated with a depolarizing shift in the voltage dependence of inactivation. Intracellular application of guanosine-5'-O-(2-thiodiphosphate) and pertussis toxin pretreatment both abolished the stimulatory effects of U-II. Dialysis of cells with the QEHA peptide, but not scrambled peptide SKEE, blocked the U-II-induced response. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin as well as the class I PI3K antagonist CH132799 blocked the U-II-induced ICa,L response. Protein kinase C antagonists calphostin C and chelerythrine chloride as well as dialysis of cells with 1,2bis(2aminophenoxy)ethaneN,N,N',N'-tetraacetic acid abolished the U-II-induced responses, whereas PKC? inhibition or PKA blockade had no effect. Exposure of ventricular myocytes to U-II markedly increased membrane PKC?1 expression, whereas inhibition of PKC?1 pharmacologically or by shRNA targeting abolished the U-II-induced ICa,L response. Functionally, we observed a significant increase in the amplitude of sarcomere shortening induced by U-II; blockade of U-IIR as well as PKC? inhibition abolished this effect, whereas Bay K8644 mimicked the U-II response. Taken together, our results indicate that U-II potentiates ICa,L through the ?? subunits of Gi/o-protein and downstream activation of the class I PI3K-dependent PKC?1 isoform. This occurred via the activation of U-IIR and contributes to the positive inotropic effect on cardiomyocytes. PMID:25678708

Zhang, Yuan; Ying, Jiaoqian; Jiang, Dongsheng; Chang, Zhigang; Li, Hua; Zhang, Guoqiang; Gong, Shan; Jiang, Xinghong; Tao, Jin

2015-03-27

46

Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for alpha-platelet-derived growth factor receptor-induced production of reactive oxygen species.  

PubMed

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47(phox), and we systematically evaluated the signal relay mechanisms by which the alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. alphaPDGFR activation is rapidly followed by the translocation of p47(phox) and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47(phox)(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs that lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cgamma, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cgamma, or SHP-2) completely abolished PDGF-dependent p47(phox) and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant alphaPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha (but not p110beta) was identified as the crucial isoform that elicits alphaPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110alpha mediates growth factor-dependent ROS production by recruiting p47(phox) and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation. PMID:18070887

Bäumer, Anselm T; Ten Freyhaus, Henrik; Sauer, Heinrich; Wartenberg, Maria; Kappert, Kai; Schnabel, Petra; Konkol, Christian; Hescheler, Jürgen; Vantler, Marius; Rosenkranz, Stephan

2008-03-21

47

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

48

Autoradiographic imaging of phosphoinositide turnover in the brain  

SciTech Connect

With ({sup 3}H)cytidine as a precursor, phosphoinositide turnover can be localized in brain slices by selective autoradiography of the product ({sup 3}H)cytidine diphosphate diacylglycerol, which is membrane-bound. In the cerebellum, glutamatergic stimulation elicits an increase of phosphoinositide turnover only in Purkinje cells and the molecular layer. In the hippocampus, both glutamatergic and muscarinic cholinergic stimulation increase phosphoinositide turnover, but with distinct localizations. Cholinergic stimulation affects CA1, CA3, CA4, and subiculum, whereas glutamatergic effects are restricted to the subiculum and CA3. Imaging phosphoinositide turnover in brain slices, which are amenable to electrophysiologic studies, will permit a dynamic localized analysis of regulation of this second messenger in response to synaptic stimulation of specific neuronal pathways.

Hwang, P.M.; Bredt, D.S.; Snyder, S.H. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA))

1990-08-17

49

Phosphoinositides in the mammalian endo-lysosomal network.  

PubMed

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

2012-01-01

50

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

51

Cellular and molecular interactions of phosphoinositides and peripheral proteins.  

PubMed

Anionic lipids act as signals for the recruitment of proteins containing cationic clusters to biological membranes. A family of anionic lipids known as the phosphoinositides (PIPs) are low in abundance, yet play a critical role in recruitment of peripheral proteins to the membrane interface. PIPs are mono-, bis-, or trisphosphorylated derivatives of phosphatidylinositol (PI) yielding seven species with different structure and anionic charge. The differential spatial distribution and temporal appearance of PIPs is key to their role in communicating information to target proteins. Selective recognition of PIPs came into play with the discovery that the substrate of protein kinase C termed pleckstrin possessed the first PIP binding region termed the pleckstrin homology (PH) domain. Since the discovery of the PH domain, more than ten PIP binding domains have been identified including PH, ENTH, FYVE, PX, and C2 domains. Representative examples of each of these domains have been thoroughly characterized to understand how they coordinate PIP headgroups in membranes, translocate to specific membrane docking sites in the cell, and function to regulate the activity of their full-length proteins. In addition, a number of novel mechanisms of PIP-mediated membrane association have emerged, such as coincidence detection-specificity for two distinct lipid headgroups. Other PIP-binding domains may also harbor selectivity for a membrane physical property such as charge or membrane curvature. This review summarizes the current understanding of the cellular distribution of PIPs and their molecular interaction with peripheral proteins. PMID:24556335

Stahelin, Robert V; Scott, Jordan L; Frick, Cary T

2014-09-01

52

Phosphoinositide metabolism and adrenergic receptors in astrocytes  

SciTech Connect

Agonist-induced phosphoinositide (PI) breakdown functions as a signal generating system. Diacylglycerol, one breakdown product of phosphotidylinositol-4,5-diphosphate hydrolysis, can stimulate protein kinase C, whereas inositol triphosphate, the other product, has been proposed to be a second messenger for Ca/sup + +/ mobilization. Using purified astrocyte cultures from neonatal rat brain, the effects of adrenergic agonists and antagonists at 10/sup -5/ M were measured on PI breakdown. Astrocytes grown in culture were prelabeled with (/sup 3/H)inositol, and basal (/sup 3/H) inositol phosphate (IP/sub 1/) accumulation was measured in the presence of Li/sup +/. Epinephrine > norepinephrine (NE) were the most active stimulants of IP/sub 1/ production. The ..cap alpha../sub 1/ adrenoreceptor blockers, phentolamine and phenoxybenzamine, added alone had no effect on IP/sub 1/ production was reduced below basal levels. Propranolol partially blocked the effects of NE. Clonidine and isoproterenol, separately added, reduced IP/sub 1/ below basal levels and when added together diminished IP/sub 1/ accumulation even further. The role of adrenergic stimulation in the production of c-AMP.

Noble, E.P.; Ritchie, T.; de Vellis, J.

1986-03-01

53

Cardioprotective Stimuli Mediate Phosphoinositide 3-Kinase and Phosphoinositide Dependent Kinase 1 Nuclear Accumulation in Cardiomyocytes  

PubMed Central

The phosphoinositide-3-kinase (PI3K) / phosphoinositide dependent kinase 1 (PDK1) signaling pathway exerts cardioprotective effects in the myocardium through activation of key proteins including Akt. Activated Akt accumulates in nuclei of cardiomyocytes suggesting that biologically relevant targets are located in that subcellular compartment. Nuclear Akt activity could be potentiated in both intensity and duration by the presence of a nuclear-associated PI3K / PDK1 signaling cascade as has been described in other non-myocyte cell types. PI3K / PDK1 distribution was determined in vitro and in vivo by immunostaining and nuclear extraction of cultured rat neonatal cardiomyocytes or transgenic mouse hearts. Results show that PI3K and PDK1 are present at a basal level in cardiomyocytes nuclei and that cardioprotective stimulation with atrial natriuretic peptide (ANP) increases their nuclear localization. In comparison, overexpression of nuclear-targeted Akt does not mediate increased translocation of either PI3K or PDK1 indicating that accumulation of Akt does not drive PI3K or PDK1 into the nuclear compartment. Furthermore, PI3K and phospho-Akt473 show parallel temporal accumulation in the nucleus following (MI) infarction challenge. These findings demonstrate the presence of a dynamically regulated nuclear-associated signaling cascade involving PI3K and PDK that presumably influences nuclear Akt activation. PMID:19269295

Rubio, Marta; Avitabile, Daniele; Fischer, Kimberlee; Emmanuel, Gregory; Gude, Natalie; Miyamoto, Shigeki; Mishra, Shikha; Schaefer, Eric M.; Brown, Joan Heller; Sussman, Mark A.

2009-01-01

54

The Phosphoinositide Kinase PIKfyve Is Vital in Early Embryonic Development  

PubMed Central

Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes PtdIns(3,5)P2 and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown. To clarify the importance of PIKfyve and PIKfyve lipid products, in this study, we have characterized the first mouse model with global deletion of the pikfyve gene using the Cre-loxP approach. We report that nearly all PIKfyveKO/KO mutant embryos died before the 32–64-cell stage. Cultured fibroblasts derived from PIKfyveflox/flox embryos and rendered pikfyve-null by Cre recombinase expression displayed severely reduced DNA synthesis, consistent with impaired cell division causing early embryo lethality. The heterozygous PIKfyveWT/KO mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyveWT/KO mice were ostensibly normal by several other in vivo, ex vivo, and in vitro criteria despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50–55% lower than those of wild-type mice. Consistently, steady-state levels of the PIKfyve products PtdIns(3,5)P2 and PtdIns5P selectively decreased, but this reduction (35–40%) was 10–15% less than that expected based on PIKfyve protein reduction. The nonlinear decrease of the PIKfyve protein versus PIKfyve lipid products, the potential mechanism(s) discussed herein, may explain how one functional allele in PIKfyveWT/KO mice is able to support the demands for PtdIns(3,5)P2/PtdIns5P synthesis during life. Our data also shed light on the known human disorder linked to PIKFYVE mutations. PMID:21349843

Ikonomov, Ognian C.; Sbrissa, Diego; Delvecchio, Khortnal; Xie, Yufen; Jin, Jian-Ping; Rappolee, Daniel; Shisheva, Assia

2011-01-01

55

Regulation of Hippo pathway by mitogenic growth factors via phosphoinositide 3-kinase and phosphoinositide-dependent kinase-1  

PubMed Central

The Hippo signaling pathway inhibits cell growth and regulates organ size through a kinase cascade that leads to the phosphorylation and nuclear exclusion of the growth-promoting transcriptional coactivator Yes-associated protein (YAP)/Yorkie. It mediates contact inhibition of cell growth downstream of cadherin adhesion molecules and other cell surface proteins. Contact inhibition is often antagonized by mitogenic growth factor signaling. We report an important mechanism for this antagonism, inhibition of Hippo pathway signaling by mitogenic growth factors. EGF treatment of immortalized mammary cells triggers the rapid translocation of YAP into the nucleus along with YAP dephosphorylation, both of which depend on Lats, the terminal kinase in the Hippo pathway. A small-molecule inhibitor screen of downstream effector pathways shows that EGF receptor inhibits the Hippo pathway through activation of PI3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK1), but independent of AKT activity. The PI3K-PDK1 pathway also mediates YAP nuclear translocation downstream of lysophosphatidic acid and serum as a result of constitutive oncogenic activation of PI3K. PDK1 associates with the core Hippo pathway-kinase complex through the scaffold protein Salvador. The entire Hippo core complex dissociates in response to EGF signaling in a PI3K-PDK1–dependent manner, leading to inactivation of Lats, dephosphorylation of YAP, and YAP nuclear accumulation and transcriptional activation of its target gene, CTGF. These findings show that an important activity of mitogenic signaling pathways is to inactivate the growth-inhibitory Hippo pathway and provide a mechanism for antagonism between contact inhibition and growth factor action. PMID:23359693

Fan, Run; Kim, Nam-Gyun; Gumbiner, Barry M.

2013-01-01

56

Nortriptyline reverses corticosteroid insensitivity by inhibition of phosphoinositide-3-kinase-?.  

PubMed

Corticosteroid insensitivity represents a major barrier to the treatment of chronic obstructive pulmonary disease (COPD) and severe asthma. It is caused by oxidative stress, leading to reduced histone deacetylase-2 (HDAC2) function through activation of phosphoinositide-3-kinase-? (PI3K?). The tricyclic antidepressant nortriptyline has been identified in high-throughput screens as an agent that increases corticosteroid responsiveness. The aim of this study was to identify the molecular mechanism whereby nortriptyline increases corticosteroid sensitivity. Phosphorylation of Akt, a footprint of PI3K activation, and HDAC activity were evaluated by Western blotting and fluorescent activity assay in U937 monocytic cells. Corticosteroid sensitivity was evaluated by the inhibition of tumor necrosis factor ? (TNF?)-induced interleukin 8 (IL-8) production by budesonide. Hydrogen peroxide (H(2)O(2)) or cigarette smoke extract (CSE) increased the level of phosphorylated Akt (pAkt) and reduced HDAC activity. Pretreatment with nortriptyline inhibited pAkt induced by CSE and H(2)O(2) as well as restored HDAC activity that had been decreased by H(2)O(2) and CSE. In addition, nortriptyline inhibited PI3K? activity, but had no effect on the PI3K? and PI3K? isoforms. Although CSE reduced the effects of budesonide on TNF?-induced IL-8 production in U937 cells, nortriptyline reversed CSE-induced corticosteroid insensitivity. Nortriptyline restores corticosteroid sensitivity induced by oxidative stress via direct inhibition of PI3K? and is a potential treatment for corticosteroid-insensitive diseases such as COPD and severe asthma. PMID:21300705

Mercado, Nicolas; To, Yasuo; Ito, Kazuhiro; Barnes, Peter J

2011-05-01

57

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

58

Multiple implications of 3-phosphoinositide-dependent protein kinase 1 in human cancer  

PubMed Central

3-phosphoinositide-dependent protein kinase-1 (PDK1) is a central mediator of cellular signaling between phosphoinositide-3 kinase and various intracellular serine/threonine kinases, including protein kinase B, p70 ribosomal S6 kinase, serum and glucocorticoid-inducible kinase, and protein kinase C. PDK1 activates members of the AGC family of protein kinases by phosphorylating serine/threonine residues in the activation loop. Here, we review the regulatory mechanisms of PDK1 and its roles in cancer. PDK1 is activated by autophosphorylation in the activation loop and other serine residues, as well as by phosphorylation of Tyr-9 and Tyr-373/376. Src appears to recognize PDK1 following tyrosine phosphorylation. The role of heat shock protein 90 in regulating PDK1 stability and PDK1-Src complex formation are also discussed. Furthermore, we summarize the subcellular distribution of PDK1. Finally, an important role for PDK1 in cancer chemotherapy is proposed. In conclusion, a better understanding of its molecular regulatory mechanisms in various signaling pathways will help to explain how PDK1 acts as an oncogenic kinase in various cancers, and will contribute to the development of novel cancer chemotherapies. PMID:21537480

Li, Yuwen; Yang, Keum-Jin; Park, Jongsun

2010-01-01

59

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

60

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

61

DAPP1: a dual adaptor for phosphotyrosine and 3-phosphoinositides.  

PubMed Central

We have identified a novel 280 amino acid protein which contains a putative myristoylation site at its N-terminus followed by an Src homology (SH2) domain and a pleckstrin homology (PH) domain at its C-terminus. It has been termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1). DAPP1 is widely expressed and exhibits high-affinity interactions with PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2), but not with other phospholipids tested. These observations predict that DAPP1 will interact with both tyrosine phosphorylated proteins and 3-phosphoinositides and may therefore play a role in regulating the location and/or activity of such proteins(s) in response to agonists that elevate PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2). PMID:10432293

Dowler, S; Currie, R A; Downes, C P; Alessi, D R

1999-01-01

62

Prospects for phosphoinositide 3-kinase inhibition as a cancer treatment  

Microsoft Academic Search

The phosphoinositide 3-kinases (PI3-kinases) are a family of lipid kinases that have a key role in the regulation of many cellular processes including proliferation, survival, carbohydrate metabolism, and motility. There is now strong evidence that some members of the PI3-kinase family have an important role in cancer. Emerging evidence for functional specialisation of PI3-kinase isoforms suggests that isoform selective inhibitors,

R C Stein

2001-01-01

63

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

64

Targeting the phosphoinositide 3-kinase pathway in cancer  

Microsoft Academic Search

The phosphoinositide 3-kinase (PI3K) pathway is a key signal transduction system that links oncogenes and multiple receptor classes to many essential cellular functions, and is perhaps the most commonly activated signalling pathway in human cancer. This pathway therefore presents both an opportunity and a challenge for cancer therapy. Even as inhibitors that target PI3K isoforms and other major nodes in

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

2009-01-01

65

Essential Role of Phosphoinositide Metabolism in Synaptic Vesicle Recycling  

Microsoft Academic Search

Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1–deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich

Ottavio Cremona; Gilbert Di Paolo; Markus R Wenk; Anita Lüthi; Warren T Kim; Kohji Takei; Laurie Daniell; Yasuo Nemoto; Stephen B Shears; Richard A Flavell; David A McCormick; Pietro De Camilli

1999-01-01

66

Nuclear phospholipid signaling: phosphatidylinositol-specific phospholipase C and phosphoinositide 3-kinase  

Microsoft Academic Search

Over the last 20 years, numerous studies have demonstrated the existence of nuclear phosphoinositide signaling distinct from\\u000a the one at the plasma membrane. The activation of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphoinositide\\u000a 3-kinase (PI3K), the generation of diacylglycerol, and the accumulation of the 3-phosphorylated phosphoinositides have been\\u000a documented in the nuclei of different cell types. In this review, we summarize some

Dora Visnjic; Hrvoje Banfic

2007-01-01

67

Transformation by the k-ras oncogene correlates with increases in phospholipase A2 activity, glycerophosphoinositol production and phosphoinositide synthesis in thyroid cells.  

PubMed

Two cell lines transformed by the k-ras oncogene (KiKi and KiMol cells) and a temperature sensitive clone (Ts), all originated from a normal rat thyroid line (FRTL5 cells), have been employed to analyse the intracellular mechanisms affected by the ras p21. In k-ras transformed cells two phosphoinositide derivatives, glycerophosphoinositol and inositol monophosphate, were markedly increased, whereas inositol bisphosphate and trisphosphate maintained the same level as in normal cells. Cytosolic Ca2+ was also unaffected. This indicates that in epithelial cells the phospholipase C activity is not altered upon ras transformation. The formation of glycerophosphoinositol involved the activation of a phosphoinositide specific phospholipase A2. The higher phospholipase A2 activity in ras transformed cells could be further demonstrated by the increase in total arachidonic acid release. In the Ts clone the increase in glycerophosphoinositol and inositol monophosphate was evident only at the permissive temperature (33 degrees C), whereas it disappeared at 39 degrees C. At 33 degrees C the cells were also characterized by an enriched membrane pool of phosphoinositides. All these changes occurred in parallel with morphological transformation. We propose that cell transformation by the k-ras oncogene affects different steps of the membrane lipid metabolism, among which the most prominent one is the activation of a phosphoinositide specific phospholipase A2. These effects could originate mitogenic metabolites. Moreover, they correlate well with the induction of the malignant phenotype. PMID:1657098

Valitutti, S; Cucchi, P; Colletta, G; Di Filippo, C; Corda, D

1991-01-01

68

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

69

Effects of thyroxine and 1-methyl, 2-mercaptoimidazol on phosphoinositides synthesis in rat liver  

Microsoft Academic Search

BACKGROUND: Phosphoinositides mediate one of the intracellular signal transduction pathways and produce a class of second messengers that are involved in the action of hormones and neurotransmitters on target cells. Thyroid hormones are well known regulators of lipid metabolism and modulators of signal transduction in cells. However, little is known about phosphoinositides cycle regulation by thyroid hormones. The present paper

Nataliya A Babenko; Oksana A Krasilnikova

2004-01-01

70

Quantitation of adenosine-5'-triphosphate used for phosphoinositide metabolism in human erythrocytes  

SciTech Connect

The human erythrocyte actively phosphorylates and dephosphorylates phosphatidylinositol present in the membrane in an apparent ''futile cycle.'' Recent reports have proposed that this phosphorylation/dephosphorylation cycle is a significant consumer of adenosine-5'-triphosphate (ATP) in the erythrocyte. This study details two independent techniques for quantitating the ATP consumed by this phosphoinositide futile cycle. With the first technique a quasi-steady-state labeling of erythrocyte ATP with TSP-phosphate was obtained, and the rate of synthesis of TSP-phosphoinositides was then monitored. The second technique used a novel labeling strategy that allowed only ATP to be labeled with TSP; the transfer of TSP from ATP to phosphoinositides was then an independent measure of the ATP consumed for phosphoinositide synthesis. These two techniques documented that 0.5% to 1.0% of net ATP produced by the erythrocyte is used for phosphoinositide synthesis.

Dale, G.L.

1985-11-01

71

Phosphoinositide isoforms determine compartment-specific ion channel activity  

PubMed Central

Phosphoinositides serve as address labels for recruiting peripheral cytoplasmic proteins to specific subcellular compartments, and as endogenous factors for modulating the activity of integral membrane proteins. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a plasma-membrane (PM)–specific phosphoinositide and a positive cofactor required for the activity of most PM channels and transporters. This requirement for phosphoinositide cofactors has been proposed to prevent PM channel/transporter activity during passage through the biosynthetic/secretory and endocytic pathways. To determine whether intracellularly localized channels are similarly “inactivated” at the PM, we studied PIP2 modulation of intracellular TRPML1 channels. TRPML1 channels are primarily localized in lysosomes, but can also be detected temporarily in the PM upon lysosomal exocytosis. By directly patch-clamping isolated lysosomes, we previously found that lysosomal, but not PM-localized, TRPML1 is active with PI(3,5)P2, a lysosome-specific PIP2, as the underlying positive cofactor. Here we found that “silent” PM-localized TRPML1 could be activated by depleting PI(4,5)P2 levels and/or by adding PI(3,5)P2 to inside-out membrane patches. Unlike PM channels, surface-expressed TRPML1 underwent a unique and characteristic run-up upon patch excision, and was potently inhibited by a low micromolar concentration of PI(4,5)P2. Conversely, depletion of PI(4,5)P2 by either depolarization-induced activation or chemically induced translocation of 5?-phosphatase potentiated whole-cell TRPML1 currents. PI(3,5)P2 activation and PI(4,5)P2 inhibition of TRPML1 were mediated by distinct basic amino acid residues in a common PIP2-interacting domain. Thus, PI(4,5)P2 may serve as a negative cofactor for intracellular channels such as TRPML1. Based on these results, we propose that phosphoinositide regulation sets compartment-specific activity codes for membrane channels and transporters. PMID:22733759

Zhang, Xiaoli; Li, Xinran; Xu, Haoxing

2012-01-01

72

Acute and long-term inhibition of agonist-stimulated phosphoinositide hydrolysis by pulse treatment of cerebellar granule cells with TPA  

Microsoft Academic Search

Acute pretreatment (30 min) of primary cultures of cerebellar granule cells with TPA (10 nM) resulted in a decrease in carbachol- and glutamate-stimulated phosphoinositide hydrolysis, but not in basal levels of PI\\u000a hydrolysis. To investigate the mechanism of TPA action, phospholipase C was assayed in membranes prepared from cerebellar\\u000a granule cells acutely treated with TPA. TPA had no effect on

Ragnhild E. Paulsen; Robert Raulli; Dennis R. Grayson; Jarda T. Wroblewski

1994-01-01

73

Phosphoinositides in the hepatitis C virus life cycle.  

PubMed

Eukaryotes possess seven different phosphoinositides (PIPs) that help form the unique signatures of various intracellular membranes. PIPs serve as docking sites for the recruitment of specific proteins to mediate membrane alterations and integrate various signaling cascades. The spatio-temporal regulation of PI kinases and phosphatases generates distinct intracellular hubs of PIP signaling. Hepatitis C virus (HCV), like other plus-strand RNA viruses, promotes the rearrangement of intracellular membranes to assemble viral replication complexes. HCV stimulates enrichment of phosphatidylinositol 4-phosphate (PI4P) pools near endoplasmic reticulum (ER) sites by activating PI4KIII?, the kinase responsible for generation of ER-specific PI4P pools. Inhibition of PI4KIII? abrogates HCV replication. PI4P, the most abundant phosphoinositide, predominantly localizes to the Golgi and plays central roles in Golgi secretory functions by recruiting effector proteins involved in transport vesicle generation. The PI4P effector proteins also include the lipid-transfer and structural proteins such as ceramide transfer protein (CERT), oxysterol binding protein (OSBP) and Golgi phosphoprotein 3 (GOLPH3) that help maintain Golgi-membrane composition and structure. Depletion of Golgi-specific PI4P pools by silencing PI4KIII?, expression of dominant negative CERT and OSBP mutants, or silencing GOLPH3 perturb HCV secretion. In this review we highlight the role of PIPs and specifically PI4P in the HCV life cycle. PMID:23202467

Bishé, Bryan; Syed, Gulam; Siddiqui, Aleem

2012-10-01

74

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

75

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

76

VISUALIZIATION OF CELLULAR PHOSPHOINOSITIDE POOLS WITH GFP-FUSED PROTEIN-DOMAINS  

PubMed Central

This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present only in tiny amounts compared to structural lipids but are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane-recruitment and activity of many protein signaling-complexes in specific membrane compartments and have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single cell level. The only available technique in live cell application is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules when fused to fluorescent proteins can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular resolution and rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. Here, we summarize the design and practical use of these constructs and also review important considerations for the interpretation of the data obtained by this technique. PMID:19283730

Balla, Tamas; Várnai, Péter

2011-01-01

77

Phosphoinositide 3-Kinase Signaling in Retinal Rod Photoreceptors  

PubMed Central

Purpose. Phosphoinositide 3-kinase (PI3K) consists of a p110 catalytic protein and a p85? regulatory protein, required for the stabilization and localization of p110-PI3K activity. The biological significance of PI3K was investigated in vertebrate rod photoreceptors by deleting its regulatory p85? protein and examining its role in photoreceptor structure, function, and protein trafficking. Methods. Mice that expressed Cre recombinase in rods were bred to mice with a floxed p85? (pik3r1) regulatory subunit of PI3K to generate a conditional deletion of pik3r1 in rods. Functional and structural changes were determined by ERG and morphometric analysis, respectively. PI3K activity was measured in retinal homogenates immunoprecipitated with an anti-PY antibody. Akt activation was determined by Western blot analysis with a pAkt antibody. Results. Light-induced stress increased PI3K activity in retinal immunoprecipitates and phosphorylation of Akt. There was no effect of pik3r1 deletion on retinal structure. However, twin flash electroretinography revealed a slight delay in recovery kinetics in pik3r1 knockout (KO) mice compared with wild-type controls. The movement of arrestin in the pik3r1 KO mice was slower than that in the wild-type mouse retinas at 5 minutes of exposure to light. At 10 minutes of exposure, the ROS localization of arrestin was almost identical between the wild-type and pik3r1 KO mice. Conclusions. The results provide the first direct evidence that rods use PI3K-generated phosphoinositides for photoreceptor function. The lack of phenotype in pik3r1 KO rod photoreceptors suggests a redundant role in controlling PIP3 synthesis. PMID:21730346

Ivanovic, Ivana; Allen, Dustin T.; Dighe, Radhika; Le, Yun Z.; Anderson, Robert E.

2011-01-01

78

Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases  

E-print Network

Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases target combinations in these two families. Crystal structures revealed that the dual selectivity for combinations of targets that are structurally divergent. Here we report the systematic discovery of molecules

Williams, Roger L.

79

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

80

D-3 phosphoinositides of the ciliate Tetrahymena: Characterization and study of their regulatory role in lysosomal enzyme secretion  

Microsoft Academic Search

Phosphatidylinositol 3-phosphate, PtdIns(3)P, is a phosphoinositide which is implicated in regulating membrane trafficking in both mammalian and yeast cells. It also serves as a precursor for the synthesis of phosphatidylinositol 3,5-bisphosphate, PtdIns(3,5)P2, a phosphoinositide, the exact functions of which remain unknown. In this report, we show that these two phosphoinositides are constitutive lipid components of the ciliate Tetrahymena. Using HPLC

George Leondaritis; Arno Tiedtke; Dia Galanopoulou

2005-01-01

81

Phosphoinositide system-linked serotonin receptor subtypes and their pharmacological properties and clinical correlates.  

PubMed Central

Serotonergic neurotransmission represents a complex mechanism involving pre- and post-synaptic events and distinct 5-HT receptor subtypes. Serotonin (5-HT) receptors have been classified into several categories, and they are termed as 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7 type receptors. 5-HT1 receptors have been further subdivided into 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E and 5-HT1F. 5-HT2 receptors have been divided into 5-HT2A, 5-HT2B and 5-HT2C receptors. All 5-HT2 receptor subtypes are linked to the multifunctional phosphoinositide (PI) signalling system. 5-HT3 receptors are considered ion-gated receptors and are also linked to the PI signalling system by an unknown mechanism. The 5-HT2A receptor subtype is the most widely studied of the 5-HT receptors in psychiatric disorders (for example, suicide, depression and schizophrenia) as well as in relation to the mechanism of action of antidepressant drugs. The roles of 5-HT2C and 5-HT3 receptors in psychiatric disorders are less clear. These 5-HT receptors also play an important role in alcoholism. It has been shown that 5-HT2A, 5-HT2C and 5-HT3 antagonists cause attenuation of alcohol intake in animals and humans. However, the exact mechanisms are unknown. The recent cloning of the cDNAs for 5-HT2A, 5-HT2C and 5-HT3 receptors provides the opportunity to explore the molecular mechanisms responsible for the alterations in these receptors during illness as well as pharmacotherapy. This review article will focus on the current research into the pharmacological properties, molecular biology, and clinical correlates of 5-HT2A, 5-HT2C and 5-HT3 receptors. PMID:7786883

Pandey, S C; Davis, J M; Pandey, G N

1995-01-01

82

Inositol Pentakisphosphate Isomers Bind PH Domains with Varying Specificity and Inhibit Phosphoinositide Interactions  

SciTech Connect

PH domains represent one of the most common domains in the human proteome. These domains are recognized as important mediators of protein-phosphoinositide and protein-protein interactions. Phosphoinositides are lipid components of the membrane that function as signaling molecules by targeting proteins to their sites of action. Phosphoinositide based signaling pathways govern a diverse range of important cellular processes including membrane remodeling, differentiation, proliferation and survival. Myo-Inositol phosphates are soluble signaling molecules that are structurally similar to the head groups of phosphoinositides. These molecules have been proposed to function, at least in part, by regulating PH domain-phosphoinositide interactions. Given the structural similarity of inositol phosphates we were interested in examining the specificity of PH domains towards the family of myo-inositol pentakisphosphate isomers. In work reported here we demonstrate that the C-terminal PH domain of pleckstrin possesses the specificity required to discriminate between different myo-inositol pentakisphosphate isomers. The structural basis for this specificity was determined using high-resolution crystal structures. Moreover, we show that while the PH domain of Grp1 does not possess this high degree of specificity, the PH domain of protein kinase B does. These results demonstrate that some PH domains possess enough specificity to discriminate between myo-inositol pentakisphosphate isomers allowing for these molecules to differentially regulate interactions with phosphoinositides. Furthermore, this work contributes to the growing body of evidence supporting myo-inositol phosphates as regulators of important PH domain-phosphoinositide interactions. Finally, in addition to expanding our knowledge of cellular signaling, these results provide a basis for developing tools to probe biological pathway.

S Jackson; S Al-Saigh; C Schultz; M Junop

2011-12-31

83

Oxysterol-binding proteins: sterol and phosphoinositide sensors coordinating transport, signaling and metabolism.  

PubMed

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of sterol and phosphoinositide binding proteins conserved in eukaryotes. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and control the activity of enzymatic effectors or assembly of protein complexes, with impacts on signaling, vesicle transport, and lipid metabolism. An increasing number of protein interaction partners of ORPs have been identified, providing clues of their involvement in multiple aspects of cell regulation. The functions assigned for mammalian ORPs include coordination of sterol and sphingolipid metabolism and mitogenic signaling (OSBP), control of ER-late endosome (LE) contacts and LE motility (ORP1L), neutral lipid metabolism (ORP2), cell adhesion (ORP3), cholesterol eggress from LE (ORP5), macrophage lipid homeostasis, migration and high-density lipoprotein metabolism (ORP8), apolipoprotein B-100 secretion (ORP10), and adipogenesis (ORP11). The anti-proliferative ORPphilin compounds target OSBP and ORP4, revealing a function of ORPs in cell proliferation and survival. The Saccharomyces cerevisiae OSBP homologue (Osh) proteins execute multifaceted functions in sterol and sphingolipid homeostasis, post-Golgi vesicle transport, as well as phosphatidylinositol-4-phosphate and target of rapamycin complex 1 (TORC1) signaling. These observations identify ORPs as coordinators of lipid signals with an unforeseen variety of cellular processes. PMID:23830809

Olkkonen, Vesa M; Li, Shiqian

2013-10-01

84

Role of Phosphoinositide 3-OH Kinase p110? in Skeletal Myogenesis.  

PubMed

Phosphoinositide 3-OH kinase (PI3K) regulates a number of developmental and physiologic processes in skeletal muscle; however, the contributions of individual PI3K p110 catalytic subunits to these processes are not well-defined. To address this question, we investigated the role of the 110-kDa PI3K catalytic subunit ? (p110?) in myogenesis and metabolism. In C2C12 cells, pharmacological inhibition of p110? delayed differentiation. We next generated mice with conditional deletion of p110? in skeletal muscle (p110? muscle knockout [p110?-mKO] mice). While young p110?-mKO mice possessed a lower quadriceps mass and exhibited less strength than control littermates, no differences in muscle mass or strength were observed between genotypes in old mice. However, old p110?-mKO mice were less glucose tolerant than old control mice. Overexpression of p110? accelerated differentiation in C2C12 cells and primary human myoblasts through an Akt-dependent mechanism, while expression of kinase-inactive p110? had the opposite effect. p110? overexpression was unable to promote myoblast differentiation under conditions of p110? inhibition, but expression of p110? was able to promote differentiation under conditions of p110? inhibition. These findings reveal a role for p110? during myogenesis and demonstrate that long-term reduction of skeletal muscle p110? impairs whole-body glucose tolerance without affecting skeletal muscle size or strength in old mice. PMID:25605332

Matheny, Ronald W; Riddle-Kottke, Melissa A; Leandry, Luis A; Lynch, Christine M; Abdalla, Mary N; Geddis, Alyssa V; Piper, David R; Zhao, Jean J

2015-04-01

85

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

86

NuMA interacts with phosphoinositides and links the mitotic spindle with the plasma membrane.  

PubMed

The positioning and the elongation of the mitotic spindle must be carefully regulated. In human cells, the evolutionary conserved proteins LGN/G?i1-3 anchor the coiled-coil protein NuMA and dynein to the cell cortex during metaphase, thus ensuring proper spindle positioning. The mechanisms governing cortical localization of NuMA and dynein during anaphase remain more elusive. Here, we report that LGN/G?i1-3 are dispensable for NuMA-dependent cortical dynein enrichment during anaphase. We further establish that NuMA is excluded from the equatorial region of the cell cortex in a manner that depends on the centralspindlin components CYK4 and MKLP1. Importantly, we reveal that NuMA can directly associate with PtdInsP (PIP) and PtdInsP2 (PIP2) phosphoinositides in vitro. Furthermore, chemical or enzymatic depletion of PIP/PIP2 prevents NuMA cortical localization during mitosis, and conversely, increasing PIP2 levels augments mitotic cortical NuMA. Overall, our study uncovers a novel function for plasma membrane phospholipids in governing cortical NuMA distribution and thus the proper execution of mitosis. PMID:24996901

Kotak, Sachin; Busso, Coralie; Gönczy, Pierre

2014-08-18

87

Stimulation of phosphoinositide hydrolysis by thrombin in embryonic chick heart cells  

SciTech Connect

The authors have shown that muscarinic receptor stimulation results in phosphoinositide (PI) hydrolysis in dissociated embryonic chick heart cells, with maximal stimulation produced by 1 mM carbachol. In an effort to identify other stimulators of PI turnover, they studied the effects of thrombin in primary cultures of 13-day embryonic chick heart cells. Thrombin increased PI hydrolysis, as measured by the accumulation of (/sup 3/H)inositol 1-phosphate in the presence of 10 mM LiCl. Stimulation occurred in a dose-dependent manner with half-maximal stimulation obtained at 0.07 U/ml and maximal stimulation at 0.3 U/ml thrombin. The effects of maximal doses of carbachol and thrombin were additive, suggesting different mechanisms of action or different populations of cells. Effects of both hormones were inhibited by pretreatment of the monolayer cultures with 4..beta..-phorbol 12 ..beta..-myristate 13..cap alpha..-acetate. The simultaneous addition of hirudin with thrombin blocked thrombin-stimulated PI turnover. An active-site-blocked derivative of thrombin was ineffective in stimulating IP formation. These data indicate a proteolytic action of thrombin in stimulation of the PI response. To their knowledge, these are the first data suggesting that there may be thrombin receptors or effects of thrombin on heart cells.

Jones, L.G.; Brown, J.H.

1986-03-05

88

Assembly of a Fab1 Phosphoinositide Kinase Signaling Complex Requires the Fig4 Phosphoinositide Phosphatase  

PubMed Central

Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] regulates several vacuolar functions, including acidification, morphology, and membrane traffic. The lipid kinase Fab1 converts phosphatidylinositol-3-phosphate [PtdIns(3)P] to PtdIns(3,5)P2. PtdIns(3,5)P2 levels are controlled by the adaptor-like protein Vac14 and the Fig4 PtdIns(3,5)P2-specific 5-phosphatase. Interestingly, Vac14 and Fig4 serve a dual function: they are both implicated in the synthesis and turnover of PtdIns(3,5)P2 by an unknown mechanism. We now show that Fab1, through its chaperonin-like domain, binds to Vac14 and Fig4 and forms a vacuole-associated signaling complex. The Fab1 complex is tethered to the vacuole via an interaction between the FYVE domain in Fab1 and PtdIns(3)P on the vacuole. Moreover, Vac14 and Fig4 bind to each other directly and are mutually dependent for interaction with the Fab1 kinase. Our observations identify a protein complex that incorporates the antagonizing Fab1 lipid kinase and Fig4 lipid phosphatase into a common functional unit. We propose a model explaining the dual roles of Vac14 and Fig4 in the synthesis and turnover of PtdIns(3,5)P2. PMID:18653468

Botelho, Roberto J.; Efe, Jem A.; Teis, David

2008-01-01

89

The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth.  

PubMed Central

Phosphoinositide 3-kinases (PI3Ks) have been identified in an evolutionarily diverse range of organisms, including mammals, Drosophila, yeast, plants and Dictyostelium. They are activated by a multitude of extracellular signals and implicated in mitogenesis, differentiation and cell survival, as well as in the control of the cytoskeleton and cell shape. Here we describe the molecular and functional analysis of Drosophila p110 (Dp110). A full-length Dp110 cDNA was isolated and found to encode a protein homologous throughout its length to the class I mammalian PI3Ks p110alpha and p110beta. Overexpression of Dp110 in wing or eye imaginal discs resulted in flies with enlarged wings or eyes respectively. In contrast, overexpression of Dp110 containing a mutation predicted to result in the loss of catalytic activity resulted in smaller wings and eyes. The alterations in wing size result from changes in both cell size and cell number, whereas in the eye only differences in cell size were detected. These data imply a role for Dp110 in growth control during Drosophila development and have implications for the function of class I PI3Ks in other organisms. Images PMID:8978685

Leevers, S J; Weinkove, D; MacDougall, L K; Hafen, E; Waterfield, M D

1996-01-01

90

Attenuation of phosphoinositide 3-kinase ? signaling restrains autoimmune disease.  

PubMed

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by the production of autoantibodies against nuclear components. Lyn-deficient mice are an excellent animal model of SLE manifesting clinical, pathological and biochemical features seen in the human disease. They develop autoreactive antibodies, glomerulonephritis and show generalized inflammation, and their B cells have a hyperactive phenotype. Since loss of Lyn confers hyper-activation of phosphoinositide 3-kinase (PI3K) signaling, we studied the effect of down-modulating PI3K in Lyn-deficient mice. We found that heterozygous inactivation of the p110? isoform of PI3K was sufficient to restrain disease in Lyn-deficient mice, leading to significantly decreased autoantibody development and autoimmune-mediated kidney pathology, and improved survival. Intriguingly, haploinsufficiency of p110? did not dampen signaling in Lyn-deficient B cells. However, plasma cell numbers, serum immunoglobulin titers, inflammation and T cell signaling and activation were significantly moderated in Lyn(-/-)p110?(+/KD) mice. Importantly, we have shown that haploinsufficiency of p110? has minor effects on the B cell compartment per se but leads to significant defects in T cell activation and B cell class-switching. These studies suggest that agents targeting p110? PI3K need not achieve full blockade of the enzyme to be of great benefit in the treatment of SLE. PMID:22537464

Maxwell, Mhairi J; Tsantikos, Evelyn; Kong, Anne M; Vanhaesebroeck, Bart; Tarlinton, David M; Hibbs, Margaret L

2012-06-01

91

Phosphoinositide 3-kinase in T cell activation and survival.  

PubMed

PI3Ks (phosphoinositide 3-kinases) regulate diverse signalling pathways involved in growth, proliferation, survival, differentiation and metabolism. In T cells, PI3Ks can be activated by a number of different receptors, including the TcR (T cell receptor), co-stimulatory receptors, cytokine receptors and chemokine receptors. However, the specific roles of PI3Ks downstream of these receptors vary. An inactivating mutation in the leucocyte-specific PI3K isoform p110delta results in impaired TcR-dependent proliferation under circumstances where CD28 co-stimulation is blocked or not required. Recruitment and activation of PI3K by CD28 promotes survival by inducing increased expression of Bcl-X(L). However, CD28 engages additional signals that regulate proliferation and interleukin-2 production independently of PI3K. Thus a model emerges whereby PI3K is involved in both TcR and CD28 signalling, but each receptor may only exploit a subset of the signalling pathways potentially controlled by PI3K activation. PMID:15046602

Okkenhaug, K; Bilancio, A; Emery, J L; Vanhaesebroeck, B

2004-04-01

92

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

93

Cholinergic stimulation of phosphoinositide hydrolysis in rabbit kidney slices  

SciTech Connect

The release of inositol phosphates (IP) from phosphoinositides (PI) by carbachol was studied in the tissue slices from cortex (C), outer medulla (OM) and inner medulla (IM) of rabbit kidneys. The method involved the incubation of the slices with (/sup 3/H)inositol for its incorporation into the PI and measurement of the release of IP in presence of lithium which prevents dephosphorylation of IP. The results of (/sup 3/H)IP formation are expressed as % of total (/sup 3/H)inositol incorporation in the tissue. No significant effect of carbachol was found on the release of IP in the C. The drug produced a 48% increase in IP release in the OM. In the IM, carbachol produced a concentration dependent increase in IP release with a maximum of 772% at 1 mM. The release of IP in the IM by 1 mM carbachol was completely blocked by 1 ..mu..M atropine. Our results indicate that IP release by carbachol is due to activation of muscarinic receptors in the IM of the rabbit kidney.

Garg, L.C.; McArdle, S.; Crews, F.T.

1986-03-01

94

Kappa opioid receptors stimulate phosphoinositide turnover in rat brain  

SciTech Connect

The effects of various subtype-selective opioid agonists and antagonists on the phosphoinositide (PI) turnover response were investigated in the rat brain. The {kappa}-agonists U-50,488H and ketocyclazocine produced a concentration-dependent increase in the accumulation of IP's in hippocampal slices. The other {kappa}-agonists Dynorphin-A (1-13) amide, and its protected analog D(Ala){sup 2}-dynorphin-A (1-13) amide also produced a significant increase in the formation of ({sup 3}H)-IP's, whereas the {mu}-selective agonists (D-Ala{sup 2}-N-Me-Phe{sup 4}-Gly{sup 5}-ol)-enkephalin and morphine and the {delta}-selective agonist (D-Pen{sup 2,5})-enkephalin were ineffective. The increase in IP's formation elicited by U-50,488H was partially antagonized by naloxone and more completely antagonized by the {kappa}-selective antagonists nor-binaltorphimine and MR 2266. The formation of IP's induced by U-50,488H varies with the regions of the brain used, being highest in hippocampus and amygdala, and lowest in striatum and pons-medullar. The results indicate that brain {kappa}- but neither {mu}- nor {delta}- receptors are coupled to the PI turnover response.

Periyasamy, S.; Hoss, W. (Univ. of Toledo, OH (USA))

1990-01-01

95

Characterization of phosphoinositide-specific phospholipase C from human platelets.  

PubMed Central

Phosphoinositide-specific phospholipase C (PI-PLC) from human platelet cytosol was purified 190-fold to a specific activity of 0.68 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein. It hydrolyses PI and phosphatidylinositol 4,5-bisphosphate (PIP2), but not phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. The enzyme exhibits an acid pH optimum of 5.5 and has a molecular mass of 98 kDa as determined by Sephacryl S-200 gel filtration. It required millimolar concentrations of Ca2+ for PI hydrolysis, whereas micromolar concentrations are optimal for PIP2 hydrolysis. Mg2+ could substitute for Ca2+ when PIP2, but not PI, was used as the substrate. EDTA was more effective than EGTA in inhibiting the basal PI-PLC activity towards PIP2. Sodium deoxycholate strongly inhibits the purified PI-PLC activity with either PI or PIP2 as substrate. Ras proteins, either alone or in the form of liposomes, have no effect on PI-PLC activity. Images Fig. 2. PMID:2821991

Manne, V; Kung, H F

1987-01-01

96

A Drosophila neuroligin regulates synaptic growth and function in response to activity and phosphoinositide-3-kinase  

PubMed Central

Neuroligins are postsynaptic neural cell adhesion molecules that mediate synaptic maturation and function in vertebrates and invertebrates, but their mechanisms of action and regulation are not well understood. At the Drosophila larval neuromuscular junction (NMJ), previous analysis demonstrated a requirement for Drosophila neuroligin 1 (dnlg1) in synaptic growth and maturation. The goal of the present study was to better understand the effects and mechanisms of loss-of-function and overexpression of dnlg1 on synapse size and function, and to identify signaling pathways that control dnlg1 expression. Consistent with a reduced synapse size, evoked excitatory junctional currents (EJCs) were diminished in dnlg1 mutants but displayed normal Ca2+ sensitivity and short-term plasticity. However, postsynaptic function was also perturbed, in that glutamate receptor staining and the distribution of amplitudes of miniature excitatory junctional currents (mEJCs) were abnormal in mutants. All the above phenotypes were rescued by a genomic transgene. Overexpression of dnlg1 in muscle resulted in synaptic overgrowth, but reduced the amplitudes of EJCs and mEJCs. Overgrowth and reduced EJC amplitude required dnrx1 function, suggesting that increased dnlg1/dnrx1 signaling attenuates synaptic transmission and regulates growth through a retrograde mechanism. In contrast, reduced mEJC amplitude was independent of dnrx1. Synaptic overgrowth, triggered by neuronal hyperactivity, absence of the E3 ubiquitin ligase highwire, and increased phosphoinositide-3-kinase (PI3K) signaling in motor neurons reduced synaptic DNlg1 levels. Likewise, postsynaptic attenuation of PI3K, which increases synaptic strength, was associated with reduced DNlg1 levels. These observations suggest that activity and PI3K signaling pathways modulate growth and synaptic transmission through dnlg1-dependent mechanisms. PMID:22954894

Mozer, Brian A.; Sandstrom, David J.

2012-01-01

97

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

98

Dynamic steps in receptor tyrosine kinase mediated activation of class IA phosphoinositide 3-kinases (PI3K)  

E-print Network

Dynamic steps in receptor tyrosine kinase mediated activation of class IA phosphoinositide 3-kinases (PI3K) captured by H/D exchange (HDX-MS) John E. Burke*, Roger L. Williams* Medical Research of all class IA phosphoinositide 3-kinases (PI3Ks) associate with identical p85-related subunits and phos

Williams, Roger L.

99

Dynamics of the Phosphoinositide 3-Kinase p110d Interaction with p85a and Membranes Reveals  

E-print Network

Structure Article Dynamics of the Phosphoinositide 3-Kinase p110d Interaction with p85a.06.003 SUMMARY Phosphoinositide 3-kinase d is upregulated in lymphocytic leukemias. Because the p85-regulatory of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2

Williams, Roger L.

100

Binding selectivity studies of phosphoinositide 3-kinases using free energy calculations.  

PubMed

Phosphoinositide 3-kinases (PI3Ks) and their phosphatidylinositol 3,4,5-triphosphate (PIP(3)) products regulate a variety of cellular processes. Of these, PI3K? is an attractive target for anticancer drug design. Mutations in the PI3K? kinase domain alter the mobility of the activation loop resulting in gain of function. We employed molecular dynamics (MD) simulations-based energetic analysis using molecular mechanics/generalized born surface area (MM/GBSA) for PI3K? and -?. MD simulations were carried out for PI3K models based on the RESP (restrained electrostatic potential) and quantum mechanics (QM)-polarized ligand docking (QPLD)-derived partial charges. Computational alanine scanning was also used to evaluate the contributions of key binding residues to ligand binding. Our results show that both RESP and QPLD charge models of PI3K? and PI3K? provide similar performance in MD simulations. For example, the predicted RESP and QPLD free energies of -9.5 and -9.3 kcal/mol for LY294002 binding to PI3K? and -10.9 and -11.7 kcal/mol for wortmannin binding to PI3K? are in good agreement with experimental values. A significant loss in binding free energy was observed when hydrophobic residues were mutated to alanine, suggesting that specific hydrophobic interactions are important to optimal ligand binding. MM/GBSA calculations suggested that residues Ser774, Gln859, and Ile932 of PI3K? might be used to design H1047R mutant-specific ligands, whereas Lys890 of PI3K? can be used for ligand design targeting PI3K?. PMID:23157418

Sabbah, Dima A; Vennerstrom, Jonathan L; Zhong, Haizhen A

2012-12-21

101

Genome-Wide Analysis of the Phosphoinositide Kinome from Two Ciliates Reveals Novel Evolutionary Links for Phosphoinositide Kinases in Eukaryotic Cells  

PubMed Central

Background The complexity of phosphoinositide signaling in higher eukaryotes is partly due to expansion of specific families and types of phosphoinositide kinases (PIKs) that can generate all phosphoinositides via multiple routes. This is particularly evident in the PI3Ks and PIPKs, and it is considered an evolutionary trait associated with metazoan diversification. Yet, there are limited comprehensive studies on the PIK repertoire of free living unicellular organisms. Methodology/Principal Findings We undertook a genome-wide analysis of putative PIK genes in two free living ciliated cells, Tetrahymena and Paramecium. The Tetrahymena thermophila and Paramecium tetraurelia genomes were probed with representative kinases from all families and types. Putative homologs were verified by EST, microarray and deep RNA sequencing database searches and further characterized for domain structure, catalytic efficiency, expression patterns and phylogenetic relationships. In total, we identified and characterized 22 genes in the Tetrahymena thermophila genome and 62 highly homologues genes in Paramecium tetraurelia suggesting a tight evolutionary conservation in the ciliate lineage. Comparison to the kinome of fungi reveals a significant expansion of PIK genes in ciliates. Conclusions/Significance Our study highlights four important aspects concerning ciliate and other unicellular PIKs. First, ciliate-specific expansion of PI4KIII-like genes. Second, presence of class I PI3Ks which, at least in Tetrahymena, are associated with a metazoan-type machinery for PIP3 signaling. Third, expansion of divergent PIPK enzymes such as the recently described type IV transmembrane PIPKs. Fourth, presence of possible type II PIPKs and presumably inactive PIKs (hence, pseudo-PIKs) not previously described. Taken together, our results provide a solid framework for future investigation of the roles of PIKs in ciliates and indicate that novel functions and novel regulatory pathways of phosphoinositides may be more widespread than previously thought in unicellular organisms. PMID:24244373

Leondaritis, George; Siokos, John; Skaripa, Irini; Galanopoulou, Dia

2013-01-01

102

Phosphoinositide-3 Kinase-Akt Pathway Controls Cellular Entry of Ebola Virus  

PubMed Central

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. A novel and critical role of the PI3K signaling pathway was demonstrated in cell entry of Zaire Ebola virus (ZEBOV). Inhibitors of PI3K and Akt significantly reduced infection by ZEBOV at an early step during the replication cycle. Furthermore, phosphorylation of Akt-1 was induced shortly after exposure of cells to radiation-inactivated ZEBOV, indicating that the virus actively induces the PI3K pathway and that replication was not required for this induction. Subsequent use of pseudotyped Ebola virus and/or Ebola virus-like particles, in a novel virus entry assay, provided evidence that activity of PI3K/Akt is required at the virus entry step. Class 1A PI3Ks appear to play a predominant role in regulating ZEBOV entry, and Rac1 is a key downstream effector in this regulatory cascade. Confocal imaging of fluorescently labeled ZEBOV indicated that inhibition of PI3K, Akt, or Rac1 disrupted normal uptake of virus particles into cells and resulted in aberrant accumulation of virus into a cytosolic compartment that was non-permissive for membrane fusion. We conclude that PI3K-mediated signaling plays an important role in regulating vesicular trafficking of ZEBOV necessary for cell entry. Disruption of this signaling leads to inappropriate trafficking within the cell and a block in steps leading to membrane fusion. These findings extend our current understanding of Ebola virus entry mechanism and may help in devising useful new strategies for treatment of Ebola virus infection. PMID:18769720

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

2008-01-01

103

Nortriptyline Reverses Corticosteroid Insensitivity by Inhibition of Phosphoinositide-3-Kinase-?S?  

PubMed Central

Corticosteroid insensitivity represents a major barrier to the treatment of chronic obstructive pulmonary disease (COPD) and severe asthma. It is caused by oxidative stress, leading to reduced histone deacetylase-2 (HDAC2) function through activation of phosphoinositide-3-kinase-? (PI3K?). The tricyclic antidepressant nortriptyline has been identified in high-throughput screens as an agent that increases corticosteroid responsiveness. The aim of this study was to identify the molecular mechanism whereby nortriptyline increases corticosteroid sensitivity. Phosphorylation of Akt, a footprint of PI3K activation, and HDAC activity were evaluated by Western blotting and fluorescent activity assay in U937 monocytic cells. Corticosteroid sensitivity was evaluated by the inhibition of tumor necrosis factor ? (TNF?)-induced interleukin 8 (IL-8) production by budesonide. Hydrogen peroxide (H2O2) or cigarette smoke extract (CSE) increased the level of phosphorylated Akt (pAkt) and reduced HDAC activity. Pretreatment with nortriptyline inhibited pAkt induced by CSE and H2O2 as well as restored HDAC activity that had been decreased by H2O2 and CSE. In addition, nortriptyline inhibited PI3K? activity, but had no effect on the PI3K? and PI3K? isoforms. Although CSE reduced the effects of budesonide on TNF?-induced IL-8 production in U937 cells, nortriptyline reversed CSE-induced corticosteroid insensitivity. Nortriptyline restores corticosteroid sensitivity induced by oxidative stress via direct inhibition of PI3K? and is a potential treatment for corticosteroid-insensitive diseases such as COPD and severe asthma. PMID:21300705

Mercado, Nicolas; To, Yasuo; Ito, Kazuhiro

2011-01-01

104

Phosphoinositide-specific phospholipase C: structural basis for catalysis and regulatory interactions  

E-print Network

-dimensional structure / tyrosine kinase receptors ©1997 Academic Press Ltd A LARGE NUMBER of extracellular signals stimulate hydrolysis of phosphatidylinositol 4,5-bisphophate (PIP2) by phosphoinositide, involved in calcium release from intracellular stores and stimulation of protein kinase C isozymes.1

Williams, Roger L.

105

Phosphoinositide-dependent Protein Kinase (PDK) Activity Regulates Phosphatidylinositol 3,4,5-Trisphosphate-dependent  

E-print Network

Phosphoinositide-dependent Protein Kinase (PDK) Activity Regulates Phosphatidylinositol 3,4,5-Trisphosphate-dependent and-independentProteinKinaseB Activation and Chemotaxis*S Received for publication of two protein kinase B (PKB) homologues, PkbA and PkbR1, in Dictyostelium discoideum by phosphorylation

Devreotes, Peter

106

Molecular Cell, Vol. 6, 909919, October, 2000, Copyright 2000 by Cell Press Structural Determinants of Phosphoinositide  

E-print Network

Determinants of Phosphoinositide 3-Kinase Inhibition by Wortmannin, LY294002, Quercetin, Myricetin, and Staurosporine for proteins such as protein kinase B (PKB) and phos- pholipid-dependent kinase 1 (PDK1 kinaseInositide Laboratory receptors. The class IB PI3K has a p101 subunit, whichBabraham Institute

Williams, Roger L.

107

IN VITRO ALUMINUM INHIBITION OF BRAIN PHOSPHOINOSITIDE METABOLISM:COMPARISON OF NEONATAL AND ADULTS RATS  

EPA Science Inventory

Recent evidence indicates that the neurotoxic metal aluminum interferes with the phosphoinositide second messenger system in adult rats both in vitro and in vivo. e have examined the age-related effects of aluminum chloride (AlCl3) on receptor-stimulated inositol phosphate (IP) a...

108

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...

109

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.

110

Human Rhinovirus 1B Exposure Induces Phosphatidylinositol 3-Kinase-dependent Airway Inflammation in Mice  

Microsoft Academic Search

Rationale: Infection with rhinovirus (RV) triggers exacerbations of asthma and chronic obstructive lung disease. Objectives: We sought to develop a mouse model of RV employing RV1B, a minor group serotype that binds to the low-density lipopro- tein receptor. Methods: C57BL\\/6 mice were inoculated intranasally with RV1B, replication-deficient ultraviolet (UV)-irradiated RV1B, or RV39, a major group virus. Measurements and Main Results:

Dawn C. Newcomb; Umadevi S. Sajjan; Deepti R. Nagarkar; Qiong Wang; Suparna Nanua; Ying Zhou; Christina L. McHenry; Kenneth T. Hennrick; Wan C. Tsai; J. Kelley Bentley; Nicholas W. Lukacs; Sebastian L. Johnston; Marc B. Hershenson

111

PI3-kinase-dependent electrogenic intestinal transport of glucose and amino acids.  

PubMed

Intestinal glucose and amino acid transport is stimulated by the serum- and glucocorticoid-inducible kinase isoforms SGK1, SGK2, and SGK3 and protein kinase B which are, in turn, stimulated following activation of the phosphoinositol-3 kinase (PI3 kinase). The present study has been performed to explore whether pharmacological inhibition of the PI3 kinase affects electrogenic jejunal transport of glucose and amino acids. In Ussing chamber experiments, glucose (20 mM), phenylalanine (20 mM), glutamine (20 mM), cysteine (20 mM), and proline (20 mM) generated lumen negative currents (I (glc), I (phe), I (gln), I (cys), and I (pro)), respectively, which gradually declined following application of the PI3 kinase inhibitor Wortmannin (1 muM). Within 40 min, Wortmannin treatment significantly decreased I (glc) by 39 +/- 10% (n = 5), I (phe) by 70 +/- 7% (n = 4), I (gln) by 69 +/- 8% (n = 4), I (cys) by 67 +/- 8% (n = 6), and I (prol) by 79 +/- 12% (n = 3). A similar decline of I (glc) was observed following application of the PI3 kinase inhibitor LY294002 (50 microM). Exposure to the inhibitors did not significantly alter transepithelial potential difference and resistance in the absence of substrates for electrogenic transport. The observations suggest that the electrogenic transport of glucose and several amino acids requires the continued activity of PI3 kinase. PMID:17051390

Rexhepaj, Rexhep; Artunc, Ferruh; Metzger, Marco; Skutella, Thomas; Lang, Florian

2007-03-01

112

D-3 phosphoinositides of the ciliate Tetrahymena: characterization and study of their regulatory role in lysosomal enzyme secretion.  

PubMed

Phosphatidylinositol 3-phosphate, PtdIns3P, is a phosphoinositide which is implicated in regulating membrane trafficking in both mammalian and yeast cells. It also serves as a precursor for the synthesis of phosphatidylinositol 3,5-bisphosphate, PtdIns3,5P2, a phosphoinositide, the exact functions of which remain unknown. In this report, we show that these two phosphoinositides are constitutive lipid components of the ciliate Tetrahymena. Using HPLC analysis, PtdIns3P and PtdIns3,5P2 were found to comprise 16% and 30-40% of their relevant phosphoinositide pools, respectively. Treatment of Tetrahymena cells with wortmannin (0.1-10 microM) resulted in the depletion of PtdIns3P and PtdIns3,5P2 without any effect on D-4 phosphoinositides. Wortmannin was further used for the investigation of D-3 phosphoinositide involvement in the regulation of lysosomal vesicular trafficking. Incubation of Tetrahymena cells with wortmannin resulted in enhanced secretion of two different lysosomal enzymes without any change in their total activities. Experiments performed with a T. thermophila secretion mutant strain verified that the wortmannin-induced secretion is specific and it is not due to a diversion of lysosomal enzymes to other secretory pathways. Moreover, experiments performed with a phagocytosis-deficient T. thermophila strain showed that a substantial fraction of wortmannin-induced secretion was dependent on the presence of functional phagosomes/phagolysosomes. PMID:16081170

Leondaritis, George; Tiedtke, Arno; Galanopoulou, Dia

2005-09-30

113

Abscisic Acid-Induced Phosphoinositide Turnover in Guard Cell Protoplasts of Vicia faba.  

PubMed Central

Guard cell protoplasts of Vicia faba treated with 10 [mu]M (+)abscisic acid (ABA) in the light exhibited a 20% decrease in diameter within 1.5 h, from 24.1 to 19.6 [mu]m. Within 10 s of administration of ABA, a 90% increase in levels of inositol 1,4,5-trisphosphate was observed, provided that cells were treated with Li+, an inhibitor of inositol phosphatase activity, prior to incubation. Concomitantly, levels of 32P-labeled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate decreased 20% compared to levels in control cells; levels of label in the membrane lipids phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol did not change significantly in response to ABA treatment. These results show that phosphoinositide turnover is activated in response to ABA in guard cells. We conclude that phosphoinositide signaling is likely to be a step in the biochemical cascade that couples ABA to guard cell shrinking and stomatal closure. PMID:12226236

Lee, Y.; Choi, Y. B.; Suh, S.; Lee, J.; Assmann, S. M.; Joe, C. O.; Kelleher, J. F.; Crain, R. C.

1996-01-01

114

Effects of inhibition of mitochondrial ATP synthesis on phosphoinositide metabolism in rabbit aorta  

Microsoft Academic Search

The authors tested a hypothesis that the plasma membrane phosphoinositide kinase reactions are sensitive to decreases in mitochondrial energy delivery. This hypothesis followed the finding of Lundberg et al of a high Km for ATP for PI and PIP kinase. In aortic rings contracted with norepinephrine (NE), hypoxia causes a decrease in J\\/sub ATP\\/ from 2.2 to 1.6 ..mu..mole\\/(min)(gram wet

R. F. Coburn; C. Baron; M. T. Papadopoulos

1987-01-01

115

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

Microsoft Academic Search

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

James R Brown; Kurt R Auger

2011-01-01

116

Activation of Phosphoinositide 3OH Kinase by the ?6?4 Integrin Promotes Carcinoma Invasion  

Microsoft Academic Search

We demonstrate that the ?6?4 integrin promotes carcinoma invasion through a preferential and localized targeting of phosphoinositide-3 OH kinase (PI3K) activity. Stable expression of ?6?4 increased carcinoma invasion in a PI3K-dependent manner, and transient expression of a constitutively active PI3K increased invasion in the absence of ?6?4. Ligation of ?6?4 stimulated significantly more PI3K activity than ligation of ?1 integrins,

Leslie M Shaw; Isaac Rabinovitz; Helen H.-F Wang; Alex Toker; Arthur M Mercurio

1997-01-01

117

Phospholipase C-dependent phosphoinositide breakdown induced by ELF-EMF in Peganum harmala calli  

Microsoft Academic Search

With the aim of examining the response of plant cells to extremely low frequency (ELF) electromagnetic fields (EMF), we investigated the behaviour of the phosphatidylinositol 4,5 bisphosphate (PtdIns 4,5-P2) molecule (the precursor of the phosphoinositide signal transduction cascade) by exposing callus cells from Peganum harmala to 50 Hz, 1 gauss EMF for 10 min and by examining the level and the fatty acid composition

Maria Piera Piacentini; Elena Piatti; Daniele Fraternale; Donata Ricci; Maria Cristina Albertini; Augusto Accorsi

2004-01-01

118

Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B ?  

Microsoft Academic Search

Background: Protein kinase B (PKB), also known as c-Akt, is activated rapidly when mammalian cells are stimulated with insulin and growth factors, and much of the current interest in this enzyme stems from the observation that it lies ‘downstream’ of phosphoinositide 3-kinase on intracellular signalling pathways. We recently showed that insulin or insulin-like growth factor 1 induce the phosphorylation of

Dario R. Alessi; Stephen R. James; C. Peter Downes; Andrew B. Holmes; Piers R. J. Gaffney; Colin B. Reese; Philip Cohen

1997-01-01

119

Diet and disease alter phosphoinositide composition and metabolism in murine polycystic kidneys.  

PubMed

Because diet can affect the progression of polycystic kidney disease (PKD) and because renal phosphoinositide metabolism is altered in mice with PKD, the effects of diet and disease on phosphoinositide composition and metabolism were examined in kidneys of mice with PKD. The phosphatidylinositol-phosphate (PIP) to phosphatidylinositol (PI) molar ratio was higher (0.034 +/- 0.003 vs. 0.023 +/- 0.001, P < 0.01) and the PI-bisphosphate (PIP2) to PIP molar ratio was lower (0.70 +/- 0.08 vs. 1.19 +/- 0.10, P < 0.05) in kidneys of mice with PKD [DBA/2FG-pcy (pcy)] compared with normal controls (DBA/2J). When initial incorporation (reflecting synthesis) of [3H]inositol into renal phosphoinositides in mice injected with [3H]inositol was measured, the [3H]PIP to [3H]PI ratio was higher in the diseased kidneys compared with normal kidneys (0.016 +/- 0.001 vs. 0.013 +/- 0.001, P < 0.05), whereas the [3H]PIP2 to [3H]PIP ratio was not significantly different. In a study using dietary manipulations that alter the progression of PKD in pcy mice (6 vs. 25% casein and sunflower seed oil vs. fish oil in a 2 x 2 design), animals were injected intraperitoneally with [3H]inositol 5 h before killing. In these animals, the [3H]PIP2 to [3H]PIP ratio seemed to be the best indicator of disease progression. In addition, kidney weight (as altered by diet) was positively correlated (r = 0.62, P = 0.02) with the level of the [3H]PI-3-P isomer relative to total [3H]PIP in the kidney. These results demonstrate that alterations in dietary protein level and lipid composition can modulate renal phosphoinositide signal transduction in mice with PKD. PMID:7738678

Aukema, H M; Yamaguchi, T; Tomobe, K; Philbrick, D J; Chapkin, R S; Takahashi, H; Holub, B J

1995-05-01

120

Importance of Phosphoinositide 3Kinase ? in the host defense against pneu- mococcal infection  

Microsoft Academic Search

Rationale: The pivotal role of phosphoinositide 3-kinase (PI3K) in leukocyte recruitment makes it an attractive target for immuno- modulatory therapy. However, interfering with PI3K signaling might increase the risk of bacterial infections in humans. Objectives: We hypothesized that deletion or pharmacologic inhibi- tion of PI3K would impair the lung inflammatory response to the prototypic gram-positive bacterial pathogen Streptococcus pneumoniae. Methods:

Ulrich A. Maus; Myriam Backi; Christine Winter; Mrigank Srivastava; Matthias K. Schwarz; James C. Paton; Matthias Mack; Tobias Welte; Regina Maus; Rainer M. Bohle; Christian Rommel; Klaus T. Preissner; Werner Seeger

121

The Class II Phosphoinositide 3Kinase C2  Is Not Essential for Epidermal Differentiation  

Microsoft Academic Search

Phosphoinositide 3-kinases (PI3Ks) regulate an array of cellular processes and are comprised of three classes. Class I PI3Ks include the well-studied agonist-sensitive p110 isoforms; however, the functions of class II and III PI3Ks are less well characterized. Of the three class II PI3Ks, C2 and C2 are widely expressed in many tissues, including the epidermis, while C2 is confined to

Kazutoshi Harada; Amy B. Truong; Ti Cai; Paul A. Khavari

2005-01-01

122

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

123

Phosphoinositide-specific phospholipase C in oat roots: association with the actin cytoskeleton  

Microsoft Academic Search

Phosphoinositide-specific phospholipase C (PI-PLC) activities are involved in mediating plant cell responses to environmental\\u000a stimuli. Two variants of PI-PLC have been partially purified from the roots of oat seedlings; one cytosolic and one particulate.\\u000a Although the cytosolic enzyme was significantly purified, the activity still co-migrated with a number of other proteins on\\u000a heparin HPLC and also on size-exclusion chromatography. The

Chiung-Hua Huang; Richard C. Crain

2009-01-01

124

Prenatal ethanol exposure reduces phosphoinositide hydrolysis stimulated by quisqualate in rat cerebellar granule cell cultures  

Microsoft Academic Search

Prenatal ethanol exposure-induced alteration in poly-phosphoinositide (PPI) hydrolysis stimulated by excitatory amino acids\\u000a (EAA) was studied in rat cerebellar granule cells previously labeled with [3H]myoinositol. The prenatal exposure to ethanol was achieved via maternal consumption of a Sustacal (chocolate flavored) liquid\\u000a diet containing either 5% ethanol (w\\/v, 35% of calories) or isocaloric sucrose (pair-fed) substituted for ethanol from gestation\\u000a d

Philip G. Rhodes; Zhengwei Cai; Nannan Zhu

1994-01-01

125

Unique cell wall abnormalities in the putative phosphoinositide phosphatase mutant AtSAC9.  

PubMed

SAC9 is a putative phosphoinositide phosphatase in Arabidopsis thaliana involved in phosphoinositide signaling. sac9-1 plants have a constitutively stressed phenotype with shorter roots which notably accumulate phosphatidylinositol 4,5-bisphosphate and its hydrolysis product inositol trisphosphate. We investigated the primary roots of sac9-1 seedlings at the cytological and ultrastructural level to determine the structural basis for this altered growth. Despite the normal appearance of organelles and cytoplasmic elements, our studies reveal extreme abnormalities of cell wall and membrane structures in sac9-1 primary root cells, regardless of cell type, position within the meristematic area, and plane of section. Cell wall material was deposited locally and in a range of abnormal shapes, sometimes completely fragmenting the cell. Simple protuberances, broad flanges, diffuse patches, elaborate folds, irregular loops and other complex three-dimensional structures were found to extend randomly from the pre-existing cell wall. Abundant vesicles and excessive membrane material were associated with these irregular wall structures. We argue that a perturbed phosphoinositide metabolism most likely induces these observed abnormalities and hypothesize that a disorganized cytoskeleton and excessive membrane trafficking mediate the cell wall defects. PMID:21698459

Vollmer, Almut H; Youssef, Nabil N; DeWald, Daryll B

2011-11-01

126

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

127

Cbl participates in shikonin-induced apoptosis by negatively regulating phosphoinositide 3-kinase/protein kinase B signaling.  

PubMed

Shikonin, a naturally occurring naphthoquinone, exhibits anti-tumorigenic activity. However, its precise mechanisms of action have remained elusive. In the present study, the involvement in the action of shikonin of the ubiquitin ligases Cbl?b and c?Cbl, which are negative regulators of phosphoinositide 3?kinase (PI3K) activation, was investigated. Shikonin was observed to reduce cell viability and induce apoptosis and G2/M phase arrest in lung cancer cells. In addition, shikonin increased the protein levels of B?cell lymphoma 2 (Bcl?2)?associated X and p53 and reduced those of Bcl?2. Additionally, shikonin inhibited PI3k/Akt activity and upregulated Cbl protein expression. In addition, a specific inhibitor of PI3K, LY294002, was observed to have a synergistic effect on the proliferation inhibition and apoptotic induction of A549 cells with shikonin. In conclusion, the results of the present study suggested that Cbl proteins promote shikonin?induced apoptosis by negatively regulating PI3K/Akt signaling in lung cancer cells. PMID:25815461

Qu, Dan; Xu, Xiao-Man; Zhang, Meng; Jiang, Ting-Shu; Zhang, Yi; Li, Sheng-Qi

2015-07-01

128

RUNX1 regulates phosphoinositide 3-kinase/AKT pathway: role in chemotherapy sensitivity in acute megakaryocytic leukemia.  

PubMed

RUNX1 (AML1) encodes the core binding factor alpha subunit of a heterodimeric transcription factor complex which plays critical roles in normal hematopoiesis. Translocations or down-regulation of RUNX1 have been linked to favorable clinical outcomes in acute leukemias, suggesting that RUNX1 may also play critical roles in chemotherapy responses in acute leukemias; however, the molecular mechanisms remain unclear. The median level of RUNX1b transcripts in Down syndrome (DS) children with acute megakaryocytic leukemia (AMkL) were 4.4-fold (P < .001) lower than that in non-DS AMkL cases. Short hairpin RNA knockdown of RUNX1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivity to cytosine arabinoside, accompanied by significantly decreased expression of PIK3CD, which encodes the delta catalytic subunit of the survival kinase, phosphoinositide 3 (PI3)-kinase. Transcriptional regulation of PIK3CD by RUNX1 was further confirmed by chromatin immunoprecipitation and promoter reporter gene assays. Further, a PI3-kinase inhibitor, LY294002, and cytosine arabinoside synergized in antileukemia effects on Meg-01 and primary pediatric AMkL cells. Our results suggest that RUNX1 may play a critical role in chemotherapy response in AMkL by regulating the PI3-kinase/Akt pathway. Thus, the treatment of AMkL may be improved by integrating PI3-kinase or Akt inhibitors into the chemotherapy of this disease. PMID:19638627

Edwards, Holly; Xie, Chengzhi; LaFiura, Katherine M; Dombkowski, Alan A; Buck, Steven A; Boerner, Julie L; Taub, Jeffrey W; Matherly, Larry H; Ge, Yubin

2009-09-24

129

p110? and p110? isoforms of phosphoinositide 3-kinase differentially regulate natural killer cell migration in health and disease  

PubMed Central

The mechanisms that regulate NK cell trafficking are unclear. Phosphoinositide-3 kinases (PI3K) control cell motility and the p110? and p110? isoforms are mostly expressed in leukocytes, where they transduce signals downstream of G protein coupled receptors (GPCR) or tyrosine kinase receptors, respectively. Here, we set out to determine the relative contribution of p110? and p110? to NK cell migration in mice. Using a combination of single-cell imaging analysis of transgenic cells reporting on PI3K activity in real time and small molecule inhibitors of p110? and p110?, we show here that the tyrosine-kinase coupled p110? is linked to GPCR signaling and, depending on the GPCR, may even be preferentially activated over p110?. Using gene-targeted mice, we showed that both isoforms were essential for NK cell chemotaxis to CXCL12 and to CCL3 and, in vivo, for normal NK cell migration during pregnancy and to the inflamed peritoneum. By contrast, only p110? was indispensable for chemotaxis to S1P and CXCL10 and for NK cell distribution throughout lymphoid and nonlymphoid tissues and for extravasation to tumors. These results implicate p110? downstream of GPCRs in NK cells and highlight its nonredundant role as a key regulator of NK cell trafficking in health and disease. PMID:19297623

Saudemont, Aurore; Garçon, Fabien; Yadi, Hakim; Roche-Molina, Marta; Kim, Nayoung; Segonds-Pichon, Anne; Martín-Fontecha, Alfonso; Okkenhaug, Klaus; Colucci, Francesco

2009-01-01

130

Phorbol esters inhibit alpha/sub 1/-adrenergic receptor stimulated phosphoinositide hydrolysis and contraction in rat aorta  

SciTech Connect

The mechanisms of pharmacomechanical coupling in vascular tissue are at the present time unclear. The authors and others have proposed that receptor-induced activation of phosphoinositide (PI) hydrolysis may be involved. To investigate this possibility they studied the actions of two biologically active phorbol esters: phorbol dibutyrate (PDB) and phorbol myristate diacetate (PMA) on receptor-stimulated PI hydrolysis in rat aortic rings. They found both PDB (IC/sub 5//sup 0/ approx. 5nM) and PMA (IC/sub 50/ approx. 30 nM) but not 4-..cap alpha..-phorbol (IC32%/sub 0/ > 10,000 nM) inhibited norepinephrine-stimulated PI hydrolysis. In the presence of the calcium channel antagonist nitrendipine, PDB potently inhibited both the phasic and tonic components of norepinephrine-induced vascular contraction. In the presence of 10/sup -7/M nitrendipine, PDB had an IC/sub 50/ for contraction of approximately 10nM. The results thus suggest a functional coupling between ..cap alpha../sub 1/-adrenergic receptor-stimulated PI hydrolysis and vascular contraction. The findings further imply a mode of feed-back regulation in vascular tissue involving phorbol ester and receptor-stimulated PI hydrolysis.

Not Available

1986-03-01

131

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

132

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

133

Modulation of phosphoinositide metabolism in aortic smooth muscle cells by allylamine  

SciTech Connect

Aortic smooth muscle cells (SMC) modulate from a contractile to a proliferative phenotype upon subchronic exposure to allylamine. The present studies were designed to determine if this phenotypic modulation is associated with alterations in the metabolism of membrane phosphoinositides. 32P incorporation into phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA) was lower by 31, 35, and 22%, respectively, in SMC from allylamine-treated animals relative to controls. In contrast, incorporation of (3H)myoinositol into inositol phosphates did not differ in allylamine cells relative to control cells. Exposure to dibutyryl (db) cAMP (0.2 mM) and theophylline (0.1 mM) reduced 32P incorporation into PIP and PIP2 in SMC from both experimental groups. Under these conditions, a decrease in (3H)myoinositol incorporation into inositol 1-phosphate was only observed in allylamine cells. The effects of db cAMP and theophylline in allylamine and control SMC correlated with a marked decrease in cellular proliferation. These results suggest that alterations in phosphoinositide synthesis and/or degradation contribute to the enhanced proliferation of SMC induced by allylamine. To further examine this concept, the effects of agents which modulate protein kinase C (PKC) activity were evaluated. Sphingosine (125-500 ng/ml), a PKC inhibitor, decreased SMC proliferation in allylamine, but not control cells. 12-O-Tetradecanoylphorbol-13-acetate (1-100 ng/ml), a PKC agonist, stimulated proliferation in control cells, but inhibited proliferation in cells from allylamine-treated animals. We conclude that allylamine-induced phenotypic modulation of SMC is associated with alterations in phosphoinositide metabolism.

Cox, L.R.; Murphy, S.K.; Ramos, K. (Philadelphia College of Pharmacy and Science, PA (USA))

1990-08-01

134

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

135

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

136

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

137

Structural basis for isoform selectivity in a class of benzothiazole inhibitors of phosphoinositide 3-kinase ?.  

PubMed

Phosphoinositide 3-kinase ? (PI3K?) is an attractive target to potentially treat a range of disease states. Herein, we describe the evolution of a reported phenylthiazole pan-PI3K inhibitor into a family of potent and selective benzothiazole inhibitors. Using X-ray crystallography, we discovered that compound 22 occupies a previously unreported hydrophobic binding cleft adjacent to the ATP binding site of PI3K?, and achieves its selectivity by exploiting natural sequence differences among PI3K isoforms in this region. PMID:24754609

Collier, Philip N; Martinez-Botella, Gabriel; Cornebise, Mark; Cottrell, Kevin M; Doran, John D; Griffith, James P; Mahajan, Sudipta; Maltais, François; Moody, Cameron S; Huck, Emilie Porter; Wang, Tiansheng; Aronov, Alex M

2015-01-01

138

Spatial regulation of membrane fusion controlled by modification of phosphoinositides.  

PubMed

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 (PLCgamma), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P(2)) 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

139

Endothelial epithelial sodium channel inhibition activates endothelial nitric oxide synthase via phosphoinositide 3-kinase/Akt in small-diameter mesenteric arteries.  

PubMed

Recent studies have shown that the epithelial sodium channel (ENaC) is expressed in vascular tissue. However, the role that ENaC may play in the responses to vasoconstrictors and NO production has yet to be addressed. In this study, the contractile responses of perfused pressurized small-diameter rat mesenteric arteries to phenylephrine and serotonin were reduced by ENaC blockade with amiloride (75.1+/-3.2% and 16.9+/-2.3% of control values, respectively; P<0.01) that was dose dependent (EC(50)=88.9+/-1.6 nmol/L). Incubation with benzamil, another ENaC blocker, had similar effects. alpha, beta, and gamma ENaC were identified in small-diameter rat mesenteric arteries using RT-PCR and Western blot with specific antibodies. In situ hybridization and immunohistochemistry localized ENaC expression to the tunica media and endothelium of small-diameter rat mesenteric arteries. Patch-clamp experiments demonstrated that primary cultures of mesenteric artery endothelial cells expressed amiloride-sensitive sodium currents. Mechanical ablation of the endothelium or inhibition of eNOS with N(omega)-nitro-L-arginine inhibited the reduction in contractility caused by ENaC blockers. ENaC inhibitors increased eNOS phosphorylation (Ser 1177) and Akt phosphorylation (Ser 473). The presence of the phosphoinositide 3-kinase inhibitor LY294002 blunted Akt phosphorylation and eNOS phosphorylation and the decrease in the response to phenylephrine caused by blockers of ENaC, indicating that the phosphoinositide 3-kinase/Akt pathway was activated after ENaC inhibition. Finally, we observed that the effects of blockers of ENaC were flow dependent and that the vasodilatory response to shear stress was enhanced by ENaC blockade. Our results identify a previously unappreciated role for ENaC as a negative modulator of eNOS and NO production in resistance arteries. PMID:19398659

Pérez, Francisco R; Venegas, Fabiola; González, Magdalena; Andrés, Sergio; Vallejos, Catalina; Riquelme, Gloria; Sierralta, Jimena; Michea, Luis

2009-06-01

140

Electrostatics of phosphoinositide bilayer membranes. Theoretical and experimental results.  

PubMed

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

1990-02-01

141

5-Hydroxytryptamine receptor-mediated phosphoinositide hydrolysis in canine cultured tracheal smooth muscle cells.  

PubMed Central

1. 5-Hydroxytryptamine (5-HT) has been shown to induce contraction of tracheal smooth muscle. However, the mechanisms of action of 5-HT are not known. We therefore investigated the effects of 5-HT on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and its regulation in canine cultured tracheal smooth muscle cells (TSMCs) labelled with [3H]-inositol. 5-HT-induced inositol phosphates (IPs) accumulation was time- and dose-dependent with a half-maximal response (EC50) and a maximal response at 0.38 +/- 0.05 and 10 microM, respectively. 2. Ketanserin and mianserin (10 and 100 nM), 5-HT2 receptor antagonists, were equipotent in blocking the 5-HT-induced IPs accumulation with pKB values of 8.46 and 8.21, respectively. In contrast, the dose-response curves of 5-HT-induced IPs accumulation were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased up to 10 microM. 3. Pretreatment of TSMCs with pertussis toxin or cholera toxin did not inhibit the 5-HT-induced IPs accumulation, but partially inhibited the AlF(4-)-induced IPs response. 4. Stimulation of IPs accumulation by 5-HT required the presence of external Ca2+ and was blocked by EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. A further Ca(2+)-dependent increase in IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphoshate) (GTP gamma S) or 5-HT. The combination of GTP gamma S and 5-HT elicited an additive effect on IPs accumulation. 5. Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM, 30 min) abolished the 5-HT-induced IPs accumulation. The concentrations of PMA that gave a half-maximal and maximal inhibition of 5-HT-induced IPs accumulation were 2.2 +/- 0.4 nM and 1 microM, n = 3, respectively. The protein kinase C (PKC) activator, 4 alpha-phorbol 12,13-didecanoate, at 1 microM, did not influence this response. The inhibitory effect of PMA was reversed by staurosporine, a PKC inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 6. 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 effect of PMA is distal to the 5-HT receptor.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8019756

Yang, C. M.; Yo, Y. L.; Hsieh, J. T.; Ong, R.

1994-01-01

142

Yeast-based methods to assess PTEN phosphoinositide phosphatase activity in vivo.  

PubMed

The PTEN phosphoinositide 3-phosphatase is a tumor suppressor commonly targeted by pathologic missense mutations. Subject to multiple complex layers of regulation, its capital role in cancer relies on its counteracting function of class I phosphoinositide 3-kinase (PI3K), a key feature in oncogenic signaling pathways. Precise assessment of the involvement of PTEN mutations described in the clinics in loss of catalytic activity requires either tedious in vitro phosphatase assays or in vivo experiments involving transfection into mammalian cell lines. Taking advantage of the versatility of the model organism Saccharomyces cerevisiae, we have developed different functional assays by reconstitution of the mammalian PI3K-PTEN switch in this lower eukaryote. This methodology is based on the fact that regulated PI3K expression in yeast cells causes conversion of PtdIns(4,5)P2 in PtdIns(3,4,5)P3 and co-expression of PTEN counteracts this effect. This can be traced by monitoring growth, given that PtdIns(4,5)P2 pools are essential for the yeast cell, or by using fluorescent reporters amenable for microscopy or flow cytometry. Here we describe the methodology and review its application to evaluate the functionality of PTEN mutations. We show that the technique is amenable to both directed and systematic structure-function relationship studies, and present an example of its use for the study of the recently discovered PTEN-L variant. PMID:25448481

Rodríguez-Escudero, Isabel; Fernández-Acero, Teresa; Bravo, Ignacio; Leslie, Nicholas R; Pulido, Rafael; Molina, María; Cid, Víctor J

2015-05-01

143

Phosphoinositides: Minor Lipids Make a Major Impact on Photoreceptor Cell Functions  

PubMed Central

Activation of the phosphoinositide (PI) cycle generates the second messengers that control various aspects of cellular signaling. We have previously shown that two PI cycle enzymes, type II phosphatidylinositol 5-phosphate 4-kinase (PIPK II?) and phosphoinositide 3-kinase (PI3K), are activated through light stimulation. In our earlier studies, we measured enzyme activities, instead of directly measuring the products, due to lack of sensitive analytical techniques. Cells have very low levels of PIs, compared to other lipids, so special techniques and sensitive analytical instruments are necessary for their identification and quantification. There are also other considerations, such as different responses in different cell types, which may complicate quantification of PIs. For example, although light activated PIPK II?, there was no increase in PI-4,5-P2 measured by liquid chromatography–mass spectrometry (LC/MS) This discrepancy is due to the heterogeneous nature of the retina, which is composed of various cell types. In this study, we examined PI generation in situ using immunohistochemistry with specific PI antibodies. PIs were generated in specific retinal cell layers, suggesting that analyzing PIs from the total retina by LC/MS underscores the significance. This suggests that PI-specific antibodies are useful tools to study the cell-specific regulation of PIs in the retina. PMID:24964953

Rajala, Raju V. S.; Rajala, Ammaji; Morris, Andrew J.; Anderson, Robert E.

2014-01-01

144

Phosphoinositides: minor lipids make a major impact on photoreceptor cell functions.  

PubMed

Activation of the phosphoinositide (PI) cycle generates the second messengers that control various aspects of cellular signaling. We have previously shown that two PI cycle enzymes, type II phosphatidylinositol 5-phosphate 4-kinase (PIPK II?) and phosphoinositide 3-kinase (PI3K), are activated through light stimulation. In our earlier studies, we measured enzyme activities, instead of directly measuring the products, due to lack of sensitive analytical techniques. Cells have very low levels of PIs, compared to other lipids, so special techniques and sensitive analytical instruments are necessary for their identification and quantification. There are also other considerations, such as different responses in different cell types, which may complicate quantification of PIs. For example, although light activated PIPK II?, there was no increase in PI-4,5-P2 measured by liquid chromatography-mass spectrometry (LC/MS) This discrepancy is due to the heterogeneous nature of the retina, which is composed of various cell types. In this study, we examined PI generation in situ using immunohistochemistry with specific PI antibodies. PIs were generated in specific retinal cell layers, suggesting that analyzing PIs from the total retina by LC/MS underscores the significance. This suggests that PI-specific antibodies are useful tools to study the cell-specific regulation of PIs in the retina. PMID:24964953

Rajala, Raju V S; Rajala, Ammaji; Morris, Andrew J; Anderson, Robert E

2014-01-01

145

Phosphoinositide Binding by the Toll Adaptor dMyD88 Controls Antibacterial Responses in Drosophila  

PubMed Central

The cell biological principles that govern innate immune responses in Drosophila are unknown. Here, we report that Toll signaling in flies was dictated by the subcellular localization of the adaptor protein dMyD88. dMyD88 was located at the plasma membrane by a process dependent on a C-terminal phosphoinositide-binding domain. In vivo analysis revealed that lipid binding by dMyD88 was necessary for its antimicrobial and developmental functions, as well as for the recruitment of the downstream cytosolic adaptor Tube to the cell surface. These data are reminiscent of the interactions between the mammalian Toll adaptors MyD88 and TIRAP with one major exception. In the mammalian system, MyD88 is the cytosolic adaptor that depends on the phosphoinositide-binding protein TIRAP for its recruitment to the cell surface. We therefore propose that dMyD88 is the functional homologue of TIRAP, and that both proteins function as sorting adaptors to recruit downstream signaling adaptors to activated receptors. PMID:22464168

Marek, Lorri R.; Kagan, Jonathan C.

2012-01-01

146

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)

2011-10-18

147

Disulfiram Treatment Facilitates Phosphoinositide 3-Kinase Inhibition in Human Breast Cancer Cells In vitro and In vivo  

PubMed Central

Frequent genetic alterations of the components in the phosphoinositide 3-kinase (PI3K)/PTEN/AKT signaling pathway contribute greatly to breast cancer initiation and progression, which makes targeting this signaling pathway a promising therapeutic strategy for breast cancer treatment. In this study, we showed that in the presence of copper (Cu), disulfiram (DSF), a clinically used antialcoholism drug, could potently inhibit breast cancer cell growth regardless of the PIK3CA status. Surprisingly, the treatment with a mixture of DSF and copper (DSF-Cu) led to the decreased expression of PTEN protein and the activation of AKT in a dose- and time-dependent manner in different cell lines with or without PIK3CA mutations. Treatment of breast cancer cell lines with a combination of DSF-Cu and LY294002, a pan-PI3K inhibitor, resulted in the significant inhibition of cell growth when compared with either drug alone. In addition, the combined treatment of DSF and LY294002 significantly inhibited the growth of the breast tumor xenograft in nude mice induced by MDA-MB-231 cells expressing mutant PIK3CA-H1047R and PIK3CA-E545K, whereas neither DSF nor LY294002 alone could significantly retard tumor growth. Finally, the observed in vivo inhibitory effects are found associated with aberrant signaling alterations and apoptosis-inducing activities in tumor samples. Thus, our finding shows for the first time that treatment of breast cancer with DSF results in a novel feedback mechanism that activates AKT signaling. Our study also suggests that the combination of DSF and a PI3K inhibitor may offer a new combinational treatment model for breast cancer, particularly for those with PIK3CA mutations. PMID:20424113

Zhang, Haijun; Chen, Di; Ringler, Jonathan; Chen, Wei; Cui, Qiuzhi Cindy; Ethier, Stephen P.; Dou, Q. Ping; Wu, Guojun

2013-01-01

148

Cell autonomous phosphoinositide 3-kinase activation in oocytes disrupts normal ovarian function through promoting survival and overgrowth of ovarian follicles.  

PubMed

In this study, we explored the effects of oocytic phosphoinositide 3-kinase (PI3K) activation on folliculogensis by generating transgenic mice, in which the oocyte-specific Cre-recombinase induces the expression of constitutively active mutant PI3K during the formation of primordial follicles. The ovaries of neonatal transgenic (Cre+) mice showed significantly reduced apoptosis in follicles, which resulted in an excess number of follicles per ovary. Thus, the elevation of phosphatidylinositol (3,4,5)-trisphosphate levels within oocytes promotes the survival of follicles during neonatal development. Despite the increase in AKT phosphorylation, primordial follicles in neonatal Cre+ mice remained dormant demonstrating a nuclear accumulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These primordial follicles containing a high level of nuclear PTEN persisted in postpubertal females, suggesting that PTEN is the dominant factor in the maintenance of female reproductive lifespan through the regulation of primordial follicle recruitment. Although the oocytic PI3K activity and PTEN levels were elevated, the activation of primordial follicles and the subsequent accumulation of antral follicles with developmentally competent oocytes progressed normally in prepubertal Cre+ mice. However, mature Cre+ female mice were anovulatory. Because postnatal day 50 Cre+ mice released cumulus-oocyte complexes with developmentally competent oocytes in response to super-ovulation treatment, the anovulatory phenotype was not due to follicular defects but rather endocrine abnormalities, which were likely caused by the excess number of overgrown follicles. Our current study has elucidated the critical role of oocytic PI3K activity in follicular function, as well as the presence of a PTEN-mediated mechanism in the prevention of immature follicle activation. PMID:25594701

Kim, So-Youn; Ebbert, Katherine; Cordeiro, Marilia H; Romero, Megan; Zhu, Jie; Serna, Vanida Ann; Whelan, Kelly A; Woodruff, Teresa K; Kurita, Takeshi

2015-04-01

149

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.

150

Repression of B-Cell Linker (BLNK) and B-Cell Adaptor for Phosphoinositide 3-Kinase (BCAP) Is Important for  

E-print Network

-Rel protein is seen in classic Hodgkin's lymphoma (cHL) and primary mediastinal large B-cell lymphoma (MLBCLRepression of B-Cell Linker (BLNK) and B-Cell Adaptor for Phosphoinositide 3-Kinase (BCAP-Rel and c-Rel proteins uncovered that Rel protein expression leads to transcriptional repression of key B-cell

Chen, Kuang-Yu

151

The yeast VAP homolog Scs2p has a phosphoinositide-binding ability that is correlated with its activity  

SciTech Connect

The yeast VAMP-associated protein (VAP) homolog Scs2p is an endoplasmic reticulum (ER)/nuclear membrane protein that binds to an FFAT (diphenylalanine in an acidic tract) motif found in various lipid-metabolic proteins, including Opi1p, a negative regulator of phospholipid biosynthesis. Here, we show that Scs2p is a novel phosphoinositide-binding protein that can bind to phosphatidylinositol monophosphates and bisphosphates in vitro. The phosphoinositide-binding domain was assigned to the N-terminal major sperm protein (MSP) domain which also contains the FFAT-binding domain. When several lysine residues in the MSP domain were substituted for alanine, the resulting mutant Scs2 proteins lost the phosphoinositide-binding ability and failed to complement the inositol auxotrophy of an scs2 deletion strain. However, the mutant proteins still localized in the ER/nuclear membrane, in a similar manner to wild-type Scs2p. These results suggest the possibility that Scs2p activity is regulated by phosphoinositides to coordinate phospholipid biosynthesis in response to changes in phospholipid composition.

Kagiwada, Satoshi [Department of Biological Sciences, Faculty of Science, Nara Women's University, Nara 630-8506 (Japan)], E-mail: kagiwada@cc.nara-wu.ac.jp; Hashimoto, Misa [Department of Biological Sciences, Faculty of Science, Nara Women's University, Nara 630-8506 (Japan)

2007-12-28

152

Structural Mapping of the Catalytic Mechanism for a Mammalian Phosphoinositide-Specific Phospholipase C,  

E-print Network

,5-bisphosphate (PIP2) to D-myo-inositol-1,4,5- trisphosphate (1,4,5-IP3) and sn-1,2-diacylglycerol (DAG) (Rhee phosphorylation states by activating various protein kinase C isozymes (Dekker et al., 1995). Three classes of - and -isozymes is regulated by G protein- coupled and tyrosine kinase-linked receptors, respectively (Lee & Rhee

Williams, Roger L.

153

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

154

MTM-6, a Phosphoinositide Phosphatase, is Required to Promote Synapse Formation in Caenorhabditis elegans  

PubMed Central

Forming the proper number of synapses is crucial for normal neuronal development. We found that loss of function of the phosphoinositide phosphatase mtm-6 results in a reduction in the number of synaptic puncta. The reduction in synapses is partially the result of MTM-6 regulation of the secretion of the Wnt ligand EGL-20 from cells in the tail and partially the result of neuronal action. MTM-6 shows relative specificity for EGL-20 over the other Wnt ligands. We suggest that the ability of MTM-6 to regulate EGL-20 secretion is a function of its expression pattern. We conclude that regulation of secretion of different Wnt ligands can use different components. Additionally, we present a novel neuronal function for MTM-6. PMID:25479419

Ericson, Vivian R.; Spilker, Kerri A.; Tugizova, Madina S.; Shen, Kang

2014-01-01

155

Phosphoinositide 3-kinase ? regulates membrane fission of Golgi carriers for selective cytokine secretion.  

PubMed

Phosphoinositide 3-kinase (PI3K) p110 isoforms are membrane lipid kinases classically involved in signal transduction. Lipopolysaccharide (LPS)-activated macrophages constitutively and abundantly secrete proinflammatory cytokines including tumor necrosis factor-? (TNF). Loss of function of the p110? isoform of PI3K using inhibitors, RNA-mediated knockdown, or genetic inactivation in mice abolishes TNF trafficking and secretion, trapping TNF in tubular carriers at the trans-Golgi network (TGN). Kinase-active p110? localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110? is inactivated. Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2. These findings demonstrate a new function for p110? as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages. PMID:20837769

Low, Pei Ching; Misaki, Ryo; Schroder, Kate; Stanley, Amanda C; Sweet, Matthew J; Teasdale, Rohan D; Vanhaesebroeck, Bart; Meunier, Frédéric A; Taguchi, Tomohiko; Stow, Jennifer L

2010-09-20

156

Essential role for the p110delta phosphoinositide 3-kinase in the allergic response.  

PubMed

Inflammatory substances released by mast cells induce and maintain the allergic response. Mast cell differentiation and activation are regulated, respectively, by stem cell factor (SCF; also known as Kit ligand) and by allergen in complex with allergen-specific immunoglobulin E (IgE). Activated SCF receptors and high-affinity receptors for IgE (FcvarepsilonRI) engage phosphoinositide 3-kinases (PI(3)Ks) to generate intracellular lipid second messenger signals. Here, we report that genetic or pharmacological inactivation of the p110delta isoform of PI(3)K in mast cells leads to defective SCF-mediated in vitro proliferation, adhesion and migration, and to impaired allergen-IgE-induced degranulation and cytokine release. Inactivation of p110delta protects mice against anaphylactic allergic responses. These results identify p110delta as a new target for therapeutic intervention in allergy and mast-cell-related pathologies. PMID:15496927

Ali, Khaled; Bilancio, Antonio; Thomas, Matthew; Pearce, Wayne; Gilfillan, Alasdair M; Tkaczyk, Christine; Kuehn, Nicolas; Gray, Alexander; Giddings, June; Peskett, Emma; Fox, Roy; Bruce, Ian; Walker, Christoph; Sawyer, Carol; Okkenhaug, Klaus; Finan, Peter; Vanhaesebroeck, Bart

2004-10-21

157

Phosphoinositide 3-kinase ? regulates membrane fission of Golgi carriers for selective cytokine secretion  

PubMed Central

Phosphoinositide 3-kinase (PI3K) p110 isoforms are membrane lipid kinases classically involved in signal transduction. Lipopolysaccharide (LPS)-activated macrophages constitutively and abundantly secrete proinflammatory cytokines including tumor necrosis factor-? (TNF). Loss of function of the p110? isoform of PI3K using inhibitors, RNA-mediated knockdown, or genetic inactivation in mice abolishes TNF trafficking and secretion, trapping TNF in tubular carriers at the trans-Golgi network (TGN). Kinase-active p110? localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110? is inactivated. Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2. These findings demonstrate a new function for p110? as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages. PMID:20837769

Low, Pei Ching; Misaki, Ryo; Schroder, Kate; Stanley, Amanda C.; Sweet, Matthew J.; Teasdale, Rohan D.; Vanhaesebroeck, Bart; Meunier, Frédéric A.; Taguchi, Tomohiko

2010-01-01

158

New Inhibitor of 3-Phosphoinositide Dependent Protein Kinase-1 Identified from Virtual Screening  

PubMed Central

3-Phosphoinositide-dependent protein kinase-1 (PDK1) has been recognized as a promising anticancer target. Thus, it is interesting to identify new inhibitors of PDK1 for anticancer drug discovery. Through a combined use of virtual screening and wet experimental activity assays, we have identified a new PDK1 inhibitor with IC50 = ~200 nM. The anticancer activities of this compound have been confirmed by the anticancer activity assays using 60 cancer cell lines. The obtained new PDK1 inhibitor and its PDK1-inhibitor binding mode should be valuable in future de novo design of novel, more potent and selective PDK1 inhibitors for future development of anticancer therapeutics. PMID:22266037

Yang, Wenchao; AbdulHameed, Mohamed Diwan M.; Hamza, Adel; Zhan, Chang-Guo

2015-01-01

159

Novel Phosphoinositides in Barley Aleurone Cells (Additional Evidence for the Presence of Phosphatidyl-scyllo-Inositol).  

PubMed Central

A novel isomer of phosphatidylinositol that differs in the structure of the head group was detected in barley (Hordeum vulgare cv Himalaya) seeds. In this paper we describe our efforts to elucidate the structure of the novel isomer. Evidence from a variety of techniques, including chemical modification of in vivo 32Pi- and myo-[3H]inositol-labeled compounds, gas chromatography-mass spectrometry analysis, in vivo incorporation of scyllo-[3H]inositol, and enzymatic studies that suggest that the structure is phosphatidylscyllo-inositol (scyllo-PI), is presented. The use of microwave energy to significantly enhance the slow rate of hydrolysis of phosphoinositides is described. The presence of scyllo-PI can be easily overlooked by the methods commonly employed; therefore, experimental considerations important for the detection of scyllo-PI are discussed. PMID:12223679

Narasimhan, B.; Pliska-Matyshak, G.; Kinnard, R.; Carstensen, S.; Ritter, M. A.; Von Weymarn, L.; Murthy, PPN.

1997-01-01

160

A phosphoinositide switch controls the maturation and signaling properties of APPL endosomes  

PubMed Central

SUMMARY The recent identification of several novel endocytic compartments has challenged our current understanding of the topological and functional organization of the endocytic pathway. Using quantitative single vesicle imaging and acute manipulation of phosphoinositides we show that APPL endosomes, which participate in growth factor receptor trafficking and signaling, represent an early endocytic intermediate common to a subset of clathrin derived endocytic vesicles and macropinosomes. Most APPL endosomes are precursors of classical PI3P positive endosomes, and PI3P plays a critical role in promoting this conversion. Depletion of PI3P causes a striking reversion of Rab5 positive endosomes to the APPL stage, and results in enhanced growth factor signaling. These findings reveal a surprising plasticity of the early endocytic pathway. Importantly, PI3P functions as a switch to dynamically regulate maturation and signaling of APPL endosomes. PMID:19303853

Zoncu, Roberto; Perera, Rushika M; Balkin, Daniel M; Pirruccello, Michelle; Toomre, Derek; De Camilli, Pietro

2009-01-01

161

Involvement of the phosphoinositide 3-kinase/Akt pathway in apoptosis induced by capsaicin in the human pancreatic cancer cell line PANC-1  

PubMed Central

Capsaicin, one of the major pungent ingredients found in red peppers, has been recently demonstrated to induce apoptosis in various malignant cell lines through an unclear mechanism. In this study, the effect of capsaicin on proliferation and apoptosis in the human pancreatic cancer cell line PANC-1 and its possible mechanism(s) of action were investigated. The results of a Cell Counting Kit-8 (CCK-8) assay revealed that capsaicin significantly decreased the viability of PANC-1 cells in a dose-dependent manner. Capsaicin induced G0/G1 phase cell cycle arrest and apoptosis in PANC-1 cells as demonstrated by a flow cytometric assessment. Caspase-3 expression at both the protein and mRNA level was promoted following capsaicin treatment. Furthermore, we revealed that phospho-PI3 Kinase p85 (Tyr458) and phospho-Akt (Ser473) in PANC-1 cells were downregulated in response to capsaicin. Moreover, capsaicin gavage significantly inhibited the growth of pancreatic cancer PANC-1 cell xenografts in athymic nude mice. An increased number of TUNEL-positive cells and cleaved caspase-3 were observed in capsaicin-treated mice. In vivo, capsaicin downregulated the expression of phospho-PI3 Kinase p85 (Tyr458) and phospho-Akt (Ser473). In conclusion, we have demonstrated that capsaicin is an inhibitor of growth of PANC-1 cells, and downregulation of the phosphoinositide 3-kinase/Akt pathway may be involved in capsaicin-induced apoptosis in vitro and in vivo. PMID:23255891

ZHANG, JIAN-HONG; LAI, FU-JI; CHEN, HUI; LUO, JIANG; ZHANG, RI-YUAN; BU, HE-QI; WANG, ZHAO-HONG; LIN, HONG-HAI; LIN, SHENG-ZHANG

2013-01-01

162

Distinctive Changes in Plasma Membrane Phosphoinositides Underlie Differential Regulation of TRPV1 in Nociceptive Neurons  

PubMed Central

Transient Receptor Potential Vanilloid 1 (TRPV1) is a polymodal, Ca2+-permeable cation channel crucial to regulation of nociceptor responsiveness. Sensitization of TRPV1 by G-protein coupled receptor (GPCR) agonists to its endogenous activators, such as low pH and noxious heat, is a key factor in hyperalgesia during tissue injury as well as pathological pain syndromes. Conversely, chronic pharmacological activation of TRPV1 by capsaicin leads to calcium influx-induced adaptation of the channel. Paradoxically, both conditions entail activation of phospholipase C (PLC) enzymes, which hydrolyze phosphoinositides. We found that in sensory neurons PLC? activation by bradykinin led to a moderate decrease in phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), but no sustained change in the levels of its precursor PI(4)P. Preventing this selective decrease in PI(4,5)P2 inhibited TRPV1 sensitization, while selectively decreasing PI(4,5)P2 independently of PLC potentiated the sensitizing effect of protein kinase C (PKC) on the channel, thereby inducing increased TRPV1 responsiveness. Maximal pharmacological TRPV1 stimulation led to a robust decrease of both PI(4,5)P2 and its precursor PI(4)P in sensory neurons. Attenuating the decrease of either lipid significantly reduced desensitization, and simultaneous reduction of PI(4,5)P2 and PI(4)P independently of PLC inhibited TRPV1. We found that, on the mRNA level, the dominant highly Ca2+-sensitive PLC isoform in dorsal root ganglia is PLC?4. Capsaicin-induced desensitization of TRPV1 currents was significantly reduced, whereas capsaicin-induced nerve impulses in the skin–nerve preparation increased in mice lacking this isoform. We propose a comprehensive model in which differential changes in phosphoinositide levels mediated by distinct PLC isoforms result in opposing changes in TRPV1 activity. PMID:23843517

Lukacs, Viktor; Yudin, Yevgen; Hammond, Gerald R.; Sharma, Esseim; Fukami, Kiyoko

2013-01-01

163

Pharmacological characterization of the phosphoinositide second messenger system in the rabbit kidney  

SciTech Connect

The cellular response to hormones and neurotransmitters is a result of receptor activation of a second messenger system to initiate the intracellular cascade. In several tissues, such as brain and liver, one of the second messenger systems involves the hydrolysis of phosphoinositides (PIs) for the formation of inositol phosphate and diacylglycerol as the intracellular messengers. In the present study, they examined the effect of various agents on the hydrolysis of PIs in the rabbit kidney. In the kidney, the effect of the various hormones and neurotransmitters was region specific. Hydrolysis of PIs was stimulated in the inner medulla by (arg{sup 8})-vasopressin, angiotensin II, and atriopeptin I, and in the outer medulla by histamine, adenosine, and secretin. Only carbachol was able to stimulate the hydrolysis of PIs in both the inner and outer medulla. None of the substances tested were able to stimulate this response in the cortex. The following agents did not have an effect in any of the three zones of the kidney: norepinephrine, dopamine, atriopeptins II, and III. They have directly demonstrated the presence of a high affinity saturable binding site on inner medullary collecting duct (IMCD) cells with studies of binding characteristics of the radiolabelled muscarinic antagonist, 1-quinuclidinyl (phenyl-4-{sup 3}H) benzilate (({sup 3}H)QNB). The K{sub d} of 0.27 nM and the B{sub max} of 27.5 fmol/mg protein were determined from Scatchard analysis of the saturation data. In summary, they have demonstrated that cholinergic muscarinic receptors are present in the rabbit kidney, specifically in the IMCD cells. These receptors, which are coupled to the hydrolysis of phosphoinositides, may be involved in the vasodilatory and/or diuretic effects of cholinergic agents.

McArdle, S.

1988-01-01

164

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

165

Excitatory amino acid receptor-stimulated phosphoinositide turnover in primary cerebrocortical cultures.  

PubMed Central

1. Characterization of excitatory amino acid-induced accumulation of [3H]-phosphoinositides was carried out in primary cerebrocortical cultures isolated from foetal rats. 2. All of the excitatory amino acid receptor agonists examined caused concentration-dependent enhancement of phosphoinositide (PI) formation. The most potent excitatory amino acid receptor agonists were quisqualate, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD), ibotenate and glutamate with mean EC50 values of 0.9 +/- 0.4 microM, 15 +/- 5 microM, 15 +/- 3 microM and 41 +/- 8 microM respectively. 3. The selective ionotropic receptor antagonists kynurenic acid (1 mM), 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX, 10 microM) and (+/-)-4-(3-phosphonopropyl)-2 piperazinecarboxylic acid (CPP, 100 microM), failed to block responses to quisqualate, (1S,3R)-ACPD or glutamate. D,L-2-Amino-3-phosphonopropionate (D,L-AP3) did not block 1S,3R-ACPD or quisqualate-induced PI turnover, but had an additive effect with quisqualate or (1S,3R)-ACPD. 4. Exposure of cultures to agonists in the absence of added extracellular calcium reduced the maximal quisqualate response by approximately 45%, revealing a two-component concentration-response curve. Concentration-response curves to ibotenate and glutamate became flattened by omission of extracellular calcium, whereas (1S,3R)-ACPD-stimulated PI turnover was unaffected. 5. Pretreatment of cultures with pertussis toxin markedly inhibited PI responses evoked by (1S,3R)-ACPD. 6. These results suggest that excitatory amino acid-stimulated PI turnover in cerebrocortical cultures is independent of ionotropic receptor activation and is mediated via specific G-protein-linked metabotropic receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8395285

Birrell, G. J.; Marcoux, F. W.

1993-01-01

166

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

PubMed Central

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 Ca2+-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 Ca2+ release, modulates defense gene expression and compromises plant defense responses. PMID:24966862

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

2014-01-01

167

The phosphoinositide 3'-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia.  

PubMed

In lymphocytes, the phosphoinositide 3'-kinase (PI3K) isoform p110? (PI3K?) transmits signals from surface receptors, including the B-cell receptor (BCR). CAL-101, a selective inhibitor of PI3K?, displays clinical activity in CLL, causing rapid lymph node shrinkage and a transient lymphocytosis. Inhibition of pro-survival pathways, the presumed mechanism of CAL-101, does not explain this characteristic pattern of activity. Therefore, we tested CAL-101 in assays that model CLL-microenvironment interactions in vitro. We found that CAL-101 inhibits CLL cell chemotaxis toward CXCL12 and CXCL13 and migration beneath stromal cells (pseudoemperipolesis). CAL-101 also down-regulates secretion of chemokines in stromal cocultures and after BCR triggering. CAL-101 reduces survival signals derived from the BCR or from nurse-like cells, and inhibits BCR- and chemokine-receptor-induced AKT and MAP kinase (ERK) activation. In stromal cocultures, CAL-101 sensitizes CLL cells toward bendamustine, fludarabine, and dexamethasone. These results are corroborated by clinical data showing marked reductions in circulating CCL3, CCL4, and CXCL13 levels, and a surge in lymphocytosis during CAL-101 treatment. Thus, CAL-101 displays a dual mechanism of action, directly decreasing cell survival while reducing interactions that retain CLL cells in protective tissue microenvironments. These data provide an explanation for the clinical activity of CAL-101, and a roadmap for future therapeutic development. PMID:21803855

Hoellenriegel, Julia; Meadows, Sarah A; Sivina, Mariela; Wierda, William G; Kantarjian, Hagop; Keating, Michael J; Giese, Neill; O'Brien, Susan; Yu, Albert; Miller, Langdon L; Lannutti, Brian J; Burger, Jan A

2011-09-29

168

Curcumin inhibits vasculogenic mimicry through the downregulation of erythropoietin-producing hepatocellular carcinoma-A2, phosphoinositide 3-kinase and matrix metalloproteinase-2  

PubMed Central

Glioblastomas (GBMs) are the most common and aggressive malignant primary brain tumors found in humans. In high-grade gliomas, vasculogenic mimicry (VM) is often detected. VM is the formation of de novo vascular networks by highly invasive tumor cells, instead of endothelial cells. An understanding of the mechanisms of VM formation will contribute to the targeted therapy of GBMs. In the present study, the efficacy of curcumin (CCM) on VM formation and its mechanisms were investigated. It was found that CCM inhibits the VM formation, proliferation, migration and invasion of human glioma U251 cells in a dose-dependent manner. Furthermore, CCM downregulated the protein and mRNA expression of erythropoietin-producing hepatocellular carcinoma-A2, phosphoinositide 3-kinase and matrix metalloproteinase-2, indicating that CCM may function through these factors for the inhibition of VM formation. These data provide novel insights into the use of CCM to antagonize VM, and may contribute to the angiogenesis-targeted therapy of malignant glioma. PMID:25202424

LIANG, YIMING; HUANG, MIN; LI, JIANWEN; SUN, XINLIN; JIANG, XIAODAN; LI, LIANGPING; KE, YIQUAN

2014-01-01

169

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

170

Protective effects of exercise and phosphoinositide 3-kinase(p110) signaling in dilated and hypertrophic cardiomyopathy  

Microsoft Academic Search

Physical activity protects against cardiovascular disease, and physiological cardiac hypertrophy associated with regular exercise is usually beneficial, in marked contrast to pathological hypertrophy associated with disease. The p110? isoform of phosphoinositide 3-kinase (PI3K) plays a critical role in the induction of exercise-induced hypertrophy. Whether it or other genes activated in the athlete's heart might have an impact on cardiac function

J. R. McMullen; Fatemeh Amirahmadi; E. A. Woodcock; Martina Schinke-Braun; R. D. Bouwman; K. A. Hewitt; J. P. Mollica; L. Zhang; Yunyu Zhang; Tetsuo Shioi; Antje Buerger; Seigo Izumo; P. Y. Jay; G. L. Jennings

2007-01-01

171

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

172

Cloning, Characterization, and Expression of a Novel Zn2+-Binding FYVE Finger-Containing Phosphoinositide Kinase in Insulin-Sensitive Cells  

PubMed Central

Signaling by phosphorylated species of phosphatidylinositol (PI) appears to regulate diverse responses in eukaryotic cells. A differential display screen for fat- and muscle-specific transcripts led to identification and cloning of the full-length cDNA of a novel mammalian 2,052-amino-acid protein (p235) from a mouse adipocyte cDNA library. Analysis of the deduced amino acid sequence revealed that p235 contains an N-terminal zinc-binding FYVE finger, a chaperonin-like region in the middle of the molecule, and a consensus for phosphoinositide 5-kinases at the C terminus. p235 mRNA appears as a 9-kb transcript, enriched in insulin-sensitive cells and tissues, likely transcribed from a single-copy gene in at least two close-in-size splice variants. Specific antibodies against mouse p235 were raised, and both the endogenously and heterologously expressed proteins were biochemically detected in 3T3-L1 adipocytes and transfected COS cells, respectively. Immunofluorescence microscopy analysis of endogenous p235 localization in 3T3-L1 adipocytes with affinity-purified anti-p235 antibodies documented a punctate peripheral pattern. In COS cells, the expressed p235 N-terminal but not the C-terminal region displayed a vesicular pattern similar to that in 3T3-L1 adipocytes that became diffuse upon Zn2+ chelation or FYVE finger truncation. A recombinant protein comprising the N-terminal but not the C-terminal region of the molecule was found to bind 2.2 mole equivalents of Zn2+. Determination of the lipid kinase activity in the p235 immunoprecipitates derived from 3T3-L1 adipocytes or from COS cells transiently expressing p235 revealed that p235 displayed unique preferences for PI substrate over already phosphorylated PI. In conclusion, the mouse p235 protein determines an important novel class of phosphoinositide kinases that seems to be targeted to specific intracellular loci by a Zn-dependent mechanism. PMID:9858586

Shisheva, Assia; Sbrissa, Diego; Ikonomov, Ognian

1999-01-01

173

Phosphoinositides and Rho Proteins Spatially Regulate Actin Polymerization to Initiate and Maintain Directed Movement in a One-Dimensional Model of a Motile Cell  

PubMed Central

Gradient sensing, polarization, and chemotaxis of motile cells involve the actin cytoskeleton, and regulatory modules, including the phosphoinositides (PIs), their kinases/phosphatases, and small GTPases (Rho proteins). Here we model their individual components (PIP1, PIP2, PIP3; PTEN, PI3K, PI5K; Cdc42, Rac, Rho; Arp2/3, and actin), their interconversions, interactions, and modular functions in the context of a one-dimensional dynamic model for protrusive cell motility, with parameter values derived from in vitro and in vivo studies. In response to a spatially graded stimulus, the model produces stable amplified internal profiles of regulatory components, and initiates persistent motility (consistent with experimental observations). By connecting the modules, we find that Rho GTPases work as a spatial switch, and that the PIs filter noise, and define the front versus back. Relatively fast PI diffusion also leads to selection of a unique pattern of Rho distribution from a collection of possible patterns. We use the model to explore the importance of specific hypothesized interactions, to explore mutant phenotypes, and to study the role of actin polymerization in the maintenance of the PI asymmetry. We also suggest a mechanism to explain the spatial exclusion of Cdc42 and PTEN and the inability of cells lacking active Cdc42 to properly detect chemoattractant gradients. PMID:17098793

Dawes, Adriana T.; Edelstein-Keshet, Leah

2007-01-01

174

Osteopontin is a myosphere-derived secretory molecule that promotes angiogenic progenitor cell proliferation through the phosphoinositide 3-kinase/Akt pathway  

SciTech Connect

We have reported that skeletal myosphere-derived progenitor cells (MDPCs) can differentiate into vascular cells, and that MDPC transplantation into cardiomyopathic hearts improves cardiac function. However, the autocrine/paracrine molecules and underlying mechanisms responsible for MDPC growth have not yet been determined. To explore the molecules enhancing the proliferation of MDPCs, we performed serial analysis of gene expression and signal sequence trap methods using RNA isolated from MDPCs. We identified osteopontin (OPN), a secretory molecule, as one of most abundant molecules expressed in MDPCs. OPN provided a proliferative effect for MDPCs. MDPCs treated with OPN showed Akt activation, and inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway repressed the proliferative effect of OPN. Furthermore, OPN-pretreated MDPCs maintained their differentiation potential into endothelial and vascular smooth muscle cells. These findings indicate an important role of OPN as an autocrine/paracrine molecule in regulating the proliferative growth of muscle-derived angiogenic progenitor cells via the PI3K/Akt pathway.

Ogata, Takehiro [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Ueyama, Tomomi [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan)]. E-mail: tueyama@kuhp.kyoto-u.ac.jp; Nomura, Tetsuya [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Asada, Satoshi [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Tagawa, Masashi [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Nakamura, Tomoyuki [Department of Pharmacology, Kansai Medical University, Moriguchi, Osaka 570-8507 (Japan); Takahashi, Tomosaburo [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Matsubara, Hiroaki [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Oh, Hidemasa [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan)]. E-mail: hidemasa@kuhp.kyoto-u.ac.jp

2007-07-27

175

Dynamics of the phosphoinositide 3-kinase p110? interaction with p85? and membranes reveals aspects of regulation distinct from p110?.  

PubMed

Phosphoinositide 3-kinase ? is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85?. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110? with p85?. Both nSH2 and cSH2 domains of p85? contribute to full inhibition of p110?, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110?. The cSH2 inhibits p110? and p110?, but not p110?, implying that p110? is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes. PMID:21827948

Burke, John E; Vadas, Oscar; Berndt, Alex; Finegan, Tara; Perisic, Olga; Williams, Roger L

2011-08-10

176

p110gamma and p110delta isoforms of phosphoinositide 3-kinase differentially regulate natural killer cell migration in health and disease.  

PubMed

The mechanisms that regulate NK cell trafficking are unclear. Phosphoinositide-3 kinases (PI3K) control cell motility and the p110gamma and p110delta isoforms are mostly expressed in leukocytes, where they transduce signals downstream of G protein coupled receptors (GPCR) or tyrosine kinase receptors, respectively. Here, we set out to determine the relative contribution of p110gamma and p110delta to NK cell migration in mice. Using a combination of single-cell imaging analysis of transgenic cells reporting on PI3K activity in real time and small molecule inhibitors of p110gamma and p110delta, we show here that the tyrosine-kinase coupled p110delta is linked to GPCR signaling and, depending on the GPCR, may even be preferentially activated over p110gamma. Using gene-targeted mice, we showed that both isoforms were essential for NK cell chemotaxis to CXCL12 and to CCL3 and, in vivo, for normal NK cell migration during pregnancy and to the inflamed peritoneum. By contrast, only p110delta was indispensable for chemotaxis to S1P and CXCL10 and for NK cell distribution throughout lymphoid and nonlymphoid tissues and for extravasation to tumors. These results implicate p110delta downstream of GPCRs in NK cells and highlight its nonredundant role as a key regulator of NK cell trafficking in health and disease. PMID:19297623

Saudemont, Aurore; Garçon, Fabien; Yadi, Hakim; Roche-Molina, Marta; Kim, Nayoung; Segonds-Pichon, Anne; Martín-Fontecha, Alfonso; Okkenhaug, Klaus; Colucci, Francesco

2009-04-01

177

Dynamics of the Phosphoinositide 3-Kinase p110? Interaction with p85? and Membranes Reveals Aspects of Regulation Distinct from p110?  

PubMed Central

Summary Phosphoinositide 3-kinase ? is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85?. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110? with p85?. Both nSH2 and cSH2 domains of p85? contribute to full inhibition of p110?, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110?. The cSH2 inhibits p110? and p110?, but not p110?, implying that p110? is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes. PMID:21827948

Burke, John E.; Vadas, Oscar; Berndt, Alex; Finegan, Tara; Perisic, Olga; Williams, Roger L.

2011-01-01

178

Effect of albumin-bound DHA on phosphoinositide phosphorylation in collagen stimulated human platelets  

SciTech Connect

The effect of exogenous albumin-bound docosahexaenoic acid (22:6n-3) (DHA), arachidonic acid (20:4n-6) (AA), and eicosapendaenoic acid (20:5n-3) (EPA) on phosphoinositide metabolism following collagen stimulation was studied using (3H)inositol prelabelled platelets. Collagen stimulation (3 min, 1.8 micrograms/ml) increased the labelling of both phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-biphosphate (PIP2). Of the fatty acids tested, only pre-incubation (2 min) with DHA (20 microM) significantly attenuated the collagen-induced increased PIP and PIP2 labelling; EPA was without effect, while AA enhanced PIP labelling. Forty microM DHA was less effective at attenuating the increased PIP and PIP2 labelling even though this concentration of DHA resulted in greater inhibition of platelet aggregation. Neither concentration of DHA attenuated the increased polyphosphoinositide labelling resulting from stimulation by the endoperoxide analogue U46619, or the phorbol ester, PMA. These data suggest that the effect of DHA on attenuating the increased PIP and PIP2 labelling following collagen stimulation likely occurs before thromboxane receptor occupancy, may not occur at the level of protein kinase C activation, and could be mediated in part via a lessened synthesis of thromboxane A2.

Gaudette, D.C.; Holub, B.J. (Univ. of Guelph, Ontario (Canada))

1990-05-15

179

Ethanol stimulates calcium mobilization, phosphoinositide turnover and shape change in human platelets  

SciTech Connect

The effect of ethanol of cytosolic free calcium levels was investigated in human platelets which were loaded with the intracellular calcium indicator FURA-2. Administration of ethanol in the concentration range of 50-300 mM resulted in a transient, dose-dependent increase in cytosolic calcium, maximal within 5 seconds and returning to near basal levels over the next 5 minutes. Parallel incubations of platelets with the same concentrations of ethanol stimulated shape change as detected in an aggregometer. Chelation of external calcium with EGTA prior to the addition of ethanol reduced the calcium burst partially, but not completely, whereas shape change was largely unaffected. Addition of ethanol to /sup 32/P-labeled platelets resulted in a 16% decrease in the level of /sup 32/P-phosphatidylinositol 4,5-bisphosphate and a 14% increase in /sup 32/P-phosphatidylinositol 4-phosphate within 2 minutes. /sup 32/P-phosphatidic acid levels increased rapidly within 30 seconds and rose linearly thereafter. Phosphatidylinositol and phosphatidylcholine were unaffected by ethanol. The results indicate that ethanol mobilizes intracellular calcium by activation of phosphoinositide-specific phospholipase C, thereby stimulating platelet shape change.

Rubin, R.; Hoek, J.B.

1987-05-01

180

Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl  

PubMed Central

Like several Rho GDP/GTP exchange factors (GEFs), Kalirin7 (Kal7) contains an N-terminal Sec14 domain and multiple spectrin repeats. A natural splice variant of Kalrn lacking the Sec14 domain and four spectrin repeats is unable to increase spine formation; our goal was to understand the function of the Sec14 and spectrin repeat domains. Kal7 lacking its Sec14 domain still increased spine formation, but the spines were short. Strikingly, Kal7 truncation mutants containing only the Sec14 domain and several spectrin repeats increased spine formation. The Sec14 domain bound phosphoinositides, a minor but crucial component of cellular membranes, and binding was increased by a phosphomimetic mutation. Expression of KalSec14-GFP in nonneuronal cells impaired receptor-mediated endocytosis, linking Kal7 to membrane trafficking. Consistent with genetic studies placing Abl, a non–receptor tyrosine kinase, and the Drosophila orthologue of Kalrn into the same signaling pathway, Abl1 phosphorylated two sites in the fourth spectrin repeat of Kalirin, increasing its sensitivity to calpain-mediated degradation. Treating cortical neurons of the wild-type mouse, but not the Kal7KO mouse, with an Abl inhibitor caused an increase in linear spine density. Phosphorylation of multiple sites in the N-terminal Sec14/spectrin region of Kal7 may allow coordination of the many signaling pathways contributing to spine morphogenesis. PMID:24600045

Ma, Xin-Ming; Miller, Megan B.; Vishwanatha, K. S.; Gross, Maegan J.; Wang, Yanping; Abbott, Thomas; Lam, TuKiet T.; Mains, Richard E.; Eipper, Betty A.

2014-01-01

181

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

PubMed

Oncogenic mutations in PIK3CA lead to an increase in instrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3K? to its plasma membrane localised substrate is also required for increased levels of downstream PIP3/Akt signalling. We have studied the subcellular localisation 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 lipid in vitro or in cells maintained in 10% FCS. Following stimulation of receptor tyrosine kinases, microinjected PI3K? was recruited to the plasma membrane, but oncogenic forms of PI3K? were not recruited to the plasma membrane to a greater extent and did not reside at the plasma membrane longer than 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; Ivetac, Ivan; Horan, Kristy A; Mitchell, Christina A; Phillips, Wayne A

2014-03-01

182

Phosphoinositide 3-kinase ? gene mutation predisposes to respiratory infection and airway damage.  

PubMed

Genetic mutations cause primary immunodeficiencies (PIDs) that predispose to infections. Here, we describe activated PI3K-? syndrome (APDS), a PID associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (E1021K) in the p110? protein, the catalytic subunit of phosphoinositide 3-kinase ? (PI3K?), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased immunoglobulin M, and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110?. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110? inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with APDS. PMID:24136356

Angulo, Ivan; Vadas, Oscar; Garçon, Fabien; Banham-Hall, Edward; Plagnol, Vincent; Leahy, Timothy R; Baxendale, Helen; Coulter, Tanya; Curtis, James; Wu, Changxin; Blake-Palmer, Katherine; Perisic, Olga; Smyth, Deborah; Maes, Mailis; Fiddler, Christine; Juss, Jatinder; Cilliers, Deirdre; Markelj, Gašper; Chandra, Anita; Farmer, George; Kielkowska, Anna; Clark, Jonathan; Kracker, Sven; Debré, Marianne; Picard, Capucine; Pellier, Isabelle; Jabado, Nada; Morris, James A; Barcenas-Morales, Gabriela; Fischer, Alain; Stephens, Len; Hawkins, Phillip; Barrett, Jeffrey C; Abinun, Mario; Clatworthy, Menna; Durandy, Anne; Doffinger, Rainer; Chilvers, Edwin R; Cant, Andrew J; Kumararatne, Dinakantha; Okkenhaug, Klaus; Williams, Roger L; Condliffe, Alison; Nejentsev, Sergey

2013-11-15

183

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

184

RAS and RHO Families of GTPases Directly Regulate Distinct Phosphoinositide 3-Kinase Isoforms  

PubMed Central

Summary RAS proteins are important direct activators of p110?, p110?, and p110? type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110? isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110?, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110? via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110?. Cells from mice carrying mutations in the p110? RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110? downstream of GPCRs. PMID:23706742

Fritsch, Ralph; de Krijger, Inge; Fritsch, Kornelia; George, Roger; Reason, Beth; Kumar, Madhu S.; Diefenbacher, Markus; Stamp, Gordon; Downward, Julian

2013-01-01

185

Phosphoinositide 3-kinase ? gene mutation predisposes to respiratory infection and airway damage  

PubMed Central

Genetic mutations cause primary immunodeficiencies (PIDs), which predispose to infections. Here we describe Activated PI3K-? Syndrome (APDS), a PID associated with a dominant gain-of-function mutation E1021K in the p110? protein, the catalytic subunit of phosphoinositide 3-kinase ? (PI3K?), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3,346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased IgM and reduced IgG2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110?. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110? inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, suggesting a therapeutic approach for patients with APDS. PMID:24136356

Angulo, Ivan; Vadas, Oscar; Garçon, Fabien; Banham-Hall, Edward; Plagnol, Vincent; Leahy, Timothy R.; Baxendale, Helen; Coulter, Tanya; Curtis, James; Wu, Changxin; Blake-Palmer, Katherine; Perisic, Olga; Smyth, Deborah; Maes, Mailis; Fiddler, Christine; Juss, Jatinder; Cilliers, Deirdre; Markelj, Gašper; Chandra, Anita; Farmer, George; Kielkowska, Anna; Clark, Jonathan; Kracker, Sven; Debré, Marianne; Picard, Capucine; Pellier, Isabelle; Jabado, Nada; Morris, James A.; Barcenas-Morales, Gabriela; Fischer, Alain; Stephens, Len; Hawkins, Phillip; Barrett, Jeffrey C.; Abinun, Mario; Clatworthy, Menna; Durandy, Anne; Doffinger, Rainer; Chilvers, Edwin; Cant, Andrew J.; Kumararatne, Dinakantha; Okkenhaug, Klaus; Williams, Roger L.; Condliffe, Alison; Nejentsev, Sergey

2014-01-01

186

Role of the phosphoinositide phosphatase FIG4 gene in familial epilepsy with polymicrogyria  

PubMed Central

Objective: The aim of this study was to identify the causal gene in a consanguineous Moroccan family with temporo-occipital polymicrogyria, psychiatric manifestations, and epilepsy, previously mapped to the 6q16–q22 region. Methods: We used exome sequencing and analyzed candidate variants in the 6q16–q22 locus, as well as a rescue assay in Fig4-null mouse fibroblasts and immunohistochemistry of Fig4-null mouse brains. Results: A homozygous missense mutation (p.Asp783Val) in the phosphoinositide phosphatase gene FIG4 was identified. Pathogenicity of the variant was supported by impaired rescue of the enlarged vacuoles in transfected fibroblasts from Fig4-deficient mice. Histologic examination of Fig4-null mouse brain revealed neurodevelopmental impairment in the hippocampus, cortex, and cerebellum as well as impaired cerebellar gyration/foliation reminiscent of human cortical malformations. Conclusions: This study extends the spectrum of phenotypes associated with FIG4 mutations to include cortical malformation associated with seizures and psychiatric manifestations, in addition to the previously described Charcot-Marie-Tooth disease type 4J and Yunis-Varón syndrome. PMID:24598713

Baulac, Stéphanie; Lenk, Guy M.; Dufresnois, Béatrice; Ouled Amar Bencheikh, Bouchra; Couarch, Philippe; Renard, Julie; Larson, Peter A.; Ferguson, Cole J.; Noé, Eric; Poirier, Karine; Hubans, Christine; Ferreira, Stéphanie; Guerrini, Renzo; Ouazzani, Reda; El Hachimi, Khalid Hamid; Meisler, Miriam H.

2014-01-01

187

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

188

Stress-inducible and constitutive phosphoinositide pools have distinctive fatty acid patterns in Arabidopsis thaliana.  

PubMed

Function and development of eukaryotic cells require tight control of diverse physiological processes. Numerous cellular processes are regulated by polyphosphoinositides, which interact with protein partners or mediate release of the second messenger, inositol 1,4,5-trisphosphate (InsP3). Emerging evidence suggests that different regulatory or signaling functions of polyphosphoinositides may be orchestrated by the establishment of distinct subcellular pools; the principles underlying pool-formation are, however, not understood. Arabidopsis plants exhibit transient increases in polyphosphoinositides with hyperosmotic stress, providing a model for comparing constitutive and stress-inducible polyphosphoinositide pools. Using a combination of thin-layer-chromatography and gas-chromatography, phospholipids from stressed and nonstressed Arabidopsis plants were analyzed for their associated fatty acids. Under nonstress conditions structural phospholipids and phosphatidylinositol contained 50-70 mol% polyunsaturated fatty acids (PUFA), whereas polyphosphoinositides were more saturated (10-20 mol% PUFA). With hyperosmotic stress polyphosphoinositides with up to 70 mol% PUFA were formed that differed from constitutive species and coincided with a transient loss in unsaturated phosphatidylinositol. The patterns indicate inducible turnover of an unsaturated phosphatidylinositol pool, which accumulates under standard conditions and is primed for phosphorylation on stimulation. Metabolic analysis of wild-type and transgenic plants disturbed in phosphoinositide metabolism suggests that, in contrast to saturated species, unsaturated polyphosphoinositides are channeled toward InsP3-production. PMID:17327357

König, Sabine; Mosblech, Alina; Heilmann, Ingo

2007-07-01

189

Cell Activation-Induced Phosphoinositide 3-Kinase Alpha/Beta Dimerization Regulates PTEN Activity  

PubMed Central

The phosphoinositide 3-kinase (PI3K)/PTEN (phosphatase and tensin homolog) pathway is one of the central routes that enhances cell survival, division, and migration, and it is frequently deregulated in cancer. PI3K catalyzes formation of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] after cell activation; PTEN subsequently reduces these lipids to basal levels. Activation of the ubiquitous p110? isoform precedes that of p110? at several points during the cell cycle. We studied the potential connections between p110? and p110? activation, and we show that cell stimulation promotes p110? and p110? association, demonstrating oligomerization of PI3K catalytic subunits within cells. Cell stimulation also promoted PTEN incorporation into this complex, which was necessary for PTEN activation. Our results show that PI3Ks dimerize in vivo and that PI3K and PTEN activities modulate each other in a complex that controls cell PI(3,4,5)P3 levels. PMID:24958106

Pérez-García, Vicente; Redondo-Muñoz, Javier; Kumar, Amit

2014-01-01

190

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

191

ANALYSIS OF 3-PHOSPHOINOSITIDE-DEPENDENT KINASE-1 SIGNALING AND FUNCTION IN ES CELLS  

PubMed Central

3-Phosphoinositide-dependent kinase-1 (PDK1) phosphorylates and activates several kinases in the cAMP-dependent, cGMP-dependent and protein kinase C(AGC) family. Many putative PDK1 substrates have been identified, but have not been analyzed following transient and specific inhibition of PDK1 activity. Here, we demonstrate that a previously characterized PDK1 inhibitor, BX-795, shows biological effects that are not consistent with PDK1 inhibition. Therefore, we describe the creation and characterization of a PDK1 mutant, L159G, which can bind inhibitor analogues containing bulky groups that hinder access to the ATP binding pocket of wild type (WT) kinases. When expressed in PDK1?/? ES cells, PDK1 L159G restored phosphorylation of PDK1 targets known to be hypophosphorylated in these cells. Screening of multiple inhibitor analogues showed that 1-NM-PP1 and 3,4-DMB-PP1 optimally inhibited the phosphorylation of PDK1 targets in PDK1?/? ES cells expressing PDK1 L159G but not WT PDK1. These compounds confirmed previously assumed PDK1 substrates, but revealed distinct dephosphorylation kinetics. While PDK1 inhibition had little effect on cell growth, it sensitized cells to apoptotic stimuli. Furthermore, PDK1 loss abolished growth of allograft tumors. Taken together we describe a model system that allows for acute and reversible inhibition of PDK1 in cells, to probe biochemical and biological consequences. PMID:18514190

Tamgüney, Tanja; Zhang, Chao; Fiedler, Dorothea; Shokat, Kevan; Stokoe, David

2008-01-01

192

Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl.  

PubMed

Like several Rho GDP/GTP exchange factors (GEFs), Kalirin7 (Kal7) contains an N-terminal Sec14 domain and multiple spectrin repeats. A natural splice variant of Kalrn lacking the Sec14 domain and four spectrin repeats is unable to increase spine formation; our goal was to understand the function of the Sec14 and spectrin repeat domains. Kal7 lacking its Sec14 domain still increased spine formation, but the spines were short. Strikingly, Kal7 truncation mutants containing only the Sec14 domain and several spectrin repeats increased spine formation. The Sec14 domain bound phosphoinositides, a minor but crucial component of cellular membranes, and binding was increased by a phosphomimetic mutation. Expression of KalSec14-GFP in nonneuronal cells impaired receptor-mediated endocytosis, linking Kal7 to membrane trafficking. Consistent with genetic studies placing Abl, a non-receptor tyrosine kinase, and the Drosophila orthologue of Kalrn into the same signaling pathway, Abl1 phosphorylated two sites in the fourth spectrin repeat of Kalirin, increasing its sensitivity to calpain-mediated degradation. Treating cortical neurons of the wild-type mouse, but not the Kal7(KO) mouse, with an Abl inhibitor caused an increase in linear spine density. Phosphorylation of multiple sites in the N-terminal Sec14/spectrin region of Kal7 may allow coordination of the many signaling pathways contributing to spine morphogenesis. PMID:24600045

Ma, Xin-Ming; Miller, Megan B; Vishwanatha, K S; Gross, Maegan J; Wang, Yanping; Abbott, Thomas; Lam, Tukiet T; Mains, Richard E; Eipper, Betty A

2014-05-01

193

Neuregulin 1-ErbB4-PI3K signaling in schizophrenia and phosphoinositide 3-kinase-p110? inhibition as a potential therapeutic strategy.  

PubMed

Neuregulin 1 (NRG1) and ErbB4, critical neurodevelopmental genes, are implicated in schizophrenia, but the mediating mechanisms are unknown. Here we identify a genetically regulated, pharmacologically targetable, risk pathway associated with schizophrenia and with ErbB4 genetic variation involving increased expression of a PI3K-linked ErbB4 receptor (CYT-1) and the phosphoinositide 3-kinase subunit, p110? (PIK3CD). In human lymphoblasts, NRG1-mediated phosphatidyl-inositol,3,4,5 triphosphate [PI(3,4,5)P3] signaling is predicted by schizophrenia-associated ErbB4 genotype and PIK3CD levels and is impaired in patients with schizophrenia. In human brain, the same ErbB4 genotype again predicts increased PIK3CD expression. Pharmacological inhibition of p110? using the small molecule inhibitor, IC87114, blocks the effects of amphetamine in a mouse pharmacological model of psychosis and reverses schizophrenia-related phenotypes in a rat neonatal ventral hippocampal lesion model. Consistent with these antipsychotic-like properties, IC87114 increases AKT phosphorylation in brains of treated mice, implicating a mechanism of action. Finally, in two family-based genetic studies, PIK3CD shows evidence of association with schizophrenia. Our data provide insight into a mechanism of ErbB4 association with schizophrenia; reveal a previously unidentified biological and disease link between NRG1-ErbB4, p110?, and AKT; and suggest that p110? is a previously undescribed therapeutic target for the treatment of psychiatric disorders. PMID:22689948

Law, Amanda J; Wang, Yanhong; Sei, Yoshitatsu; O'Donnell, Patricio; Piantadosi, Patrick; Papaleo, Francesco; Straub, Richard E; Huang, Wenwei; Thomas, Craig J; Vakkalanka, Radhakrishna; Besterman, Aaron D; Lipska, Barbara K; Hyde, Thomas M; Harrison, Paul J; Kleinman, Joel E; Weinberger, Daniel R

2012-07-24

194

Neuregulin 1-ErbB4-PI3K signaling in schizophrenia and phosphoinositide 3-kinase-p110? inhibition as a potential therapeutic strategy  

PubMed Central

Neuregulin 1 (NRG1) and ErbB4, critical neurodevelopmental genes, are implicated in schizophrenia, but the mediating mechanisms are unknown. Here we identify a genetically regulated, pharmacologically targetable, risk pathway associated with schizophrenia and with ErbB4 genetic variation involving increased expression of a PI3K-linked ErbB4 receptor (CYT-1) and the phosphoinositide 3-kinase subunit, p110? (PIK3CD). In human lymphoblasts, NRG1-mediated phosphatidyl-inositol,3,4,5 triphosphate [PI(3,4,5)P3] signaling is predicted by schizophrenia-associated ErbB4 genotype and PIK3CD levels and is impaired in patients with schizophrenia. In human brain, the same ErbB4 genotype again predicts increased PIK3CD expression. Pharmacological inhibition of p110? using the small molecule inhibitor, IC87114, blocks the effects of amphetamine in a mouse pharmacological model of psychosis and reverses schizophrenia-related phenotypes in a rat neonatal ventral hippocampal lesion model. Consistent with these antipsychotic-like properties, IC87114 increases AKT phosphorylation in brains of treated mice, implicating a mechanism of action. Finally, in two family-based genetic studies, PIK3CD shows evidence of association with schizophrenia. Our data provide insight into a mechanism of ErbB4 association with schizophrenia; reveal a previously unidentified biological and disease link between NRG1-ErbB4, p110?, and AKT; and suggest that p110? is a previously undescribed therapeutic target for the treatment of psychiatric disorders. PMID:22689948

Law, Amanda J.; Wang, Yanhong; Sei, Yoshitatsu; O’Donnell, Patricio; Piantadosi, Patrick; Papaleo, Francesco; Straub, Richard E.; Huang, Wenwei; Thomas, Craig J.; Vakkalanka, Radhakrishna; Besterman, Aaron D.; Lipska, Barbara K.; Hyde, Thomas M.; Harrison, Paul J.; Kleinman, Joel E.; Weinberger, Daniel R.

2012-01-01

195

Changes in phosphoinositide turnover, Ca sup 2+ mobilization, and protein phosphorylation in platelets from NIDDM patients  

SciTech Connect

Enhanced platelet functions have been demonstrated in patients with non-insulin-dependent diabetes mellitus (NIDDM). This study evaluated abnormalities in platelet signal transduction in diabetic patients, including turnover of phosphoinositides, mobilization of intracellular Ca2+, and phosphorylation of 20,000- and 47,000-Mr proteins (P20 and P47). Washed platelets were obtained from 6 patients with NIDDM whose platelet aggregation rates were abnormally elevated (DM-A group), 11 NIDDM patients with normal platelet aggregation rates (DM-B group), and 8 age-matched healthy control subjects. The mass and specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), and phosphatidic acid (PA) in 32P-labeled platelets were not different among the three groups. Hydrolysis of PIP2, PIP, and PI; accumulation of PA; and phosphorylation of P20 in platelets stimulated by 0.05 U/ml thrombin were significantly increased in the DM-A group compared with the control or DM-B group. There was no difference in P47 phosphorylation among the three groups. On the contrary, P20 and P47 phosphorylation induced by 50 nM of 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, was significantly decreased in the DM-A group. Additionally, the intracellular free Ca2+ concentration (( Ca2+)i) was measured with the fluorescent Ca2+ indicator fura 2. Although the basal (Ca2+)i value was similar in the three groups, the rise in (Ca2+)i induced by 0.05 U/ml thrombin in the presence and the absence of extracellular Ca2+ was significantly higher in the DM-A group than the other groups.

Ishii, H.; Umeda, F.; Hashimoto, T.; Nawata, H. (Kyushu Univ., Fukuoka (Japan))

1990-12-01

196

Effects of inhibition of mitochondrial ATP synthesis on phosphoinositide metabolism in rabbit aorta  

SciTech Connect

The authors tested a hypothesis that the plasma membrane phosphoinositide kinase reactions are sensitive to decreases in mitochondrial energy delivery. This hypothesis followed the finding of Lundberg et al of a high Km for ATP for PI and PIP kinase. In aortic rings contracted with norepinephrine (NE), hypoxia causes a decrease in J/sub ATP/ from 2.2 to 1.6 ..mu..mole/(min)(gram wet wt), and a decrease in PCr/total Cr from 0.68 to 0.23, without a detectable change in (ATP). Rings were incubated with (/sup 3/H)-myo-inositol for 30 min at 37/sup 0/C which labelled myo-inositol with only a small /sup 3/H incorporation into inositol phospholipids. Rings were then activated with 18 ..mu..M NE, either during normoxia or hypoxia. The authors measured specific activities of inositol phospholipids and myo-inositol. Hypoxia caused a decrease in the NE-activated inositol phospholipid flux rate from 0.052 +/- 0.006 to 0.023 +/- 0.004 nmoles/(min)(100 nmoles PL Pi), a decrease in the rate of increase in PI, PIP and PIP/sub 2/ specific activities, a decrease in the rate of increase in IP, IP/sub 2/, IP/sub 3/ and IP/sub 4/ radioactivities, and an increase in PIP pool size (and the ratio PIP/PIP/sub 2/). The authors conclude: inositol phospholipid metabolism is sensitive to decreases in delivery of energy to plasma membrane PIP kinase and ATP-dependent reactions involved in PI resynthesis.

Coburn, R.F.; Baron, C.; Papadopoulos, M.T.

1987-05-01

197

The Phosphoinositide 3-Kinase Pathway in Human Cancer: Genetic Alterations and Therapeutic Implications  

PubMed Central

The phosphoinositide 3-kinase (PI3K) pathway is frequently activated in human cancer and represents an attractive target for therapies based on small molecule inhibitors. PI3K isoforms play an essential role in the signal transduction events activated by cell surface receptors including receptor tyrosine kinases (RTKs) and G-protein-coupled receptors (GPCRs). There are eight known PI3K isoforms in humans, which have been subdivided into three classes (I-III). Therefore PI3Ks show considerable diversity and it remains unclear which kinases in this family should be targeted in cancer. The class IA of PI3K comprises the p110?, p110? and p110? isoforms, which associate with activated RTKs. In human cancer, recent reports have described activating mutations in the PIK3CA gene encoding p110?, and inactivating mutations in the phosphatase and tensin homologue (PTEN) gene, a tumour suppressor and antagonist of the PI3K pathway. The PIK3CA mutations described in cancer constitutively activate p110? and, when expressed in cells drive oncogenic transformation. Moreover, these mutations cause the constitutive activation of downstream signaling molecules such as Akt/protein kinase B (PKB), mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K) that is commonly observed in cancer cells. In addition to p110?, the other isoforms of the PI3K family may also play a role in human cancer, although their individual functions remain to be precisely identified. In this review we will discuss the evidence implicating individual PI3K isoforms in human cancer and their potential as drug targets in this context. PMID:19384426

Arcaro, Alexandre; Guerreiro, Ana S

2007-01-01

198

The phosphoinositide-dependent protein kinase 1 inhibitor, UCN-01, induces fragmentation: possible role of metalloproteinases.  

PubMed

Phosphoinositide-dependent protein kinase 1 (PDK1) is a key enzyme, master regulator of cellular proliferation and metabolism; it is considered a key target for pharmacological intervention. Using membranes obtained from DDT1 MF-2 cells, phospho-PDK1 was identified by Western blotting, as two major protein bands of Mr 58-68 kDa. Cell incubation with the PDK1 inhibitor, UCN-01, induced a time- and concentration-dependent decrease in the amount of phospho-PDK1 with a concomitant appearance of a ?42 kDa phosphorylated fragment. Knocking down PDK1 diminished the amount of phospho-PDK1 detected in membranes, accompanied by similarly decreased fragment generation. UCN-01-induced fragment generation was also observed in membranes from cells stably expressing a myc-tagged PDK1 construct. Other PDK1 inhibitors were also tested: OSU-03012 induced a clear decrease in phospho-PDK1 and increased the presence of the phosphorylated fragment in membrane preparations; in contrast, GSK2334470 and staurosporine induced only marginal increases in the amount of PDK1 fragment. Galardin and batimastat, two metalloproteinase inhibitors, markedly attenuated inhibitor-induced PDK1 fragment generation. Metalloproteinases 2, 3, and 9 co-immunoprecipitated with myc-PDK1 under baseline conditions and this interaction was stimulated by UCN-01; batimastat also markedly diminished this effect of the PDK1 inhibitor. Our results indicate that a series of protein kinase inhibitors, namely UCN-01 and OSU-03012 and to a lesser extent GSK2334470 and staurosporine induce PDK1 fragmentation and suggest that metalloproteinases could participate in this effect. PMID:25016091

Alcántara-Hernández, Rocío; Hernández-Méndez, Aurelio; García-Sáinz, J Adolfo

2014-10-01

199

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

200

Phosphoinositide 3-kinase couples NMDA receptors to superoxide release in excitotoxic neuronal death  

PubMed Central

Sustained activation of neuronal N-methly D-aspartate (NMDA)-type glutamate receptors leads to excitotoxic cell death in stroke, trauma, and neurodegenerative disorders. Excitotoxic neuronal death results in part from superoxide produced by neuronal NADPH oxidase (NOX2), but how NMDA receptors are coupled to neuronal NOX2 activation is not well understood. Here, we identify a signaling pathway coupling NMDA receptor activation to NOX2 activation in primary neuron cultures. Calcium influx through the NR2B subunit of NMDA receptors leads to the activation of phosphoinositide 3-kinase (PI3K). Formation of phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3) by PI3K activates the atypical protein kinase C, PKC zeta (PKC?), which in turn phosphorylates the p47phox organizing subunit of neuronal NOX2. Calcium influx through NR2B-containing NMDA receptors triggered mitochondrial depolarization, NOX2 activation, superoxide formation, and cell death. However, equivalent magnitude calcium elevations induced by ionomycin did not induce NOX2 activation or neuronal death, despite causing mitochondrial depolarization. The PI3K inhibitor wortmannin prevented NMDA-induced NOX2 activation and cell death, without preventing cell swelling, calcium elevation, or mitochondrial depolarization. The effects of wortmannin were circumvented by exogenous supply of the PI3K product, PI(3,4,5)P3, and by transfection with protein kinase M, a constitutively active form of PKC?. These findings demonstrate that superoxide formation and excitotoxic neuronal death can be dissociated from mitochondrial depolarization, and identify a novel role for PI3K in this cell death pathway. Perturbations in this pathway may either increase or decrease superoxide production in response to NMDA receptor activation, and may thereby impact neurological disorders, in which excitotoxicity is a contributing factor. PMID:23559014

Brennan-Minnella, A M; Shen, Y; Swanson, R A

2013-01-01

201

Phosphoinositide-dependent kinase PDK1 in the regulation of Ca2+ entry into mast cells.  

PubMed

The function of mast cells is modified by the phosphoinositol-3 (PI3)-kinase pathway. The kinase signals partially through the phosphoinositide-dependent kinase PDK1, which on the one hand activates the serum- and glucocorticoid- inducible kinase SGK1 and on the other hand activates protein kinase PKC?. SGK1 participates in the stimulation of Ca(2+) entry and degranulation, PKC? inhibits degranulation. The present experiments explored the role of PDK1 in mast cell function. As mice completely lacking PDK1 are not viable, experiments have been performed in mast cells isolated from bone marrow (BMMCs) of PDK1 hypomorphic mice (pdk1(hm)) and their wild-type littermates (pdk1(wt)). Antigen stimulation via the FceRI receptor was followed by Ca(2+) entry leading to increase of cytosolic Ca(2+) activity in pdk1(wt) BMMCs, an effect significantly blunted in pdk1(hm) BMMCs. In contrast, Ca(2+) release from intracellular stores was not different between BMMCs of the two genotypes. The currents through Ca(2+)-activated K(+) channels following antigen exposure were again significantly larger in pdk1(wt) than in pdk1(hm) cells. The Ca(2+) ionophore ionomycin (1 ?M) increased the K(+) channel conductance to similar values in both genotypes. ?-hexosaminidase and histamine release were similar in pdk1(wt) BMMCs and pdk1(hm) BMMCs. PKC? inhibitor rottlerin increased ?-hexosaminidase release in pdk1(wt) BMMCs but not in pdk1(hm) BMMCs. Phosphorylation of PKC? and of the SGK1 target NDRG1, was stimulated by the antigen in pdk1(wt) but not in pdk1(hm) cells. The observations reveal a role for PDK1 in the regulation of Ca(2+) entry into and degranulation of murine mast cells. PMID:21063107

Shumilina, Ekaterina; Zemtsova, Irina M; Heise, Nicole; Schmid, Evi; Eichenmüller, Melanie; Tyan, Leonid; Rexhepaj, Rexhep; Lang, Florian

2010-01-01

202

Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival  

PubMed Central

The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110? by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer. PMID:23526884

Green, Benjamin D.; Barry, Emma F.; Ma, Yuefang; Woodcock, Joanna; Fitter, Stephen; Zannettino, Andrew C. W.; Pitson, Stuart M.; Hughes, Timothy P.; Lopez, Angel F.; Shepherd, Peter R.; Wei, Andrew H.; Ekert, Paul G.; Guthridge, Mark A.

2013-01-01

203

Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes.  

PubMed

Phosphoinositides (PI) play important regulatory roles in cell physiology. Localization and quantitation of PIs within the cell is necessary to understand their precise function. Currently, ectopic expression of green fluorescent protein (GFP)-fused PI-binding domains is used to visualize PIs localized to the cell membrane. However, ectopically expressed PI-binding domains may compete with endogenous binding proteins, thus altering the physiological functions of the PIs. Here, we establish a novel method for quantification and visualization of PIs in cells and tissue samples using PI-binding domains labeled with quantum dots (Qdot) as specific probes. This method allowed us to simultaneously quantify three distinct PIs, phosphatidylinositol 3,4,5-triphosphatase [PtdIns(3,4,5)P(3)), PtdIns(3,4)P(2), and PtdIns(4,5)P(2), in crude acidic lipids extracted from insulin-stimulated cells. In addition, the method allowed the PIs to be visualized within fixed cells and tissues. Sequential and spatial changes in PI production and distribution were detected in platelet-derived growth factor (PDGF)-stimulated NRK49F cells. We also observed accumulation of PtdIns(3,4)P(2) at the dorsal ruffle in PDGF-stimulated NIH3T3 cells. Finally, we found PtdIns(3,4,5)P(3) was enriched in lung cancer tissues, which also showed high levels of phosphorylated Akt. Our new method to quantify and visualize PIs is expected to provide further insight into the role of lipid signaling in a wide range of cellular events. PMID:22308508

Irino, Yasuhiro; Tokuda, Emi; Hasegawa, Junya; Itoh, Toshiki; Takenawa, Tadaomi

2012-04-01

204

3-Phosphoinositide-dependent protein kinase-1 as an emerging target in the management of breast cancer  

PubMed Central

It should be noted that 3-phosphoinositide-dependent protein kinase-1 (PDK1) is a protein encoded by the PDPK1 gene, which plays a key role in the signaling pathways activated by several growth factors and hormones. PDK1 is a crucial kinase that functions downstream of phosphoinositide 3-kinase activation and activates members of the AGC family of protein kinases, such as protein kinase B (Akt), protein kinase C (PKC), p70 ribosomal protein S6 kinases, and serum glucocorticoid-dependent kinase, by phosphorylating serine/threonine residues in the activation loop. AGC kinases are known to play crucial roles in regulating physiological processes relevant to metabolism, growth, proliferation, and survival. Changes in the expression and activity of PDK1 and several AGC kinases have been linked to human diseases including cancer. Recent data have revealed that the alteration of PDK1 is a critical component of oncogenic phosphoinositide 3-kinase signaling in breast cancer, suggesting that inhibition of PDK1 can inhibit breast cancer progression. Indeed, PDK1 is highly expressed in a majority of human breast cancer cell lines and both PDK1 protein and messenger ribonucleic acid are overexpressed in a majority of human breast cancers. Furthermore, overexpression of PDK1 is sufficient to transform mammary epithelial cells. PDK1 plays an essential role in regulating cell migration, especially in the context of phosphatase and tensin homologue deficiency. More importantly, downregulation of PDK1 levels inhibits migration and experimental metastasis of human breast cancer cells. Thus, targeting PDK1 may be a valuable anticancer strategy that may improve the efficacy of chemotherapeutic strategies in breast cancer patients. In this review, we summarize the evidence that has been reported to support the idea that PDK1 may be a key target in breast cancer management. PMID:24039447

Fyffe, Chanse; Falasca, Marco

2013-01-01

205

AntiMalarial Drug Artesunate Attenuates Experimental Allergic Asthma via Inhibition of the Phosphoinositide 3Kinase\\/Akt Pathway  

Microsoft Academic Search

BackgroundPhosphoinositide 3-kinase (PI3K)\\/Akt pathway is linked to the development of asthma. Anti-malarial drug artesunate is a semi-synthetic derivative of artemisinin, the principal active component of a medicinal plant Artemisia annua, and has been shown to inhibit PI3K\\/Akt activity. We hypothesized that artesunate may attenuate allergic asthma via inhibition of the PI3K\\/Akt signaling pathway.Methodology\\/Principal FindingsFemale BALB\\/c mice sensitized and challenged with

Chang Cheng; W. Eugene Ho; Fera Y. Goh; Shou Ping Guan; Li Ren Kong; Wen-Qi Lai; Bernard P. Leung; W. S. Fred Wong

2011-01-01

206

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

207

Infectious Bursal Disease Virus VP5 Polypeptide: A Phosphoinositide-Binding Protein Required for Efficient Cell-to-Cell Virus Dissemination  

PubMed Central

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a major avian pathogen responsible for an immunosuppressive disease affecting juvenile chickens. The IBDV genome is formed by two dsRNA segments. The largest one harbors two partially overlapping open reading frames encoding a non-structural polypeptide, known as VP5, and a large polyprotein, respectively. VP5 is non-essential for virus replication. However, it plays a major role in IBDV pathogenesis. VP5 accumulates at the plasma membrane (PM) of IBDV-infected cells. We have analyzed the mechanism underlying the VP5 PM targeting. Updated topological prediction algorithm servers fail to identify a transmembrane domain within the VP5 sequence. However, the VP5 polycationic C-terminal region, harboring three closely spaced patches formed by two or three consecutive basic amino acid residues (lysine or arginine), might account for its PM tropism. We have found that mutations, either C-terminal VP5 deletions or replacement of basic amino acids by alanine residues, that reduce the electropositive charge of the VP5 C-terminus abolish PM targeting. Lipid overlay assays performed with an affinity-purified Flag-tagged VP5 (FVP5) protein version show that this polypeptide binds several phosphoinositides (PIP), exhibiting a clear preference for monophosphate species. Experiments performed with FVP5 mutant proteins lacking the polycationic domain demonstrate that this region is essential for PIP binding. Data gathered with IBDV mutants expressing C-terminal deleted VP5 polypeptides generated by reverse genetics demonstrate that the VP5-PIP binding domain is required both for its PM targeting in infected cells, and for efficient virus dissemination. Data presented here lead us to hypothesize that IBDV might use a non-lytic VP5-dependent cell-to-cell spreading mechanism. PMID:25886023

Rodríguez, Dolores; Rodríguez, José F.

2015-01-01

208

Substrates of semicarbazide-sensitive amine oxidase co-operate with vanadate to stimulate tyrosine phosphorylation of insulin-receptor-substrate proteins, phosphoinositide 3-kinase activity and GLUT4 translocation in adipose cells.  

PubMed Central

It has been shown that the combination of benzylamine or tyramine and low concentrations of vanadate markedly stimulates glucose transport in rat adipocytes by a mechanism that requires semicarbazide-sensitive amine oxidase (SSAO) activity and H(2)O(2) formation. Here we have further analysed the insulin-like effects of the combination of SSAO substrates and vanadate and we have studied the signal-transduction pathway activated in rat adipocytes. We found that several SSAO substrates (benzylamine, tyramine, methylamine, n-decylamine, histamine, tryptamine or beta-phenylethylamine), in combination with low concentrations of vanadate, stimulate glucose transport in isolated rat adipocytes. Furthermore, SSAO substrates together with vanadate stimulated the recruitment of GLUT4 to the cell surface in isolated rat adipocytes. Benzylamine plus vanadate also stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. Benzylamine or tyramine in combination with vanadate potently stimulated the tyrosine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-3. In contrast, benzylamine and vanadate caused only a weak stimulation of insulin receptor kinase. Benzylamine or tyramine in combination with vanadate also stimulated phosphoinositide 3-kinase activity; wortmannin abolished the stimulatory effect of benzylamine and vanadate on glucose transport in adipose cells. Furthermore, the administration of benzylamine and vanadate in vivo caused a rapid lowering of plasma glucose levels, which took place in the absence of alterations in plasma insulin. On the basis of these results we propose that SSAO activity regulates glucose transport in adipocytes. SSAO oxidative activity stimulates glucose transport via the translocation of GLUT4 carriers to the cell surface, resulting from a potent tyrosine phosphorylation of IRS-1 and IRS-3 and phosphoinositide 3-kinase activation. Our results also indicate that substrates of SSAO might regulate glucose disposal in vivo. PMID:10926841

Enrique-Tarancón, G; Castan, I; Morin, N; Marti, L; Abella, A; Camps, M; Casamitjana, R; Palacín, M; Testar, X; Degerman, E; Carpéné, C; Zorzano, A

2000-01-01

209

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

PubMed Central

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 p85? constitutively led to stress fiber disruption, cortical actin recruitment, extensive ruffling, and macropinosome formation, as measured by a selective acceleration of fluid-phase endocytosis. These alterations closely correlated with activation of PI3K and phosphatidylinositol-specific phospholipase C (PI-PLC), as assayed by 3-phosphoinositide synthesis in situ and in vitro and inositol 1,4,5 trisphosphate steady-state levels, respectively; they were abolished by stable transfection of v-Src–transformed cells for dominant-negative truncated p85? expression and by pharmacological inhibitors of PI3K and PI-PLC, indicating a requirement for both enzymes. Whereas PI3K activation resisted PI-PLC inhibition, PI-PLC activation was abolished by a PI3K inhibitor and dominant-negative transfection, thus placing PI-PLC downstream of PI3K. Together, these data suggest that permanent sequential activation of both PI3K and PI-PLC is necessary for the dramatic reorganization of the actin cytoskeleton in oncogene-transformed fibroblasts, resulting in constitutive ruffling and macropinocytosis. PMID:11029048

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

2000-01-01

210

Pharmacologic Profiling of Phosphoinositide 3-Kinase Inhibitors as Mitigators of Ionizing Radiation–Induced Cell Death  

PubMed Central

Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated ?IR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of ?IR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after ?IR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased ?IR-induced DNA damage as measured by ?H2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after IR abrogated cell death. PMID:24068833

Sharlow, Elizabeth R.; Epperly, Michael W.; Lira, Ana; Leimgruber, Stephanie; Skoda, Erin M.; Wipf, Peter; Greenberger, Joel S.

2013-01-01

211

Purification and characterization of two immunologically distinct phosphoinositide-specific phospholipases C from bovine brain.  

PubMed

We previously reported (Ryu, S. H., Cho, K. S., Lee, K. Y., Suh, P. G., and Rhee, S. G. (1986) Biochem. Biophys. Res. Commun. 141, 137-144) that cytosolic fractions of bovine brain contain two phosphoinositide-specific phospholipase C (PLC), PLC-I and PLC-II. In this paper purification procedures and properties of these two forms of enzyme are presented. The two enzymes exhibit similar substrate specificity. Both PLC-I and PLC-II catalyze the hydrolysis of phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2). Yet, they respond differently to activators such as Ca2+ and nucleotides and to inhibitory divalent metal ions such as Hg2+ and Cd2+. In addition, they are immunologically distinct as evidenced by the fact that monoclonal antibodies directed against either enzyme do not cross-react with the other. Their activities are Ca2+ concentration-dependent. PIP and PIP2 are better substrates than PI for both PLC-I and PLC-II when the concentration of Ca2+ is in the micromolar range. Study of the effect of nucleotides, such as GTP, guanosine 5'-(3-O-thio)triphosphate, guanyl-5'-yl imidodiphosphate, and ATP, on the activities of both isozymes with PIP2 as substrate revealed that (i) in the absence of Ca2+, PLC-I activity is enhanced by 400% by either GTP or ATP. In the presence of Ca2+ (a condition in which PLC-I exhibits much higher activity), the activation factor by nucleotides is diminished to approximately 140%. (ii) without Ca2+, PLC-II activity is too low to measure with or without added nucleotides. The effect of nucleotides on PLC-II activity is trivial in the presence of Ca2+. In addition, studies on the effect of metal ions on PI hydrolysis showed that the activities of both PLC-I and PLC-II are not affected by 50 microM of Mg2+, Mn2+, Ca2+, or Ni2+. However, Hg2+, Zn2+, and Cu2+ inhibited both PLC-I and PLC-II, with PLC-II exhibiting much higher sensitivity to these metal ions than PLC-I. For example, the value of I0.5 for Hg2+ inhibition is 0.2 microM for PLC-II and 1 microM for PLC-I. Cd2+ selectively inhibits PLC-II with a I0.5 value of 5 microM. Most of these metal ions' inhibition can be overcome by either dithiothreitol or EDTA. PMID:3040753

Ryu, S H; Cho, K S; Lee, K Y; Suh, P G; Rhee, S G

1987-09-15

212

d-myo-Inositol 1,4,5,6-tetrakisphosphate produced in human intestinal epithelial cells in response to Salmonella invasion inhibits phosphoinositide 3-kinase signaling?pathways  

PubMed Central

Several inositol-containing compounds play key roles in receptor-mediated cell signaling events. Here, we describe a function for a specific inositol polyphosphate, d-myo-inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P4], that is produced acutely in response to a receptor-independent process. Thus, infection of intestinal epithelial cells with the enteric pathogen Salmonella, but not with other invasive bacteria, induced a multifold increase in Ins(1,4,5,6)P4 levels. To define a specific function of Ins(1,4,5,6)P4, a membrane-permeant, hydrolyzable ester was used to deliver it to the intracellular compartment, where it antagonized epidermal growth factor (EGF)-induced inhibition of calcium-mediated chloride (Cl?) secretion (CaMCS) in intestinal epithelia. This EGF function is likely mediated through a phosphoinositide 3-kinase (PtdIns3K)-dependent mechanism because the EGF effects are abolished by wortmannin, and three different membrane-permeant esters of the PtdIns3K product phosphatidylinositol 3,4,5-trisphosphate mimicked the EGF effect on CaMCS. We further demonstrate that Ins(1,4,5,6)P4 antagonized EGF signaling downstream of PtdIns3K because Ins(1,4,5,6)P4 interfered with the PtdInsP3 effect on CaMCS without affecting PtdIns3K activity. Thus, elevation of Ins(1,4,5,6)P4 in Salmonella-infected epithelia may promote Cl? flux by antagonizing EGF inhibition mediated through PtdIns3K and PtdInsP3. PMID:9405634

Eckmann, Lars; Rudolf, Marco T.; Ptasznik, Andrzej; Schultz, Carsten; Jiang, Tao; Wolfson, Nora; Tsien, Roger; Fierer, Joshua; Shears, Stephen B.; Kagnoff, Martin F.; Traynor-Kaplan, Alexis E.

1997-01-01

213

Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells.  

PubMed

Vasodilator actions of insulin are mediated by signaling pathways involving phosphatidylinositol 3-kinase (PI 3-kinase) and Akt that lead to activation of endothelial nitric oxide synthase (eNOS) in endothelium. Signaling molecules immediately upstream and downstream from PI 3-kinase involved with production of NO in response to insulin have not been previously identified. In this study, we evaluated roles of insulin receptor substrate 1 (IRS-1) and phosphoinositide-dependent kinase 1 (PDK-1) in production of NO. The fluorescent dye 4,5-diamine fluorescein diacetate was used to directly measure NO in NIH-3T3(IR) cells transiently cotransfected with eNOS and various IRS-1 or PDK-1 constructs. In control cells, transfected with only eNOS, insulin stimulated a rapid dose-dependent increase in NO. Overexpression of wild-type IRS-1 increased the maximal insulin response 3-fold. Overexpression of IRS1-F6 (mutant that does not bind PI 3-kinase) or an antisense ribozyme against IRS-1 substantially inhibited insulin-stimulated production of NO. Likewise, overexpression of wild-type PDK-1 enhanced insulin-stimulated production of NO, whereas a kinase-inactive mutant PDK-1 inhibited this action of insulin. Qualitatively similar results were observed in vascular endothelial cells. Production of NO by a calcium-dependent mechanism in response to lysophosphatidic acid was unaffected by either wild-type or mutant IRS-1 and PDK-1. We conclude that IRS-1 and PDK-1 play necessary roles in insulin-signaling pathways leading to activation of eNOS. Furthermore, classical Ca2+-mediated pathways for activation of eNOS are separable from IRS-1- and PDK-1-dependent insulin-signaling pathways. PMID:12145346

Montagnani, Monica; Ravichandran, Lingamanaidu V; Chen, Hui; Esposito, Diana L; Quon, Michael J

2002-08-01

214

RNA interference-mediated knockdown of Aurora-B alters the metastatic behavior of A549 cells via modulation of the phosphoinositide 3-kinase/Akt signaling pathway  

PubMed Central

Accumulating evidence has revealed that an elevated expression level of Aurora-B is associated with metastasis in various types of malignant tumor. However, it is currently unclear whether this molecule is involved in non-small lung cancer (NSCLC) metastasis, and the molecular mechanisms associated with Aurora-B and metastasis remain unknown. In the present study, in order to investigate whether Aurora-B is involved in the development and metastasis of NSCLC, the Aurora-B protein expression in NSCLC tissues was detected by immunohistochemistry and its association with metastasis was analyzed. The results revealed that the expression levels of the Aurora-B protein in tissues obtained from NSCLC patients with lymph node metastasis were significantly higher than those without metastatic disease. Furthermore, the effect of Aurora-B inhibition on A549 cell migration and invasion, as well as the activity of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was evaluated. Aurora-B was inhibited in the A549 cells using short hairpin RNA, and the cell migration and invasion rates were investigated using wound healing and Transwell invasion assays. In addition, the expression of the main proteins in the PI3K/Akt/nuclear factor-?B (NF-?B) signaling pathway, and matrix metalloproteinase (MMP)-2 and -9 were measured by western blot analysis. The results demonstrated that cell migration and invasion were decreased as a result of silencing Aurora-B. Furthermore, the activity of the PI3K/Akt/NF-?B signaling pathway and the expression of MMP-2 and -9 protein were suppressed by silencing Aurora-B. The results of the present study indicate that the knockdown of Aurora-B suppresses A549 cell invasion and migration via the inhibition of the PI3K/Akt signaling pathway in vitro and thus, targeting Aurora-B may present a potential treatment strategy for NSCLC. PMID:25295091

ZHOU, LONG DIAN; XIONG, XU; LONG, XIN HUA; LIU, ZHI LI; HUANG, SHAN HU; ZHANG, WEI

2014-01-01

215

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

216

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

217

Attenuation of PTEN increases p21 stability and cytosolic localization in kidney cancer cells: a potential mechanism of apoptosis resistance  

Microsoft Academic Search

BACKGROUND: The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on

Pei-Yin Lin; Susan P Fosmire; See-Hyoung Park; Jin-Young Park; Shairaz Baksh; Jaime F Modiano; Robert H Weiss

2007-01-01

218

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

219

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

220

TfPLC1 , a gene encoding phosphoinositide-specific phospholipase C, is predominantly expressed in reproductive organs in Torenia fournieri  

Microsoft Academic Search

Phosphoinositide-specific phospholipase C (PI-PLC) is an important enzyme, which is a key player involved in eukaryotic signal\\u000a transduction pathways. In plants, it plays a key role in growth and development as well as environmental stress. However,\\u000a little is known about its roles in signal transduction during sexual reproduction process. In this study, we cloned and characterized\\u000a a gene of full-length

Meifang Song; Shijie Liu; Zhen Zhou; Yuzhen Han

2008-01-01

221

A regulatory subunit of phosphoinositide 3-kinase increases the nuclear accumulation of X-box–binding protein-1 to modulate the unfolded protein response  

Microsoft Academic Search

Class Ia phosphoinositide 3-kinase (PI3K), an essential mediator of the metabolic actions of insulin, is composed of a catalytic (p110? or p110?) and regulatory (p85??, p85?? or p55?) subunit. Here we show that p85?? interacts with X-box–binding protein-1 (XBP-1), a transcriptional mediator of the unfolded protein response (UPR), in an endoplasmic reticulum (ER) stress-dependent manner. Cell lines with knockout or

Jonathon N Winnay; Jeremie Boucher; Marcelo A Mori; Kohjiro Ueki; C Ronald Kahn

2010-01-01

222

Studies on the metabolism of metallothionein and alkaline phosphatase of adult rat primary hepatocyte cultures: role of fetal calf serum and agonists of the phosphoinositide cascade.  

PubMed

Adult rat primary hepatocytes maintained in DMEM/F12 (Ham) media were used as a model system for studying the role of fetal calf serum (FCS) and agonists of the phosphoinositide cascade in the metabolism of metallothionein (MT) and alkaline phosphatase (ALP). Experiments were performed both after a 24 h preincubation with FCS and with bovine serum albumin (BSA). Hepatocytes were treated with dexamethasone (DEX), zinc (Zn) and with the agonists of the phosphoinositide cascade A23187, 1,2-dioctanoyl-sn-glycerol (DiC8), 12-O-tetradecanoylphorbol-13-acetate (TPA), angiotensin II (AT), platelet activating factor (PAF), Arg8-vasopressin (VP) and were analyzed for MT and ALP activity in cell homogenates. Cell viability was evaluated by lactate dehydrogenase (LDH) liberation into culture medium, induction of tyrosine aminotransferase (TAT) through DEX and by trypan blue exclusion. Overall, cell viability was improved by the FCS pretreatment and by DEX. Exposure of hepatocytes to the established direct inducers Zn and DEX of MT resulted in a manifold increase in MT, independent of whether the cultures were FCS pretreated or not. The FCS preincubation produced a moderate elevation of ALP activity by stimulating cell viability. However, ALP was unaltered in response to Zn and DEX. None of the experiments conducted with agonists of the phosphoinositide cascade led to an elevation of MT and ALP. Only the incubation of hepatocytes with A23187 resulted in a concentration dependent significant decrease of MT and ALP. This observation was due to a cytotoxic effect of A 23187, displayed by LDH leakage and an increase in the number of cells stained with trypan blue. In conclusion, in primary hepatocyte cultures agonists of the phosphoinositide did not have an effect on the metabolism of MT and ALP. Previous in vivo results indicating alterations of Zn metabolism in liver, therefore seem to be caused by indirect systemic responses. PMID:8237077

Krämer, K; Markwitan, A; Pallauf, J

1993-09-01

223

A Dual Phosphoinositide3Kinase A\\/mTOR Inhibitor Cooperates with Blockade ofEpidermal Growth Factor Receptor in PTEN-Mutant Glioma  

Microsoft Academic Search

We have shown previously that blockade of epidermal growth factor receptor (EGFR) cooperates with a pan-selective inhibitor of phosphoinositide-3-kinase (PI3K) in EGFR-driven glioma. In this communication, we tested EGFR-driven glioma differing in PTEN status, treating with the EGFR inhibitor erlotinib and a novel dual inhibitor of PI3KA and mTOR (PI-103). Erlotinib blocked proliferation only in PTENwt cells expressing EGFR. Although

Qi-Wen Fan; Christine K. Cheng; Theodore P. Nicolaides; Christopher S. Hackett; Zachary A. Knight; Kevan M. Shokat

224

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

225

Modulation of heteromeric P2X1/5 receptors by phosphoinositides in astrocytes depends on the P2X1 subunit.  

PubMed

Purinergic signaling is critical for neuron-glia communication. Glial cells participate in synaptic transmission and express metabotropic P2Y as well as ionotropic P2X ATP receptors. In astrocytes, endogenous ATP-evoked currents with kinetics and pharmacology characteristic of the heteromeric P2X1/5 receptor channel have recently been reported. We investigated the interaction of major phosphoinositides with heteromeric P2X1/5 channels. Using patch-clamp electrophysiology on enhanced green fluorescent protein-expressing astrocytes acutely isolated from cortical slices of transgenic mice, we report a strong modulation of P2X1/5-like currents by phosphoinositides. Wortmannin-induced depletion of phosphoinositides decreases the amplitude of both the fast and sustained component of the P2X1/5-like currents although recovery and kinetics remain intact. In transfected human embryonic kidney cells, we provide evidence that depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] levels significantly decreases P2X1/5 currents while intracellular application of PI(4,5)P(2) completely rescued P2X1/5 currents, ruling out the involvement of phosphatidylinositol 3,4,5-trisphosphate. In contrast to P2X1, homomeric P2X5 current responses were found insensitive to phosphoinositides, and the C-terminus of P2X5 subunit lacked binding to phospholipids in an overlay assay. Our results suggest that the contribution of calcium-permeable heteromeric P2X1/5 receptor channels to the excitability of astrocytes is modulated by PI(4,5)P(2) through the P2X1 lipid-binding domain. PMID:20374427

Ase, Ariel R; Bernier, Louis-Philippe; Blais, Dominique; Pankratov, Yuriy; Séguéla, Philippe

2010-06-01

226

Repression of phosphoinositide-dependent protein kinase 1 expression by ciglitazone via Egr-1 represents a new approach for inhibition of lung cancer cell growth  

PubMed Central

Background Peroxisome proliferator-activated receptors gamma (PPAR?) ligands have been shown to inhibit the growth of non-small cell lung cancer (NSCLC) cells.?However, the mechanisms underlying this effect remain incompletely elucidated. Methods Cell proliferation and apoptosis were measured by cell viability, MTT and caspase3/7 activity assays. Phosphorylation/protein expression and gene silence/overexpression of AMPK?, phosphoinositide-dependent protein kinase 1 (PDK1), Egr-1 and PPAR? were performed by Western blot and siRNA/transfection assays. Dual-Luciferase Reporter Kit was used to measure the PPAR response elements (PPRE) reporter and PDK1 promoter activities, and ChIP assay was used to detect the Egr-1 protein binding to the DNA site in the PDK1 gene promoter. Results We found that ciglitazone, one synthetic PPAR? ligand, inhibited growth and induced apoptosis of NSCLC cells through decreased expression of PDK1, which was not blocked by GW9662 (a specific PPAR? antagonist). Overexpression of PDK1 overcame the effect of ciglitazone on cell growth and caspase 3/7 activity. Ciglitazone increased the phosphorylation of AMPK? and c-Jun N-terminal kinase (JNK), and the inhibitor of AMPK (compound C), but not JNK (SP600125), reversed the effect of ciglitazone on PDK1 protein expression. Ciglitazone reduced PDK1 gene promoter activity, which was not observed in cells exposed to compound C, but not silenced of PPAR? siRNA. Combination of ciglitazone and metformin further reduced PDK1 expression and promoter activity.?Furthermore, we showed that ciglitazone induced the protein expression of Egr-1, which was not observed in cells silencing of AMPK?. Moreover, silencing of Egr-1 abrogated the effect of ciglitazone on PDK1 promoter activity and cell growth. On the contrary, overexpression of Egr-1 enhanced the effect of ciglitazone on PDK1 gene promoter activity. ChIP assays demonstrated that ciglitazone induced Egr-1 protein bind to the specific DNA site in the PDK1 gene promoter. Conclusion Collectively, our results demonstrate that ciglitazone inhibits PDK1 expression through AMPK?-mediated induction of Egr-1 and Egr-1 binding to the specific DNA site in the PDK1 gene promoter, which is independent of PPAR?. Activation of AMPK? by metformin enhances the effect of ciglitazone. In turn, this leads to inhibition of NSCLC cell proliferation. PMID:24925061

2014-01-01

227

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

228

A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases  

PubMed Central

Voltage sensitive phosphatases (VSPs), including engineered voltage sensitive PTEN, are excellent tools to rapidly and reversibly alter the phosphoinositide (PI) content of the plasma membrane in vivo and study the tumor suppressor PTEN. However, widespread adoption of these tools is hampered by the requirement for electrophysiological instrumentation to control the activity of VSPs. Additionally, monitoring and quantifying the PI changes in living cells requires sophisticated microscopy equipment and image analysis. Here we present methods that bypass these obstacles. First, we explore technically simple means for activation of VSPs via extracellularly applied agents or light. Secondly, we characterize methods to monitor PI(4,5)P2 and PI(3,4,5)P3 levels using fluorescence microscopy or photometry in conjunction with translocation or FRET based PI probes, respectively. We then demonstrate the application of these techniques by characterizing the effect of known PTEN mutations on its enzymatic activity, analyzing the effect of PTEN inhibitors, and detecting in real time rapid inhibition of protein kinase B following depletion of PI(3,4,5)P3. Thus, we established an approach that does not only allow for rapidly manipulating and monitoring PI(4,5)P2 and PI(3,4,5)P3 levels in a population of cells, but also facilitates the study of PTEN mutants and pharmacological targeting in mammalian cells.

Mavrantoni, Angeliki; Thallmair, Veronika; Leitner, Michael G.; Schreiber, Daniela N.; Oliver, Dominik; Halaszovich, Christian R.

2015-01-01

229

Different roles of protein kinase C-beta and -delta in arachidonic acid cascade, superoxide formation and phosphoinositide hydrolysis.  

PubMed Central

In contrast with protein kinase C (PKC)-beta, PKC-delta is exclusively detectable in the membrane fraction of liver macrophages. After long-term treatment with phorbol 12-myristate 13-acetate (PMA) PKC-beta is depleted faster (within 3 h) than PKC-delta (> 7h). Simultaneously, pretreatment with PMA for 3 h inhibits the PMA- and zymosan-induced generation of superoxide and the PMA-induced formation of prostaglandin (PG) E2, whereas a preincubation of more than 7 h is required to affect the zymosan-induced release of PGE2 and inositol phosphates. These results support an involvement of PKC-beta in the PMA-induced activation of the arachidonic acid cascade and in superoxide formation and imply an involvement of PKC-delta in zymosan-induced phosphoinositide hydrolysis and PGE2 formation. Two phorbol ester derivates, sapintoxin A (SAPA) and 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA), which have been previously reported to activate preferentially PLC-beta but not PKC-delta in vitro [Ryves, Evans, Olivier, Parker and Evans (1992) FEBS Lett. 288, 5-9], induce the formation of PGE2 and superoxide, down-regulate PKC-delta and potentiate inositol phosphate formation in parallel SAPA, but not DOPPA, down-regulates PKC-beta and inhibits the PMA-induced formation of eicosanoids and superoxide. Images Figure 1 Figure 2 Figure 5 PMID:8389125

Duyster, J; Schwende, H; Fitzke, E; Hidaka, H; Dieter, P

1993-01-01

230

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

231

Phosphoinositide-dependent kinase 1 regulates leukemia stem cell maintenance in MLL-AF9-induced murine acute myeloid leukemia.  

PubMed

Although great efforts have been made to improve available therapies, the mortality rate of acute myeloid leukemia (AML) remains high due to poor treatment response and frequent relapse after chemotherapy. Leukemia stem cells (LSCs) are thought to account for this poor prognosis and relapse. Phosphoinositide-dependent kinase 1 (PDK1) is a critical regulator of the PI3K/Akt pathway and has been shown to be frequently activated in leukemia. However, the role of PDK1 in the regulation of LSCs in AML is still not clear. Using a PDK1 conditional deletion MLL-AF9 murine AML model, we revealed that the deletion of PDK1 prolonged the survival of AML mice by inducing LSC apoptosis. This was accompanied by the increased expression of the pro-apoptotic genes Bax and p53 and the reduced expression of Stat5, which has been shown to be constitutively activated in leukemia. Thus, our findings suggest that PDK1 plays an essential role in maintaining LSCs. Further delineating the function of PDK1 in LSCs may provide a new strategy for the improved treatment of AML relapse. PMID:25769952

Hu, Tianyuan; Li, Cong; Zhang, Yingchi; Wang, Le; Peng, Luyun; Cheng, Hui; Wang, Weili; Chu, Yajing; Xu, Mingjiang; Cheng, Tao; Yuan, Weiping

2015-04-17

232

The p85? regulatory subunit of phosphoinositide 3-kinase has unique and redundant functions in B cells  

PubMed Central

Phosphoinositide kinase (PI3K) is activated by various receptors on lymphocytes and regulates development, activation, and tolerance. Genetic ablation of PI3K function in T cells leads to the appearance of autoimmune disorders. In B cells, loss of the class IA regulatory subunit p85? causes a partial defect in B cell development and proliferation, whereas loss of p85? alone causes no apparent changes in B cell function. Here we investigate further the consequences of p85? deletion in B cells, in the presence or absence of p85?. We demonstrate that p85? partially compensates for loss of p85? in B cell development and peripheral survival, with greater defects observed when both isoforms are absent. BCR-mediated AKT phosphorylation is partially reduced in p85?-deficient B cells and further diminished with concomitant loss of p85?. Unexpectedly, loss of p85? results in increased BCR-mediated proliferation and ERK phosphorylation. These results indicate that the p85? regulatory isoform has partially overlapping functions with p85? in B cells as well as a unique role in opposing BCR responses. PMID:19811262

OAK, JEAN S.; CHEN, JING; PERALTA, RAECHEL Q.; DEANE, JONATHAN A.; FRUMAN, DAVID A.

2009-01-01

233

Overexpression of a rat kinase-deficient phosphoinositide 3-kinase, Vps34p, inhibits cathepsin D maturation.  

PubMed Central

Lipid kinases and their phosphorylated products are important regulators of many cellular processes, including intracellular membrane traffic. The best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p, which is required for correct targeting of newly synthesized carboxypeptidase Y to the yeast vacuole. A probable mammalian Vps34p orthologue has been previously identified, but its function in the trafficking of lysosomal enzymes has not been resolved. To investigate the possible role(s) of mammalian Vps34p in protein targeting to lysosomes, we have cloned the rat orthologue and overexpressed a kinase-deficient mutant in HeLa cells. Expression of the mutant protein inhibited both maturation of procathepsin D and basal secretion of the precursor. In contrast wortmannin, which also inhibited maturation, caused hypersecretion of the precursor. We propose that mammalian Vps34p plays a direct role in targeting lysosomal enzyme precursors to the endocytic pathway in an analogous fashion to its role in the fusion of early endocytic vesicles with endosomes. We further suggest that inhibition of a wortmannin-sensitive enzyme, other than mammalian Vps34p, is responsible for the failure to recycle unoccupied mannose 6-phosphate receptors to the trans-Golgi network, and consequent hypersecretion of lysosomal enzyme precursors observed in the presence of this drug. PMID:11171063

Row, P E; Reaves, B J; Domin, J; Luzio, J P; Davidson, H W

2001-01-01

234

Expression analysis of a stress-related phosphoinositide-specific phospholipase C gene in wheat (Triticum aestivum L.).  

PubMed

Plant phosphoinositide-specific phospholipases C (PI-PLCs) function in several essential plant processes associated with either development or environmental stress. In this report, we examined the expression patterns of TaPLC1 under drought and high salinity stress at the transcriptional and post-transcriptional levels. TaPLC1 mRNA was expressed in all wheat organs examined. U73122 and edelfosine, the PLC inhibitor, impaired seedling growth and enhanced seedling sensitivity to drought and high salinity stress. Though TaPLC1 expression in wheat was lowest at the seedling stage, it was strongly induced under conditions of stress. When 6-day-old wheat seedlings were treated with 200 mM NaCl or 20% (w/v) PEG 6000 for 6 or 12 h, respectively, the TaPLC1 transcript level increased by 16-fold compared to the control. Western blotting showed that the TaPLC protein concentration was also maintained at a high level from 24 to 48 h during stress treatment. Together, our results indicate the possible biological functions of TaPLC1 in regulating seedling growth and the response to drought and salinity stress. PMID:25121594

Zhang, Ke; Jin, Congcong; Wu, Lizhu; Hou, Mingyu; Dou, Shijuan; Pan, Yanyun

2014-01-01

235

Propofol pretreatment attenuates lipopolysaccharide-induced acute lung injury in rats by activating the phosphoinositide-3-kinase/Akt pathway  

PubMed Central

The aim of this study was to investigate the effect of propofol pretreatment on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the role of the phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathway in this procedure. Survival was determined 48 h after LPS injection. At 1 h after LPS challenge, the lung wet- to dry-weight ratio was examined, and concentrations of protein, tumor necrosis factor-? (TNF-?), and interleukin-6 (IL-6) in bronchoalveolar lavage fluid (BALF) were determined using the bicinchoninic acid method or ELISA. Lung injury was assayed via lung histological examination. PI3K and p-Akt expression levels in the lung tissue were determined by Western blotting. Propofol pretreatment prolonged survival, decreased the concentrations of protein, TNF-?, and IL-6 in BALF, attenuated ALI, and increased PI3K and p-Akt expression in the lung tissue of LPS-challenged rats, whereas treatment with wortmannin, a PI3K/Akt pathway specific inhibitor, blunted this effect. Our study indicates that propofol pretreatment attenuated LPS-induced ALI, partly by activation of the PI3K/Akt pathway. PMID:25387673

Zhao, L.L.; Hu, G.C.; Zhu, S.S.; Li, J.F.; Liu, G.J.

2014-01-01

236

Expression Analysis of a Stress-Related Phosphoinositide-Specific Phospholipase C Gene in Wheat (Triticum aestivum L.)  

PubMed Central

Plant phosphoinositide-specific phospholipases C (PI-PLCs) function in several essential plant processes associated with either development or environmental stress. In this report, we examined the expression patterns of TaPLC1 under drought and high salinity stress at the transcriptional and post-transcriptional levels. TaPLC1 mRNA was expressed in all wheat organs examined. U73122 and edelfosine, the PLC inhibitor, impaired seedling growth and enhanced seedling sensitivity to drought and high salinity stress. Though TaPLC1 expression in wheat was lowest at the seedling stage, it was strongly induced under conditions of stress. When 6-day-old wheat seedlings were treated with 200 mM NaCl or 20% (w/v) PEG 6000 for 6 or 12 h, respectively, the TaPLC1 transcript level increased by 16-fold compared to the control. Western blotting showed that the TaPLC protein concentration was also maintained at a high level from 24 to 48 h during stress treatment. Together, our results indicate the possible biological functions of TaPLC1 in regulating seedling growth and the response to drought and salinity stress. PMID:25121594

Wu, Lizhu; Hou, Mingyu; Dou, Shijuan; Pan, Yanyun

2014-01-01

237

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

238

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

PubMed Central

Lipid signaling pathways define central mechanisms of cellular regulation. Productive lipid signaling requires an orchestrated coupling0020between 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, where 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

2009-01-01

239

Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase  

NASA Technical Reports Server (NTRS)

To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.; Brown, C. S. (Principal Investigator)

2002-01-01

240

Multiple forms of p55PIK, a regulatory subunit of phosphoinositide 3-kinase, are generated by alternative initiation of translation.  

PubMed Central

A cDNA encoding p55PIK, one of the regulatory subunits of phosphoinositide (phosphatidylinositol) 3-kinase, was cloned from a cDNA library derived from the mouse mammary epithelial cell line C57MG. The cDNA coding for full-length p55PIK was transiently expressed in COS-7 cells. Western blot analysis of p55PIK expression using a specific antibody against p55PIK revealed that multiple protein products with different molecular masses were detected in COS-7 cell extracts. Experiments presented here demonstrate that multiple forms of p55PIK detected in COS-7 cells were produced by alternative initiation of translation. We also show that at least two in-frame start codons (AUG#2 and AUG#5) in p55PIK mRNA are used in COS-7 cells for the initiation of translation of p55PIK into proteins of 54 kDa and 50 kDa respectively. p55PIK mRNA was also alternatively translated into two proteins in PC cells, a mouse teratoma cell line, indicating that the alternative initiation of translation of p55PIK is not restricted to COS-7 cells. Results from immunoprecipitation and Western blot analysis showed that two forms (54 kDa and 50 kDa protein species) of p55PIK were detected in C57MG cells. Interestingly, when C57MG cells were treated with insulin, only p55PIK, but not p50PIK, bound to insulin receptor substrate-1 protein, providing evidence that different forms of p55PIKs may have specific distinct roles in signal transduction pathways. PMID:10417350

Xia, X; Serrero, G

1999-01-01

241

Effect of ethanol administration and withdrawal on serotonin receptor subtypes and receptor-mediated phosphoinositide hydrolysis in rat brain.  

PubMed

The effect of short-term (15 days) and long-term (60 days) ethanol treatment and withdrawal on agonist-stimulated phosphoinositide (Pl) hydrolysis, serotonin receptor subtypes (5HT1A and 5HT2), and alpha 1-adrenergic receptors were studied in rat cerebral cortex. Short-term ethanol treatment had no significant effect on serotonin (5HT), norepinephrine (NE), and calcium ionophore (A23187)-stimulated [3H]-inositol-1-phosphate ([3H]-IP1) formation and 5-HT2 receptors as measured by 125I-lysergic acid diethylamide (125I-LSD) binding, in rat cerebral cortex. However, 15 days of ethanol treatment, followed by 24 hr of withdrawal resulted in a decrease in Bmax of 125I-LSD binding without significant change in KD, as well as a decrease in 5HT-stimulated [3H]-IP1 formation in rat cerebral cortex. 5HT1A and alpha 1-adrenergic receptors were determined by using [3H]-8-hydroxy-2-(di-N-propylamino)tetralin and [3H]-prazosin as radioligand, respectively. We also observed that long-term ethanol treatment had no significant effect on Bmax and KD of 5HT2, 5HT1A, and alpha 1-adrenergic receptors, as well as NE and A23187-stimulated [3H]-IP1 formation, but significantly decreased the 5HT-stimulated [3H]-IP1 formation in rat cerebral cortex. It is possible that a decrease in 5HT-induced PI turnover after long-term ethanol exposure may be due to a decrease in coupling of 5HT2 receptors to G protein or PLC enzyme, whereas the decrease in 5HT-induced PI turnover after withdrawal may be due to a decrease in functional 5HT2 receptor number. PMID:1335222

Pandey, S C; Piano, M R; Schwertz, D W; Davis, J M; Pandey, G N

1992-12-01

242

Suppression of the {alpha}-isoform of class II phosphoinositide 3-kinase gene expression leads to apoptotic cell death  

SciTech Connect

Phosphoinositide 3-kinases (PI3Ks) have known to be key enzymes activating intracellular signaling molecules when a number of growth factors bind to their cell surface receptors. PI3Ks are divided into three classes (I, II, and III) and enzymes of each class have different tissue-specificities and physiological functions. Class II PI3Ks consist of three isoforms ({alpha}, {beta}, {gamma}). Although the {alpha}-isoform (PI3K-C2{alpha}) is considered ubiquitous and preferentially activated by insulin rather than the {beta}-isoform, the physiological significance of PI3K-C2{alpha} is poorly understood. The present study aimed to determine whether PI3K-C2{alpha} is associated with the suppression of apoptotic cell death. Different sense- and antisense oligonucleotides (ODNs) were synthesized based on the sequence of C2 domain of PI3K-C2{alpha} gene. Transfection of CHO-IR cells with two different antisense ODNs clearly reduced the protein content as well as mRNA levels of PI3K-C2{alpha} whereas neither the nonspecific mock- nor sense ODNs affected. The decrease of PI3K-C2{alpha} gene expression was paralleled by cellular changes indicating apoptotic cell death such as nuclear condensation, formation of apoptotic bodies, and DNA fragmentation. PI3K-C2{alpha} mRNA levels were also reduced when cells were incubated in growth factor-deficient medium. Supplementing growth factors (serum or insulin) into medium lead to an increase of PI3K-C2{alpha} mRNA levels. This finding strongly suggests that PI3K-C2{alpha} is a crucial survival factor.

Kang, Shinhae [Technology Innovation Center, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Song, Jihoon [Department of Life Science, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Kang, Jihoon [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Kang, Heekyoung [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Lee, Daeho [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Lee, Youngki [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Park, Deokbae [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of)]. E-mail: parkdb@cheju.ac.kr

2005-04-01

243

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

244

The phosphoinositide 3'-kinase p110? modulates contractile protein production and IL-6 release in human airway smooth muscle.  

PubMed

Transforming growth factor (TGF) ?1 increases pro-inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3' kinase (PI3K) is one of the signaling pathways implicated in TGF?1 stimulation, and may be altered in asthmatic airways. This study compared the expression of PI3K isoforms by ASM cells from donors with asthma (A), chronic obstructive pulmonary disease (COPD), or neither disease (NA), and investigated the role of PI3K isoforms in the production of TGF?1 induced pro-inflammatory cytokine and contractile proteins in ASM cells. A cells expressed higher basal levels of p110? mRNA compared to NA and COPD cells; however COPD cells produced more p110? protein. TGF?1 increased 110? mRNA expression to the same extent in the three groups. Neither the p110? inhibitor IC87114 (1, 10, 30?µM), the p110? inhibitor TGX221 (0.1, 1, 10 µM) nor the PI3K pan inhibitor LY294002 (3, 10?µM) had any effect on basal IL-6, calponin or smooth muscle ?-actin (?-SMA) expression. However, TGF?1 increased calponin and ?-SMA expression was inhibited by IC87114 and LY294002 in all three groups. IC87114, TGX221, and LY294002 reduced TGF?1 induced IL-6 release in a dose related manner in all groups of ASM cells. PI3K p110? is important for TGF?1 induced production of the contractile proteins calponin and ?-SMA and the proinflammatory cytokine IL-6 in ASM cells, and may therefore be relevant as a potential therapeutic target to treat both inflammation and airway remodeling. PMID:22015454

Ge, Qi; Moir, Lyn M; Trian, Thomas; Niimi, Kyoko; Poniris, Maree; Shepherd, Peter R; Black, Judith L; Oliver, Brian G; Burgess, Janette K

2012-08-01

245

The p110delta subunit of phosphoinositide 3-kinase is required for the lipopolysaccharide response of mouse B cells.  

PubMed

PI3K (phosphoinositide 3-kinase) I(A) family members contain a regulatory subunit and a catalytic subunit. The p110delta catalytic subunit is expressed predominantly in haematopoietic cells. There, among other functions, it regulates antigen receptor-mediated responses. Using mice deficient in the p110delta subunit of PI3K, we investigated the role of this subunit in LPS (lipopolysaccharide)-induced B cell responses, which are mediated by Toll-like receptor 4 and RP105. After injection of DNP-LPS (where DNP stands for 2,4-dinitrophenol), p110delta(-/-) mice produced reduced levels of DNP-specific IgM and IgG when compared with wild-type mice. In vitro, the proliferation and up-regulation of surface activation markers such as CD86 and CD25 induced by LPS and an antibody against RP105 were decreased. We analysed the activation state of key components of the LPS pathway in B cells to determine whether there was a defect in signalling in p110delta(-/-) B cells. They showed normal extracellular-signal-regulated kinase phosphorylation, but anti-RP105-induced protein kinase B, IkappaB (inhibitor of nuclear factor kappaB) and c-Jun N-terminal kinase activation was severely reduced. This demonstrates that the p110delta subunit of PI3K is involved in the LPS response in B cells and may represent a link between the innate and the adaptive immune system. PMID:15494016

Hebeis, B J; Vigorito, E; Turner, M

2004-11-01

246

Genetic association of the Phosphoinositide-3 kinase in schizophrenia and bipolar disorder and interaction with a BDNF gene polymorphism.  

PubMed

Phosphoinositide-3-kinase, class III (PIK3C3) is a member of the phosphoinosite-3-kinases family, involved in cell signaling, membrane trafficking, and neurodevelopment. Previous studies have indeed shown an association between PIK3C3 gene variants and both bipolar disorder (BD) and schizophrenia (SZ). Brain-derived neurotrophic factor (BDNF) is a neurodevelopmental factor, which can regulate the PI3K signaling pathway. Associations have been reported between BDNF gene polymorphisms and affective and psychotic disorders. The aim of the present study was to replicate an association between PIK3C3 and BDNF gene variants in SZ and BD and a putative epistasis between the two genes. Patients meeting the DSM-IV criteria of BD and SZ were included in this study (98 BD and 79 SZ) as well as 158 healthy controls. Blood DNA was extracted and genotyping was performed either by the polymerase chain reaction (PCR) technique followed by enzymatic digestion or by the high-resolution melt (HRM) method. Genotype and haplotype association was assessed with the UNPHASED statistical program.The results showed one nominal association with BD (P < 0.02) and two risk haplotypes in both SZ (P < 0.001) and BP (P < 0.0005), which survived multiple testing correction. A modest interaction between a BDNF variant and PI3KC3 polymorphism was observed (P < 0.04).These preliminary results confirm the genetic association of PI3K gene variants with both SZ and BD, and support the hypothesis that SZ and BD share a genetic background. PMID:22399091

Carrard, Anthony; Salzmann, Annick; Perroud, Nader; Gafner, Jérémie; Malafosse, Alain; Karege, Félicien

2011-11-01

247

Protective effects of exercise and phosphoinositide 3-kinase(p110alpha) signaling in dilated and hypertrophic cardiomyopathy.  

PubMed

Physical activity protects against cardiovascular disease, and physiological cardiac hypertrophy associated with regular exercise is usually beneficial, in marked contrast to pathological hypertrophy associated with disease. The p110alpha isoform of phosphoinositide 3-kinase (PI3K) plays a critical role in the induction of exercise-induced hypertrophy. Whether it or other genes activated in the athlete's heart might have an impact on cardiac function and survival in a setting of heart failure is unknown. To examine whether progressive exercise training and PI3K(p110alpha) activity affect survival and/or cardiac function in two models of heart disease, we subjected a transgenic mouse model of dilated cardiomyopathy (DCM) to swim training, genetically crossed cardiac-specific transgenic mice with increased or decreased PI3K(p110alpha) activity to the DCM model, and subjected PI3K(p110alpha) transgenics to acute pressure overload (ascending aortic constriction). Life-span, cardiac function, and molecular markers of pathological hypertrophy were examined. Exercise training and increased cardiac PI3K(p110alpha) activity prolonged survival in the DCM model by 15-20%. In contrast, reduced PI3K(p110alpha) activity drastically shortened lifespan by approximately 50%. Increased PI3K(p110alpha) activity had a favorable effect on cardiac function and fibrosis in the pressure-overload model and attenuated pathological growth. PI3K(p110alpha) signaling negatively regulated G protein-coupled receptor stimulated extracellular responsive kinase and Akt (via PI3K, p110gamma) activation in isolated cardiomyocytes. These findings suggest that exercise and enhanced PI3K(p110alpha) activity delay or prevent progression of heart disease, and that supraphysiologic activity can be beneficial. Identification of genes important for hypertrophy in the athlete's heart could offer new strategies for treating heart failure. PMID:17202264

McMullen, Julie R; Amirahmadi, Fatemeh; Woodcock, Elizabeth A; Schinke-Braun, Martina; Bouwman, Russell D; Hewitt, Kimberly A; Mollica, Janelle P; Zhang, Li; Zhang, Yunyu; Shioi, Tetsuo; Buerger, Antje; Izumo, Seigo; Jay, Patrick Y; Jennings, Garry L

2007-01-01

248

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

249

A drug targeting only p110? can block phosphoinositide 3-kinase signalling and tumour growth in certain cell types  

PubMed Central

Genetic alterations in PI3K (phosphoinositide 3-kinase) signalling are common in cancer and include deletions in PTEN (phosphatase and tensin homologue deleted on chromosome 10), amplifications of PIK3CA and mutations in two distinct regions of the PIK3CA gene. This suggests drugs targeting PI3K, and p110? in particular, might be useful in treating cancers. Broad-spectrum inhibition of PI3K is effective in preventing growth factor signalling and tumour growth, but suitable inhibitors of p110? have not been available to study the effects of inhibiting this isoform alone. In the present study we characterize a novel small molecule, A66, showing the S-enantiomer to be a highly specific and selective p110? inhibitor. Using molecular modelling and biochemical studies, we explain the basis of this selectivity. Using a panel of isoform-selective inhibitors, we show that insulin signalling to Akt/PKB (protein kinase B) is attenuated by the additive effects of inhibiting p110?/p110?/p110? in all cell lines tested. However, inhibition of p110? alone was sufficient to block insulin signalling to Akt/PKB in certain cell lines. The responsive cell lines all harboured H1047R mutations in PIK3CA and have high levels of p110? and class-Ia PI3K activity. This may explain the increased sensitivity of these cells to p110? inhibitors. We assessed the activation of Akt/PKB and tumour growth in xenograft models and found that tumours derived from two of the responsive cell lines were also responsive to A66 in vivo. These results show that inhibition of p110? alone has the potential to block growth factor signalling and reduce growth in a subset of tumours. PMID:21668414

Jamieson, Stephen; Flanagan, Jack U.; Kolekar, Sharada; Buchanan, Christina; Kendall, Jackie D.; Lee, Woo-Jeong; Rewcastle, Gordon W.; Denny, William A.; Singh, Ripudaman; Dickson, James; Baguley, Bruce C.; Shepherd, Peter R.

2011-01-01

250

Cloning of a human phosphoinositide 3-kinase with a C2 domain that displays reduced sensitivity to the inhibitor wortmannin.  

PubMed Central

The generation of phosphatidylinositide 3-phosphates has been observed in a variety of cellular responses. The enzymes that mediate synthesis are the phosphoinositide 3-kinases (PI3-Ks) that form a family of structurally diverse enzymes with distinct substrate specificities. In this paper, we describe the cloning of a novel human PI3-K, namely PI3-K-C2 alpha, which contains a C-terminal C2 domain. This enzyme can be assigned to the class II PI3-Ks, which was defined by characterization of the Drosophila 68D enzyme and includes the recently described murine enzymes m-cpk and p170. Despite the overall similarity in the amino acid sequence of the murine and human enzymes, which suggests that they are encoded by closely related genes, these molecules show marked sequence heterogeneity at their N-termini. Biochemical analysis of recombinant PI3-K-C2 alpha demonstrates a restricted lipid substrate specificity. As reported for other members of this class, the enzyme only phosphorylates PtdIns and PtdIns4P when the lipids are presented alone. However, when lipids were presented together with phosphatidylserine acting as a carrier, phosphorylation of PtdIns(4,5)P2 was also observed. The catalytic activity of PI3-K-C2 alpha is refractory to concentrations of wortmannin and LY294002 which inhibit the PI3-K activity of other family members. The comparative insensitivity of PI3-K-C2 alpha to these inhibitors suggests that their use should be reevaluated in the study of PI3-Ks. PMID:9337861

Domin, J; Pages, F; Volinia, S; Rittenhouse, S E; Zvelebil, M J; Stein, R C; Waterfield, M D

1997-01-01

251

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

252

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

253

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

254

Acquired PIK3CA amplification causes resistance to selective phosphoinositide 3-kinase inhibitors in breast cancer  

PubMed Central

Agents targeting the PI3K/mTOR signaling axis have shown promise in early-phase clinical trials and are currently being studied in later stages of clinical development in multiple indications. Experience with other targeted agents suggests that clinical responses may be short-lived because of acquired resistance to therapy. Here, we report preclinical modeling of acquired resistance in a HER2-positive, PIK3CA mutant breast cancer cell line, KPL-4. We identified a heretofore-unreported mechanism of resistance, specifically high-level amplification of the mutant allele of PIK3CA, which resulted in a marked upregulation of PI3K signaling, enabling resistant cells to regain proliferative capacity at clinically relevant concentrations of the PI3K inhibitor, GDC-0941. We show that knockdown of the amplified PIK3CA mutant allele in these cells by small interfering RNA restored pathway signaling and sensitivity to PI3K inhibition at levels comparable to parental cells. These novel preclinical findings suggest that, in addition to assessment of other previously reported mechanisms of resistance, evaluation of PI3K copy number variation should be integrated into the exploratory analysis of biopsies obtained at disease progression. PMID:24366379

Huw, L-Y; O'Brien, C; Pandita, A; Mohan, S; Spoerke, J M; Lu, S; Wang, Y; Hampton, G M; Wilson, T R; Lackner, M R

2013-01-01

255

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

256

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

257

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 p85? PI3K regulatory subunit reveals a previously unknown p85?-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)

2006-11-21

258

ADH-dependent phosphoinositide signalling system and prostaglandin E production in the frog urinary bladder.  

PubMed

Antidiuretic hormone (ADH) stimulated formation of inositol 1,4,5-trisphosphate (IP3), 1,2-diaclyglycerol (DAG) and an increase of phosphatidylinositol 4,5-biphosphate (PIP2) breakdown in the frog urinary bladder 20 s after addition. ADH also increased the prostaglandin E (PGE) secretion into serosal medium 3.5-fold and the release of arachidonic acid (AA) from 1,2-DAG, which was intensified in the presence of DAG kinase inhibitor R59022. Neomycin sulphate (10(-5) M) from the serosal side blocked ADH-stimulated PIP2 hydrolysis, IP3 production and increased the hydro-osmotic response to ADH. It also inhibited the ADH-stimulated PGE production (55%) and release of AA from 1,2-DAG. This data suggest that PIP2 breakdown is involved in the mechanism of feedback regulation of ADH action and is associated with PGE production via (i) the increase of AA release from PIP2-generated 1,2-DAG and (ii) possible activation of phospholipase A2 by IP3-induced elevation of cytosol Ca2+. PMID:1652269

Parnova, R G; Firsov, D L

1991-01-01

259

A HIGHLY DYNAMIC ER-DERIVED PHOSPHATIDYLINOSITOL SYNTHESIZING ORGANELLE SUPPLIES PHOSPHOINOSITIDES TO CELLULAR MEMBRANES  

PubMed Central

SUMMARY Polyphosphoinositides are lipid signaling molecules generated from phosphatidylinositol (PtdIns) with critical roles in vesicular trafficking and signaling. It is poorly understood where PtdIns is located within cells and how it moves around between membranes. Here we identify a hitherto unrecognized highly mobile membrane compartment as the site of PtdIns synthesis and a likely source of PtdIns of all membranes. We show that the PtdIns synthesizing enzyme, PIS associates with a rapidly moving compartment of ER origin that makes ample contacts with other membranes. In contrast, CDP-diacylglycerol synthases that provide PIS with its substrate reside in the tubular ER. Expression of a PtdIns-specific bacterial PLC generates diacylglycerol also in rapidly moving cytoplasmic objects. We propose a model in which PtdIns is synthesized in a highly mobile lipid distribution platform and is delivered to other membranes during multiple contacts by yet to be defined lipid transfer mechanisms. PMID:22075145

Kim, Yeun Ju; Guzman-Hernandez, Maria Luisa; Balla, Tamas

2011-01-01

260

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

261

Rotavirus Replication in Intestinal Cells Differentially Regulates Integrin Expression by a Phosphatidylinositol 3-Kinase-Dependent Pathway, Resulting in Increased Cell Adhesion and Virus Yield?  

PubMed Central

Changes in the interactions between intestinal cells and their surrounding environment during virus infection have not been well documented. The growth and survival of intestinal epithelial cells, the main targets of rotavirus infection, are largely dependent on the interaction of cell surface integrins with the extracellular matrix. In this study, we detected alterations in cellular integrin expression following rotavirus infection, identified the signaling components required, and analyzed the subsequent effects on cell binding to the matrix component collagen. After rotavirus infection of intestinal cells, expression of ?2?1 and ?2 integrins was up-regulated, whereas that of ?V?3, ?V?5, and ?5?1 integrins, if present, was down-regulated. This differential regulation of integrins was reflected at the transcriptional level. It was unrelated to the use of integrins as rotavirus receptors, as both integrin-using and integrin-independent viruses induced integrin regulation. Using pharmacological agents that inhibit kinase activity, integrin regulation was shown to be dependent on phosphatidylinositol 3-kinase (PI3K) but independent of the activities of the mitogen-activated protein kinases p38 and ERK1/2, and cyclooxygenase-2. Replication-dependent activation of the PI3K/Akt pathway was observed following infection of intestinal and nonintestinal cell lines. Rotavirus activation of PI3K was important for regulation of ?2?1 expression. Blockade of integrin regulation by PI3K inhibition led to decreased adherence of infected intestinal cells to collagen and a concomitant decrease in virus titer. These findings indicate that rotavirus-induced PI3K activation causes regulation of integrin expression in intestinal cells, leading to prolonged adherence of infected cells to collagen and increased virus production. PMID:17942548

Halasz, Peter; Holloway, Gavan; Turner, Stephen J.; Coulson, Barbara S.

2008-01-01

262

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

263

Class IA phosphoinositide 3-kinase ? and ? regulate neutrophil oxidase activation in response to Aspergillus fumigatus hyphae.  

PubMed

An effective immune response to the ubiquitous fungus Aspergillus fumigatus is dependent upon production of reactive oxygen species (ROS) by the NADPH oxidase. This is evidenced by the acute sensitivity of oxidase-deficient humans and mice to invasive aspergillosis. Neutrophils are recruited to the lungs shortly postinfection and respond by phagocytosing conidia and mediating extracellular killing of germinated hyphae in a ROS-dependent manner. However, the signaling mechanisms regulating the generation of ROS in response to hyphae are poorly understood. PI3Ks are important regulators of numerous cellular processes, with much recent work describing unique roles for the different class I PI3K isoforms. We showed by live-cell imaging that the lipid products of class I PI3Ks accumulated at the hyphal-bound neutrophil plasma membrane. Further, we used pharmacological and genetic approaches to demonstrate essential, but overlapping, roles for PI3K? and PI3K? in the ROS and spreading responses of murine neutrophils to Aspergillus hyphae. Hyphal-induced ROS responses were substantially inhibited by deletion of the common ?2-integrin subunit CD18, with only a minor, redundant role for Dectin-1. However, addition of soluble algal glucans plus the genetic deletion of CD18 were required to significantly inhibit activation of the PI3K-effector protein kinase B. Hyphal ROS responses were also totally dependent on the presence of Syk, but not its ITAM-containing adaptor proteins FcR? or DAP12, and the Vav family of Rac-guanine nucleotide exchange factors. These results start to define the signaling network controlling neutrophil ROS responses to A. fumigatus hyphae. PMID:21257963

Boyle, Keith B; Gyori, David; Sindrilaru, Anca; Scharffetter-Kochanek, Karin; Taylor, Philip R; Mócsai, Attila; Stephens, Len R; Hawkins, Phillip T

2011-03-01

264

Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides.  

PubMed

Combinations of direct-acting antivirals (DAAs) against the hepatitis C virus (HCV) have the potential to revolutionize the HCV therapeutic regime. An integral component of DAA combination therapies is HCV NS5A inhibitors. It has previously been proposed that NS5A DAAs inhibit two functions of NS5A: RNA replication and virion assembly. In this study, we characterize the impact of a prototype NS5A DAA, daclatasvir (DCV), on HCV replication compartment formation. DCV impaired HCV replicase localization and NS5A motility. In order to characterize the mechanism behind altered HCV replicase localization, we examined the impact of DCV on the interaction of NS5A with its essential cellular cofactor, phosphatidylinositol-4-kinase III ? (PI4KA). We observed that DCV does not inhibit PI4KA directly, nor does it impair early events of the NS5A-PI4KA interaction that can occur when NS5A is expressed alone. NS5A functions that are unaffected by DCV include PI4KA binding, as determined by co-immunoprecipitation, and a basal accumulation of the PI4KA product, PI4P. However, DCV impairs late steps in PI4KA activation that requires NS5A expressed in the context of the HCV polyprotein. These NS5A functions include hyper-stimulation of PI4P levels and appropriate replication compartment formation. The data are most consistent with a model wherein DCV inhibits conformational changes in the NS5A protein or protein complex formations that occur in the context of HCV polyprotein expression and stimulate PI4P hyper-accumulation and replication compartment formation. PMID:25546252

Chukkapalli, Vineela; Berger, Kristi L; Kelly, Sean M; Thomas, Meryl; Deiters, Alexander; Randall, Glenn

2015-02-01

265

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

266

The reno-protective effect of a phosphoinositide 3-kinase inhibitor wortmannin on streptozotocin-induced proteinuric renal disease rats  

PubMed Central

Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1 and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was 3.55 ± 0.56 mg/day, whereas wortmannin group was 1.77 ± 0.48 mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was 53.08 ± 10.82 mg/g, whereas wortmannin group was 20.27 ± 6.41 mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The expression levels of nephrin (79.66 ± 0.02), podocin (87.81 ± 0.03) and Rac1/Cdc42 (86.12 ± 0.02) in the wortmannin group were higher than the expression levels of nephrin (55.32 ± 0.03), podocin (53.40 ± 0.06) and Rac1/Cdc42 (54.05 ± 0.04) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage. PMID:22056625

Kim, Sang-Hoon; Jang, Young-Woo; Hwang, Patrick; Kim, Hyun-Jung; Han, Gi-Yeon

2012-01-01

267

Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction.  

PubMed Central

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 wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes. PMID:8657148

Wymann, M P; Bulgarelli-Leva, G; Zvelebil, M J; Pirola, L; Vanhaesebroeck, B; Waterfield, M D; Panayotou, G

1996-01-01

268

Early phosphoinositide 3-kinase activity is required for late activation of protein kinase Cepsilon in platelet-derived-growth-factor-stimulated cells: evidence for signalling across a large temporal gap.  

PubMed Central

At least two signalling systems have the potential to contribute to the activation of protein kinase C (PKC) family members such as PKCepsilon. One of these is phosphoinositide 3-kinase (PI 3-kinase), whose lipid products activate PKCepsilon in vitro and in living cells. The recent observation that there are multiple waves of PI 3-kinase and PKCepsilon activity within the G(0)-to-S phase interval provides a new opportunity to investigate the relationship between these two signalling enzymes in vivo. We have assessed the relative importance of the early and late waves of PI 3-kinase activity for the corresponding waves of PKCepsilon activity. Blocking the first phase of PI 3-kinase activity inhibited both early and late activation of PKCepsilon. In contrast, the second wave of PI 3-kinase activity was dispensable for late activation of PKCepsilon. These findings suggested that early PI 3-kinase activation induced a stable change in PKCepsilon, which predisposed it to subsequent activation by lipid cofactors. Indeed, partial proteolysis of PKCepsilon indicated that early activation of PI 3-kinase led to a conformation change in PKCepsilon that persisted as the activity of PKCepsilon cycled. We propose a two-step hypothesis for the activation of PKCepsilon in vivo. One step is stable and depends on PI 3-kinase, whereas the other is transient and may depend on the availability of lipid cofactors. Finally, these studies reveal that PI 3-kinase and PKCepsilon are capable of communicating over a relatively long time interval and begin to elucidate the mechanism. PMID:11513725

Balciunaite, E; Kazlauskas, A

2001-01-01

269

Inositol 1,4,5-triphosphate-induced granule secretion in platelets. Evidence that the activation of phospholipase C mediated by platelet thromboxane receptors involves a guanine nucleotide binding protein-dependent mechanism distinct from that of thrombin.  

PubMed Central

Phosphoinositide hydrolysis in platelets stimulated by thrombin is thought to be regulated by a pertussis toxin-sensitive guanine nucleotide binding protein (G protein) referred to as Gp. The present studies examine the role of Gp in platelet responses to the thromboxane A2 analogue U46619 and in the pathway by which the phosphoinositide hydrolysis product inositol 1,4,5-triphosphate (IP3) causes secretion. In permeabilized platelets, U46619 caused phosphatidic acid formation and secretion, which were abolished by the G protein inhibitor, guanosine 5'-O-(2-thiophosphate) (GDP beta S). Unlike thrombin, however, U46619-induced phosphoinositide hydrolysis was unaffected by pertussis toxin, and U46619 was unable to inhibit the [32P]ADP ribosylation of the 42-kD pertussis toxin substrate in platelets. IP3-induced secretion, which is known to depend upon intracellular Ca release and subsequent arachidonic acid metabolism, was also inhibited by GDP beta S, as was Ca-induced secretion. These observations suggest that platelet thromboxane A2 (TxA2) receptors are coupled to a toxin-resistant form of Gp distinct from the one that is coupled to thrombin receptors, and that TxA2-stimulated phosphoinositide hydrolysis may serve as a feedback mechanism by which stimuli for arachidonic acid release, such as IP3 and Ca, amplify responses to agonists. Images PMID:3031135

Brass, L F; Shaller, C C; Belmonte, E J

1987-01-01

270

A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation.  

PubMed Central

We identified a putative Saccharomyces cerevisiae homolog of a phosphoinositide-specific phospholipase C (PI-PLC) gene, PLC1, which encodes a protein most similar to the delta class of PI-PLC enzymes. The PLC1 gene was isolated during a study of yeast strains that exhibit defects in chromosome segregation. plc1-1 cells showed a 10-fold increase in aberrant chromosome segregation compared with the wild type. Molecular analysis revealed that PLC1 encodes a predicted protein of 101 kDa with approximately 50 and 26% identity to the highly conserved X and Y domains of PI-PLC isozymes from humans, bovines, rats, and Drosophila melanogaster. The putative yeast protein also contains a consensus EF-hand domain that is predicted to bind calcium. Interestingly, the temperature-sensitive and chromosome missegregation phenotypes exhibited by plc1-1 cells were partially suppressed by exogenous calcium. Images PMID:8391635

Payne, W E; Fitzgerald-Hayes, M

1993-01-01

271

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

272

Exendin-4 enhances the migration of adipose-derived stem cells to neonatal rat ventricular cardiomyocyte-derived conditioned medium via the phosphoinositide 3-kinase/Akt-stromal cell-derived factor-1?/CXC chemokine receptor 4 pathway.  

PubMed

Adipose?derived stem cells (ADSCs) are considered a suitable source of cells for the repair of tissue following acute myocardial infarction (AMI); however, the transplantation efficiency of ADSCs remains low. Therefore, identification of an efficient method to enhance the migration of engrafted cells to the target site is required. The present study used exendin?4 (Ex?4), a glucagon?like peptide?1 receptor agonist, to optimize the migratory capacity of ADSCs. The aim was to determine the effect and mechanisms of Ex?4 on the migration of ADSCs to neonatal rat ventricular cardiomyocyte?derived conditioned medium (NRVC?CM). The ADSCs and cardiomyocytes were cultured in vitro. Following incubation of the ADSCs with Ex?4, cell proliferation was measured using an MTT assay and the expression levels of CXC chemokine receptor 4 (CXCR4) were investigated by reverse transctiption quantitative polymerase chain reaction (RT?qPCR), western blot analysis and flow cytometry. In addition, the expression levels of stromal cell?derived factor?1? (SDF?1?) were evaluated in the NRVC?CM treated with Ex?4 by ELISA, RT?qPCR and western blot analysis. The migration of the ADSCs to the NRVC?CM was examined using a Transwell assay. Changes in the protein expression levels of phosphorylated (p?)Akt were examined in the two types of cell by western blot analysis. The results suggested that Ex?4 promoted the proliferation and expression of CXCR4 in the ADSCs, increased the secretion of SDF?1? in the cardiomyocytes and increased the expression levels of p?Akt in both cells. However, the alterations to the SDF?1?/CXCR4 cascade in the cells were abrogated following pretreatment with LY?294002, a phosphoinositide 3?kinase(PI3K) inhibitor. Furthermore, a Transwell migration assay revealed marked translocation of the ADSCs through the membranes, towards the NRVC?CM, following treatment with Ex?4. However, these effects were reduced significantly by pretreatment of the cells with the SDF?1?/CXCR4 cascade antagonist, AMD3100, and the PI3K inhibitor, LY?294002. These results indicated that Ex?4 augmented the SDF?1?/CXCR4 cascade by activating the PI3K/Akt pathways in the ADSCs and NRVCs. Furthermore, enhancement of the PI3K/Akt-SDF-1?/CXCR4 pathway may be important in the migratory response of ADSCs to NRVC?CM in vitro. PMID:25625935

Zhou, Hao; Yang, Junjie; Xin, Ting; Zhang, Tao; Hu, Shunyin; Zhou, Shanshan; Chen, Guanghui; Chen, Yundai

2015-06-01

273

A novel regulatory mechanism links PLC?1 to PDK1  

PubMed Central

Summary 3-Phosphoinositide-dependent protein kinase-1 (PDK1) and phospholipase C (PLC)?1 are two key enzymes in signal transduction that control several intracellular processes. Despite the fact that PLC?1 has been investigated for several years, the mechanisms of activation of this enzyme are still not completely clear. Similarly, although PDK1 has been mostly investigated for its role in activation of Akt, a crucial enzyme in regulation of several cellular processes, it has become evident recently that the role of PDK1 in physiological and pathological conditions is not limited to Akt activation. Here we demonstrate that PDK1 regulates PLC?1 activation in a mechanism involving association of the two enzymes and modulation of PLC?1 tyrosine phosphorylation. We further show that this novel PDK1–PLC?1 pathway is important for cancer cell invasion. The identification of a PDK1–PLC?1 pathway reveals the existence of a previously undetected link between two of the most important enzymes in signal transduction. This is likely to have profound consequences for our understanding of several cellular functions that are dependent on phosphoinositides and controlled by PDK1 and PLC?1. PMID:22454520

Raimondi, Claudio; Chikh, Anissa; Wheeler, Ann P.; Maffucci, Tania; Falasca, Marco

2012-01-01

274

Muscarinic M1 receptors activate phosphoinositide turnover and Ca2+ mobilisation in rat sympathetic neurones, but this signalling pathway does not mediate M-current inhibition.  

PubMed

1. The relationship between muscarinic receptor activation, phosphoinositide turnover, calcium mobilisation and M-current inhibition has been studied in rat superior cervical ganglion (SCG) neurones in primary culture. 2. Phosphoinositide-specific phospholipase C (PLC) stimulation was measured by the accumulation of [3H]-cytidine monophosphate phosphatidate (CMP-PA) after incubation with [3H]-cytidine in the presence of Li+. The muscarinic agonist oxotremorine methiodide (oxo-M) stimulated PLC in a dose-dependent manner with an EC50 of approximately 3.5 microM. 3. The concentration-response curve for oxo-M was shifted to the right by a factor of about 10 by pirenzepine (100 nM), suggesting a pKB (-log of the apparent dissociation constant) of 7.9 +/- 0.4, while himbacine (1 microM) shifted the curve by a factor of about 13 (pKB approximately 7.1 +/- 0.6). This indicates involvement of the M1 muscarinic receptor in this response. 4. The accumulation of CMP-PA was localised by in situ autoradiography to SCG principal neurones, with no detectable signal in glial cells present in the primary cultures. 5. The ability of oxo-M to release Ca2+ from inositol(1,4, 5)trisphosphate (InsP3)-sensitive stores was determined by fura-2 microfluorimetry of SCG neurones voltage clamped in perforated patch mode. Oxo-M failed to evoke intracellular Ca2+ (Ca2+i) mobilisation in SCG neurones voltage clamped at -60 mV, but produced a significant Ca2+i rise (67 +/- 15 nM, n = 9) in cells voltage clamped at -25 mV. 6. Thapsigargin (0.5-1 microM) caused a 70 % inhibition of the oxo-M-induced Ca2+i increase, indicating its intracellular origin, while oxo-M-induced inhibition of M-current in the same cells was unaffected by thapsigargin. 7. Our results do not support the involvement of InsP3-sensitive calcium mobilisation in M-current inhibition. PMID:10517804

del Río, E; Bevilacqua, J A; Marsh, S J; Halley, P; Caulfield, M P

1999-10-01

275

CD40 ligand exhibits a direct antiviral effect on Herpes Simplex Virus type-1 infection via a PI3K-dependent, autophagy-independent mechanism.  

PubMed

The interaction between CD40 and its ligand, CD40L/CD154, is crucial for the efficient initiation and regulation of immune responses against viruses. Herpes Simplex Virus type-1 (HSV-1) is a neurotropic virus capable of manipulating host responses and exploiting host proteins to establish productive infection. Herein we have examined the impact of CD40L-mediated CD40 activation on HSV-1 replication in U2OS cells stably expressing the CD40 receptor. Treatment of these cells with CD40L significantly reduced the HSV-1 progeny virus compared to non-treated cells. The activation of CD40 signaling did not affect the binding of HSV-1 virions on the cell surface but rather delayed the translocation of VP16 to the nucleus, affecting all stages of viral life cycle. Using pharmacological inhibitors and RNAi we show that inhibition of PI3 kinase but not autophagy reverses the effects of CD40L on HSV-1 replication. Collectively, these data demonstrate that CD40 activation exerts a direct inhibitory effect on HSV-1, initiating from the very early stages of the infection by exploiting PI3 kinase-dependent but autophagy-independent mechanisms. PMID:25765994

Vlahava, Virginia-Maria; Eliopoulos, Aristides G; Sourvinos, George

2015-06-01

276

An essential function of a phosphoinositide-specific phospholipase C is relieved by inhibition of a cyclin-dependent protein kinase in the yeast Saccharomyces cerevisiae.  

PubMed Central

The PLC1 gene product of Saccharomyces cerevisiae is a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C (PI-PLC). We found that two genes (SPL1 and SPL2), when overexpressed, can bypass the temperature-sensitive growth defect of a plc1delta cell. SPL1 is identical to the PHO81 gene, which encodes an inhibitor of a cyclin (Pho80p)-dependent protein kinase (Pho85p) complex (Cdk). In addition to overproduction of Pho81p, two other conditions that inactivate this Cdk, a cyclin (pho80delta) mutation and growth on low-phosphate medium, also permitted growth of plc1delta cells at the restrictive temperature. Suppression of the temperature sensitivity of plc1delta cells by pho80delta does not depend upon the Pho4p transcriptional regulator, the only known substrate of the Pho80p/Pho85p Cdk. The second suppressor, SPL2, encodes a small (17-kD) protein that bears similarity to the ankyrin repeat regions present in Pho81p and in other known Cdk inhibitors. Both pho81delta and spl2delta show a synthetic phenotype in combination with plc1delta. Unlike single mutants, plc1delta pho81delta and plc1delta spl2delta double mutants were unable to grow on synthetic complete medium, but were able to grow on rich medium. PMID:9475719

Flick, J S; Thorner, J

1998-01-01

277

Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway.  

PubMed Central

Upon detachment from the extracellular matrix, epithelial cells enter into programmed cell death, a phenomenon known as anoikis, ensuring that they are unable to survive in an inappropriate location. Activated ras oncogenes protect cells from this form of apoptosis. The nature of the survival signals activated by integrin engagement and usurped by oncogenic Ras are unknown: here we show that in both cases phosphoinositide 3-OH kinase (PI 3-kinase), but not Raf, mediates this protection, acting through protein kinase B/Akt (PKB/Akt). Constitutively activated PI 3-kinase or PKB/Akt block anoikis, while inhibition of PI 3-kinase abrogates protection by Ras, but not PKB/Akt. Inhibition of either PI 3-kinase or PKB/Akt induces apoptosis in adherent epithelial cells. Attachment of cells to matrix leads to rapid elevation of the levels of PI 3-kinase lipid products and PKB/Akt activity, both of which remain high in Ras-transformed cells even in suspension. PI 3-kinase acting through PKB/Akt is therefore implicated as a key mediator of the aberrant survival of Ras-transformed epithelial cells in the absence of attachment, and mediates matrix-induced survival of normal epithelial cells. PMID:9184223

Khwaja, A; Rodriguez-Viciana, P; Wennström, S; Warne, P H; Downward, J

1997-01-01

278

Phosphoinositide 3-kinase/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.  

PubMed

In this study, it was found that undifferentiated myoblasts were more vulnerable to menadione-induced oxidative stress than differentiated myotubes. Cell death occurred with a relatively low concentration of menadione in myoblasts compared to myotubes. With the same concentration of menadione, the Bcl-2/Bax ratio decreased and nuclei containing condensed chromatin were observed in myoblasts to a greater extent than in myotubes. However, myotubes became increasingly susceptible to menadione when phosphoinositide 3-kinase (PI3-K) was blocked by pre-incubation with LY294002, a PI3-K inhibitor. Actually, PI3-K activity was reduced by menadione in myoblasts but not in myotubes. In addition, the phosphorylation of Akt, a downstream effector of PI3-K, was inhibited in myoblasts by menadione but increased in myotubes. Both LY294002 and API-2, an Akt inhibitor, decreased the Bcl-2/Bax ratio in menadione-exposed myotubes. These results suggest that the differential activity of PI3-K/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress. PMID:19051078

Lim, Jeong A; Woo, Joo Hong; Kim, Hye Sun

2008-09-01

279

Functional Redundancy of Class I Phosphoinositide 3-Kinase (PI3K) Isoforms in Signaling Growth Factor-Mediated Human Neutrophil Survival  

PubMed Central

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.

2012-01-01

280

Selective class I phosphoinositide 3-kinase inhibitors: optimization of a series of pyridyltriazines leading to the identification of a clinical candidate, AMG 511.  

PubMed

The phosphoinositide 3-kinase family catalyzes the phosphorylation of phosphatidylinositol-4,5-diphosphate to phosphatidylinositol-3,4,5-triphosphate, a secondary messenger which plays a critical role in important cellular functions such as metabolism, cell growth, and cell survival. Our efforts to identify potent, efficacious, and orally available phosphatidylinositol 3-kinase (PI3K) inhibitors as potential cancer therapeutics have resulted in the discovery of 4-(2-((6-methoxypyridin-3-yl)amino)-5-((4-(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine (1). In this paper, we describe the optimization of compound 1, which led to the design and synthesis of pyridyltriazine 31, a potent pan inhibitor of class I PI3Ks with a superior pharmacokinetic profile. Compound 31 was shown to potently block the targeted PI3K pathway in a mouse liver pharmacodynamic model and inhibit tumor growth in a U87 malignant glioma glioblastoma xenograft model. On the basis of its excellent in vivo efficacy and pharmacokinetic profile, compound 31 was selected for further evaluation as a clinical candidate and was designated AMG 511. PMID:22897589

Norman, Mark H; Andrews, Kristin L; Bo, Yunxin Y; Booker, Shon K; Caenepeel, Sean; Cee, Victor J; D'Angelo, Noel D; Freeman, Daniel J; Herberich, Bradley J; Hong, Fang-Tsao; Jackson, Claire L M; Jiang, Jian; Lanman, Brian A; Liu, Longbin; McCarter, John D; Mullady, Erin L; Nishimura, Nobuko; Pettus, Liping H; Reed, Anthony B; Miguel, Tisha San; Smith, Adrian L; Stec, Markian M; Tadesse, Seifu; Tasker, Andrew; Aidasani, Divesh; Zhu, Xiaochun; Subramanian, Raju; Tamayo, Nuria A; Wang, Ling; Whittington, Douglas A; Wu, Bin; Wu, Tian; Wurz, Ryan P; Yang, Kevin; Zalameda, Leeanne; Zhang, Nancy; Hughes, Paul E

2012-09-13

281

Loss of Cardiac Phosphoinositide 3-kinase p110? Results in Contractile Dysfunction: Lu et al PI3K p110? Regulates Myocyte Contractility  

PubMed Central

Background Phosphoinositide 3-kinase (PI3K) p110? plays a key role in insulin action and tumorigenesis. Myocyte contraction is initiated by an inward Ca2+ current (ICa,L) through the voltage-dependent L-type Ca2+ channel (LTCC). The aim of this study was to evaluate if p110? also controls cardiac contractility by regulating the LTCC. Methods and Results Genetic ablation of p110? (also known as Pik3ca), but not p110? (also known as Pik3cb), in cardiac myocytes of adult mice reduced ICa,L and blocked insulin signaling in the heart. p110?-null myocytes had a reduced number of LTCCs on the cell surface and a contractile defect that decreased cardiac function in vivo. Similarly, pharmacological inhibition of p110? decreased ICa,L and contractility in canine myocytes. Inhibition of p110? did not reduce ICa,L. Conclusions PI3K p110? but not p110? regulates the LTCC in cardiac myocytes. Decreased signaling to p110? reduces the number of LTCCs on the cell surface and thus attenuates ICa,L and contractility. PMID:19597047

Lu, Zhongju; Jiang, Ya-Ping; Wang, Wei; Xu, Xin-Hua; Mathias, Richard T.; Entcheva, Emilia; Ballou, Lisa M.; Cohen, Ira S.; Lin, Richard Z.

2009-01-01

282

New Insights into Tumor Suppression: PTEN Suppresses Tumor Formation by Restraining the Phosphoinositide 3-kinase\\/AKT Pathway  

Microsoft Academic Search

The most recently discovered PTEN tumor suppressor gene has been found to be defective in a large number of human cancers. In addition, germ-line mutations in PTEN result in the dominantly inherited disease Cowden syndrome, which is characterized by multiple hamartomas and a high proclivity for developing cancer. A series of publications over the past year now suggest a mechanism

Lewis C. Cantley; Benjamin G. Neel

1999-01-01

283

The inhibition of phosphoinositide synthesis and muscarinic-receptor-mediated phospholipase C activity by Li+ as secondary, selective, consequences of inositol depletion in 1321N1 cells.  

PubMed Central

Conditions are described for culture of 1321N1 cells under which cellular inositol is decreased from approximately 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [3H]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [3H]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations of [3H]inositol, [3H]InsP1, [3H]InsP2, [3H]InsP3 and [3H]InsP4, which were largely sustained over 90 min, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased only to approximately 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was prevented and, although accumulation of [3H]InsP1, [3H]InsP2 and [3H]InsP3 was initially (0-30 min) potentiated, rates of accumulation of [3H]InsP1 and concentrations of [3H]polyphosphates later (> 30-60 min) declined, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased respectively to approximately 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, stimulated PLC activity was decreased by approximately 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected by changes in the stimulated concentrations of [3H]Ins(1,3,4)P3 but not of [3H]Ins(1,4,5)P3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimulated accumulation of [3H]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively impaired by Li+ as a secondary consequence of inositol monophosphatase inhibition in cells which are highly dependent on inositol re-cycling, but imply that, although Li+ attenuation of PLC activity correlates closely with parameters indicative of limiting inositol supply, it is not readily attributed to decreased PtdInsP2 availability. The potential for complex regulation of PLC and PtdIns synthase is discussed. PMID:8110190

Batty, I H; Downes, C P

1994-01-01

284

Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase.  

PubMed

Integrin-linked kinase (ILK) is an ankyrin-repeat containing serine-threonine protein kinase capable of interacting with the cytoplasmic domains of integrin beta1, beta2, and beta3 subunits. Overexpression of ILK in epithelial cells disrupts cell-extracellular matrix as well as cell-cell interactions, suppresses suspension-induced apoptosis (also called Anoikis), and stimulates anchorage-independent cell cycle progression. In addition, ILK induces nuclear translocation of beta-catenin, where the latter associates with a T cell factor/lymphocyte enhancer-binding factor 1 (TCF/LEF-1) to form an activated transcription factor. We now demonstrate that ILK activity is rapidly, but transiently, stimulated upon attachment of cells to fibronectin, as well as by insulin, in a phosphoinositide-3-OH kinase [Pi(3)K]-dependent manner. Furthermore, phosphatidylinositol(3,4,5)trisphosphate specifically stimulates the activity of ILK in vitro, and in addition, membrane targetted constitutively active Pi(3)K activates ILK in vivo. We also demonstrate here that ILK is an upstream effector of the Pi(3)K-dependent regulation of both protein kinase B (PKB/AKT) and glycogen synthase kinase 3 (GSK-3). Specifically, ILK can directly phosphorylate GSK-3 in vitro and when stably, or transiently, overexpressed in cells can inhibit GSK-3 activity, whereas the overexpression of kinase-deficient ILK enhances GSK-3 activity. In addition, kinase-active ILK can phosphorylate PKB/AKT on serine-473, whereas kinase-deficient ILK severely inhibits endogenous phosphorylation of PKB/AKT on serine-473, demonstrating that ILK is involved in agonist stimulated, Pi(3)K-dependent, PKB/AKT activation. ILK is thus a receptor-proximal effector for the Pi(3)K-dependent, extracellular matrix and growth factor mediated, activation of PKB/AKT, and inhibition of GSK-3. PMID:9736715

Delcommenne, M; Tan, C; Gray, V; Rue, L; Woodgett, J; Dedhar, S

1998-09-15

285

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

286

Phosphoinositide 3-Kinase Alpha-Dependent Regulation of Branching Morphogenesis in Murine Embryonic Lung: Evidence for a Role in Determining Morphogenic Properties of FGF7  

PubMed Central

Branching morphogenesis is a critical step in the development of many epithelial organs. The phosphoinositide-3-kinase (PI3K) pathway has been identified as a central component of this process but the precise role has not been fully established. Herein we sought to determine the role of PI3K in murine lung branching using a series of pharmacological inhibitors directed at this pathway. The pan-class I PI3K inhibitor ZSTK474 greatly enhanced the branching potential of whole murine lung explants as measured by an increase in the number of terminal branches compared with controls over 48 hours. This enhancement of branching was also observed following inhibition of the downstream signalling components of PI3K, Akt and mTOR. Isoform selective inhibitors of PI3K identified that the alpha isoform of PI3K is a key driver in branching morphogenesis. To determine if the effect of PI3K inhibition on branching was specific to the lung epithelium or secondary to an effect on the mesenchyme we assessed the impact of PI3K inhibition in cultures of mesenchyme-free lung epithelium. Isolated lung epithelium cultured with FGF7 formed large cyst-like structures, whereas co-culture with FGF7 and ZSTK474 induced the formation of defined branches with an intact lumen. Together these data suggest a novel role for PI3K in the branching program of the murine embryonic lung contradictory to that reported in other branching organs. Our observations also point towards PI3K acting as a morphogenic switch for FGF7 signalling. PMID:25460003

Carter, Edward; Miron-Buchacra, Gabriela; Goldoni, Silvia; Danahay, Henry; Westwick, John; Watson, Malcolm L.; Tosh, David; Ward, Stephen G.

2014-01-01

287

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

288

Increased serum leptin protects from adiposity despite the increased glucose uptake in white adipose tissue in mice lacking p85alpha phosphoinositide 3-kinase.  

PubMed

Mice lacking the p85alpha regulatory subunit of phosphoinositide (PI) 3-kinase (Pik3r1(-/-)) showed increased glucose uptake in white adipose tissue (WAT) and skeletal muscle due to increased phosphatidylinositol (3,4,5)-triphosphate [PtdIns(3,4,5)P3] production and on a normal diet had a body weight and fat mass similar to wild-type mice. After 3 months on a high-fat diet, Pik3r1(-/-) mice still had increased insulin sensitivity and better glucose tolerance than wild-type mice, but showed markedly greater increases in body weight and WAT mass than wild-type mice. On the normal diet, serum leptin levels of Pik3r1(-/-) mice were significantly higher than in wild-type mice as a result of increased leptin secretion from adipocytes, presumably due to the increased PtdIns(3,4,5)P3 production in adipocytes. Leptin (5 microg/g body wt per day) caused a reduction in food intake and decrease in body weight by the wild-type mice as well as Pik3r1(-/-) mice, suggesting Pik3r1(-/-) mice having leptin sensitivity similar to wild-type mice. The slightly increased serum leptin compensated for the increased glucose uptake by adipocytes in Pik3r1(-/-) mice, thereby preventing adiposity on the normal diet. On the high-fat diet, leptin (5 microg/g body wt per day) failed to decrease food intake or body weight in either genotype, indicating that both genotypes had indeed become severely leptin resistant. Consequently, leptin secretion was unable to sufficiently compensate for the severe leptin resistance caused by the high-fat diet, thereby failing to prevent obesity in Pik3r1(-/-) mice. Our findings suggest that primary increase in serum leptin on the normal diet play a role in the protection from adiposity in Pik3r1(-/-) mice. PMID:15331535

Terauchi, Yasuo; Matsui, Junji; Kamon, Junji; Yamauchi, Toshimasa; Kubota, Naoto; Komeda, Kajuro; Aizawa, Shinichi; Akanuma, Yasuo; Tomita, Motowo; Kadowaki, Takashi

2004-09-01

289

The phosphoinositide PI(3,5)P? mediates activation of mammalian but not plant TPC proteins: functional expression of endolysosomal channels in yeast and plant cells.  

PubMed

Two-pore channel proteins (TPC) encode intracellular ion channels in both animals and plants. In mammalian cells, the two isoforms (TPC1 and TPC2) localize to the endo-lysosomal compartment, whereas the plant TPC1 protein is targeted to the membrane surrounding the large lytic vacuole. Although it is well established that plant TPC1 channels activate in a voltage- and calcium-dependent manner in vitro, there is still debate on their activation under physiological conditions. Likewise, the mode of animal TPC activation is heavily disputed between two camps favoring as activator either nicotinic acid adenine dinucleotide phosphate (NAADP) or the phosphoinositide PI(3,5)P?. Here, we investigated TPC current responses to either of these second messengers by whole-vacuole patch-clamp experiments on isolated vacuoles of Arabidopsis thaliana. After expression in mesophyll protoplasts from Arabidopsis tpc1 knock-out plants, we detected the Arabidopsis TPC1-EGFP and human TPC2-EGFP fusion proteins at the membrane of the large central vacuole. Bath (cytosolic) application of either NAADP or PI(3,5)P? did not affect the voltage- and calcium-dependent characteristics of AtTPC1-EGFP. By contrast, PI(3,5)P? elicited large sodium currents in hTPC2-EGFP-containing vacuoles, while NAADP had no such effect. Analogous results were obtained when PI(3,5)P? was applied to hTPC2 expressed in baker's yeast giant vacuoles. Our results underscore the fundamental differences in the mode of current activation and ion selectivity between animal and plant TPC proteins and corroborate the PI(3,5)P?-mediated activation and Na(+) selectivity of mammalian TPC2. PMID:24770793

Boccaccio, Anna; Scholz-Starke, Joachim; Hamamoto, Shin; Larisch, Nina; Festa, Margherita; Gutla, Paul Vijay Kanth; Costa, Alex; Dietrich, Petra; Uozumi, Nobuyuki; Carpaneto, Armando

2014-11-01

290

Endogenous expression of histamine H1 receptors functionally coupled to phosphoinositide hydrolysis in C6 glioma cells: regulation by cyclic AMP.  

PubMed Central

1. The effects of histamine receptor agonists and antagonists on phospholipid hydrolysis in rat-derived C6 glioma cells have been investigated. 2. Histamine H1 receptor-stimulation caused a concentration-dependent increase in the accumulation of total [3H]-inositol phosphates in cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was histamine (EC50 = 24 microM) > N alpha-methylhistamine (EC50 = 31 microM) > 2-thiazolylethylamine (EC50 = 91 microM). 3. The response to 0.1 mM histamine was antagonized in a concentration-dependent manner by the H1-antagonists, mepyramine (apparent Kd = 1 nM) and (+)-chlorpheniramine (apparent Kd = 4 nM). In addition, (-)-chlorpheniramine was more than two orders of magnitude less potent than its (+)-stereoisomer. 4. Elevation of intracellular cyclic AMP accumulation with forskolin (10 microM, EC50 = 0.3 microM), isoprenaline (1 microM, EC50 = 4 nM) or rolipram (0.5 mM), significantly reduced the histamine-mediated (0.1 mM) inositol phosphate response by 37%, 43% and 26% respectively. In contrast, 1,9-dideoxyforskolin did not increase cyclic AMP accumulation and had no effect on the phosphoinositide response to histamine. 5. These data indicate the presence of functionally coupled, endogenous histamine H1 receptors in C6 glioma cells. Furthermore, the results also indicate that H1 receptor-mediated phospholipid hydrolysis is inhibited by the elevation of cyclic AMP levels in these cells. PMID:7889313

Peakman, M C; Hill, S J

1994-01-01

291

The N-terminal fragment of the ?-amyloid precursor protein of Alzheimer's disease (N-APP) binds to phosphoinositide-rich domains on the surface of hippocampal neurons.  

PubMed

The function of the ?-amyloid precursor protein (APP) of Alzheimer's disease is poorly understood. The secreted ectodomain fragment of APP (sAPP?) can be readily cleaved to produce a small N-terminal fragment (N-APP) that contains heparin-binding and metal-binding domains and that has been found to have biological activity. In the present study, we examined whether N-APP can bind to lipids. We found that N-APP binds selectively to phosphoinositides (PIPs) but poorly to most other lipids. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 )-rich microdomains were identified on the extracellular surface of neurons and glia in primary hippocampal cultures. N-APP bound to neurons and colocalized with PIPs on the cell surface. Furthermore, the binding of N-APP to neurons increased the level of cell-surface PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate. However, PIPs were not the principal cell-surface binding site for N-APP, because N-APP binding to neurons was not inhibited by a short-acyl-chain PIP analogue, and N-APP did not bind to glial cells which also possessed PI(4,5)P2 on the cell surface. The data are explained by a model in which N-APP binds to two distinct components on neurons, one of which is an unidentified receptor and the second of which is a PIP lipid, which binds more weakly to a distinct site within N-APP. Our data provide further support for the idea that N-APP may be an important mediator of APP's biological activity. PMID:24916405

Dawkins, Edgar; Gasperini, Robert; Hu, Yanling; Cui, Hao; Vincent, Adele J; Bolós, Marta; Young, Kaylene M; Foa, Lisa; Small, David H

2014-11-01

292

A novel role for c-Jun N-terminal kinase and phosphoinositide 3-kinase in the liver X receptor-mediated induction of macrophage gene expression  

PubMed Central

Liver X receptors (LXRs) are ligand-dependent transcription factors that are activated by metabolites of cholesterol, oxysterols, and a number of synthetic agonists. LXRs play potent anti-atherogenic roles in part by stimulating the efflux of cholesterol from macrophage foam cells. The LXR-induced expression of ATP-binding cassette transporter (ABC)-A1 and Apolipoprotein E (ApoE) in macrophages is essential for the stimulation of cholesterol efflux and the prevention of atherosclerotic development. Unfortunately, the signaling pathways underlying such regulation are poorly understood and were therefore investigated in human macrophages. The expression of ApoE and ABCA1 induced by synthetic or natural LXR ligands [TO901317, GW3965, and 22-(R)-hydroxycholesterol (22-(R)-HC), respectively] was attenuated by inhibitors of c-Jun N-terminal kinase (JNK) (curcumin and SP600125) and phosphoinositide 3-kinase (PI3K) (LY294002). Similar results were obtained with ABCG1 and LXR-?, two other LXR target genes. LXR agonists activated several components of the JNK pathway (SEK1, JNK and c-Jun) along with AKT, a downstream target for PI3K. In addition, dominant negative mutants of JNK and PI3K pathways inhibited the LXR-agonists-induced activity of the ABCA1 and LXR-? gene promoters in transfected cells. LXR agonists also induced the binding of activator protein-1 (AP-1), a key transcription factor family regulated by JNK, to recognition sequences present in the regulatory regions of the ApoE and ABCA1 genes. These studies reveal a novel role for JNK and PI3K/AKT signaling in the LXR-regulated expression in macrophages of several key genes implicated in atherosclerosis. PMID:21070853

Huwait, Etimad A.; Greenow, Kirsty R.; Singh, Nishi N.; Ramji, Dipak P.

2011-01-01

293

Overexpression of phospholipase D prevents actinomycin D-induced apoptosis through potentiation of phosphoinositide 3-kinase signalling pathways in Chinese-hamster ovary cells.  

PubMed Central

To examine the roles of PLD (phospholipase D) in the regulation of the apoptotic process, PLD1 and PLD2 were stably overexpressed in S1P3-CHO cells [CHO (Chinese-hamster ovary) cells expressing the S1P (sphingosine 1-phosphate) receptor S1P3]. Treatment of S1P3-CHO cells with ActD (actinomycin D) induced apoptosis, as shown by the occurrence of nuclear fragmentation and the caspase-dependent proteolytic cleavage of PARP [poly(ADP-ribose) polymerase] and protein kinase Cd. Overexpression of either PLD1 or PLD2 protected S1P3-CHO cells from ActD-induced apoptosis, as demonstrated by an increased number of viable cells and inhibition of PARP and protein kinase Cd cleavage. However, in the early phase of apoptosis, ActD induced an increase in PLD activity and activation of key factors in the cell-survival signalling pathways, such as PI3K (phosphoinositide 3-kinase), Akt, p70S6K (p70 S6 kinase) and ERK (extracellular-signal-regulated kinase). Furthermore, the ActD-induced activation of these survival signalling enzymes was potentiated by overexpression of either PLD1 or PLD2. The PI3K inhibitor LY294002 inhibited the ActD-induced activation of Akt and p70S6K, and completely abolished the effects of PLD1 or PLD2, whereas inhibition of ERK activity by the MEK inhibitor U0126 had a milder effect. The ActD-induced activation of p70S6K and ERKs was blocked by 1-butanol, but not by t-butanol; similar to S1P, exogenous PLD suppressed the ActD-induced events in the apoptosis signalling pathways. These results show that, in S1P3-CHO cells, increased expression of PLDs prevents ActD-induced apoptosis by enhanced activation of the PI3K signalling pathways. PMID:14640974

Yamada, Momoko; Banno, Yoshiko; Takuwa, Yoh; Koda, Masahiro; Hara, Akira; Nozawa, Yoshinori

2004-01-01

294

Muscarinic-receptor-mediated inhibition of insulin-like growth factor-1 receptor-stimulated phosphoinositide 3-kinase signalling in 1321N1 astrocytoma cells.  

PubMed Central

In 1321N1 astrocytoma cells, stimulation of the IGF-1 (insulin-like growth factor-1) receptor increased the association of PI3K [phosphoinositide (PI) 3-kinase] activity with IRS-1 (insulin re-ceptor substrate 1), and increased the cellular concentration of PtdIns(3,4,5)P3. Carbachol, acting on M3 muscarinic receptors, inhibited insulin-, but not PDGF (platelet-derived growth factor)-, stimulated responses by approximately 50%. The inhibition of IRS-1-associated PI3K activity by carbachol (i) was rapid (<1 min), persistent (> or =60 min) and potent (half-maximal concentration approximately 1 microM); (ii) was reproduced by stimuli for several phospholipase-C-coupled receptors; (iii) was prevented by the inhibition of protein kinase C, but not by chelation of intracellular Ca2+; and (iv) was not blocked or reproduced by inhibitors or stimuli respectively of mitogen-activated protein kinase, PI3K, protein kinase B or the mammalian target of rapamycin. However, the effects of carbachol were prevented by sodium vanadate, a protein tyrosine phosphatase inhibitor, and were accompanied by reduced insulin-stimulated IRS-1 tyrosine phosphorylation and recruitment of the 85 kDa regulatory subunit of PI3K to IRS-1, but not by reduced IGF-1 receptor kinase activity. The inhibitory effect of carbachol was reproduced by okadaic acid, a protein serine/threonine phosphatase inhibitor, but not by PDGF, yet all three agents stimulated the serine phosphorylation of IRS-1 at residues Ser312, Ser616 and Ser636/639, albeit to different extents. Thus muscarinic receptors may inhibit insulin signalling by promoting IRS-1 tyrosine dephosphorylation and/or by uncoupling IRS-1 from the stimulated IGF-1 receptor by stimulating IRS-1 serine phosphorylation. However, the proportion of IRS-1 molecules phosphorylated at a particular site or the phosphorylation of additional IRS-1 serine residues other than those noted above must be important. PMID:14769130

Batty, Ian H; Fleming, Ian N; Downes, C Peter

2004-01-01

295

Arabidopsis thaliana phosphoinositide-specific phospholipase C isoform 3 (AtPLC3) and AtPLC9 have an additive effect on thermotolerance.  

PubMed

The heat stress response is an important adaptation, enabling plants to survive challenging environmental conditions. Our previous work demonstrated that Arabidopsis thaliana Phosphoinositide-Specific Phospholipase C Isoform 9 (AtPLC9) plays an important role in thermotolerance. During prolonged heat treatment, mutants of AtPLC3 showed decreased heat resistance. We observed no obvious phenotypic differences between plc3 mutants and wild type (WT) seedlings under normal growth conditions, but after heat shock, the plc3 seedlings displayed a decline in thermotolerance compared with WT, and also showed a 40-50% decrease in survival rate and chlorophyll contents. Expression of AtPLC3 in plc3 mutants rescued the heat-sensitive phenotype; the AtPLC3-overexpressing lines also exhibited much higher heat resistance than WT and vector-only controls. The double mutants of plc3 and plc9 displayed increased sensitivity to heat stress, compared with either single mutant. In transgenic lines containing a AtPLC3:GUS promoter fusion, GUS staining showed that AtPLC3 expresses in all tissues, except anthers and young root tips. Using the Ca(2+)-sensitive fluorescent probe Fluo-3/AM and aequorin reconstitution, we showed that plc3 mutants show a reduction in the heat-induced Ca(2+) increase. The expression of HSP genes (HSP18.2, HSP25.3, HSP70-1 and HSP83) was down-regulated in plc3 mutants and up-regulated in AtPLC3-overexpressing lines after heat shock. These results indicated that AtPLC3 also plays a role in thermotolerance in Arabidopsis, and that AtPLC3 and AtPLC9 function additionally to each other. PMID:25149227

Gao, Kang; Liu, Yu-Liang; Li, Bing; Zhou, Ren-Gang; Sun, Da-Ye; Zheng, Shu-Zhi

2014-11-01

296

Phosphoinositide metabolism in intestinal brush borders: stimulation of IP3 formation by guanine nucleotides and Ca2+.  

PubMed

The potential role of polyphosphoinositide (PPI) metabolism as a signal-transduction mechanism in apical membranes of polarized epithelial cells was evaluated by examining the formation and breakdown of PPI in rat intestinal brush-border membranes (BBM) prelabeled by intraluminal injection of [3H]inositol in vivo or by [gamma-32P]ATP in vitro. Freshly isolated BBM prelabeled with [3H]inositol contained higher amounts of [3H]phosphatidylinositol 4,5-diphosphate compared with a basolateral membrane (BLM) preparation (approximately 14 and 6.8% of total [3H]PPIs, respectively) and were enriched in inositol lipid kinases, diacylglycerol (DAG) kinase, and phosphomonoesterases degrading PPI, inositol bisphosphate, and inositol triphosphate (IP3). In the presence of nonhydrolysable GTP analogues and low Ca2+ (pCa 6-8) or at high Ca2+ alone (pCa 4) endogenous pools of PPI were rapidly depleted by an intrinsic PPI-specific phospholipase C apparently coupled to a GTP-binding protein (G protein). Surprisingly, despite the assignment of most G protein-coupled hormone receptors to the BLM, the capacity of isolated BBM to release [3H]IP3 in response to Ca2+ or GTP gamma S appeared comparable to that in a BLM preparation. Intestinal secretagogues acting through apical membrane receptors (adenosine, heat-stable Escherichia coli toxin), however, were unable to promote [3H]IP3 release in isolated BBM in the presence of GTP. PPI metabolism in BBM may be coupled to receptors for as yet unidentified secretagogues or may serve as an amplification mechanism for hormone-stimulated PPI breakdown in BLM. The local release of DAG and IP3 at the interior of the intestinal microvilli likely plays a role in the regulation of ion transport systems in microvillar membranes. PMID:2169204

Vaandrager, A B; Ploemacher, M C; De Jonge, H R

1990-09-01

297

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

298

Biomaterials differentially regulate Src kinases and phosphoinositide 3-kinase-? in polymorphonuclear leukocyte primary and tertiary granule release.  

PubMed

In the foreign body response, infiltrating PMNs exocytose granule subsets to influence subsequent downstream inflammatory and wound healing events. In previous studies, we found that PMNs cultured on poly(ethylene glycol) (PEG)-containing hydrogels (i.e., PEG and gelatin + PEG hydrogels) had enhanced primary granule release, yet similar tertiary granule release compared with PMNs cultured on polydimethylsiloxane or tissue culture polystyrene. PMN primary granules contain microbicidal proteins and proteases, which can potentially injure bystander cells, degrade the extracellular matrix, and promote inflammation. Here, we sought to understand the mechanism of the enhanced primary granule release from PMNs on PEG hydrogels. We found that primary granule release from PMNs on PEG hydrogels was adhesion mediated and involved Src family kinases and PI3K-?. The addition of gelatin to PEG hydrogels did not further enhance PMN primary granule release. Using stable-isotope dimethyl labeling-based shotgun proteomics, we identified many serum proteins - including Ig gamma constant chain region proteins and alpha-1-acid glycoprotein 1 - that were absorbed/adsorbed in higher quantities on PEG hydrogels than on TCPS, and may be involved in mediating PMN primary granule release. Ultimately, this mechanistic knowledge can be used to direct inflammation and wound healing following biomaterial implantation to promote a more favorable healing response. PMID:25736495

Cohen, Hannah Caitlin; Frost, Dustin C; Lieberthal, Tyler Jacob; Li, Lingjun; Kao, W John

2015-05-01

299

Alpha 1-adrenergic receptor-mediated phosphoinositide hydrolysis and prostaglandin E2 formation in Madin-Darby canine kidney cells. Possible parallel activation of phospholipase C and phospholipase A2  

SciTech Connect

alpha 1-Adrenergic receptors mediate two effects on phospholipid metabolism in Madin-Darby canine kidney (MDCK-D1) cells: hydrolysis of phosphoinositides and arachidonic acid release with generation of prostaglandin E2 (PGE2). The similarity in concentration dependence for the agonist (-)-epinephrine in eliciting these two responses implies that they are mediated by a single population of alpha 1-adrenergic receptors. However, we find that the kinetics of the two responses are quite different, PGE2 production occurring more rapidly and transiently than the hydrolysis of phosphoinositides. The antibiotic neomycin selectively decreases alpha 1-receptor-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis without decreasing alpha 1-receptor-mediated arachidonic acid release and PGE2 generation. In addition, receptor-mediated inositol trisphosphate formation is independent of extracellular calcium, whereas release of labeled arachidonic acid is largely calcium-dependent. Moreover, based on studies obtained with labeled arachidonic acid, receptor-mediated generation of arachidonic acid cannot be accounted for by breakdown of phosphatidylinositol monophosphate, phosphatidylinositol bisphosphate, or phosphatidic acid. Further studies indicate that epinephrine produces changes in formation or turnover of several classes of membrane phospholipids in MDCK cells. We conclude that alpha 1-adrenergic receptors in MDCK cells appear to regulate phospholipid metabolism by the parallel activation of phospholipase C and phospholipase A2. This parallel activation of phospholipases contrasts with models described in other systems which imply sequential activation of phospholipase C and diacylglycerol lipase or phospholipase A2.

Slivka, S.R.; Insel, P.A.

1987-03-25

300

Phosphoinositide 3-kinase as a novel functional target for the regulation of the insulin signaling pathway by SIRT1.  

PubMed

The protein deacetylase SIRT1, and its activator resveratrol, exert beneficial effects on glucose metabolism. Different SIRT1 targets have been identified, including PTP1B, AMPK, FOXO, PGC-1? and IRS2. The latter may underscore a tight link between SIRT1 and insulin signaling components. However, whether SIRT1 has a direct effect on insulin resistance and whether resveratrol acts directly or indirectly in this context is still a matter of controversy and this question has not been addressed in muscle cells. Here, we show that SIRT1 protein expression is decreased in muscle biopsies and primary myotubes derived from type 2 diabetic patients, suggesting a contribution of diminished SIRT1 in the determination of muscle insulin resistance. To investigate the functional impact of SIRT1 on the insulin pathway, the activation of insulin downstream effector PKB was evaluated after SIRT1 inactivation by RNAi, SIRT1 overexpression, or resveratrol treatments. In muscle cells and HEK293 cells, downregulation of SIRT1 reduced, while overexpression increased, insulin-induced PKB activatory phosphorylation. Further molecular characterisation revealed that SIRT1 interacts in an insulin-independent manner with the PI3K adapter subunit p85. We then investigated whether resveratrol may improve insulin signaling in muscle cells via SIRT1, or alternative targets. Incubation of muscle cells with resveratrol reverted the insulin-resistant state induced by prolonged TNF? or insulin treatment. Resveratrol-dependent improvement of insulin-resistance occurred through inhibition of serine phosphorylation of IRS1/2, implicating resveratrol as a serine kinase inhibitor. Finally, a functional interaction between PI3K and SIRT1 was demonstrated in C. elegans, where constitutively active PI3K - mimicking increased IIS signaling - lead to shortened lifespan, while removal of sir-2.1 abolished PI3K-induced lifespan shortening. Our data identify SIRT1 as a positive modulator of insulin signaling in muscle cells through PI3K, and this mechanism appears to be conserved from C. elegans through humans. PMID:21241768

Fröjdö, Sara; Durand, Christine; Molin, Laurent; Carey, Andrew L; El-Osta, Assam; Kingwell, Bronwyn A; Febbraio, Mark A; Solari, Florence; Vidal, Hubert; Pirola, Luciano

2011-03-30

301

The N-terminal 34 residues of the 55 kDa regulatory subunits of phosphoinositide 3-kinase interact with tubulin.  

PubMed Central

There are five regulatory subunit isoforms of phosphoinositide 3-kinase (PI 3-kinase), which are classified into three groups: proteins of 85 kDa (p85alpha and p85beta), 55 kDa (p55alpha and p55gamma) and 50 kDa (p50alpha). Structural differences between the three groups reside in the N-terminus. To elucidate the unique functional role of the 55 kDa regulatory subunits, GST (glutathione S-transferase) fusion proteins containing a unique N-terminal portion consisting of a 34-amino-acid sequence of p55alpha or p55gamma (GST-p55alpha/gammaN(1-34)) were used as affinity matrices to screen rat brain cell extracts for proteins to which this portion binds specifically. A protein that bound was identified as beta-tubulin by protein sequencing. In addition, not only the beta isoform of tubulin, but also the alpha and gamma isoforms, were detected in the protein absorbed from cell lysates with GST-p55gammaN(1-34) and GST-p55alphaN(1-34) by immunoblotting. Indeed, the only regulatory subunit present in the purified microtubule assembly from rat brain was the 55 kDa isoform; neither 85 kDa nor 50 kDa subunits were detected. These results indicate endogenous binding of 55 kDa regulatory subunits of PI 3-kinase to tubulin in the brain. Finally, we measured tubulin-associated PI 3-kinase activity in CHO/IR cells overexpressing each of the five regulatory subunit isoforms. Only in cells expressing p55alpha or p55gamma was there a significant elevation of tubulin-associated PI 3-kinase activity in response to insulin. These results suggest that the p55alpha and p55gamma regulatory subunits have important roles in regulating PI 3-kinase activity, particularly for microtubules at the cell periphery. PMID:10677370

Inukai, K; Funaki, M; Nawano, M; Katagiri, H; Ogihara, T; Anai, M; Onishi, Y; Sakoda, H; Ono, H; Fukushima, Y; Kikuchi, M; Oka, Y; Asano, T

2000-01-01

302

Physical process Mechanical mechanisms  

E-print Network

1 Physical process Generation · Mechanical mechanisms F = m·a · Electric/Magnetic mechanisms F = B·i·l · Fluid dynamic/Hydraulic mechanisms q, p, ij · Thermal/Optical #12;2 Source unit and source mechanisms ­ Monopoles......volume fluctuations ­ Dipoles ......pressure fluctuations

Berlin,Technische Universität

303

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

304

Molecular Mechanism behind Rotavirus NSP1-Mediated PI3 Kinase Activation: Interaction between NSP1 and the p85 Subunit of PI3 Kinase  

PubMed Central

Our previous study had reported on the interaction of rotavirus NSP1 with cellular phosphoinositide 3-kinase (PI3K) during activation of the PI3K pathway (P. Bagchi et al., J. Virol. 84:6834–6845, 2010). In this study, we have analyzed the molecular mechanism behind this interaction. Results showed that this interaction is direct and that both ? and ? isomers of the PI3K regulatory subunit p85 and full-length NSP1 are important for this interaction, which results in efficient activation of the PI3K/Akt pathway during rotavirus infection. PMID:23221569

Bagchi, Parikshit; Nandi, Satabdi; Nayak, Mukti Kant

2013-01-01

305

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

306

Epidermal growth factor up-regulates the transcription of mouse lon homology ATP-dependent protease through extracellular signal-regulated protein kinase- and phosphatidylinositol-3-kinase-dependent pathways.  

PubMed

Epidermal growth factor (EGF) induces tumorigenic transformation of mouse epidermal cells (JB6 P(+)). We cloned a full-length EGF-responsive cDNA in JB6P(+) cells; EGF up-regulated mRNA expression of this gene 5- to 6-fold. The deduced amino acid sequence of this cDNA exhibited 84 and 96% homology with human and rat Lon homology ATP-dependent protease, respectively, and all conserved domains of Lon, such as ATPase/protease domains, are present in the mouse gene, indicating that this gene is mouse Lon. EGF increased the transcriptional rate without affecting the mRNA stability of m-Lon. The level of m-Lon in irreversibly transformed mouse epidermal cells (JB7) was 3.4-fold higher than that in parental JB6 P(+) cells. Similarly, human mammary epithelial cells overexpressing the proto-oncogene ErbB2 expressed significantly higher levels of Lon than normal mammary epithelial cells. EGF failed to regulate Lon expression in ERK-deficient JB6 P(-) cells or cells that expressed the dominant-negative p85 P13-K regulatory unit. Furthermore, selective chemical blockers for MEK1 and P13-K (PD98059 and LY294002) inhibited EGF-mediated induction. Mitochondria-localized Lon protease plays a critical role in the regulation of mitochondrial gene expression and genome integrity. Disruption of mitochondrial homeostasis is a general characteristic of tumorigenic transformation. Thus, the role of Lon in tumor promotion warrants further study. PMID:12372343

Zhu, Yunfeng; Wang, Mei; Lin, Hong; Huang, Chuanshu; Shi, Xianglin; Luo, Jia

2002-10-15

307

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

308

Retinoic acid signaling in axonal regeneration.  

PubMed

Following an acute central nervous system (CNS) injury, axonal regeneration and functional recovery are extremely limited. This is due to an extrinsic inhibitory growth environment and the lack of intrinsic growth competence. Retinoic acid (RA) signaling, essential in developmental dorsoventral patterning and specification of spinal motor neurons, has been shown through its receptor, the transcription factor RA receptor ?2 (RAR?2), to induce axonal regeneration following spinal cord injury (SCI). Recently, it has been shown that in dorsal root ganglion neurons (DRGs), cAMP levels were greatly increased by lentiviral RAR?2 expression and contributed to neurite outgrowth. Moreover, RAR?agonists, in cerebellar granule neurons (CGN) and in the brain in vivo, induced phosphoinositide 3-kinase dependent phosphorylation of AKT that was involved in RAR?-dependent neurite outgrowth. More recently, RA-RAR?pathways were shown to directly transcriptionally repress a member of the inhibitory Nogo receptor (NgR) complex, Lingo-1, under an axonal growth inhibitory environment in vitro as well as following spinal injury in vivo. This perspective focuses on these newly discovered molecular mechanisms and future directions in the field. PMID:22287943

Puttagunta, Radhika; Di Giovanni, Simone

2011-01-01

309

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

310

MECHANICAL ENGINEERING What is Mechanical  

E-print Network

MECHANICAL ENGINEERING What is Mechanical Engineering? Mechanical engineering is one of the broadest engineering fields. Mechanical engineers are found in virtually all productive industries, from aircraft and automotive to consumer products and building equipment. In these jobs, mechanical engineers

311

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)

2002-08-06

312

Repression of B-Cell Linker (BLNK) and B-Cell Adaptor for Phosphoinositide 3Kinase (BCAP) Is Important for Lymphocyte Transformation by Rel Proteins  

Microsoft Academic Search

Persistent Rel\\/nuclear factor-KB (NF-KB) activity is a hallmark of many human cancers, and the Rel proteins are implicated in leukemia\\/lymphomagenesis but the mechanism is not fully understood.Microarray analysis to identify transformation- impacting genes regulated by NF-KB's oncogenic v-Rel and c-Rel proteins uncovered that Rel protein expression leads to transcriptional repression of key B-cell receptor (BCR) components and signaling molecules like

Jeffrey Delrow; Amar Drawid; Anirvan M. Sengupta; Celine Gelinas

2008-01-01

313

REGULATION OF CA2+ ENTRY BY INOSITOL LIPIDS IN MAMMALIAN CELLS BY MULTIPLE MECHANISMS  

PubMed Central

Increased phosphoinositide turnover was first identified as an early signal transduction event initiated by cell surface receptors that were linked to calcium signaling. Subsequently, the generation of inositol 1,4,5-trisphosphate by phosphoinositide-specific phospholipase C enzymes was defined as the major link between inositide turnover and the cytosolic Ca2+ rise in response to external stimulation. However, in the last decades, phosphoinositides have been emerging as major regulatory lipids involved in virtually every membrane-associated signaling process. Phosphoinositides regulate both the activity and the trafficking of almost all ion channels and transporters contributing to the maintenance of the ionic gradients that are essential for the proper functioning of all eukaryotic cells. Here we summarize the various means by which phosphoinositides affect ion channel functions with special emphasis on Ca2+ signaling and outline the principles that govern the highly compartmentalized roles of these regulatory lipids. PMID:19395084

Balla, Tamas

2009-01-01

314

Signalling mechanisms regulating phenotypic changes in breast cancer cells  

PubMed Central

In MCF-7 breast cancer cells epidermal growth factor (EGF) induces cell proliferation, whereas heregulin (HRG)/neuregulin (NRG) induces irreversible phenotypic changes accompanied by lipid accumulation. Although these changes in breast cancer cells resemble processes that take place in the tissue, there is no understanding of signalling mechanisms regulating it. To identify molecular mechanisms mediating this cell-fate decision process, we applied different perturbations to pathways activated by these growth factors. The results demonstrate that phosphoinositide 3 (PI3) kinase (PI3K) and mammalian target of rapamycin (mTOR) complex (mTORC)1 activation is necessary for lipid accumulation that can also be induced by insulin, whereas stimulation of the extracellular-signal-regulated kinase (ERK) pathway is surprisingly dispensable. Interestingly, insulin exposure, as short as 4 h, was sufficient for triggering the lipid accumulation, whereas much longer treatment with HRG was required for achieving similar cellular response. Further, activation patterns of ATP citrate lyase (ACLY), an enzyme playing a central role in linking glycolytic and lipogenic pathways, suggest that lipids accumulated within cells are produced de novo rather than absorbed from the environment. In the present study, we demonstrate that PI3K pathway regulates phenotypic changes in breast cancer cells, whereas signal intensity and duration is crucial for cell fate decisions and commitment. Our findings reveal that MCF-7 cell fate decisions are controlled by a network of positive and negative regulators of both signalling and metabolic pathways. PMID:25643809

Volinsky, Natalia; McCarthy, Cormac J.; von Kriegsheim, Alex; Saban, Nina; Okada-Hatakeyama, Mariko; Kolch, Walter; Kholodenko, Boris N.

2015-01-01

315

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

316

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

317

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

318

Possible mechanisms of epinephrine actions in quin-2-loaded platelets refractory to arachidonic acid.  

PubMed

We evaluated the effect of Quin-2 loading (> 20 microM) on platelet responses such as phosphoinositide turnover, elevation of cytosolic Ca2+, phosphorylation of myosin light chain (MLC) and a 47-kDa protein, and aggregation in human platelets stimulated with arachidonic acid (AA) and epinephrine. The formation of inositol phosphates (IP, IP2, and IP3) in platelets stimulated with AA was inhibited by 50.4, 59.5, and 61%, respectively, in the presence of Quin-2 (40 microM). A similar degree of inhibition was observed in platelets stimulated with epinephrine (50 microM) and thrombin (0.1 U/ml). Even though Quin-2-induced inhibition of aggregation in response to AA was reversed by epinephrine, its effect on phosphoinositide turnover remained unaffected. Monitoring of cytosolic Ca2+ changes further indicates that the ability of epinephrine to restore aggregation in Quin-2-loaded (40 microM) and AA-stimulated platelets is not coupled to an increase in cytosolic Ca2+. Quin-2 loading (40 microM) caused a significant inhibition of MLC phosphorylation (20 kDa) in platelets stimulated by AA. However, it had no effect on the phosphorylation of the 47-kDa protein induced by AA. Furthermore, Quin-2 loading (40 microM) exerted no significant effect on shape change, actin filament assembly, and spreading, but caused a significant inhibition of secretion and clot retraction. We conclude that the formation of inositol phosphates, increases in cytosolic Ca2+, and phosphorylation of MLC affected by Quin-2 are not coupled to the mechanisms by which platelets develop stickiness, undergo shape change, spreading, and aggregation in response to epinephrine and AA. It appears that the effect of epinephrine in restoring the aggregation response of refractory platelets is coupled to a calcium-mediated alpha-adrenergic receptor, and it may serve as a critical salvage pathway in platelets with compromised functions. PMID:8123296

Rao, G H; Gerrard, J M; Murthy, M; White, J G

1993-12-01

319

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

320

Mechanical down jar mechanism  

SciTech Connect

This paper describes a mechanical down jar mechanism for freeing stuck objects within a well bore and for conducting other down hole activities. It comprises: an elongate tubular housing having anvil means; mandrel means adapted for connection to an object to be moved downwardly within the well bore and being disposed in telescoping relation with the anvil means and the elongate tubular housing, the mandrel means adapted to be struck by the anvil means to impart a downwardly directed jarring force to the object; the elongate tubular housing having internal firing and recocking detent groove means located in axially spaced relation and forming a firing lug support land therebetween; a radially expandable and retractable firing lug assembly being disposed within the elongate tubular housing and in absence of force being applied axially thereto being radially restrained by the firing lug support land; load spring means being disposed within the elongate tubular housing and being in downward force transmitting relation with the firing lug assembly; recocking spring means being disposed within the elongate tubular housing and having upward axial force transmitting relation with the firing lug assembly.

Taylor, W.T.

1991-12-03

321

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

322

Bohmian mechanics contradicts quantum mechanics  

E-print Network

Bohmian mechanics contradicts quantum mechanics Arnold Neumaier Institut fur Mathematik, Universit and quantum mechanics predict values of opposite sign for certain time correlations. The discrepancy can no loophole for claiming that Bohmian mechanics reproduces all predictions of quantum mechanics exactly

Neumaier, Arnold

323

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

324

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

325

Neuregulin-1 Regulates Cell Adhesion via an ErbB2/Phosphoinositide-3 Kinase/Akt-Dependent Pathway: Potential Implications for Schizophrenia and Cancer  

PubMed Central

Background Neuregulin-1 (NRG1) is a putative schizophrenia susceptibility gene involved extensively in central nervous system development as well as cancer invasion and metastasis. Using a B lymphoblast cell model, we previously demonstrated impairment in NRG1?-mediated migration in cells derived from patients with schizophrenia as well as effects of risk alleles in NRG1 and catechol-O-methyltransferase (COMT), a second gene implicated both in schizophrenia susceptibility and in cancer. Methodology/Principal Findings Here, we examine cell adhesion, an essential component process of cell motility, using an integrin-mediated cell adhesion assay based on an interaction between ICAM-1 and the CD11a/CD18 integrin heterodimer expressed on lymphoblasts. In our assay, NRG1? induces lymphoblasts to assume varying levels of adhesion characterized by time-dependent fluctuations in the firmness of attachment. The maximum range of variation in adhesion over sixty minutes correlates strongly with NRG1?-induced migration (r2?=?0.61). NRG1?-induced adhesion variation is blocked by erbB2, PI3K, and Akt inhibitors, but not by PLC, ROCK, MLCK, or MEK inhibitors, implicating the erbB2/PI3K/Akt1 signaling pathway in NRG1-stimulated, integrin-mediated cell adhesion. In cell lines from 20 patients with schizophrenia and 20 normal controls, cells from patients show a significant deficiency in the range of NRG1?-induced adhesion (p?=?0.0002). In contrast, the response of patient-derived cells to phorbol myristate acetate is unimpaired. The COMT Val108/158Met genotype demonstrates a strong trend towards predicting the range of the NRG1?-induced adhesion response with risk homozygotes having decreased variation in cell adhesion even in normal subjects (p?=?0.063). Conclusion/Significance Our findings suggest that a mechanism of the NRG1 genetic association with schizophrenia may involve the molecular biology of cell adhesion. PMID:18159252

Kanakry, Christopher G.; Li, Zhen; Nakai, Yoko; Sei, Yoshitatsu; Weinberger, Daniel R.

2007-01-01

326

Control of Cerebellar Long-Term Potentiation by P-Rex-Family Guanine-Nucleotide Exchange Factors and Phosphoinositide 3-Kinase  

PubMed Central

Background Long-term potentiation (LTP) at the parallel fibre–Purkinje cell synapse in the cerebellum is a recently described and poorly characterized form of synaptic plasticity. The induction mechanism for LTP at this synapse is considered reciprocal to “classical” LTP at hippocampal CA1 pyramidal neurons: kinases promote increased trafficking of AMPA receptors into the postsynaptic density in the hippocampus, whereas phosphatases decrease internalization of AMPA receptors in the cerebellum. In the hippocampus, LTP occurs in overlapping phases, with the transition from early to late phases requiring the consolidation of initial induction processes by structural re-arrangements at the synapse. Many signalling pathways have been implicated in this process, including PI3 kinases and Rho GTPases. Principal Findings We hypothesized that analogous phases are present in cerebellar LTP, and took as the starting point for investigation our recent discovery that P-Rex – a Rac guanine nucleotide exchange factor which is activated by PtdIns(3,4,5)P3 – is highly expressed in mouse cerebellar Purkinje neurons and plays a role in motor coordination. We found that LTP evoked at parallel fibre synapses by 1 Hz stimulation or by NO donors was not sustained beyond 30 min when P-Rex was eliminated or Rac inhibited, suggesting that cerebellar LTP exhibits a late phase analogous to hippocampal LTP. In contrast, inhibition of PI3 kinase activity eliminated LTP at the induction stage. Conclusions Our data suggest that a PI3K/P-Rex/Rac pathway is required for late phase LTP in the mouse cerebellum, and that other PI3K targets, which remain to be discovered, control LTP induction. PMID:20694145

Jackson, Claire; Welch, Heidi C.; Bellamy, Tomas C.

2010-01-01

327

NGF-induces Axonal Filopodia through Localized Microdomains of Phosphoinositide 3-kinase (PI3K) Activity that Drive the Formation of Cytoskeletal Precursors to Filopodia  

PubMed Central

The initiation of axonal filopodia is the first step in the formation of collateral branches and synaptic structures. In sensory neurons, nerve growth factor (NGF) promotes the formation of axonal filopodia and branches. However, the signaling and cytoskeletal mechanisms of NGF-induced initiation of axonal filopodia are not clear. Axonal filopodia arise from precursor axonal cytoskeletal structures termed F-actin patches. Patches form spontaneously and are transient. Although filopodia emerge from patches, only a fraction of patches normally gives rise to filopodia. Using chicken sensory neurons and live imaging of eYFP-actin dynamics, we report that NGF promotes the formation of axonal filopodia by increasing the rate of F-actin patch formation, but not the fraction of patches that give rise to filopodia. We also demonstrate that activation of the PI3K-Akt pathway is sufficient and required for driving the formation of axonal F-actin patches, filopodia and axon branches. Using the GFP-PH domain of Akt, which targets to PI3K-generated PIP3, we report localized microdomains of PIP3 accumulation that form in synchrony with F-actin patches, and that NGF promotes the formation of microdomains of PIP3 and patches. Finally, we find that in NGF F-actin patches form in association with axonal mitochondria and oxidative phosphorylation is required for patch formation. This investigation demonstrates that surprisingly NGF promotes formation of axonal filopodia by increasing the formation of cytoskeletal filopodial precursors (patches) through localized microdomains of PI3K signaling but not the emergence of filopodia from patches. PMID:20826681

Ketschek, Andrea; Gallo, Gianluca

2010-01-01

328

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

329

SOEP: Mechanics.  

ERIC Educational Resources Information Center

A series of articles examines aspects of supervised occupational experience programs (SOEP) in agricultural mechanics, including production agriculture, horticulture, technical institute programs, mechanized agriculture, safety education, point guide system for developing competencies, and teacher education's responsibility. (SK)

Jacobs, Clinton O.; And Others

1984-01-01

330

Computational mechanics  

Microsoft Academic Search

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

Raboin

1998-01-01

331

Inhibition of mechanical stress-induced hypertrophic scar inflammation by emodin.  

PubMed

At least 50% of hypertrophic scarring (HS) is characterized by inflammation, for which there is currently no effective treatment available. Emodin is a major component of the widely used Chinese herb, rhubarb, which has been used to treat inflammation in several types of disease. However, few studies have investigated the efficacy of emodin in the treatment of HS. In the present study, a mouse model with mechanical stress?induced HS was used to investigate the effects of emodin (20, 40, 80, or 120 mg/ml) on HS, and to determine the potential underlying mechanisms. Treatment with emodin significantly attenuated HS inflammation, as determined by histopathological assessment of the scar elevation index, collagen structure and inflammation (P<0.05). Furthermore, treatment with emodin (40 mg/ml) markedly inhibited phosphoinositide 3?kinase (PI3K)/Akt activity (P<0.01) and this attenuation was associated with reduced expression levels of tumor necrosis factor??, interleukin?6 and monocyte chemoattractant protein?1 (P<0.05) in the HS tissue. The results of the present study indicated that administration of emodin had therapeutic effects on the progression of HS and the underlying mechanism of this may be due to inhibition of the PI3K/Akt signaling pathway. PMID:25634255

Liu, Cheng

2015-06-01

332

Phosphorylation of phosphoinositides in human platelets  

SciTech Connect

32P-labelling of phosphatidylinositol (PI), PI-4-monophosphate (PIP), PI-4,5-bisphosphate (PIP2) and phosphatidic acid (PA) in /sup 32/P-labelled intact human platelets was investigated in the presence of various agents which alter intracellular level of cAMP or Ca/sup 2 +/. Addition of dibutyryl cAMP to intact platelets pre- or pulse labelled with /sup 32/P resulted in increased /sup 32/P-labelling of PIP and in concomitant decreased /sup 32/P-labelling of PI without affecting that of PIP2 or PA. Similar changes were observed in intact platelets treated by prostaglandin I2 (PGI2) or a new low Km phosphodiesterase inhibitor (DN-9693). When intracellular Ca/sup 2 +/ was chelated by loading quin 2-AM to intact platelets, /sup 32/P-labelling of PIP was significantly increased in a dose related manner. From these observations it was concluded that PI kinase is activated by elevation of cAMP or chelation of Ca/sup 2 +/ in intact platelets.

Suga, K.; Kambayashi, J.; Kawasaki, T.; Sakon, M.; Mori, T.

1986-10-15

333

Phosphoinositide turnover in cell growth and transformation  

SciTech Connect

Interaction of cells with various stimuli triggers a common signal transduction pathway involving breakdown and resynthesis of the minor membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP/sub 2/). Hydrolysis of PIP/sub 2/ by phospholipase C generates two key catabolites-inositol-1,4,5-trisphosphate (IP/sub 3/) and diacylglycerol (DAG)-which mediate and amplify cellular responses. These studies provide evidence for potential involvement of this pathway in oncogenic transformation and cell cycle progression. Altered levels of PIP/sub 2/ and its breakdown products were found in cells transformed by ras oncogenes, in contrast to untransformed counterparts. Steady-state levels of PIP/sub 2/, DAG and inositol phosphates were measured in NIH 3T3 and NRK cells metabolically labelled with /sup 3/H-glycerol and /sup 3/H-inositol. DAG and inositol phosphate levels were significantly elevated by 2.5-3 fold in the transformed cells while levels of PIP/sub 2/ were decreased. These findings suggest that the ras protein may activate phospholipase C. Elevated DAG content in the transformed cells was also measured by phosphorylation of DAG using a partially purified DAG kinase, indicating that the differences seen could not be attributed to differences in labelling between the cell lines.

Fleischman, L.F.

1987-01-01

334

Translocation of the Na+/H+ exchanger 1 (NHE1) in cardiomyocyte responses to insulin and energy-status signalling  

PubMed Central

The Na+/H+ exchanger NHE1 is a highly regulated membrane protein that is required for pH homoeostasis in cardiomyocytes. The activation of NHE1 leads to proton extrusion, which is essential for counteracting cellular acidity that occurs following increased metabolic activity or ischaemia. The activation of NHE1 intrinsic catalytic activity has been well characterized and established experimentally. However, we have examined in the present study whether a net translocation of NHE1 to the sarcolemma of cardiomyocytes may also be involved in the activation process. We have determined the distribution of NHE1 by means of immunofluorescence microscopy and cell-surface biotinylation. We have discovered changes in the distribution of NHE1 that occur when cardiomyocytes are stimulated with insulin that are PI3K (phosphoinositide 3-kinase)-dependent. Translocation of NHE1 also occurs when cardiomyocytes are challenged by hypoxia, or inhibition of mitochondrial oxidative metabolism or electrically induced contraction, but these responses occur through a PI3K-independent process. As the proposed additional level of control of NHE1 through translocation was unexpected, we have compared this process with the well-established translocation of the glucose transporter GLUT4. In immunofluorescence microscopy comparisons, the translocation of NHE1 and GLUT4 to the sarcolemma that occur in response to insulin appear to be very similar. However, in basal unstimulated cells the two proteins are mainly located, with the exception of some co-localization in the perinuclear region, in distinct subcellular compartments. We propose that the mechanisms of translocation of NHE1 and GLUT4 are linked such that they provide spatially and temporally co-ordinated responses to cardiac challenges that necessitate re-adjustments in glucose transport, glucose metabolism and cell pH. PMID:20868366

Lawrence, Scott P.; Holman, Geoffrey D.; Koumanov, Françoise

2010-01-01

335

Geometric Mechanics  

NASA Astrophysics Data System (ADS)

Mechanics for the nonmathematician-a modern approach For physicists, mechanics is quite obviously geometric, yet the classical approach typically emphasizes abstract, mathematical formalism. Setting out to make mechanics both accessible and interesting for nonmathematicians, Richard Talman uses geometric methods to reveal qualitative aspects of the theory. He introduces concepts from differential geometry, differential forms, and tensor analysis, then applies them to areas of classical mechanics as well as other areas of physics, including optics, crystal diffraction, electromagnetism, relativity, and quantum mechanics. For easy reference, Dr. Talman treats separately Lagrangian, Hamiltonian, and Newtonian mechanics-exploring their geometric structure through vector fields, symplectic geometry, and gauge invariance respectively. Practical perturbative methods of approximation are also developed. Geometric Mechanics features illustrative examples and assumes only basic knowledge of Lagrangian mechanics. Of related interest . . . APPLIED DYNAMICS With Applications to Multibody and Mechatronic Systems Francis C. Moon A contemporary look at dynamics at an intermediate level, including nonlinear and chaotic dynamics. 1998 (0-471-13828-2) 504 pp. MATHEMATICAL PHYSICS Applied Mathematics for Scientists and Engineers Bruce Kusse and Erik Westwig A comprehensive treatment of the mathematical methods used to solve practical problems in physics and engineering. 1998 (0-471-15431-8) 680 pp.

Talman, Richard

1999-10-01

336

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

337

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

338

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

339

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,

340

Mechanical Drives  

NSDL National Science Digital Library

This is a web page with learning objects on Mechanical Drives with lessons in: Belt & Chain Drives, and Shaft Couplings, Clutches & Brakes. Each lesson links to an interactive resource which helps to further explain the concepts.

341

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

342

Quantum Mechanics  

NASA Astrophysics Data System (ADS)

The Manchester Physics Series General Editors: D. J. Sandiford; F. Mandl; A. C. Phillips Department of Physics and Astronomy, University of Manchester Properties of Matter B. H. Flowers and E. Mendoza Optics Second Edition F. G. Smith and J. H. Thomson Statistical Physics Second Edition F. Mandl Electromagnetism Second Edition I. S. Grant and W. R. Phillips Statistics R. J. Barlow Solid State Physics Second Edition J. R. Hook and H. E. Hall Quantum Mechanics F. Mandl Particle Physics Second Edition B. R. Martin and G. Shaw The Physics of Stars Second Edition A. C. Phillips Computing for Scientists R. J. Barlow and A. R. Barnett Quantum Mechanics aims to teach those parts of the subject which every physicist should know. The object is to display the inherent structure of quantum mechanics, concentrating on general principles and on methods of wide applicability without taking them to their full generality. This book will equip students to follow quantum-mechanical arguments in books and scientific papers, and to cope with simple cases. To bring the subject to life, the theory is applied to the all-important field of atomic physics. No prior knowledge of quantum mechanics is assumed. However, it would help most readers to have met some elementary wave mechanics before. Primarily written for students, it should also be of interest to experimental research workers who require a good grasp of quantum mechanics without the full formalism needed by the professional theorist. Quantum Mechanics features: A flow diagram allowing topics to be studied in different orders or omitted altogether. Optional "starred" and highlighted sections containing more advanced and specialized material for the more ambitious reader. Sets of problems at the end of each chapter to help student understanding. Hints and solutions to the problems are given at the end of the book.

Mandl, F.

1992-07-01

343

Cellular Mechanisms of Gravitropic Response in Higher Plants  

NASA Astrophysics Data System (ADS)

The evolutionary success of land plants in adaptation to the vectorial environmental factors was based mainly on the development of polarity systems. In result, normal plant ontogenesis is based on the positional information. Polarity is a tool by which the developing plant organs and tissues are mapped and the specific three-dimensional structure of the organism is created. It is due to their polar organization plants are able to orient themselves relative to the gravity vector and different vectorial cues, and to respond adequately to various stimuli. Gravitation is one of the most important polarized environmental factor that guides the development of plant organisms in space. Every plant can "estimate" its position relative to the gravity vector and correct it, if necessary, by means of polarized growth. The direction and the magnitude of gravitational stimulus are constant during the whole plant ontogenesis. The key plant response to the action of gravity is gravitropism, i.e. the directed growth of organs with respect to the gravity vector. This response is a very convenient model to study the mechanisms of plant orientation in space. The present report is focused on the main cellular mechanisms responsible for graviropic bending in higher plants. These mechanisms and structures include electric polarization of plant cells, Ca ({2+) }gradients, cytoskeleton, G-proteins, phosphoinositides and the machinery responsible for asymmetric auxin distribution. Those mechanisms tightly interact demonstrating some hierarchy and multiple feedbacks. The Ca (2+) gradients provide the primary physiological basis of polarity in plant cells. Calcium ions influence on the bioelectric potentials, the organization of actin cytoskeleton, the activity of Ca (2+) -binding proteins and Ca (2+) -dependent protein kinases. Protein kinases modulate transcription factors activity thereby regulating the gene expression and switching the developmental programs. Actin cytoskeleton affects the molecular machinery of polar auxin transport. It results in the changes of auxin gradients in plant organs and tissues, which modulate all cellular mechanisms of polarity via multiple feedback loops. The understanding of the mechanisms of plant organism orientation relative to the gravity vector will allow us to develop efficient technologies for plant growing in microgravity conditions at orbital space stations and during long piloted space flights. This work was supported by the grant of Russian Foundation for Basic Research (N 14-04-01-624) and by the grant of St.-Petersburg State University (N 1.38.233.2014).

Medvedev, Sergei; Smolikova, Galina; Pozhvanov, Gregory; Suslov, Dmitry

344

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

345

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

346

Mural propagation of descending vasa recta responses to mechanical stimulation  

PubMed Central

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 [DiBAC4(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

2013-01-01

347

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

348

Soil mechanics  

NASA Technical Reports Server (NTRS)

Preliminary results are presented of an investigation of the physical and mechanical properties of lunar soil on the Descartes slopes, and the Cayley Plains in the vicinity of the LM for Apollo 16. The soil mechanics data were derived form (1) crew commentary and debriefings, (2) television, (3) lunar surface photography, (4) performance data and observations of interactions between soil and lunar roving vehicle, (5) drive-tube and deep drill samples, (6) sample characteristics, and (7) measurements using the SRP. The general characteristics, stratigraphy and variability are described along with the core samples, penetrometer test results, density, porosity and strength.

Mitchell, J. K.; Carrier, W. D., III; Houston, W. N.; Scott, R. F.; Bromwell, L. G.; Durgunoglu, H. T.; Hovland, H. J.; Treadwell, D. D.; Costes, N. C.

1972-01-01

349

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

350

Mechanical Drafting.  

ERIC Educational Resources Information Center

This publication, the third in a series on drafting, is intended to strengthen students' competence in the specialized field of mechanical drafting. The text consists of instructional materials for both teacher and students, written in terms of student performance using measurable objectives. The course includes 11 units. Each instructional unit…

McClain, Gerald R.

351

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

352

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.

353

Automotive Mechanics.  

ERIC Educational Resources Information Center

This curriculum guide provides materials for a competency-based course in automotive mechanics at the secondary level. The curriculum design uses the curriculum infused model for the teaching of basic skills as part of vocational education and demonstrates the relationship of vocationally related skills to communication, mathematics, and science…

Brown, Desmond

354

Fluid Mechanics.  

ERIC Educational Resources Information Center

Outlines the contents of Volume II of "Principia" by Sir Isaac Newton. Reviews the contributions of subsequent scientists to the physics of fluid dynamics. Discusses the treatment of fluid mechanics in physics curricula. Highlights a few of the problems of modern research in fluid dynamics. Shows that problems still remain. (CW)

Drazin, Philip

1987-01-01

355

Introduction Amoeboid motility is a widespread cell propulsive mechanism  

E-print Network

,4,5-trisphosphate (PIP3) gradient through phosphoinositide 3-kinase (PI3K) activity. Since forces applied binding to actin through formin and Rho kinase (ROCK) activation (Gasteier et al., 2003). Efficient actin is regulated by phosphatidylinositol 4,5-bisphosphate (PIP2), whose turnover is very active at the leading edge

Devreotes, Peter

356

Kinetics and mechanism of the interaction of the C1 domain of protein kinase C with lipid membranes  

E-print Network

PIP 3 ), the product of phosphoinositide-3-kinase (PI3K), asPIP 3 to regulate PKC? activity would be through activation of PKC?’s upstream kinasekinase (PDK) (21). However, one report has shown that PIP 3

Dries, Daniel Robert

2007-01-01

357

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

358

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

359

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

360

Impact Mechanics  

NASA Astrophysics Data System (ADS)

Impact mechanics is concerned with the reaction forces that develop during a collision and the dynamic response of structures to these reaction forces. The subject has a wide range of engineering applications, from designing sports equipment to improving the crashworthiness of automobiles. This book develops several different methodologies for analysing collisions between structures. These range from rigid body theory for structures that are stiff and compact, to vibration and wave analyses for flexible structures. The emphasis is on low-speed impact where damage is local to the small region of contact between the colliding bodies. The analytical methods presented give results that are more robust or less sensitive to initial conditions than have been achieved hitherto. As a text, Impact Mechanics builds upon foundation courses in dynamics and strength of materials. It includes numerous industrially relevant examples and end-of-chapter homework problems drawn from industry and sports. Practising engineers will also find the methods presented in this book useful in calculating the response of a mechanical system to impact.

Stronge, W. J.

2004-03-01

361

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

362

The mechanism of mechanical alloying  

Microsoft Academic Search

The mechanical alloying process is a new method for producing composite metal powders with controlled microstructures. It\\u000a is unique in that it is an entirely solid state process, permitting dispersion of insoluble phases such as refractory oxides\\u000a and addition of reactive alloying elements such as aluminum and titanium. Interdispersion of the ingredients occurs by repeated\\u000a cold welding and fracture of

J. S. Benjamin; T. E. Volin

1974-01-01

363

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

364

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

365

Mechanics Mania  

NSDL National Science Digital Library

Through 10 lessons and numerous activities, students explore the natural universal rules engineers and physicists use to understand how things move and stay still. Together, these rules are called "mechanics." The study of mechanics is a way to improve our understanding of everyday movements, such as how gravity pulls things together, how objects balance, spin and twirl, and how things fly and fall. While studying Newton's three laws of motion, students gain hands-on experience with the concepts of forces, changes in motion, and action and reaction. Through hands-on activities, students model the behavior of parachutes and helicopters, closely examine falling objects, build and use a spring scale, examine collisions between skateboards, make model rockets with balloons and string, collect data from cotton ball catapults, study friction with small hovercrafts made from old CDs and balloons, experiment with center of mass by balancing objects on coat hangers and strings, compete to design clay beams with the best strength-to-weight ratio, experiment with weight distribution on homemade spinning tops, experiment with string length, weight and angle of release of pendulums made from fishing weights and string, and use marshmallows and spaghetti to construct their own structures to see which can hold the most weight. For each lesson, associated literacy activities provide additional student engagement. See the Unit Overview section for a list of topics by lesson and descriptions of the associated literacy activities.

2014-09-18

366

Fracture mechanics  

NASA Technical Reports Server (NTRS)

The application of fracture mechanics to the design of ceramic structures will require the precise measurement of crack growth and fracture resistance of these materials over their entire range of anticipated service temperatures and standardized test methods for making such measurements. The development of a standard test for measuring the plane strain fracture toughness is sought. Stress intensity factor coefficients were determined for three varieties of chevron-notch specimens, and fracture toughness measurements were made on silicon nitrides, silicon carbides, and aluminum oxides to assess the performance of each specimen variety. It was determined that silicon nitride and silicon carbides have flat crack growth resistance curves, but aluminum oxide does not. Additionally, batch-to-batch differences were noticed for the aluminum oxide. Experiments are continuing to explain the rising crack growth resistance and batch-to-batch variations for the aluminum oxide.

Shannon, John L., Jr.

1986-01-01

367

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

368

Multiple Mutations and Bypass Mechanisms Can Contribute to Development of Acquired Resistance to MET Inhibitors  

PubMed Central

Therapies targeting receptor tyrosine kinases have shown efficacy in molecularly defined subsets of cancers. Unfortunately, cancers invariably develop resistance, and overcoming or preventing resistance will ultimately be key to unleashing their full therapeutic potential. In this study, we examined how cancers become resistant to MET inhibitors, a class of drugs currently under clinical development. We utilized the highly sensitive gastric carcinoma cell line, SNU638, and two related MET inhibitors PHA-665752 and PF-2341066. To our surprise, we observed at least two mechanisms of resistance that arose simultaneously. Both resulted in maintenance of downstream PI3K (phosphoinositide 3-kinase)-AKT and MEK (MAP/ERK kinase)-ERK signaling in the presence of inhibitor. One mechanism, observed by modeling resistance both in vitro and in vivo, involved the acquisition of a mutation in the MET activation loop (Y1230). Structural analysis indicates that this mutation destabilizes the autoinhibitory conformation of MET and abrogates an important aromatic stacking interaction with the inhibitor. The other cause of resistance was activation of the epidermal growth factor receptor (EGFR) pathway due to increased expression of transforming growth factor ?. Activation of EGFR bypassed the need for MET signaling to activate downstream signaling in these cells. This resistance could be overcome by combined EGFR and MET inhibition. Thus, therapeutic strategies that combine MET inhibitors capable of inhibiting Y1230 mutant MET in combination with anti-EGFR–based therapies may enhance clinical benefit for patients with MET-addicted cancers. Importantly, these results also underscore the notion that a single cancer can simultaneously develop resistance induced by several mechanisms and highlight the daunting challenges associated with preventing or overcoming resistance. PMID:21266357

Qi, Jie; McTigue, Michele A.; Rogers, Andrew; Lifshits, Eugene; Christensen, James G.; Jänne, Pasi A.; Engelman, Jeffrey A.

2011-01-01

369

Mechanisms of insulin resistance in the amygdala: influences on food intake.  

PubMed

Obesity is increasing worldwide and is triggered, at least in part, by enhanced caloric intake. Food intake is regulated by a complex mechanism involving the hypothalamus and hindbrain circuitries. However, evidences have showing that reward systems are also important in regulating feeding behavior. In this context, amygdala is considered a key extra-hypothalamic area regulating feeding behavior in human beings and rodents. This review focuses on the regulation of food intake by amygdala and the mechanisms of insulin resistance in this brain area. Similar to the hypothalamus the anorexigenic effect of insulin is mediated via PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B) pathway in the amygdala. Insulin decreases NPY (neuropeptide Y) and increases oxytocin mRNA levels in the amygdala. High fat diet and saturated fatty acids induce inflammation, ER (endoplasmic reticulum) stress and the activation of serine kinases such as PKC? (protein kinase C theta), JNK (c-Jun N-terminal kinase) and IKK? (inhibitor of nuclear factor kappa-B kinase beta) in the amygdala, which have an important role in insulin resistance in this brain region. Overexpressed PKC? in the CeA (central nucleus of amygdala) of rats increases weight gain, food intake, insulin resistance and hepatic triglycerides content. The inhibition of ER stress ameliorates insulin action/signaling, increases oxytocin and decreases NPY gene expression in the amygdala of high fat feeding rodents. Those data suggest that PKC? and ER stress are main mechanisms of insulin resistance in the amygdala of obese rats and play an important role regulating feeding behavior. PMID:25601576

Areias, Maria Fernanda Condes; Prada, Patricia Oliveira

2015-04-01

370

Department of Mechanical Engineering-  

E-print Network

Department of Mechanical Engineering- Engineering Mechanics Presidential Council of Alumnae Click #12;The Department of Mechanical Engineering ­ Engineering Mechanics honors its outstanding women Bachelor's of Science Degree in Mechanical Engineering in 1978. She earned her Master's of Science Degree

Endres. William J.

371

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

372

Gelation Mechanisms  

NASA Astrophysics Data System (ADS)

In this paper, we survey the gelation mechanisms for various polymeric systems which are classified by the type and the strength of the cross-linkages. These are the "irreversible" gels that are cross-linked chemically by covalent bonds and the "reversible" gels that are cross-linked physically by hydrogen or ionic bonds and by the physical entanglement of polymer chains. Some of the natural polymer gels fall into the class of physical gels, among which the red algae that has attracted attention for various applications is discussed in detail. Various composite gels, formed from mixture of physical and chemical gels are also discussed in the last section of the article. Theoretical models describe the gelation as a process of random linking of subunits to larger and larger molecules by formation of an infinite network, where no matter what type of objects are linked, there is always a critical "gel point" at which the system behaves neither as a liquid nor as a solid on any length scale. The Flory-Stockmayer theory and percolation theory provide bases for modeling this sol-gel phase transition. The experimental techniques for measuring the critical exponents for sol-gel phase transitions in different polymeric systems are introduced and the validation of various theoretical predictions are surveyed.

Pekcan, Önder; Kara, Selim

2012-10-01

373

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

374

TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77*  

PubMed Central

T cell immunoglobulin and mucin domain (TIM) proteins are cell-surface signaling receptors in T cells and scavenger receptors in antigen-presenting cells and kidney tubular epithelia. Here, we demonstrated a function for TIM proteins in mediating the degradation of NUR77, a nuclear receptor implicated in the cellular response to injury. TIM proteins interacted with and mediated the lysosomal degradation of NUR77 in a phosphoinositide 3-kinase–dependent pathway. We also showed dynamic cycling of TIM-1 to and from the cell surface through clathrin-dependent constitutive endocytosis. Blocking this process or mutating the phosphatidylserine-binding pocket in TIM-1 abrogated TIM-1–mediated degradation of NUR77. In an in vitro model of kidney injury, silencing TIM-1 increased NUR77 abundance and decreased epithelial cell survival. These results show that TIM proteins may affect immune cell function and the response of the kidney to injury. PMID:23233528

Balasubramanian, Savithri; Kota, Satya Keerthi; Kuchroo, Vijay K; Humphreys, Benjamin D; Strom, Terry B

2013-01-01

375

Mechanical engineering Department Seminar  

E-print Network

Mechanical engineering Department Seminar James Bird Department of Mechanical Engineering Boston ­ are discussed. James Bird is an Assistant Professor in the Mechanical Engineering Department at Boston completed post-doctoral research at MIT. His research interests include experimental fluid mechanics

376

Role of a Novel PH-Kinase Domain Interface in PKB/Akt Regulation: Structural Mechanism for Allosteric Inhibition  

PubMed Central

Protein kinase B (PKB/Akt) belongs to the AGC superfamily of related serine/threonine protein kinases. It is a key regulator downstream of various growth factors and hormones and is involved in malignant transformation and chemo-resistance. Full-length PKB protein has not been crystallised, thus studying the molecular mechanisms that are involved in its regulation in relation to its structure have not been simple. Recently, the dynamics between the inactive and active conformer at the molecular level have been described. The maintenance of PKB's inactive state via the interaction of the PH and kinase domains prevents its activation loop to be phosphorylated by its upstream activator, phosphoinositide-dependent protein kinase-1 (PDK1). By using a multidisciplinary approach including molecular modelling, classical biochemical assays, and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM), a detailed model depicting the interaction between the different domains of PKB in its inactive conformation was demonstrated. These findings in turn clarified the molecular mechanism of PKB inhibition by AKT inhibitor VIII (a specific allosteric inhibitor) and illustrated at the molecular level its selectivity towards different PKB isoforms. Furthermore, these findings allude to the possible function of the C-terminus in sustaining the inactive conformer of PKB. This study presents essential insights into the quaternary structure of PKB in its inactive conformation. An understanding of PKB structure in relation to its function is critical for elucidating its mode of activation and discovering how to modulate its activity. The molecular mechanism of inhibition of PKB activation by the specific drug AKT inhibitor VIII has critical implications for determining the mechanism of inhibition of other allosteric inhibitors and for opening up opportunities for the design of new generations of modulator drugs. PMID:19166270

Parker, Peter J; Larijani, Banafshé

2009-01-01

377

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

378

Mechanical & Aerospace Engineering  

E-print Network

verification of physics-based prognosis of mechanical damage, such as fatigue. The proposed experimentalMechanical & Aerospace Engineering An experimental methodology is presented for mechanism methodology includes multi-resolution in-situ mechanical testing, advanced imaging analysis, and mechanism

379

Mechanical Engineering Undergraduate  

E-print Network

.................................................................................................................12 2.6 AP PHYSICS C (MECHANICS) CREDIT AND 530.103/104 INTRO TO MECHANICS.....................13 2Mechanical Engineering Department Undergraduate Advising Manual for Bachelor of Science Degrees in Mechanical Engineering and Engineering Mechanics 2012-2013 - Updated July 14, 2013 #12;Johns Hopkins

Ghosh, Somnath

380

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

381

Mechanical Engineering & Thermal Group  

E-print Network

Mechanical Engineering & Thermal Group The Mechanical Engineering (ME) & Thermal Group at LASP has evolved with the complexity of instrument design demands, LASP mechanical engineers develop advanced the expertise you need to solve your mechanical engineering design challenges. Mechanical Analysis The LASP ME

Mojzsis, Stephen J.

382

Zanthoxylum schinifolium leaf ethanol extract inhibits adipocyte differentiation through inactivation of the extracellular signal regulated kinase and phosphoinositide 3-kinase/Akt signaling pathways in 3T3-L1 pre-adipocytes.  

PubMed

Zanthoxylum schinifolium is widely used as a food flavoring in east Asia. Although this plant has also been used in traditional oriental medicine for the treatment of the common cold, toothache, stomach ache, diarrhea and jaundice, its anti?obesity activity remains to be elucidated. The present study investigated the effects of ethanol extract from the leaves of Z. schinifolium (EEZS) on adipocyte differentiation, and its underlying mechanism, in 3T3?L1 pre?adipocytes. The results demonstrated that EEZS effectively suppressed intracellular lipid accumulation at non?toxic concentrations, and was associated with the downregulation of several adipocyte?specific transcription factors, including peroxisome proliferation?activity receptor ? (PPAR?), CCAAT/enhancer binding protein (C/EBP)? and C/EBP?, in a concentration?dependent manner. Furthermore, it was observed that EEZS markedly inactivated the extracellular signal?regulated protein kinase (ERK) and phosphatidylinositide 3?kinase (PI3K)/Akt pathways, which act upstream of PPAR? and C/EBPs in adipogenesis. These results suggested that EEZS inhibited lipid accumulation by downregulating the major transcription factors involved in the pathway of adipogenesis, including PPAR?, C/EBP? and C/EBP?, via regulation of the ERK and PI3K/Akt signaling pathways in 3T3?L1 adipocyte differentiation. This indicated the potential use of EEZS as an anti?obesity agent. PMID:25760758

Choi, Eun-Ok; Park, Cheol; Shin, Soon Shik; Cho, Eun-Ju; Kim, Byung Woo; Hwang, Jin Ah; Hwang, Hye-Jin; Choi, Yung Hyun

2015-07-01

383

MECHANICAL ENGINEERING UNDERGRADUATE MAJOR  

E-print Network

HANDBOOK FOR MECHANICAL ENGINEERING UNDERGRADUATE MAJOR Old Dominion University Department OF CONTENTS MECHANICAL ENGINEERING HANDBOOK............................................................ 14 #12;1 MECHANICAL ENGINEERING HANDBOOK This handbook has been designed to help you earn a Bachelor

384

Mechanical engineering Department Seminar  

E-print Network

Mechanical engineering Department Seminar Robert J. Hannemann The Gordon Institute and the Department of Mechanical Engineering Tufts University Retooling Our Energy Ecosystem: challenges and Chair of the Tufts Department of Mechanical Engineering. His technical and academic interests

385

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

386

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.

387

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

388

Quantum Mechanics + Open Systems  

E-print Network

Quantum Mechanics + Open Systems = Thermodynamics ? Jochen Gemmer T¨ubingen, 09.02.2006 #12., World Scientific) #12;Fundamental Law or Emergent Description? Quantum Mechanics i t = (- 2 2m + V or Emergent Description? Quantum Mechanics i t = (- 2 2m + V ) "Heisenberg Cut" Classical Mechanics: m d2

Steinhoff, Heinz-Jürgen

389

Mechanical Engineering Graduate Student  

E-print Network

Mechanical Engineering Graduate Student Handbook January 2014 Department of Mechanical Engineering University of Wisconsin-Madison #12;Mechanical Engineering Web Page: http://www.engr.wisc.edu/me Graduate & Terrace Chairs) and Samantha Stepp (ME Building) 1 #12;Department of Mechanical Engineering AGUIDE

Wisconsin at Madison, University of

390

Mechanical & Biomedical Engineering  

E-print Network

Mechanical & Biomedical Engineering Department BACHELOR OF SCIENCE IN MECHANICAL ENGINEERING COURSE 105 Mechanical Engineering Graphics 3 CHEM 111L College Chemistry Lab (DLN) 1 ENGL 102 English Experimental Methods Lab (CID) 2 ME, CE, or ENGR 350 Engineering Mechanics of Materials 3 ME 352 Machine Design

Barrash, Warren

391

Mechanical Engineer Company Description  

E-print Network

Mechanical Engineer Company Description Control Solutions Inc. is a small, dynamic, and rapidly. Position Description The Mechanical Engineer is responsible for all aspects associated with the mechanicalE Mechanism, when applicable. · Perform static, dynamic, vibration, thermal, and other engineering analysis

Kostic, Milivoje M.

392

Mechanical Engineering Undergraduate  

E-print Network

Mechanical Engineering Department Undergraduate Advising Manual for Bachelor of Science Degrees in Mechanical Engineering and Engineering Mechanics 2011-2012 - Updated April 15, 2012 #12;Johns Hopkins University ­ Department of Mechanical Engineering 2011-2012 Undergraduate Student Advising Manual Page 2

Ghosh, Somnath

393

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.

394

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.

395

Treponema denticola major outer sheath protein induces actin assembly at free barbed ends by a PIP2-dependent uncapping mechanism in fibroblasts.  

PubMed

The major outer sheath protein (Msp) of Treponema denticola perturbs actin dynamics in fibroblasts by inducing actin reorganization, including subcortical actin filament assembly, leading to defective calcium flux, diminished integrin engagement of collagen, and retarded cell migration. Yet, its mechanisms of action are unknown. We challenged Rat-2 fibroblasts with enriched native Msp. Msp activated the small GTPases Rac1, RhoA and Ras, but not Cdc42, yet only Rac1 localized to areas of actin rearrangement. We used Rac1 dominant negative transfection and chemical inhibition of phosphatidylinositol-3 kinase (PI3K) to show that even though Rac1 activation was PI3K-dependent, neither was required for Msp-induced actin rearrangement. Actin free barbed end formation (FBE) by Msp was also PI3K-independent. Immunoblotting experiments showed that gelsolin and CapZ were released from actin filaments, whereas cofilin remained in an inactive state. Msp induced phosphatidylinositol (4,5)-bisphosphate (PIP2) formation through activation of a phosphoinositide 3-phosphatase and its recruitment to areas of actin assembly at the plasma membrane. Using a PIP2 binding peptide or lipid phosphatase inhibitor, PIP2 was shown to be required for Msp-mediated actin uncapping and FBE formation. Evidently, Msp induces actin assembly in fibroblasts by production and recruitment of PIP2 and release of the capping proteins CapZ and gelsolin from actin barbed ends. PMID:21901132

Visser, Michelle B; Koh, Adeline; Glogauer, Michael; Ellen, Richard P

2011-01-01

396

Integrating molecular mechanisms and clinical evidence in the management of trastuzumab resistant or refractory HER-2? metastatic breast cancer.  

PubMed

Human epidermal growth factor receptor (HER)-2(+) breast cancer is a distinct molecular and clinical entity, the prognosis of which is improved by trastuzumab. However, primary resistance to trastuzumab is observed in >50% of patients with HER-2(+) advanced breast cancer, and the majority of patients who initially respond to treatment eventually develop disease progression. To facilitate crosstrial comparisons and the understanding of resistance mechanisms, we propose a unifying definition of trastuzumab resistance as progression at first radiological reassessment at 8-12 weeks or within 3 months after first-line trastuzumab in the metastatic setting or new recurrences diagnosed during or within 12 months after adjuvant trastuzumab. In contrast, we define trastuzumab-refractory breast cancer as disease progression after two or more lines of trastuzumab-containing regimens that initially achieved disease response or stabilization at first radiological assessment. We review mechanisms of trastuzumab resistance mediated by p95HER-2 overexpression, phosphoinositide 3-kinase pathway activation, and signaling pathway activation driven by HER-3, epidermal growth factor receptor, and insulin-like growth factor 1 receptor. We distinguish in vitro from in vivo evidence, highlighting that most data describing trastuzumab resistance are derived from preclinical studies or small retrospective patient cohorts, and discuss targeted therapeutic approaches to overcome resistance. Prospective analysis through clinical trials with robust tissue collection procedures, prior to and following acquisition of resistance, integrated with next-generation tumor genome sequencing technologies, is identified as a priority area for development. The identification of predictive biomarkers is of paramount importance to optimize health economic costs and enhance stratification of anti-HER-2 targeted therapies. PMID:22020213

Wong, Hilda; Leung, Roland; Kwong, Ava; Chiu, Joanne; Liang, Raymond; Swanton, Charles; Yau, Thomas

2011-01-01

397

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

398

Internal pipe attachment mechanism  

DOEpatents

An attachment mechanism 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.

Bast, Richard M. (Livermore, CA); Chesnut, Dwayne A. (Pleasanton, CA); Henning, Carl D. (Livermore, CA); Lennon, Joseph P. (Livermore, CA); Pastrnak, John W. (Livermore, CA); Smith, Joseph A. (Livermore, CA)

1994-01-01

399

Central neural mechanisms governing postural cardiovascular mechanisms  

NASA Technical Reports Server (NTRS)

The results of the vestibular apparatus and cerebellum in orthostatic reflex control are summarized. Mechanisms within the brain which govern circulation reflexes and the consequences of disturbances in their function are also included.

Reis, D. J.

1976-01-01

400

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

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