Science.gov

Sample records for phosphoinositide 3-kinase-dependent mechanism

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

    PubMed

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

    2000-07-15

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

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

    SciTech Connect

    Yamaki, Nao; Negishi, Manabu; Katoh, Hironori . E-mail: hirokato@pharm.kyoto-u.ac.jp

    2007-08-01

    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.

  3. Integrin β1-mediated acquired gefitinib resistance in non-small cell lung cancer cells occurs via the phosphoinositide 3-kinase-dependent pathway

    PubMed Central

    DENG, QIN-FANG; SU, BO; ZHAO, YIN-MIN; TANG, LIANG; ZHANG, JIE; ZHOU, CAI-CUN

    2016-01-01

    The present study aimed to explore the role of integrin β1 and the relevant signaling pathways in acquired gefitinib resistance in non-small cell lung cancer (NSCLC). The inhibitory effects of gefitinib, with or without LY294002, on cellular proliferation were evaluated by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide assay. Cell cycle progression and apoptosis were analyzed by flow cytometry, while western blotting was used to evaluate the expression of EGFR, phosphorylated (phospho)-EGFR, protein kinase B (Akt), phospho-Akt, extracellular signal-regulated kinase (Erk) and phospho-Erk. The gene expression profiles of PC9 and PC9/G cells were determined by DNA microarray. Integrin β1 was knocked down in PC9/G cells by transiently transfected short interfering RNA (siRNA). A scrambled siRNA sequence was used as a control. Apoptosis of transfected cells was determined by Annexin V-phycoerythrin-Cy5/propidium iodide staining. Sequencing products were amplified by nested PCR. The resistant index of PC9/G cells to gefitinib was ~138- to 256-fold higher than that of PC9 cells, and this resistance was accompanied by significant increase in integrin β1 expression in PC9/G cells. Knockdown of integrin β1 with short hairpin RNA in PC9/G cells markedly inhibited proliferation and enhanced apoptosis in response to gefitinib, restoring the sensitivity of PC9/G cells gefitinib. Phosphoinositide 3-kinase (PI3K)/Akt activation was observed in PC9/G cells in the presence of gefitinib and the sensitivity of PC9/G cells to gefitinib was also able to be restored by PI3K/Akt pathway inhibitor LY294002. Finally, knockdown of integrin β1 significantly reduced the levels of phospho-Akt. These findings suggest that integrin β1 signaling via the PI3K/Akt pathway may be a significant mechanism underlying gefitinib resistance, and may potentially present an alternative therapeutic target for the treatment of NSCLC unresponsive to EGFR inhibitors. PMID:26870244

  4. Polycystin-1 Induces Cell Migration by Regulating Phosphatidylinositol 3-kinase-dependent Cytoskeletal Rearrangements and GSK3?-dependent CellCell Mechanical Adhesion

    PubMed Central

    Boca, Manila; D'Amato, Lisa; Distefano, Gianfranco; Polishchuk, Roman S.; Germino, Gregory G.

    2007-01-01

    Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1?/? mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and ?-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated ?-catenin through activation of GSK3?. Expression of a nondegradable form of ?-catenin in PC-1 MDCK cells restores strong cellcell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis. PMID:17671167

  5. Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals

    PubMed Central

    Mckenzie, Grahame; Ward, George; Stallwood, Yvette; Briend, Emmanuel; Papadia, Sofia; Lennard, Andrew; Turner, Martin; Champion, Brian; Hardingham, Giles E

    2006-01-01

    Background Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways. Results We show that cellular responsiveness to Notch signals depends on the activity of the PI3K-Akt pathway in cells as diverse as CHO cells, primary T-cells and hippocampal neurons. Induction of the endogenous PI3K-Akt pathway in CHO cells (by the insulin pathway), in T-cells (via TCR activation) or in neurons (via TrKB activation) potentiates Notch-dependent responses. We propose that the PI3K-Akt pathway exerts its influence on Notch primarily via inhibition of GSK3-beta, a kinase known to phosphorylate and regulate Notch signals. Conclusion The PI3K-Akt pathway acts as a "gain control" for Notch signal responses. Since physiological levels of intracellular Notch are often low, coincidence with PI3K-activation may be crucial for induction of Notch-dependent responses. PMID:16507111

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

    PubMed Central

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

    1998-01-01

    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

  7. Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels

    PubMed Central

    Ufret-Vincenty, Carmen A.; Klein, Rebecca M.; Collins, Marcus D.; Rosasco, Mario G.; Martinez, Gilbert Q.

    2015-01-01

    Although PI(4,5)P2 is believed to play an essential role in regulating the activity of numerous ion channels and transporters, the mechanisms by which it does so are unknown. Here, we used the ability of the TRPV1 ion channel to discriminate between PI(4,5)P2 and PI(4)P to localize the region of TRPV1 sequence that interacts directly with the phosphoinositide. We identified a point mutation in the proximal C-terminal region after the TRP box, R721A, that inverted the selectivity of TRPV1. Although the R721A mutation produced only a 30% increase in the EC50 for activation by PI(4,5)P2, it decreased the EC50 for activation by PI(4)P by more than two orders of magnitude. We used chemically induced and voltage-activated phosphatases to determine that PI(4)P continued to support TRPV1 activity even after depletion of PI(4,5)P2 from the plasma membrane. Our data cannot be explained by a purely electrostatic mechanism for interaction between the phosphoinositide and the protein, similar to that of the MARCKS (myristoylated alanine-rich C kinase substrate) effector domain or the EGF receptor. Rather, conversion of a PI(4,5)P2-selective channel to a PI(4)P-selective channel indicates that a structured phosphoinositide-binding site mediates the regulation of TRPV1 activity and that the amino acid at position 721 likely interacts directly with the moiety at the 5′ position of the phosphoinositide. PMID:25918361

  8. TRPM7 ion channels are required for sustained phosphoinositide 3-kinase signaling in lymphocytes.

    PubMed

    Sahni, Jaya; Scharenberg, Andrew M

    2008-07-01

    Lymphocytes lacking the TRPM7 (transient receptor potential cation channel, subfamily M, member 7) dual function ion channel/protein kinase exhibit a unique phenotype: they are unable to proliferate in regular media, but proliferate normally in media supplemented with 10-15 mM extracellular Mg(2+). Here, we have analyzed the molecular mechanisms underlying this phenotype. We find that upon transition from proliferation-supporting Mg(2+)-supplemented media to regular media, TRPM7-deficient cells rapidly downregulate their rate of growth, resulting in a secondary arrest in proliferation. The downregulated growth rate of transitioning cells is associated with a deactivation of signaling downstream from phosphoinositide 3-kinase, and expression of constitutively active p110 phosphoinositide 3-kinase is sufficient to support growth and proliferation of TRPM7-deficient cells in regular media. Together, these observations indicate that TRPM7 channels are required for sustained phosphoinositide 3-kinase-dependent growth signaling and therefore, that TRPM7 is positioned alongside phosphoinositide 3-kinases as a central regulator of lymphocyte growth and proliferation. PMID:18590694

  9. Sac1--Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus

    SciTech Connect

    Cai, Yiying; Deng, Yongqiang; Horenkamp, Florian; Reinisch, Karin M.; Burd, Christopher G.

    2014-08-25

    Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot–Marie–Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1–Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.

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

    PubMed

    Métais, C; Hughes, B; Herron, C E

    2015-04-16

    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

  11. Phosphoinositide phosphatases in cell biology and disease.

    PubMed

    Liu, Yang; Bankaitis, Vytas A

    2010-07-01

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

  12. 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...

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

    PubMed Central

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

    2014-01-01

    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

  14. Angiopoietin-like protein 2 renders colorectal cancer cells resistant to chemotherapy by activating spleen tyrosine kinase–phosphoinositide 3-kinase-dependent anti-apoptotic signaling

    PubMed Central

    Horiguchi, Haruki; Endo, Motoyoshi; Miyamoto, Yuji; Sakamoto, Yasuo; Odagiri, Haruki; Masuda, Tetsuro; Kadomatsu, Tsuyoshi; Tanoue, Hironori; Motokawa, Ikuyo; Terada, Kazutoyo; Morioka, Masaki Suimye; Manabe, Ichiro; Baba, Hideo; Oike, Yuichi

    2014-01-01

    Angiopoietin-like protein 2 (ANGPTL2) plays an important role in inflammatory carcinogenesis and tumor metastasis by activating tumor angiogenesis and tumor cell chemotaxis and invasiveness. However, it is unclear whether ANGPTL2 expression has an effect on tumor cell survival. Here, we explored that possibility by determining whether ANGPTL2 expression altered survival of human colorectal cancer cell lines treated with antineoplastic drugs. To do so, we generated SW480 cells expressing ANGPTL2 (SW480/ANGPTL2) and control (SW480/Ctrl) cells. Apoptosis induced by antineoplastic drug treatment was significantly decreased in SW480/ANGPTL2 compared to control cells. Expression of anti-apoptotic BCL-2 family genes was upregulated in SW480/ANGPTL2 compared to SW480/Ctrl cells. To assess signaling downstream of ANGPTL2 underlying this effect, we carried out RNA sequencing analysis of SW480/ANGPTL2 and SW480/Ctrl cells. That analysis, combined with in vitro experiments, indicated that Syk-PI3K signaling induced expression of BCL-2 family genes in SW480/ANGPTL2 cells. Furthermore, ANGPTL2 increased its own expression in a feedback loop by activating the spleen tyrosine kinase–nuclear factor of activated T cells (Syk–NFAT) pathway. Finally, we observed a correlation between higher ANGPTL2 expression in primary unresectable tumors from colorectal cancer patients who underwent chemotherapy with a lower objective response rate. These findings suggest that attenuating ANGPTL2 signaling in tumor cells may block tumor cell resistance to antineoplastic therapies. PMID:25287946

  15. What a Tangled Web We Weave: Emerging Resistance Mechanisms to Inhibition of the Phosphoinositide 3-kinase Pathway

    PubMed Central

    Klempner, Samuel J.; Myers, Andrea P.; Cantley, Lewis C.

    2013-01-01

    The phosphoinositide 3-kinase (PI3K) pathway is one of the most frequently mutated pathways in cancer, and is actively being pursued as a therapeutic target. Despite the importance of the PI3K pathway in cancer, durable responses to PI3K-pathway targeted therapies are uncommon with monotherapy. Several in vitro and xenograft models have elucidated compensatory signaling and genomic changes which may limit the therapeutic effectiveness of PI3K inhibitors in the clinic. Future clinical trials with prospective evaluation of tumor signaling and genomic changes are likely to identify novel resistance mechanisms as well as subsets of patients who may derive maximal benefit from PI3K pathway inhibitors. PMID:24265156

  16. Raft-dependent endocytosis of autocrine motility factor is phosphatidylinositol 3-kinase-dependent in breast carcinoma cells.

    PubMed

    Kojic, Liliana D; Joshi, Bharat; Lajoie, Patrick; Le, Phuong U; Cox, Michael E; Turbin, Dmitry A; Wiseman, Sam M; Nabi, Ivan R

    2007-10-01

    Autocrine motility factor (AMF) is internalized via a receptor-mediated, dynamin-dependent, cholesterol-sensitive raft pathway to the smooth endoplasmic reticulum that is negatively regulated by caveolin-1. Expression of AMF and its receptor (AMFR) is associated with tumor progression and malignancy; however, the extent to which the raft-dependent uptake of AMF is tumor cell-specific has yet to be addressed. By Western blot and cell surface fluorescence-activated cell sorter (FACS) analysis, AMFR expression is increased in tumorigenic MCF7 and metastatic MDA-231 and MDA-435 breast cancer cell lines relative to dysplastic MCF10A mammary epithelial cells. AMF uptake, determined by FACS measurement of protease-insensitive internalized fluorescein-conjugated AMF, was increased in MCF7 and MDA-435 cells relative to MCF-10A and caveolin-1-expressing MDA-231 cells. Uptake of fluorescein-conjugated AMF was dynamin-dependent, methyl-beta-cyclodextrin- and genistein-sensitive, reduced upon overexpression of caveolin-1 in MDA-435 cells, and increased upon short hairpin RNA reduction of caveolin-1 in MDA-231 cells. Tissue microarray analysis of invasive primary human breast carcinomas showed that AMFR expression had no impact on survival but did correlate significantly with expression of phospho-Akt. Phospho-Akt expression was increased in AMF-internalizing MCF7 and MDA-435 breast carcinoma cells. AMF uptake in these cells was reduced by phosphatidylinositol 3-kinase inhibition but not by regulators of macropinocytosis such as amiloride, phorbol ester, or actin cytoskeleton disruption by cytochalasin D. The raft-dependent endocytosis of AMF therefore follows a distinct phosphatidylinositol 3-kinase-dependent pathway that is up-regulated in more aggressive tumor cells. PMID:17690101

  17. Phosphoinositide dynamics in the postsynaptic membrane compartment: Mechanisms and experimental approach.

    PubMed

    Leitner, Michael G; Halaszovich, Christian R; Ivanova, Olga; Oliver, Dominik

    2015-01-01

    Phosphoinositides (PIs) are minor constituents of eukaryotic membranes that control a plethora of cellular functions through direct modulation of membrane-associated proteins and through membrane recruitment of enzymes or signaling molecules. It is well established that in neurons PIs play essential roles in the pre-synapse, especially during exocytotic neurotransmitter release and recycling of synaptic vesicles. In contrast, the physiological importance of PIs in postsynaptic membranes is far less understood. The extent and the spatiotemporal characteristics of dynamic changes in the concentrations of PIs caused by synaptic activity are largely unknown. Recent work suggests that postsynaptic PI dynamics are involved in the induction and maintenance of synaptic plasticity, but the general principles are far from clear. This review summarizes current knowledge on the relevance of PIs for postsynaptic processes, focussing on PI signaling in the control of electrical activity and synaptic plasticity. We highlight the state-of-the-art of methods to study PI dynamics and discuss recent technical improvements that should help to define the role of PIs in postsynaptic physiology. PMID:26092197

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

    PubMed Central

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

    2012-01-01

    The phosphoinositide 3-kinase (PI3K) pathway is a central signal transduction axis controlling normal B cell homeostasis and activation in humoral immunity. The p110δ 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

  19. Phosphoinositide-dependent kinase 1 targets protein kinase A in a pathway that regulates interleukin 4.

    PubMed

    Nirula, Ajay; Ho, Mary; Phee, Hyewon; Roose, Jeroen; Weiss, Arthur

    2006-07-10

    CD28 plays a critical role in T cell immune responses. Although the kinase Akt has been shown to act downstream of CD28 in T helper (Th)1 cytokine induction, it does not induce Th2 cytokines such as interleukin 4 (IL-4). We recently reported that phosphoinositide-dependent kinase 1 (PDK1) partially corrects the defect in IL-4 production present in CD28-deficient T cells, suggesting that PDK1 regulates IL-4 independently of Akt. We now describe a signaling pathway in which PDK1 targets IL-4 in the murine Th2 cell line D10. PDK1-mediated activation of this pathway is dependent on protein kinase A (PKA) and the nuclear factor of activated T cells (NFAT) P1 transcriptional element in the IL-4 promoter. PDK1 localizes to the immune synapse in a phosphatidylinositol 3-kinase-dependent manner, partially colocalizes with PKA at the synapse, and physically interacts with PKA. In RNA interference knockdown experiments, PDK1 is necessary for phosphorylation of PKA in T cells, as well as for activation of the IL-4 NFAT P1 element by the T cell receptor (TCR) and CD28. Phosphorylation of the critical PKA threonine residue is stimulated by engagement of TCR/CD28 via a PDK1-dependent mechanism. These findings together define a pathway linking the kinases PDK1 and PKA in the induction of the Th2 cytokine IL-4. PMID:16785309

  20. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1.

    PubMed

    Le Good, J A; Ziegler, W H; Parekh, D B; Alessi, D R; Cohen, P; Parker, P J

    1998-09-25

    Phosphorylation sites in members of the protein kinase A (PKA), PKG, and PKC kinase subfamily are conserved. Thus, the PKB kinase PDK1 may be responsible for the phosphorylation of PKC isotypes. PDK1 phosphorylated the activation loop sites of PKCzeta and PKCdelta in vitro and in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner in vivo in human embryonic kidney (293) cells. All members of the PKC family tested formed complexes with PDK1. PDK1-dependent phosphorylation of PKCdelta in vitro was stimulated by combined PKC and PDK1 activators. The activation loop phosphorylation of PKCdelta in response to serum stimulation of cells was PI 3-kinase-dependent and was enhanced by PDK1 coexpression. PMID:9748166

  1. Calcium receptor-induced serotonin secretion by parafollicular cells: role of phosphatidylinositol 3-kinase-dependent signal transduction pathways.

    PubMed

    Liu, Kuo-peing; Russo, Andrew F; Hsiung, Shu-chi; Adlersberg, Mella; Franke, Thomas F; Gershon, Michael D; Tamir, Hadassah

    2003-03-15

    Elevation of extracellular Ca2+ (increase[Ca2+]e) stimulates the Ca2+ receptor (CaR) to induce secretion of 5-hydroxytryptamine (5-HT) from the calcium-sensing parafollicular (PF) cells. The CaR has been reported to couple to Galpha(q) with subsequent activation of protein kinase C-gamma (PKCgamma). We have identified a parallel transduction pathway in primary cultures of sheep PF cells by using a combinatorial approach in which we expressed adenoviral-encoded dominant-negative signaling proteins and performed in vitro kinase assays. The role of the CaR was established by expression of a dominant-negative CaR that eliminated calcium-induced 5-HT secretion but not secretion in response to KCl or phorbol esters. The calcium-induced secretion was inhibited by a dominant-negative p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K). PI3-K activity was also assayed using isoform-specific antibodies. The activity of p85/p110beta (PI3-Kbeta) immunocomplexes was elevated by increase[Ca2+]e and activated by Gbetagamma subunits. In addition, secretion of 5-HT was antagonized by the expression of a minigene encoding a peptide scavenger of Gbetagamma subunits (C-terminal fragment peptide of bovine beta-adrenergic receptor kinase). One target of PI3-K activity is phosphoinositide-dependent kinase-1 (PDK1), which in turn activated PKCzeta. Expression of a dominant-negative PKCzeta in PF cells reduced 5-HT secretion. Together, these observations establish that increase[Ca2+]e evokes 5-HT secretion from PF cells by stimulating both Galpha(q)- and Gbetagamma-signaling pathways downstream of the CaR. The betagamma cascade subsequently activates PI3-Kbeta-dependent signaling that is coupled to PDK1 and the downstream effector PKCzeta, and results in an increase in 5-HT release. PMID:12657663

  2. Targeting the phosphoinositide 3-kinase pathway in hematologic malignancies

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    2013-01-01

    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

  4. Channelopathies linked to plasma membrane phosphoinositides

    PubMed Central

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

    2014-01-01

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

  5. Role of phosphoinositides at the neuronal synapse

    PubMed Central

    Frere, Samuel G.; Chang-Ileto, Belle; Di Paolo, Gilbert

    2013-01-01

    Synaptic transmission is amongst the most sophisticated and tightly controlled biological phenomena in higher eukaryotes. In the past few decades, tremendous progress has been made in our understanding of the molecular mechanisms underlying multiple facets of neurotransmission, both pre- and postsynaptically. Brought under the spotlight by pioneer studies in the areas of secretion and signal transduction, phosphoinositides and their metabolizing enzymes have been increasingly recognized as key protagonists in fundamental aspects of neurotransmission. Not surprisingly, dysregulation of phosphoinositide metabolism has also been implicated in synaptic malfunction associated with a variety of brain disorders. In the present chapter, we summarize current knowledge on the role of phosphoinositides at the neuronal synapse and highlight some of the outstanding questions in this research field. PMID:22374090

  6. The emerging role of phosphoinositide clustering in intracellular trafficking and signal transduction

    PubMed Central

    Picas, Laura; Gaits-Iacovoni, Frederique; Goud, Bruno

    2016-01-01

    Phosphoinositides are master regulators of multiple cellular processes: from vesicular trafficking to signaling, cytoskeleton dynamics, and cell growth. They are synthesized by the spatiotemporal regulated activity of phosphoinositide-metabolizing enzymes. The recent observation that some protein modules are able to cluster phosphoinositides suggests that alternative or complementary mechanisms might operate to stabilize the different phosphoinositide pools within cellular compartments. Herein, we discuss the different known and potential molecular players that are prone to engage phosphoinositide clustering and elaborate on how such a mechanism might take part in the regulation of intracellular trafficking and signal transduction. PMID:27092250

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

    PubMed Central

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

    2011-01-01

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

  8. Phosphoinositide regulation of non-canonical Transient Receptor Potential channels

    PubMed Central

    Rohacs, Tibor

    2009-01-01

    Transient Receptor Potential (TRP) channels are involved in a wide range of physiological processes, and characterized by diverse activation mechanisms. Phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PIP2, or PtdIns(4,5)P2] recently emerged as regulators of many TRP channels. Several TRP channels require PIP2 for activity, and depletion of the lipid inhibits them. For some TRP channels, however, phosphoinositide regulation seems more complex, both activating and inhibitory effects have been reported. This review will discuss phosphoinositide regulation of members of the TRPM (Melastatin), TRPV (Vanilloid), TRPA (Ankyrin) and TRPP (Polycystin) families. Lipid regulation of TRPC (Canonical) channels is discussed elsewhere in this volume. PMID:19376575

  9. Rho GTPases, phosphoinositides, and actin

    PubMed Central

    Croisé, Pauline; Estay-Ahumada, Catherine; Gasman, Stéphane; Ory, Stéphane

    2014-01-01

    Rho GTPases are well known regulators of the actin cytoskeleton that act by binding and activating actin nucleators. They are therefore involved in many actin-based processes, including cell migration, cell polarity, and membrane trafficking. With the identification of phosphoinositide kinases and phosphatases as potential binding partners or effectors, Rho GTPases also appear to participate in the regulation of phosphoinositide metabolism. Since both actin dynamics and phosphoinositide turnover affect the efficiency and the fidelity of vesicle transport between cell compartments, Rho GTPases have emerged as critical players in membrane trafficking. Rho GTPase activity, actin remodeling, and phosphoinositide metabolism need to be coordinated in both space and time to ensure the progression of vesicles along membrane trafficking pathways. Although most molecular pathways are still unclear, in this review, we will highlight recent advances made in our understanding of how Rho-dependent signaling pathways organize actin dynamics and phosphoinositides and how phosphoinositides potentially provide negative feedback to Rho GTPases during endocytosis, exocytosis and membrane exchange between intracellular compartments. PMID:24914539

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

    PubMed Central

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

    2003-01-01

    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

  11. Phosphoinositides and host-pathogen interactions.

    PubMed

    Pizarro-Cerdá, Javier; Kühbacher, Andreas; Cossart, Pascale

    2015-06-01

    Phosphoinositides control key cellular processes including vesicular trafficking and actin polymerization. Intracellular bacterial pathogens manipulate phosphoinositide metabolism in order to promote their uptake by target cells and to direct in some cases the biogenesis of their replication compartments. In this chapter, we review the molecular strategies that major pathogens including Listeria, Mycobacterium, Shigella, Salmonella, Legionella and Yersinia use to hijack phosphoinositides during infection. This article is part of a Special Issue entitled Phosphoinositides. PMID:25241942

  12. Phosphoinositides in membrane contact sites.

    PubMed

    Raiborg, Camilla; Wenzel, Eva M; Pedersen, Nina M; Stenmark, Harald

    2016-04-15

    Cellular membranes communicate extensively via contact sites that form between two membranes. Such sites allow exchange of specific ions, lipids or proteins between two compartments without content mixing, thereby preserving organellar architecture during the transfer process. Even though the molecular compositions of membrane contact sites are diverse, it is striking that several of these sites, including contact sites between the endoplasmic reticulum (ER) and endosomes, Golgi and the plasma membrane (PM), and contact sites between lysosomes and peroxisomes, contain phosphorylated derivatives of phosphatidylinositol known as phosphoinositides. In this mini-review we discuss the involvement and functions of phosphoinositides in membrane contact sites. PMID:27068950

  13. Phosphoinositide signaling in somatosensory neurons.

    PubMed

    Rohacs, Tibor

    2016-05-01

    Somatosensory neurons of the dorsal root ganglia (DRG) and trigeminal ganglia (TG) are responsible for detecting thermal and tactile stimuli. They are also the primary neurons mediating pain and itch. A large number of cell surface receptors in these neurons couple to phospholipase C (PLC) enzymes leading to the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and the generation of downstream signaling molecules. These neurons also express many different ion channels, several of which are regulated by phosphoinositides. This review will summarize the knowledge on phosphoinositide signaling in DRG neurons, with special focus on effects on sensory and other ion channels. PMID:26724974

  14. IL-21 Promotes CD4 T Cell Responses by Phosphatidylinositol 3-KinaseDependent Upregulation of CD86 on B Cells

    PubMed Central

    Attridge, Kesley; Kenefeck, Rupert; Wardzinski, Lukasz; Qureshi, Omar S.; Wang, Chun Jing; Manzotti, Claire; Okkenhaug, Klaus

    2014-01-01

    The cytokine IL-21 is a potent immune modulator with diverse mechanisms of action on multiple cell types. IL-21 is in clinical use to promote tumor rejection and is an emerging target for neutralization in the setting of autoimmunity. Despite its clinical potential, the biological actions of IL-21 are not yet fully understood and the full range of effects of this pleiotropic cytokine are still being uncovered. In this study, we identify a novel role for IL-21 as an inducer of the costimulatory ligand CD86 on B lymphocytes. CD86 provides critical signals through T cellexpressed CD28 that promote T cell activation in response to Ag engagement. Expression levels of CD86 are tightly regulated in vivo, being actively decreased by regulatory T cells and increased in response to pathogen-derived signals. In this study, we demonstrate that IL-21 can trigger potent and sustained CD86 upregulation through a STAT3 and PI3K-dependent mechanism. We show that elevated CD86 expression has functional consequences for the magnitude of CD4 T cell responses both in vitro and in vivo. These data pinpoint CD86 upregulation as an additional mechanism by which IL-21 can elicit immunomodulatory effects. PMID:24470500

  15. Insulin promotes Rip11 accumulation at the plasma membrane by inhibiting a dynamin- and PI3-kinase-dependent, but Akt-independent, internalisation event.

    PubMed

    Boal, Frdric; Hodgson, Lorna R; Reed, Sam E; Yarwood, Sophie E; Just, Victoria J; Stephens, David J; McCaffrey, Mary W; Tavar, Jeremy M

    2016-01-01

    Rip11 is a Rab11 effector protein that has been shown to be important in controlling the trafficking of several intracellular cargoes, including the fatty acid transporter FAT/CD36, V-ATPase and the glucose transporter GLUT4. We have previously demonstrated that Rip11 translocates to the plasma membrane in response to insulin and here we examine the basis of this regulated phenomenon in more detail. We show that Rip11 rapidly recycles between the cell interior and surface, and that the ability of insulin to increase the appearance of Rip11 at the cell surface involves an inhibition of Rip11 internalisation from the plasma membrane. By contrast the hormone has no effect on the rate of Rip11 translocation towards the plasma membrane. The ability of insulin to inhibit Rip11 internalisation requires dynamin and class I PI3-kinases, but is independent of the activation of the protein kinase Akt; characteristics which are very similar to the mechanism by which insulin inhibits GLUT4 endocytosis. PMID:26515129

  16. Insulin promotes Rip11 accumulation at the plasma membrane by inhibiting a dynamin- and PI3-kinase-dependent, but Akt-independent, internalisation event

    PubMed Central

    Boal, Frédéric; Hodgson, Lorna R.; Reed, Sam E.; Yarwood, Sophie E.; Just, Victoria J.; Stephens, David J.; McCaffrey, Mary W.; Tavaré, Jeremy M.

    2016-01-01

    Rip11 is a Rab11 effector protein that has been shown to be important in controlling the trafficking of several intracellular cargoes, including the fatty acid transporter FAT/CD36, V-ATPase and the glucose transporter GLUT4. We have previously demonstrated that Rip11 translocates to the plasma membrane in response to insulin and here we examine the basis of this regulated phenomenon in more detail. We show that Rip11 rapidly recycles between the cell interior and surface, and that the ability of insulin to increase the appearance of Rip11 at the cell surface involves an inhibition of Rip11 internalisation from the plasma membrane. By contrast the hormone has no effect on the rate of Rip11 translocation towards the plasma membrane. The ability of insulin to inhibit Rip11 internalisation requires dynamin and class I PI3-kinases, but is independent of the activation of the protein kinase Akt; characteristics which are very similar to the mechanism by which insulin inhibits GLUT4 endocytosis. PMID:26515129

  17. The anti-apoptotic effect of IGF-1 on tissue resident stem cells is mediated via PI3-kinase dependent secreted frizzled related protein 2 (Sfrp2) release

    SciTech Connect

    Gehmert, Sebastian; Sadat, Sanga; Song Yaohua; Yan Yasheng; Alt, Eckhard

    2008-07-11

    Previous studies suggest that IGF-1 may be used as an adjuvant to stem cell transfer in order to improve cell engraftment in ischemic tissue. In the current study, we investigated the effect of IGF-1 on serum deprivation and hypoxia induced stem cell apoptosis and the possible mechanisms involved. Exposure of adipose tissue derived stem cells (ASCs) to serum deprivation and hypoxia resulted in significant apoptosis in ASC which is partially prevented by IGF-1. IGF-1's anti-apoptotic effect was abolished in ASCs transfected with Sfrp2 siRNA but not by the control siRNA. Using Western blot analysis, we demonstrated that serum deprivation and hypoxia reduced the expression of nuclear {beta}-catenin, which is reversed by IGF-1. IGF-1's effect on {beta}-catenin expression was abolished by the presence of PI3-kinase inhibitor LY294002 or in ASCs transfected with Sfrp2 siRNA. These results suggest that IGF-1, through the release of the Sfrp2, contributes to cell survival by stabilizing {beta}-catenin.

  18. PHOSPHOINOSITIDE SIGNALING: NEW TOOLS AND INSIGHTS

    PubMed Central

    Balla, Tamas; Szentpetery, Zsofia; Kim, Yeun Ju

    2011-01-01

    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. PMID:19675354

  19. Biochemistry and structure of phosphoinositide phosphatases

    PubMed Central

    Kim, Young Jun; Jahan, Nusrat; Bahk, Young Yil

    2013-01-01

    Phosphoinositides are the phosphorylated derivatives of phosphatidylinositol, and play a very significant role in a diverse range of signaling processes in eukaryotic cells. A number of phosphoinositide-metabolizing enzymes, including phosphoinositide-kinases and phosphatases are involved in the synthesis and degradation of these phospholipids. Recently, the function of various phosphatases in the phosphatidylinositol signaling pathway has been of great interest. In the present review we summarize the structural insights and biochemistry of various phosphatases in regulating phosphoinositide metabolism. [BMB Reports 2013; 46(1): 1-8] PMID:23351377

  20. Mechanism of hyperhomocysteinemia-induced vascular endothelium dysfunction - possible dysregulation of phosphatidylinositol-3-kinase and its downstream phosphoinositide dependent kinase and protein kinase B.

    PubMed

    Sharma, Saurabh; Singh, Manjeet; Sharma, Pyare Lal

    2013-12-01

    Imbalance of l-arginine/endothelial nitric oxide synthatase (eNOS) activity is the hallmark of vascular endothelium dysfunction. Hyperhomocysteinemia (Hhy) has been identified as a potential risk factor for vascular endothelium dysfunction that leads to cardiovascular disorders. Phosphatidylinositol-3 kinase (PI3K) is a ubiquitous enzyme involved in plethora of cell signaling including the endothelial cells and it has been reported that signaling through this enzyme and its downstream pathway viz phosphoinositide-dependent kinase (PDK)/protein kinase B (Akt) and eNOS is impaired in diseased conditions. Thus present study was designed to investigate the role of PI3K and PDK/Akt in vascular endothelium dysfunction produced by Hhy. Hhy was produced by administering l-methionine (1.7%w/w, p.o). After four weeks of l-methionine administration, vascular endothelium dysfunction was assessed in terms of attenuation of acetylcholine-induced endothelium dependent relaxation (Isolated aortic ring preparation), a decrease in serum nitrite level, mRNA expression of eNOS (rtPCR) and disruption of integrity of vascular endothelium (Electron microscopy). Administration of insulin (0.6 IU/kg/day, s.c), YS-49 (1.6 mg/kg/day, i.p), DAQB1 (5mg/kg/day, i.p) and atorvastatin (30 mg/kg/day, p.o) significantly improved acetylcholine-induced endothelium-dependent relaxation, serum nitrate/nitrite level, mRNA expression of eNOS and integrity of vascular endothelium. This ameliorative effect of insulin was blocked by wortmannin (inhibitor of PI3K), UCN-01(PDK inhibitor), API-2 (Akt inhibitor) and l-NAME (eNOS inhibitor). Thus, it may be concluded that activation of PI3K and its downstream pathways viz. PDK/Akt and eNOS improve Hhy-induced vascular endothelium dysfunction and that therapeutic interventions designed for these pathways may provide potential therapeutic strategies to combat vascular complications. PMID:24021535

  1. Phosphoinositide 3-kinase and Akt are essential for Sonic Hedgehog signaling.

    PubMed

    Riob, Natalia A; Lu, Ke; Ai, Xingbin; Haines, Gwendolyn M; Emerson, Charles P

    2006-03-21

    Hedgehogs (Hhs) are key signaling regulators of stem cell maintenance and tissue patterning in embryos, and activating mutations in the pathway that increase Gli transcriptional activity are causal in a diversity of cancers. Here, we report that phosphoinositide 3-kinase (PI3-kinase)-dependent Akt activation is essential for Sonic Hedgehog (Shh) signaling in the specification of neuronal fates in chicken neural explants, chondrogenic differentiation of 10T1/2 cells, and Gli activation in NIH 3T3 cells. Stimulation of PI3-kinase/Akt by insulin-like growth factor I potentiates Gli activation induced by low levels of Shh; however, insulin-like growth factor I alone is insufficient to induce Gli-dependent transcription. Protein kinase A (PKA) and glycogen synthase kinase 3beta sequentially phosphorylate Gli2 at multiple sites, identified by mutagenesis, thus resulting in a reduction of its transcriptional activity. Gli2 mutant proteins in which the major PKA and glycogen synthase kinase 3beta phosphorylation sites were mutated to alanine remain fully transcriptionally active; however, PKA-mutant Gli2 functions independently of Akt signaling, indicating that Akt positively regulates Shh signaling by controlling PKA-mediated Gli inactivation. Our findings provide a basis for the synergistic role of PI3-kinase/Akt in Hh signaling in embryonic development and Hh-dependent tumors. PMID:16537363

  2. Detection and manipulation of phosphoinositides.

    PubMed

    Idevall-Hagren, Olof; De Camilli, Pietro

    2015-06-01

    Phosphoinositides (PIs) are minor components of cell membranes, but play key roles in cell function. Recent refinements in techniques for their detection, together with imaging methods to study their distribution and changes, have greatly facilitated the study of these lipids. Such methods have been complemented by the parallel development of techniques for the acute manipulation of their levels, which in turn allow bypassing the long-term adaptive changes implicit in genetic perturbations. Collectively, these advancements have helped elucidate the role of PIs in physiology and the impact of the dysfunction of their metabolism in disease. Combining methods for detection and manipulation enables the identification of specific roles played by each of the PIs and may eventually lead to the complete deconstruction of the PI signaling network. Here, we review current techniques used for the study and manipulation of cellular PIs and also discuss advantages and disadvantages associated with the various methods. This article is part of a Special Issue entitled Phosphoinositides. PMID:25514766

  3. The Phox homology (PX) domain, a new player in phosphoinositide signalling.

    PubMed Central

    Xu, Y; Seet, L F; Hanson, B; Hong, W

    2001-01-01

    Phosphoinositides are key regulators of diverse cellular processes. The pleckstrin homology (PH) domain mediates the action of PtdIns(3,4)P(2), PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), while the FYVE domain relays the pulse of PtdIns3P. The recent establishment that the Phox homology (PX) domain interacts with PtdIns3P and other phosphoinositides suggests another mechanism by which phosphoinositides can regulate/integrate multiple cellular events via a spectrum of PX domain-containing proteins. Together with the recent discovery that the epsin N-terminal homologue (ENTH) domain interacts with PtdIns(4,5)P(2), it is becoming clear that phosphoinositides regulate diverse cellular events through interactions with several distinct structural motifs present in many different proteins. PMID:11736640

  4. Modulation of agonist-induced phosphoinositide metabolism, Ca2+ signalling and contraction of airway smooth muscle by cyclic AMP-dependent mechanisms.

    PubMed Central

    Hoiting, B. H.; Meurs, H.; Schuiling, M.; Kuipers, R.; Elzinga, C. R.; Zaagsma, J.

    1996-01-01

    1. The effects of increased cellular cyclic AMP levels induced by isoprenaline, forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cyclic AMP) on phosphoinositide metabolism and changes in intracellular Ca2+ elicited by methacholine and histamine were examined in bovine isolated tracheal smooth muscle (BTSM) cells. 2. Isoprenaline (pD2 (-log10 EC50) = 6.32 +/- 0.24) and forskolin (pD2 = 5.6 +/- 0.05) enhanced cyclic AMP levels in a concentration-dependent fashion in these cells, while methacholine (pD2 = 5.64 +/- 0.12) and histamine (pD2 = 4.90 +/- 0.04) caused a concentration-related increase in [3H]-inositol phosphates (IP) accumulation in the presence of 10 mM LiCl. 3. Preincubation of the cells (5 min, 37 degrees C) with isoprenaline (1 microM), forskolin (10 microM) and 8-Br-cyclic AMP (1 mM) did not affect the IP accumulation induced by methacholine, but significantly reduced the maximal IP production by histamine (1 mM). However, the effect of isoprenaline was small (15.0 +/- 0.6% inhibition) and insignificant at histamine concentrations between 0.1 and 100 microM. 4. Both methacholine and histamine induced a fast (max. in 0.5-2 s) and transient increase of intracellular Ca2+ concentration ([Ca2+]i) followed by a sustained phase lasting several minutes. EGTA (5 mM) attenuated the sustained phase, indicating that this phase depends on extracellular Ca2+. 5. Preincubation of the cells (5 min, 37 degrees C) with isoprenaline (1 microM), forskolin (10 microM) and 8-Br-cyclic AMP (1 microM) significantly attenuated both the Ca(2+)-transient and the sustained phase generated at equipotent IP producing concentrations of 1 microM methacholine and 100 microM histamine (approx. 40% of maximal methacholine-induced IP response), but did not affect changes in [Ca2+]i induced by 100 microM methacholine (95.2 +/- 3.5% of maximal methacholine-induced IP response). 6. Significant correlations were found between the isoprenaline-induced inhibition of BTSM contraction and inhibition of Ca2+ mobilization or influx induced by methacholine and histamine, that were similar for each contractile agonist. 7. These data indicate that (a) cyclic AMP-dependent inhibition of Ca2+ mobilization in BTSM cells is not primarily caused by attenuation of IP production, suggesting that cyclic AMP induced protein kinase A (PKA) activation is effective at a different level in the [Ca2+]i homeostasis, (b) that attenuation of intracellular Ca2+ concentration plays a major role in beta-adrenoceptor-mediated relaxation of methacholine- and histamine-induced airway smooth muscle contraction, and (c) that the relative resistance of the muscarinic agonist-induced contraction to beta-adrenoceptor agonists, especially at (supra) maximal contractile concentrations is largely determined by its higher potency in inducing intracellular Ca2+ changes. PMID:8821529

  5. Mass spectrometry based cellular phosphoinositides profiling and phospholipid analysis: A brief review

    PubMed Central

    Kim, Youngjun; Shanta, Selina Rahman; Zhou, Li-Hua

    2010-01-01

    Phospholipids are key components of cellular membrane and signaling. Among cellular phospholipids, phosphoinositides, phosphorylated derivatives of phosphatidylinositol are important as a participant in essential metabolic processes in animals. However, due to its low abundance in cells and tissues, it is difficult to identify the composition of phosphoinositides. Recent advances in mass spectrometric techniques, combined with established separation methods, have allowed the rapid and sensitive detection and quantification of a variety of lipid species including phosphoinositides. In this mini review, we briefly introduce progress in profiling of cellular phosphoinositides using mass spectrometry. We also summarize current progress of matrices development for the analysis of cellular phospholipids using matrix-assisted laser desorption/ionization mass spectrometry. The phosphoinositides profiling and phospholipids imaging will help us to understand how they function in a biological system and will provide a powerful tool for elucidating the mechanism of diseases such as diabetes, cancer and neurodegenerative diseases. The investigation of cellular phospholipids including phosphoinositides using electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry will suggest new insights on human diseases, and on clinical application through drug development of lipid related diseases. PMID:19887898

  6. Regulation of Golgi function via phosphoinositide lipids

    PubMed Central

    Mayinger, Peter

    2009-01-01

    Phosphoinositides play important roles in Golgi traffic and structural integrity. Specific lipid kinases and phosphatases associate with the Golgi complex and regulate the multiplicity of trafficking routes from this organelle. Work in different model systems showed that the basic elements that regulate lipid signaling at the Golgi are conserved form yeast to humans. Many of the enzymes involved in Golgi phosphoinositide metabolism are essential for viability or cause severe human disease when malfunctioning. Phosphoinositide effectors at the Golgi control both non-vesicular transfer of lipids and sorting of secretory and membrane proteins. In addition, Golgi phosphoinositides were recently implicated in the metabolic and cell growth-dependent regulation of the secretory pathway. PMID:19508852

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

    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.

  8. Serotonin-stimulated phosphoinositide turnover: mediation by the S2 binding site in rat cerebral cortex but not in subcortical regions

    SciTech Connect

    Conn, P.J.; Sanders-Bush, E.

    1985-07-01

    In rat cerebral cortex, serotonin (5-HT) stimulates phosphoinositide turnover with an EC50 of 1 microM in the presence of pargyline. The EC50 is 16-fold higher in the absence of pargyline. Selective S2 antagonists inhibit 5-HT-stimulated phosphoinositide turnover. Schild analysis of the blockade by ketanserin of the 5-HT effect gives an estimated Kd of ketanserin for the phosphoinositide-linked receptor of 11.7 nM, which agrees with the Kd (3.5 nM) of (/sup 3/H)ketanserin for the S2 site. Furthermore, MK-212, 5-HT and 5-fluorotryptamine stimulate phosphoinositide turnover with potencies that resemble their potencies at the S2 but not the S1 binding site. Of 11 agonists tested, the tryptamine derivatives tend to be more efficacious than the piperazine derivatives. The selective S1 agonist 8-hydroxy-2-(di-N-propylamino)tetralin is inactive at stimulating phosphoinositide turnover. No significant relationship exists between the regional distributions of 5-HT-stimulated phosphoinositide turnover and S2 binding sites. Furthermore, the S2 antagonist ketanserin is less potent and less efficacious in hippocampus and limbic forebrain than in cerebral cortex. These data suggest that 5-HT-stimulated phosphoinositide turnover is linked to the S2 binding site in rat cerebral cortex. However, 5-HT increases phosphoinositide turnover in subcortical regions by mechanisms other than stimulation of the S2 receptor.

  9. Ion Induced Changes in Phosphoinositide Monolayers at Phisiological Concentrations

    NASA Astrophysics Data System (ADS)

    Kazadi Badiambile, Adolphe; Forstner, Martin

    2013-03-01

    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. NSF

  10. Maitotoxin: Effects on calcium channels, phosphoinositide breakdown, and arachidonate release in pheochromocytoma PC12 cells

    SciTech Connect

    Choi, O.H.; Padgett, W.L.; Nishizawa, Y.; Gusovsky, F.; Yasumoto, T.; Daly, J.W. )

    1990-02-01

    Maitotoxin (MTX) increases formation of (3H)inositol phosphates from phosphoinositides and release of (3H)arachidonic acid from phospholipids in pheochromocytoma PC12 cells. Formation of (3H)inositol phosphates is detected within 1 min of incubation even with concentrations as low as 0.3 ng/ml (90 pm) MTX, whereas release of (3H)arachidonic acid is not detected until 20 min even with concentrations as high as 1 ng/ml (300 pm) MTX. Stimulation of arachidonic acid release can be detected at 0.03 ng/ml (9 pm) MTX, whereas 0.1 ng/ml (30 pm) MTX is the threshold for detection of phosphoinositide breakdown. Organic and inorganic calcium channel blockers, except Cd2+ and a high concentration of Mn2+, have no effect on MTX-elicited phosphoinositide breakdown, whereas inorganic blockers (e.g., Co2+, Mn2+, Cd2+), but not organic blockers (nifedipine, verapamil, diltiazem), inhibit MTX-stimulated arachidonic acid release. All calcium channel blockers, however, inhibited MTX-elicited influx of 45Ca2+ and the MTX-elicited increase in internal Ca2+ measured with fura-2 was markedly reduced by nifedipine. MTX-elicited phosphoinositide breakdown and arachidonic acid release are abolished or reduced, respectively, in the absence of extracellular calcium plus chelating agent. The calcium ionophore A23187 has little or no effect alone but, in combination with MTX, A23187 inhibits MTX-elicited phosphoinositide breakdown and enhances arachidonic acid release, the latter even in the absence of extracellular calcium. The results suggest that different sites and/or mechanisms are involved in stimulation of calcium influx, breakdown of phosphoinositides, and release of arachidonic acid by MTX.

  11. Decreased platelet phosphoinositide turnover and enhanced platelet activation in IDDM

    SciTech Connect

    Bastyr, E.J. 3d.; Kadrofske, M.M.; Dershimer, R.C.; Vinik, A.I. )

    1989-09-01

    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.

  12. HGF/c-Met signaling promotes liver progenitor cell migration and invasion by an epithelial-mesenchymal transition-independent, phosphatidyl inositol-3 kinase-dependent pathway in an in vitro model.

    PubMed

    Surez-Causado, A; Caballero-Daz, D; Bertrn, E; Roncero, C; Addante, A; Garca-lvaro, M; Fernndez, M; Herrera, B; Porras, A; Fabregat, I; Snchez, A

    2015-10-01

    Oval cells constitute an interesting hepatic cell population. They contribute to sustain liver regeneration during chronic liver damage, but in doing this they can be target of malignant conversion and become tumor-initiating cells and drive hepatocarcinogenesis. The molecular mechanisms beneath either their pro-regenerative or pro-tumorigenic potential are still poorly understood. In this study, we have investigated the role of the HGF/c-Met pathway in regulation of oval cell migratory and invasive properties. Our results show that HGF induces c-Met-dependent oval cell migration both in normal culture conditions and after in vitro wounding. HGF-triggered migration involves F-actin cytoskeleton reorganization, which is also evidenced by activation of Rac1. Furthermore, HGF causes ZO-1 translocation from cell-cell contact sites to cytoplasm and its concomitant activation by phosphorylation. However, no loss of expression of cell-cell adhesion proteins, including E-cadherin, ZO-1 and Occludin-1, is observed. Additionally, migration does not lead to cell dispersal but to a characteristic organized pattern in rows, in turn associated with Golgi compaction, providing strong evidence of a morphogenic collective migration. Besides migration, HGF increases oval cell invasion through extracellular matrix, a process that requires PI3K activation and is at least partly mediated by expression and activation of metalloproteases. Altogether, our findings provide novel insights into the cellular and molecular mechanisms mediating the essential role of HGF/c-Met signaling during oval cell-mediated mouse liver regeneration. PMID:26001768

  13. Phosphoinositide kinase signaling controls ER-PM cross-talk.

    PubMed

    Omnus, Deike J; Manford, Andrew G; Bader, Jakob M; Emr, Scott D; Stefan, Christopher J

    2016-04-01

    Membrane lipid dynamics must be precisely regulated for normal cellular function, and disruptions in lipid homeostasis are linked to the progression of several diseases. However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress. We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca(2+)-regulated lipid biogenesis, upon plasma membrane (PM) stress. Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress. Thus PI kinase-mediated ER-PM cross-talk comprises a regulatory system that ensures cellular integrity under membrane stress conditions. PMID:26864629

  14. Urotensin-II Receptor Stimulation of Cardiac L-type Ca2+ Channels Requires the βγ Subunits of Gi/o-protein and Phosphatidylinositol 3-Kinase-dependent Protein Kinase C β1 Isoform*

    PubMed Central

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

    2015-01-01

    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 Ca2+ 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 PKCα inhibition or PKA blockade had no effect. Exposure of ventricular myocytes to U-II markedly increased membrane PKCβ1 expression, whereas inhibition of PKCβ1 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 PKCβ 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 βγ subunits of Gi/o-protein and downstream activation of the class I PI3K-dependent PKCβ1 isoform. This occurred via the activation of U-IIR and contributes to the positive inotropic effect on cardiomyocytes. PMID:25678708

  15. Effectors of animal and plant pathogens use a common domain to bind host phosphoinositides.

    PubMed

    Salomon, Dor; Guo, Yirui; Kinch, Lisa N; Grishin, Nick V; Gardner, Kevin H; Orth, Kim

    2013-01-01

    Bacterial Type III Secretion Systems deliver effectors into host cells to manipulate cellular processes to the advantage of the pathogen. Many host targets of these effectors are found on membranes. Therefore, to identify their targets, effectors often use specialized membrane-localization domains to localize to appropriate host membranes. However, the molecular mechanisms used by many domains are unknown. Here we identify a conserved bacterial phosphoinositide-binding domain (BPD) that is found in functionally diverse Type III effectors of both plant and animal pathogens. We show that members of the BPD family functionally bind phosphoinositides and mediate localization to host membranes. Moreover, NMR studies reveal that the BPD of the newly identified Vibrio parahaemolyticus Type III effector VopR is unfolded in solution, but folds into a specific structure upon binding its ligand phosphatidylinositol-(4,5)-bisphosphate. Thus, our findings suggest a possible mechanism for promoting refolding of Type III effectors after delivery into host cells. PMID:24346350

  16. Implication of phosphoinositide phosphatases in genetic diseases: the case of myotubularin.

    PubMed

    Tronchère, H; Buj-Bello, A; Mandel, J-L; Payrastre, B

    2003-10-01

    Phosphoinositides play a central role in the control of major eukaryotic cell signaling mechanisms. Accordingly, the list of phosphoinositide-metabolizing enzymes implicated in human diseases has considerably increased these last years. Here we will focus on myotubularin, the protein mutated in the X-linked myotubular myopathy (XLMTM) and the founding member of a family of 13 related proteins. Recent data demonstrate that myotubularin and several other members of the family are potent lipid phosphatases showing a marked specificity for phosphatidylinositol 3-phosphate [PtdIns(3)P]. This finding has raised considerable interest as PtdIns(3)P is implicated in vesicular trafficking and sorting through its binding to specific protein domains. The structure of myotubularin, the molecular mechanisms of its function and its implication in the etiology of XLMTM will be discussed, as well as the potential function and role of the other members of the family. PMID:14618257

  17. Phosphoinositides: Important lipids in the coordination of cell dynamics.

    PubMed

    Viaud, Julien; Mansour, Rana; Antkowiak, Adrien; Mujalli, Abdulrahman; Valet, Colin; Chicanne, Gaëtan; Xuereb, Jean-Marie; Terrisse, Anne-Dominique; Séverin, Sonia; Gratacap, Marie-Pierre; Gaits-Iacovoni, Frédérique; Payrastre, Bernard

    2016-06-01

    By interacting specifically with proteins, phosphoinositides organize the spatiotemporal formation of protein complexes involved in the control of intracellular signaling, vesicular trafficking and cytoskeleton dynamics. A set of specific kinases and phosphatases ensures the production, degradation and inter-conversion of phosphoinositides to achieve a high level of precision in the regulation of cellular dynamics coordinated by these lipids. The direct involvement of these enzymes in cancer, genetic or infectious diseases, and the recent arrival of inhibitors targeting specific phosphoinositide kinases in clinic, emphasize the importance of these lipids and their metabolism in the biomedical field. PMID:26391221

  18. Phosphoinositides of sheep platelets plasma membranes.

    PubMed

    Kumar, V; Srivastava, N; Grover, S K; Jaiswal, A S; Bansal, S K; Misra, U K

    1991-04-01

    Phospholipid composition of sheep blood platelets and its various plasma membrane fractions have been analyzed. Based on their flotation rates in discontinuous sucrose density gradient centrifugation, three membrane fractions were isolated. 5'-Nucelotidase and alkaline phosphatase were distributed nearly equally in all the three membrane fractions. However these membrane fractions showed differences in the distribution of phosphatidyl ethanolamine, phosphatidyl choline and phosphoinositides. Phosphatidyl ethanolamine was predominant in fraction I (11.05 micrograms PLP/mg protein) while phosphatidyl choline was predominant in fractions II and III (110.10 and 68.30 micrograms PLP/mg protein respectively). Phosphatidyl inositol (Ptd-InsP) was equally distributed in all three membrane fractions. However, both Ptd-InsP and phosphatidyl inositol 4,5-bisphosphate were about 4-fold higher in fraction II (73.55 and 89.89 micrograms PLP/mg protein respectively). PMID:1652550

  19. Structure and function of phosphoinositide 3-kinases.

    PubMed

    Wymann, M P; Pirola, L

    1998-12-01

    Phosphoinositide kinases (PI3Ks) play an important role in mitogenic signaling and cell survival, cytoskeletal remodeling, metabolic control and vesicular trafficking. Here we summarize the structure-function relationships delineating the activation process of class I PI3Ks involving various domains of adapter subunits, Ras, and interacting proteins. The resulting product, PtdIns(3,4,5)P3, targets Akt/protein kinase B (PKB), Bruton's tyrosine kinase (Btk), phosphoinositide-dependent kinases (PDK), integrin-linked kinase (ILK), atypical protein kinases C (PKC), phospholipase Cgamma and more. Surface receptor-activated PI3Ks function in mammals, insects, nematodes and slime mold, but not yeast. While many members of the class II family have been identified and characterized biochemically, it is presently unknown how these C2-domain containing PI3Ks are activated, and which PI substrate they phosphorylate in vivo. PtdIns 3-P is produced by Vps34p/class III PI3Ks and operates via the PtdIns 3-P-binding proteins early endosomal antigen (EEA1), yeast Vac1p, Vps27p, Pip1p in lysosomal protein targeting. Besides the production of D3 phosphorylated lipids, PI3Ks have an intrinsic protein kinase activity. For trimeric GTP-binding protein-activated PI3Kgamma, protein kinase activity seems to be sufficient to trigger mitogen-activated protein kinase (MAPK). Recent disruption of PI3K genes in slime mold, Caenorhabditis elegans, Drosophila melanogaster and mice further underlines the importance of PI3K signaling systems and elucidates the role of PI3K signaling in multicellular organisms. PMID:9838078

  20. The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

    SciTech Connect

    Zhang, Yanfeng; Varnum, Susan M.

    2012-03-01

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.

  1. Autoradiographic imaging of phosphoinositide turnover in the brain

    SciTech Connect

    Hwang, P.M.; Bredt, D.S.; Snyder, S.H. )

    1990-08-17

    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.

  2. PITPs as Targets for Selectively Interfering With Phosphoinositide Signaling in Cells

    PubMed Central

    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

    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

  3. Chromosomal Instability and Phosphoinositide Pathway Gene Signatures in Glioblastoma Multiforme.

    PubMed

    Waugh, Mark G

    2016-01-01

    Structural rearrangements of chromosome 10 are frequently observed in glioblastoma multiforme and over 80 % of tumour samples archived in the catalogue of somatic mutations in cancer database had gene copy number loss for PI4K2A which encodes phosphatidylinositol 4-kinase type IIalpha. PI4K2A loss of heterozygosity mirrored that of PTEN, another enzyme that regulates phosphoinositide levels and also PIK3AP1, MINPP1, INPP5A and INPP5F. These results indicated a reduction in copy number for a set of phosphoinositide signalling genes that co-localise to chromosome 10q. This analysis was extended to a panel of phosphoinositide pathway genes on other chromosomes and revealed a number of previously unreported associations with glioblastoma multiforme. Of particular note were highly penetrant copy number losses for a group of X-linked phosphoinositide phosphatase genes OCRL, MTM1 and MTMR8; copy number amplifications for the chromosome 19 genes PIP5K1C, AKT2 and PIK3R2, and also for the phospholipase C genes PLCB1, PLCB4 and PLCG1 on chromosome 20. These mutations are likely to affect signalling and trafficking functions dependent on the PI(4,5)P2, PI(3,4,5)P3 and PI(3,5)P2 lipids as well as the inositol phosphates IP3, IP5 and IP6. Analysis of flanking genes with functionally unrelated products indicated that chromosomal instability as opposed to a phosphoinositide-specific process underlay this pattern of copy number variation. This in silico study suggests that in glioblastoma multiforme, karyotypic changes have the potential to cause multiple abnormalities in sets of genes involved in phosphoinositide metabolism and this may be important for understanding drug resistance and phosphoinositide pathway redundancy in the advanced disease state. PMID:25502460

  4. Phosphoinositide 3-kinase: friend and foe in cardiovascular disease

    PubMed Central

    Ghigo, Alessandra; Li, Mingchuan

    2015-01-01

    Class I phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases activated by cell membrane receptors, either receptor tyrosine kinases (RTKs) or G protein–coupled receptors (GPCRs), to catalyze the production of the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3). These enzymes engage multiple downstream intracellular signaling pathways controlling cell proliferation, survival and migration. In the cardiovascular system, the four class I PI3K isoforms, PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ are differentially expressed in distinct cell subsets which include cardiomyocytes, fibroblasts, endothelial, and vascular smooth muscle cells as well as leukocytes, suggesting specific functions for distinct PI3K isoenzymes. During the last decades, genetic disruption studies targeting different PI3K genes have elucidated the contribution of specific isoenzymes to cardiac and vascular function regulation, highlighting both beneficial and maladaptive roles. New layers of complexity in the function of PI3Ks have recently emerged, indicating that distinct PI3K isoforms are interconnected by various crosstalk events and can function not only as kinases, but also as scaffold proteins coordinating key signalosomes in cardiovascular health and disease. In this review, we will summarize major breakthroughs in the comprehension of detrimental and beneficial actions of PI3K signaling in cardiovascular homeostasis, and we will discuss recently unraveled cross-talk and scaffold mechanisms as well as the role of the less characterized class II and III PI3K isoforms. PMID:26321955

  5. Structure, function, and control of phosphoinositide-specific phospholipase C.

    PubMed

    Rebecchi, M J; Pentyala, S N

    2000-10-01

    Phosphoinositide-specific phospholipase C (PLC) subtypes beta, gamma, and delta comprise a related group of multidomain phosphodiesterases that cleave the polar head groups from inositol lipids. Activated by all classes of cell surface receptor, these enzymes generate the ubiquitous second messengers inositol 1,4, 5-trisphosphate and diacylglycerol. The last 5 years have seen remarkable advances in our understanding of the molecular and biological facets of PLCs. New insights into their multidomain arrangement and catalytic mechanism have been gained from crystallographic studies of PLC-delta(1), while new modes of controlling PLC activity have been uncovered in cellular studies. Most notable is the realization that PLC-beta, -gamma, and -delta isoforms act in concert, each contributing to a specific aspect of the cellular response. Clues to their true biological roles were also obtained. Long assumed to function broadly in calcium-regulated processes, genetic studies in yeast, slime molds, plants, flies, and mammals point to specific and conditional roles for each PLC isoform in cell signaling and development. In this review we consider each subtype of PLC in organisms ranging from yeast to mammals and discuss their molecular regulation and biological function. PMID:11015615

  6. Phosphoinositide 3-kinase p85beta regulates invadopodium formation

    PubMed Central

    Cariaga-Martínez, Ariel E.; Cortés, Isabel; García, Esther; Pérez-García, Vicente; Pajares, María J.; Idoate, Miguel A.; Redondo-Muñóz, Javier; Antón, Inés M.; Carrera, Ana C.

    2014-01-01

    ABSTRACT The acquisition of invasiveness is characteristic of tumor progression. Numerous genetic changes are associated with metastasis, but the mechanism by which a cell becomes invasive remains unclear. Expression of p85β, a regulatory subunit of phosphoinositide-3-kinase, markedly increases in advanced carcinoma, but its mode of action is unknown. We postulated that p85β might facilitate cell invasion. We show that p85β localized at cell adhesions in complex with focal adhesion kinase and enhanced stability and maturation of cell adhesions. In addition, p85β induced development at cell adhesions of an F-actin core that extended several microns into the cell z-axis resembling the skeleton of invadopodia. p85β lead to F-actin polymerization at cell adhesions by recruiting active Cdc42/Rac at these structures. In accordance with p85β function in invadopodium-like formation, p85β levels increased in metastatic melanoma and p85β depletion reduced invadopodium formation and invasion. These results show that p85β enhances invasion by inducing cell adhesion development into invadopodia-like structures explaining the metastatic potential of tumors with increased p85β levels. PMID:25217619

  7. Integration of Phosphoinositide and Calmodulin Mediated Regulation of TRPC6

    PubMed Central

    Kwon, Young; Hofmann, Thomas; Montell, Craig

    2007-01-01

    Multiple TRP channels are regulated by phosphoinositides (PIs). However, it is not known whether PIs bind directly to TRP channels. Furthermore, the mechanisms through which PIs regulate TRP channels are obscure. To analyze the role of PI/TRP interactions, we used a biochemical approach, focusing on TRPC6. TRPC6 bound directly to PIs, and with highest potency to phosphatidylinositol 3,4,5-trisphosphate (PIP3). We found that PIP3 binding disrupted the association of calmodulin (CaM) with TRPC6. We identified the PIP3 binding site and found that mutations that increased or decreased the affinity of the PIP3/TRPC6 interaction enhanced or reduced the TRPC6-dependent current respectively. PI mediated disruption of CaM binding appears to be a theme that applies to other TRP channels, such as TRPV1, as well as to the voltage-gated channels, KCNQ1 and Cav1.2. We propose that regulation of CaM binding by PIs provides a mode for integration of channel regulation by Ca2+ and PIs. PMID:17317623

  8. Role of phosphoinositides in STIM1 dynamics and store-operated calcium entry

    PubMed Central

    Walsh, Ciara M.; Chvanov, Michael; Haynes, Lee P.; Petersen, Ole H.; Tepikin, Alexei V.; Burgoyne, Robert D.

    2009-01-01

    Ca2+ entry through store-operated Ca2+ channels involves the interaction at ER–PM (endoplasmic reticulum–plasma membrane) junctions of STIM (stromal interaction molecule) and Orai. STIM proteins are sensors of the luminal ER Ca2+ concentration and, following depletion of ER Ca2+, they oligomerize and translocate to ER–PM junctions where they form STIM puncta. Direct binding to Orai proteins activates their Ca2+ channel function. It has been suggested that an additional interaction of the C-terminal polybasic domain of STIM1 with PM phosphoinositides could contribute to STIM1 puncta formation prior to binding to Orai. In the present study, we investigated the role of phosphoinositides in the formation of STIM1 puncta and SOCE (store-operated Ca2+ entry) in response to store depletion. Treatment of HeLa cells with inhibitors of PI3K (phosphatidylinositol 3-kinase) and PI4K (phosphatidylinositol 4-kinase) (wortmannin and LY294002) partially inhibited formation of STIM1 puncta. Additional rapid depletion of PtdIns(4,5)P2 resulted in more substantial inhibition of the translocation of STIM1–EYFP (enhanced yellow fluorescent protein) into puncta. The inhibition was extensive at a concentration of LY294002 (50 μM) that should primarily inhibit PI3K, consistent with a major role for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in puncta formation. Depletion of phosphoinositides also inhibited SOCE based on measurement of the rise in intracellular Ca2+ concentration after store depletion. Overexpression of Orai1 resulted in a recovery of translocation of STMI1 into puncta following phosphoinositide depletion and, under these conditions, SOCE was increased to above control levels. These observations support the idea that phosphoinositides are not essential but contribute to STIM1 accumulation at ER–PM junctions with a second translocation mechanism involving direct STIM1–Orai interactions. PMID:19843011

  9. Phosphoinositides Regulate Ciliary Protein Trafficking to Modulate Hedgehog Signaling.

    PubMed

    Garcia-Gonzalo, Francesc R; Phua, Siew Cheng; Roberson, Elle C; Garcia, Galo; Abedin, Monika; Schurmans, Stphane; Inoue, Takanari; Reiter, Jeremy F

    2015-08-24

    Primary cilia interpret vertebrate Hedgehog (Hh) signals. Why cilia are essential for signaling is unclear. One possibility is that some forms of signaling require a distinct membrane lipid composition, found at cilia. We found that the ciliary membrane contains a particular phosphoinositide, PI(4)P, whereas a different phosphoinositide, PI(4,5)P2, is restricted to the membrane of the ciliary base. This distribution is created by Inpp5e, a ciliary phosphoinositide 5-phosphatase. Without Inpp5e, ciliary PI(4,5)P2 levels are elevated and Hh signaling is disrupted. Inpp5e limits the ciliary levels of inhibitors of Hh signaling, including Gpr161 and the PI(4,5)P2-binding protein Tulp3. Increasing ciliary PI(4,5)P2 levels or conferring the ability to bind PI(4)P on Tulp3 increases the ciliary localization of Tulp3. Lowering Tulp3 in cells lacking Inpp5e reduces ciliary Gpr161 levels and restores Hh signaling. Therefore, Inpp5e regulates ciliary membrane phosphoinositide composition, and Tulp3 reads out ciliary phosphoinositides to control ciliary protein localization, enabling Hh signaling. PMID:26305592

  10. Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation

    PubMed Central

    Sauer, Karsten; Huang, Yina Hsing; Lin, Hongying; Sandberg, Mark; Mayr, Georg W.

    2015-01-01

    Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5-trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+ signaling in lymphocytes. IP3 phosphorylation by IP3 3-kinases generates inositol 1,3,4,5-tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3 in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high-performance liquid chromatographic separation methods followed by radiolabeling or non-radioactive metal-dye detection. Finally, less broadly applicable nonchromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+ thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development. PMID:19918943

  11. Role of phosphoinositide 3-kinase in the autophagic death of serum-deprived PC12 cells.

    PubMed

    Guillon-Munos, A; van Bemmelen, M X P; Clarke, P G H

    2005-10-01

    The death of serum-deprived undifferentiated PC12 cells shows both autophagic and apoptotic features. Since it is still controversial whether the autophagy is instrumental in the cell death or a mere epiphenomenon, we tested the effects of inhibiting the autophagy by a variety of phosphoinositide 3-kinase inhibitors, and provided evidence that the autophagy, or a related trafficking event, is indeed instrumental in the cell death. Furthermore, by comparing the effects of PI3-K inhibition and caspase-inhibition on autophagic and apoptotic cellular events, we showed that in this case the autophagic and apoptotic mechanisms mediate cell death by parallel pathways and do not act in series. PMID:16151638

  12. Arabidopsis AtPLC2 Is a Primary Phosphoinositide-Specific Phospholipase C in Phosphoinositide Metabolism and the Endoplasmic Reticulum Stress Response

    PubMed Central

    Kanehara, Kazue; Yu, Chao-Yuan; Cho, Yueh; Cheong, Wei-Fun; Torta, Federico; Shui, Guanghou; Wenk, Markus R; Nakamura, Yuki

    2015-01-01

    Abstract Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response. PMID:26401841

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

    PubMed Central

    Strahl, Thomas; Thorner, Jeremy

    2007-01-01

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

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

    PubMed Central

    Dai, Gucan; Peng, Changhong; Liu, Chunming

    2013-01-01

    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 (PIPn), including phosphatidylinositol 3,4,5-triphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2), 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 PIPn application. However, PIPn induced an ∼2.5-fold increase in cAMP efficacy for A3 channels. The PIPn-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 PIPn 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 PIPn regulation of heteromeric A3+B3 channels. B3 subunits were not sufficient to confer PIPn sensitivity to heteromeric channels formed with PIPn-insensitive A subunits. Finally, channels formed by mixtures of PIPn-insensitive A3 subunits, having complementary mutations in N- and/or C-terminal regions, restored PIPn regulation, implying that intersubunit N–C interactions help control the phosphoinositide sensitivity of cone CNG channels. PMID:23530136

  15. Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis

    PubMed Central

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

    2012-01-01

    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 2105 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

  16. Phenylephrine stimulated breakdown of phosphoinositides in brown adipocytes is attenuated by adenosine

    SciTech Connect

    Schimmel, R.J.

    1986-03-01

    Selective activation of alpha adrenergic receptors on brown adipocytes brings about increased mitochondrial respiration. This response is associated with a rapid breakdown of phosphoinositides in the plasma membrane. The authors have shown that respiration increased by alpha receptor activation can be inhibited by adenosine but the mechanisms underlying this effect are unknown. The present study probes the possibility that adenosine inhibition of alpha receptor stimulated respiration is secondary to an inhibition of stimulated breakdown of inositol phospholipids. Phospholipids were labeled with (/sup 32/P) by incubation with (/sup 32/P)-Pi for up to four hours. Phenylephrine and other ligands were then added and the radioactivity present in individual lipids determined following their resolution by thin layer chromatography. Addition of 2-chloroadenosine or phenylisopropyl adenosine, but not 2',5'-dideoxyadenosine, inhibited phenylephrine promoted breakdown of phosphoinositides. The dose response relation for this effect was similar to that for attenuation of stimulated respiration. This finding demonstrates adenosine inhibition of a phospholipase in brown fat cells and suggests the possibility that breakdown of inositol phospholipids is a critical control site for stimulation and attenuation of respiration.

  17. Sec14-nodulin proteins and the patterning of phosphoinositide landmarks for developmental control of membrane morphogenesis

    PubMed Central

    Ghosh, Ratna; de Campos, Marília K. F.; Huang, Jin; Huh, Seong K.; Orlowski, Adam; Yang, Yuan; Tripathi, Ashutosh; Nile, Aaron; Lee, Hsin-Chieh; Dynowski, Marek; Schäfer, Helen; Róg, Tomasz; Lete, Marta G.; Ahyayauch, Hasna; Alonso, Alicia; Vattulainen, Ilpo; Igumenova, Tatyana I.; Schaaf, Gabriel; Bankaitis, Vytas A.

    2015-01-01

    Polarized membrane morphogenesis is a fundamental activity of eukaryotic cells. This process is essential for the biology of cells and tissues, and its execution demands exquisite temporal coordination of functionally diverse membrane signaling reactions with high spatial resolution. Moreover, mechanisms must exist to establish and preserve such organization in the face of randomizing forces that would diffuse it. Here we identify the conserved AtSfh1 Sec14-nodulin protein as a novel effector of phosphoinositide signaling in the extreme polarized membrane growth program exhibited by growing Arabidopsis root hairs. The data are consistent with Sec14-nodulin proteins controlling the lateral organization of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) landmarks for polarized membrane morphogenesis in plants. This patterning activity requires both the PtdIns(4,5)P2 binding and homo-oligomerization activities of the AtSfh1 nodulin domain and is an essential aspect of the polarity signaling program in root hairs. Finally, the data suggest a general principle for how the phosphoinositide signaling landscape is physically bit mapped so that eukaryotic cells are able to convert a membrane surface into a high-definition lipid-signaling screen. PMID:25739452

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

    PubMed Central

    Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

    2015-01-01

    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 PI(4)P from the plasma membrane through Ca2+-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ 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 or 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

  19. Sec14-nodulin proteins and the patterning of phosphoinositide landmarks for developmental control of membrane morphogenesis.

    PubMed

    Ghosh, Ratna; de Campos, Marlia K F; Huang, Jin; Huh, Seong K; Orlowski, Adam; Yang, Yuan; Tripathi, Ashutosh; Nile, Aaron; Lee, Hsin-Chieh; Dynowski, Marek; Schfer, Helen; Rg, Tomasz; Lete, Marta G; Ahyayauch, Hasna; Alonso, Alicia; Vattulainen, Ilpo; Igumenova, Tatyana I; Schaaf, Gabriel; Bankaitis, Vytas A

    2015-05-01

    Polarized membrane morphogenesis is a fundamental activity of eukaryotic cells. This process is essential for the biology of cells and tissues, and its execution demands exquisite temporal coordination of functionally diverse membrane signaling reactions with high spatial resolution. Moreover, mechanisms must exist to establish and preserve such organization in the face of randomizing forces that would diffuse it. Here we identify the conserved AtSfh1 Sec14-nodulin protein as a novel effector of phosphoinositide signaling in the extreme polarized membrane growth program exhibited by growing Arabidopsis root hairs. The data are consistent with Sec14-nodulin proteins controlling the lateral organization of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) landmarks for polarized membrane morphogenesis in plants. This patterning activity requires both the PtdIns(4,5)P2 binding and homo-oligomerization activities of the AtSfh1 nodulin domain and is an essential aspect of the polarity signaling program in root hairs. Finally, the data suggest a general principle for how the phosphoinositide signaling landscape is physically bit mapped so that eukaryotic cells are able to convert a membrane surface into a high-definition lipid-signaling screen. PMID:25739452

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

    PubMed Central

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

    2015-01-01

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

  1. Signalling by the phosphoinositide 3-kinase family in immune cells

    PubMed Central

    Okkenhaug, Klaus

    2015-01-01

    Phosphoinositide 3-kinases control many important aspects of immune cell development, differentiation and function. Mammals have eight PI3K catalytic subunits which are divided into 3 classes based on similarities in structure and function. Specific roles for the class I PI3Ks have been broadly investigated and are relatively well understood as is the function of their corresponding phosphatases. More recently, specific roles for the class II and class III PI3Ks have emerged. Through vertebrate evolution and in parallel with the evolution of adaptive immunity, there has been a dramatic increase not only in the genes for PI3K subunits but also in genes for phosphatases that act on 3-phopshoinositides and on 3-phosphoinositide binding proteins. Our understanding of the PI3Ks in immunity is guided by fundamental discoveries made in simpler model organisms as well as by appreciating new adaptions of this signalling module in mammals in general and immune cells in particular. PMID:23330955

  2. Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

    PubMed Central

    2013-01-01

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

  3. Effect of chronic ethanol exposure on myocardial phosphoinositide turnover.

    PubMed

    Piano, M R; Schwertz, D W

    1997-06-01

    The effect of chronic ethanol consumption (2 months) on atrial contractility and the myocardial phosphoinositide signaling system was examined in rat heart. Two months of ethanol consumption was not associated with changes in heart weight-to-body weight ratios; however, developed twitch tension was significantly lower in the ethanol atria compared with the control atria. Cytosolic and membrane-associated phospholipase C activity in atrial and ventricular tissue was measured and ethanol consumption was only associated with changes in ventricular cytosolic phospholipase C activity. When examining alpha(1)-adrenergic stimulated phosphoinositide turnover in [3H]inositol radiolabeled left atria, no differences in phenylephrine (10 microM)-stimulated inositol monophosphate, inositol bisphosphate, inositol trisphosphate, and inositol tetrakisphosphate were found between groups before or at various times after the addition of phenylephrine. It is concluded that short-term ethanol consumption is associated with depressed contractile function, but not the development of hypertrophy or changes in alpha(1)-adrenoreceptor-stimulated phosphoinositide turnover. PMID:9194930

  4. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation.

    PubMed

    Pritchard, Rory A; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated. PMID:26313408

  5. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation

    PubMed Central

    Pritchard, Rory A.; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S.

    2016-01-01

    Abstract Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated. PMID:26313408

  6. INPP5E interacts with AURKA, linking phosphoinositide signaling to primary cilium stability

    PubMed Central

    Plotnikova, Olga V.; Seo, Seongjin; Cottle, Denny L.; Conduit, Sarah; Hakim, Sandra; Dyson, Jennifer M.; Mitchell, Christina A.; Smyth, Ian M.

    2015-01-01

    ABSTRACT Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) cause the ciliopathies known as Joubert and MORM syndromes; however, the role of INPP5E in ciliary biology is not well understood. Here, we describe an interaction between INPP5E and AURKA, a centrosomal kinase that regulates mitosis and ciliary disassembly, and we show that this interaction is important for the stability of primary cilia. Furthermore, AURKA phosphorylates INPP5E and thereby increases its 5-phosphatase activity, which in turn promotes transcriptional downregulation of AURKA, partly through an AKT-dependent mechanism. These findings establish the first direct link between AURKA and phosphoinositide signaling and suggest that the function of INPP5E in cilia is at least partly mediated by its interactions with AURKA. PMID:25395580

  7. Stimulatory effects of maitotoxin on insulin release in insulinoma HIT cells: Role of calcium uptake and phosphoinositide breakdown

    SciTech Connect

    Soergel, D.G.; Gusovsky, F.; Yasumoto, T.; Daly, J.W. )

    1990-12-01

    In hamster insulinoma (HIT) cells, maitotoxin (MTX) induces a time-dependent and concentration-dependent release of insulin that requires the presence of extracellular calcium. The response is nearly completely blocked by cinnarizine and cadmium, but is not inhibited by the L-type calcium channel blocker nifedipine or by manganese. MTX induces 45Ca+ uptake in these cells in a dose-dependent mode, and the uptake is blocked with cinnarizine, nifedipine and cadmium, and is partially inhibited by manganese. MTX induces phosphoinositide breakdown in HIT cells, and the response is partially blocked by cadmium, but is not affected by nifedipine, cinnarizine or manganese. High concentrations of potassium ions also induce insulin release and calcium uptake in HIT cells. Both effects of potassium are blocked partially by nifedipine, cadmium and cinnarizine. High concentrations of potassium do not induce phosphoinositide breakdown in HIT cells. The results suggest that MTX-elicited release of insulin is attained by two mechanisms: (1) a nifedipine-sensitive action, which results from MTX-induced activation of L-type calcium channels, which can be mimicked with high potassium concentrations; and (2) a nifedipine-insensitive action, which may be initiated by the activation of phosphoinositide breakdown by MTX. Such an activation of phospholipase C would result in the formation of 1,4,5-inositol trisphosphate, a release of intracellular calcium and then release of insulin to the extracellular space. Cinnarizine is proposed to block both MTX-elicited mechanisms, the first by blockade of calcium channels and the second by blocking 1,4,5-inositol trisphosphate-induced release of internal calcium. Either mechanism alone appears capable of eliciting release of insulin.

  8. Phosphoinositide and Erk signaling pathways mediate activity-driven rodent olfactory sensory neuronal survival and stress mitigation.

    PubMed

    Kim, So Yeun; Mammen, Alex; Yoo, Seung-Jun; Cho, Bongki; Kim, Eun-Kyoung; Park, Jong-In; Moon, Cheil; Ronnett, Gabriele V

    2015-08-01

    Olfactory sensory neurons (OSNs) are the initial site for olfactory signal transduction. Therefore, their survival is essential to olfactory function. In the current study, we demonstrated that while odorant stimulation promoted rodent OSN survival, it induced generation of reactive oxygen species in a dose- and time-dependent manner as well as loss of membrane potential and fragmentation of mitochondria. The MEK-Erk pathway played a critical role in mediating these events, as its inhibition decreased odorant stimulation-dependent OSN survival and exacerbated intracellular stress measured by reactive oxygen species generation and heat-shock protein 70 expression. The phosphoinositide pathway, rather than the cyclic AMP pathway, mediated the odorant-induced activation of the MEK-Erk pathway. These findings provide important insights into the mechanisms of activity-driven OSN survival, the role of the phosphoinositide pathway in odorant signaling, and demonstrate that odorant detection and odorant stimulation-mediated survival proceed via independent signaling pathways. This mechanism, which permits independent regulation of odorant detection from survival signaling, may be advantageous if not diminished by repeated or prolonged odor exposure. We investigated the role of odorant stimulation in generating cellular stress and the molecular mechanisms mitigating such stress and promoting neuronal survival. Odorant stimulation promoted olfactory sensory neuron (OSN) survival and also induced intracellular oxidative stress, which was exacerbated when MEK/Erks pathway was inhibited. Sensory stimulation simultaneously activated at least two parallel pathways, the AC/cAMP cascade responsible for odorant detection, and phosphoinositide hydrolysis to promote odorant stimulation-dependent neuronal survival odorants may activate parallel signaling cascades to mediate sensory detection and sensory stimulation-dependent survival. AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; Erk, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase. PMID:25903517

  9. Alcohol induced changes in phosphoinositide signaling system in rat brain

    SciTech Connect

    Pandey, S.; Piano, M.; Schwertz, D.; Davis, J.; Pandey, G. )

    1991-03-11

    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.

  10. Phosphoinositide-3-Kinase Is the Primary Mediator of Phosphoinositide-Dependent Inhibition in Mammalian Olfactory Receptor Neurons

    PubMed Central

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

    2016-01-01

    Odorants inhibit as well as excite primary olfactory receptor neurons (ORNs) in many animal species. Growing evidence suggests that inhibition of mammalian ORNs is mediated by phosphoinositide (PI) signaling through activation of phosphoinositide 3-kinase (PI3K), and that canonical adenylyl cyclase III signaling and PI3K signaling interact to provide the basis for ligand-induced selective signaling. As PI3K is known to act in concert with phospholipase C (PLC) in some cellular systems, the question arises as to whether they work together to mediate inhibitory transduction in mammalian ORNs. The present study is designed to test this hypothesis. While we establish that multiple PLC isoforms are expressed in the transduction zone of rat ORNs, that odorants can activate PLC in ORNs in situ, and that pharmacological blockade of PLC enhances the excitatory response to an odorant mixture in some ORNs in conjunction with PI3K blockade, we find that by itself PLC does not account for an inhibitory response. We conclude that PLC does not make a measurable independent contribution to odor-evoked inhibition, and that PI3K is the primary mediator of PI-dependent inhibition in mammalian ORNs. PMID:27147969

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

    PubMed Central

    Balla, Tamas; Várnai, Péter

    2011-01-01

    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

  12. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks. © 2016 Wiley Periodicals, Inc. PMID:26915738

  13. Identification of Components of the Host Type IA Phosphoinositide 3-Kinase Pathway That Promote Internalization of Listeria monocytogenes

    PubMed Central

    Jiwani, Shahanawaz; Wang, Yi; Dowd, Georgina C.; Gianfelice, Antonella; Pichestapong, Phannipha; Gavicherla, Balramakrishna; Vanbennekom, Neyda

    2012-01-01

    The bacterial pathogen Listeria monocytogenes causes food-borne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria promotes its internalization into some human cells through binding of the bacterial surface protein InlB to the host receptor tyrosine kinase Met. The interaction of InlB with the Met receptor stimulates host signaling pathways that promote cell surface changes driving bacterial uptake. One human signaling protein that plays a critical role in Listeria entry is type IA phosphoinositide 3-kinase (PI 3-kinase). The molecular mechanism by which PI 3-kinase promotes bacterial internalization is not understood. Here we perform an RNA interference (RNAi)-based screen to identify components of the type IA PI 3-kinase pathway that control the entry of Listeria into the human cell line HeLa. The 64 genes targeted encode known upstream regulators or downstream effectors of type IA PI 3-kinase. The results of this screen indicate that at least 9 members of the PI 3-kinase pathway play important roles in Listeria uptake. These 9 human proteins include a Rab5 GTPase, several regulators of Arf or Rac1 GTPases, and the serine/threonine kinases phosphoinositide-dependent kinase 1 (PDK1), mammalian target of rapamycin (mTor), and protein kinase C-ζ. These findings represent a key first step toward understanding the mechanism by which type IA PI 3-kinase controls bacterial internalization. PMID:22158742

  14. A Role for Phosphoinositides in Regulating Plant Nuclear Functions

    PubMed Central

    Dieck, Catherine B.; Boss, Wendy F.; Perera, Imara Y.

    2012-01-01

    Nuclear localized inositol phospholipids and inositol phosphates are important for regulating many essential processes in animal and yeast cells such as DNA replication, recombination, RNA processing, mRNA export and cell cycle progression. An overview of the current literature indicates the presence of a plant nuclear phosphoinositide (PI) pathway. Inositol phospholipids, inositol phosphates, and enzymes of the PI pathway have been identified in plant nuclei and are implicated in DNA replication, chromatin remodeling, stress responses and hormone signaling. In this review, the potential functions of the nuclear PI pathway in plants are discussed within the context of the animal and yeast literature. It is anticipated that future research will help shed light on the functional significance of the nuclear PI pathway in plants. PMID:22645589

  15. In vivo tracking of phosphoinositides in Drosophila photoreceptors

    PubMed Central

    Hardie, Roger C.; Liu, Che-Hsiung; Randall, Alexander S.; Sengupta, Sukanya

    2015-01-01

    ABSTRACT In order to monitor phosphoinositide turnover during phospholipase C (PLC)-mediated Drosophila phototransduction, fluorescently tagged lipid probes were expressed in photoreceptors and imaged both in dissociated cells, and in eyes of intact living flies. Of six probes tested, TbR332H (a mutant of the Tubby protein pleckstrin homology domain) was judged the best reporter for phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], and the P4M domain from Legionella SidM for phosphatidylinositol 4-phosphate (PtdIns4P). Using accurately calibrated illumination, we found that only ∼50% of PtdIns(4,5)P2 and very little PtdIns4P were depleted by full daylight intensities in wild-type flies, but both were severely depleted by ∼100-fold dimmer intensities in mutants lacking Ca2+-permeable transient receptor potential (TRP) channels or protein kinase C (PKC). Resynthesis of PtdIns4P (t½ ∼12 s) was faster than PtdIns(4,5)P2 (t½ ∼40 s), but both were greatly slowed in mutants of DAG kinase (rdgA) or PtdIns transfer protein (rdgB). The results indicate that Ca2+- and PKC-dependent inhibition of PLC is required for enabling photoreceptors to maintain phosphoinositide levels despite high rates of hydrolysis by PLC, and suggest that phosphorylation of PtdIns4P to PtdIns(4,5)P2 is the rate-limiting step of the cycle. PMID:26483384

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

    SciTech Connect

    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

    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.

  17. Regulation of phosphoinositide hydrolysis and fibrinogen receptor expression by GTP regulatory protein(s) in isolated platelet membranes

    SciTech Connect

    Baldassare, J.J.; Fisher, G.

    1986-05-01

    Thrombin through a GTP-dependent mechanism, stimulated fibrinogen binding and indogenous (/sup 3/H) inositol- and (/sup 32/P)-labeled phosphoinositide hydrolysis in membranes prepared from platelets. Incubation of isolated membranes with GTP enhanced both fibrinogen binding and phosphoinositide metabolism in a dose dependent fashion with half-maximal activation at approximately 50..mu..m and 25..mu..m respectively. Addition of thrombin (l..mu../ml) or ADP (10..mu..M) and GTP enhanced fibrinogen lending 10-fold compared to GTP alone. Inositol triphosphate formation was increased by 300 to 500% within 60 seconds after the addition of thrombin (l..mu../ml). Addition of thrombin plus GTP, however, did not lead to the formation of inositol on glycerol phosphoinositol. These data suggest that in a cell free system these processes are mediated by guanine nucleotide binding regulatory protein(s). Although a general role for GTP-binding proteins in receptor-regulated processes is currently a popular idea, this data is the most direct evidence to date for a role in platelets other than the well established mediation of adenylate cyclase activity.

  18. Phosphoinositide kinases and the synthesis of polyphosphoinositides in higher plant cells

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  19. Computational studies of the binding profile of phosphoinositide PtdIns (3,4,5) P3 with the pleckstrin homology domain of an oomycete cellulose synthase.

    PubMed

    Kuang, Guanglin; Bulone, Vincent; Tu, Yaoquan

    2016-01-01

    Saprolegnia monoica is a model organism to investigate Saprolegnia parasitica, an important oomycete which causes considerable loss in aquaculture every year. S. monoica contains cellulose synthases vital for oomycete growth. However, the molecular mechanism of the cellulose biosynthesis process in the oomycete growth is still poorly understood. Some cellulose synthases of S. monoica, such as SmCesA2, are found to contain a plecsktrin homology (PH) domain, which is a protein module widely found in nature and known to bind to phosphoinositides, a class of signaling compounds involved in many biological processes. Understanding the molecular interactions between the PH domain and phosphoinositides would help to unravel the cellulose biosynthesis process of oomycetes. In this work, the binding profile of PtdIns (3,4,5) P3, a typical phosphoinositide, with SmCesA2-PH was studied by molecular docking, molecular dynamics and metadynamics simulations. PtdIns (3,4,5) P3 is found to bind at a specific site located at β1, β2 and β1-β2 loop of SmCesA2-PH. The high affinity of PtdIns (3,4,5) P3 to SmCesA2-PH is contributed by the free phosphate groups, which have electrostatic and hydrogen-bond interactions with Lys88, Lys100 and Arg102 in the binding site. PMID:26857031

  20. Computational studies of the binding profile of phosphoinositide PtdIns (3,4,5) P3 with the pleckstrin homology domain of an oomycete cellulose synthase

    PubMed Central

    Kuang, Guanglin; Bulone, Vincent; Tu, Yaoquan

    2016-01-01

    Saprolegnia monoica is a model organism to investigate Saprolegnia parasitica, an important oomycete which causes considerable loss in aquaculture every year. S. monoica contains cellulose synthases vital for oomycete growth. However, the molecular mechanism of the cellulose biosynthesis process in the oomycete growth is still poorly understood. Some cellulose synthases of S. monoica, such as SmCesA2, are found to contain a plecsktrin homology (PH) domain, which is a protein module widely found in nature and known to bind to phosphoinositides, a class of signaling compounds involved in many biological processes. Understanding the molecular interactions between the PH domain and phosphoinositides would help to unravel the cellulose biosynthesis process of oomycetes. In this work, the binding profile of PtdIns (3,4,5) P3, a typical phosphoinositide, with SmCesA2-PH was studied by molecular docking, molecular dynamics and metadynamics simulations. PtdIns (3,4,5) P3 is found to bind at a specific site located at β1, β2 and β1-β2 loop of SmCesA2-PH. The high affinity of PtdIns (3,4,5) P3 to SmCesA2-PH is contributed by the free phosphate groups, which have electrostatic and hydrogen-bond interactions with Lys88, Lys100 and Arg102 in the binding site. PMID:26857031

  1. Computational studies of the binding profile of phosphoinositide PtdIns (3,4,5) P3 with the pleckstrin homology domain of an oomycete cellulose synthase

    NASA Astrophysics Data System (ADS)

    Kuang, Guanglin; Bulone, Vincent; Tu, Yaoquan

    2016-02-01

    Saprolegnia monoica is a model organism to investigate Saprolegnia parasitica, an important oomycete which causes considerable loss in aquaculture every year. S. monoica contains cellulose synthases vital for oomycete growth. However, the molecular mechanism of the cellulose biosynthesis process in the oomycete growth is still poorly understood. Some cellulose synthases of S. monoica, such as SmCesA2, are found to contain a plecsktrin homology (PH) domain, which is a protein module widely found in nature and known to bind to phosphoinositides, a class of signaling compounds involved in many biological processes. Understanding the molecular interactions between the PH domain and phosphoinositides would help to unravel the cellulose biosynthesis process of oomycetes. In this work, the binding profile of PtdIns (3,4,5) P3, a typical phosphoinositide, with SmCesA2-PH was studied by molecular docking, molecular dynamics and metadynamics simulations. PtdIns (3,4,5) P3 is found to bind at a specific site located at β1, β2 and β1-β2 loop of SmCesA2-PH. The high affinity of PtdIns (3,4,5) P3 to SmCesA2-PH is contributed by the free phosphate groups, which have electrostatic and hydrogen-bond interactions with Lys88, Lys100 and Arg102 in the binding site.

  2. Control of cardiac repolarization by phosphoinositide 3-kinase signaling to ion channels.

    PubMed

    Ballou, Lisa M; Lin, Richard Z; Cohen, Ira S

    2015-01-01

    Upregulation of phosphoinositide 3-kinase (PI3K) signaling is a common alteration in human cancer, and numerous drugs that target this pathway have been developed for cancer treatment. However, recent studies have implicated inhibition of the PI3K signaling pathway as the cause of a drug-induced long-QT syndrome in which alterations in several ion currents contribute to arrhythmogenic drug activity. Surprisingly, some drugs that were thought to induce long-QT syndrome by direct block of the rapid delayed rectifier (IKr) also seem to inhibit PI3K signaling, an effect that may contribute to their arrhythmogenicity. The importance of PI3K in regulating cardiac repolarization is underscored by evidence that QT interval prolongation in diabetes mellitus also may result from changes in multiple currents because of decreased insulin activation of PI3K in the heart. How PI3K signaling regulates ion channels to control the cardiac action potential is poorly understood. Hence, this review summarizes what is known about the effect of PI3K and its downstream effectors, including Akt, on sodium, potassium, and calcium currents in cardiac myocytes. We also refer to some studies in noncardiac cells that provide insight into potential mechanisms of ion channel regulation by this signaling pathway in the heart. Drug development and safety could be improved with a better understanding of the mechanisms by which PI3K regulates cardiac ion channels and the extent to which PI3K inhibition contributes to arrhythmogenic susceptibility. PMID:25552692

  3. Control of Cardiac Repolarization by Phosphoinositide 3-kinase Signaling to Ion Channels

    PubMed Central

    Ballou, Lisa M.; Lin, Richard Z.; Cohen, Ira S.

    2014-01-01

    Upregulation of phosphoinositide 3-kinase (PI3K) signaling is a common alteration in human cancer, and numerous drugs that target this pathway have been developed for cancer treatment. However, recent studies have implicated inhibition of the PI3K signaling pathway as the cause of a drug-induced long QT syndrome in which alterations in several ion currents contribute to arrhythmogenic drug activity. Surprisingly, some drugs that were thought to induce long QT syndrome by direct block of the rapid delayed rectifier (IKr) also appear to inhibit PI3K signaling, an effect that may contribute to their arrhythmogenicity. The importance of PI3K in regulating cardiac repolarization is underscored by evidence that QT interval prolongation in diabetes also may result from changes in multiple currents due to decreased insulin activation of PI3K in the heart. How PI3K signaling regulates ion channels to control the cardiac action potential is poorly understood. Hence, this review summarizes what is known about the impact of PI3K and its downstream effectors including Akt on sodium, potassium and calcium currents in cardiac myocytes. We also refer to some studies in non-cardiac cells that provide insight into potential mechanisms of ion channel regulation by this signaling pathway in the heart. Drug development and safety could be improved with a better understanding of the mechanisms by which PI3K regulates cardiac ion channels and the extent to which PI3K inhibition contributes to arrhythmogenic susceptibility. PMID:25552692

  4. A Screen for Novel Phosphoinositide 3-kinase Effector Proteins*

    PubMed Central

    Dixon, Miles J.; Gray, Alexander; Boisvert, François-Michel; Agacan, Mark; Morrice, Nicholas A.; Gourlay, Robert; Leslie, Nicholas R.; Downes, C. Peter; Batty, Ian H.

    2011-01-01

    Class I phosphoinositide 3-kinases exert important cellular effects through their two primary lipid products, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). As few molecular targets for PtdIns(3,4)P2 have yet been identified, a screen for PI 3-kinase-responsive proteins that is selective for these is described. This features a tertiary approach incorporating a unique, primary recruitment of target proteins in intact cells to membranes selectively enriched in PtdIns(3,4)P2. A secondary purification of these proteins, optimized using tandem pleckstrin homology domain containing protein-1 (TAPP-1), an established PtdIns(3,4)P2 selective ligand, yields a fraction enriched in proteins of potentially similar lipid binding character that are identified by liquid chromatography-tandem MS. Thirdly, this approach is coupled to stable isotope labeling with amino acids in cell culture using differential isotope labeling of cells stimulated in the absence and presence of the PI 3-kinase inhibitor wortmannin. This provides a ratio-metric readout that distinguishes authentically responsive components from copurifying background proteins. Enriched fractions thus obtained from astrocytoma cells revealed a subset of proteins that exhibited ratios indicative of their initial, cellular responsiveness to PI 3-kinase activation. The inclusion among these of tandem pleckstrin homology domain containing protein-1, three isoforms of Akt, switch associated protein-70, early endosome antigen-1 and of additional proteins expressing recognized lipid binding domains demonstrates the utility of this strategy and lends credibility to the novel candidate proteins identified. The latter encompass a broad set of proteins that include the gene product of TBC1D2A, a putative Rab guanine nucleotide triphosphatase activating protein (GAP) and IQ motif containing GAP1, a potential tumor promoter. A sequence comparison of the former protein indicates the presence of a pleckstrin homology domain whose lipid binding character remains to be established. IQ motif containing GAP1 lacks known lipid interacting components and a preliminary analysis here indicates that this may exemplify a novel class of atypical phosphoinositide (aPI) binding domain. PMID:21263009

  5. Ethanol stimulates phosphoinositide-specific phospholipase C in rat hepatocytes

    SciTech Connect

    Rubin, R.; Thomas, A.; Hoek, J.B.

    1986-05-01

    Ethanol (50-300 mM) causes a transient mobilization of calcium from hormone-sensitive pools in rat hepatocytes. They have studied the action of ethanol on polyphosphoinositide and inositol phosphate levels in these cells. Addition of ethanol to isolated hepatocytes prelabelled with /sup 32/Pi resulted in a small (3-5%) decrease in the level of (/sup 32/P)-phosphatidylinositol 4,5-bisphosphate and a 10-15% increase in (/sup 32/P) phosphatidylinositol 4-phosphate and (/sup 32/P) phosphatidic acid. Maximal changes were seen within 30 sec after ethanol. (/sup 32/P) phosphatidylcholine was unaffected. The changes were concentration dependent over a range of 50-500 mM ethanol. Phorbol esters, which inhibit ethanol-induced calcium mobilization, also inhibited these effects of ethanol on polyphosphoinositides. In intact hepatocytes labeled with myo-(2-/sup 3/H)inositol a significant ethanol-induced increase in (/sup 3/H)inositol phosphates could not be detected. After permeabilization of the cells with digitonin, however, ethanol caused a time and concentration dependent release of (/sup 3/H) inositol trisphosphate and (/sup 3/H)inositol bisphosphate. These results indicate that ethanol-induced calcium mobilization in hepatocytes is due to the activation by ethanol of the phosphoinositide-specific phospholipase C.

  6. Kappa opioid receptors stimulate phosphoinositide turnover in rat brain

    SciTech Connect

    Periyasamy, S.; Hoss, W. )

    1990-01-01

    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.

  7. Requirement of Phosphoinositides Containing Stearic Acid To Control Cell Polarity.

    PubMed

    Doignon, François; Laquel, Patricia; Testet, Eric; Tuphile, Karine; Fouillen, Laetitia; Bessoule, Jean-Jacques

    2015-01-01

    Phosphoinositides (PIPs) are present in very small amounts but are essential for cell signaling, morphogenesis, and polarity. By mass spectrometry, we demonstrated that some PIPs with stearic acyl chains were strongly disturbed in a psi1Δ Saccharomyces cerevisiae yeast strain deficient in the specific incorporation of a stearoyl chain at the sn-1 position of phosphatidylinositol. The absence of PIPs containing stearic acid induced disturbances in intracellular trafficking, although the total amount of PIPs was not diminished. Changes in PIPs also induced alterations in the budding pattern and defects in actin cytoskeleton organization (cables and patches). Moreover, when the PSI1 gene was impaired, a high proportion of cells with bipolar cortical actin patches that occurred concomitantly with the bipolar localization of Cdc42p was specifically found among diploid cells. This bipolar cortical actin phenotype, never previously described, was also detected in a bud9Δ/bud9Δ strain. Very interestingly, overexpression of PSI1 reversed this phenotype. PMID:26711260

  8. Ciliary Phosphoinositide Regulates Ciliary Protein Trafficking in Drosophila.

    PubMed

    Park, Jina; Lee, Nayoung; Kavoussi, Adriana; Seo, Jeong Taeg; Kim, Chul Hoon; Moon, Seok Jun

    2015-12-29

    Cilia are highly specialized antennae-like cellular organelles. Inositol polyphosphate 5-phosphatase E (INPP5E) converts PI(4,5)P2 into PI4P and is required for proper ciliary function. Although Inpp5e mutations are associated with ciliopathies in humans and mice, the precise molecular role INPP5E plays in cilia remains unclear. Here, we report that Drosophila INPP5E (dINPP5E) regulates ciliary protein trafficking by controlling the phosphoinositide composition of ciliary membranes. Mutations in dInpp5e lead to hearing deficits due to the mislocalization of dTULP and mechanotransduction channels, Inactive and NOMPC, in chordotonal cilia. Both loss of dINPP5E and ectopic expression of the phosphatidylinositol-4-phosphate 5-kinase Skittles increase PI(4,5)P2 levels in the ciliary base. The fact that Skittles expression phenocopies the dInpp5e mutants confirms a central role for PI(4,5)P2 in the regulation of dTULP, Inactive, and NOMPC localization. These data suggest that the spatial localization and levels of PI(4,5)P2 in ciliary membranes are important regulators of ciliary trafficking and function. PMID:26723017

  9. Tools for visualization of phosphoinositides in the cell nucleus.

    PubMed

    Kalasova, Ilona; Fáberová, Veronika; Kalendová, Alžběta; Yildirim, Sukriye; Uličná, Lívia; Venit, Tomáš; Hozák, Pavel

    2016-04-01

    Phosphoinositides (PIs) are glycerol-based phospholipids containing hydrophilic inositol ring. The inositol ring is mono-, bis-, or tris-phosphorylated yielding seven PIs members. Ample evidence shows that PIs localize both to the cytoplasm and to the nucleus. However, tools for direct visualization of nuclear PIs are limited and many studies thus employ indirect approaches, such as staining of their metabolic enzymes. Since localization and mobility of PIs differ from their metabolic enzymes, these approaches may result in incomplete data. In this paper, we tested commercially available PIs antibodies by light microscopy on fixed cells, tested their specificity using protein-lipid overlay assay and blocking assay, and compared their staining patterns. Additionally, we prepared recombinant PIs-binding domains and tested them on both fixed and live cells by light microscopy. The results provide a useful overview of usability of the tools tested and stress that the selection of adequate tools is critical. Knowing the localization of individual PIs in various functional compartments should enable us to better understand the roles of PIs in the cell nucleus. PMID:26847181

  10. Phosphoinositide 3-kinase-gamma induces Xenopus oocyte maturation via lipid kinase activity.

    PubMed Central

    Hehl, S; Stoyanov, B; Oehrl, W; Schönherr, R; Wetzker, R; Heinemann, S H

    2001-01-01

    Type-I phosphoinositide 3-kinases (PI3Ks) were characterized as a group of intracellular signalling proteins expressing both protein and lipid kinase activities. Recent studies implicate PI3Ks as mediators of oocyte maturation, but the molecular mechanisms are poorly defined. Here we used the Xenopus oocyte expression system as a model to investigate a possible contribution of the gamma-isoform of PI3K (PI3Kgamma) in the different pathways leading to cell-cycle progression by monitoring the time course of germinal vesicle breakdown (GVBD). Expression of a constitutive active PI3Kgamma (PI3Kgamma-CAAX) induced GVBD and increased the levels of phosphorylated Akt/protein kinase B and mitogen-activated protein kinase (MAPK). Furthermore, PI3Kgamma-CAAX accelerated progesterone-induced GVBD, but had no effect on GVBD induced by insulin. The effects of PI3Kgamma-CAAX could be suppressed by pre-incubation of the oocytes with LY294002, PD98059 or roscovitine, inhibitors of PI3K, MEK (MAPK/extracellular-signal-regulated protein kinase kinase) and cdc2/cyclin B kinase, respectively. Mutants of PI3Kgamma-CAAX, in which either lipid kinase or both lipid and protein kinase activities were altered or eliminated, did not induce significant GVBD. Our data demonstrate that expression of PI3Kgamma in Xenopus oocytes accelerates their progesterone-induced maturation and that lipid kinase activity is required to induce this effect. PMID:11736661

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

    PubMed Central

    Kotak, Sachin; Busso, Coralie; Gönczy, Pierre

    2014-01-01

    The positioning and the elongation of the mitotic spindle must be carefully regulated. In human cells, the evolutionary conserved proteins LGN/Gαi1-3 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/Gαi1-3 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

  12. Impairment and reorganization of the phosphoinositide-specific phospholipase C enzymes in suicide brains.

    PubMed

    Lo Vasco, Vincenza Rita; Leopizzi, M; Della Rocca, C; Fais, P; Montisci, M; Cecchetto, G

    2015-03-15

    A number of studies suggested that suicide may be associated with specific neurobiological abnormalities. Neurobiology studies focused upon abnormalities of signalling mechanisms with special regard to the serotonin system and the related Phosphoinositide (PI) signalling system. Previous data suggested the involvement of the PI-specific phospholipase C (PLC) family in neuropsychiatric disorders. By using PCR and morphological microscopy observation we examined the whole panel of expression of PLC isoforms in the brains of 28 individuals who committed suicide and in normal controls in order to evaluate the involvement of specific PLC isoforms. The overall PLC expression was reduced and a complex reorganization of the isoforms was observed. The knowledge of the complex network of neurobiological molecules and interconnected signal transduction pathways in the brain of suicide victims might be helpful to understand the natural history and the pathogenesis of the suicidal behavior. That might lead to obtain prognostic suggestions in order to prevent suicide and to new therapeutic agents targeting specific sites in this signalling cascade. PMID:25532079

  13. Specific interactions among transmembrane 4 superfamily (TM4SF) proteins and phosphoinositide 4-kinase.

    PubMed Central

    Yauch, R L; Hemler, M E

    2000-01-01

    In earlier work we established that phosphoinositide 4-kinase (PI 4-kinase) may associate with transmembrane 4 superfamily (TM4SF, tetraspanin) proteins, but critical specificity issues were not addressed. Here we demonstrate that at least five different TM4SF proteins (CD9, CD63, CD81, CD151 and A15/TALLA1) can associate with a similar or identical 55 kDa type II PI 4-kinase. These associations were specific, since we found no evidence for other phosphoinositide kinases (e.g. phosphoinositide 3-kinase and phosphoinositide-4-phosphate 5-kinase) associating with TM4SF proteins, and many other TM4SF proteins (including CD82 and CD53) did not associate with PI 4-kinase. CD63-PI 4-kinase complexes were almost entirely intracellular, and thus are distinct from other TM4SF-PI 4-kinase complexes (e.g. involving CD9), which are largely located in the plasma membrane. These results suggest that a specific subset of TM4SF proteins may recruit PI 4-kinase to specific membrane locations, and thereby influence phosphoinositide-dependent signalling. PMID:11042117

  14. Phosphoinositides in the nucleus and myogenic differentiation: how a nuclear turtle with a PHD builds muscle.

    PubMed

    Divecha, Nullin

    2016-02-15

    Phosphoinositides are a family of phospholipid messenger molecules that control various aspects of cell biology in part by interacting with and regulating downstream protein partners. Importantly, phosphoinositides are present in the nucleus. They form part of the nuclear envelope and are present within the nucleus in nuclear speckles, intra nuclear chromatin domains, the nuclear matrix and in chromatin. What their exact role is within these compartments is not completely clear, but the identification of nuclear specific proteins that contain phosphoinositide interaction domains suggest that they are important regulators of DNA topology, chromatin conformation and RNA maturation and export. The plant homeo domain (PHD) finger is a phosphoinositide binding motif that is largely present in nuclear proteins that regulate chromatin conformation. In the present study I outline how changes in the levels of the nuclear phosphoinositide PtdIns5P impact on muscle cell differentiation through the PHD finger of TAF3 (TAF, TATA box binding protein (TBP)-associated factor), which is a core component of a number of different basal transcription complexes. PMID:26862219

  15. Effect of dual agonists on phosphoinositide pools in WRK-1 cells.

    PubMed Central

    Monaco, M E; Attinasi, M; Koréh, K

    1990-01-01

    Both vasopressin and bradykinin activate the phosphoinositide cycle in WRK-1 rat mammary tumour cells. When the two agonists are added simultaneously, partial additivity is observed with respect to disappearance of prelabelled phosphoinositides and accumulation of inositol phosphates; no additivity is observed with respect to resynthesis of phosphatidylinositol as assessed by monitoring [32P]Pi incorporation. Lack of complete additivity can be explained, at least in part, by heterologous desensitization. In order to determine whether the two agonists were accessing a common or individual hormone-sensitive phosphoinositide pools, cells were incubated with [32P]Pi in the presence of either vasopressin or bradykinin and subsequently restimulated with the alternative agonist. The lipid pool labelled in the presence of either agonist was sensitive to subsequent treatment by the other ligand, suggesting a common phosphoinositide pool. However, when cells were incubated with [32P]Pi in the absence of agonists, the time course of labelling of the hormone-sensitive pool was different for bradykinin and vasopressin, with that for bradykinin becoming labelled within a much shorter time. Thus although there is a significant overlap between the phosphoinositide pools responding to vasopressin and bradykinin, there is a small fraction of the hormone-sensitive lipid which responds only to bradykinin. PMID:2167661

  16. [Phosphoinositides: lipidic essential actors in the intracellular traffic].

    PubMed

    Bertazzi, Dimitri L; De Craene, Johan-Owen; Bär, Séverine; Sanjuan-Vazquez, Myriam; Raess, Matthieu A; Friant, Sylvie

    2015-01-01

    Phosphoinositides (PPIn) are lipids involved in the vesicular transport of proteins between the different intracellular compartments. They act by recruiting and/or activating effector proteins and are thus involved in crucial cellular functions including vesicle budding, fusion and dynamics of membranes and regulation of the cytoskeleton. Although they are present in low concentrations in membranes, their activity is essential for cell survival and needs to be tightly controlled. Therefore, phosphatases and kinases specific of the various cellular membranes can phosphorylate/dephosphorylate their inositol ring on the positions D3, D4 and/or D5. The differential phosphorylation determines the intracellular localisation and the activity of the PPIn. Indeed, non-phosphorylated phosphatidylinositol (PtdIns) is the basic component of the PPIn and can be found in all eukaryotic cells at the cytoplasmic face of the ER, the Golgi, mitochondria and microsomes. It can get phosphorylated on position D4 to obtain PtdIns4P, a PPIn enriched in the Golgi compartment and involved in the maintenance of this organelle as well as anterograde and retrograde transport to and from the Golgi. PtdIns phosphorylation on position D3 results in PtdIns3P that is required for endosomal transport and multivesicular body (MVB) formation and sorting. These monophosphorylated PtdIns can be further phosphorylated to produce bisphophorylated PtdIns. Thus, PtdIns(4,5)P2, mainly produced by PtdIns4P phosphorylation, is enriched in the plasma membrane and involved in the regulation of actin cytoskeleton and endocytosis. PtdIns(3,5)P2, mainly produced by PtdIns3P phosphorylation, is enriched in late endosomes, MVBs and the lysosome/vacuole and plays a role in endosome to vacuole transport. PtdIns(3,4)P2 is absent in yeast, cells and mainly produced by PtdIns4P phosphorylation in human cells; PtdIns(3,4)P2 is localised in the plasma membrane and plays an important role as a second messenger by recruiting specific protein kinases (Akt and PDK1). Finally the triple phosphorylated PPIn, PtdIns(3,4,5)P3 also absent in yeast, is produced by the phosphorylation of PtdIns(3,4)P2 and localized at the plasma membrane of human cells where it binds proteins via their PH domain. Interaction partners include members of the Arf (ADP-ribosylation factors) family, PDK1 (Phosphoinositide Dependent Kinase 1) and Akt. Therefore this last PPIn is essential for the control of cell proliferation and its deregulation leads to the development of numerous cancers. In conclusion, the regulation of PPIn phosphorylation/dephosphorylation is complex and needs to be very precisely regulated. Indeed phosphatases and kinases allow the maintenance of the equilibrium between the different forms. PPIn play a crucial role in numerous cellular functions and a loss in their synthesis or regulation results in severe genetic diseases. PMID:26115715

  17. Ca2+ Regulation of Trypanosoma brucei Phosphoinositide Phospholipase C

    PubMed Central

    King-Keller, Sharon; Moore, Christina A.; Docampo, Roberto

    2015-01-01

    We characterized a phosphoinositide phospholipase C (PI-PLC) from the procyclic form (PCF) of Trypanosoma brucei. The protein contains a domain organization characteristic of typical PI-PLCs, such as X and Y catalytic domains, an EF-hand calcium-binding motif, and a C2 domain, but it lacks a pleckstrin homology (PH) domain. In addition, the T. brucei PI-PLC (TbPI-PLC) contains an N-terminal myristoylation consensus sequence found only in trypanosomatid PI-PLCs. A peptide containing this N-terminal domain fused to green fluorescent protein (GFP) was targeted to the plasma membrane. TbPI-PLC enzymatic activity was stimulated by Ca2+ concentrations below the cytosolic levels in the parasite, suggesting that the enzyme is constitutively active. TbPI-PLC hydrolyzes both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2), with a higher affinity for PIP2. We found that modification of a single amino acid in the EF-hand motif greatly affected the protein's Ca2+ sensitivity and substrate preference, demonstrating the role of this motif in Ca2+ regulation of TbPI-PLC. Endogenous TbPI-PLC localizes to intracellular vesicles and might be using an intracellular source of PIP2. Knockdown of TbPI-PLC expression by RNA interference (RNAi) did not result in growth inhibition, although enzymatic activity was still present in parasites, resulting in hydrolysis of PIP2 and a contribution to the inositol 1,4,5-trisphosphate (IP3)/diacylglycerol (DAG) pathway. PMID:25769297

  18. Carbachol-induced phosphoinositide turnover in NCB-20 cells

    SciTech Connect

    Chuang, D.M.; Dillon-Carter, O.

    1986-03-01

    NCB-20 cells (fetal Chinese hamster brain cell x neuroblastoma hybrids) have been shown to contain a variety of neurotransmitter receptors. The authors now report that this cloned cell line also contains acetylcholne receptors which are linked to phospholipase C. Confluent cell cultures were preincubated with /sup 3/H-myo-inositol to label endogenous phosphoinositide (PI) and the accumulation of a PI metabolite, inositol monophosphate (IP/sub 1/), was measured in the presence of LiCl. Carbachol increased IP/sub 1/), accumulation be more than 400% with a EC/sub 50/ of about 50 ..mu..M. Acetylcholine and muscarine were also effective, whereas oxotremorine and McN-A-343 were weak in both potency and efficacy. The carbachol-induced IP/sub 1/ accumulation was completely blocked by atropine (Ki approx. 0.6 nM) and pirenzepine (Ki approx. 15 nM). The presence of KCl was not required for the carbachol-induced effect. The formation of inositol bis- and triphosphate was also increased carbachol; these increases occurred earlier but were of much smaller magnitude. Pretreatment of cells with 4 ..beta..-phorbol dibutyrate or 4 ..beta..-phorbol myristate acetate was found to attenuate the carbachol-induced formation of IP/sub 1/ (IC/sub 50/ in the low nanomolar concentration ranges), however 4 ..beta..-phorbol, the biologically inactive phorbol ester, was ineffective in causing this attenuation. These results suggest a feedback inhibition of PI turnover in NCB-20 cells through the action of protein kinase C.

  19. The role of phosphoinositide-regulated actin reorganization in chemotaxis and cell migration

    PubMed Central

    Wu, C-Y; Lin, M-W; Wu, D-C; Huang, Y-B; Huang, H-T; Chen, C-L

    2014-01-01

    Reorganization of the actin cytoskeleton is essential for cell motility and chemotaxis. Actin-binding proteins (ABPs) and membrane lipids, especially phosphoinositides PI(4,5)P2 and PI(3,4,5)P3 are involved in the regulation of this reorganization. At least 15 ABPs have been reported to interact with, or regulated by phosphoinositides (PIPs) whose synthesis is regulated by extracellular signals. Recent studies have uncovered several parallel intracellular signalling pathways that crosstalk in chemotaxing cells. Here, we review the roles of ABPs and phosphoinositides in chemotaxis and cell migration. Linked Articles This article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-24 PMID:25420930

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

    PubMed Central

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

    2013-01-01

    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

  1. Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity.

    PubMed

    Park, J; Hill, M M; Hess, D; Brazil, D P; Hofsteenge, J; Hemmings, B A

    2001-10-01

    3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased approximately 6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family. PMID:11481331

  2. Phosphoinositide 3-kinase-δ regulates fungus-induced allergic lung inflammation through endoplasmic reticulum stress

    PubMed Central

    Lee, Kyung Sun; Jeong, Jae Seok; Kim, So Ri; Cho, Seong Ho; Kolliputi, Narasaiah; Ko, Yun Hee; Lee, Kyung Bae; Park, Suk Chul; Park, Hae Jin; Lee, Yong Chul

    2016-01-01

    Background Sensitisation with Aspergillus fumigatus (Af) is known to be associated with severe allergic lung inflammation, but the mechanism remains to be clarified. Phosphoinositide 3-kinase (PI3K)-δ and endoplasmic reticulum (ER) stress are suggested to be involved in steroid-resistant lung inflammation. We aimed to elucidate the role of PI3K-δ and its relationship with ER stress in fungus-induced allergic lung inflammation. Methods Using Af-exposed in vivo and in vitro experimental systems, we examined whether PI3K-δ regulates ER stress, thereby contributing to steroid resistance in fungus-induced allergic lung inflammation. Moreover, we checked expression of an ER stress marker in lung tissues isolated from patients with allergic bronchopulmonary aspergillosis. Results Af-exposed mice showed that ER stress markers, unfolded protein response (UPR)-related proteins, phosphorylated Akt, generation of mitochondrial reactive oxygen species (mtROS), eosinophilic allergic inflammation, and airway hyperresponsiveness (AHR) were increased in the lung. Similarly, glucose-regulated protein 78 was increased in lung tissues of patients with ABPA. A PI3K-δ inhibitor reduced Af-induced increases in ER stress markers, UPR-related proteins, allergic inflammation and AHR in mice. However, dexamethasone failed to reduce Af-induced allergic inflammation, AHR and elevation of ER stress. Administration of an ER stress inhibitor or a mtROS scavenger improved Af-induced allergic inflammation. The PI3K-δ inhibitor reduced Af-induced mtROS generation and the mtROS scavenger ameliorated ER stress. In primary cultured tracheal epithelial cells, Af-induced ER stress was inhibited by blockade of PI3K-δ. Conclusions These findings suggest that PI3K-δ regulates Af-induced steroid-resistant eosinophilic allergic lung inflammation through ER stress. PMID:26543090

  3. Role of phosphoinositide 3-OH kinase p110β in skeletal myogenesis.

    PubMed

    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

    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 β (p110β) in myogenesis and metabolism. In C2C12 cells, pharmacological inhibition of p110β delayed differentiation. We next generated mice with conditional deletion of p110β in skeletal muscle (p110β muscle knockout [p110β-mKO] mice). While young p110β-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 p110β-mKO mice were less glucose tolerant than old control mice. Overexpression of p110β accelerated differentiation in C2C12 cells and primary human myoblasts through an Akt-dependent mechanism, while expression of kinase-inactive p110β had the opposite effect. p110β overexpression was unable to promote myoblast differentiation under conditions of p110α inhibition, but expression of p110α was able to promote differentiation under conditions of p110β inhibition. These findings reveal a role for p110β during myogenesis and demonstrate that long-term reduction of skeletal muscle p110β impairs whole-body glucose tolerance without affecting skeletal muscle size or strength in old mice. PMID:25605332

  4. Phosphoinositide-dependent protein kinase 1 (PDK1) mediates potent inhibitory effects on eosinophils.

    PubMed

    Sturm, Eva M; Parzmair, Gerald P; Radnai, Balázs; Frei, Robert B; Sturm, Gunter J; Hammer, Astrid; Schuligoi, Rufina; Lippe, Irmgard Th; Heinemann, Akos

    2015-05-01

    Prostaglandin E2 (PGE2 ) protects against allergic responses via binding to prostanoid receptor EP4, which inhibits eosinophil migration in a PI3K/PKC-dependent fashion. The phosphoinositide-dependent protein kinase 1 (PDK1) is known to act as a downstream effector in PI3K signaling and has been implicated in the regulation of neutrophil migration. Thus, here we elucidate whether PDK1 mediates inhibitory effects of E-type prostanoid receptor 4 (EP4) receptors on eosinophil function. Therefore, eosinophils were isolated from human peripheral blood or differentiated from mouse BM. PDK1 signaling was investigated in shape change, chemotaxis, CD11b, respiratory burst, and Ca(2+) mobilization assays. The specific PDK1 inhibitors BX-912 and GSK2334470 prevented the inhibition by prostaglandin E2 and the EP4 agonist ONO-AE1-329. Depending on the cellular function, PDK1 seemed to act through PI3K-dependent or PI3K-independent mechanisms. Stimulation of EP4 receptors caused PDK1 phosphorylation at Ser396 and induced PI3K-dependent nuclear translocation of PDK1. EP4-induced inhibition of shape change and chemotaxis was effectively reversed by the Akt inhibitor triciribine. In support of this finding, ONO-AE1-329 induced a PI3K/PDK1-dependent increase in Akt phosphorylation. In conclusion, our data illustrate a critical role for PDK1 in transducing inhibitory signals on eosinophil effector function. Thus, our results suggest that PDK1 might serve as a novel therapeutic target in diseases involving eosinophilic inflammation. PMID:25645675

  5. Coordinated Expression of Phosphoinositide Metabolic Genes during Development and Aging of Human Dorsolateral Prefrontal Cortex

    PubMed Central

    Rapoport, Stanley I.; Primiani, Christopher T.; Chen, Chuck T.; Ahn, Kwangmi; Ryan, Veronica H.

    2015-01-01

    Background Phosphoinositides, lipid-signaling molecules, participate in diverse brain processes within a wide metabolic cascade. Hypothesis Gene transcriptional networks coordinately regulate the phosphoinositide cascade during human brain Development and Aging. Methods We used the public BrainCloud database for human dorsolateral prefrontal cortex to examine age-related expression levels of 49 phosphoinositide metabolic genes during Development (0 to 20+ years) and Aging (21+ years). Results We identified three groups of partially overlapping genes in each of the two intervals, with similar intergroup correlations despite marked phenotypic differences between Aging and Development. In each interval, ITPKB, PLCD1, PIK3R3, ISYNA1, IMPA2, INPPL1, PI4KB, and AKT1 are in Group 1, PIK3CB, PTEN, PIK3CA, and IMPA1 in Group 2, and SACM1L, PI3KR4, INPP5A, SYNJ1, and PLCB1 in Group 3. Ten of the genes change expression nonlinearly during Development, suggesting involvement in rapidly changing neuronal, glial and myelination events. Correlated transcription for some gene pairs likely is facilitated by colocalization on the same chromosome band. Conclusions Stable coordinated gene transcriptional networks regulate brain phosphoinositide metabolic pathways during human Development and Aging. PMID:26168237

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

    PubMed Central

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

    2012-01-01

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

  7. Structure-Function Analysis of Vaccinia Virus H7 Protein Reveals a Novel Phosphoinositide Binding Fold Essential for Poxvirus Replication

    PubMed Central

    Kolli, Swapna; Meng, Xiangzhi; Wu, Xiang; Shengjuler, Djoshkun; Cameron, Craig E.

    2014-01-01

    ABSTRACT Phosphoinositides and phosphoinositide binding proteins play a critical role in membrane and protein trafficking in eukaryotes. Their critical role in replication of cytoplasmic viruses has just begun to be understood. Poxviruses, a family of large cytoplasmic DNA viruses, rely on the intracellular membranes to develop their envelope, and poxvirus morphogenesis requires enzymes from the cellular phosphoinositide metabolic pathway. However, the role of phosphoinositides in poxvirus replication remains unclear, and no poxvirus proteins show any homology to eukaryotic phosphoinositide binding domains. Recently, a group of poxvirus proteins, termed viral membrane assembly proteins (VMAPs), were identified as essential for poxvirus membrane biogenesis. A key component of VMAPs is the H7 protein. Here we report the crystal structure of the H7 protein from vaccinia virus. The H7 structure displays a novel fold comprised of seven α-helices and a highly curved three-stranded antiparallel β-sheet. We identified a phosphoinositide binding site in H7, comprised of basic residues on a surface patch and the flexible C-terminal tail. These residues were found to be essential for viral replication and for binding of H7 to phosphatidylinositol-3-phosphate (PI3P) and phosphatidylinositol-4-phosphate (PI4P). Our studies suggest that phosphoinositide binding by H7 plays an essential role in poxvirus membrane biogenesis. IMPORTANCE Poxvirus viral membrane assembly proteins (VMAPs) were recently shown to be essential for poxvirus membrane biogenesis. One of the key components of VMAPs is the H7 protein. However, no known structural motifs could be identified from its sequence, and there are no homologs of H7 outside the poxvirus family to suggest a biochemical function. We have determined the crystal structure of the vaccinia virus (VACV) H7 protein. The structure displays a novel fold with a distinct and positively charged surface. Our data demonstrate that H7 binds phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate and that the basic surface patch is indeed required for phosphoinositide binding. In addition, mutation of positively charged residues required for lipid binding disrupted VACV replication. Phosphoinositides and phosphoinositide binding proteins play critical roles in membrane and protein trafficking in eukaryotes. Our study demonstrates that VACV H7 displays a novel fold for phosphoinositide binding, which is essential for poxvirus replication. PMID:25473060

  8. Characterization of the binding between a 70-kDa heat shock protein, HspA1A, and phosphoinositides.

    PubMed

    McCallister, Chelsea; Kdeiss, Brianna; Oliverio, Ryan; Nikolaidis, Nikolas

    2016-03-25

    HspA1A, a seventy-kilodalton heat shock protein, binds to specific anionic lipids and this interaction regulates important physiological phenomena like apoptosis, tumor growth, and lysosomal rescue. However, whether HspA1A binds to phosphoinositides has yet to be established and quantified. Therefore, in this study, we determined the binding affinity of HspA1A to several phosphoinositides and characterized five aspects of their molecular interaction. First, we established that HspA1A binds phosphatidylinositol monophosphates with higher affinity than di- and triphosphorylated inositides. Second, using high concentrations of potassium we found that HSPA1A embeds within the lipid bilayer of all phosphoinositides tested. However, the effects of the high salt concentrations were significantly different between the different phosphoinositides. Third, using calcium and reaction buffers equilibrated at different pH values we found that these differentially affected HspA1A-phosphoinositide binding, revealing a lipid-specific pattern of binding. Fourth, by assessing the binding properties of the two HspA1A domains, the nucleotide-binding domain and the substrate-binding domain, we determined that in most cases the full-length protein is necessary for binding to phosphoinositides. Fifth, by including in the reactions nucleotides and protein substrates we determined that they minimally and differentially affected phosphoinositide-binding. Collectively, these findings strongly suggest that the HspA1A-phosphoinositide binding is complex yet specific, is mediated by both electrostatic and hydrophobic interactions, is not related to the lipid-head charge, and depends on the physicochemical properties of the lipid. PMID:26923070

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

    PubMed

    Leondaritis, George; Tiedtke, Arno; Galanopoulou, Dia

    2005-09-30

    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

  10. The p110α isoform of phosphoinositide 3-kinase is essential for cone photoreceptor survival.

    PubMed

    Rajala, Raju V S; Ranjo-Bishop, Michelle; Wang, Yuhong; Rajala, Ammaji; Anderson, Robert E

    2015-05-01

    Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylates the 3'OH of the inositol ring of phosphoinositides (PIs). They are responsible for coordinating a diverse range of cellular functions. Class IA PI3K is a heterodimeric protein composed of a regulatory p85 and a catalytic p110 subunit. In this study, we conditionally deleted the p110α-subunit of PI3K in cone photoreceptor cells using the Cre-loxP system. Cone photoreceptors allow for color vision in bright light (daylight vision). Cone-specific deletion of p110α resulted in cone degeneration. Our studies suggest that PI3K signaling is essential for cone photoreceptor functions. PMID:25742742

  11. Exploiting the ubiquitin and phosphoinositide pathways by the Legionella pneumophila effector, SidC.

    PubMed

    Wasilko, David J; Mao, Yuxin

    2016-02-01

    Intracellular bacterial pathogens use secreted effector proteins to alter host cellular processes, with the goal of subverting host defenses and allowing the infection to progress. One such pathogen, Legionella pneumophila, secretes ~300 proteins into its host to alter a number of pathways including intracellular trafficking, phosphoinositide metabolism, and cell signaling. The Legionella effector SidC was previously found to bind to PI(4)P and was responsible for the enrichment of ER proteins and ubiquitinated species on the Legionella-containing vacuoles. Through our recent work, we have discovered that SidC contains a unique N-terminal E3 ubiquitin ligase domain and a C-terminal novel PI(4)P-binding domain. Our results demonstrate that SidC serves to link two distinct cellular pathways, ubiquitin and phosphoinositide. However, how the ubiquitin ligase activity regulates host membrane trafficking events remains to be investigated. PMID:26433729

  12. Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS.

    PubMed

    Ludolphs, Michaela; Schneeberger, Daniela; Soykan, Tolga; Schäfer, Jonas; Papadopoulos, Theofilos; Brose, Nils; Schindelin, Hermann; Steinem, Claudia

    2016-01-01

    The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally "opens" CB2SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering. PMID:26546675

  13. Phosphoinositide 3-Kinase-C2α Regulates Polycystin-2 Ciliary Entry and Protects against Kidney Cyst Formation.

    PubMed

    Franco, Irene; Margaria, Jean Piero; De Santis, Maria Chiara; Ranghino, Andrea; Monteyne, Daniel; Chiaravalli, Marco; Pema, Monika; Campa, Carlo Cosimo; Ratto, Edoardo; Gulluni, Federico; Perez-Morga, David; Somlo, Stefan; Merlo, Giorgio R; Boletta, Alessandra; Hirsch, Emilio

    2016-04-01

    Signaling from the primary cilium regulates kidney tubule development and cyst formation. However, the mechanism controlling targeting of ciliary components necessary for cilium morphogenesis and signaling is largely unknown. Here, we studied the function of class II phosphoinositide 3-kinase-C2α(PI3K-C2α) in renal tubule-derived inner medullary collecting duct 3 cells and show that PI3K-C2αresides at the recycling endosome compartment in proximity to the primary cilium base. In this subcellular location, PI3K-C2αcontrolled the activation of Rab8, a key mediator of cargo protein targeting to the primary cilium. Consistently, partial reduction of PI3K-C2αwas sufficient to impair elongation of the cilium and the ciliary transport of polycystin-2, as well as to alter proliferation signals linked to polycystin activity. In agreement, heterozygous deletion of PI3K-C2αin mice induced cilium elongation defects in kidney tubules and predisposed animals to cyst development, either in genetic models of polycystin-1/2 reduction or in response to ischemia/reperfusion-induced renal damage. These results indicate that PI3K-C2αis required for the transport of ciliary components such as polycystin-2, and partial loss of this enzyme is sufficient to exacerbate the pathogenesis of cystic kidney disease. PMID:26271513

  14. TbFRP, a novel FYVE-domain containing phosphoinositide-binding Ras-like GTPase from trypanosomes

    PubMed Central

    Adung’a, Vincent O.; Field, Mark C.

    2013-01-01

    Ras-like small GTPases are regulatory proteins that control multiple aspects of cellular function, and are particularly prevalent in vesicular transport. A proportion of GTPase paralogs appear restricted to certain eukaryote lineages, suggesting roles specific to a restricted lineage, and hence potentially reflecting adaptation to individual lifestyles or ecological niche. Here we describe the role of a GTPase, TbFRP, a FYVE domain N-terminally fused to a Ras-like GTPase, originally identified in Trypanosoma brucei. As FYVE-domains specifically bind phosphoinositol 3-phosphate (PI3P), which associates with endosomes, we suggest that TbFRP may unite phosphoinositide and small G protein endosomal signaling in trypanosomatids. TbFRP orthologs are present throughout the Euglenazoa suggesting that FRP has functions throughout the group. We show that the FYVE domain of TbFRP is functional in PI3P-dependent membrane targeting and localizes at the endosomal region. Further, while TbFRP is apparently non-essential, knockdown and immunochemical evidence indicates that TbFRP is rapidly cleaved upon synthesis, releasing the GTPase and FYVE-domains. Finally, TbFRP expression at both mRNA and protein levels is cell density-dependent. Together, these data suggest that TbFRP is an endocytic GTPase with a highly unusual mechanism of action that involves proteolysis of the nascent protein and membrane targeting via PI3P. PMID:23220323

  15. TbFRP, a novel FYVE-domain containing phosphoinositide-binding Ras-like GTPase from trypanosomes.

    PubMed

    Adung'a, Vincent O; Field, Mark C

    2013-03-01

    Ras-like small GTPases are regulatory proteins that control multiple aspects of cellular function, and are particularly prevalent in vesicular transport. A proportion of GTPase paralogs appear restricted to certain eukaryote lineages, suggesting roles specific to a restricted lineage, and hence potentially reflecting adaptation to individual lifestyles or ecological niche. Here we describe the role of a GTPase, TbFRP, a FYVE domain N-terminally fused to a Ras-like GTPase, originally identified in Trypanosoma brucei. As FYVE-domains specifically bind phosphoinositol 3-phosphate (PI3P), which associates with endosomes, we suggest that TbFRP may unite phosphoinositide and small G protein endosomal signaling in trypanosomatids. TbFRP orthologs are present throughout the Euglenazoa suggesting that FRP has functions throughout the group. We show that the FYVE domain of TbFRP is functional in PI3P-dependent membrane targeting and localizes at the endosomal region. Further, while TbFRP is apparently non-essential, knockdown and immunochemical evidence indicates that TbFRP is rapidly cleaved upon synthesis, releasing the GTPase and FYVE-domains. Finally, TbFRP expression at both mRNA and protein levels is cell density-dependent. Together, these data suggest that TbFRP is an endocytic GTPase with a highly unusual mechanism of action that involves proteolysis of the nascent protein and membrane targeting via PI3P. PMID:23220323

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

    SciTech Connect

    Yarbrough, Wendell G. Whigham, Amy; Brown, Brandee; Roach, Michael; Slebos, Robbert

    2007-10-01

    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.

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

    SciTech Connect

    Not Available

    1986-03-01

    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.

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

    PubMed

    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

    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

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

    PubMed Central

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

    2013-01-01

    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

  20. Stromal resistance of fibroblasts against oxidative damage: involvement of tumor cell-secreted platelet-derived growth factor (PDGF) and phosphoinositide 3-kinase (PI3K) activation.

    PubMed

    Werth, Christel; Stuhlmann, Dominik; Cat, Bahar; Steinbrenner, Holger; Alili, Lirija; Sies, Helmut; Brenneisen, Peter

    2008-02-01

    A critical step in tumor progression is the interaction of malignant and stromal cells via paracrine mechanisms. Stromal cells, particularly fibroblasts, support cancer cells in invasion of the surrounding tissue for access to the vascular system. Here, the question is addressed of whether tumor cells induce 'stromal resistance', i.e. protect the microenvironment from oxidative damage. The supernatant of cultured skin-derived tumor cells was added to fibroblasts and was shown to protect the fibroblasts from hydrogen peroxide-mediated cell toxicity. The platelet-derived growth factor secreted from the cancer cells was identified as trigger of this protection in fibroblasts via the phosphoinositide 3-kinase pathway. These data suggest that prosurvival signals in stromal fibroblasts as initiated by tumor cells constitute a strategy of 'stromal resistance', illustrating a novel biological role of fibroblasts for the tumor microenvironment. PMID:18174235

  1. The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian target of rapamycin following eccentric contractions.

    PubMed

    O'Neil, T K; Duffy, L R; Frey, J W; Hornberger, T A

    2009-07-15

    Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K-PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K-PKB is a transient event (<15 min), while the activation of mTOR is sustained for a long duration (>12 h). Furthermore, inhibition of PI3K-PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K-PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K-PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K-PKB-independent mechanism that requires PLD and PA. PMID:19470781

  2. Phosphoinositide-mediated oligomerization of a defensin induces cell lysis

    PubMed Central

    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

    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

  3. Phosphoinositide signalling in type 2 diabetes: a β-cell perspective.

    PubMed

    Rameh, Lucia E; Deeney, Jude T

    2016-02-15

    Type 2 diabetes is a complex disease. It results from a failure of the body to maintain energy homoeostasis. Multicellular organisms have evolved complex strategies to preserve a relatively stable internal nutrient environment, despite fluctuations in external nutrient availability. This complex strategy involves the co-ordinated responses of multiple organs to promote storage or mobilization of energy sources according to the availability of nutrients and cellular bioenergetics needs. The endocrine pancreas plays a central role in these processes by secreting insulin and glucagon. When this co-ordinated effort fails, hyperglycaemia and hyperlipidaemia develops, characterizing a state of metabolic imbalance and ultimately overt diabetes. Although diabetes is most likely a collection of diseases, scientists are starting to identify genetic components and environmental triggers. Genome-wide association studies revealed that by and large, gene variants associated with type 2 diabetes are implicated in pancreatic β-cell function, suggesting that the β-cell may be the weakest link in the chain of events that results in diabetes. Thus, it is critical to understand how environmental cues affect the β-cell. Phosphoinositides are important 'decoders' of environmental cues. As such, these lipids have been implicated in cellular responses to a wide range of growth factors, hormones, stress agents, nutrients and metabolites. Here we will review some of the well-established and potential new roles for phosphoinositides in β-cell function/dysfunction and discuss how our knowledge of phosphoinositide signalling could aid in the identification of potential strategies for treating or preventing type 2 diabetes. PMID:26862218

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

    SciTech Connect

    Balduini, W.; Murphy, S.D.; Costa, L.G. )

    1990-05-01

    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%.

  5. Modulation of phosphoinositide metabolism in aortic smooth muscle cells by allylamine

    SciTech Connect

    Cox, L.R.; Murphy, S.K.; Ramos, K. )

    1990-08-01

    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.

  6. Phosphoinositides, kinases and adaptors coordinating endocytosis in Trypanosoma brucei

    PubMed Central

    Manna, Paul T; Field, Mark C

    2015-01-01

    In the kinetoplastid parasite Trypanosoma brucei clathrin-mediated endocytosis is essential for survival and aids immune evasion in the mammalian host. The formation of endocytic clathrin coated vesicles in T. brucei is via a unique mechanism owing to an evolutionarily recent loss of the adaptor protein (AP)2 complex, a central hub in endocytic vesicle assembly. Despite this loss, recent studies examining endocytic clathrin coat assembly have highlighted a high degree of conservation between trypanosomes and their mammalian hosts. In particular phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and its putative effectors, TbCALM and TbEpsinR, are central to clathrin-mediated endocytosis in the trypanosome, just as they are in animal cells. In addition to providing insights into the cell biology of T. brucei, these studies also suggest an ancient, possibly pan-eukaryotic connection between PtdIns(4,5)P2 and endocytosis. PMID:27064836

  7. Spatial Regulation of Membrane Fusion Controlled by Modification of Phosphoinositides

    PubMed Central

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

    2010-01-01

    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 (PLCγ), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P2) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism. PMID:20808914

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

    SciTech Connect

    Van Aller, Glenn S.; Carson, Jeff D.; Tang, Wei; Peng, Hao; Zhao, Lin; Copeland, Robert A.; Tummino, Peter J.; Luo, Lusong

    2011-03-11

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

  9. Modulation of Ciliary Phosphoinositide Content Regulates Trafficking and Sonic Hedgehog Signaling Output.

    PubMed

    Chvez, Marcelo; Ena, Sabrina; Van Sande, Jacqueline; de Kerchove d'Exaerde, Alban; Schurmans, Stphane; Schiffmann, Serge N

    2015-08-10

    Ciliary transport is required for ciliogenesis, signal transduction, and trafficking of receptors to the primary cilium. Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) have been associated with ciliary dysfunction; however, its role in regulating ciliary phosphoinositides is unknown. Here we reportthat in neural stem cells, phosphatidylinositol 4-phosphate (PI4P) is found in high levels in cilia whereas phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is not detectable. Upon INPP5E inactivation, PI(4,5)P2 accumulates at the ciliary tip whereas PI4P is depleted. This is accompanied by recruitment of the PI(4,5)P2-interacting protein TULP3 to the ciliary membrane, along with Gpr161. This results in an increased production of cAMP and a repression of the Shh transcription gene Gli1. Our results reveal the link between ciliary regulation of phosphoinositides by INPP5E and Shh regulation via ciliary trafficking of TULP3/Gpr161 and also provide mechanistic insight into ciliary alterations found in Joubert and MORM syndromes resulting from INPP5E mutations. PMID:26190144

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

    PubMed

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

    2015-05-01

    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

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

    PubMed

    Ortega, X; Pérez, L M

    2001-01-01

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

  12. Dynamic formation of ER-PM junctions presents a lipid phosphatase to regulate phosphoinositides.

    PubMed

    Dickson, Eamonn J; Jensen, Jill B; Vivas, Oscar; Kruse, Martin; Traynor-Kaplan, Alexis E; Hille, Bertil

    2016-04-11

    Endoplasmic reticulum-plasma membrane (ER-PM) contact sites play an integral role in cellular processes such as excitation-contraction coupling and store-operated calcium entry (SOCE). Another ER-PM assembly is one tethered by the extended synaptotagmins (E-Syt). We have discovered that at steady state, E-Syt2 positions the ER and Sac1, an integral ER membrane lipid phosphatase, in discrete ER-PM junctions. Here, Sac1 participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2 Activation of G protein-coupled receptors that deplete PM PI(4,5)P2disrupts E-Syt2-mediated ER-PM junctions, reducing Sac1's access to the PM and permitting PM PI(4)P and PI(4,5)P2to recover. Conversely, depletion of ER luminal calcium and subsequent activation of SOCE increases the amount of Sac1 in contact with the PM, depleting PM PI(4)P. Thus, the dynamic presence of Sac1 at ER-PM contact sites allows it to act as a cellular sensor and controller of PM phosphoinositides, thereby influencing many PM processes. PMID:27044890

  13. Late Simian virus 40 transcription factor is a target of the phosphoinositide 3-kinase/Akt pathway in anti-apoptotic Alzheimer's amyloid precursor protein signalling.

    PubMed Central

    Kashour, Tarek; Burton, Teralee; Dibrov, Alexander; Amara, Francis M

    2003-01-01

    The association of familial Alzheimer's disease (FAD) with mutations in Alzheimer's amyloid precursor protein (APP) suggests important functions for APP in the central nervous system. Mutations in APP impair its function to confer resistance to apoptosis in cells under stress, and this may contribute to neurodegeneration in Alzheimer's disease (AD) brain, but the mechanisms involved are unknown. We examined the role of the late Simian virus 40 transcription factor (LSF), in anti-apoptotic APP pathways. We show that in APP-deficient B103 cells, expression of wild-type human APP (hAPPwt), but not of FAD-mutant APP, inhibited staurosporine (STS)-induced apoptosis. This inhibition was further enhanced by expression of LSFwt, although LSFwt alone was not sufficient to inhibit STS-induced apoptosis. In contrast, expression of dominant-negative LSF led to a marked increase in STS-induced cell death that was significantly blocked by hAPPwt. These effects of APP were accompanied by LSF nuclear translocation and dependent gene transcription. The activation of LSF is dependent on the expression of hAPPwt and is inhibited by the expression of dominant-negative forms of either phosphoinositide 3-kinase or Akt. These results demonstrate that LSF activation is required for the neuroprotective effects of APP via phosphoinositide 3-kinase/Akt signalling. Alterations in this pathway by aberrations in APP and/or LSF could promote neuronal loss in AD brain, due to secondary insults. Thus a link is established between APP and LSF and AD. PMID:12472467

  14. Pulmonary administration of phosphoinositide 3-kinase inhibitor is a curative treatment for chronic obstructive pulmonary disease by alveolar regeneration.

    PubMed

    Horiguchi, Michiko; Oiso, Yuki; Sakai, Hitomi; Motomura, Tomoki; Yamashita, Chikamasa

    2015-09-10

    Chronic obstructive pulmonary disease (COPD) is an intractable pulmonary disease, causing widespread and irreversible alveoli collapse. The discovery of a low-molecular-weight compound that induces regeneration of pulmonary alveoli is of utmost urgency to cure intractable pulmonary diseases such as COPD. However, a practically useful compound for regenerating pulmonary alveoli is yet to be reported. Previously, we have elucidated that Akt phosphorylation is involved in a differentiation-inducing molecular mechanism of human alveolar epithelial stem cells, which play a role in regenerating pulmonary alveoli. In the present study, we directed our attention to phosphoinositide 3-kinase (PI3K)-Akt signaling and examined whether PI3K inhibitors display the pulmonary alveolus regeneration. Three PI3K inhibitors with different PI3K subtype specificities (Wortmannin, AS605240, PIK-75 hydrochloride) were tested for the differentiation-inducing effect on human alveolar epithelial stem cells, and Wortmannin demonstrated the most potent differentiation-inducing activity. We evaluated Akt phosphorylation in pulmonary tissues of an elastase-induced murine COPD model and found that Akt phosphorylation in the pulmonary tissue was enhanced in the murine COPD model compared with normal mice. Then, the alveolus-repairing effect of pulmonary administration of Wortmannin to murine COPD model was evaluated using X-ray CT analysis and hematoxylin-eosin staining. As a result, alveolar damages were repaired in the Wortmannin-administered group to a similar level of normal mice. Furthermore, pulmonary administration of Wortmannin induced a significant recovery of the respiratory function, compared to the control group. These results indicate that Wortmannin is capable of inducing differentiation of human alveolar epithelial stem cells and represents a promising drug candidate for curative treatment of pulmonary alveolar destruction in COPD. PMID:26160307

  15. Euphorbia fischeriana Steud inhibits malignant melanoma via modulation of the phosphoinositide-3-kinase/Akt signaling pathway

    PubMed Central

    DONG, MENG-HUA; ZHANG, QIAN; WANG, YUAN-YUAN; ZHOU, BAI-SUI; SUN, YU-FEI; FU, QIANG

    2016-01-01

    Euphorbia fischeriana Steud, a traditional Chinese medicine, has been shown to inhibit the growth of various cancers by the induction of apoptosis and cell cycle arrest. The purpose of the present study was to investigate the association between the phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway and the inhibitory effect of Euphorbia fischeriana Steud on the growth and metastasis of melanoma B16 cells in vitro, and the underlying mechanisms. MTT assay results indicated that Euphorbia fischeriana Steud inhibited the growth of B16 cells in a time- and dose-dependent manner. Flow cytometric analysis revealed that Euphorbia fischeriana Steud markedly induced apoptosis of the B16 cells, with arrest at the G0/G1 phase of the cell cycle. In addition, in a Transwell assay Euphorbia fischeriana Steud significantly suppressed the migration of B16 cells. Western blot analysis revealed that the expression levels of phosphatase and tensin homolog (PTEN) were upregulated, and the phosphorylation of Akt was downregulated, which resulted in inhibition of the PI3K/Akt signaling pathway and the eventual suppression of its downstream targets, such as matrix metalloproteinase-2 mRNA, in B16 cells. The results demonstrated that Euphorbia fischeriana Steud inhibited the growth and migration of B16 cells, possibly via modulation of the PI3K/Akt signaling pathway and upregulation of PTEN expression levels, in addition to downregulation of p-Akt expression. The aforementioned findings suggest that Euphorbia fischeriana Steud may have broad therapeutic applications in the treatment of malignant melanoma. PMID:27073468

  16. Dual 4- and 5-phosphatase activities regulate SopB-dependent phosphoinositide dynamics to promote bacterial entry.

    PubMed

    Piscatelli, Heather L; Li, Menghan; Zhou, Daoguo

    2016-05-01

    Salmonella are able to invade non-phagocytic cells such as intestinal epithelial cells by modulating the host actin cytoskeleton to produce membrane ruffles. Two type III effector proteins SopB and SopE play key roles to this modulation. SopE is a known guanine nucleotide exchange factor (GEF) capable of activating Rac1 and CDC42. SopB is a phosphatidylinositol 4-phosphatase and 5-phosphatase promoting membrane ruffles and invasion of Salmonella through undefined mechanisms. Previous studies have demonstrated that the 4-phosphatase activity of SopB is required for PtdIns-3-phosphate (PtdIns(3)P) accumulation and SopB-mediated invasion. We show here that both the 4-phosphatase as well as the 5-phosphatase activities of SopB are essential in ruffle formation and subsequent invasion. We found that the 5-phosphatase activity of SopB is likely responsible for generating PtdIns-3,4-bisphosphate (PtdIns(3,4)P2 ) and subsequent recruitment of sorting nexin 9 (SNX9), an actin modulating protein. Intriguingly, the 4-phosphatase activity is responsible for the dephosphorylation of PtdIns(3,4)P2 into PtdIns(3)P. Alone, neither activity is sufficient for ruffling but when acting in conjunction with one another, the 4-phosphatase and 5-phosphatase activities led to SNX9-mediated ruffling and Salmonella invasion. This work reveals the unique ability of bacterial effector protein SopB to utilize both its 4- and 5-phosphatase activities to regulate phosphoinositide dynamics to promote bacterial entry. PMID:26537021

  17. Microscopic modes and free energies of 3-phosphoinositide-dependent kinase-1 (PDK1) binding with celecoxib and other inhibitors.

    PubMed

    Abdulhameed, Mohamed Diwan M; Hamza, Adel; Zhan, Chang-Guo

    2006-12-28

    Celecoxib, also known as Celebrex (approved by FDA in 1998) and remembered as the fastest-selling drug in history, was used as a cyclooxygenase-2 (COX-2) selective inhibitor having both anti-inflammatory and anticancer activities. Most recent studies have revealed that the apoptotic activity of celecoxib (and its derivatives) is actually independent of the COX-2 inhibitory activity and that celecoxib also inhibits the kinase activity of 3-phosphoinositide-dependent protein kinase-1 (PDK1), suggesting that the well-known anticancer activity of celecoxib is not due to the inhibition of COX-2, but possibly is due to the inhibition of PDK1. It is highly desirable to develop new celecoxib derivatives as PDK1-specifc inhibitors to avoid the side effects of COX-2 inhibitors. To understand how PDK1 binds with celecoxib and its derivatives, we have performed extensive molecular docking and combined molecular dynamics (MD) simulations and molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations on eight representative PDK1 inhibitors, leading to the finding of a new, more favorable binding mode which is remarkably different from the previously proposed binding mode. Based on the determined most stable binding structures, the calculated binding free energies are all in good agreement with the corresponding experimental data, and the biological activity data available for celecoxib and its derivatives can be better interpreted. The obtained new insights, concerning both the binding mode and computational protocol, will be valuable not only for future rational design of novel, more potent PDK1-specific inhibitors as promising anticancer therapeutics, but also for rational design of drugs targeting other proteins. PMID:17181296

  18. Depletion of plasma membrane PtdIns(4,5)P2 reveals essential roles for phosphoinositides in flagellar biogenesis

    PubMed Central

    Wei, Ho-Chun; Rollins, Janet; Fabian, Lacramioara; Hayes, Madeline; Polevoy, Gordon; Bazinet, Christopher; Brill, Julie A.

    2011-01-01

    Summary Axonemes are microtubule-based organelles of crucial importance in the structure and function of eukaryotic cilia and flagella. Despite great progress in understanding how axonemes are assembled, the signals that initiate axoneme outgrowth remain unknown. Here, we identified phosphatidylinositol phosphates (phosphoinositides) as key regulators of early stages of axoneme outgrowth in Drosophila melanogaster spermatogenesis. In a study of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] function in developing Drosophila male germ cells, we depleted PtdIns(4,5)P2 by expression of a potent phosphoinositide phosphatase. Phosphatase expression dramatically inhibited sperm tail formation and perturbed microtubule organization in a manner reversible by co-expression of a PtdIns 4-phosphate 5-kinase. Depletion of PtdIns(4,5)P2 caused increased levels of basal body γ-tubulin and altered the distribution of proteins known to be required for axoneme assembly. Examination of PtdIns(4,5)P2-depleted spermatids by transmission electron microscopy revealed defects in basal body docking to the nuclear envelope, and in axoneme architecture and integrity of the developing flagellar axoneme and axial sheath. Our results provide the first evidence that phosphoinositides act at several steps during flagellar biogenesis, coordinately regulating microtubule and membrane organization. They further suggest that phosphoinositides play evolutionarily conserved roles in flagella and cilia, across phyla and in structurally diverse cell types. PMID:18334551

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

    SciTech Connect

    Kagiwada, Satoshi Hashimoto, Misa

    2007-12-28

    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.

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

    PubMed Central

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

    2014-01-01

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

  1. Phosphoinositide 3-kinase signalling in breast cancer: how big a role might it play?

    PubMed Central

    Fry, Michael John

    2001-01-01

    Phosphoinositide 3-kinase (PI3K) was first identified as a lipid kinase activity associated with the products of viral oncogenes and with activated protein-tyrosine kinases. Since those early studies, the PI3K superfamily has grown to embrace at least 12 structurally and functionally related enzymes present in the human genome, some of which have protein kinase activity but not lipid kinase activity. Evidence is emerging that PI3K superfamily members, and components of PI3K signalling, play a role in the development of many human cancers. In this review, the PI3K family of enzymes and their signalling is reviewed, with particular reference to possible involvement in breast cancer. PMID:11597319

  2. Platelet alpha-2 adrenergic receptor-mediated phosphoinositide responses in endogenous depression

    SciTech Connect

    Mori, Hideki ); Koyama, Tsukasa; Yamashita, Itaru )

    1991-01-01

    We have previously indicated that epinephrine stimulates phosphoinositide (PI) hydrolysis by activating alpha-2 adrenergic receptors in human platelets. This method involves the measurement of the accumulation of ({sup 3}H)-inositol-1-phosphate (IP-1) as an index of Pl hydrolysis; lithium is added to inhibit the metabolism of IP-1, thus giving an enhanced signal. In the present study, we assessed the platelet alpha-2 adrenergic receptor-mediated PI responses in samples from 15 unmedicated patients with endogenous depression and 15 age- and sex-matched control subjects. The responses to epinephrine in the depressed patients were significantly higher than those of the controls, whereas the basal values did not differ significantly. These results support the hypothesis that platelet alpha-2 adrenergic receptors may be supersensitive in patients with endogenous depression.

  3. The serine kinase phosphoinositide-dependent kinase 1 (PDK1) regulates T cell development.

    PubMed

    Hinton, Heather J; Alessi, Dario R; Cantrell, Doreen A

    2004-05-01

    T lymphocyte activation is associated with activation of diverse AGC serine kinases (named after family members protein kinase A, protein kinase G and protein kinase C). It has been difficult to assess the function of these molecules in T cell development with simple gene-deletion strategies because different isoforms of AGC kinases are coexpressed in the thymus and have overlapping, redundant functions. To circumvent these problems, we explored the consequences of genetic manipulation of phosphoinositide-dependent kinase 1 (PDK1), a rate-limiting 'upstream' activator of AGC kinases. Here we analyzed the effect of PDK1 deletion on T lineage development. We also assessed the consequences of reducing PDK1 levels to 10% of normal. Complete PDK1 loss blocked T cell differentiation in the thymus, whereas reduced PDK1 expression allowed T cell differentiation but blocked proliferative expansion. These studies show that AGC family kinases are essential for T cell development. PMID:15077109

  4. Regulation of calcium and phosphoinositides at endoplasmic reticulum-membrane junctions.

    PubMed

    Dickson, Eamonn J; Jensen, Jill B; Hille, Bertil

    2016-04-15

    Effective cellular function requires both compartmentalization of tasks in space and time, and coordination of those efforts. The endoplasmic reticulum's (ER) expansive and ramifying structure makes it ideally suited to serve as a regulatory platform for organelle-organelle communication through membrane contacts. These contact sites consist of two membranes juxtaposed at a distance less than 30 nm that mediate the exchange of lipids and ions without the need for membrane fission or fusion, a process distinct from classical vesicular transport. Membrane contact sites are positioned by organelle-specific membrane-membrane tethering proteins and contain a growing number of additional proteins that organize information transfer to shape membrane identity. Here we briefly review the role of ER-containing membrane junctions in two important cellular functions: calcium signalling and phosphoinositide processing. PMID:27068956

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

    PubMed Central

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

    2014-01-01

    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

  6. Alternative Splicing Governs Cone Cyclic Nucleotide-gated (CNG) Channel Sensitivity to Regulation by Phosphoinositides*

    PubMed Central

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

    2014-01-01

    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

  7. Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

    SciTech Connect

    Martinson, E.A.; Goldstein, D.; Brown, J.H. )

    1989-09-05

    We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of (3H)choline and (3H)phosphorylcholine ((3H)Pchol) from cells containing (3H)choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of (3H)phosphatidic acid ((3H)PA) in cells containing (3H)myristate-labeled PC. (3H)Diacylglycerol ((3H)DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with (3H)myristate and (14C)arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol.

  8. 8-(Tosylamino)quinoline inhibits tumour progression through targeting phosphoinositide-3-kinase/Akt pathway.

    PubMed

    Jung, Yongwoo; Yi, Young-Su; Yoo, Dae Sung; Kim, Ji Hye; Yang, Woo Seok; Lee, Jongsung; Park, Kye Won; Kweon, Dae-Hyuk; Hong, Sungyoul; Cho, Jae Youl

    2013-02-01

    We examined whether 8-(tosylamino)quinoline (8-TQ), a structural analogue of BAY 11-7082, is able to modulate various tumourigenic responses using various in vitro and in vivo experimental conditions. 8-TQ exhibited the strongest suppressive activity on the proliferation of C6, A431, HeLa and MDA-MB-231 cells with IC550 values ranging from 10 to 30 microM. According to the analysis of level of active caspase-3, and morphologies of C6, HeLa and MDA-MB-231 cells, it was revealed that 8-TQ is able to induce apoptosis. Furthermore, this compound strongly diminished the invasion of MDA-MB-231 cells, the migration of HeLa cells, and the new generation of blood vessels under non-toxic conditions. Reduction of the phospho-form levels of intracellular signalling enzymes by 8-TQ strongly indicated that molecular signalling machineries composed of phosphoinositide 3-kinase (PI3K)/phosphoinositide-dependent kinase-1 (PDK1)/Akt and extracellular-signal-regulated kinase (ERK) could be targeted by 8-TQ treatment. Indeed, the specific inhibitors (LY294002 and U0126) of PI3K/PDK1/Akt and ERK showed similar anti-cancer properties to 8-TQ. Finally, 8-TQ intraperitoneally injected suppressed the increase of tumour volume up to 40% compared to vehicle-treated control. Taken together, our results clearly suggest that 8-TQ might have applications as a novel anti-cancer drug or may be served as a lead compound to be further optimized. PMID:23469688

  9. Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation

    SciTech Connect

    Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

    2014-07-18

    Highlights: • Lithium suppresses Akt activity by reducing PI3K-mediated Akt phosphorylation. • Lithium enhances GSK-3β activity by reducing Akt-mediated GSK-3β phosphorylation. • Lithium suppresses GSK-3β activity through its direct inhibition. - Abstract: Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li{sub 2}CO{sub 3} significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li{sub 2}CO{sub 3} did not affect PI3K-mediated PI(3,4,5)P{sub 3} production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li{sub 2}CO{sub 3} on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li{sub 2}CO{sub 3} significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li{sub 2}CO{sub 3} significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity.

  10. Characterization of phosphoinositide-specific phospholipase C in rat colonocyte membranes.

    PubMed Central

    Bolt, M J; Bissonnette, B M; Wali, R K; Hartmann, S C; Brasitus, T A; Sitrin, M D

    1993-01-01

    The phosphoinositide signal transduction pathway mediates important processes in intestinal physiology, yet the key enzyme, phosphoinositide-specific phospholipase C (PI-PLC), is not well-characterized in the colon. PI-PLC activity was examined in rat colonic membranes using exogenous [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate, and beta-glycerophosphate to suppress degradation of substrate or product. The activity of membrane PI-PLC increased 6-fold with the addition of alamethicin, and a further 2-3-fold enhancement was observed with 10 microM guanosine 5'-[gamma-thio]triphosphate (GTP[S]), suggesting the involvement of G-protein(s). The effect of GTP[S] appeared to be specific, as up to 100 microM adenosine 5'-[gamma-thio]-triphosphate failed to stimulate PI-PLC activity, and guanosine 5'-[beta-thio]diphosphate inhibited activity. The response of membrane PI-PLC to Ca2+ was biphasic, while > 0.5 mM Mg2+ was inhibitory with or without GTP[S]. Comparable total PI-PLC activities and responses to GTP[S] and Ca2+ were observed in purified brush-border and basolateral membranes. Western immunoblots probed with monoclonal antibodies to PLC isoenzymes PLC-beta 1, -gamma 1 and -delta 1 demonstrated that these antipodal plasma membranes contain predominantly the PLC-delta 1 isoform, with small amounts of PLC-gamma 1 present but no detectable PLC-beta 1. PLC-gamma 1 was the major isoform detected in cytosol. Images Figure 5 PMID:8389128

  11. Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with lipopolysaccharide.

    PubMed

    Lo Vasco, Vincenza Rita; Fabrizi, Cinzia; Fumagalli, Lorenzo; Cocco, L

    2010-04-01

    Signal transduction pathways, involved in cell cycle and activities, depend on various components including lipid signalling molecules, such as phosphoinositides and related enzymes. Many evidences support the hypothesis that inositol lipid cycle is involved in astrocytes activation during neurodegeneration. Previous studies investigated the pattern of expression of phosphoinositide-specific phospholipase C (PI-PLC) family isoforms in astrocytes, individuating in cultured neonatal rat astrocytes, supposed to be quiescent cells, the absence of some isoforms, accordingly to their well known tissue specificity. The same study was conducted in cultured rat astrocytoma C6 cells and designed a different pattern of expression of PI-PLCs in the neoplastic counterpart, accordingly to literature suggesting a PI signalling involvement in tumour progression. It is not clear the role of PI-PLC isoforms in inflammation; recent data demonstrate they are involved in cytokines production, with special regard to IL-6. PI-PLCs expression in LPS treated neonatal rat astrocytes performed by using RT-PCR, observed at 3, 6, 18 and 24 h intervals, expressed: PI-PLC beta1, beta4 and gamma1 in all intervals analysed; PI-PLC delta1 at 6, 18 and 24 h; PI-PLC delta3 at 6 h after treatment. PI-PLC beta3, delta4 and epsilon, present in untreated astrocytes, were not detected after LPS treatment. Immunocytochemical analysis, performed to visualize the sub-cellular distribution of the expressed isoforms, demonstrated different patterns of localisation at different times of exposure. These observations suggest that PI-PLCs expression and distribution may play a role in ongoing inflammation process of CNS. PMID:20082315

  12. Phosphatidylinositol-3-kinase-dependent phosphorylation of SLP-76 by the lymphoma-associated ITK-SYK fusion-protein

    SciTech Connect

    Hussain, Alamdar; Department of Biosciences, COMSATS Institute of Information Technology, Chak Shazad Campus, Islamabad ; Faryal, Rani; Department of Biosciences, COMSATS Institute of Information Technology, Chak Shazad Campus, Islamabad ; Nore, Beston F.; Mohamed, Abdalla J.; Smith, C.I. Edvard

    2009-12-18

    Recurrent chromosomal translocations have long been implicated in various types of lymphomas and other malignancies. Novel recurrent t(5;9)(q33;q22) has been recently discovered in un-specified peripheral T-cell lymphoma. To elucidate the role of this translocation, the corresponding fusion construct encoding the N-terminal portion of the ITK kinase and the C-terminal catalytic region of the SYK kinase was generated. We herein show that the ITK-SYK fusion-protein is constitutively active. Moreover, we demonstrate that ITK-SYK is phosphorylated on key tyrosine residues and is capable of potently phosphorylating the related adapter proteins BLNK and SLP-76. In transiently transfected cells, SYK was phosphorylated at Y352 but not detectably at the activation-loop tyrosines Y525/Y526. In contrast, ITK-SYK was phosphorylated both at Y212 and the activation-loop tyrosines Y385/Y386, corresponding to Y352 and Y525/Y526 in SYK, respectively. In resting primary lymphocytes, ITK-SYK predominantly localizes to the cell surface. In addition, we demonstrate that following stimulation, the ITK-SYK fusion-protein in cell lines translocates to the cell membrane and, moreover, that this phenomenon as well as SLP-76 phosphorylation are blocked upon phosphatidylinositol-3-kinase (PI3-kinase) inhibition.

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

    PubMed

    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

    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

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

    PubMed Central

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

    2013-01-01

    We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ) and p50–101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN−/− cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN−/− MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110β function in the absence of PTEN. PMID:24124465

  15. Importance of phosphoinositide-dependent signaling pathways in the control of gene expression in resting cells and in response to phytohormones

    PubMed Central

    Kalachova, Tetiana; Kravets, Volodymyr; Zachowski, Alain; Ruelland, Eric

    2015-01-01

    “Phosphoinositide” refers to phosphorylated forms of phosphatidylinositol, including phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. Both of these molecules could be in vivo substrates of plant phospholipase C. These phosphoinositides can also be biologically active “per se,” by directly binding to proteins and thus altering their location and/or activity. The use of pharmacological agents in Arabidopsis suspension cells allowed us to identify genes whose expression was positively or negatively controlled, in the basal state, by products of phosphoinositide-dependent phospholipase C. In this basal state, it seems that no genes exhibit a phosphoinositide-dependent expression “per se.” However, many genes whose expression is altered in the presence of phospholipase C inhibitors appeared to be responsive to salicylic acid. This allowed us to show that salicylic acid acts both by increasing the phosphoinositide pool and by inhibiting the phospholipase C. In response to salicylic acid it is possible to identify genes whose expression is controlled by products of PI-PLC, but also genes whose expression is controlled by phosphoinositides “per se.” Our data highlight the importance of phosphoinositide-dependent pathways in gene expression in resting cells and in response to phytohormones. PMID:26039482

  16. Effects of acetylcholine and other agents on /sup 32/P-prelabeled phosphoinositides and phosphatidate in crude synaptosomal preparations

    SciTech Connect

    White, H.L.

    1988-05-01

    Experimental conditions are described which permit effects of various agents on polyphosphoinositides and phosphatidic acid (PA) to be evaluated simultaneously in crude nerve-ending preparations from rat brain. Acetylcholine (3-100 microM) or carbachol (30-1,000 microM) induced the hydrolysis of prelabeled polyphosphoinositides and, at the same time, stimulated the net label incorporated in phosphatidic acid. All muscarinic effects were blocked by atropine or pirenzepine. Non-muscarinic agonists (glutamate, adenosine, norepinephrine) stimulated polyphosphoinositide hydrolysis in this preparation, but of these only norepinephrine affected phosphatidic acid turnover. A potentiation of acetylcholine-induced phosphoinositide turnover by KCl was observed, as well as an apparent selective inhibition of PIP2 hydrolysis by LiCl. Acetylcholine-stimulated turnover of PA was not necessarily coupled to phosphoinositide hydrolysis.

  17. Effects of glucose on sorbitol pathway activation, cellular redox, and metabolism of myo-inositol, phosphoinositide, and diacylglycerol in cultured human retinal pigment epithelial cells.

    PubMed Central

    Thomas, T P; Porcellati, F; Kato, K; Stevens, M J; Sherman, W R; Greene, D A

    1994-01-01

    Sorbitol (aldose reductase) pathway flux in diabetes perturbs intracellular metabolism by two putative mechanisms: reciprocal osmoregulatory depletion of other organic osmolytes e.g., myo-inositol, and alterations in NADPH/NADP+ and/or NADH/NAD+. The "osmolyte" and "redox" hypotheses predict secondary elevations in CDP-diglyceride, the rate-limiting precursor for phosphatidylinositol synthesis, but through different mechanisms: the "osmolyte" hypothesis via depletion of intracellular myo-inositol (the cosubstrate for phosphatidylinositol-synthase) and the "redox" hypothesis through enhanced de novo synthesis from triose phosphates. The osmolyte hypothesis predicts diminished phosphoinositide-derived arachidonyl-diacylglycerol, while the redox hypothesis predicts increased total diacylglycerol and phosphatidic acid. In high aldose reductase expressing retinal pigment epithelial cells, glucose-induced, aldose reductase inhibitor-sensitive CDP-diglyceride accumulation and inhibition of 32P-incorporation into phosphatidylinositol paralleled myo-inositol depletion (but not cytoplasmic redox, that was unaffected by glucose) and depletion of arachidonyl-diacylglycerol. 3 mM pyruvate added to the culture medium left cellular redox unaltered, but stimulated Na(+)-dependent myo-inositol uptake, accumulation, and incorporation into phosphatidylinositol. These results favor myo-inositol depletion rather than altered redox as the primary cause of glucose-induced aldose reductase-related defects in phospholipid metabolism in cultured retinal pigment epithelial cells. Images PMID:8201009

  18. Bipolar Plasma Membrane Distribution of Phosphoinositides and Their Requirement for Auxin-Mediated Cell Polarity and Patterning in Arabidopsis[W

    PubMed Central

    Tejos, Ricardo; Sauer, Michael; Vanneste, Steffen; Palacios-Gomez, Miriam; Li, Hongjiang; Heilmann, Mareike; van Wijk, Ringo; Vermeer, Joop E.M.; Heilmann, Ingo; Munnik, Teun; Friml, Jiří

    2014-01-01

    Cell polarity manifested by asymmetric distribution of cargoes, such as receptors and transporters, within the plasma membrane (PM) is crucial for essential functions in multicellular organisms. In plants, cell polarity (re)establishment is intimately linked to patterning processes. Despite the importance of cell polarity, its underlying mechanisms are still largely unknown, including the definition and distinctiveness of the polar domains within the PM. Here, we show in Arabidopsis thaliana that the signaling membrane components, the phosphoinositides phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as well as PtdIns4P 5-kinases mediating their interconversion, are specifically enriched at apical and basal polar plasma membrane domains. The PtdIns4P 5-kinases PIP5K1 and PIP5K2 are redundantly required for polar localization of specifically apical and basal cargoes, such as PIN-FORMED transporters for the plant hormone auxin. As a consequence of the polarity defects, instructive auxin gradients as well as embryonic and postembryonic patterning are severely compromised. Furthermore, auxin itself regulates PIP5K transcription and PtdIns4P and PtdIns(4,5)P2 levels, in particular their association with polar PM domains. Our results provide insight into the polar domain–delineating mechanisms in plant cells that depend on apical and basal distribution of membrane lipids and are essential for embryonic and postembryonic patterning. PMID:24876254

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

    PubMed

    Liang, Yiming; Huang, Min; Li, Jianwen; Sun, Xinlin; Jiang, Xiaodan; Li, Liangping; Ke, Yiquan

    2014-10-01

    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

  20. Fucoidan inhibits the migration and proliferation of HT-29 human colon cancer cells via the phosphoinositide-3 kinase/Akt/mechanistic target of rapamycin pathways

    PubMed Central

    HAN, YONG-SEOK; LEE, JUN HEE; LEE, SANG HUN

    2015-01-01

    Fucoidan, a sulfated polysaccharide, has a variety of biological activities, including anti-cancer, anti-angiogenic and anti-inflammatory effects. However, the underlying mechanisms of fucoidan as an anti-cancer agent remain to be elucidated. The present study examined the anti-cancer effect of fucoidan on HT-29 human colon cancer cells. The cell growth of HT29 cells was significantly decreased following treatment with fucoidan (200 μg/ml). In addition, fucoidan inhibited the migration of HT-29 cells by decreasing the expression levels of matrix metalloproteinase-2 in a dose-dependent manner (0–200 μg/ml). The underlying mechanism of these inhibitory effects included the downregulation of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) by treatment with fucoidan. Furthermore, fucoidan increased the expression of cleaved caspase-3 and decreased cancer sphere formation. The present study suggested that fucoidan exerts an anti-cancer effect on HT-29 human colon cancer cells by downregulating the PI3K-Akt-mTOR signaling pathway. Therefore, fucoidan may be a potential therapeutic reagent against the growth of human colon cancer cells. PMID:25998232

  1. Structural basis for different phosphoinositide specificities of the PX domains of sorting nexins regulating G-protein signaling.

    PubMed

    Mas, Caroline; Norwood, Suzanne J; Bugarcic, Andrea; Kinna, Genevieve; Leneva, Natalya; Kovtun, Oleksiy; Ghai, Rajesh; Ona Yanez, Lorena E; Davis, Jasmine L; Teasdale, Rohan D; Collins, Brett M

    2014-10-10

    Sorting nexins (SNXs) or phox homology (PX) domain containing proteins are central regulators of cell trafficking and signaling. A subfamily of PX domain proteins possesses two unique PX-associated domains, as well as a regulator of G protein-coupled receptor signaling (RGS) domain that attenuates Gαs-coupled G protein-coupled receptor signaling. Here we delineate the structural organization of these RGS-PX proteins, revealing a protein family with a modular architecture that is conserved in all eukaryotes. The one exception to this is mammalian SNX19, which lacks the typical RGS structure but preserves all other domains. The PX domain is a sensor of membrane phosphoinositide lipids and we find that specific sequence alterations in the PX domains of the mammalian RGS-PX proteins, SNX13, SNX14, SNX19, and SNX25, confer differential phosphoinositide binding preferences. Although SNX13 and SNX19 PX domains bind the early endosomal lipid phosphatidylinositol 3-phosphate, SNX14 shows no membrane binding at all. Crystal structures of the SNX19 and SNX14 PX domains reveal key differences, with alterations in SNX14 leading to closure of the binding pocket to prevent phosphoinositide association. Our findings suggest a role for alternative membrane interactions in spatial control of RGS-PX proteins in cell signaling and trafficking. PMID:25148684

  2. Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P2 affinity, phosphoinositide selectivity, and PI(4,5)P2 pool accessibility

    PubMed Central

    Rjasanow, Alexandra; Leitner, Michael G.; Thallmair, Veronika; Halaszovich, Christian R.; Oliver, Dominik

    2015-01-01

    The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P2 to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P2. Because cellular PI(4,5)P2 is resynthesized rapidly, steady state PI(4,5)P2 changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P2 with Ci-VSP allows for the quantification of relative PI(4,5)P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K+ channels to Ci-VSP allowed for comparison of PI(4,5)P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P2 and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P2 that differ in their accessibility to PLC and VSPs. PMID:26150791

  3. Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy.

    PubMed

    Yu, Xinlei; Long, Yun Chau; Shen, Han-Ming

    2015-10-01

    Autophagy is an evolutionarily conserved and exquisitely regulated self-eating cellular process with important biological functions. Phosphatidylinositol 3-kinases (PtdIns3Ks) and phosphoinositide 3-kinases (PI3Ks) are involved in the autophagic process. Here we aim to recapitulate how 3 classes of these lipid kinases differentially regulate autophagy. Generally, activation of the class I PI3K suppresses autophagy, via the well-established PI3K-AKT-MTOR (mechanistic target of rapamycin) complex 1 (MTORC1) pathway. In contrast, the class III PtdIns3K catalytic subunit PIK3C3/Vps34 forms a protein complex with BECN1 and PIK3R4 and produces phosphatidylinositol 3-phosphate (PtdIns3P), which is required for the initiation and progression of autophagy. The class II enzyme emerged only recently as an alternative source of PtdIns3P and autophagic initiator. However, the orthodox paradigm is challenged by findings that the PIK3CB catalytic subunit of class I PI3K acts as a positive regulator of autophagy, and PIK3C3 was thought to be an amino acid sensor for MTOR, which curbs autophagy. At present, a number of PtdIns3K and PI3K inhibitors, including specific PIK3C3 inhibitors, have been developed for suppression of autophagy and for clinical applications in autophagy-related human diseases. PMID:26018563

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

    PubMed Central

    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

    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

  5. Inhibition of phosphoinositide 3-kinase delta attenuates experimental autoimmune encephalomyelitis in mice

    PubMed Central

    Shang, Shi-Qiang; Li, Jian; Li, Shu-Qing; Cao, Yu-Ling

    2015-01-01

    The phosphoinositide 3-kinase delta (PI3Kδ) has been implicated in multiple signaling pathways involved in autoimmune diseases. We here aimed to test the hypothesis that selective inhibition of PI3Kδ may promote anti-inflammatory effects by inhibiting Th1 and Th17 cells. We investigated the therapeutic efficacy of a selective PI3Kδ inhibitor IC87114 in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). The efficacy was evaluated based on clinical scores, histopathology, serum cytokines and inflammatory infiltrations in the central nervous system (CNS). Treatment of EAE mice with IC87114 reduced the clinical symptoms, histopathology and cellular infiltration into the CNS. And treatment of EAE with IC87114 suppressed the Th1 and Th17 cell ratios. Consistently, the serum levels of IL-1β, IL-6, IL-17 and INF-γ were markedly reduced by IC87114. Taken together, our studies demonstrate that inhibition of PI3Kδ may serve as novel therapy to suppress neuroinflammation seen during EAE. PMID:26884985

  6. Tyrosol Suppresses Allergic Inflammation by Inhibiting the Activation of Phosphoinositide 3-Kinase in Mast Cells

    PubMed Central

    Kim, Sung-Wan; Lee, Soyoung; Lee, Hyun-Shik; Park, Eui Kyun; Khang, Dongwoo; Kim, Sang-Hyun

    2015-01-01

    Allergic diseases such as atopic dermatitis, rhinitis, asthma, and anaphylaxis are attractive research areas. Tyrosol (2-(4-hydroxyphenyl)ethanol) is a polyphenolic compound with diverse biological activities. In this study, we investigated whether tyrosol has anti-allergic inflammatory effects. Ovalbumin-induced active systemic anaphylaxis and immunoglobulin E-mediated passive cutaneous anaphylaxis models were used for the immediate-type allergic responses. Oral administration of tyrosol reduced the allergic symptoms of hypothermia and pigmentation in both animal models. Mast cells that secrete allergic mediators are key regulators on allergic inflammation. Tyrosol dose-dependently decreased mast cell degranulation and expression of inflammatory cytokines. Intracellular calcium levels and activation of inhibitor of ?B kinase (IKK) regulate cytokine expression and degranulation. Tyrosol blocked calcium influx and phosphorylation of the IKK complex. To define the molecular target for tyrosol, various signaling proteins involved in mast cell activation such as Lyn, Syk, phosphoinositide 3-kinase (PI3K), and Akt were examined. Our results showed that PI3K could be a molecular target for tyrosol in mast cells. Taken together, these findings indicated that tyrosol has anti-allergic inflammatory effects by inhibiting the degranulation of mast cells and expression of inflammatory cytokines; these effects are mediated via PI3K. Therefore, we expect tyrosol become a potential therapeutic candidate for allergic inflammatory disorders. PMID:26068872

  7. Interaction of aluminum ions with phosphoinositide metabolism in rat cerebral cortical membranes

    SciTech Connect

    Candura, S.M.; Castoldi, A.F.; Costa, L.G. Fondazione Clinica del Lavoro, Pavia ); Manzo, L. Fondazione Clinica del Lavoro, Pavia )

    1991-01-01

    Al, complexed with fluoride to form fluoroaluminate (AlF{sub 4}-), can activate the GTP-binding (G) proteins of the adenylate cyclase and retinal cyclic GMP phosphodiesterase systems. Since an involvement of G-proteins with cerebral phosphoinositide (PtdIns) metabolism has also been suggested, in this study the authors investigated the interaction of the stable GTP analogue GTP(S), Al salts and NaF with this system. In rat cerebral cortical membranes, GTP(S) dose-dependently stimulated ({sup 3}H)inositol phosphates (({sup 3}H)InsPs) accumulation. This effect was potentiated by carbachol and was partially prevented by the GTP-binding antagonist GDP(S), indicating that CNS muscarinic receptor activation is coupled to PtdIns hydrolysis via putative G-protein(s). GTP(S) stimulation was also inhibited by phorbol 12-myristate 13-acetate (PMA). Both Al salts and NaF mimicked the action of GTP(S) in stimulating PtdIns turnover. Their actions were highly synergistic. However, the stimulatory effects of AlCl{sub 3} and/or NaF were not potentiated by carbachol and were not inhibited by GDP(S) and PMA. In the nervous tissue, activation of PtdIns hydrolysis by Al may be mediated by activating a regulatory G-protein at a location distinct from the GTP-binding site or by a direct stimulation of phospholipase C.

  8. Inducible costimulator promotes helper T-cell differentiation through phosphoinositide 3-kinase.

    PubMed

    Gigoux, Mathieu; Shang, Jijun; Pak, Youngshil; Xu, Minghong; Choe, Jongseon; Mak, Tak W; Suh, Woong-Kyung

    2009-12-01

    The T-cell costimulatory receptors, CD28 and the inducible costimulator (ICOS), are required for the generation of follicular B helper T cells (T(FH)) and germinal center (GC) reaction. A common signal transducer used by CD28 and ICOS is the phosphoinositide 3-kinase (PI3K). Although it is known that CD28-mediated PI3K activation is dispensable for GC reaction, the role of ICOS-driven PI3K signaling has not been defined. We show here that knock-in mice that selectively lost the ability to activate PI3K through ICOS had severe defects in T(FH) generation, GC reaction, antibody class switch, and antibody affinity maturation. In preactivated CD4(+) T cells, ICOS delivered a potent PI3K signal that was critical for the induction of the key T(FH) cytokines, IL-21 and IL-4. Under the same settings, CD28 was unable to activate PI3K but supported a robust secondary expansion of T cells. Thus, our results demonstrate a nonredundant function of ICOS-PI3K pathway in the generation of T(FH) and suggest that CD28 and ICOS play differential roles during a multistep process of T(FH) differentiation. PMID:19915142

  9. Aluminium differentially modifies lipid metabolism from the phosphoinositide pathway in Coffea arabica cells.

    PubMed

    Martínez-Estévez, Manuel; Racagni-Di Palma, Graciela; Muñoz-Sánchez, J Armando; Brito-Argáez, Ligia; Loyola-Vargas, Víctor M; Hernández-Sotomayor, S M

    2003-11-01

    The effect of aluminium (Al) on phosphoinositide-specific phospholipase C (PLC) and lipid kinase activities was examined in a cellular suspension of coffee. Two main effects were seen when cells were treated with AlCl3. In periods as short as 1 minute, Al-exposed cells increased the activity of PLC and IP3 formation up to two fold. Over longer periods PLC activity was inhibited by more than 50%. The activity of phosphatidylinositol 4-kinase (Pl 4-K), phosphatidylinositol phosphate 5-kinase (PIP 5-K) and diacylglycerol kinase increased when cells were incubated in the presence of different concentrations of AlCl3. The present study reports for the first time that Al may have different effects on the Pl-signaling pathway depending on the time of exposure. Our results strongly support the hypothesis that Al disrupts the metabolism of membrane phospholipids regulating not only PLC but also other enzymes that have key roles in signal-transduction pathways. PMID:14658381

  10. Alpha-1 adrenergic receptor: Binding and phosphoinositide breakdown in human myometrium

    SciTech Connect

    Breuiller-Fouche, M.; Doualla-Bell Kotto Maka, F.; Geny, B.; Ferre, F. )

    1991-07-01

    Alpha-1 adrenergic receptors were examined in both inner and outer layers of human pregnant myometrium using radioligand binding of (3H)prazosin. (3H)prazosin bound rapidly and reversibly to a single class of high affinity binding sites in myometrial membrane preparations. Scatchard analysis gave similar values of equilibrium dissociation constants in both myometrial layers. In contrast, more alpha-1 adrenergic receptors were detected in the outer layer than in the inner layer. Antagonist inhibited (3H)prazosin binding with an order of potency of prazosin greater than phentolamine greater than idazoxan. Competition experiments have also revealed that a stable guanine nucleotide decreases the apparent affinity of norepinephrine for myometrial (3H)prazosin binding sites. The functional status of these alpha-1 adrenergic receptors was also assessed by measuring the norepinephrine-induced accumulation of inositol phosphates in myometrial tissue. Norepinephrine produced a concentration-dependent accumulation of inositol phosphates in both myometrial layers. However, norepinephrine-induced increases in inositol 1,4,5-triphosphate were only observed in the outer layer. These results indicate that alpha-1 adrenergic receptors in human myometrium at the end of pregnancy are linked to phosphoinositide hydrolysis and that this response occurs mainly in the outer layer.

  11. Short-Form Ron Promotes Spontaneous Breast Cancer Metastasis through Interaction with Phosphoinositide 3-Kinase

    PubMed Central

    Liu, Xuemei; Zhao, Ling; DeRose, Yoko S.; Lin, Yi-Chun; Bieniasz, Magdalena; Eyob, Henok; Buys, Saundra S.; Neumayer, Leigh

    2011-01-01

    Receptor tyrosine kinases (RTKs) have been the subject of intense investigation due to their widespread deregulation in cancer and the prospect of developing targeted therapeutics against these proteins. The Ron RTK has been implicated in tumor aggressiveness and is a developing target for therapy, but its function in tumor progression and metastasis is not fully understood. We examined Ron activity in human breast cancers and found striking predominance of an activated Ron isoform known as short-form Ron (sfRon), whose function in breast tumors has not been explored. We found that sfRon plays a significant role in aggressiveness of breast cancer in vitro and in vivo. sfRon expression was sufficient to convert slow-growing, nonmetastatic tumors into rapidly growing tumors that spontaneously metastasized to liver and bones. Mechanistic studies revealed that sfRon promotes epithelial-mesenchymal transition, invasion, tumor growth, and metastasis through interaction with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K). Inhibition of PI3K activity, or introduction of a single mutation in the p85 docking site on sfRon, completely eliminated the ability of sfRon to promote tumor growth, invasion, and metastasis. These findings reveal sfRon as an important new player in breast cancer and validate Ron and PI3K as therapeutic targets in this disease. PMID:22207901

  12. Molecular and Enzymatic Characterization of Three Phosphoinositide-Specific Phospholipase C Isoforms from Potato1

    PubMed Central

    Kopka, Joachim; Pical, Christophe; Gray, Julie E.; Mller-Rber, Bernd

    1998-01-01

    Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 ?m Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner. PMID:9449844

  13. Essential Role of the p110β Subunit of Phosphoinositide 3-OH Kinase in Male Fertility

    PubMed Central

    Ciraolo, Elisa; Morello, Fulvio; Hobbs, Robin M.; Wolf, Frieder; Marone, Romina; Iezzi, Manuela; Lu, Xiaoyun; Mengozzi, Giulio; Altruda, Fiorella; Sorba, Giovanni; Guan, Kaomei; Pandolfi, Pier Paolo; Wymann, Matthias P.

    2010-01-01

    Phosphoinositide 3-kinases (PI3K) are key molecular players in male fertility. However, the specific roles of different p110 PI3K catalytic subunits within the spermatogenic lineage have not been characterized so far. Herein, we report that male mice expressing a catalytically inactive p110β develop testicular hypotrophy and impaired spermatogenesis, leading to a phenotype of oligo-azoospermia and defective fertility. The examination of testes from p110β-defective tubules demonstrates a widespread loss in spermatogenic cells, due to defective proliferation and survival of pre- and postmeiotic cells. In particular, p110β is crucially needed in c-Kit–mediated spermatogonial expansion, as c-Kit–positive cells are lost in the adult testis and activation of Akt by SCF is blocked by a p110β inhibitor. These data establish that activation of the p110β PI3K isoform by c-Kit is required during spermatogenesis, thus opening the way to new treatments for c-Kit positive testicular cancers. PMID:20053680

  14. Calcium mobilization and phosphoinositide turnover in fluoride-activated human neutrophils

    SciTech Connect

    Strnad, C.F.; Wong, K.

    1986-05-01

    Fluoride ion, at concentrations above 10 mM, has been found to activate a superoxide production response in human neutrophils which is strongly dependent on the presence of extracellular calcium. In an attempt to further explore the calcium requirement of fluoride-induced neutrophil activation, intracellular calcium concentrations were monitored through use of the fluorescent calcium probe, Quin 2. Fluoride ion, at concentrations between 10 and 20 mM, was found to elicit a rise in intracellular calcium levels which was characterized by a lag period of 4 to 10 min and a prolonged duration of action (greater than 20 min). In contrast, the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (FMLP), induced a rise in intracellular calcium concentration which peaked within 1 min. Preincubation of cells with 1 ..mu..g/ml pertussis toxin resulted in inhibition of the FMLP-induced response, but not that elicited by fluoride. Furthermore, anion exchange chromatography indicated that inositol phosphate accumulation occurred in fluoride-treated cells in association with calcium mobilization. Recent evidence suggests that the FMLP receptor is coupled to phospholipase C and phosphoinositide turnover through a guanine nucleotide binding protein susceptible to inhibition by pertussis toxin. Present results suggest that fluoride ion may serve to activate this protein in a manner resistant to inhibition by pertussis toxin.

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

    PubMed Central

    Angulo, Ivan; Vadas, Oscar; Garon, 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, Gaper; Chandra, Anita; Farmer, George; Kielkowska, Anna; Clark, Jonathan; Kracker, Sven; Debr, 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

    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

  16. Phosphoinositide 3-kinase family in channel catfish and their regulated expression after bacterial infection.

    PubMed

    Li, Zhaoxia; Yao, Jun; Xie, Yangjie; Geng, Xin; Liu, Zhanjiang

    2016-02-01

    The phosphoinositide-3-kinase (PI3Ks) family of lipid kinases is widely conserved from yeast to mammals. In this work, we identified a total of 14 members of the PI3Ks from the channel catfish genome and transcriptome and conducted phylogenetic and syntenic analyses of these genes. The expression profiles after infection with Edwardsiella ictaluri and Flavobacterium columnare were examined to determine the involvement of PI3Ks in immune responses after bacterial infection in catfish. The results indicated that PI3Ks genes including all of the catalytic subunit and several regulatory subunits genes were widely regulated after bacterial infection. The expression patterns were quite different when challenged with different bacteria. The PI3Ks were up-regulated rapidly at the early stage after ESC infection, but their induced expression was much slower, at the middle stage after columnaris infection. RNA-Seq datasets indicated that PI3K genes may be expressed at different levels in different catfish differing in their resistance levels against columnaris. Future studies are required to confirm and validate these observations. Taken together, this study indicated that PI3K genes may be involved as a part of the defense responses of catfish after infections, and they could be one of the determinants for disease resistance. PMID:26772478

  17. Morphological characterization of Class III phosphoinositide 3-kinase during mouse brain development.

    PubMed

    Inaguma, Yutaka; Ito, Hidenori; Iwamoto, Ikuko; Matsumoto, Ayumi; Yamagata, Takanori; Tabata, Hidenori; Nagata, Koh-Ichi

    2016-03-01

    The mammalian Class III phosphoinositide 3-kinase (PIK3C3, also known as mammalian vacuolar protein sorting 34 homologue, Vps34) is a regulator of vesicular trafficking, autophagy, and nutrient sensing. In this study, we generated a specific antibody against PIK3C3, and carried out expression and morphological analyses of PIK3C3 during mouse brain development. In Western blotting, PIK3C3 was detected throughout the developmental process with higher expression in the early embryonic stage. In immunohistochemical analyses with embryonic day 16 mouse brain, PIK3C3 was detected strongly in the axon of cortical neurons. While PIK3C3 was distributed at the soma, nucleus, axon, and dendrites in primary cultured mouse hippocampal neurons at 3 days in vitro (div), it was also found in a punctate distribution with partial colocalization with synaptic marker, synaptophysin, at 21 div. The obtained results indicate that PIK3C3 is expressed and may have a physiological role in central nervous system during corticogenesis. PMID:26242203

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

    SciTech Connect

    Ng, Stanley K.L.; Neo, Soek-Ying; Yap, Yann-Wan; Karuturi, R. Krishna Murthy; Loh, Evelyn S.L.; Liau, Kui-Hin; Ren, Ee-Chee; Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore

    2009-09-18

    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.

  19. A Toxoplasma gondii phosphoinositide phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol

    PubMed Central

    Fang, Jianmin; Marchesini, Norma; Moreno, Silvia N. J.

    2005-01-01

    The Toxoplasma gondii phosphoinositide-specific phospholipase C gene (TgPI-PLC) was cloned, sequenced and expressed in Escherichia coli and its enzymatic characteristics were investigated. TgPI-PLC is present in the genome as a single-copy gene consisting of 22 exons interrupted by 21 introns, and encodes a polypeptide of 1097 amino acids with a predicted molecular mass of 121 kDa. In addition to the conserved catalytic X and Y domains, TgPI-PLC contains an apparent N-terminal PH domain, an EF hand motif and a C-terminal C2 domain. When compared with mammalian δ-type PI-PLC, TgPI-PLC has an additional extended N-terminus and two insertions in the region between the X and Y domains, with a 31–35% identity over the whole sequence. Recombinant TgPI-PLC, as well as the native enzyme obtained from crude membrane extracts of the parasite, was more active with phosphatidylinositol than with phosphatidylinositol 4,5-bisphosphate as substrate. Indirect immunofluorescence analysis using an affinity-purified antibody against TgPI-PLC revealed that this enzyme localizes in the plasma membrane of the parasites. PMID:16288600

  20. Chronic ethanol inhibits receptor-stimulated phosphoinositide hydrolysis in rat liver slices

    SciTech Connect

    Gonzales, R.A.; Crews, F.T. )

    1991-03-01

    The effects of chronic ethanol feeding on norepinephrine (NE)- and arginine-vasopressin (AVP)-stimulated phosphoinositide (PI) hydrolysis in rat liver slices was determined. The maximum NE-stimulated PI response was significantly reduced by 40% in liver slices from 8-month-old rats which had been treated for 5 months with a liquid diet containing ethanol compared to pair-fed controls. The maximum AVP-stimulated PI response was decreased by 39% in liver slices from the ethanol-fed rats compared to control. EC50 values for NE- and AVP-stimulated PI hydrolysis in liver slices were not affected by the chronic ethanol treatment. Similar reductions in the maximal NE- and AVP-stimulated PI hydrolysis (28% and 27%, respectively) were found in 22-month-old rats which had been maintained on an ethanol containing diet for 5 months compared to pair-fed controls. The binding of (3H)prazosin and (3H)AVP to liver plasma membranes from 8-month-old ethanol-fed rats was not significantly different from binding to liver membranes from sucrose-fed controls. Our data suggest that chronic ethanol ingestion may lead to a reduction in PI-linked signal transduction in liver.

  1. Voltage sensitive phosphoinositide phosphatases of Xenopus: their tissue distribution and voltage dependence

    PubMed Central

    Ratzan, William J.; Evsikov, Alexei V.; Okamura, Yasushi; Jaffe, Laurinda A.

    2011-01-01

    Voltage sensitive phosphatases (VSPs) are unique proteins in which membrane potential controls enzyme activity. They are comprised of the voltage sensor domain of an ion channel coupled to a lipid phosphatase specific for phosphoinositides, and for ascidian and zebrafish VSPs, the phosphatase activity has been found to be activated by membrane depolarization. The physiological functions of these proteins are unknown, but their expression in testis and embryos suggests a role in fertilization or development. Here we investigate the expression pattern and voltage dependence of VSPs in two frog species, Xenopus laevis and Xenopus tropicalis, that are well suited for experimental studies of these possible functions. X. laevis has two VSP genes (Xl-VSP1 and Xl-VSP2), whereas X. tropicalis has only one gene (Xt-VSP). The highest expression of these genes was observed in testis, ovary, liver, and kidney. Our results show that while Xl-VSP2 activates only at positive membrane potentials outside of the physiological range, Xl-VSP1 and Xt-VSP phosphatase activity is regulated in the voltage range that regulates sperm-egg fusion at fertilization. PMID:21618529

  2. Unique serotonin receptor (5HT-1C) in choroid plexus is linked to phosphoinositide hydrolysis

    SciTech Connect

    Sanders-Bush, E.; Conn, P.J.; Hoffman, B.J.; Hartig, P.R.

    1986-03-01

    The binding of /sup 125/I-LSD to the 5HT-1C site and of /sup 3/H-ket-anserin to the 5HT-2 site was determined in choroid plexus and cerebral cortex of male Sprague-Dawley rats, respectively. As an index of phosphoinositide (PI) hydrolysis, whole choroid plexus and cerebral cortex slices were prelabelled with /sup 3/H-inositol and serotonin (5HT) stimulated release of /sup 3/H-inositol-1-phosphate was measured. 5HT stimulated PI hydrolysis in choroid plexus (6-fold) and in cerebral cortex (2.5-fold). 5HT was more potent in choroid plexus (EC/sub 50/ = 46 nM) consistent with the involvement of the 5HT-1C site. 5HT antagonists, ketanserin, mianserin and spiperone, inhibited the response to 5HT with different potencies in the two tissues. In cerebral cortex all 3 antagonists had nM affinities and a rank order (spiperone > ketanserin > mianserin) consistent with the 5HT-2 site. In choroid plexus, however, the rank order (mianserin > ketanserin > spiperone) and absolute potencies agreed with binding to the 5HT-1C site. These data suggest that the 5HT-1C site is a functional receptor which utilizes PI hydrolysis as its biochemical effector system.

  3. Phosphoinositide 3-kinase and Bruton's tyrosine kinase regulate overlapping sets of genes in B lymphocytes

    PubMed Central

    Fruman, David A.; Ferl, Gregory Z.; An, Sam S.; Donahue, Amber C.; Satterthwaite, Anne B.; Witte, Owen N.

    2002-01-01

    Bruton's tyrosine kinase (Btk) acts downstream of phosphoinositide 3-kinase (PI3K) in a pathway required for B cell receptor (BCR)-dependent proliferation. We used DNA microarrays to determine what fraction of genes this pathway influences and to investigate whether PI3K and Btk mediate distinct gene regulation events. As complete loss-of-function mutations in PI3K and Btk alter B cell subpopulations and may cause compensatory changes in gene expression, we used B cells with partial loss of function in either PI3K or Btk. Only about 5% of the BCR-dependent gene expression changes were significantly affected by reduced PI3K or Btk. The results indicate that PI3K and Btk share target genes, and that PI3K influences additional genes independently of Btk. These data are consistent with PI3K acting through Btk and other effectors to regulate expression of a critical subset of BCR target genes that determine effective entry into the cell cycle. PMID:11756681

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

    SciTech Connect

    Gaudette, D.C.; Holub, B.J. )

    1990-05-15

    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.

  5. Coincidence detection in a neural correlate of classical conditioning is initiated by bidirectional 3-phosphoinositide-dependent kinase-1 signalling and modulated by adenosine receptors.

    PubMed

    Keifer, Joyce; Zheng, Zhaoqing

    2015-04-01

    How the neural substrates for detection of paired stimuli are distinct from unpaired stimuli is poorly understood and a fundamental question for understanding the signalling mechanisms for coincidence detection during associative learning. To address this question, we used a neural correlate of eyeblink classical conditioning in an isolated brainstem from the turtle, in which the cranial nerves are directly stimulated in place of using a tone or airpuff. A bidirectional response is activated in <5 min of training, in which phosphorylated 3-phosphoinositide-dependent kinase-1 (p-PDK1) is increased in response to paired and decreased in response to unpaired nerve stimulation and is mediated by the opposing actions of neurotrophin receptors TrkB and p75(NTR) . Surprisingly, blockade of adenosine 2A (A2A ) receptors inhibits both of these responses. Pairing also induces substantially increased surface expression of TrkB that is inhibited by Src family tyrosine kinase and A2A receptor antagonists. Finally, the acquisition of conditioning is blocked by a PDK1 inhibitor. The unique action of A2A receptors to function directly as G proteins and in receptor transactivation to control distinct TrkB and p75(NTR) signalling pathways allows for convergent activation of PDK1 and protein kinase A during paired stimulation to initiate classical conditioning. PMID:25639253

  6. Inhibition of class II phosphoinositide 3-kinase γ expression by p185Bcr-Abl contributes to impaired chemotaxis and aberrant homing of leukemic cells

    PubMed Central

    Yu, Weidong; Sun, Xiaolin; Tang, Hongxing; Tao, Yunxia; Dai, Zonghan

    2010-01-01

    Expression of p185Bcr-Abl in Ba/F3 cells inhibits chemotactic response of these cells to SDF1α. A mutant p185Bcr-Abl with deletion of amino acids from 176 to 426 (p185Δ176–426) is deficient in suppressing SDF1α-stimulated chemotaxis. Comparison of the gene expression profiles among parental Ba/F3 cells and the cells transformed by p185Bcr-Abl and p185Δ176–426 reveals that class II phosphoinositide 3-kinase γ (PI3KC2γ) expression is markedly down-regulated by p185Bcr-Abl but not p185Δ176–426. Furthermore, knockdown of PI3KC2γ expression in p185Δ176–426 cells is sufficient to suppress SDF1α-stimulated chemotaxis and to promote infiltration of these cells into liver. Together, these studies suggest that inhibition of PI3KC2γ expression may represent a mechanism by which Bcr-Abl suppresses SDF1α-induced chemotaxis and induces abnormal homing of leukemic cells. PMID:20536348

  7. Roles of Pdk1p, a Fission Yeast Protein Related to Phosphoinositide-dependent Protein Kinase, in the Regulation of Mitosis and Cytokinesis

    PubMed Central

    Bimbó, Andrea; Liu, Jianhua; Balasubramanian, Mohan K.

    2005-01-01

    Proteins related to the phosphoinositide-dependent protein kinase family have been identified in the majority of eukaryotes. Although much is known about upstream mechanisms that regulate the PDK1-family of kinases in metazoans, how these kinases regulate cell growth and division remains unclear. Here, we characterize a fission yeast protein related to members of this family, which we have termed Pdk1p. Pdk1p localizes to the spindle pole body and the actomyosin ring in early mitotic cells. Cells deleted for pdk1 display multiple defects in mitosis and cytokinesis, all of which are exacerbated when the function of fission yeast polo kinase, Plo1p, is partially compromised. We conclude that Pdk1p functions in concert with Plo1p to regulate multiple processes such as the establishment of a bipolar mitotic spindle, transition to anaphase, placement of the actomyosin ring and proper execution of cytokinesis. We also present evidence that the effects of Pdk1p on cytokinesis are likely mediated via the fission yeast anillin-related protein, Mid1p, and the septation initiation network. PMID:15857958

  8. Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins

    PubMed Central

    Schlam, Daniel; Bagshaw, Richard D.; Freeman, Spencer A.; Collins, Richard F.; Pawson, Tony; Fairn, Gregory D.; Grinstein, Sergio

    2015-01-01

    Phagocytosis is responsible for the elimination of particles of widely disparate sizes, from large fungi or effete cells to small bacteria. Though superficially similar, the molecular mechanisms involved differ: engulfment of large targets requires phosphoinositide 3-kinase (PI3K), while that of small ones does not. Here, we report that inactivation of Rac and Cdc42 at phagocytic cups is essential to complete internalization of large particles. Through a screen of 62 RhoGAP-family members, we demonstrate that ARHGAP12, ARHGAP25 and SH3BP1 are responsible for GTPase inactivation. Silencing these RhoGAPs impairs phagocytosis of large targets. The GAPs are recruited to large—but not small—phagocytic cups by products of PI3K, where they synergistically inactivate Rac and Cdc42. Remarkably, the prominent accumulation of phosphatidylinositol 3,4,5-trisphosphate characteristic of large-phagosome formation is less evident during phagocytosis of small targets, accounting for the contrasting RhoGAP distribution and the differential requirement for PI3K during phagocytosis of dissimilarly sized particles. PMID:26465210

  9. Autotaxin stimulates urokinase-type plasminogen activator expression through phosphoinositide 3-kinase-Akt-nuclear [corrected] factor kappa B signaling cascade in human melanoma cells.

    PubMed

    Lee, Jangsoon; Duk Jung, In; Gyo Park, Chang; Han, Jeung-Whan; Young Lee, Hoi

    2006-10-01

    Autotaxin, a lysophospholipase D producing lysophosphatidic acid, augments invasive and metastatic potential of tumor cells. Current investigations have focused on understanding the molecular mechanisms by which autotaxin regulates the expression of a major mediator of tumor invasion and metastasis, urokinase-type plasminogen activator (uPA) in human A2058 melanoma cells. Autotaxin induced uPA expression in a dose-dependent manner that was inhibited by pharmacological inhibitors for Gi (pertussis toxin), phosphoinositide 3-kinase (PI3K, LY294002), Akt inhibitor (AktI), proteosome activity and IkappaB phosphorylation (pyrrolidine dithiocarbamate), and by a dominant negative mutant (DN) of Akt. Autotaxin phosphorylated Akt and induced the translocation of nuclear [corrected] factor-kappaB (NF-kappaB) to the nucleus that were inhibited by AktI or by overexpressing DN-Akt. Consistently, green fluorescence protein-tagged p65 of NF-kappaB accumulated in the nucleus by autotaxin that was abrogated when the cells were transfected with DN-Akt. Moreover, autotaxin increased the DNA binding ability of NF-kappaB and promoter activity of uPA. Collectively, these data strongly suggest autotaxin induces uPA expression via the Gi-PI3K-Akt-NF-kappaB signaling pathway that might be critical for autotaxin-induced tumor cell invasion and metastasis. PMID:17013094

  10. Evolution of the Voltage Sensor Domain of the Voltage-Sensitive Phosphoinositide Phosphatase VSP/TPTE Suggests a Role as a Proton Channel in Eutherian Mammals

    PubMed Central

    Sutton, Keith A.; Jungnickel, Melissa K.; Jovine, Luca; Florman, Harvey M.

    2012-01-01

    The voltage-sensitive phosphoinositide phosphatases provide a mechanism to couple changes in the transmembrane electrical potential to intracellular signal transduction pathways. These proteins share a domain architecture that is conserved in deuterostomes. However, gene duplication events in primates, including humans, give rise to the paralogs TPTE and TPTE2 that retain protein domain organization but, in the case of TPTE, have lost catalytic activity. Here, we present evidence that these human proteins contain a functional voltage sensor, similar to that in nonmammalian orthologs. However, domains of these human proteins can also generate a noninactivating outward current that is not observed in zebra fish or tunicate orthologs. This outward current has the anticipated characteristics of a voltage-sensitive proton current and is due to the appearance of a single histidine residue in the S4 transmembrane segment of the voltage sensor. Histidine is observed at this position only during the eutherian radiation. Domains from both human paralogs generate proton currents. This apparent gain of proton channel function during the evolution of the TPTE protein family may account for the conservation of voltage sensor domains despite the loss of phosphatase activity in some human paralogs. PMID:22396523

  11. Evolution of the voltage sensor domain of the voltage-sensitive phosphoinositide phosphatase VSP/TPTE suggests a role as a proton channel in eutherian mammals.

    PubMed

    Sutton, Keith A; Jungnickel, Melissa K; Jovine, Luca; Florman, Harvey M

    2012-09-01

    The voltage-sensitive phosphoinositide phosphatases provide a mechanism to couple changes in the transmembrane electrical potential to intracellular signal transduction pathways. These proteins share a domain architecture that is conserved in deuterostomes. However, gene duplication events in primates, including humans, give rise to the paralogs TPTE and TPTE2 that retain protein domain organization but, in the case of TPTE, have lost catalytic activity. Here, we present evidence that these human proteins contain a functional voltage sensor, similar to that in nonmammalian orthologs. However, domains of these human proteins can also generate a noninactivating outward current that is not observed in zebra fish or tunicate orthologs. This outward current has the anticipated characteristics of a voltage-sensitive proton current and is due to the appearance of a single histidine residue in the S4 transmembrane segment of the voltage sensor. Histidine is observed at this position only during the eutherian radiation. Domains from both human paralogs generate proton currents. This apparent gain of proton channel function during the evolution of the TPTE protein family may account for the conservation of voltage sensor domains despite the loss of phosphatase activity in some human paralogs. PMID:22396523

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

    SciTech Connect

    Ogata, Takehiro; Ueyama, Tomomi . E-mail: tueyama@kuhp.kyoto-u.ac.jp; Nomura, Tetsuya; Asada, Satoshi; Tagawa, Masashi; Nakamura, Tomoyuki; Takahashi, Tomosaburo; Matsubara, Hiroaki; Oh, Hidemasa . E-mail: hidemasa@kuhp.kyoto-u.ac.jp

    2007-07-27

    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.

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

    PubMed Central

    Arcaro, Alexandre; Guerreiro, Ana S

    2007-01-01

    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 p110α, p110β and p110δ isoforms, which associate with activated RTKs. In human cancer, recent reports have described activating mutations in the PIK3CA gene encoding p110α, 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 p110α 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 p110α, 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

  14. Phosphoinositides Play Differential Roles in Regulating Phototropin1- and Phototropin2-Mediated Chloroplast Movements in Arabidopsis

    PubMed Central

    Aggarwal, Chhavi; Łabuz, Justyna; Gabryś, Halina

    2013-01-01

    Phototropins are UVA/blue-light receptors involved in controlling the light-dependent physiological responses which serve to optimize the photosynthetic activity of plants and promote growth. The phototropin-induced phosphoinositide (PI) metabolism has been shown to be essential for stomatal opening and phototropism. However, the role of PIs in phototropin-induced chloroplast movements remains poorly understood. The aim of this work is to determine which PI species are involved in the control of chloroplast movements in Arabidopsis and the nature of their involvement. We present the effects of the inactivation of phospholipase C (PLC), PI3-kinase (PI3K) and PI4-kinase (PI4K) on chloroplast relocations in Arabidopsis. The inhibition of the phosphatidylinositol 4,5-bisphospahte [PI(4,5)P2]-PLC pathway, using neomycin and U73122, suppressed the phot2-mediated chloroplast accumulation and avoidance responses, without affecting movement responses controlled by phot1. On the other hand, PI3K and PI4K activities are more restricted to phot1- and phot2-induced weak-light responses. The inactivation of PI3K and PI4K by wortmannin and LY294002 severely affected the weak blue-light-activated accumulation response but had little effect on the strong blue-light-activated avoidance response. The inhibitory effect observed with PI metabolism inhibitors is, at least partly, due to a disturbance in Ca2+(c) signaling. Using the transgenic aequorin system, we show that the application of these inhibitors suppresses the blue-light-induced transient Ca2+(c) rise. These results demonstrate the importance of PIs in chloroplast movements, with the PI(4,5)P2-PLC pathway involved in phot2 signaling while PI3K and PI4K are required for the phot1- and phot2-induced accumulation response. Our results suggest that these PIs modulate cytosolic Ca2+ signaling during movements. PMID:23405144

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

    PubMed

    Rhodes, P G; Cai, Z; Zhu, N

    1994-09-01

    Prenatal ethanol exposure-induced alteration in poly-phosphoinositide (PPI) hydrolysis stimulated by excitatory amino acids (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 diet containing either 5% ethanol (w/v, 35% of calories) or isocaloric sucrose (pair-fed) substituted for ethanol from gestation d 11 until the day of parturition. The ionotropic glutamate receptor agonists, N-methyl-D-aspartate, kainate or (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) (100 microM each) induced a two- to four-fold increase in PPI hydrolysis over the basal level, regardless of the liquid dietary treatment. Stimulation with quisqualate (QA), an agonist activating both metabotropic and ionotropic glutamate receptors, resulted in a much stronger and dose-dependent response in PPI hydrolysis and exposure in utero to ethanol significantly reduced this response. Tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or (+/-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) had no effect on QA-stimulated PPI hydrolysis nor on the suppression of this hydrolysis by ethanol. Exposure in utero to ethanol did not affect PPI hydrolysis stimulated by a selective metabotropic glutamate receptor agonist, trans-(+/-)-l-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD). Although the PPI hydrolysis stimulated by t-ACPD could be blocked by (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG), an antagonist of the metabotropic glutamate receptor, MCPG was incapable of affecting QA-induced PPI hydrolysis and the suppressive effects of prenatal ethanol exposure on this hydrolysis. Taken together, the data suggest that the long-lasting suppressive effects of prenatal ethanol exposure on QA-stimulated PPI hydrolysis in cerebellar granule cell cultures is through a metabotropic QA receptor pathway that may be different from the one activated by t-ACPD. PMID:7893331

  16. Peroxovanadate induces tyrosine phosphorylation of phosphoinositide-dependent protein kinase-1 potential involvement of src kinase.

    PubMed

    Grillo, S; Grémeaux, T; Casamayor, A; Alessi, D R; Le Marchand-Brustel, Y; Tanti, J F

    2000-11-01

    Phosphoinositide-dependent protein kinase-1 (PDK1) is a recently identified kinase that phosphorylates and activates protein kinase B (PKB). Activation of PKB by insulin is linked to its translocation from the cytosol to the plasma membrane. However, no data are available yet concerning the localization of PDK1 in insulin-sensitive tissue. Using isolated adipocytes, we studied the effect of insulin and of an insulin-mimicking agent peroxovanadate on the subcellular localization of PDK1. In unstimulated adipocytes, overexpressed PDK1 was mostly cytosolic with a low amount associated to membranes. Peroxovanadate stimulation induced the redistribution of PDK1 to the membranes while insulin was without effect. This peroxovanadate effect was dependent on phosphatidylinositol 3,4,5 triphosphate [PtdIns(3,4,5)P3] production as inhibition of PtdIns 3-kinase by wortmannin or deletion of the PH domain of PDK1 prevented the peroxovanadate-induced translocation of PDK1. Further, peroxovanadate-treatment induced a tyrosine phosphorylation of PDK1 which was wortmannin insensitive and did not require the PH domain of PDK1. An inhibitor of Src kinase (PP2) decreased the peroxovanadate-induced PDK1 tyrosine phosphorylation and overexpression of v-Src stimulated this phosphorylation. Mutation of tyrosine 373 of PDK1 abolished the v-Src induced PDK1 tyrosine phosphorylation and partially reduced the effect of peroxovanadate. Our findings suggest that PDK1 could be a substrate for tyrosine kinases and identify Src kinase as one of the tyrosine kinases able to phosphorylate PDK1. PMID:11054117

  17. Role of Phosphoinositide 3-Kinase β in Glycoprotein VI-mediated Akt Activation in Platelets*

    PubMed Central

    Kim, Soochong; Mangin, Pierre; Dangelmaier, Carol; Lillian, Rivka; Jackson, Shaun P.; Daniel, James L.; Kunapuli, Satya P.

    2009-01-01

    Glycoprotein (GP) VI is a critical platelet collagen receptor. Phosphoinositide 3-kinase (PI3K) plays an important role in GPVI-mediated platelet activation, yet the major PI3K isoforms involved in this process have not been identified. In addition, stimulation of GPVI results in the activation of Akt, a downstream effector of PI3K. Thus, we investigated the contribution of PI3K isoforms to GPVI-mediated platelet activation and Akt activation. A protein kinase C inhibitor GF 109203X or a P2Y12 receptor antagonist AR-C69931MX partly reduced GPVI-induced Akt phosphorylation. Platelets from mice dosed with clopidogrel also showed partial Akt phosphorylation, indicating that GPVI-mediated Akt phosphorylation is regulated by both secretion-dependent and -independent pathways. In addition, GPVI-induced Akt phosphorylation in the presence of ADP antagonists was completely inhibited by PI3K inhibitor LY294002 and PI3Kβ inhibitor TGX-221 indicating an essential role of PI3Kβ in Akt activation directly downstream of GPVI. Moreover, GPVI-mediated platelet aggregation, secretion, and intracellular Ca2+ mobilization were significantly inhibited by TGX-221, and less strongly inhibited by PI3Kα inhibitor PIK75, but were not affected by PI3Kγ inhibitor AS252424 and PI3Kδ inhibitor IC87114. Consistently, GPVI-induced integrin αIIbβ3 activation of PI3Kγ−/− and PI3Kδ−/− platelets also showed no significant difference compared with wild-type platelets. These results demonstrate that GPVI-induced Akt activation in platelets is dependent in part on Gi stimulation through P2Y12 receptor activation by secreted ADP. In addition, a significant portion of GPVI-dependent, ADP-independent Akt activation also exists, and PI3Kβ plays an essential role in GPVI-mediated platelet aggregation and Akt activation. PMID:19700402

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

    SciTech Connect

    Ishii, H.; Umeda, F.; Hashimoto, T.; Nawata, H. )

    1990-12-01

    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.

  19. Agonist-induced calcium flux, phosphoinositide metabolism, aggregation and enzyme secretion in human neutrophils.

    PubMed

    Rossi, A G; McMillan, R M; MacIntyre, D E

    1988-07-01

    Formyl-methionyl-leucyl-phenylalanine (FMLP), platelet activating factor (PAF) and leukotriene B4 (LTB4) are potent activators of human neutrophils. Using human neutrophils prelabelled with the fluorescent indicator dye, Quin 2, or with [32P]-orthophosphate, we examined the effects of these stimuli on intracellular free calcium concentration, [Ca2+]i, and on various indices of phosphoinositide metabolism, including [32P]-phosphatidic acid (PtdA) formation. The concentration-dependence of the observed changed in [Ca2+]i or [32P]-PtdA were then compared to stimulus-induced aggregation and enzyme release (beta-N-acetylglucosaminidase (NAG) and lysozyme). FMLP, PAF and LTB4 caused a concentration-dependent elevation of [Ca2+]i, aggregation and enzyme release. However, unlike FMLP and PAF, LTB4 (less than or equal to 2.5 microM) did not cause significant formation of [32P]-PtdA. The concentration response curves for agonist-induced elevation of [Ca2+]i lie to the left of those for aggregation and enzyme release. FMLP and PAF also caused an elevation of [Ca2+]i at concentrations lower than those required to elicit [32P]-PtdA formation. These observations suggest that [Ca2+]i elevation per se cannot mediate human neutrophil functional responses to FMLP, PAF and LTB4. Consequently there may exist other mediator(s) that act in concert with [Ca2+]i or are triggered by [Ca2+]i elevation to promote human neutrophil activation. Both the elevation of [Ca2+]i and the formation of these putative mediator(s) in response to LTB4 apparently occur independently of inositol phospholipid hydrolysis. PMID:2845744

  20. Initiation of human astrovirus type 1 infection was blocked by inhibitors of phosphoinositide 3-kinase

    PubMed Central

    2013-01-01

    Background Upon initial contact with a virus, host cells activate a series of cellular signaling cascades that facilitate viral entry and viral propagation within the cell. Little is known about how the human astrovirus (HAstV) exploits signaling cascades to establish an infection in host cells. Recent studies showed that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is important for HAstV infection, though the involvement of other signaling cascades remains unclear. Methods A panel of kinase blockers was used to search for cellular signaling pathways important for HAstV1 infection. To determine their impact on the infectious process, we examined viral gene expression, RNA replication, and viral RNA and capsid protein release from host cells. Results Inhibitors of phosphoinositide 3-kinase (PI3K) activation interfered with the infection, independent of their effect on ERK 1/2 activation. Activation of the PI3K signaling cascade occurred at an early phase of the infection, judging from the timeframe of Akt phosphorylation. PI3K inhibition at early times, but not at later times, blocked viral gene expression. However, inhibiting the downstream targets of PI3K activation, Akt and Rac1, did not block infection. Inhibition of protein kinase A (PKA) activation was found to block a later phase of HAstV1 production. Conclusions Our results reveal a previously unknown, essential role of PI3K in the life cycle of HAstV1. PI3K participates in the early stage of infection, possibly during the viral entry process. Our results also reveal the role of PKA in viral production. PMID:23680019

  1. Triggering actin polymerization in Xenopus egg extracts from phosphoinositide-containing lipid bilayers.

    PubMed

    Walrant, Astrid; Saxton, Daniel S; Correia, Guilherme Pereira; Gallop, Jennifer L

    2015-01-01

    Xenopus egg extracts are a powerful tool to reconstitute complex cell biological processes using a cell-free strategy. When used in conjunction with liposomes and supported lipid bilayers, they can recapitulate the biochemical activities occurring at the cytosol/plasma membrane interface of the cell that underlie remodeling of the actin cytoskeleton. We use these in vitro systems to elucidate how membranes and proteins collaborate to make the appropriate actin structure at a given time and place. We have recently broadened the types of membrane substrate used, and also optimized protocols for preparation of Xenopus egg extracts for actin assembly assays from membranes. Tuning the lipid composition and curvature appropriately demands an appreciation of the native phospholipid and curvature environments that can form transiently in cells. Supported lipid bilayers on glass coverslips that contain phosphatidylserine and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) make actin bundles termed filopodia-like structures that contain fascin and have vasodilator-stimulated phosphoprotein (VASP) at their growing tips, indicating that these resemble filopodia growing from the plasma membrane. The combination of PI(4,5)P2 and phosphatidylinositol 3-phosphate in curved liposomes or supported bilayers on glass nanospheres uses Snx9, Cdc42, N-WASP (neuronal-Wiskott-Aldrich syndrome protein), and Arp2/3 complex for actin polymerization suggesting that this membrane may mimic the progression from plasma membrane to endosomes. Here we describe how to prepare high-speed supernatant frog egg extracts and phosphoinositide-containing liposomes and supported lipid bilayers that can assemble actin structures. We also describe the methods we use to assay actin polymerization using microscopy and spectrofluorometry and our protocol for immunodepleting specific proteins from extracts. PMID:25997346

  2. Phosphoinositide 3-Kinase γ Inhibits Cardiac GSK-3 Independently of Akt

    PubMed Central

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

    2014-01-01

    Activation of cardiac phosphoinositide 3-kinase a (PI3Kα) by growth factors, such as insulin, or activation of PI3Kγ downstream of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors stimulates the activity of the kinase Akt, which phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). We found that PI3Kγ inhibited GSK-3 independently of the insulin-PI3Ka-Akt axis. Although insulin treatment activated Akt in PI3Kγ knockout mice, phosphorylation of GSK-3 was decreased compared to control mice. GSK-3 is activated when dephosphorylated by the protein phosphatase 2A (PP2A), which is activated when methylated by the PP2A methyltransferase PPMT-1. PI3Kγ knockout mice showed increased activity of PPMT-1 and PP2A and enhanced nuclear export of the GSK-3 substrate NFATc3. GSK-3 inhibits cardiac hypertrophy, and the hearts of PI3Kγ knockout mice were smaller compared to those of wild-type mice. Cardiac overexpression of a catalytically inactive PI3Kγ (PI3Kγinact) transgene in PI3Kγ knockout mice reduced the activities of PPMT-1 and PP2A and increased phosphorylation of GSK-3. Furthermore, PI3Kγ knockout mice expressing the PI3Kγinact transgene had larger hearts than wild-type or PI3Kγ knockout mice. Our studies show that a kinase-independent function of PI3Kγ could directly inhibit GSK-3 function by preventing the PP2A–PPMT-1 interaction and that this inhibition of GSK-3 was independent of Akt. PMID:23354687

  3. Phosphoinositides play differential roles in regulating phototropin1- and phototropin2-mediated chloroplast movements in Arabidopsis.

    PubMed

    Aggarwal, Chhavi; Labuz, Justyna; Gabryś, Halina

    2013-01-01

    Phototropins are UVA/blue-light receptors involved in controlling the light-dependent physiological responses which serve to optimize the photosynthetic activity of plants and promote growth. The phototropin-induced phosphoinositide (PI) metabolism has been shown to be essential for stomatal opening and phototropism. However, the role of PIs in phototropin-induced chloroplast movements remains poorly understood. The aim of this work is to determine which PI species are involved in the control of chloroplast movements in Arabidopsis and the nature of their involvement. We present the effects of the inactivation of phospholipase C (PLC), PI3-kinase (PI3K) and PI4-kinase (PI4K) on chloroplast relocations in Arabidopsis. The inhibition of the phosphatidylinositol 4,5-bisphospahte [PI(4,5)P2]-PLC pathway, using neomycin and U73122, suppressed the phot2-mediated chloroplast accumulation and avoidance responses, without affecting movement responses controlled by phot1. On the other hand, PI3K and PI4K activities are more restricted to phot1- and phot2-induced weak-light responses. The inactivation of PI3K and PI4K by wortmannin and LY294002 severely affected the weak blue-light-activated accumulation response but had little effect on the strong blue-light-activated avoidance response. The inhibitory effect observed with PI metabolism inhibitors is, at least partly, due to a disturbance in Ca(2+) ((c)) signaling. Using the transgenic aequorin system, we show that the application of these inhibitors suppresses the blue-light-induced transient Ca(2+) ((c)) rise. These results demonstrate the importance of PIs in chloroplast movements, with the PI(4,5)P2-PLC pathway involved in phot2 signaling while PI3K and PI4K are required for the phot1- and phot2-induced accumulation response. Our results suggest that these PIs modulate cytosolic Ca(2+) signaling during movements. PMID:23405144

  4. IQGAP proteins reveal an atypical phosphoinositide (aPI) binding domain with a pseudo C2 domain fold.

    PubMed

    Dixon, Miles J; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R; van Aalten, Daan M F; Downes, C Peter; Batty, Ian H

    2012-06-29

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)). The binding affinity for PtdInsP(3), together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP(3) effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules. PMID:22493426

  5. IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

    SciTech Connect

    Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M.F.; Downes, C. Peter; Batty, Ian H.

    2012-10-16

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.

  6. 3-Phosphoinositide-dependent protein kinase-1 as an emerging target in the management of breast cancer

    PubMed Central

    Fyffe, Chanse; Falasca, Marco

    2013-01-01

    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

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

    PubMed Central

    Rodrguez, Dolores; Rodrguez, Jos F.

    2015-01-01

    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

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

    PubMed

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

    2013-05-01

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

  9. Effect of aging on alpha-1 adrenergic stimulation of phosphoinositide hydrolysis in various regions of rat brain

    SciTech Connect

    Burnett, D.M.; Bowyer, J.F.; Masserano, J.M.; Zahniser, N.R. )

    1990-12-01

    The effects of aging were examined on the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis in three brain regions. Tissue minces of thalamus, cerebral cortex and hippocampus from 3-, 18- and 28-month-old male Fischer 344 rats were prelabeled with ({sup 3}H)myoinositol. Exposure of these prelabeled minces to phenylephrine and (-)-norepinephrine revealed that accumulation of ({sup 3}H)inositol phosphates was selectively reduced by 20 to 30% in the thalamus and cerebral cortex of the oldest age group. Analysis of concentration-response and competition binding curves indicated that this decrease was due to diminished agonist efficacy rather than diminished receptor affinity. The reduction in responsiveness to phenylephrine and (-)-norepinephrine in the cerebral cortex and the lack of any changes in the hippocampus parallel previously reported changes in the density of alpha-1 adrenergic receptors with aging. These data indicate that the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis is reduced in some, but not all, brain regions of aged Fischer 344 rats.

  10. Yunis-Varón Syndrome Is Caused by Mutations in FIG4, Encoding a Phosphoinositide Phosphatase

    PubMed Central

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

    2013-01-01

    Yunis-Varón syndrome (YVS) is an autosomal-recessive disorder with cleidocranial dysplasia, digital anomalies, and severe neurological involvement. Enlarged vacuoles are found in neurons, muscle, and cartilage. By whole-exome sequencing, we identified frameshift and missense mutations of FIG4 in affected individuals from three unrelated families. FIG4 encodes a phosphoinositide phosphatase required for regulation of PI(3,5)P2 levels, and thus endosomal trafficking and autophagy. In a functional assay, both missense substitutions failed to correct the vacuolar phenotype of Fig4-null mouse fibroblasts. Homozygous Fig4-null mice exhibit features of YVS, including neurodegeneration and enlarged vacuoles in neurons. We demonstrate that Fig4-null mice also have small skeletons with reduced trabecular bone volume and cortical thickness and that cultured osteoblasts accumulate large vacuoles. Our findings demonstrate that homozygosity or compound heterozygosity for null mutations of FIG4 is responsible for YVS, the most severe known human phenotype caused by defective phosphoinositide metabolism. In contrast, in Charcot-Marie-Tooth disease type 4J (also caused by FIG4 mutations), one of the FIG4 alleles is hypomorphic and disease is limited to the peripheral nervous system. This genotype-phenotype correlation demonstrates that absence of FIG4 activity leads to central nervous system dysfunction and extensive skeletal anomalies. Our results describe a role for PI(3,5)P2 signaling in skeletal development and maintenance. PMID:23623387

  11. Cloning, expression, purification, and characterization of the human Class Ia phosphoinositide 3-kinase isoforms.

    PubMed

    Meier, Timothy I; Cook, James A; Thomas, James E; Radding, Jeffrey A; Horn, Candice; Lingaraj, Trupti; Smith, Michele C

    2004-06-01

    The Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositides. Although closely related, experimental evidence suggests that the four Class I PI3Ks may be functionally distinct. To further study their unique biochemical properties, the three human Class Ia PI3K (alpha, beta, and delta) p110 catalytic domains were cloned and co-expressed with the p85alpha regulatory domain in Sf9 cells. None of the p110 subunits were successfully expressed in the absence of p85alpha. Successful expression and purification of each p85alpha/p110 protein required using an excess of the p110 vector over the p85 vector during co-infection of Sf9 cells. Proteins were purified as the p85alpha/p110 complex by nickel affinity chromatography through an N-terminal His-tag on the p110 subunit using an imidazole gradient. The purification yields were high using the optimized ratio of p85/p110 vector and small culture volumes, with 24mg/L cell culture media for p85alpha/p110alpha, 17.5mg/L for p85alpha/p110delta, and 3.5mg/L for p85alpha/p110beta. The identity of each purified isoform was confirmed by mass spectral analysis and immunoblotting. The activities of the three p85alpha/p110 proteins and the Class Ib p110gamma catalytic domain were investigated using phosphatidylinositol 4,5-bisphosphate (PIP2) as the substrate in a PIP2/phosphatidylserine (PS) liposome. All four enzymes exhibited reaction velocities that were dependent on the surface concentration of PIP2. The surface concentrations that gave maximal activity for each human isoform with 0.5mM PIP2 were 2.5mol% PIP2 for p110gamma, 7.5mol% for p85alpha/p110beta, and 10mol% PIP2 for p85alpha/p110alpha and p85alpha/p110delta. The specific activity of p85alpha/p110alpha was three to five times higher than that of the other human isoforms. These kinetic differences may contribute to the unique roles of these isoforms in cells. PMID:15135396

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

    PubMed Central

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

    2013-01-01

    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 γIR-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 γIR, 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 γIR 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 γIR-induced DNA damage as measured by γH2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after IR abrogated cell death. PMID:24068833

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

    PubMed

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

    2007-04-01

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

  14. The Forkhead Transcription Factor FOXO3a Increases Phosphoinositide-3 Kinase/Akt Activity in Drug-Resistant Leukemic Cells through Induction of PIK3CA Expression▿

    PubMed Central

    Hui, Rosaline C.-Y.; Gomes, Ana R.; Constantinidou, Demetra; Costa, Joana R.; Karadedou, Christina T.; Fernandez de Mattos, Silvia; Wymann, Matthias P.; Brosens, Jan J.; Schulze, Almut; Lam, Eric W.-F.

    2008-01-01

    The phosphoinositide-3 kinase (PI3K)/Akt signal pathway plays a key role in the tumorigenesis of many cancers and in the subsequent development of drug resistance. Using the K562 chronic myelogenous leukemia (CML) cell line and the doxorubicin-resistant derivative lines KD30 and KD225 as models, we observed that enhanced PI3K/Akt activity and the acquisition of chemoresistance correlated unexpectedly with the increased expression and nuclear accumulation of FOXO3a. Moreover, we found that the induction of FOXO3a activity in naïve K562 cells was sufficient to enhance PI3K/Akt activity and to confer resistance to the cytotoxic effects of doxorubicin. Conversely, the knockdown of endogenous FOXO3a expression reduced PI3K/Akt activity and sensitized these cells to doxorubicin. Further chromatin immunoprecipitation and promoter mutation analyses demonstrated that FOXO3a regulates the expression of the PI3K catalytic subunit p110α through the activation of a promoter region proximal to a novel untranslated exon upstream from the reported transcription start site of the p110α gene PIK3CA. As was the case for FOXO3a, the expression or knockdown of p110α was sufficient to amplify or reduce PI3K/Akt activity, respectively. Thus, our results suggest that the chronic activation of FOXO3a by doxorubicin in CML cells can enhance survival through a feedback mechanism that involves enhanced p110α expression and hyperactivation of the PI3K/Akt pathway. PMID:18644865

  15. 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

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

    2014-01-01

    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-κB (NF-κB) 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-κB 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

  16. The phosphoinositide 3-kinase inhibitor PI-103 downregulates choline kinase alpha leading to phosphocholine and total choline decrease detected by magnetic resonance spectroscopy.

    PubMed

    Al-Saffar, Nada M S; Jackson, L Elizabeth; Raynaud, Florence I; Clarke, Paul A; Ramírez de Molina, Ana; Lacal, Juan C; Workman, Paul; Leach, Martin O

    2010-07-01

    The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus ((31)P)- and proton ((1)H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103-induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase alpha (ChoK(alpha)), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r(2) = 0.9, P = 0.009) was found only between PC concentrations and ChoK(alpha) expression but not with FAS or pACL. This study identified inhibition of ChoK(alpha) as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of (1)H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with (31)P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors. PMID:20551061

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

    SciTech Connect

    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.

    2014-10-02

    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.

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

    PubMed Central

    Dai, Gucan

    2013-01-01

    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

  19. Purification of two distinct types of phosphoinositide-specific phospholipase C from rat liver. Enzymological and structural studies.

    PubMed Central

    Nakanishi, O; Homma, Y; Kawasaki, H; Emori, Y; Suzuki, K; Takenawa, T

    1988-01-01

    Two kinds of phosphoinositide-specific phospholipase C (PLC) were purified from rat liver by acid precipitation and several steps of column chromatography. About 50% of the activity could be precipitated when the pH of the liver homogenate was lowered to pH 4.7. The redissolved precipitate yielded two peaks, PLC I and PLC II, in an Affi-gel Blue column, and each was further purified to homogeneity by three sequential h.p.l.c. steps, which were different for the two enzymes. The purified PLC I and PLC II had estimated Mr values of 140,000 and 71,000 respectively on SDS/polyacrylamide-gel electrophoresis. Both enzymes hydrolysed phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in a Ca2+- and pH-dependent manner. PLC I was most active at 10 microM- and 0.1 mM-Ca2+ for hydrolysis of PI and PIP2 respectively, whereas PLC II showed the highest activity at 5 mM- and 10 microM-Ca2+ for that of PI and PIP2 respectively. The optimal pH of the two enzymes also differed with substrates or Ca2+ concentration, in the range pH 5.0-6.0. Hydrolysis of phosphoinositides by these enzymes was completely inhibited by Hg2+ and was affected by other bivalent cations. From data obtained by peptide mapping and partial amino acid sequencing, it was clarified that PLC I and PLC II had distinct structures. Moreover, partial amino acid sequences of three proteolytic fragments of PLC I completely coincided with those of PLC-148 [Stahl, Ferenz, Kelleher, Kriz & Knopf (1988) Nature (London) 332, 269-272]. Images Fig. 3. Fig. 6. PMID:2851991

  20. Down-regulation of class II phosphoinositide 3-kinase alpha expression below a critical threshold induces apoptotic cell death.

    PubMed

    Elis, Winfried; Triantafellow, Ellen; Wolters, Natalie M; Sian, Katie R; Caponigro, Giordano; Borawski, Jason; Gaither, L Alex; Murphy, Leon O; Finan, Peter M; Mackeigan, Jeffrey P

    2008-04-01

    Members of the phosphoinositide 3-kinase (PI3K) family collectively control multiple cellular responses, including proliferation, growth, chemotaxis, and survival. These diverse effects can partly be attributed to the broad range of downstream effectors being regulated by the products of these lipid kinases, the 3'-phosphoinositides. However, an additional layer of complexity is introduced by the existence of multiple PI3K enzyme isoforms. Much has been learned over the last years on the roles of the classes I and III PI3K members in cellular signaling, but little is known about the isoform-specific tasks done by the class II PI3Ks (C2alpha, beta, and gamma). In this study, we used quantitative reverse transcription-PCR and RNA interference in mammalian cells to gain further insight into the function of these lesser studied PI3K enzymes. We find that PI3K-C2alpha, but not PI3K-C2beta, has an important role in controlling cell survival and by using a panel of RNA interference reagents, we were able to determine a critical threshold of PI3K-C2alpha mRNA levels, below which the apoptotic program is switched on, via the intrinsic cell death pathway. In addition, knockdown of PI3K-C2alpha to levels that by themselves do not induce apoptosis sensitize cells to the anticancer agent Taxol (paclitaxel). Lastly, we report that lowering the levels of PI3K-C2alpha in a number of cancer cell lines reduces their proliferation and cell viability, arguing that PI3K inhibitors targeting not only the class Ialpha isoform but also class IIalpha may contribute to an effective anticancer strategy. PMID:18403640

  1. Molecular cytogenetic interphase analysis of Phosphoinositide-specific Phospholipase C ?1 gene in paraffin-embedded brain samples of major depression patients.

    PubMed

    Lo Vasco, Vincenza Rita; Polonia, Patrizia

    2012-01-01

    Mood disorders represent a major medical need, as their chronic treatments are not effective in all patients. Literature data suggested that phosphoinositides (PI) signal transduction pathway and related molecules such as the Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes, might be involved in the pathophysiology of mood disorders, including major depression. By using interphase fluorescent in situ hybridization methodology, we analyzed PLCB1 gene, which codifies for the PI-PLC ?1 enzyme, in paraffin embedded samples of orbito-frontal cortex of 15 patients affected with major depression and in 15 normal controls. No deletions of PLCB1 were identified with the methodology used, which allows to exclude wide gene deletions. The results, the technical aspects of the FISH methodology, and its limitations are discussed. PMID:21880371

  2. Opposing effects of a ras oncogene on growth factor-stimulated phosphoinositide hydrolysis: desensitization to platelet-derived growth factor and enhanced sensitivity to bradykinin

    SciTech Connect

    Parries, G.; Hoebel, R.; Racker, E.

    1987-05-01

    Expression of a transforming Harvey or Kirsten ras gene caused opposing effects in the ability of platelet-derived growth factor (PDGF) and bradyknin to activate phospholipase C-mediated phosphoinositide hydrolysis. In (/sup 3/H)inositol-labeled rat-1 fibroblasts, PDGF resulted in a 2-fold increase in the level of (/sup 3/H)inositol trisphosphate (InsP/sub 3/) after 2 min and, in the presence of LiCl, a 3- to 8-fold increase in the level of (/sup 3/H)inositol monophosphate (InsP/sub 1/) after 30 min. However, in EJ-ras-transfected rat-1 cells, which exhibit near normal levels of PDGF receptors, PDGF resulted in little or no accumulation of either (/sup 3/H)InsP/sub 3/ or (/sup 3/H)InsP/sub 1/. Similarly, marked stimulations by PDGF were observed in NIH 3T3 cells, as well as in v-src-transformed 3T3 cells, but not in 3T3 cells transformed by Kirsten sarcoma virus or by transfection with v-Ha-ras DNA. This diminished phosphoinositide response in ras-transformed cells was associated with a markedly attenuated mitogenic response to PDGF. On the other hand, both phosphoinositide metabolism and DNA synthesis in ras-transformed fibroblasts were stimulated several-fold by serum. In NIH 3T3 cells carrying a glucocorticoid-inducible v-Ha-ras gene, a close correlation was found between the expression of p21/sup ras/ and the loss of PDGF-stimulated (/sup 3/H)InsP/sub 1/ accumulation. The authors propose that a ras gene product (p21) can, directly or indirectly, influence growth factor-stimulated phosphoinositide hydrolysis, as well as DNA synthesis, via alterations in the properties of specific growth factor receptors.

  3. Ligand-based Pharmacophore Modeling; Atom-based 3D-QSAR Analysis and Molecular Docking Studies of Phosphoinositide-Dependent Kinase-1 Inhibitors

    PubMed Central

    Kirubakaran, P.; Muthusamy, K.; Singh, K. H. D.; Nagamani, S.

    2012-01-01

    Phosphoinositide-dependent kinase-1 plays a vital role in the PI3-kinase signaling pathway that regulates gene expression, cell cycle growth and proliferation. The common human cancers include lung, breast, blood and prostate possess over stimulation of the phosphoinositide-dependent kinase-1 signaling and making phosphoinositide-dependent kinase-1 an interesting therapeutic target in oncology. A ligand-based pharmacophore and atom-based 3D-QSAR studies were carried out on a set of 82 inhibitors of PDK1. A six point pharmacophore with two hydrogen bond acceptors (A), three hydrogen bond donors (D) and one hydrophobic group (H) was obtained. The pharmacophore hypothesis yielded a 3D-QSAR model with good partial least square statistics results. The training set correlation is characterized by partial least square factors (R2 = 0.9557, SD = 0.2334, F = 215.5, P = 1.407e-32). The test set correlation is characterized by partial least square factors (Q2 ext = 0.7510, RMSE = 0.5225, Pearson-R =0.8676). The external validation indicated that our QSAR model possess high predictive power with good value of 0.99 and value of 0.88. The docking results show the binding orientations of these inhibitors at active site amino acid residues (Ala162, Thr222, Glu209 and Glu166) of phosphoinositide-dependent kinase-1 protein. The binding free energy interactions of protein-ligand complex have been calculated, which plays an important role in molecular recognition and drug design approach. PMID:23325995

  4. A switch of G protein-coupled receptor binding preference from phosphoinositide 3-kinase (PI3K)-p85 to filamin A negatively controls the PI3K pathway.

    PubMed

    Najib, Souad; Saint-Laurent, Nathalie; Estve, Jean-Pierre; Schulz, Stefan; Boutet-Robinet, Elisa; Fourmy, Daniel; Lttig, Jens; Mollereau, Catherine; Pyronnet, Stphane; Susini, Christiane; Bousquet, Corinne

    2012-03-01

    Frequent oncogenic alterations occur in the phosphoinositide 3-kinase (PI3K) pathway, urging identification of novel negative controls. We previously reported an original mechanism for restraining PI3K activity, controlled by the somatostatin G protein-coupled receptor (GPCR) sst2 and involving a ligand-regulated interaction between sst2 with the PI3K regulatory p85 subunit. We here identify the scaffolding protein filamin A (FLNA) as a critical player regulating the dynamic of this complex. A preexisting sst2-p85 complex, which was shown to account for a significant basal PI3K activity in the absence of ligand, is disrupted upon sst2 activation. FLNA was here identified as a competitor of p85 for direct binding to two juxtaposed sites on sst2. Switching of GPCR binding preference from p85 toward FLNA is determined by changes in the tyrosine phosphorylation of p85- and FLNA-binding sites on sst2 upon activation. It results in the disruption of the sst2-p85 complex and the subsequent inhibition of PI3K. Knocking down FLNA expression, or abrogating FLNA recruitment to sst2, reversed the inhibition of PI3K and of tumor growth induced by sst2. Importantly, we report that this FLNA inhibitory control on PI3K can be generalized to another GPCR, the mu opioid receptor, thereby providing an unprecedented mechanism underlying GPCR-negative control on PI3K. PMID:22203038

  5. Activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network in human hepatocellular carcinoma by systems-theoretic analysis.

    PubMed

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

    2012-01-01

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

  6. Regulation of the phosphoinositide pathway in cultured Sertoli cells from immature rats: effects of follicle-stimulating hormone and fluoride

    SciTech Connect

    Quirk, S.M.; Reichert, L.E. Jr.

    1988-07-01

    Many hormones elicit effects on target cells by stimulating the enzyme phospholipase-C, which catalyzes the hydrolysis of phosphoinositides to the intracellular second messengers diacylglycerol and inositol phosphates. The present study examined the roles of FSH and guanine nucleotide-binding proteins (G-proteins) in regulating the hydrolysis of phosphoinositides in Sertoli cells. Sertoli cell cultures prepared from 16- to 18-day-old rats were incubated for 24 h with myo-(2-3H) inositol to label endogenous phospholipids. Treatment of cells from 0.5-20 min with preparations of ovine FSH ranging in potency from 1-60 times that of NIH FSH S1 did not affect accumulation of inositol phosphates. Levels of total (3H)inositol phosphates ((3H)inositol mono-, di-, and triphosphates (IP, IP2, and IP3)) in FSH-treated cultures was 75-120% the levels in control cultures over the various time intervals studied. Addition of testosterone and the combination of testosterone plus retinoic acid, agents that have been shown to potentiate effects of FSH in other systems, did not affect accumulation of inositol phosphates in response to FSH. In contrast to the lack of effect on accumulation of inositol phosphates, FSH stimulated 4- to 11-fold increases in estradiol secretion over 24 h of culture, indicating that Sertoli cells were viable and responsive to FSH. AIF4- has been shown to activate G-proteins involved in regulation of adenylate cyclase activity. In the present study, AIF4- induced 4- to 5-fold increases in IP, IP2, and IP3 in experiments wherein FSH had no effect. Pretreatment of Sertoli cells with pertussis toxin (100 and 1000 ng/ml) for 24 h inhibited fluoride-induced generation of IP, IP2, and IP3 by 24-51%. Similar treatment with cholera toxin had no effect on basal or fluoride-induced generation of IP2 or IP3, but increased fluoride-induced generation of IP by 20-34%.

  7. Nicotine enhances hepatocyte growth factor-mediated lung cancer cell migration by activating the α7 nicotine acetylcholine receptor and phosphoinositide kinase-3-dependent pathway

    PubMed Central

    YONEYAMA, REMI; AOSHIBA, KAZUTETSU; FURUKAWA, KINYA; SAITO, MAKOTO; KATABA, HIROAKI; NAKAMURA, HIROYUKI; IKEDA, NORIHIKO

    2016-01-01

    Cigarette smoking not only promotes lung carcinogenesis, but it has also been demonstrated to promote the progression of lung cancer. Despite nicotine being a major component of cigarette smoke, it is not carcinogenic when acting alone. Instead, it is believed to function as a tumor promoter. Due to the fatal consequences of lung cancer being primarily associated with the processes of invasion and metastasis, the present study aimed to determine the effect of nicotine on the migratory activity of lung cancer cells. The effect of nicotine on the migration of lung cancer A549 cells was evaluated by a wound healing assay. Hepatocyte growth factor (HGF) was used as a pro-migratory stimulus. During several of the experiments, specific inhibitors of α7-nicotine acetylcholine receptor (α7-nAchR), phosphoinositide kinase-3 (PI3K) and extracellular signal-related kinase (ERK)1/2 were included. The phosphorylation levels of Akt and ERK1/2 were examined using a cell-based protein phosphorylation assay. It was observed that nicotine did not induce cell migration by itself, but that it instead promoted HGF-induced cell migration. The effects of nicotine were inhibited by the pretreatment of the cells with the α7-nAchR inhibitor, methyllycaconitine, and the PI3K inhibitor, LY294002. The mitogen-activated protein kinase/ERK kinase kinase inhibitor exerted modest, but non-significant inhibitory activity on the effect of nicotine. Nicotine did not induce Akt phosphorylation by itself, but instead promoted the HGF-induced phosphorylation of Akt. It was also observed that nicotine had no effect on ERK1/2 phosphorylation. The results from the present study indicate that nicotine, when alone, does not have a pro-migratory function, but instead enhances responsiveness to the pro-migratory stimulus emitted by HGF. The current study provides an insight into the mechanism of tumor promotion by demonstrating that nicotine and α7-nAchRs act in synergy with the HGF-induced PI3K/Akt signaling pathway, increasing the sensitivity of lung cancer cells to HGF, and thereby promoting cell migration, a vital step in invasion and metastasis. PMID:26870265

  8. Phosphoinositides in Ca(2+) signaling and excitation-contraction coupling in skeletal muscle: an old player and newcomers.

    PubMed

    Csernoch, Laszlo; Jacquemond, Vincent

    2015-12-01

    Since the postulate, 30 years ago, that phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2) as the precursor of inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3) would be critical for skeletal muscle excitation-contraction (EC) coupling, the issue of whether phosphoinositides (PtdInsPs) may have something to do with Ca(2+) signaling in muscle raised limited interest, if any. In recent years however, the PtdInsP world has expanded considerably with new functions for PtdIns(4,5)P 2 but also with functions for the other members of the PtdInsP family. In this context, the discovery that genetic deficiency in a PtdInsP phosphatase has dramatic consequences on Ca(2+) homeostasis in skeletal muscle came unanticipated and opened up new perspectives in regards to how PtdInsPs modulate muscle Ca(2+) signaling under normal and disease conditions. This review intends to make an update of the established, the questioned, and the unknown regarding the role of PtdInsPs in skeletal muscle Ca(2+) homeostasis and EC coupling, with very specific emphasis given to Ca(2+) signals in differentiated skeletal muscle fibers. PMID:26377756

  9. Platelet P-selectin expression: requirement for protein kinase C, but not protein tyrosine kinase or phosphoinositide 3-kinase.

    PubMed

    Libersan, Danielle; Merhi, Yahye

    2003-06-01

    P-selectin is translocated from the alpha-granules to the surface of activated platelets where it participates in thrombosis and inflammation. We investigated the signaling pathways involved in thrombin-induced human platelet P-selectin expression. Assessed by flow cytometry, inhibition of protein kinase C (PKC) with chelerythrine reduced P-selectin expression by 66%, platelet/neutrophil binding, GPIIb/IIIa activation and aggregation (p<0.05). G 6976, an inhibitor of the conventional PKCs (alpha and beta), did not alter P-selectin expression. However, rottlerin inhibited by 50% its expression (p<0.05), but only at doses that interfere with the novel (epsilon eta) and atypical (zeta) PKCs. Inhibition of protein tyrosine kinase (PTK) and phosphoinosi-tide 3-kinase (PI3-K) did not significantly affect P-selectin expression. In conclusion, thrombin-induced P-selectin expression is PKC-sensitive, but PTK and PI3-K-insensitive. The novel epsilon and eta and atypical zeta, but not the conventional alpha and beta and the novel delta PKCs, may be involved in this process. PMID:12783114

  10. Role of calcium in phosphoinositide metabolism and inhibition of norepinephrine transport into synaptic vesicles by amphetamine analogs

    SciTech Connect

    Knepper, S.M.

    1985-01-01

    Norepinephrine-(NE) and calcium ionophore A23187-stimulated phosphoinositide (PIn) metabolism in rat brain slices was studied under varying calcium conditions. Tissue was labelled with /sup 3/H-myo-inositol and /sup 3/H-inositol phosphates (IPn), products of PIn metabolism were measured. In the absence of media calcium the response to NE was decreased while that to A23187 was little affected A23187 can release calcium from intracellular stores. Basal and stimulated accumulation of /sup 3/H-IPn was reversibly antagonized with EGTA by addition of calcium. Using calcium buffers, approximately 10/sup -7/ M free calcium was required to support hydrolysis. Free intracellular calcium is maintained at approximately this level. Thus calcium is required for PIn hydrolysis but appears to play a permissive role, basal levels being sufficient to support metabolism. Conformationally-defined (rigid) and -restricted (semi-rigid) analogs of the most stable conformations of amphetamine, antiperiplanar (exo) and gauche (endo), were utilized to probe the conformational requirements of vesicular NE transport. Analogs tested were 2-aminotetralin (2AT), 3-methyltetrahydroisoquinoline, anti- and syn-9-aminobenzobicyclo(2.2.1)heptene, and endo and exo conformers of 2-aminobenzobicyclo(2.2.1)heptene and 2-aminobenzobicyclo(2.2.2)octene.

  11. Regulatory roles of phosphoinositides in membrane trafficking and their potential impact on cell-wall synthesis and re-modelling

    PubMed Central

    Krishnamoorthy, Praveen; Sanchez-Rodriguez, Clara; Heilmann, Ingo; Persson, Staffan

    2014-01-01

    Background Plant cell walls are complex matrices of carbohydrates and proteins that control cell morphology and provide protection and rigidity for the plant body. The construction and maintenance of this intricate system involves the delivery and recycling of its components through a precise balance of endomembrane trafficking, which is controlled by a plethora of cell signalling factors. Phosphoinositides (PIs) are one class of signalling molecules with diverse roles in vesicle trafficking and cytoskeleton structure across different kingdoms. Therefore, PIs may also play an important role in the assembly of plant cell walls. Scope The eukaryotic PI pathway is an intricate network of different lipids, which appear to be divided in different pools that can partake in vesicle trafficking or signalling. Most of our current understanding of how PIs function in cell metabolism comes from yeast and mammalian systems; however, in recent years significant progress has been made towards a better understanding of the plant PI system. This review examines the current state of knowledge of how PIs regulate vesicle trafficking and their potential influence on plant cell-wall architecture. It considers first how PIs are formed in plants and then examines their role in the control of vesicle trafficking. Interactions between PIs and the actin cytoskeleton and small GTPases are also discussed. Future challenges for research are suggested. PMID:24769536

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

    PubMed Central

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

    2001-01-01

    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

  13. Shiga toxin type-2 (Stx2) induces glutamate release via phosphoinositide 3-kinase (PI3K) pathway in murine neurons

    PubMed Central

    Obata, Fumiko; Hippler, Lauren M.; Saha, Progyaparamita; Jandhyala, Dakshina M.; Latinovic, Olga S.

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) can cause central nervous system (CNS) damage resulting in paralysis, seizures, and coma. The key STEC virulence factors associated with systemic illness resulting in CNS impairment are Shiga toxins (Stx). While neurons express the Stx receptor globotriaosylceramide (Gb3) in vivo, direct toxicity to neurons by Stx has not been studied. We used murine neonatal neuron cultures to study the interaction of Shiga toxin type 2 (Stx2) with cell surface expressed Gb3. Single molecule imaging three dimensional STochastic Optical Reconstruction Microscopy—Total Internal Reflection Fluorescence (3D STORM-TIRF) allowed visualization and quantification of Stx2-Gb3 interactions. Furthermore, we demonstrate that Stx2 increases neuronal cytosolic Ca2+, and NMDA-receptor inhibition blocks Stx2-induced Ca2+ influx, suggesting that Stx2-mediates glutamate release. Phosphoinositide 3-kinase (PI3K)-specific inhibition by Wortmannin reduces Stx2-induced intracellular Ca2+ indicating that the PI3K signaling pathway may be involved in Stx2-associated glutamate release, and that these pathways may contribute to CNS impairment associated with STEC infection. PMID:26236186

  14. Structural basis for UCN-01 (7-hydroxystaurosporine) specificity and PDK1 (3-phosphoinositide-dependent protein kinase-1) inhibition.

    PubMed

    Komander, David; Kular, Gursant S; Bain, Jennifer; Elliott, Matthew; Alessi, Dario R; Van Aalten, Daan M F

    2003-10-15

    PDK1 (3-phosphoinositide-dependent protein kinase-1) is a member of the AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family of protein kinases, and has a key role in insulin and growth-factor signalling through phosphorylation and subsequent activation of a number of other AGC kinase family members, such as protein kinase B. The staurosporine derivative UCN-01 (7-hydroxystaurosporine) has been reported to be a potent inhibitor for PDK1, and is currently undergoing clinical trials for the treatment of cancer. Here, we report the crystal structures of staurosporine and UCN-01 in complex with the kinase domain of PDK1. We show that, although staurosporine and UCN-01 interact with the PDK1 active site in an overall similar manner, the UCN-01 7-hydroxy group, which is not present in staurosporine, generates direct and water-mediated hydrogen bonds with active-site residues. Inhibition data from UCN-01 tested against a panel of 29 different kinases show a different pattern of inhibition compared with staurosporine. We discuss how these differences in inhibition could be attributed to specific interactions with the additional 7-hydroxy group, as well as the size of the 7-hydroxy-group-binding pocket. This information could lead to opportunities for structure-based optimization of PDK1 inhibitors. PMID:12892559

  15. Characterization of PROPPIN-Phosphoinositide Binding and Role of Loop 6CD in PROPPIN-Membrane Binding

    PubMed Central

    Busse, Ricarda A.; Scacioc, Andreea; Krick, Roswitha; Pérez-Lara, Ángel; Thumm, Michael; Kühnel, Karin

    2015-01-01

    PROPPINs (β-propellers that bind polyphosphoinositides) are a family of PtdIns3P- and PtdIns(3,5)P2-binding proteins that play an important role in autophagy. We analyzed PROPPIN-membrane binding through isothermal titration calorimetry (ITC), stopped-flow measurements, mutagenesis studies, and molecular dynamics (MD) simulations. ITC measurements showed that the yeast PROPPIN family members Atg18, Atg21, and Hsv2 bind PtdIns3P and PtdIns(3,5)P2 with high affinities in the nanomolar to low-micromolar range and have two phosphoinositide (PIP)-binding sites. Single PIP-binding site mutants have a 15- to 30-fold reduced affinity, which explains the requirement of two PIP-binding sites in PROPPINs. Hsv2 bound small unilamellar vesicles with a higher affinity than it bound large unilamellar vesicles in stopped-flow measurements. Thus, we conclude that PROPPIN membrane binding is curvature dependent. MD simulations revealed that loop 6CD is an anchor for membrane binding, as it is the region of the protein that inserts most deeply into the lipid bilayer. Mutagenesis studies showed that both hydrophobic and electrostatic interactions are required for membrane insertion of loop 6CD. We propose a model for PROPPIN-membrane binding in which PROPPINs are initially targeted to membranes through nonspecific electrostatic interactions and are then retained at the membrane through PIP binding. PMID:25954880

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

    PubMed Central

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

    2015-01-01

    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. PMID:25873899

  17. Novel roles for class II Phosphoinositide 3-Kinase C2β in signalling pathways involved in prostate cancer cell invasion

    PubMed Central

    Mavrommati, Ioanna; Cisse, Ouma; Falasca, Marco; Maffucci, Tania

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) regulate several cellular functions such as proliferation, growth, survival and migration. The eight PI3K isoforms are grouped into three classes and the three enzymes belonging to the class II subfamily (PI3K-C2α, β and γ) are the least investigated amongst all PI3Ks. Interest on these isoforms has been recently fuelled by the identification of specific physiological roles for class II PI3Ks and by accumulating evidence indicating their involvement in human diseases. While it is now established that these isoforms can regulate distinct cellular functions compared to other PI3Ks, there is still a limited understanding of the signalling pathways that can be specifically regulated by class II PI3Ks. Here we show that PI3K-C2β regulates mitogen-activated protein kinase kinase (MEK1/2) and extracellular signal-regulated kinase (ERK1/2) activation in prostate cancer (PCa) cells. We further demonstrate that MEK/ERK and PI3K-C2β are required for PCa cell invasion but not proliferation. In addition we show that PI3K-C2β but not MEK/ERK regulates PCa cell migration as well as expression of the transcription factor Slug. These data identify novel signalling pathways specifically regulated by PI3K-C2β and they further identify this enzyme as a key regulator of PCa cell migration and invasion. PMID:26983806

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

    SciTech Connect

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

    1988-02-01

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

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

    PubMed Central

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

    2014-01-01

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

  20. Crystal structure of hGEF-H1 PH domain provides insight into incapability in phosphoinositide binding.

    PubMed

    Jiang, Yan; Jiang, Heli; Zhou, Shaoyang; Meng, Bing; Liu, Zhi-Jie; Ouyang, Songying

    2016-03-18

    The guanine nucleotide exchange factor GEF-H1 (also known as ARHGEF2) is identified as a member of the Dbl family of GEFs. It regulates RhoA-dependent cell signaling pathways and plays important roles in biological processes. GEF-H1 contains an N-terminal zinc finger domain, a Dbl-homologous (DH) domain followed by a Pleckstrin homology (PH) domain, and a C-terminal domain. The specific roles of its PH domain are poorly understood. Here we report the crystal structure of human GEF-H1 PH domain to 2.45 Å resolution. A conserved surface is formed by β8, β9, β10, and it may mediate protein-protein interactions. Although the folding resembles other PH domains that have defined structures, superposition of different PH domains clearly shows that the loop between β6/β7 and the loop between β3/β4 are so close that they will prevent its binding with phosphoinositide due to steric hindrance, and this has been proved by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). Our studies provide a structural framework for further work on the function of GEF-H1. PMID:26820534

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

    PubMed Central

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

    2002-01-01

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

  2. Activation of sonic hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and -9 via the phosphoinositide-3 kinase/AKT signaling pathway in glioblastoma

    PubMed Central

    CHANG, LIANG; ZHAO, DAN; LIU, HUI-BIN; WANG, QIU-SHI; ZHANG, PING; LI, CHEN-LONG; DU, WEN-ZHONG; WANG, HONG-JUN; LIU, XING; ZHANG, ZHI-REN; JIANG, CHUAN-LU

    2015-01-01

    Aberrant hedgehog signaling contributes to the development of various malignancies, including glioblastoma (GBM). However, the potential mechanism of hedgehog signaling in GBM migration and invasion has remained to be elucidated. The present study showed that enhanced hedgehog signaling by recombinant human sonic hedgehog N-terminal peptide (rhSHH) promoted the adhesion, invasion and migration of GBM cells, accompanied by increases in mRNA and protein levels of matrix metalloproteinase-2 (MMP-2) and MMP-9. However, inhibition of hedgehog signaling with cyclopamine suppressed the adhesion, invasion and migration of GBM cells, accompanied by decreases in mRNA and protein levels of MMP-2 and -9. Furthermore, it was found that MMP-2- and MMP-9-neutralizing antibodies or GAM6001 reversed the inductive effects of rhSHH on cell migration and invasion. In addition, enhanced hedgehog signaling by rhSHH increased AKT phosphorylation, whereas blockade of hedgehog signaling decreased AKT phosphorylations. Further experiments showed that LY294002, an inhibitor of phosphoinositide-3 kinase (PI3K), decreased rhSHH-induced upregulation of MMP-2 and -9. Finally, the protein expression of glioblastoma-associated oncogene 1 was positively correlated with levels of phosphorylated AKT as well as protein expressions of MMP-2 and -9 in GBM tissue samples. In conclusion, the present study indicated that the hedgehog pathway regulates GBM-cell migration and invasion by increasing MMP-2 and MMP-9 production via the PI3K/AKT pathway. PMID:26299938

  3. Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts.

    PubMed Central

    Goruppi, S; Ruaro, E; Varnum, B; Schneider, C

    1997-01-01

    Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, Gas6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival. PMID:9234702

  4. Echinacea purpurea root extract inhibits TNF release in response to Pam3Csk4 in a phosphatidylinositol-3-kinase dependent manner.

    PubMed

    Fast, David J; Balles, John A; Scholten, Jeffrey D; Mulder, Timothy; Rana, Jatinder

    2015-10-01

    Polysaccharides derived from Echinacea have historically been shown to be immunostimulatory. We describe in this work however the anti-inflammatory effect of a water extract of Echinacea purpurea roots (EPRW) that inhibited Pam3Csk4 stimulated production of TNFα by human monocytic THP-1 cells. The polyphenols and alkylamides typically found in Echinacea extracts were absent in EPRW suggesting that the anti-inflammatory component(s) was a polysaccharide. This anti-inflammatory activity was shown to be mediated by the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway as chemical inhibition of PI3K abolished the EPRW anti-inflammatory effect. Demonstration of phosphorylation of Akt and ribosomal S6 proteins, downstream targets of PI3K confirmed EPRW-mediated activation of this pathway. In conclusion, this observation suggests that non-alkylamide/non-polyphenolic phytochemicals from Echinacea may contribute in part to some of the anti-inflammatory therapeutic effects such as reduced severity of symptoms that have been observed in vivo in the treatment of upper respiratory tract infections with Echinacea. PMID:26190752

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. 3-Phosphoinositide-dependent kinase 1 potentiates upstream lesions on the phosphatidylinositol 3-kinase pathway in breast carcinoma.

    PubMed

    Maurer, Matthew; Su, Tao; Saal, Lao H; Koujak, Susan; Hopkins, Benjamin D; Barkley, Christina R; Wu, Jiaping; Nandula, Subhadra; Dutta, Bhaskar; Xie, Yuli; Chin, Y Rebecca; Kim, Da-In; Ferris, Jennifer S; Gruvberger-Saal, Sofia K; Laakso, Mervi; Wang, Xiaomei; Memeo, Lorenzo; Rojtman, Albert; Matos, Tulio; Yu, Jennifer S; Cordon-Cardo, Carlos; Isola, Jorma; Terry, Mary Beth; Toker, Alex; Mills, Gordon B; Zhao, Jean J; Murty, Vundavalli V V S; Hibshoosh, Hanina; Parsons, Ramon

    2009-08-01

    Lesions of ERBB2, PTEN, and PIK3CA activate the phosphatidylinositol 3-kinase (PI3K) pathway during cancer development by increasing levels of phosphatidylinositol-3,4,5-triphosphate (PIP(3)). 3-Phosphoinositide-dependent kinase 1 (PDK1) is the first node of the PI3K signal output and is required for activation of AKT. PIP(3) recruits PDK1 and AKT to the cell membrane through interactions with their pleckstrin homology domains, allowing PDK1 to activate AKT by phosphorylating it at residue threonine-308. We show that total PDK1 protein and mRNA were overexpressed in a majority of human breast cancers and that 21% of tumors had five or more copies of the gene encoding PDK1, PDPK1. We found that increased PDPK1 copy number was associated with upstream pathway lesions (ERBB2 amplification, PTEN loss, or PIK3CA mutation), as well as patient survival. Examination of an independent set of breast cancers and tumor cell lines derived from multiple forms of human cancers also found increased PDK1 protein levels associated with such upstream pathway lesions. In human mammary cells, PDK1 enhanced the ability of upstream lesions to signal to AKT, stimulate cell growth and migration, and rendered cells more resistant to PDK1 and PI3K inhibition. After orthotopic transplantation, PDK1 overexpression was not oncogenic but dramatically enhanced the ability of ERBB2 to form tumors. Our studies argue that PDK1 overexpression and increased PDPK1 copy number are common occurrences in cancer that potentiate the oncogenic effect of upstream lesions on the PI3K pathway. Therefore, we conclude that alteration of PDK1 is a critical component of oncogenic PI3K signaling in breast cancer. PMID:19602588

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

    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.

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

    PubMed Central

    Edelstein-Keshet, Leah

    2012-01-01

    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

  9. Differential interaction of β2e with phosphoinositides: A comparative study between β2e and MARCKS.

    PubMed

    Kim, Dong-Il; Suh, Byung-Chang

    2016-05-01

    Voltage-gated calcium (CaV) channels are responsible for Ca(2+) influx in excitable cells. As one of the auxiliary subunits, the CaV β subunit plays a pivotal role in the membrane expression and receptor modulation of CaV channels. In particular, the subcellular localization of the β subunit is critical for determining the biophysical properties of CaV channels. Recently, we showed that the β2e isotype is tethered to the plasma membrane. Such a feature of β2e is due to the reversible electrostatic interaction with anionic membrane phospholipids. Here, we further explored the membrane interaction property of β2e by comparing it with that of myristoylated alanine-rich C kinase substrate (MARCKS). First, the charge neutralization of the inner leaf of the plasma membrane induced the translocation of both β2e and MARCKS to the cytosol, while the transient depletion of poly-phosphoinositides (poly-PIs) by translocatable pseudojanin (PJ) systems induced the cytosolic translocation of β2e but not MARCKS. Second, the activation of protein kinase C (PKC) induced the translocation of MARCKS but not β2e. We also found that after the cytosolic translocation of MARCKS by receptor activation, depletion of poly-PIs slowed the recovery of MARCKS to the plasma membrane. Together, our data demonstrate that both β2e and MARCKS bind to the membrane through electrostatic interaction but with different binding affinity, and thus, they are differentially regulated by enzymatic degradation of membrane PIs. PMID:26650714

  10. The Phosphoinositide-3-Kinase–Akt Signaling Pathway Is Important for Staphylococcus aureus Internalization by Endothelial Cells ▿

    PubMed Central

    Oviedo-Boyso, Javier; Cortés-Vieyra, Ricarda; Huante-Mendoza, Alejandro; Yu, Hong B.; Valdez-Alarcón, Juan J.; Bravo-Patiño, Alejandro; Cajero-Juárez, Marcos; Finlay, B. Brett; Baizabal-Aguirre, Víctor M.

    2011-01-01

    Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)–Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus. This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus. Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3β on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3β, and NF-κB. PMID:21844240

  11. The anti-esophageal cancer cell activity by a novel tyrosine/phosphoinositide kinase inhibitor PP121

    SciTech Connect

    Peng, Yi; Zhou, Yajuan; Cheng, Long; Hu, Desheng; Zhou, Xiaoyi; Wang, Zhaohua; Xie, Conghua; Zhou, Fuxiang

    2015-09-11

    Here we explored the potential effect of PP121, a novel dual inhibitor of tyrosine and phosphoinositide kinases, against human esophageal cancer cells. We showed that PP121 exerted potent cytotoxic effect in primary (patient-derived) and established (Eca-109, TE-1 and TE-3 lines) esophageal cancer cells, possibly through activating caspase-3-dependnent apoptosis. PP121 was, however, non-cytotoxic to the normal human esophageal epithelial cells (EECs). At the molecular level, we showed that PP121 blocked Akt-mTOR (mammalian target of rapamycin) activation in esophageal cancer cells, which was restored by introducing a constitutively-active Akt (CA-Akt). Yet, CA-Akt only partly inhibited cytotoxicity by PP121 in Eca-109 cells. Importantly, we showed that PP121 inhibited nuclear factor kappa B (NFκB) signaling activation in esophageal cancer cells, which appeared independent of Akt-mTOR blockage. In vivo, oral administration of PP121 remarkably inhibited Eca-109 xenograft growth in nude mice, and significantly improved mice survival. Further, the immunohistochemistry (IHC) and Western blot assays analyzing xenografted tumors showed that PP121 inhibited Akt-mTOR and NFκB activations in vivo. Together, we demonstrate that PP121 potently inhibits esophageal cancer cells in vitro and in vivo, possibly through concurrently inhibiting Akt-mTOR and NFκB signalings. - Highlights: • PP121 is cytotoxic against primary and established esophageal cancer cells. • PP121 induces caspase-3-dependnent apoptosis in esophageal cancer cells. • PP121 blocks Akt-mTOR activation in esophageal cancer cells. • PP121 inhibits NFκB activation, independent of Akt-mTOR blockage. • PP121 inhibits Eca-109 xenograft growth and Akt-mTOR/NFκB activation in vivo.

  12. Contrasting effects of phorbol ester and agonist-mediated activation of protein kinase C on phosphoinositide and Ca2+ signalling in a human neuroblastoma.

    PubMed Central

    Willars, G B; Challiss, R A; Stuart, J A; Nahorski, S R

    1996-01-01

    The effects of protein kinase C (PKC) activation on muscarinic receptor-mediated phosphoinositide and Ca2+ signalling were examined in the human neuroblastoma, SH-SY5Y. Carbachol evoked rapid transient elevations of Ins(1,4,5)P3 and intracellular [Ca2+] followed by lower sustained elevations. Phorbol 12,13-dibutyrate (PDBu) preferentially attenuated transient phases. Removal of the transplasmalemmal Ca2+ gradient coupled with depletion of intracellular Ca2+ stores with thapsigargin also reduced carbachol-mediated Ins(1,4,5)P3 accumulation. Under these conditions, PDBu virtually abolished Ins(1,4,5)P3 responses to carbachol thereby implicating both Ca(2+)- and PKC-sensitive components. PDBu also reduced agonist-mediated accumulation of inositol phosphates and depletion of lipids, thereby eliminating an effect of PKC on Ins(1,4,5)P3 metabolism or phosphoinositide synthesis. In electroporated cells, PDBu inhibited Ins(1,4,5)P3 accumulation mediated by carbachol or guanosine 5'-[gamma-thio]-triphosphate, the latter indicating that some PDBu-sensitive elements were downstream of the receptor. The PKC inhibitor, Ro-318220, protected against PDBu but did not enhance responses to maximal concentrations of carbachol, indicating no feedback inhibition by agonist-activated PKC. Muscarinic antagonist activity of Ro-318220 complicated such assessment at low agonist concentrations. Carbachol or PDBu induced cytosol to membrane translocation of PKC alpha. This was faster and possibly greater with PDBu, which may explain the lack of feedback by agonist-activated PKC. These results indicate that, in SH-SY5Y cells, PDBu activation of PKC preferentially inhibits rapid muscarinic receptor-mediated phosphoinositide and Ca2+ responses via suppression of PtdIns(4,5)P2 hydrolysis. This is at least partially through inhibition of Gq-protein/phosphoinositidase C coupling. However, at least at high agonist concentrations, a major agonist-mediated PKC feedback is not present in these cells. PMID:8670170

  13. Phosphoinositide-mediated clathrin adaptor progression at the trans Golgi network

    PubMed Central

    Daboussi, Lydia; Costaguta, Giancarlo; Payne, Gregory S.

    2016-01-01

    Clathrin coated vesicles mediate endocytosis and transport between the trans Golgi network (TGN) and endosomes in eukaryotic cells. Clathrin adaptors play central roles in coat assembly, interacting with clathrin, cargo, and membranes. Two major types of clathrin adaptors act in TGN-endosome traffic, Gga proteins and the AP-1 complex. Here we characterize the relationship between Gga proteins, AP-1, and other TGN clathrin adaptors using live cell and superresolution microscopy in yeast. We present evidence that Gga proteins and AP-1 are recruited sequentially in two waves of coat assembly at the TGN. Mutations that decrease phosphatidylinositol 4-phosphate (PI4P) levels at the TGN slow or uncouple AP-1 coat assembly from Gga coat assembly. Conversely, enhanced PI4P synthesis shortens the time between adaptor waves. Gga2p binds directly to the TGN PI4-kinase Pik1p and contributes to Pik1p recruitment. These results identify a PI4P-based mechanism for regulating progressive assembly of adaptor-specific clathrin coats at the TGN. PMID:22344030

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

    SciTech Connect

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

    1986-03-05

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

  15. Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

    PubMed Central

    Awwad, Hibah O.; Iyer, Varsha; Rosenfeld, Jennifer L.; Millman, Ellen E.; Foster, Estrella; Moore, Robert H.; Knoll, Brian J.

    2007-01-01

    Phosphatidylinositol 3-kinase inhibitors have been shown to affect the endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated β2-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate β2-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the β-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of β2-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of β2-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of β2-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes. PMID:17553490

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

    PubMed

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

    2012-04-15

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

  17. The interferon-gamma-mediated inhibition of lipoprotein lipase gene transcription in macrophages involves casein kinase 2- and phosphoinositide-3-kinase-mediated regulation of transcription factors Sp1 and Sp3.

    PubMed

    Harris, Sandra M; Harvey, Elizabeth J; Hughes, Timothy R; Ramji, Dipak P

    2008-12-01

    The mechanisms underlying transcriptional inhibition by interferon-gamma (IFN-gamma) are poorly understood despite the existence of a large number of genes that are regulated in this manner and the key role of this cytokine in inflammatory disorders such as atherosclerosis. We have previously identified a novel mechanism for transcriptional inhibition by IFN-gamma that involves a reduction in the binding of transcription factors Sp1 and Sp3 to regulatory sequences in the lipoprotein lipase (LPL) gene. In the present study, we have investigated the signalling pathways that impact on the IFN-gamma-mediated regulation of Sp1/Sp3 binding and LPL gene transcription in macrophages. The IFN-gamma-mediated inhibition of LPL promoter activity was prevented by expression of dominant negative forms of casein kinase 2 (CK2) and protein kinase B (PKB), a key downstream component of the phosphoinositide-3-kinase (PI3K) pathway. IFN-gamma activated both the catalytic subunits of CK2 without affecting their expression. CK2 interacted with both Sp1 and Sp3 and this association was increased by IFN-gamma. Electrophoretic mobility shift assays showed that a CK2-mediated phosphorylation of either cellular extracts or recombinant Sp1 reduced binding to the regulatory region in the LPL gene. The action of PKB was potentially mediated through mammalian target for rapamycin proteins. Taken together, these results suggest a key role for CK2 and PI3K signalling pathways in the IFN-gamma-mediated inhibition of macrophage LPL gene transcription through the regulation of Sp1/Sp3 binding. PMID:18793716

  18. 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

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

    2014-09-01

    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-3β) activity which in turn prevented the accumulation of oncoproteins β-catenin, c-jun and c-myc. Taken together, hesperidin supplementation initiated apoptosis via targeted inhibition of constitutively activated Aurora-A mediated PI3K/Akt/GSK-3β and mTOR pathways coupled with autophagic stimulation against AOM induced colon carcinogenesis. PMID:25047426

  19. Integrin αvβ3 mediates the synergetic regulation of core-binding factor α1 transcriptional activity by gravity and insulin-like growth factor-1 through phosphoinositide 3-kinase signaling.

    PubMed

    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

    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 αvβ3 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 α1 (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 αvβ3 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 αvβ3 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 β3 interacted with insulin receptor substrate 1, and that this interaction was decreased under SMG and increased under HG conditions. These results suggest that integrin αvβ3 mediates the synergetic regulation of Cbfa1 transcription activity by gravity and IGF-1 via PI3K signaling. PMID:25263523

  20. Dietary omega-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils.

    PubMed Central

    Sperling, R I; Benincaso, A I; Knoell, C T; Larkin, J K; Austen, K F; Robinson, D R

    1993-01-01

    Earlier studies demonstrated that dietary omega-3 polyunsaturated fatty acid (PUFA) supplementation attenuates the chemotactic response of neutrophils and the generation of leukotriene (LT) B4 by neutrophils stimulated with calcium ionophore; however, the mechanisms and relationship of these effects were not examined. Neutrophils and monocytes from eight healthy individuals were examined before and after 3 and 10 wk of dietary supplementation with 20 g SuperEPA daily, which provides 9.4 g eicosapentaenoic acid (EPA) and 5 g docosahexaenoic acid. The maximal neutrophil chemotactic response to LTB4, assessed in Boyden microchambers, decreased by 69% after 3 wk and by 93% after 10 wk from prediet values. The formation of [3H]inositol tris-phosphate (IP3) by [3H]inositol-labeled neutrophils stimulated by LTB4 decreased by 71% after 3 wk (0.033 +/- 0.013% [3H] release, mean +/- SEM) and by 90% after 10 wk (0.011 +/- 0.011%) from predict values (0.114 +/- 0.030%) as quantitated by beta-scintillation counting after resolution on HPLC. LTB4-stimulated neutrophil chemotaxis and IP3 formation correlated significantly (P < 0.0001); each response correlated closely and negatively with the EPA content of the neutrophil phosphatidylinositol (PI) pool (P = 0.0003 and P = 0.0005, respectively). Neither the affinities and densities of the high and low affinity LTB4 receptors on neutrophils nor LTB4-mediated diglyceride formation changed appreciably during the study. Similar results were observed in neutrophils activated with platelet-activating factor (PAF). The summed formation of LTB4 plus LTB5 was selectively inhibited in calcium ionophore-stimulated neutrophils and was also inhibited in zymosan-stimulated neutrophils. The inhibition of the summed formation of LTB4 plus LTB5 in calcium ionophore-stimulated neutrophils and in zymosan-stimulated neutrophils did not correlate significantly with the EPA content of the PI pool. The data indicate that dietary omega-3 PUFA supplementation inhibits the autoamplification of the neutrophil inflammatory response by decreasing LTB4 formation through the inactivation of the LTA epoxide hydrolase and independently by inhibiting LTB4- (and PAF) stimulated chemotaxis by attenuating the formation of IP3 by the PI-selective phospholipase C. This is the initial demonstration that dietary omega-3 PUFA supplementation can suppress signal transduction at the level of the PI-specific phospholipase C in humans. Images PMID:8381824

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

    PubMed Central

    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

    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

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

    SciTech Connect

    Ren, J.; Pei-Chen Lin, C.; Pathak, M. C.; Temple, B. R. S.; Nile, A. H.; Mousley, C. J.; Duncan, M. C.; Eckert, D. M.; Leiker, T. J.; Ivanova, P. T.; Myers, D. S.; Murphy, R. C.; Brown, H. A.; Verdaasdonk, J.; Bloom, K. S.; Ortlund, E. A.; Neiman, A. M.; Bankaitis, V. A.

    2014-01-08

    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.

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

    SciTech Connect

    Ren, Jihui; Lin, Coney Pei-Chen; 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.

    2014-07-11

    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.

  4. Emergence of the Phosphoinositide 3-Kinase-Akt-Mammalian Target of Rapamycin Axis in Transforming Growth Factor-β-Induced Epithelial-Mesenchymal Transition

    PubMed Central

    Lamouille, Samy; Derynck, Rik

    2010-01-01

    During development and in pathological contexts such as fibrosis and cancer progression, epithelial cells can initiate a complex transcriptional reprogramming, accompanied by dramatic morphological changes, in a process named ‘epithelial-mesenchymal transition’ (EMT). In this transition, epithelial cells lose their epithelial characteristics to acquire mesenchymal properties and increased motile and invasive behavior. Transforming growth factor-β (TGF-β) has emerged as a major inducer of EMT through activation of downstream signaling pathways, including Smad and non-Smad signaling pathways. Among the non-Smad pathways, increasing evidence is emerging that the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin axis plays a major role in TGF-β-induced EMT, notably through the regulation of translation and cell invasion. Pharmacological inhibitors of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin pathway may therefore represent an opportunity to selectively target essential aspects of TGF-β-induced EMT and provide an approach to prevent cancer cell dissemination toward metastasis, without the need to fully inactivate TGF-β signaling. PMID:21041997

  5. Variation in the Phosphoinositide 3-Kinase Gamma Gene Affects Plasma HDL-Cholesterol without Modification of Metabolic or Inflammatory Markers

    PubMed Central

    Kächele, Martin; Hennige, Anita M.; Machann, Jürgen; Hieronimus, Anja; Lamprinou, Apostolia; Machicao, Fausto; Schick, Fritz; Fritsche, Andreas; Stefan, Norbert; Nürnberg, Bernd; Häring, Hans-Ulrich; Staiger, Harald

    2015-01-01

    Objective Phosphoinositide 3-kinase γ (PI3Kγ) is a G-protein-coupled receptor-activated lipid kinase mainly expressed in leukocytes and cells of the cardiovascular system. PI3Kγ plays an important signaling role in inflammatory processes. Since subclinical inflammation is a hallmark of atherosclerosis, obesity-related insulin resistance, and pancreatic β-cell failure, we asked whether common genetic variation in the PI3Kγ gene (PIK3CG) contributes to body fat content/distribution, serum adipokine/cytokine concentrations, alterations in plasma lipid profiles, insulin sensitivity, insulin release, and glucose homeostasis. Study Design Using a tagging single nucleotide polymorphism (SNP) approach, we analyzed genotype-phenotype associations in 2,068 German subjects genotyped for 10 PIK3CG SNPs and characterized by oral glucose tolerance tests. In subgroups, data from hyperinsulinaemic-euglycaemic clamps, magnetic resonance spectroscopy of the liver, whole-body magnetic resonance imaging, and intravenous glucose tolerance tests were available, and peripheral blood mononuclear cells (PBMCs) were used for gene expression analysis. Results After appropriate adjustment, none of the PIK3CG tagging SNPs was significantly associated with body fat content/distribution, adipokine/cytokine concentrations, insulin sensitivity, insulin secretion, or blood glucose concentrations (p>0.0127, all; Bonferroni-corrected α-level: 0.0051). However, six non-linked SNPs displayed at least nominal associations with plasma HDL-cholesterol concentrations, two of them (rs4288294 and rs116697954) reaching the level of study-wide significance (p = 0.0003 and p = 0.0004, respectively). More precisely, rs4288294 and rs116697954 influenced HDL2-, but not HDL3-, cholesterol. With respect to the SNPs’ in vivo functionality, rs4288294 was significantly associated with PIK3CG mRNA expression in PBMCs. Conclusions We could demonstrate that common genetic variation in the PIK3CG locus, possibly via altered PIK3CG gene expression, determines plasma HDL-cholesterol concentrations. Since HDL2-, but not HDL3-, cholesterol is influenced by PIK3CG variants, PI3Kγ may play a role in HDL clearance rather than in HDL biogenesis. Even though the molecular pathways connecting PI3Kγ and HDL metabolism remain to be further elucidated, this finding could add a novel aspect to the pathophysiological role of PI3Kγ in atherogenesis. PMID:26658747

  6. Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes.

    PubMed Central

    Crljen, Vladiana; Volinia, Stefano; Banfic, Hrvoje

    2002-01-01

    Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH(3) and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2 beta activity, which is sensitive to wortmannin (10 nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices with HGF and could be mimicked by the Ca(2+) ionophore A23187 and blocked by the cell-penetrant Ca(2+) chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2 beta revealed a single immunoreactive band of 180 kDa in BLM and BBM, while after stimulation with HGF a gel shift of 18 kDa was noticed only in BBM, suggesting that the observed enzyme activation is achieved by proteolysis. When BBM were subjected to short-term (15 min) exposure to mu-calpain, a similar gel shift together with an increase in PI3K-C2 beta activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2 beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that in renal cells there is a spatial separation of the inositol lipid signalling system between BLM and BBM, and that HGF causes activation of PLC and PI3K primarily in BLM, which leads to calpain-mediated activation of PI3K-C2 beta in BBM with a concomitant increase in PtdIns3P. PMID:11931646

  7. Sann-Joong-Kuey-Jian-Tang induces autophagy in HepG2 cells via regulation of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and p38 mitogen-activated protein kinase pathways

    PubMed Central

    CHUANG, WAN-LING; SU, CHIN-CHENG; LIN, PING-YI; LIN, CHI-CHEN; CHEN, YAO-LI

    2015-01-01

    Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicine, was previously reported to induce autophagy and inhibit the proliferation of the human HepG2 hepatocellular carcinoma cell line via an extrinsic pathway. In the present study, the effects of SJKJT-induced autophagy and the cytotoxic mechanisms mediating these effects were investigated in HepG2 cells. The cytotoxicity of SJKJT in the HepG2 cells was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results demonstrated that the half-maximal inhibitory concentration of SJKJT was 2.91 mg/ml at 24 h, 1.64 mg/ml at 48 h and 1.26 mg/ml at 72 h. The results of confocal fluorescence microscopy indicated that SJKJT resulted in the accumulation of green fluorescent protein-LC3 and vacuolation of the cytoplasm. Flow cytometric analysis revealed the accumulation of acidic vesicular organelles. Furthermore, western blot analysis, used to determine the expression levels of autophagy-associated proteins, demonstrated that the HepG2 cells treated with SJKJT exhibited LC3B-I/LC3B-II conversion, increased expression levels of Beclin, Atg-3 and Atg-5 and reduced expression levels of p62 and decreased signaling of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and the p38 mitogen-activated protein kinase pathways. Taken together, these findings may assist in the development of novel chemotherapeutic agents for the treatment of malignant types of liver cancer. PMID:25847489

  8. Involvement of p21racA, phosphoinositide 3-kinase, and vacuolar ATPase in phagocytosis of bacteria and erythrocytes by Entamoeba histolytica: suggestive evidence for coincidental evolution of amebic invasiveness.

    PubMed Central

    Ghosh, S K; Samuelson, J

    1997-01-01

    Trophozoites of Entamoeba histolytica, the protozoan parasite that causes amebic dysentery, phagocytose bacteria in the colonic lumen and erythrocytes (RBC) in host tissues. Because tissue invasion is an evolutionary dead end, it is likely that amebic pathogenicity is coincidentally selected, i.e., the same methods used to kill bacteria in the colonic lumen are used by parasites to damage host cells and cause disease. In support of this idea, the amebic lectin and pore-forming peptide are involved in binding and killing, respectively, bacteria and host epithelial cells. Here amebic phagocytosis of bacteria, RBC, and mucin-coated beads was disrupted by overexpression of E. histolytica p21(racA-V12), a ras-family protein involved in selection of sites of actin polymerization, which had been mutated to eliminate its GTPase activity. p21(racA-V12) transformants were also defective in capping and cytokinesis, while pinocytosis of fluorescent dextrans was not affected. Wortmannin, a fungal inhibitor of phosphoinositide 3-kinase, markedly inhibited phagocytosis of bacteria, RBC, and mucin-coated beads by wild-type amebae. In contrast to p21(racA-V12) overexpression, wortmannin abolished amebic pinocytosis of dextrans but had no inhibitory effects on capping. Inhibition of amebic vacuolar acidification by bafilomycin also decreased bacterial and RBC uptake. These results, which demonstrate similarities between mechanisms of phagocytosis of bacteria and RBC by amebae and macrophages, support the idea of coincidental selection of amebic genes encoding proteins that mediate destruction of host cells. PMID:9317033

  9. Implication of the calcium sensing receptor and the Phosphoinositide 3-kinase/Akt pathway in the extracellular calcium-mediated migration of RAW 264.7 osteoclast precursor cells.

    PubMed

    Boudot, Cédric; Saidak, Zuzana; Boulanouar, Abdel Krim; Petit, Laurent; Gouilleux, Fabrice; Massy, Ziad; Brazier, Michel; Mentaverri, Romuald; Kamel, Saïd

    2010-05-01

    While the processes involved in the formation, maturation and apoptosis of osteoclasts have been investigated extensively in previous studies, little is known about the mechanisms responsible for the localization and homing of osteoclast precursor cells to the bone environment in order to initiate the bone remodeling process. Recent studies have suggested that the extracellular Ca(2+) (Ca(2+)(o)) concentration gradient present near the bone environment may be one of the participating factors, producing a chemoattractant effect on osteoclast precursors. Using the murine osteoclast precursor cells of the monocyte-macrophage lineage, the RAW 264.7 cell line, we have shown that Ca(2+)(o) increases the migration of these cells in a directional manner. The participation of the calcium sensing receptor (CaR) in this effect was tested by knocking down its expression through RNA interference, which resulted in an abolition of the migratory response. By the use of specific pathway inhibitors and western blot analysis, the phosphoinositide 3-kinase (PI3K)/Akt and phospholipase Cbeta pathways were shown to be implicated in the migratory effect. The implication of the Akt pathway in the Ca(2+)(o)-induced chemoattraction of RAW 264.7 cells was also confirmed by transducing the cells with the fusion protein TAT-dominant negative-Akt, which decreased the migratory effect. In contrast, the MAPK pathways (ERK1/2, p38 and JNK) were not involved in the production of the migratory effect. We conclude that through the activation of the CaR and subsequent signaling via the PI3K/Akt pathway, Ca(2+)(o) produces a chemoattractant effect on the osteoclast precursor RAW 264.7 cells. These results suggest that the Ca(2+)(o) gradient present near the bone may be one of the initiating factors for the homing of osteoclast precursors to bone, thus possibly playing a role in the initiation of bone remodeling. PMID:20149906

  10. PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse swiss 3T3 and human skin fibroblasts

    SciTech Connect

    Sturani, E.; Vicentini, L.M.; Zippel, R.; Toschi, L.; Pandiella-Alonso, A.; Comoglio, P.M.; Meldolesi, J.

    1986-05-29

    Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts it is demonstrated that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA.

  11. Progress in the preclinical discovery and clinical development of class I and dual class I/IV phosphoinositide 3-kinase (PI3K) inhibitors.

    PubMed

    Shuttleworth, S J; Silva, F A; Cecil, A R L; Tomassi, C D; Hill, T J; Raynaud, F I; Clarke, P A; Workman, P

    2011-01-01

    The phosphoinositide 3-kinases (PI3Ks) constitute an important family of lipid kinase enzymes that control a range of cellular processes through their regulation of a network of signal transduction pathways, and have emerged as important therapeutic targets in the context of cancer, inflammation and cardiovascular diseases. Since the mid-late 1990s, considerable progress has been made in the discovery and development of small molecule ATP-competitive PI3K inhibitors, a number of which have entered early phase human trials over recent years from which key clinical results are now being disclosed. This review summarizes progress made to date, primarily on the discovery and characterization of class I and dual class I/IV subtype inhibitors, together with advances that have been made in translational and clinical research, notably in cancer. PMID:21649578

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

    PubMed Central

    Payne, W E; Fitzgerald-Hayes, M

    1993-01-01

    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

  13. Targeted Inhibition of Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Sensitizes Pancreatic Cancer Cells to Doxorubicin without Exacerbating Cardiac Toxicity.

    PubMed

    Durrant, David E; Das, Anindita; Dyer, Samya; Tavallai, Seyedmehrad; Dent, Paul; Kukreja, Rakesh C

    2015-09-01

    Pancreatic cancer has the lowest 5-year survival rate of all major cancers despite decades of effort to design and implement novel, more effective treatment options. In this study, we tested whether the dual phosphoinositide 3-kinase/mechanistic target of rapamycin inhibitor BEZ235 (BEZ) potentiates the antitumor effects of doxorubicin (DOX) against pancreatic cancer. Cotreatment of BEZ235 with DOX resulted in dose-dependent inhibition of the phosphoinositide 3-kinase/mechanistic target of rapamycin survival pathway, which corresponded with an increase in poly ADP ribose polymerase cleavage. Moreover, BEZ cotreatment significantly improved the effects of DOX toward both cell viability and cell death in part through reduced Bcl-2 expression and increased expression of the shorter, more cytotoxic forms of BIM. BEZ also facilitated intracellular accumulation of DOX, which led to enhanced DNA damage and reactive oxygen species generation. Furthermore, BEZ in combination with gemcitabine reduced MiaPaca2 cell proliferation but failed to increase reactive oxygen species generation or BIM expression, resulting in reduced necrosis and apoptosis. Treatment with BEZ and DOX in mice bearing tumor xenographs significantly repressed tumor growth as compared with BEZ, DOX, or gemcitabine. Additionally, in contrast to the enhanced expression seen in MiaPaca2 cells, BEZ and DOX cotreatment reduced BIM expression in H9C2 cardiomyocytes. Also, the Bcl-2/Bax ratio was increased, which was associated with a reduction in cell death. In vivo echocardiography showed decreased cardiac function with DOX treatment, which was not improved by combination treatment with BEZ. Thus, we propose that combining BEZ with DOX would be a better option for patients than current standard of care by providing a more effective tumor response without the associated increase in toxicity. PMID:26101222

  14. Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites.

    PubMed

    Brochet, Mathieu; Collins, Mark O; Smith, Terry K; Thompson, Eloise; Sebastian, Sarah; Volkmann, Katrin; Schwach, Frank; Chappell, Lia; Gomes, Ana Rita; Berriman, Matthew; Rayner, Julian C; Baker, David A; Choudhary, Jyoti; Billker, Oliver

    2014-03-01

    Many critical events in the Plasmodium life cycle rely on the controlled release of Ca²⁺ from intracellular stores to activate stage-specific Ca²⁺-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca²⁺ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca²⁺ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5)-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca²⁺ effectors, PKG emerges as a unifying factor to control multiple cellular Ca²⁺ signals essential for malaria parasite development and transmission. PMID:24594931

  15. Modulatory effects of NMDA on phosphoinositide responses evoked by the metabotropic glutamate receptor agonist 1S,3R-ACPD in neonatal rat cerebral cortex.

    PubMed Central

    Challiss, R. A.; Mistry, R.; Gray, D. W.; Nahorski, S. R.

    1994-01-01

    1. The effect of NMDA-receptor stimulation on phosphoinositide signalling in response to the metabotropic glutamate receptor agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) has been examined in neonatal rat cerebral cortex slices. 2. Total [3H]-inositol phosphate ([3H]-InsPx) accumulation, in the presence of 5 mM LiCl, in [3H]-inositol pre-labelled slices was concentration-dependently increased by 1S,3R-ACPD (EC50 16.6 microM) and, at a maximally effective concentration, 1S,3R-ACPD (300 microM) increased [3H]-InsPx accumulation by 12.8 fold over basal values. 3. [3H]-InsPx accumulation stimulated by 1S,1R-ACPD was enhanced by low concentrations of NMDA (3-30 microM), but not by higher concentrations (> 30 microM). [3H]-InsPx accumulations stimulated by 1S,3R-ACPD in the absence or presence of 10 microM NMDA were linear with time, at least over the 15 min period examined; however, in the presence of 100 microM NMDA the initial enhancement of 1S,3R-ACPD-stimulated phosphoinositide hydrolysis progressively decreased with time. 4. In the presence of a maximal enhancing concentration of NMDA (10 microM), the response to 1S,3R-ACPD (300 microM) was increased 1.9 fold and the EC50 for agonist-stimulated [3H]-InsPx accumulation decreased about 4 fold. The enhanced response to the metabotropic agonist was concentration-dependently inhibited by competitive and uncompetitive antagonists of NMDA-receptor activation. 5. 1S,3R-ACPD also stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation with an initial peak response (5-6 fold over basal) at 15 s decaying to a smaller (2 fold), but persistent elevated accumulation (1-10 min).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7913380

  16. Phosphoinositide 3-kinase γ affects LPS-induced disturbance of blood-brain barrier via lipid kinase-independent control of cAMP in microglial cells.

    PubMed

    Frister, Adrian; Schmidt, Caroline; Schneble, Nadine; Brodhun, Michael; Gonnert, Falk A; Bauer, Michael; Hirsch, Emilio; Müller, Jörg P; Wetzker, Reinhard; Bauer, Reinhard

    2014-12-01

    The breakdown of the blood-brain barrier (BBB) is a key event in the development of sepsis-induced brain damage. BBB opening allows blood-born immune cells to enter the CNS to provoke a neuroinflammatory response. Abnormal expression and activation of matrix metalloproteinases (MMP) was shown to contribute to BBB opening. Using different mouse genotypes in a model of LPS-induced systemic inflammation, our present report reveals phosphoinositide 3-kinase γ (PI3Kγ) as a mediator of BBB deterioration and concomitant generation of MMP by microglia. Unexpectedly, microglia expressing lipid kinase-deficient mutant PI3Kγ exhibited similar MMP regulation as wild-type cells. Our data suggest kinase-independent control of cAMP phosphodiesterase activity by PI3Kγ as a crucial mediator of microglial cell activation, MMP expression and subsequent BBB deterioration. The results identify the suppressive effect of PI3Kγ on cAMP as a critical mediator of immune cell functions. PMID:25033932

  17. Phosphorylation-independent dual-site binding of the FHA domain of KIF13 mediates phosphoinositide transport via centaurin [alpha]1

    SciTech Connect

    Tong, Yufeng; Tempel, Wolfram; Wang, Hui; Yamada, Kaori; Shen, Limin; Senisterra, Guillermo A.; MacKenzie, Farrell; Chishti, Athar H.; Park, Hee-Won

    2011-11-07

    Phosphatidylinositol 3,4,5-triphosphate (PIP3) plays a key role in neuronal polarization and axon formation. PIP3-containing vesicles are transported to axon tips by the kinesin KIF13B via an adaptor protein, centaurin {alpha}1 (CENTA1). KIF13B interacts with CENTA1 through its forkhead-associated (FHA) domain. We solved the crystal structures of CENTA1 in ligand-free, KIF13B-FHA domain-bound, and PIP3 head group (IP4)-bound conformations, and the CENTA1/KIF13B-FHA/IP4 ternary complex. The first pleckstrin homology (PH) domain of CENTA1 specifically binds to PIP3, while the second binds to both PIP3 and phosphatidylinositol 3,4-biphosphate (PI(3,4)P2). The FHA domain of KIF13B interacts with the PH1 domain of one CENTA1 molecule and the ArfGAP domain of a second CENTA1 molecule in a threonine phosphorylation-independent fashion. We propose that full-length KIF13B and CENTA1 form heterotetramers that can bind four phosphoinositide molecules in the vesicle and transport it along the microtubule.

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

    SciTech Connect

    Zeitler, P.; Handwerger, S. ); Wu, Y.Q. )

    1991-01-01

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

  19. Ca2+ influx and phosphoinositide signalling are essential for the establishment and maintenance of cell polarity in monospores from the red alga Porphyra yezoensis

    PubMed Central

    Saga, Naotsune; Mikami, Koji

    2009-01-01

    The asymmetrical distribution of F-actin directed by cell polarity has been observed during the migration of monospores from the red alga Porphyra yezoensis. The significance of Ca2+ influx and phosphoinositide signalling during the formation of cell polarity in migrating monospores was analysed pharmacologically. The results indicate that the inhibition of the establishment of cell polarity, as judged by the ability of F-actin to localize asymmetrically, cell wall synthesis, and development into germlings, occurred when monospores were treated with inhibitors of the Ca2+ permeable channel, phospholipase C (PLC), diacylglycerol kinase, and inositol-1,4,5-trisphosphate receptor. Moreover, it was also found that light triggered the establishment of cell polarity via photosynthetic activity but not its direction, indicating that the Ca2+ influx and PLC activation required for the establishment of cell polarity are light dependent. By contrast, inhibition of phospholipase D (PLD) prevented the migration of monospores but not the asymmetrical localization of F-actin. Taken together, these findings suggest that there is functional diversity between the PLC and PLD signalling systems in terms of the formation of cell polarity; the former being critical for the light-dependent establishment of cell polarity and the latter playing a role in the maintenance of established cell polarity. PMID:19531546

  20. Possible involvement of phosphoinositide-Ca2+ signaling in the regulation of alpha-amylase expression and germination of rice seed (Oryza sativa L.).

    PubMed

    Kashem, M A; Itoh, K; Iwabuchi, S; Hori, H; Mitsui, T

    2000-04-01

    We have studied the effects of neomycin, a potent inhibitor of inositol phospholipid-specific phospholipase C (PLC), on the germination of rice seed and the gibberellin-induced expression of alpha-amylase in the aleurone layer and the scutellar tissues. It was shown that, in the absence of exogenous Ca2+, neomycin markedly reduced the germination speed and seedling growth of rice seeds and inhibited the gibberellin-induced expression of alpha-amylase in both secretory tissues. In addition, neomycin decreased the formation of inositol 1,4,5-trisphosphate (IP3) in the gibberellin-treated aleurone layer and the scutellar tissues. However, the inhibitory effects on the germination speed and the expression of alpha-amylase activity were overcome by supplementation of Ca2+. In addition, gibberellin elevated the level of IP3, and ABA prevented the gibberellin-induced formation of IP3, although ABA alone did not alter the IP3 level. The expression of a membrane-bound PLC molecule in rice aleurone layer was shown to be induced by gibberellin, and the gibberellin-induced expression of PLC was markedly delayed by treatment with ABA. These results strongly suggest that the phosphoinositide-Ca2+ signal transduction pathway may play an important role in the gibberellin-induced expression of alpha-amylase molecules closely related to the germination processes of rice seed. PMID:10845452

  1. Cdc42 and phosphoinositide 3-kinase drive Rac-mediated actin polymerization downstream of c-Met in distinct and common pathways.

    PubMed

    Bosse, Tanja; Ehinger, Julia; Czuchra, Aleksandra; Benesch, Stefanie; Steffen, Anika; Wu, Xunwei; Schloen, Kathrin; Niemann, Hartmut H; Scita, Giorgio; Stradal, Theresia E B; Brakebusch, Cord; Rottner, Klemens

    2007-10-01

    Activation of c-Met, the hepatocyte growth factor (HGF)/scatter factor receptor induces reorganization of the actin cytoskeleton, which drives epithelial cell scattering and motility and is exploited by pathogenic Listeria monocytogenes to invade nonepithelial cells. However, the precise contributions of distinct Rho-GTPases, the phosphatidylinositol 3-kinases, and actin assembly regulators to c-Met-mediated actin reorganization are still elusive. Here we report that HGF-induced membrane ruffling and Listeria invasion mediated by the bacterial c-Met ligand internalin B (InlB) were significantly impaired but not abrogated upon genetic removal of either Cdc42 or pharmacological inhibition of phosphoinositide 3-kinase (PI3-kinase). While loss of Cdc42 or PI3-kinase function correlated with reduced HGF- and InlB-triggered Rac activation, complete abolishment of actin reorganization and Rac activation required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N-WASP. Instead, actin polymerization was driven by Arp2/3 complex activation through the WAVE complex downstream of Rac. Together, our data establish an intricate signaling network comprising as key molecules Cdc42 and PI3-kinase, which converge on Rac-mediated actin reorganization essential for Listeria invasion and membrane ruffling downstream of c-Met. PMID:17682062

  2. Platelet alpha IIb-beta 3 integrin engagement induces the tyrosine phosphorylation of Cbl and its association with phosphoinositide 3-kinase and Syk.

    PubMed Central

    Saci, A; Rendu, F; Bachelot-Loza, C

    2000-01-01

    Agonist-induced platelet activation triggers 'inside-out' signalling which activates alpha IIb-beta 3, the most abundant integrin in platelet membranes. The engagement of activated alpha IIb-beta 3 integrin by linking fibrinogen is necessary for platelet aggregation, and this induces subsequent outside-in signalling, which enhances platelet activation. Here we studied the involvement of Cbl during alpha IIb-beta 3-integrin-mediated signal transduction. During thrombin-induced platelet activation, Cbl was tyrosine phosphorylated, and phosphoinositide 3-kinase (PI 3-kinase) activity measured in Cbl immunoprecipitates was increased. Both Cbl phosphorylation and its association with PI 3-kinase were dependent on alpha IIb-beta 3 engagement by linking fibrinogen. The P256 and anti-LIBS6 (ligand-induced binding site 6) antibodies, which activate platelets directly through alpha IIb-beta 3, induced Cbl phosphorylation and increased the PI 3-kinase activity associated with Cbl. Both thrombin and antibodies to alpha IIb-beta 3 induced association of Cbl with the tyrosine kinase, Syk. Experiments performed with inhibitors of tyrosine kinases indicated that both Src-family kinases and Syk contribute to phosphorylation of Cbl and its consequent association with PI 3-kinase. The results show that, following integrin alpha IIb-beta 3 engagement, Cbl is tyrosine phosphorylated, recruits PI 3-kinase to this integrin signalling pathway and possibly enhances PI 3-kinase activity, downstream of Src-family tyrosine kinases and Syk activation. PMID:11042121

  3. Expression pattern and sub-cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U-73122 in rat astrocytoma cells.

    PubMed

    Lo Vasco, Vincenza Rita; Fabrizi, Cinzia; Panetta, Barbara; Fumagalli, Lorenzo; Cocco, Lucio

    2010-07-01

    Phosphoinositide specific phospholipase C (PI-PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI-PLC family display different expression and/or sub cellular distribution under non-physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellular distribution of PI-PLCs compared to untreated cells. Special attention require PI-PLC beta3 and PI-PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U-73122 treatment. The meaning of these modifications is unclear, also because the use of this N-aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells. PMID:20564200

  4. miR-502 inhibits cell proliferation and tumor growth in hepatocellular carcinoma through suppressing phosphoinositide 3-kinase catalytic subunit gamma

    SciTech Connect

    Chen, Suling; Li, Fang; Chai, Haiyun; Tao, Xin; Wang, Haili; Ji, Aifang

    2015-08-21

    MicroRNAs (miRNAs) play a key role in carcinogenesis and tumor progression in hepatocellular carcinoma (HCC). In the present study, we demonstrated that miR-502 significantly inhibits HCC cell proliferation in vitro and tumor growth in vivo. G1/S cell cycle arrest and apoptosis of HCC cells were induced by miR-502. Phosphoinositide 3-kinase catalytic subunit gamma (PIK3CG) was identified as a direct downstream target of miR-502 in HCC cells. Notably, overexpression of PIK3CG reversed the inhibitory effects of miR-502 in HCC cells. Our findings suggest that miR-502 functions as a tumor suppressor in HCC via inhibition of PI3KCG, supporting its utility as a promising therapeutic gene target for this tumor type. - Highlights: • miR-502 suppresses HCC cell proliferation in vitro and tumorigenicity in vivo. • miR-502 regulates cell cycle and apoptosis in HCC cells. • PIK3CG is a direct target of miR-502. • miR-502 and PIK3CG expression patterns are inversely correlated in HCC tissues.

  5. Stimulation of endothelin-1 gene expression by insulin via phosphoinositide-3 kinase-glycogen synthase kinase-3beta signaling in endothelial cells.

    PubMed

    Yang, Zeran; Li, Ji-Cheng

    2008-02-27

    Insulin stimulates secretion of the potent vasoactive and mitogenic peptide endothelin-1 (ET-1) from endothelial cells. We sought to investigate whether phosphoinositide-3 kinase (PI3K)-dependent inactivation of glycogen synthase kinase-3beta (GSK3beta) by insulin leads to elevation of ET-1 gene expression in endothelial cells. Inhibition of GSK3beta activity by LiCl or siRNA technique mimicked insulin action to stimulate ET-1 gene expression. Luciferase reporter assay showed insulin stimulated-elevation of ET-1 promoter activity can be abolished by the PI3K inhibitor Wortmannin, but not by the mitogen activated protein kinase (MAPK) inhibitor PD-98059. To further investigate whether the transcription factor vascular endothelial zinc finger-1 (Vezf1) is involved in ET-1 regulation, site-mutated reporter plasmid was used in luciferase reporter assay. A 2-bp mutation in Vezf1 binding element abolished insulin-stimulated elevation of ET-1 promoter activity. Furthermore, siRNA inhibition of Vezf1 led to decline in the levels of ET-1 mRNA and ET-1 peptides. These observations indicate that PI3K-dependent inactivation of GSK3beta by insulin leads to upregulation of ET-1 gene expression and Vezf1 may be a target for ET-1 regulation by insulin. PI3K-GSK3beta signaling may be responsible for insulin stimulation of ET-1 production associated with insulin resistance and hyperinsulinemia. PMID:18201727

  6. Phosphoinositide 3-OH kinase p85α and p110β are essential for androgen receptor transactivation and tumor progression in prostate cancers

    PubMed Central

    Zhu, Q; Youn, H; Tang, J; Tawfik, O; Dennis, K; Terranova, PF; Du, J; Raynal, P; Thrasher, JB; Li, B

    2008-01-01

    Phosphoinositide 3-OH kinases (PI3Ks) are a group of major intracellular signaling molecules. In our previous study, we found that inhibition of PI3K activity suppressed the androgen receptor (AR)-mediated gene expression in prostate cancer cells. The AR has been considered as a critical determinant for the development and progression of human prostate cancers. In this study, we sought to identify the PI3K isoforms involved in AR transactivation. Using a gene-specific small interference RNA (siRNA) approach, we determined that the regulatory isoform p85α and the catalytic isoform p110β, but not p110α, were required for androgen-stimulated AR trans-activation and cell proliferation in prostate cancer cells. Consistently, overexpression of wild-type p110β but not p110α gene led to androgen-independent AR transactivation. Silencing p110β gene in prostate cancer cells abolished tumor growth in nude mice. Of the dual (lipid and protein) kinase activities, p110β’s lipid kinase activity was required for AR transactivation. Further analysis by a chromatin immunoprecipitation assay showed that p110β is indispensable for androgen-induced AR–DNA interaction. Finally, gene expression analysis of clinical specimens showed that both p85α and p110β were highly expressed in malignant prostate tissues compared to the nonmalignant compartments, and their expression levels correlated significantly with disease progression. Taken together, our data demonstrated that p85α and p110β are essential for androgen-stimulated AR transactivation, and their aberrant expression or activation might play an important role in prostate cancer progression. PMID:18372911

  7. Histone deacetylase 3 expression correlates with vasculogenic mimicry through the phosphoinositide3-kinase/ERKMMPlaminin5?2 signaling pathway

    PubMed Central

    Liu, Xiao; Wang, Ji-Hui; Li, Shun; Li, Lin-Lin; Huang, Min; Zhang, Yong-Hong; Liu, Yang; Yang, Yuan-Tao; Ding, Rui; Ke, Yi-Quan

    2015-01-01

    Vasculogenic mimicry (VM) refers to the process by which highly aggressive tumor cells mimic endothelial cells to form vessel-like structures that aid in supplying enough nutrients to rapidly growing tumors. Histone deacetylases (HDACs) regulate the expression and activity of numerous molecules involved in cancer initiation and progression. Notably, HDAC3 is overexpressed in the majority of carcinomas. However, thus far, no data are available to support the role of HDAC3 in VM. In this study, we subjected glioma specimens to immunohistochemical and histochemical double-staining methods and found that VM and HDAC3 expression were related to the pathological grade of gliomas. The presence of VM correlated with HDAC3 expression in glioma tissues. The formation of tubular structures, as determined by the tube formation assay to evaluate VM, was impaired in U87MG cells when transfected by siRNA or treated with an HDAC3 inhibitor. Importantly, the expression of VM-related molecules such as MMP-2/14 and laminin5?2 was also affected when HDAC3 expression was altered. Furthermore, U87MG cells were treated with a phosphoinositide 3-kinase (PI3K) inhibitor or/and ERK inhibitor and found that the PI3K and ERK signaling pathways play key roles in VM; whereas, in VM, the two signaling pathways did not act upstream or downstream from each other. Taken together, our findings showed that HDAC3 contributed to VM in gliomas, possibly through the PI3K/ERKMMPslaminin5?2 signaling pathway, which could potentially be a novel therapeutic target for gliomas. PMID:25940092

  8. Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells

    SciTech Connect

    Okajima, F.; Sho, K.; Kondo, Y.

    1988-08-01

    Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrations below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.

  9. Non-redundant Roles of Phosphoinositide 3-Kinase Isoforms α and β in Glycoprotein VI-induced Platelet Signaling and Thrombus Formation*

    PubMed Central

    Gilio, Karen; Munnix, Imke C. A.; Mangin, Pierre; Cosemans, Judith M. E. M.; Feijge, Marion A. H.; van der Meijden, Paola E. J.; Olieslagers, Servé; Chrzanowska-Wodnicka, Magdalena B.; Lillian, Rivka; Schoenwaelder, Simone; Koyasu, Shigeo; Sage, Stewart O.; Jackson, Shaun P.; Heemskerk, Johan W. M.

    2009-01-01

    Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cγ2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kα or -β strongly and selectively suppressed GPVI-induced Ca2+ mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cγ2 by PI3Kα/β. That PI3Kα and -β have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca2+ increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kα/β was blocked or p85α was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kδ or -γ. Furthermore, PI3Kα/β, but not PI3Kγ, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kα and -β isoforms are required for full GPVI-dependent platelet Ca2+ signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kα an interesting new target for anti-platelet therapy. PMID:19815551

  10. Histone deacetylase 3 expression correlates with vasculogenic mimicry through the phosphoinositide3-kinase / ERK-MMP-laminin5γ2 signaling pathway.

    PubMed

    Liu, Xiao; Wang, Ji-Hui; Li, Shun; Li, Lin-Lin; Huang, Min; Zhang, Yong-Hong; Liu, Yang; Yang, Yuan-Tao; Ding, Rui; Ke, Yi-Quan

    2015-07-01

    Vasculogenic mimicry (VM) refers to the process by which highly aggressive tumor cells mimic endothelial cells to form vessel-like structures that aid in supplying enough nutrients to rapidly growing tumors. Histone deacetylases (HDACs) regulate the expression and activity of numerous molecules involved in cancer initiation and progression. Notably, HDAC3 is overexpressed in the majority of carcinomas. However, thus far, no data are available to support the role of HDAC3 in VM. In this study, we subjected glioma specimens to immunohistochemical and histochemical double-staining methods and found that VM and HDAC3 expression were related to the pathological grade of gliomas. The presence of VM correlated with HDAC3 expression in glioma tissues. The formation of tubular structures, as determined by the tube formation assay to evaluate VM, was impaired in U87MG cells when transfected by siRNA or treated with an HDAC3 inhibitor. Importantly, the expression of VM-related molecules such as MMP-2/14 and laminin5γ2 was also affected when HDAC3 expression was altered. Furthermore, U87MG cells were treated with a phosphoinositide 3-kinase (PI3K) inhibitor or/and ERK inhibitor and found that the PI3K and ERK signaling pathways play key roles in VM; whereas, in VM, the two signaling pathways did not act upstream or downstream from each other. Taken together, our findings showed that HDAC3 contributed to VM in gliomas, possibly through the PI3K/ERK-MMPs-laminin5γ2 signaling pathway, which could potentially be a novel therapeutic target for gliomas. PMID:25940092

  11. The phosphoinositide PI(3,5)P₂ mediates activation of mammalian but not plant TPC proteins: functional expression of endolysosomal channels in yeast and plant cells.

    PubMed

    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

    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)P₂. 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)P₂ did not affect the voltage- and calcium-dependent characteristics of AtTPC1-EGFP. By contrast, PI(3,5)P₂ elicited large sodium currents in hTPC2-EGFP-containing vacuoles, while NAADP had no such effect. Analogous results were obtained when PI(3,5)P₂ 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)P₂-mediated activation and Na(+) selectivity of mammalian TPC2. PMID:24770793

  12. Functional phenotype of phosphoinositide 3-kinase p85alpha-null platelets characterized by an impaired response to GP VI stimulation.

    PubMed

    Watanabe, Naohide; Nakajima, Hideaki; Suzuki, Hidenori; Oda, Atsushi; Matsubara, Yumiko; Moroi, Masaaki; Terauchi, Yasuo; Kadowaki, Takashi; Suzuki, Harumi; Koyasu, Shigeo; Ikeda, Yasuo; Handa, Makoto

    2003-07-15

    Phosphoinositide 3-kinases (PI3Ks), a family of lipid kinases comprising 3 classes with multiple isoforms, have been shown to participate in different phases of platelet signaling. To investigate the roles that enzymes play in platelet function in vivo and determine which isoforms are important for particular signaling events, we analyzed platelet function of gene knockout mice deficient in the p85alpha regulatory subunit of heterodimeric class IA PI3K. The kinase activity of p85alpha-/- platelets was only 5% of the activity of platelets from wild-type littermates. Platelet aggregation induced by adenosine diphosphate (ADP), thrombin, U46619, phorbol 12-myristate 13-acetate (PMA), or botrocetin was not defective in p85alpha-/- mice, compared with wild-type animals. In contrast, aggregation induced by collagen and collagen-related peptide (CRP) was partially but readily impaired in p85alpha-/- mice. Both P-selectin expression and fibrinogen binding in response to CRP were also decreased to a similar extent in p85alpha-/- platelets. Platelets from p85alpha-/- mice appeared to spread poorly over a CRP-coated surface with intact filopodial protrusions. Significant attenuation of CRP-induced tyrosine phosphorylation in known PI3K effectors such as Btk, Tec, PKB/Akt, and phospholipase Cgamma2 were observed with p85alpha-/- platelets, whereas no alteration was noted in upstream molecules of Syk, LAT, and SLP-76. Considered as a whole, these results provide the first genetic evidence that PI3K p85alpha plays a significant role in platelet function, almost exclusively in the glycoprotein (GP) VI/Fc receptor gamma chain complex-mediated signaling pathway. PMID:12649157

  13. Phosphoinositide 3-Kinases Upregulate System xc− via Eukaryotic Initiation Factor 2α and Activating Transcription Factor 4 – A Pathway Active in Glioblastomas and Epilepsy

    PubMed Central

    Baxter, Paul; Kassubek, Rebecca; Albrecht, Philipp; Van Liefferinge, Joeri; Westhoff, Mike-Andrew; Halatsch, Marc-Eric; Karpel-Massler, Georg; Meakin, Paul J.; Hayes, John D.; Aronica, Eleonora; Smolders, Ilse; Ludolph, Albert C.; Methner, Axel; Conrad, Marcus; Massie, Ann; Hardingham, Giles E.

    2014-01-01

    Abstract Aims: Phosphoinositide 3-kinases (PI3Ks) relay growth factor signaling and mediate cytoprotection and cell growth. The cystine/glutamate antiporter system xc− imports cystine while exporting glutamate, thereby promoting glutathione synthesis while increasing extracellular cerebral glutamate. The aim of this study was to analyze the pathway through which growth factor and PI3K signaling induce the cystine/glutamate antiporter system xc− and to demonstrate its biological significance for neuroprotection, cell growth, and epilepsy. Results: PI3Ks induce system xc− through glycogen synthase kinase 3β (GSK-3β) inhibition, general control non-derepressible-2-mediated eukaryotic initiation factor 2α phosphorylation, and the subsequent translational up-regulation of activating transcription factor 4. This pathway is essential for PI3Ks to modulate oxidative stress resistance of nerve cells and insulin-induced growth in fibroblasts. Moreover, the pathway is active in human glioblastoma cells. In addition, it is induced in primary cortical neurons in response to robust neuronal activity and in hippocampi from patients with temporal lobe epilepsy. Innovation: Our findings further extend the concepts of how growth factors and PI3Ks induce neuroprotection and cell growth by adding a new branch to the signaling network downstream of GSK-3β, which, ultimately, leads to the induction of the cystine/glutamate antiporter system xc−. Importantly, the induction of this pathway by neuronal activity and in epileptic hippocampi points to a potential role in epilepsy. Conclusion: PI3K-regulated system xc− activity is not only involved in the stress resistance of neuronal cells and in cell growth by increasing the cysteine supply and glutathione synthesis, but also plays a role in the pathophysiology of tumor- and non-tumor-associated epilepsy by up-regulating extracellular cerebral glutamate. Antioxid. Redox Signal. 20: 2907–2922. PMID:24219064

  14. Regulation of Phosphoinositide Levels by the Phospholipid Transfer Protein Sec14p Controls Cdc42p/p21-Activated Kinase-Mediated Cell Cycle Progression at Cytokinesis▿

    PubMed Central

    Howe, Alicia G.; Fairn, Gregory D.; MacDonald, Kendra; Bankaitis, Vytas A.; McMaster, Christopher R.

    2007-01-01

    Sec14p is an essential phosphatidylcholine/phosphatidylinositol transfer protein with a well-described role in the regulation of Golgi apparatus-derived vesicular transport in yeast. Inactivation of the CDP-choline pathway for phosphatidylcholine synthesis allows cells to survive in the absence of Sec14p function through restoration of Golgi vesicular transport capability. In this study, Saccharomyces cerevisiae cells containing a SEC14 temperature-sensitive allele along with an inactivated CDP-choline pathway were transformed with a high-copy-number yeast genomic library. Genes whose increased expression inhibited cell growth in the absence of Sec14p function were identified. Increasing levels of the Rho GTPase Cdc42p and its direct effector kinases Cla4p and Ste20p prevented the growth of cells lacking Sec14p and CDP-choline pathway function. Growth suppression was accompanied by an increase in large and multiply budded cells. This effect on polarized cell growth did not appear to be due to an inability to establish cell polarity, since both the actin cytoskeleton and localization of the septin Cdc12p were unaffected by increased expression of Cdc42p, Cla4p, or Ste20p. Nuclei were present in both the mother cell and the emerging bud, consistent with Sec14p regulation of the cell cycle subsequent to anaphase but prior to cytokinesis/septum breakdown. Increased expression of phosphatidylinositol 4-kinases and phosphatidylinositol 4-phosphate 5-kinase prevented growth arrest by CDC42, CLA4, or STE20 upon inactivation of Sec14p function. Sec14p regulation of phosphoinositide levels affects cytokinesis at the level of the Cdc42p/Cla4p/Ste20p signaling cascade. PMID:17601877

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

    PubMed Central

    Peakman, M C; Hill, S J

    1994-01-01

    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

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

    PubMed Central

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

    2014-01-01

    Chemoattractant-induced reactive oxygen species (ROS) generation by adherent neutrophils occurs in two phases: the first is very rapid and transient, and the second one is delayed and lasts up to 30–40 min. We examined the role of phosphoinositide 3-kinases (PI3Ks) and Src-family kinases (SFKs) in these responses using human neutrophils treated with inhibitory compounds or murine neutrophils deficient of PI3Kγ or Hck, Fgr, and Lyn. Our studies show that PI3Kγ is indispensable for the early, fMLF-induced ROS generation and AKT and ERK phosphorylation, but is dispensable for the late response to fMLF. Additionally, the response to TNF, an agonist triggering only the delayed phase of ROS generation, was also unaffected in PI3Kγ-deficient neutrophils. In contrast, inhibition of SFKs by a selective inhibitor in human, or SFK deficiency in murine, neutrophils resulted in the inhibition of both the early and late phase of ROS generation, without affecting the early phase of AKT phosphorylation, but inhibiting the late one. Selective inhibitors of PI3Kα and PI3Kδ markedly reduced both the early and late response to fMLF and TNF in human neutrophils. These findings suggest that class IA PI3Ks may be activated by PI3Kγ via Ras in the early phase of the response and by SFKs in the late phase. The evidence that inhibition of SFKs in human, or SFK deficiency in murine, neutrophils results in suppression of Vav phosphorylation at all time points of the response to fMLF or TNF suggests that SFKs are indispensable for Vav phosphorylation. PMID:23447687

  17. Compartment-specific regulation of phosphoinositide 3-kinase by platelet-derived growth factor and insulin in 3T3-L1 adipocytes.

    PubMed Central

    Navé, B T; Haigh, R J; Hayward, A C; Siddle, K; Shepherd, P R

    1996-01-01

    To understand how the stimulation of phosphoinositide 3-kinase (PI 3-kinase) by different growth factors can activate different subsets of downstream responses, growth-factor regulation of PI 3-kinase activity at different intracellular locations was investigated in 3T3-L1 adipocytes. Insulin caused a large stimulation of glucose transport and stimulated recruitment of transferrin receptors to the plasma membrane (PM) in these cells, whereas platelet-derived growth factor (PDGF)-bb was virtually without effect on these responses. Subcellular fractionation studies after stimulation with PDGF-bb or insulin revealed a differential effect of these growth factors on subcellular localization of PI 3-kinase activity. PDGF was more effective than insulin in stimulating PI 3-kinase activity and recruiting the p85 alpha PI 3-kinase adaptor subunit in the fraction containing the PM. However, in the microsomal fraction insulin significantly increased PI 3-kinase activity and p85 alpha levels, whereas PDGF was almost without effect. In the microsomal membrane fraction the insulin-stimulated recruitment of p85 alpha closely matched the increase PI 3-kinase activity, indicating that insulin stimulation of PI 3-kinase in this fraction is largely due to recruitment of PI 3-kinase enzyme rather than alterations in specific activity. Insulin-stimulated recruitment of p85 alpha to the microsomal membranes was not inhibited by wortmannin, indicating that PI 3-kinase activity was not required for this process. A further level of compartment-specific regulation of PI 3-kinase in response to PDGF was revealed by the finding that tyrosine phosphorylation of the p85 alpha adaptor was restricted to the PM-containing fraction. Insulin had no effect on p85 tyrosine phosphorylation in either fraction. In summary, these results suggest a basis by which insulin and PDGF could both use PI 3-kinase signalling cascades but achieve different signalling outcomes. PMID:8761452

  18. Genomic organization and complete cDNA sequence of the human phosphoinositide-specific phospholipase C {beta}3 gene (PLCB3)

    SciTech Connect

    Lagercrantz, J.; Carson, E.; Phelan, C.

    1995-04-10

    We have characterized the complete cDNA sequence, genomic structure, and expression of the human phosphoinositide-specific phospholipase C {beta}3 (PLC {beta}3) gene (gene symbol PLCB3). PLC {beta}3 plays an important role in initiating receptor-mediated signal transduction. Activation of PLC takes place in many cells as a response to stimulation by hormones, growth factors, neurotransmitters, and other ligands. The partial cDNA sequence of PLC {beta}3, previously published, was extended with 876 bp in the 5{prime} direction, giving a transcript of 4400 bp and a total open reading frame of 1234 amino acids. This was in accordance with expression analysis by Northern blotting that revealed a single 4.4-kb transcript in all tissues tested. Genomic data were obtained by sequencing plasmid subclones of a cosmid that contained the whole gene. The size of the complete transcription unit was estimated to be on the order of 15 kb. The gene contains 31 exons, with all splice donor and acceptor sites conforming to the GT/AG rule. No exon exceeds 571 bp in length, and the shortest exon spans only 36 bp. More than half of the introns are smaller than 200 bp, with the smallest being only 79 bp long. The transcription initiation site was determined to be within an 8-bp cluster 328-321 bp upstream of the translation initiation site. The 5{prime} flanking region is highly GC rich, with multiple CpG doublets, and contains multiple binding sites for Sp1. Lacking typical transcriptional regulatory sequences such as TATA and CAAT boxes, the putative promoter region conforms to the group of housekeeping promoters. 28 refs., 4 figs., 1 tab.

  19. Calcineurin activation is not necessary for Doxorubicin-induced hypertrophy in H9c2 embryonic rat cardiac cells: involvement of the phosphoinositide 3-kinase-Akt pathway.

    PubMed

    Merten, Kevyn E; Jiang, Youchun; Feng, Wenke; Kang, Y James

    2006-11-01

    The calcium/calmodulin-dependent phosphatase calcineurin has been shown to be both necessary and sufficient to induce cardiac hypertrophy in vivo and in vitro. Treatment with the antineoplastic agent doxorubicin (DOX) was shown to activate calcineurin signaling in H9c2 rat cardiac muscle cells; however, the effect of this activation on hypertrophy was not investigated. Therefore, the present study was undertaken to examine the involvement of calcineurin activation in DOX-induced cardiac cell hypertrophy. H9c2 cells were treated with 1 microM DOX for 2 h following pretreatment with and in the presence of calcineurin inhibitors cyclosporine A (CsA) or FK506 (tacrolimus). Subsequent analysis of calcineurin signaling and cellular hypertrophy was performed 8 to 48 h after the treatment. DOX treatment activated calcineurin signaling and resulted in cellular hypertrophy as assessed by an increase in cell volume and protein content per cell. Inhibition of calcineurin with CsA or FK506 blocked DOX-induced calcineurin signaling. However, this inhibition did not prevent the DOX-induced hypertrophic response in H9c2 cells. Further evaluation of the possible signaling pathways involved in DOX-induced H9c2 cellular hypertrophy revealed that DOX treatment resulted in phosphorylation of the serine/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K). Moreover, the DOX-induced hypertrophic response was blunted by LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a specific inhibitor for PI3K. These results demonstrate that, although calcineurin is activated by DOX treatment, it is not necessary for DOX-induced hypertrophy in H9c2 cells. Rather, the PI3K-Akt signaling pathway seems to be more critically involved in DOX-induced hypertrophy. PMID:16926266

  20. Nuclear Phosphoinositide-Specific Phospholipase C ?1 Controls Cytoplasmic CCL2 mRNA Levels in HIV-1 gp120-Stimulated Primary Human Macrophages

    PubMed Central

    Purificato, Cristina; Sabbatucci, Michela; Podo, Franca; Ramoni, Carlo; Gessani, Sandra; Fantuzzi, Laura

    2013-01-01

    HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of CCL2 from macrophages. In turn, this chemokine acts as an autocrine factor enhancing viral replication. In this study, we show for the first time that phosphoinositide-specific phospholipase C (PI-PLC) is required for the production of CCL2 triggered by gp120 in macrophages. Using a combination of confocal laser-scanner microscopy, pharmacologic inhibition, western blotting and fluorescence-activated cell sorter analysis, we demonstrate that gp120 interaction with CCR5 leads to nuclear localization of the PI-PLC ?1 isozyme mediated by mitogen-activated protein kinase ERK-1/2. Notably, phosphatidylcholine-specific phospholipase C (PC-PLC), previously reported to be required for NF-kB-mediated CCL2 production induced by gp120 in macrophages, drives both ERK1/2 activation and PI-PLC ?1 nuclear localization induced by gp120. PI-PLC ?1 activation through CCR5 is also triggered by the natural chemokine ligand CCL4, but independently of ERK1/2. Finally, PI-PLC inhibition neither blocks gp120-mediated NF-kB activation nor overall accumulation of CCL2 mRNA, whereas it decreases CCL2 transcript level in the cytoplasm. These results identify nuclear PI-PLC ?1 as a new intermediate in the gp120-triggered PC-PLC-driven signal transduction pathway leading to CCL2 secretion in macrophages. The finding that a concerted gp120-mediated signaling involving both PC- and PI-specific PLCs is required for the expression of CCL2 in macrophages suggests that this signal transduction pathway may also be relevant for the modulation of viral replication in these cells. Thus, this study may contribute to identify novel targets for therapeutic intervention in HIV-1 infection. PMID:23555755

  1. The Na+/H+ Exchanger-3 (NHE3) Activity Requires Ezrin Binding to Phosphoinositide and Its Phosphorylation

    PubMed Central

    Babich, Victor; Di Sole, Francesca

    2015-01-01

    Na+/H+ exchanger-3 (NHE3) plays an essential role in maintaining sodium and fluid homeostasis in the intestine and kidney epithelium. Thus, NHE3 is highly regulated and its function depends on binding to multiple regulatory proteins. Ezrin complexed with NHE3 affects its activity via not well-defined mechanisms. This study investigates mechanisms by which ezrin regulates NHE3 activity in epithelial Opossum Kidney cells. Ezrin is activated sequentially by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and phosphorylation of threonine 567. Expression of ezrin lacking PIP2 binding sites inhibited NHE3 activity (-40%) indicating that ezrin binding to PIP2 is required for preserving NHE3 activity. Expression of a phosphomimetic ezrin mutated at the PIP2 binding region was sufficient not only to reverse NHE3 activity to control levels but also to increase its activity (+80%) similar to that of the expression of ezrin carrying the phosphomimetic mutation alone. Calcineurin Homologous Protein-1 (CHP1) is part, with ezrin, of the NHE3 regulatory complex. CHP1-mediated activation of NHE3 activity was blocked by expression of an ezrin variant that could not be phosphorylated but not by an ezrin variant unable to bind PIP2. Thus, for NHE3 activity under baseline conditions not only ezrin phosphorylation, but also ezrin spatial-temporal targeting on the plasma membrane via PIP2 binding is required; however, phosphorylation of ezrin appears to overcome the control of NHE3 transport. CHP1 action on NHE3 activity is not contingent on ezrin binding to PIP2 but rather on ezrin phosphorylation. These findings are important in understanding the interrelation and dynamics of a CHP1-ezrin-NHE3 regulatory complex. PMID:26042733

  2. The Na+/H+ Exchanger-3 (NHE3) Activity Requires Ezrin Binding to Phosphoinositide and Its Phosphorylation.

    PubMed

    Babich, Victor; Di Sole, Francesca

    2015-01-01

    Na+/H+ exchanger-3 (NHE3) plays an essential role in maintaining sodium and fluid homeostasis in the intestine and kidney epithelium. Thus, NHE3 is highly regulated and its function depends on binding to multiple regulatory proteins. Ezrin complexed with NHE3 affects its activity via not well-defined mechanisms. This study investigates mechanisms by which ezrin regulates NHE3 activity in epithelial Opossum Kidney cells. Ezrin is activated sequentially by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and phosphorylation of threonine 567. Expression of ezrin lacking PIP2 binding sites inhibited NHE3 activity (-40%) indicating that ezrin binding to PIP2 is required for preserving NHE3 activity. Expression of a phosphomimetic ezrin mutated at the PIP2 binding region was sufficient not only to reverse NHE3 activity to control levels but also to increase its activity (+80%) similar to that of the expression of ezrin carrying the phosphomimetic mutation alone. Calcineurin Homologous Protein-1 (CHP1) is part, with ezrin, of the NHE3 regulatory complex. CHP1-mediated activation of NHE3 activity was blocked by expression of an ezrin variant that could not be phosphorylated but not by an ezrin variant unable to bind PIP2. Thus, for NHE3 activity under baseline conditions not only ezrin phosphorylation, but also ezrin spatial-temporal targeting on the plasma membrane via PIP2 binding is required; however, phosphorylation of ezrin appears to overcome the control of NHE3 transport. CHP1 action on NHE3 activity is not contingent on ezrin binding to PIP2 but rather on ezrin phosphorylation. These findings are important in understanding the interrelation and dynamics of a CHP1-ezrin-NHE3 regulatory complex. PMID:26042733

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

    PubMed

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

    2015-05-01

    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-γ. 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

  4. Phosphoinositide interacting regulator of TRP (Pirt) enhances TRPM8 channel activity in vitro via increasing channel conductance

    PubMed Central

    Tang, Min; Wu, Guang-yi; Dong, Xin-zhong; Tang, Zong-xiang

    2016-01-01

    Aim: Pirt is a two-transmembrane domain protein that regulates the function of a variety of ion channels. Our previous study indicated that Pirt acts as a positive endogenous regulator of the TRPM8 channel. The aim of this study was to investigate the mechanism underlying the regulation of TRPM8 channel by Pirt. Methods: HEK293 cells were transfected with TRPM8+Pirt or TRPM8 alone. Menthol (1 mmol/L) was applied through perfusion to induce TRPM8-mediated voltage-dependent currents, which were recorded using a whole-cell recording technique. PIP2 (10 μmol/L) was added into the electrode pipettes (PI was taken as a control). Additionally, cell-attached single-channel recordings were conducted in CHO cells transfected with TRPM8+Pirt or TRPM8 alone, and menthol (1 mmol/L) was added into the pipette solution. Results: Either co-transfection with Pirt or intracellular application of PIP2 (but not PI) significantly enhanced menthol-induced TRPM8 currents. Furthermore, Pirt and PIP2 synergistically modulated menthol-induced TRPM8 currents. Single-channel recordings revealed that co-transfection with Pirt significantly increased the single channel conductance. Conclusion: Pirt and PIP2 synergistically enhance TRPM8 channel activity, and Pirt regulates TRPM8 channel activity by increasing the single channel conductance. PMID:26657057

  5. 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

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

    1987-03-25

    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.

  6. A novel HPLC-based approach makes possible the spatial characterization of cellular PtdIns5P and other phosphoinositides.

    PubMed

    Sarkes, Deborah; Rameh, Lucia E

    2010-06-15

    PtdIns5P was discovered in 1997 [Rameh, Tolias, Duckworth and Cantley (1997) Nature 390, 192-196], but still very little is known about its regulation and function. Hitherto, studies of PtdIns5P regulation have been hindered by the inability to measure cellular PtdIns5P using conventional HPLC, owing to poor separation from PtdIns4P. In the present paper we describe a new HPLC method for resolving PtdIns5P from PtdIns4P, which makes possible accurate measurements of basal and inducible levels of cellular PtdIns5P in the context of other phosphoinositides. Using this new method, we found that PtdIns5P is constitutively present in all cells examined (epithelial cells, fibroblasts and myoblasts, among others) at levels typically 1-2% of PtdIns4P levels. In the beta-pancreatic cell line BTC6, which is specialized in insulin secretion, PtdIns5P levels were higher than in most cells (2.5-4% of PtdIns4P). Using subcellular fractionation, we found that the majority of the basal PtdIns5P is present in the plasma membrane, but it is also enriched in intracellular membrane compartments, especially in SER (smooth endoplasmic reticulum) and/or Golgi, where high levels of PtdIns3P were also detected. Unlike PtdIns3P, PtdIns5P was also found in fractions containing very-low-density vesicles. Knockdown of PIP4K (PtdIns5P 4-kinase) leads to accumulation of PtdIns5P in light fractions and fractions enriched in SER/Golgi, whereas treatment with Brefeldin A results in a subtle, but reproducible, change in PtdIns5P distribution. These results indicate that basal PtdIns5P and the PtdIns5P pathway for PtdIns(4,5)P(2) synthesis may play a role in Golgi-mediated vesicle trafficking. PMID:20370717

  7. ETP-46321, a dual p110α/δ class IA phosphoinositide 3-kinase inhibitor modulates T lymphocyte activation and collagen-induced arthritis.

    PubMed

    Aragoneses-Fenoll, L; Montes-Casado, M; Ojeda, G; Acosta, Y Y; Herranz, J; Martínez, S; Blanco-Aparicio, C; Criado, G; Pastor, J; Dianzani, U; Portolés, P; Rojo, J M

    2016-04-15

    Class IA phosphoinositide 3-kinases (PI3Ks) are essential to function of normal and tumor cells, and to modulate immune responses. T lymphocytes express high levels of p110α and p110δ class IA PI3K. Whereas the functioning of PI3K p110δ in immune and autoimmune reactions is well established, the role of p110α is less well understood. Here, a novel dual p110α/δ inhibitor (ETP-46321) and highly specific p110α (A66) or p110δ (IC87114) inhibitors have been compared concerning T cell activation in vitro, as well as the effect on responses to protein antigen and collagen-induced arthritis in vivo. In vitro activation of naive CD4(+) T lymphocytes by anti-CD3 and anti-CD28 was inhibited more effectively by the p110δ inhibitor than by the p110α inhibitor as measured by cytokine secretion (IL-2, IL-10, and IFN-γ), T-bet expression and NFAT activation. In activated CD4(+) T cells re-stimulated through CD3 and ICOS, IC87114 inhibited Akt and Erk activation, and the secretion of IL-2, IL-4, IL-17A, and IFN-γ better than A66. The p110α/δ inhibitor ETP-46321, or p110α plus p110δ inhibitors also inhibited IL-21 secretion by differentiated CD4(+) T follicular (Tfh) or IL-17-producing (Th17) helper cells. In vivo, therapeutic administration of ETP-46321 significantly inhibited responses to protein antigen as well as collagen-induced arthritis, as measured by antigen-specific antibody responses, secretion of IL-10, IL-17A or IFN-γ, or clinical symptoms. Hence, p110α as well as p110δ Class IA PI3Ks are important to immune regulation; inhibition of both subunits may be an effective therapeutic approach in inflammatory autoimmune diseases like rheumatoid arthritis. PMID:26883061

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

    PubMed Central

    Puri, Kamal D.; Gold, Michael R.

    2012-01-01

    The delta isoform of the p110 catalytic subunit (p110δ) of phosphoinositide 3-kinase is expressed primarily in hematopoietic cells and plays an essential role in B-cell development and function. Studies employing mice lacking a functional p110δ protein, as well as the use of highly-selective chemical inhibitors of p110δ, have revealed that signaling via p110δ-containing PI3K complexes (PI3Kδ) is critical for B-cell survival, migration, and activation, functioning downstream of key receptors on B cells including the B-cell antigen receptor, chemokine receptors, pro-survival receptors such as BAFF-R and the IL-4 receptor, and co-stimulatory receptors such as CD40 and Toll-like receptors (TLRs). Similarly, this PI3K isoform plays a key role in the survival, proliferation, and dissemination of B-cell lymphomas. Herein we summarize studies showing that these processes can be inhibited in vitro and in vivo by small molecule inhibitors of p110δ enzymatic activity, and that these p110δ inhibitors have shown efficacy in clinical trials for the treatment of several types of B-cell malignancies including chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). PI3Kδ also plays a critical role in the activation, proliferation, and tissue homing of self-reactive B cells that contribute to autoimmune diseases, in particular innate-like B-cell populations such as marginal zone (MZ) B cells and B-1 cells that have been strongly linked to autoimmunity. We discuss the potential utility of p110δ inhibitors, either alone or in combination with B-cell depletion, for treating autoimmune diseases such as lupus, rheumatoid arthritis, and type 1 diabetes. Because PI3Kδ plays a major role in both B-cell-mediated autoimmune inflammation and B-cell malignancies, PI3Kδ inhibitors may represent a promising therapeutic approach for treating these diseases. PMID:22936933

  9. The Essential Phosphoinositide Kinase MSS-4 Is Required for Polar Hyphal Morphogenesis, Localizing to Sites of Growth and Cell Fusion in Neurospora crassa

    PubMed Central

    Stenzel, Irene; Hempel, Franziska; Seiler, Stephan; Heilmann, Ingo

    2012-01-01

    Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P2. In N. crassa hyphae, PtdIns(4,5)P2 localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P2 that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms. PMID:23272106

  10. The essential phosphoinositide kinase MSS-4 is required for polar hyphal morphogenesis, localizing to sites of growth and cell fusion in Neurospora crassa.

    PubMed

    Mhs, Anette; Ischebeck, Till; Heilig, Yvonne; Stenzel, Irene; Hempel, Franziska; Seiler, Stephan; Heilmann, Ingo

    2012-01-01

    Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P(2). In N. crassa hyphae, PtdIns(4,5)P(2) localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P(2) that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms. PMID:23272106

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

    PubMed Central

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

    2013-01-01

    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 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ 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 14-3-3γ 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

  12. Cellular and Molecular Mechanisms of Chronic Kidney Disease with Diabetes Mellitus and Cardiovascular Diseases as Its Comorbidities

    PubMed Central

    Gajjala, Prathibha Reddy; Sanati, Maryam; Jankowski, Joachim

    2015-01-01

    Chronic kidney disease (CKD), diabetes mellitus (DM), and cardiovascular diseases (CVD) are complex disorders of partly unknown genesis and mostly known progression factors. CVD and DM are the risk factors of CKD and are strongly intertwined since DM can lead to both CKD and/or CVD, and CVD can lead to kidney disease. In recent years, our knowledge of CKD, DM, and CVD has been expanded and several important experimental, clinical, and epidemiological associations have been reported. The tight cellular and molecular interactions between the renal, diabetic, and cardiovascular systems in acute or chronic disease settings are becoming increasingly evident. However, the (patho-) physiological basis of the interactions of CKD, DM, and CVD with involvement of multiple endogenous and environmental factors is highly complex and our knowledge is still at its infancy. Not only single pathways and mediators of progression of these diseases have to be considered in these processes but also the mutual interactions of these factors are essential. The recent advances in proteomics and integrative analysis technologies have allowed rapid progress in analyzing complex disorders and clearly show the opportunity for new efficient and specific therapies. More than a dozen pathways have been identified so far, including hyperactivity of the renin–angiotensin (RAS)–aldosterone system, osmotic sodium retention, endothelial dysfunction, dyslipidemia, RAS/RAF/extracellular-signal-regulated kinase pathway, modification of the purinergic system, phosphatidylinositol 3-kinase (PI 3-kinase)-dependent signaling pathways, and inflammation, all leading to histomorphological alterations of the kidney and vessels of diabetic and non-diabetic patients. Since a better understanding of the common cellular and molecular mechanisms of these diseases may be a key to successful identification of new therapeutic targets, we review in this paper the current literature about cellular and molecular mechanisms of CKD. PMID:26217336

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

    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.

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

    PubMed Central

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

    2013-01-01

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

  15. A phosphoinositide 3-kinase (PI3K)-serum- and glucocorticoid-inducible kinase 1 (SGK1) pathway promotes Kv7.1 channel surface expression by inhibiting Nedd4-2 protein.

    PubMed

    Andersen, Martin Nybo; Krzystanek, Katarzyna; Petersen, Frederic; Bomholtz, Sofia Hammami; Olesen, Søren-Peter; Abriel, Hugues; Jespersen, Thomas; Rasmussen, Hanne Borger

    2013-12-27

    Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells. PMID:24214981

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

    PubMed

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

    2006-11-01

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

  17. Baculovirus production of fully-active phosphoinositide 3-kinase alpha as a p85alpha-p110alpha fusion for X-ray crystallographic analysis with ATP competitive enzyme inhibitors.

    PubMed

    Sinnamon, Robert H; McDevitt, Patrick; Pietrak, Beth L; Leydon, Vaughan R; Xue, Yu; Lehr, Ruth; Qi, Hongwei; Burns, Matthew; Elkins, Patricia; Ward, Paris; Vincentini, Giorgia; Fisher, Donald; Grimes, Maggie; Brandt, Martin; Auger, Kurt R; Ho, Thau; Johanson, Kyung; Jones, Christopher S; Schwartz, Benjamin; Sweitzer, Thomas D; Kirkpatrick, Robert B

    2010-10-01

    Phosphoinositide 3-kinases have been targeted for therapeutic research because they are key components of a cell signaling cascade controlling proliferation, growth, and survival. Direct activation of the PI3Kalpha pathway contributes to the development and progression of solid tumors in breast, endometrial, colon, ovarian, and gastric cancers. In the context of a drug discovery effort, the availability of a robust crystallographic system is a means to understand the subtle differences between ATP competitive inhibitor interactions with the active site and their selectivity against other PI3Kinase enzymes. To generate a suitable recombinant design for this purpose, a p85alpha-p110alpha fusion system was developed which enabled the expression and purification of a stoichiometrically homogeneous, constitutively active enzyme for structure determination with potent ATP competitive inhibitors (Raha et al., in preparation) [56]. This approach has yielded preparations with activity and inhibition characteristics comparable to those of the full-length PI3Kalpha from which X-ray diffracting crystals were grown with inhibitors bound in the active site. PMID:20457255

  18. Imidazo[1,2-a]pyrazine inhibitors of phosphoinositide 3-kinase alpha (PI3K?): 3D-QSAR analysis utilizing the Hybrid Monte Carlo algorithm to refine receptor-ligand complexes for molecular alignment.

    PubMed

    Chadha, N; Jasuja, H; Kaur, M; Singh Bahia, M; Silakari, O

    2014-01-01

    Phosphoinositide 3-kinase alpha (PI3K?) is a lipid kinase involved in several cellular functions such as cell growth, proliferation, differentiation and survival, and its anomalous regulation leads to cancerous conditions. PI3K? inhibition completely blocks the cancer signalling pathway, hence it can be explored as an important therapeutic target for cancer treatment. In the present study, docking analysis of 49 selective imidazo[1,2-a]pyrazine inhibitors of PI3K? was carried out using the QM-Polarized ligand docking (QPLD) program of the Schrdinger software, followed by the refinement of receptor-ligand conformations using the Hybrid Monte Carlo algorithm in the Liaison program, and alignment of refined conformations of inhibitors was utilized for the development of an atom-based 3D-QSAR model in the PHASE program. Among the five generated models, the best model was selected corresponding to PLS factor 2, displaying the highest value of Q(2)test (0.650). The selected model also displayed high values of r(2)train (0.917), F-value (166.5) and Pearson-r (0.877) and a low value of SD (0.265). The contour plots generated for the selected 3D-QSAR model were correlated with the results of docking simulations. Finally, this combined information generated from 3D-QSAR and docking analysis was used to design new congeners. PMID:24601789

  19. Retinoic acid signaling in axonal regeneration.

    PubMed

    Puttagunta, Radhika; Di Giovanni, Simone

    2011-01-01

    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 β2 (RARβ2), 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 RARβ2 expression and contributed to neurite outgrowth. Moreover, RARβagonists, in cerebellar granule neurons (CGN) and in the brain in vivo, induced phosphoinositide 3-kinase dependent phosphorylation of AKT that was involved in RARβ-dependent neurite outgrowth. More recently, RA-RARβpathways 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

  20. 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

    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

    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

  1. Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110alpha and p110beta catalytic subunits of class-Ia phosphoinositide 3-kinases.

    PubMed Central

    Beeton, C A; Chance, E M; Foukas, L C; Shepherd, P R

    2000-01-01

    Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110alpha and p110beta isoforms. The lipid-kinase activity did not display Michaelis-Menten kinetics but modelling the kinetic data demonstrated that p110alpha has a higher V(max) and a 25-fold higher K(m) for PtdIns than p110beta. A similar situation occurs with PtdIns(4,5)P(2), because at low concentration of PtdIns(4,5)P(2) p110beta is a better PtdIns(4,5)P(2) kinase than p110alpha, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110beta functions better in areas of membranes containing low levels of substrate whereas p110alpha would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110beta phosphorylated p85 to a lower degree than did p110alpha. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110alpha and p110beta. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110alpha has a higher K(m) (550 microM) than p110beta (K(m) 8 microgM). Similarly, the relative V(max) towards peptide substrate of p110alpha was three times higher than that of p110beta. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110alpha and p110beta in vivo. PMID:10947948

  2. Involvement of phosphoinositide 3-kinase class IA (PI3K 110α) and NADPH oxidase 1 (NOX1) in regulation of vascular differentiation induced by vascular endothelial growth factor (VEGF) in mouse embryonic stem cells.

    PubMed

    Bekhite, Mohamed M; Müller, Veronika; Tröger, Sebastian H; Müller, Jörg P; Figulla, Hans-Reiner; Sauer, Heinrich; Wartenberg, Maria

    2016-04-01

    The impact of reactive oxygen species and phosphoinositide 3-kinase (PI3K) in differentiating embryonic stem (ES) cells is largely unknown. Here, we show that the silencing of the PI3K catalytic subunit p110α and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) by short hairpin RNA or pharmacological inhibition of NOX and ras-related C3 botulinum toxin substrate 1 (Rac1) abolishes superoxide production by vascular endothelial growth factor (VEGF) in mouse ES cells and in ES-cell-derived fetal liver kinase-1(+) (Flk-1(+)) vascular progenitor cells, whereas the mitochondrial complex I inhibitor rotenone does not have an effect. Silencing p110α or inhibiting Rac1 arrests vasculogenesis at initial stages in embryoid bodies, even under VEGF treatment, as indicated by platelet endothelial cell adhesion molecule-1 (PECAM-1)-positive areas and branching points. In the absence of p110α, tube-like structure formation on matrigel and cell migration of Flk-1(+) cells in scratch migration assays are totally impaired. Silencing NOX1 causes a reduction in PECAM-1-positive areas, branching points, cell migration and tube length upon VEGF treatment, despite the expression of vascular differentiation markers. Interestingly, silencing p110α but not NOX1 inhibits the activation of Rac1, Ras homologue gene family member A (RhoA) and Akt leading to the abrogation of VEGF-induced lamellipodia structure formation. Thus, our data demonstrate that the PI3K p110α-Akt/Rac1 and NOX1 signalling pathways play a pivotal role in VEGF-induced vascular differentiation and cell migration. Rac1, RhoA and Akt phosphorylation occur downstream of PI3K and upstream of NOX1 underscoring a role of PI3K p110α in the regulation of cell polarity and migration. PMID:26553657

  3. The gene for a novel protein, a member of the protein disulphide isomerase/form I phosphoinositide-specific phospholipase C family, is amplified in hydroxyurea-resistant cells.

    PubMed Central

    Chaudhuri, M M; Tonin, P N; Lewis, W H; Srinivasan, P R

    1992-01-01

    Cell lines selected in multiple steps for increasing resistance to hydroxyurea have been shown to have corresponding increases in ribonucleotide reductase activity. We have isolated a number of cDNA clones from a cDNA library constructed from a highly hydroxyurea-resistant hamster cell line, 600H, in which the activity of ribonucleotide reductase is elevated more than 80-fold. These clones correspond to genomic DNA sequences amplified in the 600H cell line compared with the V79 parental line. One of these cDNA clones, termed P5, codes for a 50 kDa protein detected by in vitro translation of poly(A)+ RNA isolated by hybridization/selection. The cDNA sequence contains a single open reading frame of 1317 nucleotides which encodes a polypeptide of 439 amino acids. The amino acid sequence deduced from the cDNA insert contains two copies of the 11-amino-acid sequence Val-Glu-Phe-Tyr-Ala-Pro-Trp-Cys-Gly-His-Cys. Duplicate copies of this sequence also occur in the active site of rat and human protein disulphide isomerase (also known as the beta-subunit of human prolyl 4-hydroxylase, tri-iodothyronine-binding protein) and in Form I phosphoinositide-specific phospholipase C, indicating that P5 falls into this newly defined superfamily of proteins. Genomic sequences similar to the cDNA clone are amplified 10-20-fold in hamster cells selected for resistance to increasing concentrations of hydroxyurea, a phenomenon observed earlier with cDNA clones for the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. RNA blots probed with P5 cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells. Images Fig. 1. Fig. 4. Fig. 5. Fig. 6. PMID:1311171

  4. A Potent Inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen Activated Protein (MAP) Kinase Signalling, Quercetin (3, 3', 4', 5, 7-Pentahydroxyflavone) Promotes Cell Death in Ultraviolet (UV)-B-Irradiated B16F10 Melanoma Cells

    PubMed Central

    Rafiq, Rather A.; Quadri, Afnan; Nazir, Lone A.; Peerzada, Kaiser; Ganai, Bashir A.; Tasduq, Sheikh A.

    2015-01-01

    Ultraviolet (UV) radiation–induced skin damage contributes strongly to the formation of melanoma, a highly lethal form of skin cancer. Quercetin (Qu), the most widely consumed dietary bioflavonoid and well known inhibitor of phosphoinositide 3-kinase (PI3K) and mitogen activated protein (MAP) kinase signalling, has been reported to be chemopreventive in several forms of non-melanoma skin cancers. Here, we report that the treatment of ultraviolet (UV)-B-irradiated B16F10 melanoma cells with quercetin resulted in a dose dependent reduction in cell viability and increased apoptosis. The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM). Promotion of UVB-induced cell death by quercetin was further revealed by cleavage of chromosomal DNA, caspase activation, poly (ADP) ribose polymerase (PARP) cleavage, and an increase in sub-G1 cells. Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation. Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF). Overall, these results suggest the possibility of using quercetin in combination with UVB as a possible treatment option for melanoma in future. PMID:26148186

  5. Interleukin-8 Induces the Endothelial Cell Migration through the Activation of Phosphoinositide 3-Kinase-Rac1/RhoA Pathway

    PubMed Central

    Lai, Yi; Shen, Yang; Liu, Xiao-Heng; Zhang, Yi; Zeng, Ye; Liu, Ying-Fen

    2011-01-01

    Endothelial cell migration is essential for tumor angiogenesis, and interleukin-8 (IL-8) has been shown to play an important role in tumor growth, angiogenesis, and metastasis. This study aimed to investigate the molecular mechanism of IL-8 induced endothelial cell migration. Our results indicated that IL-8 induced a rapid rearrangement of the actin cytoskeleton in EA.Hy926 cells, generating extensions resembling membrane ruffling and stress fibers. These processes required parallel upregulation of the small GTPases Rac1 and RhoA. Moreover, we demonstrated that IL-8 activated PI3K following the same kinetics observed from IL-8 induction of cytoskeletal rearrangement, suggesting the participation of PI3K in these processes. Taken together, our study demonstrates that PI3K-Rac1/RhoA signaling pathway plays a vital role in IL-8 induced endothelial cell migration, and provides new insight into the molecular mechanisms by which IL-8 contributes to tumor angiogenesis and metastasis. PMID:21750647

  6. Signalling mechanisms regulating phenotypic changes in breast cancer cells.

    PubMed

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

    2015-01-01

    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

  7. Signalling mechanisms regulating phenotypic changes in breast cancer cells

    PubMed Central

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

    2015-01-01

    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

  8. Puzzling Mechanisms

    ERIC Educational Resources Information Center

    van Deventer, M. Oskar

    2009-01-01

    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…

  9. Mechanical Systems

    NASA Technical Reports Server (NTRS)

    Davis, Robert E.

    2002-01-01

    The presentation provides an overview of requirement and interpretation letters, mechanical systems safety interpretation letter, design and verification provisions, and mechanical systems verification plan.

  10. The p110δ crystal structure uncovers mechanisms for selectivity and potency of novel PI3K inhibitors

    PubMed Central

    Berndt, Alex; Miller, Simon; Williams, Olusegun; Le, Daniel D.; Houseman, Benjamin T.; Pacold, Joseph I.; Gorrec, Fabrice; Hon, Wai-Ching; Liu, Yi; Rommel, Christian; Gaillard, Pascale; Ruckle, Thomas; Schwarz, Matthias K.; Shokat, Kevan M.; Shaw, Jeffrey P.; Williams, Roger L.

    2009-01-01

    Deregulation of the phosphoinositide 3-kinase (PI3K) pathway has been implicated in numerous pathologies like cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small molecule and ATP-competitive PI3K inhibitors with a wide range of selectivities have entered clinical development. In order to understand mechanisms underlying isoform selectivity of these inhibitors, we developed a novel expression strategy that enabled us to determine the first crystal structure of the catalytic subunit of the class IA PI3K p110δ. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI3K inhibitors reveal that selectivity towards p110δ can be achieved by exploiting its conformational flexibility and the sequence diversity of active-site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110δ with greatly improved potencies. PMID:20081827

  11. Multiple metabolic pools of phosphoinositides and phosphatidate in human erythrocytes incubated in a medium that permits rapid transmembrane exchange of phosphate.

    PubMed Central

    King, C E; Stephens, L R; Hawkins, P T; Guy, G R; Michell, R H

    1987-01-01

    1. A Hepes-based medium has been devised which allows rapid Pi exchange across the plasma membrane of the human erythrocyte. This allows the metabolically labile phosphate pools of human erythrocytes to come to equilibrium with [32P]Pi in the medium after only 5 h in vitro. 2. After 5-7 h incubation with [32P]Pi in this medium, only three phospholipids, phosphatidic acid (PtdOH), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) are radioactively labelled. The concentrations of PtdIns4P and PtdIns4,5P2 remain constant throughout the incubation, so this labelling process is a reflection of the steady-state turnover of their monoester phosphate groups. 3. During such incubations, the specific radioactivities of the monoesterified phosphates of PtdIns4, PtdIns4,5P2 and PtdOH come to a steady value after 5 h that is only 25-30% of the specific radioactivity of the gamma-phosphate of ATP at that time. We suggest that this is a consequence of metabolic heterogeneity. This heterogeneity is not a result of the heterogeneous age distribution of the erythrocytes in human blood. Thus it appears that there is metabolic compartmentation of these lipids within cells, such that within a time-scale of a few hours only 25-30% of these three lipids are actively metabolized. 4. The phosphoinositidase C of intact human erythrocytes, when activated by Ca2+-ionophore treatment, only hydrolyses 50% of the total PtdIns4,5P2 and 50% of 32P-labelled PtdIns4,5P2 present in the cells: this enzyme does not discriminate between the metabolically active and inactive compartments of lipids in the erythrocyte membrane. Hence at least four metabolic pools of PtdIns4P and PtdIns4,5P2 are distinguishable in the human erythrocyte plasma membrane. 5. The mechanisms by which multiple non-mixing metabolic pools of PtdOH, PtdIns4P and PtdIns4,5P2 are sustained over many hours in the plasma membranes of intact erythrocytes are unknown, although some possible explanations are considered. PMID:2821998

  12. Phosphoinositide 3-Kinase Pathway Activation in Phosphate and Tensin Homolog (PTEN)-deficient Prostate Cancer Cells Is Independent of Receptor Tyrosine Kinases and Mediated by the p110β and p110δ Catalytic Subunits*

    PubMed Central

    Jiang, Xinnong; Chen, Sen; Asara, John M.; Balk, Steven P.

    2010-01-01

    Class IA phosphoinositide 3-kinase (PI3K) p110 catalytic subunits are activated upon Src homology 2 domain-mediated binding of their p85 regulatory subunits to tyrosine-phosphorylated pYXXM motifs in receptor tyrosine kinases (RTKs) or adaptor proteins. The PI3K pathway is activated by phosphate and tensin homolog (PTEN) loss in most prostate cancers (PCa), but the contribution of upstream RTKs that may be targeted therapeutically has not been assessed. Immunoblotting of p85-associated proteins in serum-starved PTEN-deficient LNCaP and C4-2 PCa cells showed a small set of discrete tyrosine-phosphorylated proteins, but these proteins were not recognized by an anti-pYXXM motif antibody and were not found in PTEN-deficient PC3 PCa cells. LC/MS/MS using label-free proteomics and immunoblotting showed that p85 was associated primarily with p110β and p110δ. An interaction with ErbB3 was also detected but was independent of ErbB3 tyrosine phosphorylation and was not required for basal PI3K activity. Basal tyrosine phosphorylation of p110β and p110δ could be blocked by c-Src inhibitors, but this did not suppress PI3K activity, which was similarly independent of Ras. Basal PI3K activity was mediated by p110β in PC3 cells and by both p110β and p110δ in LNCaP cells, whereas p110α was required for PI3K activation in response to RTK stimulation by heregulin-β1. These findings show that basal PI3K activity in PTEN-deficient PCa cells is RTK-independent and can be mediated by p110β and p110δ. Increased p110β expression in PCa may be required for RTK-independent PI3K pathway activation in adult prostate epithelium with genetic or epigenetic PTEN down-regulation. PMID:20231295

  13. Galanin-like peptide (GALP) neurone-specific phosphoinositide 3-kinase signalling regulates GALP mRNA levels in the hypothalamus of males and luteinising hormone levels in both sexes.

    PubMed

    Aziz, R; Beymer, M; Negrón, A L; Newshan, A; Yu, G; Rosati, B; McKinnon, D; Fukuda, M; Lin, R Z; Mayer, C; Boehm, U; Acosta-Martínez, M

    2014-07-01

    Galanin-like peptide (GALP) neurones participate in the metabolic control of reproduction and are targets of insulin and leptin regulation. Phosphoinositide 3-kinase (PI3K) is common to the signalling pathways utilised by both insulin and leptin. Therefore, we investigated whether PI3K signalling in neurones expressing GALP plays a role in the transcriptional regulation of the GALP gene and in the metabolic control of luteinising hormone (LH) release. Accordingly, we deleted PI3K catalytic subunits p110α and p110β via conditional gene targeting (cKO) in mice (GALP-p110α/β cKO). To monitor PI3K signalling in GALP neurones, these animals were also crossed with Cre-dependent FoxO1GFP reporter mice. Compared to insulin-infused control animals, the PI3K-Akt-dependent FoxO1GFP nuclear exclusion in GALP neurones was abolished in GALP-p110α/β cKO mice. We next used food deprivation to investigate whether the GALP-neurone specific ablation of PI3K activity affected the susceptibility of the gonadotrophic axis to negative energy balance. Treatment did not affect LH levels in either sex. However, a significant genotype effect on LH levels was observed in females. By contrast, no genotype effect on LH levels was observed in males. A sex-specific genotype effect on hypothalamic GALP mRNA was observed, with fed and fasted GALP-p110α/β cKO males having lower GALP mRNA expression compared to wild-type fed males. Finally, the effects of gonadectomy and steroid hormone replacement on GALP mRNA levels were investigated. Compared to vehicle-treated mice, steroid hormone replacement reduced mediobasal hypothalamus GALP expression in wild-type and GALP-p110α/β cKO animals. In addition, within the castrated and vehicle-treated group and compared to wild-type mice, LH levels were lower in GALP-p110α/β cKO males. Double immunofluorescence using GALP-Cre/R26-YFP mice showed androgen and oestrogen receptor co-localisation within GALP neurones. Our data demonstrate that GALP neurones are direct targets of steroid hormones and that PI3K signalling regulates hypothalamic GALP mRNA expression and LH levels in a sex-specific fashion. PMID:24796383

  14. 3-D structure and dynamics of protein kinase B-new mechanism for the allosteric regulation of an AGC kinase.

    PubMed

    Calleja, Vronique; Laguerre, Michel; Larijani, Banafsh

    2009-03-01

    New developments regarding the structure and in vivo dynamics of protein kinase B (PKB/Akt) have been recently exposed. Here, we specifically review how the use of multi-disciplinary approaches has resulted in reaching the recent progress made to relate the quaternary structure of PKB to its in vivo function. Using X-ray crystallography, the structure of PKB pleckstrin homology (PH) and kinase domains was determined separately. The molecular mechanisms involved in (a) the binding of the phosphoinositides to the PH domain and (b) the activation of the kinase with the rearrangement of the catalytic site and substrate binding were determined. In vitro, nuclear magnetic resonance and circular dychroism studies gave complementary information on the interaction of the PH domain with the phosphoinositides. However, the molecular nature and the function of the interactions between the PKB domains could not be deduced from the X-ray data since the full-length PKB has not been crystallised. In vitro, dynamic information on the inter-domain conformational changes related to PKB activation states emerged with the use of tandem mass spectrometry. Cell imaging and Frster resonance energy transfer provided in vivo dynamics. Molecular modelling and dynamic simulations in conjunction with mutagenesis and biochemical analysis were used to investigate the complex interactions between the PKB domains in vivo and understand at the molecular level how it linked to its activity. The compilation of the information obtained on the 3-D structure and the spatiotemporal dynamics of this widely studied oncogene could be applied to the study of other proteins. This inter-disciplinary approach led to a more profound understanding of PKB complex activation mechanism in vivo that will shed light onto new ideas and possibilities for modulating its activity. PMID:19568789

  15. Investigation of molecular mechanisms and regulatory pathways of pro-angiogenic nanorods

    NASA Astrophysics Data System (ADS)

    Nethi, Susheel Kumar; Veeriah, Vimal; Barui, Ayan Kumar; Rajendran, Saranya; Mattapally, Saidulu; Misra, Sanjay; Chatterjee, Suvro; Patra, Chitta Ranjan

    2015-05-01

    Angiogenesis, a process involving the growth of new blood vessels from the pre-existing vasculature, plays a crucial role in various pathophysiological conditions. We have previously demonstrated that europium hydroxide [EuIII(OH)3] nanorods (EHNs) exhibit pro-angiogenic properties through the generation of reactive oxygen species (ROS) and mitogen activated protein kinase (MAPK) activation. Considering the enormous implication of angiogenesis in cardiovascular diseases (CVDs) and cancer, it is essential to understand in-depth molecular mechanisms and signaling pathways in order to develop the most efficient and effective alternative treatment strategy for CVDs. However, the exact underlying mechanism and cascade signaling pathways behind the pro-angiogenic properties exhibited by EHNs still remain unclear. Herein, we report for the first time that the hydrogen peroxide (H2O2), a redox signaling molecule, generated by these EHNs activates the endothelial nitric oxide synthase (eNOS) that promotes the nitric oxide (NO) production in a PI3K (phosphoinositide 3-kinase)/Akt dependent manner, eventually triggering angiogenesis. We intensely believe that the investigation and understanding of the in-depth molecular mechanism and signaling pathways of EHNs induced angiogenesis will help us in developing an effective alternative treatment strategy for cardiovascular related and ischemic diseases where angiogenesis plays an important role.Angiogenesis, a process involving the growth of new blood vessels from the pre-existing vasculature, plays a crucial role in various pathophysiological conditions. We have previously demonstrated that europium hydroxide [EuIII(OH)3] nanorods (EHNs) exhibit pro-angiogenic properties through the generation of reactive oxygen species (ROS) and mitogen activated protein kinase (MAPK) activation. Considering the enormous implication of angiogenesis in cardiovascular diseases (CVDs) and cancer, it is essential to understand in-depth molecular mechanisms and signaling pathways in order to develop the most efficient and effective alternative treatment strategy for CVDs. However, the exact underlying mechanism and cascade signaling pathways behind the pro-angiogenic properties exhibited by EHNs still remain unclear. Herein, we report for the first time that the hydrogen peroxide (H2O2), a redox signaling molecule, generated by these EHNs activates the endothelial nitric oxide synthase (eNOS) that promotes the nitric oxide (NO) production in a PI3K (phosphoinositide 3-kinase)/Akt dependent manner, eventually triggering angiogenesis. We intensely believe that the investigation and understanding of the in-depth molecular mechanism and signaling pathways of EHNs induced angiogenesis will help us in developing an effective alternative treatment strategy for cardiovascular related and ischemic diseases where angiogenesis plays an important role. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01327e

  16. New molecular mechanisms of inter-organelle lipid transport.

    PubMed

    Drin, Guillaume; von Filseck, Joachim Moser; Čopič, Alenka

    2016-04-15

    Lipids are precisely distributed in cell membranes, along with associated proteins defining organelle identity. Because the major cellular lipid factory is the endoplasmic reticulum (ER), a key issue is to understand how various lipids are subsequently delivered to other compartments by vesicular and non-vesicular transport pathways. Efforts are currently made to decipher how lipid transfer proteins (LTPs) work either across long distances or confined to membrane contact sites (MCSs) where two organelles are at close proximity. Recent findings reveal that proteins of the oxysterol-binding protein related-proteins (ORP)/oxysterol-binding homology (Osh) family are not all just sterol transporters/sensors: some can bind either phosphatidylinositol 4-phosphate (PtdIns(4)P) and sterol or PtdIns(4)P and phosphatidylserine (PS), exchange these lipids between membranes, and thereby use phosphoinositide metabolism to create cellular lipid gradients. Lipid exchange is likely a widespread mechanism also utilized by other LTPs to efficiently trade lipids between organelle membranes. Finally, the discovery of more proteins bearing a lipid-binding module (SMP or START-like domain) raises new questions on how lipids are conveyed in cells and how the activities of different LTPs are coordinated. PMID:27068959

  17. Dynamics of molecular mechanisms underlying ovarian oxytocin secretion.

    PubMed

    Stormshak, F; Orwig, K E; Bertrand, J E

    1995-01-01

    In the ruminant ovary, synthesis and secretion of oxytocin begin in the granulosa cells of the preovulatory follicle and are markedly stimulated by the surge of LH and FSH. Luteinization of the granulosa cells results in a further increase in oxytocin gene expression, but translation of mRNA appears to be retarded because the peak concentration of luteal oxytocin occurs later than the maximal accumulation of the message. Several hormones have been shown to stimulate oxytocin secretion from granulosa and luteal cells in vivo or in vitro. However, the role of prostaglandin F2 alpha (PGF2 alpha) in regulating luteal oxytocin secretion has perhaps received more study than other hormones. The mechanism of action of PGF2 alpha has been shown to encompass a phosphoinositide cascade and activation of protein kinase C, events that are associated with luteal secretion of oxytocin. Protein kinase C phosphorylation of the actin-binding protein myristolated alanine-rich C kinase substrate (MARCKS) may be required for exocytosis of oxytocin. PMID:7623328

  18. SOEP: Mechanics.

    ERIC Educational Resources Information Center

    Jacobs, Clinton O.; And Others

    1984-01-01

    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)

  19. Mechanical Kinesiology.

    ERIC Educational Resources Information Center

    Barham, Jerry N.

    Mechanical kinesiology is defined as a study of the mechanical factors affecting human movement, i.e., applying the physical laws of mechanics to the study of human motor behavior. This textbook on the subject is divided into thirty lessons. Each lesson is organized into three parts: a part on the text proper; a part entitled "study guidelines,"…

  20. Mechanical Selection.

    ERIC Educational Resources Information Center

    Brownson, Charles W.

    1988-01-01

    Defines mechanical and expert selection of library materials and investigates the usefulness of mechanical selection in three test cases involving popular novels, German literature, and contemporary poetry. The cost of mechanical selection is examined and more quantification in selection practice is recommended. Data are presented in eight tables,…

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

    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.

  2. [Defense mechanisms].

    PubMed

    Chabrol, Henri

    2005-09-01

    Defence mechanisms are mental operations that are involuntary and unconscious and contribute to reduce internal and external stresses. The concept of defensive organisation or style, defined as a set of defence mechanisms relatively stable and characteristic of personality appears to be a major dimension of personality, from normal to pathology. Studies on defence mechanisms have gained the interest of clinicians, largely outside the psychoanalytical field. However, the lack of reliability and validity of the assessment instruments still limits the empirical studies of the relation between defence mechanisms and psychological health as well as of their therapeutic implications. PMID:16231612

  3. Predicting the structures of complexes between phosphoinositide 3-kinase (PI3K) and romidepsin-related compounds for the drug design of PI3K/histone deacetylase dual inhibitors using computational docking and the ligand-based drug design approach.

    PubMed

    Oda, Akifumi; Saijo, Ken; Ishioka, Chikashi; Narita, Koichi; Katoh, Tadashi; Watanabe, Yurie; Fukuyoshi, Shuichi; Takahashi, Ohgi

    2014-11-01

    Predictions of the three-dimensional (3D) structures of the complexes between phosphoinositide 3-kinase (PI3K) and two inhibitors were conducted using computational docking and the ligand-based drug design approach. The obtained structures were refined by structural optimizations and molecular dynamics (MD) simulations. The ligands were located deep inside the ligand binding pocket of the p110α subunit of PI3K, and the hydrogen bond formations and hydrophobic effects of the surrounding amino acids were predicted. Although rough structures were obtained for the PI3K-inhibitor complexes before the MD simulations, the refinement of the structures by these simulations clarified the hydrogen bonding patterns of the complexes. PMID:25254927

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

    PubMed Central

    LIU, CHENG

    2015-01-01

    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-α, 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

  5. Investigation of molecular mechanisms and regulatory pathways of pro-angiogenic nanorods†

    PubMed Central

    Nethi, Susheel Kumar; Veeriah, Vimal; Barui, Ayan Kumar; Rajendran, Saranya; Mattapally, Saidulu; Misra, Sanjay

    2016-01-01

    Angiogenesis, a process involving the growth of new blood vessels from the pre-existing vasculature, plays a crucial role in various pathophysiological conditions. We have previously demonstrated that europium hydroxide [EuIII(OH)3] nanorods (EHNs) exhibit pro-angiogenic properties through the generation of reactive oxygen species (ROS) and mitogen activated protein kinase (MAPK) activation. Considering the enormous implication of angiogenesis in cardiovascular diseases (CVDs) and cancer, it is essential to understand in-depth molecular mechanisms and signaling pathways in order to develop the most efficient and effective alternative treatment strategy for CVDs. However, the exact underlying mechanism and cascade signaling pathways behind the pro-angiogenic properties exhibited by EHNs still remain unclear. Herein, we report for the first time that the hydrogen peroxide (H2O2), a redox signaling molecule, generated by these EHNs activates the endothelial nitric oxide synthase (eNOS) that promotes the nitric oxide (NO) production in a PI3K (phosphoinositide 3-kinase)/Akt dependent manner, eventually triggering angiogenesis. We intensely believe that the investigation and understanding of the in-depth molecular mechanism and signaling pathways of EHNs induced angiogenesis will help us in developing an effective alternative treatment strategy for cardiovascular related and ischemic diseases where angiogenesis plays an important role. PMID:25963768

  6. Actuator mechanism

    NASA Technical Reports Server (NTRS)

    Stange, W. C. (Inventor)

    1978-01-01

    An actuator mechanism is described, having a frame with a rotatable shaft supported in the frame, a positioning mechanism coupled to the shaft for rotating the shaft in two rotary positions, disposed approximately 180 degrees apart, and a pair of plungers coupled to the shaft. Each plunger is responsive to a control signal for applying bi-directional rotation to the shaft.

  7. Mechanism problems

    NASA Technical Reports Server (NTRS)

    Riedel, J. K.

    1972-01-01

    It is pointed out that too frequently during the design and development of mechanisms, problems occur that could have been avoided if the right question had been asked before, rather than after, the fact. Several typical problems, drawn from actual experience, are discussed and analyzed. The lessons learned are used to generate various suggestions for minimizing mistakes in mechanism design.

  8. Mechanical memory

    DOEpatents

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

    2006-08-15

    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.

  9. Mechanical memory

    DOEpatents

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

    2006-05-16

    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.

  10. Molecular and Cellular Mechanisms of Shigella flexneri Dissemination

    PubMed Central

    Agaisse, Hervé

    2016-01-01

    The intracellular pathogen Shigella flexneri is the causative agent of bacillary dysentery in humans. The disease is characterized by bacterial invasion of intestinal cells, dissemination within the colonic epithelium through direct spread from cell to cell, and massive inflammation of the intestinal mucosa. Here, we review the mechanisms supporting S. flexneri dissemination. The dissemination process primarily relies on actin assembly at the bacterial pole, which propels the pathogen throughout the cytosol of primary infected cells. Polar actin assembly is supported by polar expression of the bacterial autotransporter family member IcsA, which recruits the N-WASP/ARP2/3 actin assembly machinery. As motile bacteria encounter cell-cell contacts, they form plasma membrane protrusions that project into adjacent cells. In addition to the ARP2/3-dependent actin assembly machinery, protrusion formation relies on formins and myosins. The resolution of protrusions into vacuoles occurs through the collapse of the protrusion neck, leading to the formation of an intermediate membrane-bound compartment termed vacuole-like protrusions (VLPs). VLP formation requires tyrosine kinase and phosphoinositide signaling in protrusions, which relies on the integrity of the bacterial type 3 secretion system (T3SS). The T3SS is also required for escaping double membrane vacuoles through the activity of the T3SS translocases IpaB and IpaC, and the effector proteins VirA and IcsB. Numerous factors supporting envelope biogenesis contribute to IcsA exposure and maintenance at the bacterial pole, including LPS synthesis, membrane proteases, and periplasmic chaperones. Although less characterized, the assembly and function of the T3SS in the context of bacterial dissemination also relies on factors supporting envelope biogenesis. Finally, the dissemination process requires the adaptation of the pathogen to various cellular compartments through transcriptional and post-transcriptional mechanisms. PMID:27014639

  11. Molecular and Cellular Mechanisms of Shigella flexneri Dissemination.

    PubMed

    Agaisse, Hervé

    2016-01-01

    The intracellular pathogen Shigella flexneri is the causative agent of bacillary dysentery in humans. The disease is characterized by bacterial invasion of intestinal cells, dissemination within the colonic epithelium through direct spread from cell to cell, and massive inflammation of the intestinal mucosa. Here, we review the mechanisms supporting S. flexneri dissemination. The dissemination process primarily relies on actin assembly at the bacterial pole, which propels the pathogen throughout the cytosol of primary infected cells. Polar actin assembly is supported by polar expression of the bacterial autotransporter family member IcsA, which recruits the N-WASP/ARP2/3 actin assembly machinery. As motile bacteria encounter cell-cell contacts, they form plasma membrane protrusions that project into adjacent cells. In addition to the ARP2/3-dependent actin assembly machinery, protrusion formation relies on formins and myosins. The resolution of protrusions into vacuoles occurs through the collapse of the protrusion neck, leading to the formation of an intermediate membrane-bound compartment termed vacuole-like protrusions (VLPs). VLP formation requires tyrosine kinase and phosphoinositide signaling in protrusions, which relies on the integrity of the bacterial type 3 secretion system (T3SS). The T3SS is also required for escaping double membrane vacuoles through the activity of the T3SS translocases IpaB and IpaC, and the effector proteins VirA and IcsB. Numerous factors supporting envelope biogenesis contribute to IcsA exposure and maintenance at the bacterial pole, including LPS synthesis, membrane proteases, and periplasmic chaperones. Although less characterized, the assembly and function of the T3SS in the context of bacterial dissemination also relies on factors supporting envelope biogenesis. Finally, the dissemination process requires the adaptation of the pathogen to various cellular compartments through transcriptional and post-transcriptional mechanisms. PMID:27014639

  12. Fault mechanics

    SciTech Connect

    Segall, P. )

    1991-01-01

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

  13. Computational mechanics

    SciTech Connect

    Goudreau, G.L.

    1993-03-01

    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.

  14. Repair Mechanisms

    SciTech Connect

    Lacks, Sanford A.

    1999-07-09

    Maintaining the integrity of its genetic blueprint is of central importance for a living cell and the organism of which it is a part. To preserve the function of the genetic material within the cell and to ensure its accurate transmission to future generations, numerous mechanisms have evolved to repair errors and damage in DNA. As an example of the cellular resources devoted to this end, consider that of 1709 proteins encoded by the first bacterial genome to be sequenced, that of Haemophilus influenzae, at least 45 function in DNA repair mechanisms. The complementary, double-stranded structure of DNA, a crucial feature that allows it to be readily replicated, also facilitates its repair. The objects of repair range from mismatched bases resulting from errors in DNA replication to base damage and even gross distortion of the DNA structure by physical and chemical agents. In a few instances damage is directly reversed. Most repair mechanisms, however, first remove the damaged region together with a segment of the DNA strand in which it occurred and then resynthesize that segment correctly using the complementary strand as a template. Depending on the component initially removed or recognized, these mechanisms have been categorized as base excision, nucleotide (oroligonucleotide) excision, and mismatch repair. When the damage cannot be so simply repaired, a mechanism of recombinational repair, requiring interaction with another copy of the genome, may intervene.

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

    NASA Astrophysics Data System (ADS)

    Volotovski, Igor D.

    1995-01-01

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

  16. Molecular mechanisms whereby immunomodulatory drugs activate natural killer cells: clinical application.

    PubMed

    Hayashi, Toshiaki; Hideshima, Teru; Akiyama, Masaharu; Podar, Klaus; Yasui, Hiroshi; Raje, Noopur; Kumar, Shaji; Chauhan, Dharminder; Treon, Steven P; Richardson, Paul; Anderson, Kenneth C

    2005-01-01

    Thalidomide and immunomodulatory drugs (IMiDs), which target multiple myeloma (MM) cells and the bone marrow microenvironment, can overcome drug resistance. These agents also have immunomodulatory effects. Specifically, we have reported that thalidomide increased serum interleukin-2 (IL-2) levels and natural killer (NK) cell numbers in the peripheral blood of responding MM patients. In this study, we investigated the mechanisms whereby IMiDs augment NK cell cytotoxicity. NK cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) of peripheral blood mononuclear cells cultured with IMiDs were examined in the presence or absence of anti-IL-2 antibody, ciclosporin A or depletion of CD56-positive cells. IMiDs-induced signalling pathways, triggering IL-2 transcription in T cells, were also delineated. IMiDs facilitated the nuclear translocation of nuclear factor of activated T cells-2 and activator protein-1 via activation of phosphoinositide-3 kinase signalling, with resultant IL-2 secretion. IMiDs enhanced both NK cell cytotoxicity and ADCC induced by triggering IL-2 production from T cells. These studies defined the mechanisms whereby IMiDs trigger NK cell-mediated tumour-cell lysis, further supporting their therapeutic use in MM. PMID:15638853

  17. Computational mechanics

    SciTech Connect

    Raboin, P J

    1998-01-01

    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.

  18. Soil mechanics

    NASA Technical Reports Server (NTRS)

    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

    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.

  19. Cellular Mechanisms of Gravitropic Response in Higher Plants

    NASA Astrophysics Data System (ADS)

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

    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).

  20. Mechanical Technician.

    ERIC Educational Resources Information Center

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

    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…

  1. Automotive Mechanics.

    ERIC Educational Resources Information Center

    Brown, Desmond

    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

  2. Transcription mechanisms.

    PubMed Central

    Blackwell, T Keith; Walker, Amy K

    2006-01-01

    Appropriate regulation of mRNA transcription is central to the differentiation and functions of eukaryotic cells, and to the development of complex organisms. mRNAs are synthesized by the coordinated action of a set of general transcription and mRNA modification factors. These factors and the fundamental mechanisms involved in transcription are conserved among eukaryotes, including C. elegans. Recent studies in various systems have revealed that this apparatus is not controlled through a simple on/off "switch" at the promoter, and that the factors and mechanisms involved in transcription are instead subject to regulation at a surprising number of different levels. In this chapter we will discuss examples in which regulation involving the general mRNA transcription apparatus or other transcription co-factors plays a central role in C. elegans development, and in which C. elegans studies have provided new insights into eukaryotic transcription mechanisms. Together, these studies have shown that regulatory mechanisms that involve the general Pol II machinery are a central participant in many aspects of C. elegans biology. PMID:18050436

  3. Automotive Mechanics.

    ERIC Educational Resources Information Center

    Brown, Desmond

    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…

  4. Mechanical Drafting.

    ERIC Educational Resources Information Center

    McClain, Gerald R.

    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…

  5. Cratering mechanics

    NASA Technical Reports Server (NTRS)

    Ivanov, B. A.

    1986-01-01

    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.

  6. Mechanical Technician.

    ERIC Educational Resources Information Center

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

    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

  7. Classical Mechanics

    NASA Astrophysics Data System (ADS)

    Chow, Tai L.

    1995-05-01

    Bring Classical Mechanics To Life With a Realistic Software Simulation! You can enhance the thorough coverage of Chow's Classical Mechanics with a hands-on, real-world experience! John Wiley & Sons, Inc. is proud to announce a new computer simulation for classical mechanics. Developed by the Consortium for Upper-Level Physics Software (CUPS), this simulation offers complex, often realistic calculations of models of various physical systems. Classical Mechanics Simulations (54881-2) is the perfect complement to Chow's text. Like all of the CUPS simulations, it is remarkably easy to use, yet sophisticated enough for explorations of new ideas. Other Important Features Include: * Six powerful simulations include: The Motion Generator, Rotation of Three-Dimensional Objects, Coupled Oscillators, Anharmonic Oscillators, Gravitational Orbits, and Collisions * Pascal source code for all programs is supplied and a number of exercises suggest specific ways the programs can be modified. * Simulations usually include graphical (often animated) displays. The entire CUPS simulation series consists of nine book/software simulations which comprise most of the undergraduate physics major's curriculum.

  8. Fluid Mechanics.

    ERIC Educational Resources Information Center

    Drazin, Philip

    1987-01-01

    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)

  9. Mechanical capacitor

    NASA Technical Reports Server (NTRS)

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

    1976-01-01

    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.

  10. Mechanical clutch

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

    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.

  11. Biofluid Mechanics

    NASA Astrophysics Data System (ADS)

    Oertel, Herbert

    In contrast to the topics discussed in previous chapters, biofluid mechanics is concerned with flows that are influenced by flexible biological surfaces. We distinguish between flows past living bodies in air or in water, such as bird flight or the swimming of fish, and internal flows, such as the closed blood circulation of living beings. In the previous millions of years, evolution has developed crawling, running, swimming, gliding, and flying as methods of motion of living beings, depending on their size and weight.

  12. ELEVATING MECHANISM

    DOEpatents

    Frederick, H.S.; Kinsella, M.A.

    1959-02-24

    An elevator is described, which is arranged for movement both in a horizontal and in a vertical direction so that the elevating mechanism may be employed for servicing equipment at separated points in a plant. In accordance with the present invention, the main elevator chassis is suspended from a monorail. The chassis, in turn supports a vertically moveable carriage, a sub- carriage vertically moveable on the carriage, and a turntable carried by the sub- carriage and moveable through an arc of 90 with the equipment attached thereto. In addition, the chassis supports all the means required to elevate or rotate the equipment.

  13. Molecular Mechanics

    PubMed Central

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

    2014-01-01

    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

  14. A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation.

    PubMed

    Li, Xinran; Rydzewski, Nicholas; Hider, Ahmad; Zhang, Xiaoli; Yang, Junsheng; Wang, Wuyang; Gao, Qiong; Cheng, Xiping; Xu, Haoxing

    2016-04-01

    To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca(2+) channel TRPML1 cause lysosomal storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca(2+)-dependent centripetal movement of lysosomes towards the perinuclear region (where autophagosomes accumulate) following autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca(2+) sensor that associates physically with the minus-end-directed dynactin-dynein motor, while PtdIns(3,5)P2, a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P2-TRPML1-ALG-2-dynein signalling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely to be caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Ca(2+) release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation. PMID:26950892

  15. Amphiregulin and PTEN evoke a multimodal mechanism of acquired resistance to PI3K inhibition

    PubMed Central

    Edgar, Kyle A.; Crocker, Lisa; Cheng, Eric; Wagle, Marie-Claire; Wongchenko, Matthew; Yan, Yibing; Wilson, Timothy R.; Dompe, Nicholas; Neve, Richard M.; Belvin, Marcia; Sampath, Deepak; Friedman, Lori S.; Wallin, Jeffrey J.

    2014-01-01

    Phosphoinositide-3 kinase (PI3K) signaling pathway alterations occur broadly in cancer and PI3K is a promising therapeutic target. Here, we investigated acquired resistance to GDC-0941, a PI3K inhibitor in clinical trials. Colorectal cancer (CRC) cells made to be resistant to GDC-0941 were discovered to secrete amphiregulin, which resulted in increased EGFR/MAPK signaling. Moreover, prolonged PI3K pathway inhibition in cultured cells over a period of months led to a secondary loss of PTEN in 40% of the CRC lines with acquired resistance to PI3K inhibition. In the absence of PI3K inhibitor, these PTEN-null PI3K inhibitor-resistant clones had elevated PI3K pathway signaling and decreased sensitivity to MAPK pathway inhibitors. Importantly, PTEN loss was not able to induce resistance to PI3K inhibitors in the absence of amphiregulin, indicating a multimodal mechanism of acquired resistance. The combination of PI3K and MAPK pathway inhibitors overcame acquired resistance in vitro and in vivo. PMID:25053989

  16. Mechanism of prolactin action on Nb/sub 2/ node lymphoma cells

    SciTech Connect

    Ofenstein, J.P.

    1987-01-01

    The post-receptor mechanism of action by which the pituitary hormone prolactin stimulates cell division was investigated in the Nb/sub 2/ rat lymphoma cell. The replication of Nb/sub 2/ rat lymphoma cells is specifically stimulated by prolactin in a dose dependent manner in the concentration range from 50 pg/ml to 1 ng/ml. Prolactin stimulates proteins synthesis in these cells between 0 and 2 hrs; DNA synthesis between 12 and 16 hrs, and cell replication between 16 and 24 hrs. The effect of prolactin on phospholipid metabolism was tested by measuring the rate of incorporation of (/sup 3/H) myo-inositol, (/sup 3/H) choline and (/sup 14/C) acetate over a 14 hr period. Two significant increases in phospholipid metabolism occur from 30-90 min and from 3-8 hrs. Only the increase in phosphoinositide metabolism was shown to be non-transient. The role of the phospholipase enzymes in the prolactin stimulation of mitogenesis was investigated in the Nb/sub 2/ lymphoma cells.

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

    PubMed Central

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

    2014-01-01

    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

  18. Why increased nuchal translucency is associated with congenital heart disease: a systematic review on genetic mechanisms.

    PubMed

    Burger, Nicole B; Bekker, Mireille N; de Groot, Christianne J M; Christoffels, Vincent M; Haak, Monique C

    2015-06-01

    This overview provides insight into the underlying genetic mechanism of the high incidence of cardiac defects in fetuses with increased nuchal translucency (NT). Nuchal edema, the morphological equivalent of increased NT, is likely to result from abnormal lymphatic development and is strongly related to cardiac defects. The underlying genetic pathways are, however, unknown. This study aims to present a systematic overview of genes involved in both cardiac and lymphatic development in mouse embryos. A search of PubMed and the Mammalian Phenotype Browser was performed. Fifteen candidate genes involved in both cardiac and lymphatic development were identified: Adrenomedullin; Chicken ovalbumin upstream promoter-transcription factor 2 (COUP-TFII); Cyp51; Ephrin-B2; Forkhead box protein C2 (Foxc2); Nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1); Neurofibromatosis type 1 (Nf1); Phosphoinositide 3-kinase encoding isoform p110α (Pik3ca); Podoplanin; Prospero-related homeobox 1 (Prox1); T-box 1 (Tbx1); Tyrosine kinase with immunoglobulin-like and endothelial growth factor-like domains 1 (Tie1); vascular endothelial growth factor (Vegf)-A; Vegf receptor-3 (Vegfr-3); and Vascular endothelial zinc finger 1 (Vezf1). Mutations in all but one gene (Pik3ca) resulted in both a cardiac defect and nuchal edema. Candidate genes - mainly encoding for endothelium - are involved in both cardiac and lymphatic development. Alterations in candidate genes are associated with the strong relation between increased NT and cardiac defects. PMID:25728762

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

    PubMed Central

    Pingret, JL; Journet, EP; Barker, DG

    1998-01-01

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

  20. Mechanical embolectomy.

    PubMed

    Katz, Jeffrey M; Gobin, Y Pierre; Segal, Alan Z; Riina, Howard A

    2005-07-01

    Mechanical embolectomy in acute ischemic stroke employs the use of novel endovascular devices to revascularize occluded intracerebral arteries. Devices like the Merci Retiever and other endovascular snares, laser thrombectomy and rheolytic/obliterative microcatheters, intracranial balloon angioplasty and stenting, and intra-arterial and transcranial ultrasound-enhanced chemical thrombolysis are intended to improve tissue rescue and diminish reperfusion hemorrhage while broadening the population eligible for therapy. Patient selection with MRI- and CT-based stroke protocols can detect tissue at risk and may obviate the classic limitations of the stroke therapeutic time window. These devices are being developed and modified at a rapid pace, requiring mounting endovascular expertise, and are being used successfully alone or in conjunction with chemical thrombolysis with relative safety. PMID:15990038

  1. quantum mechanics

    PubMed Central

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

    2013-01-01

    -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

  2. Ribosome Mechanics Informs about Mechanism.

    PubMed

    Zimmermann, Michael T; Jia, Kejue; Jernigan, Robert L

    2016-02-27

    The essential aspects of the ribosome's mechanism can be extracted from coarse-grained simulations, including the ratchet motion, the movement together of critical bases at the decoding center, and movements of the peptide tunnel lining that assist in the expulsion of the synthesized peptide. Because of its large size, coarse graining helps to simplify and to aid in the understanding of its mechanism. Results presented here utilize coarse-grained elastic network modeling to extract the dynamics, and both RNAs and proteins are coarse grained. We review our previous results, showing the well-known ratchet motions and the motions in the peptide tunnel and in the mRNA tunnel. The motions of the lining of the peptide tunnel appear to assist in the expulsion of the growing peptide chain, and clamps at the ends of the mRNA tunnel with three proteins ensure that the mRNA is held tightly during decoding and essential for the helicase activity at the entrance. The entry clamp may also assist in base recognition to ensure proper selection of the incoming tRNA. The overall precision of the ribosome machine-like motions is remarkable. PMID:26687034

  3. The Rational Design of Selective Benzoxazepin Inhibitors of the α-Isoform of Phosphoinositide 3-Kinase Culminating in the Identification of (S)-2-((2-(1-Isopropyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)oxy)propanamide (GDC-0326).

    PubMed

    Heffron, Timothy P; Heald, Robert A; Ndubaku, Chudi; Wei, BinQing; Augistin, Martin; Do, Steven; Edgar, Kyle; Eigenbrot, Charles; Friedman, Lori; Gancia, Emanuela; Jackson, Philip S; Jones, Graham; Kolesnikov, Aleksander; Lee, Leslie B; Lesnick, John D; Lewis, Cristina; McLean, Neville; Mörtl, Mario; Nonomiya, Jim; Pang, Jodie; Price, Steve; Prior, Wei Wei; Salphati, Laurent; Sideris, Steve; Staben, Steven T; Steinbacher, Stefan; Tsui, Vickie; Wallin, Jeffrey; Sampath, Deepak; Olivero, Alan G

    2016-02-11

    Inhibitors of the class I phosphoinositide 3-kinase (PI3K) isoform PI3Kα have received substantial attention for their potential use in cancer therapy. Despite the particular attraction of targeting PI3Kα, achieving selectivity for the inhibition of this isoform has proved challenging. Herein we report the discovery of inhibitors of PI3Kα that have selectivity over the other class I isoforms and all other kinases tested. In GDC-0032 (3, taselisib), we previously minimized inhibition of PI3Kβ relative to the other class I insoforms. Subsequently, we extended our efforts to identify PI3Kα-specific inhibitors using PI3Kα crystal structures to inform the design of benzoxazepin inhibitors with selectivity for PI3Kα through interactions with a nonconserved residue. Several molecules selective for PI3Kα relative to the other class I isoforms, as well as other kinases, were identified. Optimization of properties related to drug metabolism then culminated in the identification of the clinical candidate GDC-0326 (4). PMID:26741947

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

    PubMed Central

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

    2013-01-01

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

  5. Windmill mechanism

    SciTech Connect

    Yang, W. H.

    1985-07-23

    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.

  6. INDEXING MECHANISM

    DOEpatents

    Kock, L.J.

    1959-09-22

    A device is presented for loading and unloading fuel elements containing material fissionable by neutrons of thermal energy. The device comprises a combination of mechanical features Including a base, a lever pivotally attached to the base, an Indexing plate on the base parallel to the plane of lever rotation and having a plurality of apertures, the apertures being disposed In rows, each aperture having a keyway, an Index pin movably disposed to the plane of lever rotation and having a plurality of apertures, the apertures being disposed in rows, each aperture having a keyway, an index pin movably disposed on the lever normal to the plane rotation, a key on the pin, a sleeve on the lever spaced from and parallel to the index pin, a pair of pulleys and a cable disposed between them, an open collar rotatably attached to the sleeve and linked to one of the pulleys, a pin extending from the collar, and a bearing movably mounted in the sleeve and having at least two longitudinal grooves in the outside surface.

  7. Cyberspatial mechanics.

    PubMed

    Bayne, Jay S

    2008-06-01

    In support of a generalization of systems theory, this paper introduces a new approach in modeling complex distributed systems. It offers an analytic framework for describing the behavior of interactive cyberphysical systems (CPSs), which are networked stationary or mobile information systems responsible for the real-time governance of physical processes whose behaviors unfold in cyberspace. The framework is predicated on a cyberspace-time reference model comprising three spatial dimensions plus time. The spatial domains include geospatial, infospatial, and sociospatial references, the latter describing relationships among sovereign enterprises (rational agents) that choose voluntarily to organize and interoperate for individual and mutual benefit through geospatial (physical) and infospatial (logical) transactions. Of particular relevance to CPSs are notions of timeliness and value, particularly as they relate to the real-time governance of physical processes and engagements with other cooperating CPS. Our overarching interest, as with celestial mechanics, is in the formation and evolution of clusters of cyberspatial objects and the federated systems they form. PMID:18558529

  8. Mechanical Design

    SciTech Connect

    Shook, Richard; /Marquette U. /SLAC

    2010-08-25

    The particle beam of the SXR (soft x-ray) beam line in the LCLS (Linac Coherent Light Source) has a high intensity in order to penetrate through samples at the atomic level. However, the intensity is so high that many experiments fail because of severe damage. To correct this issue, attenuators are put into the beam line to reduce this intensity to a level suitable for experimentation. Attenuation is defined as 'the gradual loss in intensity of any flux through a medium' by [1]. It is found that Beryllium and Boron Carbide can survive the intensity of the beam. At very thin films, both of these materials work very well as filters for reducing the beam intensity. Using a total of 12 filters, the first 9 being made of Beryllium and the rest made of Boron Carbide, the beam's energy range of photons can be attenuated between 800 eV and 9000 eV. The design of the filters allows attenuation for different beam intensities so that experiments can obtain different intensities from the beam if desired. The step of attenuation varies, but is relative to the thickness of the filter as a power function of 2. A relationship for this is f(n) = x{sub 0}2{sup n} where n is the step of attenuation desired and x{sub 0} is the initial thickness of the material. To allow for this desired variation, a mechanism must be designed within the test chamber. This is visualized using a 3D computer aided design modeling tool known as Solid Edge.

  9. Phosphoinositide-specific Phospholipase C β 1b (PI-PLCβ1b) Interactome: Affinity Purification-Mass Spectrometry Analysis of PI-PLCβ1b with Nuclear Protein*

    PubMed Central

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

    2013-01-01

    Two isoforms of inositide-dependent phospholipase C β1 (PI-PLCβ1) are generated by alternative splicing (PLCβ1a and PLCβ1b). Both isoforms are present within the nucleus, but in contrast to PLCβ1a, the vast majority of PLCβ1b is nuclear. In mouse erythroid leukemia cells, PI-PLCβ1 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-PLCβ1, 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-PLCβ1 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-PLCβ1b, 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

  10. EGF raises cytosolic Ca sup 2+ in A431 and Swiss 3T3 cells by a dual mechanism

    SciTech Connect

    Pandiella, A.; Malgaroli, A.; Meldolesi, J.; Vicentini, L.M. )

    1987-05-01

    The changes in Ca{sup 2+} homeostasis and phosphoinositide hydrolysis induced by EGF were studied in human epidermoid carcinoma A431 cells both when attached to a substratum and after detachment and suspension. The cytosolic Ca{sup 2+} concentration was measured by the conventional fluorimetric technique, using the specific probe, quin2, as well as by a new microscopic technique in which single cells are investigated after loading with another probe, fura-2. EGF applied in the complete, Ca{sup 2+}-containing medium caused a rapid rise in the cytosolic {sup 45}Ca{sup 2+} concentration, that remained elevated for several minutes. In Ca{sup 2+}-free, EGTA-containing medium, part of this response persisted, as revealed by quin2 results in suspended cells and microscopic results with fura-2. These results, as well as additional microscopic fura-2 results in Swiss 3T3 fibroblasts, demonstrate that the Ca{sup 2+} signal elicited by EGF is due to two components: redistribution from an intracellular store and stimulated influx across the plasmalemma. This latter process was not detected in 3T3 cells treated with either PDGF or bombesin. It is therefore suggested that the {sup 45}Ca{sup 2+} influx effect of EGF is under the control of a separate, as yet unidentified mechanism.

  11. Cellular and molecular mechanisms of HGF/Met in the cardiovascular system.

    PubMed

    Gallo, Simona; Sala, Valentina; Gatti, Stefano; Crepaldi, Tiziana

    2015-12-01

    Met tyrosine kinase receptor, also known as c-Met, is the HGF (hepatocyte growth factor) receptor. The HGF/Met pathway has a prominent role in cardiovascular remodelling after tissue injury. The present review provides a synopsis of the cellular and molecular mechanisms underlying the effects of HGF/Met in the heart and blood vessels. In vivo, HGF/Met function is particularly important for the protection of the heart in response to both acute and chronic insults, including ischaemic injury and doxorubicin-induced cardiotoxicity. Accordingly, conditional deletion of Met in cardiomyocytes results in impaired organ defence against oxidative stress. After ischaemic injury, activation of Met provides strong anti-apoptotic stimuli for cardiomyocytes through PI3K (phosphoinositide 3-kinase)/Akt and MAPK (mitogen-activated protein kinase) cascades. Recently, we found that HGF/Met is also important for autophagy regulation in cardiomyocytes via the mTOR (mammalian target of rapamycin) pathway. HGF/Met induces proliferation and migration of endothelial cells through Rac1 (Ras-related C3 botulinum toxin substrate 1) activation. In fibroblasts, HGF/Met antagonizes the actions of TGFβ1 (transforming growth factor β1) and AngII (angiotensin II), thus preventing fibrosis. Moreover, HGF/Met influences the inflammatory response of macrophages and the immune response of dendritic cells, indicating its protective function against atherosclerotic and autoimmune diseases. The HGF/Met axis also plays an important role in regulating self-renewal and myocardial regeneration through the enhancement of cardiac progenitor cells. HGF/Met has beneficial effects against myocardial infarction and endothelial dysfunction: the cellular and molecular mechanisms underlying repair function in the heart and blood vessels are common and include pro-angiogenic, anti-inflammatory and anti-fibrotic actions. Thus administration of HGF or HGF mimetics may represent a promising therapeutic agent for the treatment of both coronary and peripheral artery disease. PMID:26561593

  12. Signal transduction mechanism of biased ligands at histamine H2 receptors.

    PubMed

    Alonso, Natalia; Monczor, Federico; Echeverría, Emiliana; Davio, Carlos; Shayo, Carina; Fernández, Natalia

    2014-04-01

    7TMRs (seven-transmembrane receptors) exist as conformational collections in which different conformations would lead to differential downstream behaviours such as receptor phosphorylation, G-protein activation and receptor internalization. In this context, a ligand may cause differential activation of some, but not all, of the signalling events, which are associated to a particular receptor, and it would lead to biased agonism. The aim of the present study was to investigate whether H2R (histamine H2 receptor) ligands, described as inverse agonists because of their negative efficacy at modulating adenylate cyclase, could display some positive efficacy concerning receptor desensitization, internalization or even signalling through an adenylate-cyclase-independent pathway. Our present findings indicate that treatment with H2R inverse agonists leads to receptor internalization in HEK (human embryonic kidney)-293T transfected cells, by a mechanism mediated by arrestin and dynamin, but independent of GRK2 (G-protein-coupled receptor kinase 2)-mediated phosphorylation. On the other hand, we prove that two of the H2R inverse agonists tested, ranitidine and tiotidine, also induce receptor desensitization. Finally, we show that these ligands are able to display positive efficacy towards the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway by a mechanism that involves Gβγ and PI3K (phosphoinositide 3-kinase)-mediated signalling in both transfected HEK-293T cells and human gastric adenocarcinoma cells. These results point to the aspect of pluridimensional efficacy at H2R as a phenomenon that could be extended to naïve cells, and challenge previous classification of pharmacologically relevant histaminergic ligands. PMID:24417223

  13. 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

    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

    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 γ (PPARγ), CCAAT/enhancer binding protein (C/EBP)α and C/EBPβ, 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 PPARγ 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 PPARγ, C/EBPα and C/EBPβ, 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

  14. Reusable Mechanical Pin Puller

    NASA Technical Reports Server (NTRS)

    Ngo, Son; Farley, Rodger; Devine, ED

    1991-01-01

    Reusable mechanical pin puller relatively simple spring-loaded trigger mechanism. Designed to save money and increase safety as substitute for costly and potentially dangerous pyrotechnic pin pullers used in development and testing of deployment mechanisms.

  15. Mechanical systems: A compilation

    NASA Technical Reports Server (NTRS)

    1975-01-01

    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.

  16. Distinct Dasatinib-Induced Mechanisms of Apoptotic Response and Exosome Release in Imatinib-Resistant Human Chronic Myeloid Leukemia Cells

    PubMed Central

    Liu, Juan; Zhang, Yujing; Liu, Aichun; Wang, Jinghua; Li, Lianqiao; Chen, Xi; Gao, Xinyu; Xue, Yanming; Zhang, Xiaomin; Liu, Yao

    2016-01-01

    Although dasatinib is effective in most imatinib mesylate (IMT)-resistant chronic myeloid leukemia (CML) patients, the underlying mechanism of its effectiveness in eliminating imatinib-resistant cells is only partially understood. This study investigated the effects of dasatinib on signaling mechanisms driving-resistance in imatinib-resistant CML cell line K562 (K562RIMT). Compared with K562 control cells, exsomal release, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling and autophagic activity were increased significantly in K562RIMT cells and mTOR-independent beclin-1/Vps34 signaling was shown to be involved in exosomal release in these cells. We found that Notch1 activation-mediated reduction of phosphatase and tensin homolog (PTEN) was responsible for the increased Akt/mTOR activities in K562RIMT cells and treatment with Notch1 γ-secretase inhibitor prevented activation of Akt/mTOR. In addition, suppression of mTOR activity by rapamycin decreased the level of activity of p70S6K, induced upregulation of p53 and caspase 3, and led to increase of apoptosis in K562RIMT cells. Inhibition of autophagy by spautin-1 or beclin-1 knockdown decreased exosomal release, but did not affect apoptosis in K562RIMT cells. In summary, in K562RIMT cells dasatinib promoted apoptosis through downregulation of Akt/mTOR activities, while preventing exosomal release and inhibiting autophagy by downregulating expression of beclin-1 and Vps34. Our findings reveal distinct dasatinib-induced mechanisms of apoptotic response and exosomal release in imatinib-resistant CML cells. PMID:27070592

  17. Distinct Dasatinib-Induced Mechanisms of Apoptotic Response and Exosome Release in Imatinib-Resistant Human Chronic Myeloid Leukemia Cells.

    PubMed

    Liu, Juan; Zhang, Yujing; Liu, Aichun; Wang, Jinghua; Li, Lianqiao; Chen, Xi; Gao, Xinyu; Xue, Yanming; Zhang, Xiaomin; Liu, Yao

    2016-01-01

    Although dasatinib is effective in most imatinib mesylate (IMT)-resistant chronic myeloid leukemia (CML) patients, the underlying mechanism of its effectiveness in eliminating imatinib-resistant cells is only partially understood. This study investigated the effects of dasatinib on signaling mechanisms driving-resistance in imatinib-resistant CML cell line K562 (K562R(IMT)). Compared with K562 control cells, exsomal release, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling and autophagic activity were increased significantly in K562R(IMT) cells and mTOR-independent beclin-1/Vps34 signaling was shown to be involved in exosomal release in these cells. We found that Notch1 activation-mediated reduction of phosphatase and tensin homolog (PTEN) was responsible for the increased Akt/mTOR activities in K562R(IMT) cells and treatment with Notch1 γ-secretase inhibitor prevented activation of Akt/mTOR. In addition, suppression of mTOR activity by rapamycin decreased the level of activity of p70S6K, induced upregulation of p53 and caspase 3, and led to increase of apoptosis in K562R(IMT) cells. Inhibition of autophagy by spautin-1 or beclin-1 knockdown decreased exosomal release, but did not affect apoptosis in K562R(IMT) cells. In summary, in K562R(IMT) cells dasatinib promoted apoptosis through downregulation of Akt/mTOR activities, while preventing exosomal release and inhibiting autophagy by downregulating expression of beclin-1 and Vps34. Our findings reveal distinct dasatinib-induced mechanisms of apoptotic response and exosomal release in imatinib-resistant CML cells. PMID:27070592

  18. Molecular mechanism of apoptosis induction in skin cancer cells by the centipedegrass extract

    PubMed Central

    2013-01-01

    Background Centipedegrass extract (CGE) is mainly composed of maysin and its derivatives, which are recognized internationally as natural compounds. Compared to other flavonoids, maysin has a unique structure in that mannose is bound to the flavonoid backbone. CGE exhibits some biological properties in that it can function as an anti-oxidant, anti-inflammatory, anti-adipogenic, and insecticidal. Whether CGE has other biological functions, such as anti-cancer activity, is unknown. Methods B16F1 (mouse) and SKMEL-5 (human) cells were treated with CGE, and their subsequent survival was determined using MTT assay. We performed a cell cycle analysis using propidium iodide (PI), and detected apoptosis using double staining with annexin V-FITC/PI. In addition, we examined mitochondrial membrane potentials using flow cytometry, as well as signaling mechanisms with an immunoblotting analysis. Results CGE inhibited skin cancer cell growth by arresting the cell cycle in the G2/M phase, and increased both early and late apoptotic cell populations without affecting normal cells. Furthermore, we observed mitochondrial transmembrane depolarization, increased cytochrome-c release, caspase-3 and caspase-7 activation, and increased poly ADP-ribose polymerase degradation. CGE also downregulated activation of p-AKT, p-glycogen synthase kinase-3β (GSK-3β), and p-BAD in a time-dependent manner. LY294002 inhibition of phosphoinositide 3-kinase (PI3K) significantly sensitized skin cancer cells, which led to an increase in CGE-induced apoptosis. Conclusions CGE controlled skin cancer cell growth by inhibiting the PI3K/AKT/GSK-3β signaling pathway and activating the effector caspases. This study is the first to demonstrate anti-cancer properties for CGE, and that CGE may be an effective therapeutic agent for treating skin cancer. PMID:24325618

  19. Statistical mechanics of distributed resource sharing mechanisms

    SciTech Connect

    Yemini, Y.

    1983-01-01

    The problem of analyzing the performance of large-scale interacting distributed resource sharing mechanisms arises in packet broadcast networks as well as multiprocessor switching mechanisms, VLSI chip communications and distributed databases. Queueing theory has major limitations in analyzing such systems. This paper proposes an alternative approach to the problem based upon an analogy with statistical mechanics. Using this approach it is possible to analyze the performance of intricate distributed resource sharing mechanisms with a relative ease. Moreover, the analogy to physical phenomena offers new performance measures and physical insights to the behavior of such systems. 20 references.

  20. Defense Mechanisms: A Bibliography.

    ERIC Educational Resources Information Center

    Pedrini, D. T.; Pedrini, Bonnie C.

    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.…

  1. Internal pipe attachment mechanism

    DOEpatents

    Bast, Richard M.; Chesnut, Dwayne A.; Henning, Carl D.; Lennon, Joseph P.; Pastrnak, John W.; Smith, Joseph A.

    1994-01-01

    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.

  2. Internal pipe attachment mechanism

    DOEpatents

    Bast, R.M.; Chesnut, D.A.; Henning, C.D.; Lennon, J.P.; Pastrnak, J.W.; Smith, J.A.

    1994-12-13

    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.

  3. Central neural mechanisms governing postural cardiovascular mechanisms

    NASA Technical Reports Server (NTRS)

    Reis, D. J.

    1976-01-01

    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.

  4. Phosphoinositide metabolism and metabolism-contraction coupling in rabbit aorta

    SciTech Connect

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

    1988-12-01

    The authors tested a hypothesis that metabolism-contraction coupling in vascular smooth muscle is controlled by the rate of delivery of energy to ATP-dependent reactions in the inositol phospholipid transduction system that generate second messengers exerting control on smooth muscle force. Rabbit aorta was contracted by norepinephrine (NOR) under conditions of normoxia and hypoxia, and changes in inositol phospholipid pool sizes and metabolic flux rates (J{sub F}) were determined. J{sub F} was determined by labeling free cytosolic myo-inositol by incubation of unstimulated muscle with myo-({sup 3}H)inositol and then measuring rates of incorporation of this isotope into inositol phospholipids and inositol phosphates when the muscle was activated by NOR. J{sub F} measured during maintenance of NOR-induced force was markedly inhibited during hypoxia to 40-50% of that determined during normoxia; rates of increases in inositol phosphate radioactivities were similarly depressed during NOR activation under hypoxia. The hypoxia-induced decrease in J{sub F} was associated with four- to fivefold increase in phosphatidylinositol 4-phosphate (PIP) total pool size, suggesting PIP kinase was inhibited and rate limiting. These data suggest that activation of inositol phospholipid metabolism, which generates inositol 1,4,5-trisphosphate (IP{sub 3}) and diacylglycerol, is blunted under conditions where aerobic energy production is inhibited. Data are consistent with rate-limiting effects of decreased ATP delivery, or decreased phosphate potential, on PIP kinase and reactions that control resynthesis of phosphatidylinositol.

  5. Analysis of plasma membrane phosphoinositides from fusogenic carrot cells

    SciTech Connect

    Wheeler, J.J.; Boss, W.F.

    1987-04-01

    Phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP/sub 2/) were found to be associated with the plasma membrane-rich fractions isolated by aqueous polymer two-phase partitioning from fusogenic cells. They represented at least 5% and 0.7% of the total inositol-labeled lipids in the plasma membrane-rich fractions, respectively, and were present in a ratio of about 7:1 (PIP:PIP/sub 2/). In addition, two unidentified inositol-labeled compounds, which together were approximately 3% of the inositol-labeled lipids, were found predominantly in the plasma membrane-rich fractions and migrated between PIP/sub 2/ and PIP. The R/sub f/s of these compounds were approximately 0.31 and 0.34 in the solvent system CHCl/sub 3/:MeOH:15N NH/sub 4/OH:H/sub 2/O (90:90:7:22) using LK5 plates presoaked in 1% potassium oxalate. These compounds incorporated /sup 32/P/sub i/, (/sup 3/H)inositol and were hydrolyzed in mild base. These data suggested that they were glycero-phospholipids. Although the compounds did not comigrate with lysoPIP obtained from bovine brain (R/sub f/ approx. 0.35), when endogenous PIP was hydrolyzed to lysoPIP, the breakdown product migrated in the region of the unidentified inositol lipids.

  6. Ninteenth Aerospace Mechanisms Symposium

    NASA Technical Reports Server (NTRS)

    1985-01-01

    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.

  7. Mechanical Properties of Polymers.

    ERIC Educational Resources Information Center

    Aklonis, J. J.

    1981-01-01

    Mechanical properties (stress-strain relationships) of polymers are reviewed, taking into account both time and temperature factors. Topics include modulus-temperature behavior of polymers, time dependence, time-temperature correspondence, and mechanical models. (JN)

  8. Learning in quantum mechanics

    NASA Astrophysics Data System (ADS)

    Gardner, David E.

    This thesis describes qualitative research conducted to understand the problems students have when learning quantum mechanics. It differs from previous studies on educational issues associated with quantum mechanics in that I have examined the difficulties from the students' perspective. Three questions guided this research: What are the experiences of students learning quantum mechanics? What conceptual difficulties do students have with quantum mechanics? and, How do students approach learning quantum mechanics? From these questions, two themes emerged. First, students do not consider the quantum mechanical concepts of wave-particle duality or the uncertainty principle to be important sources of difficulties for them. Second, many of the difficulties students encounter are not related to conceptual understanding of specific topics, but stem from a mindset that is incongruent with the nature and structure of quantum mechanics. The implications for teaching are that the nature and structure of quantum mechanics should be emphasized and be an explicit part of instruction.

  9. Missing Mechanism Information

    ERIC Educational Resources Information Center

    Tryon, Warren W.

    2009-01-01

    The first recommendation Kazdin made for advancing the psychotherapy research knowledge base, improving patient care, and reducing the gulf between research and practice was to study the mechanisms of therapeutic change. He noted, "The study of mechanisms of change has received the least attention even though understanding mechanisms may well be…

  10. Mechanical Engineering Technology Curriculum.

    ERIC Educational Resources Information Center

    Georgia State Univ., Atlanta. Dept. of Vocational and Career Development.

    This guide offers information and procedures necessary to train mechanical engineering technicians. Discussed first are the rationale and objectives of the curriculum. The occupational field of mechanical engineering technology is described. Next, a curriculum model is set forth that contains information on the standard mechanical engineering…

  11. Solar array deployment mechanism

    NASA Technical Reports Server (NTRS)

    Calassa, Mark C.; Kackley, Russell

    1995-01-01

    This paper describes a Solar Array Deployment Mechanism (SADM) used to deploy a rigid solar array panel on a commercial spacecraft. The application required a deployment mechanism design that was not only lightweight, but also could be produced and installed at the lowest possible cost. This paper covers design, test, and analysis of a mechanism that meets these requirements.

  12. Basic Engineer Equipment Mechanic.

    ERIC Educational Resources Information Center

    Marine Corps Inst., Washington, DC.

    This student guide, one of a series of correspondence training courses designed to improve the job performance of members of the Marine Corps, deals with the skills needed by basic engineer equipment mechanics. Addressed in the four individual units of the course are the following topics: mechanics and their tools (mechanics, hand tools, and power…

  13. HYDRAULIC SERVO CONTROL MECHANISM

    DOEpatents

    Hussey, R.B.; Gottsche, M.J. Jr.

    1963-09-17

    A hydraulic servo control mechanism of compact construction and low fluid requirements is described. The mechanism consists of a main hydraulic piston, comprising the drive output, which is connected mechanically for feedback purposes to a servo control piston. A control sleeve having control slots for the system encloses the servo piston, which acts to cover or uncover the slots as a means of controlling the operation of the system. This operation permits only a small amount of fluid to regulate the operation of the mechanism, which, as a result, is compact and relatively light. This mechanism is particuiarly adaptable to the drive and control of control rods in nuclear reactors. (auth)

  14. Phospholipase A2 is involved in the mechanism of activation of neutrophils by polychlorinated biphenyls.

    PubMed Central

    Tithof, P K; Schiamberg, E; Peters-Golden, M; Ganey, P E

    1996-01-01

    Aroclor 1242, a mixture of polychlorinated biphenyls (PCBs), activates neutrophils to produce superoxide anion (O2-) by a mechanism that involves phospholipase C-dependent hydrolysis of membrane phosphoinositides; however, subsequent signal transduction mechanisms are unknown. We undertook this study to determine whether phospholipase A2-dependent release of arachidonic acid is involved in PCB-induced O2- production. We measured O2- production in vitro in glycogen-elicited, rat neutrophils in the presence and absence of the inhibitors of phospholipase A2: quinacrine, 4-bromophenacyl bromide (BPB), and manoalide. All three agents significantly decreased the amount of O2- 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, although data suggest that quinacrine is an O2- scavenger. Significant release of 3H-arachidonic acid preceded O2- production in neutrophils stimulated with Aroclor 1242 or fMLP. Manoalide, at a concentration that abolished O2- production, also inhibited the release of 3H-arachidonate. Aspirin, zileuton, or WEB 2086 did not affect Aroclor 1242-induced O2- production, suggesting that eicosanoids and platelet-activating factor are not needed for neutrophil activation by PCBs. Activation of phospholipase A2 and O2- production do not appear to involve the Ah receptor because a congener with low affinity, but not one with high affinity for this receptor, stimulated the release of arachidonic acid and O2-. These data suggest that Aroclor 1242 stimulates neutrophils to produce O2- by a mechanism that involves phospholipase A2-dependent release of arachidonic acid. Images Figure 1. A Figure 1. B Figure 2. Figure 3. Figure 4. A Figure 4. B Figure 4. C Figure 5. A Figure 5. B Figure 5. C Figure 6. A Figure 6. B Figure 6. C Figure 6. D PMID:8834862

  15. Mechanical seal assembly

    DOEpatents

    Kotlyar, Oleg M.

    2002-01-01

    An improved mechanical seal assembly is provided for sealing rotating shafts with respect to their shaft housings, wherein the rotating shafts are subject to substantial axial vibrations. The mechanical seal assembly generally includes a rotating sealing ring fixed to the shaft, a non-rotating sealing ring adjacent to and in close contact with the rotating sealing ring for forming an annular seal about the shaft, and a mechanical diode element that applies a biasing force to the non-rotating sealing ring by means of hemispherical joint. The alignment of the mechanical diode with respect to the sealing rings is maintained by a series of linear bearings positioned axially along a desired length of the mechanical diode. Alternative embodiments include mechanical or hydraulic amplification components for amplifying axial displacement of the non-rotating sealing ring and transfering it to the mechanical diode.

  16. Mechanical seal assembly

    DOEpatents

    Kotlyar, Oleg M.

    2001-01-01

    An improved mechanical seal assembly is provided for sealing rotating shafts with respect to their shaft housings, wherein the rotating shafts are subject to substantial axial vibrations. The mechanical seal assembly generally includes a rotating sealing ring fixed to the shaft, a non-rotating sealing ring adjacent to and in close contact with the rotating sealing ring for forming an annular seal about the shaft, and a mechanical diode element that applies a biasing force to the non-rotating sealing ring by means of hemispherical joint. The alignment of the mechanical diode with respect to the sealing rings is maintained by a series of linear bearings positioned axially along a desired length of the mechanical diode. Alternative embodiments include mechanical or hydraulic amplification components for amplifying axial displacement of the non-rotating sealing ring and transferring it to the mechanical diode.

  17. GFP's Mechanical Intermediate States

    PubMed Central

    Saeger, John; Hytönen, Vesa P.; Klotzsch, Enrico; Vogel, Viola

    2012-01-01

    Green fluorescent protein (GFP) mutants have become the most widely used fluorescence markers in the life sciences, and although they are becoming increasingly popular as mechanical force or strain probes, there is little direct information on how their fluorescence changes when mechanically stretched. Here we derive high-resolution structural models of the mechanical intermediate states of stretched GFP using steered molecular dynamics (SMD) simulations. These structures were used to produce mutants of EGFP and EYFP that mimic GFP's different mechanical intermediates. A spectroscopic analysis revealed that a population of EGFP molecules with a missing N-terminal α-helix was significantly dimmed, while the fluorescence lifetime characteristic of the anionic chromophore state remained unaffected. This suggests a mechanism how N-terminal deletions can switch the protonation state of the chromophore, and how the fluorescence of GFP molecules in response to mechanical disturbance might be turned off. PMID:23118864

  18. Mechanism of enzymatic reaction and protein-protein interactions of PLD from a 3D structural model.

    PubMed

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

    2015-01-01

    The phospholipase D (PLD) superfamily catalyzes the hydrolysis of cell membrane phospholipids generating the key intracellular lipid second messenger phosphatidic acid. However, there is not yet any resolved structure either from a crystallized protein or from NMR of any mammalian PLDs. We propose here a 3D model of the PLD2 by combining homology and ab initio 3 dimensional structural modeling methods, and docking conformation. This model is in agreement with the biochemical and physiological behavior of PLD in cells. For the lipase activity, the N- and C-terminal histidines of the HKD motifs (His 442/His 756) form a catalytic pocket, which accommodates phosphatidylcholine head group (but not phosphatidylethanolamine or phosphatidyl serine). The model explains the mechanism of the reaction catalysis, with nucleophilic attacks of His 442 and water, the latter aided by His 756. Further, the secondary structure regions superimposed with bacterial PLD crystal structure, which indicated an agreement with the model. It also explains protein-protein interactions, such as PLD2-Rac2 transmodulation (with a 1:2 stoichiometry) and PLD2 GEF activity both relevant for cell migration, as well as the existence of binding sites for phosphoinositides such as PIP2. These consist of R236/W238 and R557/W563 and a novel PIP2 binding site in the PH domain of PLD2, specifically R210/R212/W233. In each of these, the polar inositol ring is oriented towards the basic amino acid Arginine. Since tumor-aggravating properties have been found in mice overexpressing PLD2 enzyme, the 3D model of PLD2 will be also useful, to a large extent, in developing pharmaceuticals to modulate its in vivo activity. PMID:25308783

  19. The mechanism of fracture

    SciTech Connect

    Goel, V.S.

    1985-01-01

    This book presents the papers given at a conference on the fracture mechanics of metals. Topics considered at the conference included microcrack mechanics, pressurized thermal shock behavior of LWR pressure vessels, stress intensity factors, submerged arc welding, weldments in power plants, pipeline weld quality, natural gas tanks, cast iron for spent nuclear fuel shipping casks, pipe ruptures, physical radiation effects, pressure tubes, hydrogen embrittlement, critical flaw size curves, and the fracture mechanics of steels in turbines of power stations.

  20. Mechanical devices: A compilation

    NASA Technical Reports Server (NTRS)

    1976-01-01

    A collection of new technology items that should be of interest to mechanical engineers, machinists, and others who design or work with mechanical devices was described. Section 1 contains articles on several new or modified tools, Section 2 describes a number of specialized mechanical systems, and the last section is devoted to valves, bearings, and other parts that might be used with larger systems. The last patent information available is also given.

  1. Space Mechanisms Technology Workshop

    NASA Technical Reports Server (NTRS)

    Oswald, Fred B. (Editor)

    2002-01-01

    The Mechanical Components Branch at NASA Glenn Research Center hosted a workshop on Tuesday, May 14, 2002, to discuss space mechanisms technology. The theme for this workshop was 'Working in the Cold,' a focus on space mechanisms that must operate at low temperatures. We define 'cold' as below -60C (210 K), such as would be found near the equator of Mars. However, we are also concerned with much colder temperatures such as in permanently dark craters of the Moon (about 40 K).

  2. Vehicular transmission shift mechanism

    SciTech Connect

    Okubo, K.

    1988-12-27

    This patent describes a vehicular transmission having a main speed change mechanism and a sub speed change mechanism both housed within a transmission case. The main speed change mechanism has gear trains in plural shifting stages provided between input and output shafts and capable of being established selectively and also has a plurality of synchronizing mechanisms operable by shift forks to establish the gear trains selectively. The sub speed change mechanism has a reduction gear train for obtaining a still lower speed than the lowest shifting stage in the main speed change mechanism and also has a sub speed gear shifting synchronizing mechanism operable by a sub speed gear shift fork to establish the reduction gear train. This sub speed change mechanism is disposed in parallel with the main speed change mechanism, the sub speed gear shift fork having means connected to one end of a lever pivotably supported at an intermediate part thereof by a pivot pin, the pivot pin being mounted on the transmission case from the outside thereof.

  3. Mechanical processes in biochemistry.

    PubMed

    Bustamante, Carlos; Chemla, Yann R; Forde, Nancy R; Izhaky, David

    2004-01-01

    Mechanical processes are involved in nearly every facet of the cell cycle. Mechanical forces are generated in the cell during processes as diverse as chromosomal segregation, replication, transcription, translation, translocation of proteins across membranes, cell locomotion, and catalyzed protein and nucleic acid folding and unfolding, among others. Because force is a product of all these reactions, biochemists are beginning to directly apply external forces to these processes to alter the extent or even the fate of these reactions hoping to reveal their underlying molecular mechanisms. This review provides the conceptual framework to understand the role of mechanical force in biochemistry. PMID:15189157

  4. Potential mechanisms of action of lithium in bipolar disorder. Current understanding.

    PubMed

    Malhi, Gin S; Tanious, Michelle; Das, Pritha; Coulston, Carissa M; Berk, Michael

    2013-02-01

    Lithium has been used for over half a century for the treatment of bipolar disorder as the archetypal mood stabilizer, and has a wealth of empirical evidence supporting its efficacy in this role. Despite this, the specific mechanisms by which lithium exerts its mood-stabilizing effects are not well understood. Given the inherently complex nature of the pathophysiology of bipolar disorder, this paper aims to capture what is known about the actions of lithium ranging from macroscopic changes in mood, cognition and brain structure, to its effects at the microscopic level on neurotransmission and intracellular and molecular pathways. A comprehensive literature search of databases including MEDLINE, EMBASE and PsycINFO was conducted using relevant keywords and the findings from the literature were then reviewed and synthesized. Numerous studies report that lithium is effective in the treatment of acute mania and for the long-term maintenance of mood and prophylaxis; in comparison, evidence for its efficacy in depression is modest. However, lithium possesses unique anti-suicidal properties that set it apart from other agents. With respect to cognition, studies suggest that lithium may reduce cognitive decline in patients; however, these findings require further investigation using both neuropsychological and functional neuroimaging probes. Interestingly, lithium appears to preserve or increase the volume of brain structures involved in emotional regulation such as the prefrontal cortex, hippocampus and amygdala, possibly reflecting its neuroprotective effects. At a neuronal level, lithium reduces excitatory (dopamine and glutamate) but increases inhibitory (GABA) neurotransmission; however, these broad effects are underpinned by complex neurotransmitter systems that strive to achieve homeostasis by way of compensatory changes. For example, at an intracellular and molecular level, lithium targets second-messenger systems that further modulate neurotransmission. For instance, the effects of lithium on the adenyl cyclase and phospho-inositide pathways, as well as protein kinase C, may serve to dampen excessive excitatory neurotransmission. In addition to these many putative mechanisms, it has also been proposed that the neuroprotective effects of lithium are key to its therapeutic actions. In this regard, lithium has been shown to reduce the oxidative stress that occurs with multiple episodes of mania and depression. Further, it increases protective proteins such as brain-derived neurotrophic factor and B-cell lymphoma 2, and reduces apoptotic processes through inhibition of glycogen synthase kinase 3 and autophagy. Overall, it is clear that the processes which underpin the therapeutic actions of lithium are sophisticated and most likely inter-related. PMID:23371914

  5. The Mechanization of Work.

    ERIC Educational Resources Information Center

    Ginzberg, Eli

    1982-01-01

    Discusses how mechanization of work has been treated by economists, what its effect has been on the past U.S. economy, and what its future effect is likely to be. Emphasizes the impact of mechanization on the shifting structure and character of the labor force and evolution of the work environment. (Author/JN)

  6. Engineer Equipment Mechanic.

    ERIC Educational Resources Information Center

    Marine Corps Inst., Washington, DC.

    Developed as part of the Marine Corps Institute (MCI) correspondence training program, this course on engineer equipment mechanics is designed to advance the professional competence of privates through sergeants as equipment mechanics, Military Occupation Specialty 1341, and is adaptable for nonmilitary instruction. Introductory materials include…

  7. INJECTION WELL MECHANICAL INTEGRITY

    EPA Science Inventory

    EPA's underground injection control regulations require that all injection wells demonstrate mechanical integrity. The regulations state that an injection well has mechanical integrity if (1) there is no significant leak in the casing, tubing or packer and (2) there is no signifi...

  8. Thermally responsive mechanical actuator

    NASA Technical Reports Server (NTRS)

    Madey, J. M.

    1973-01-01

    Device built for use in heat control, heat measurement, and mechanical actuation by heat include thermometers, thermostats, safety switches, circuit breakers, and mechanical actuators. Silicon rubber has highest coefficient of expansion of any known material and seems suitable for most of these devices.

  9. The Clementine mechanisms

    NASA Technical Reports Server (NTRS)

    Purdy, William; Hurley, Michael

    1995-01-01

    The Clementine spacecraft was developed under the 'faster, better, cheaper' theme. The constraints of a low budget coupled with an unusually tight schedule forced many departures from the normal spacecraft development methods. This paper discusses technical lessons learned about several of the mechanisms on the Clementine spacecraft as well as managerial lessons learned for the entire mechanisms subsystem. A quick overview of the Clementine mission is included; the mission schedule and environment during the mechanisms releases and deployment are highlighted. This paper then describes the entire mechanisms subsystem. The design and test approach and key philosophies for a fast-track program are discussed during the description of the mechanisms subsystem. The mechanism subsystem included a marman clamp separation system, a separation nut separation system, a solar panel deployment and pointing system, a high gain antenna feed deployment system, and two separate sensor cover systems. Each mechanism is briefly discussed. Additional technical discussion is given on the marman clamp design, the sensor cover designs, and the design and testing practices for systems driven by heated actuators (specifically paraffin actuators and frangibolts). All of the other mechanisms were of conventional designs and will receive less emphasis. Lessons learned are discussed throughout the paper as they applied to the systems being discussed. Since there is information on many different systems, this paper is organized so that information on a particular topic can be quickly referenced.

  10. Schwinger mechanism revisited

    NASA Astrophysics Data System (ADS)

    Gelis, François; Tanji, Naoto

    2016-03-01

    In this article, we review recent theoretical works on the Schwinger mechanism of particle production in external electrical fields. Although the non-perturbative Schwinger mechanism is at the center of this discussion, many of the approaches that we discuss can cope with general time and space dependent fields, and therefore also capture the perturbative contributions to particle production.

  11. Ann Wagner, Mechanical Engineer.

    ERIC Educational Resources Information Center

    Bennett, Betsy K.

    1996-01-01

    Presents a profile of Ann Wagner, a mechanical engineer at the Goddard Space Flight Center in Maryland, and her job responsibilities there. Also includes a brief history of mechanical engineering as well as a sample graph and data activity sheet with answers. (AIM)

  12. Safety Critical Mechanisms

    NASA Technical Reports Server (NTRS)

    Robertson, Brandan

    2008-01-01

    Spaceflight mechanisms have a reputation for being difficult to develop and operate successfully. This reputation is well earned. Many circumstances conspire to make this so: the environments in which the mechanisms are used are extremely severe, there is usually limited or no maintenance opportunity available during operation due to this environment, the environments are difficult to replicate accurately on the ground, the expense of the mechanism development makes it impractical to build and test many units for long periods of time before use, mechanisms tend to be highly specialized and not prone to interchangeability or off-the-shelf use, they can generate and store a lot of energy, and the nature of mechanisms themselves, as a combination of structures, electronics, etc. designed to accomplish specific dynamic performance, makes them very complex and subject to many unpredictable interactions of many types. In addition to their complexities, mechanism are often counted upon to provide critical vehicle functions that can result in catastrophic events should the functions not be performed. It is for this reason that mechanisms are frequently subjected to special scrutiny in safety processes. However, a failure tolerant approach, along with good design and development practices and detailed design reviews, can be developed to allow such notoriously troublesome mechanisms to be utilized confidently in safety-critical applications.

  13. The resupply interface mechanism

    NASA Technical Reports Server (NTRS)

    Gorin, Barney F.

    1990-01-01

    Viewgraphs on the resupply interface mechanism are presented. The major servicing activities for any major asset are normal maintenance, consumable replenishment, repair of failed components, and preplanned product improvement. Spacecraft propulsion system resupply can be divided into three major issues: design for servicing, servicing technology, and mechanization.

  14. Mechanics: Statics; A Syllabus.

    ERIC Educational Resources Information Center

    Compo, Louis

    The instructor's guide presents material for structuring an engineering fundamentals course covering the basic laws of statistics as part of a mechanical technology program. Detailed behavioral objectives are described for the following five areas of course content: principles of mechanics, two-dimensional equilibrium, equilibrium of internal

  15. The Clementine mechanisms

    NASA Astrophysics Data System (ADS)

    Purdy, William; Hurley, Michael

    1995-05-01

    The Clementine spacecraft was developed under the 'faster, better, cheaper' theme. The constraints of a low budget coupled with an unusually tight schedule forced many departures from the normal spacecraft development methods. This paper discusses technical lessons learned about several of the mechanisms on the Clementine spacecraft as well as managerial lessons learned for the entire mechanisms subsystem. A quick overview of the Clementine mission is included; the mission schedule and environment during the mechanisms releases and deployment are highlighted. This paper then describes the entire mechanisms subsystem. The design and test approach and key philosophies for a fast-track program are discussed during the description of the mechanisms subsystem. The mechanism subsystem included a marman clamp separation system, a separation nut separation system, a solar panel deployment and pointing system, a high gain antenna feed deployment system, and two separate sensor cover systems. Each mechanism is briefly discussed. Additional technical discussion is given on the marman clamp design, the sensor cover designs, and the design and testing practices for systems driven by heated actuators (specifically paraffin actuators and frangibolts). All of the other mechanisms were of conventional designs and will receive less emphasis. Lessons learned are discussed throughout the paper as they applied to the systems being discussed. Since there is information on many different systems, this paper is organized so that information on a particular topic can be quickly referenced.

  16. Goryachkin's agricultural mechanics

    NASA Astrophysics Data System (ADS)

    Chinenova, Vera

    2016-03-01

    The paper contributes to the development of applied mechanics by establishing a new discipline, namely, agricultural mechanics by academician Vasilii Prohorovich Goryachkin (1868-1935) who was an apprentice of Nikolay Yegorovich Zhukovsky and a graduate of the Moscow University (current known as Moscow State University) and the Imperial Higher Technical School.

  17. Magnetic capture docking mechanism

    NASA Technical Reports Server (NTRS)

    Howard, Nathan (Inventor); Nguyen, Hai D. (Inventor)

    2010-01-01

    A mechanism uses a magnetic field to dock a satellite to a host vehicle. A docking component of the mechanism residing on the host vehicle has a magnet that is used to induce a coupled magnetic field with a docking component of the mechanism residing on the satellite. An alignment guide axially aligns the docking component of the satellite with the docking component of the host device dependent on the coupled magnetic field. Rotational alignment guides are used to rotationally align the docking component of the satellite with the docking component of the host device. A ball-lock mechanism is used to mechanically secure the docking component of the host vehicle and the docking component of the satellite.

  18. Mechanical code comparator

    DOEpatents

    Peter, Frank J.; Dalton, Larry J.; Plummer, David W.

    2002-01-01

    A new class of mechanical code comparators is described which have broad potential for application in safety, surety, and security applications. These devices can be implemented as micro-scale electromechanical systems that isolate a secure or otherwise controlled device until an access code is entered. This access code is converted into a series of mechanical inputs to the mechanical code comparator, which compares the access code to a pre-input combination, entered previously into the mechanical code comparator by an operator at the system security control point. These devices provide extremely high levels of robust security. Being totally mechanical in operation, an access control system properly based on such devices cannot be circumvented by software attack alone.

  19. Mechanisms in biomedical ontology.

    PubMed

    Röhl, Johannes

    2012-09-21

    The concept of a mechanism has become a standard proposal for explanations in biology. It has been claimed that mechanistic explanations are appropriate for systems biology, because they occupy a middle ground between strict reductionism and holism. Because of their importance in the field a formal ontological description of mechanisms is desirable. The standard philosophical accounts of mechanisms are often ambiguous and lack the clarity that can be provided by a formal-ontological framework. The goal of this paper is to clarify some of these ambiguities and suggest such a framework for mechanisms. Taking some hints from an "ontology of devices" I suggest as a general approach for this task the introduction of functional kinds and functional parts by which the particular relations between a mechanism and its components can be captured. PMID:23046727

  20. The orbital mechanics of flight mechanics

    NASA Technical Reports Server (NTRS)

    Dunning, R. S.

    1973-01-01

    A reference handbook on modern dynamic orbit theory is presented. Starting from the most basic inverse-square law, the law of gravity for a sphere is developed, and the motion of point masses under the influence of a sphere is considered. The reentry theory and the orbital theory are discussed along with the relative motion between two bodies in orbit about the same planet. Relative-motion equations, rectangular coordinates, and the mechanics of simple rigid bodies under the influence of a gravity gradient field are also discussed.

  1. Cytoskeletal Bundle Mechanics

    PubMed Central

    Bathe, Mark; Heussinger, Claus; Claessens, Mireille M. A. E.; Bausch, Andreas R.; Frey, Erwin

    2008-01-01

    The mechanical properties of cytoskeletal actin bundles play an essential role in numerous physiological processes, including hearing, fertilization, cell migration, and growth. Cells employ a multitude of actin-binding proteins to actively regulate bundle dimensions and cross-linking properties to suit biological function. The mechanical properties of actin bundles vary by orders of magnitude depending on diameter and length, cross-linking protein type and concentration, and constituent filament properties. Despite their importance to cell function, the molecular design principles responsible for this mechanical behavior remain unknown. Here, we examine the mechanics of cytoskeletal bundles using a molecular-based model that accounts for the discrete nature of constituent actin filaments and their distinct cross-linking proteins. A generic competition between filament stretching and cross-link shearing determines three markedly different regimes of mechanical response that are delineated by the relative values of two simple design parameters, revealing the universal nature of bundle-bending mechanics. In each regime, bundle-bending stiffness displays distinct scaling behavior with respect to bundle dimensions and molecular composition, as observed in reconstituted actin bundles in vitro. This mechanical behavior has direct implications on the physiological bending, buckling, and entropic stretching behavior of cytoskeletal processes, as well as reconstituted actin systems. Results are used to predict the bending regimes of various in vivo cytoskeletal bundles that are not easily accessible to experiment and to generate hypotheses regarding implications of the isolated behavior on in vivo bundle function. PMID:18055529

  2. Facing quantum mechanical reality.

    PubMed

    Rohrlich, F

    1983-09-23

    Two recent precision experiments provide conclusive evidence against any local hidden variables theory and in favor of standard quantum mechanics. Therefore the epistemology and the ontology of quantum mechanics must now be taken more seriously than ever before. The consequences of the standard interpretation of quantum mechanics are summarized in nontechnical language. The implications of the finiteness of Planck's constant (h > 0) for the quantum world are as strange as the implications of the finiteness of the speed of light (c < infinity for space and time in relativity theory. Both lead to realities beyond our common experience that cannot be rejected. PMID:17776308

  3. MECHANISMS OF RECEPTOR ADAPTATION.

    PubMed

    MENDELSON, M; LOWENSTEIN, W R

    1964-05-01

    Two determinants of adaptation in a mechanoreceptor (Pacinian corpuscle) are described. Both are filters of non-transients. One, a mechanical filter, prevents static components of the mechanical stimulus from reaching the transducer element (nerve ending). This filter is represented by the laminar capsule of the receptor. When the capsule is eliminated by dissection, the generator potential of the nerve ending in response to a sustained stimulus is markedly prolonged. Another filter is represented by an inactivation process of the mechanisms of nerve impulse initiation which prevents a steady outward current from producing repetitive impulses. Both filters cut the duration of the receptor response to a few milliseconds. PMID:14194104

  4. Mechanisms of cardiac arrhythmias

    PubMed Central

    Tse, Gary

    2015-01-01

    Blood circulation is the result of the beating of the heart, which provides the mechanical force to pump oxygenated blood to, and deoxygenated blood away from, the peripheral tissues. This depends critically on the preceding electrical activation. Disruptions in the orderly pattern of this propagating cardiac excitation wave can lead to arrhythmias. Understanding of the mechanisms underlying their generation and maintenance requires knowledge of the ionic contributions to the cardiac action potential, which is discussed in the first part of this review. A brief outline of the different classification systems for arrhythmogenesis is then provided, followed by a detailed discussion for each mechanism in turn, highlighting recent advances in this area. PMID:27092186

  5. Understand mechanical seals

    SciTech Connect

    Godse, A.G.

    1995-04-01

    Most pump failures (about 70%) are attributed to malfunctioning mechanical seals. Rotating machinery consists primarily of centrifugal pumps. The regular revision of the API standard on pumps and the increasing role of environmental regulations bring mechanical seals into sharp focus for centrifugal pumps. A integrated process plant brings a variety of influences into the selection process: end user; process consultant; engineering and construction contractors and pump/seal manufacturer. The paper discusses seal selection, understanding the seal mechanism, vapor pressure, forces acting on the seal faces and the concept of balance ratio, importance of PV curves, types of seals, heat balance, flushing plans, materials of construction, and other factors.

  6. Mechanisms of genomic imprinting.

    PubMed

    Brannan, C I; Bartolomei, M S

    1999-04-01

    A small number of mammalian genes undergo the process of genomic imprinting whereby the expression level of the alleles of a gene depends upon their parental origin. In the past year, attention has focused on the mechanisms that determine parental-specific expression patterns. Many imprinted genes are located in conserved clusters and, although it is apparent that imprinting of adjacent genes is jointly regulated, multiple mechanisms among and within clusters may operate. Recent developments have also refined the timing of the gametic imprints and further defined the mechanism by which DNA methyltransferases confer allelic methylation patterns. PMID:10322141

  7. Molecular mechanisms of epilepsy

    PubMed Central

    Staley, Kevin

    2015-01-01

    Decades of experimental work have established an imbalance of excitation and inhibition as the leading mechanism of the transition from normal brain function to seizure. In epilepsy, these transitions are rare and abrupt. Transition processes incorporating positive feedback, such as activity-dependent disinhibition, could provide these unique timing features. A rapidly expanding array of genetic etiologies will help delineate the molecular mechanism(s). This delineation will entail quite a bit of cell biology. The genes discovered to date are currently more remarkable for their diversity than their similarities. PMID:25710839

  8. Rotary mechanical latch

    DOEpatents

    Spletzer, Barry L.; Martinez, Michael A.; Marron, Lisa C.

    2012-11-13

    A rotary mechanical latch for positive latching and unlatching of a rotary device with a latchable rotating assembly having a latching gear that can be driven to latched and unlatched states by a drive mechanism such as an electric motor. A cam arm affixed to the latching gear interfaces with leading and trailing latch cams affixed to a flange within the drive mechanism. The interaction of the cam arm with leading and trailing latch cams prevents rotation of the rotating assembly by external forces such as those due to vibration or tampering.

  9. Central mechanisms of itch.

    PubMed

    Mochizuki, Hideki; Kakigi, Ryusuke

    2015-09-01

    Itch is a complex sensory and emotional experience. Functional brain imaging studies have been performed to identify brain regions associated with this complex experience, and these studies reported that several brain regions are activated by itch stimuli. The possible roles of these regions in itch perception and difference in cerebral mechanism between healthy subjects and chronic itch patients are discussed in this review article. Additionally, the central itch modulation system and cerebral mechanisms of contagious itch, pleasurable sensation evoked by scratching have also been investigated in previous brain imaging studies. We also discuss how these studies advance our understanding of these mechanisms. PMID:25534483

  10. Electronic door locking mechanism

    DOEpatents

    Williams, G.L.; Kirby, P.G.

    1997-10-21

    The invention is a motorized linkage for engaging a thumb piece in a door mechanism. The device has an exterior lock assembly with a small battery cell and combination lock. Proper entry by a user of a security code allows the battery to operate a small motor within the exterior lock assembly. The small motor manipulates a cam-plunger which moves an actuator pin into a thumb piece. The user applies a force on to the thumb piece. This force is transmitted by the thumb piece to a latch engagement mechanism by the actuator pin. The latch engagement mechanism operates the door latch. 6 figs.

  11. Electronic door locking mechanism

    DOEpatents

    Williams, Gary Lin; Kirby, Patrick Gerald

    1997-01-01

    The invention is a motorized linkage for engaging a thumb piece in a door mechanism. The device has an exterior lock assembly with a small battery cell and combination lock. Proper entry by a user of a security code allows the battery to operate a small motor within the exterior lock assembly. The small motor manipulates a cam-plunger which moves an actuator pin into a thumb piece. The user applies a force on to the thumb piece. This force is transmitted by the thumb piece to a latch engagement mechanism by the actuator pin. The latch engagement mechanism operates the door latch.

  12. Mechanics: Ideas, problems, applications

    NASA Astrophysics Data System (ADS)

    Ishlinskii, A. Iu.

    The book contains the published articles and reports by academician Ishlinskii which deal with the concepts and ideas of modern mechanics, its role in providing a general understanding of the natural phenomena, and its applications to various problems in science and engineering. Attention is given to the methodological aspects of mechanics, to the history of the theories of plasticity, friction, gyroscopic and inertial systems, and inertial navigation, and to mathematical methods in mechanics. The book also contains essays on some famous scientists and engineers.

  13. Docking and retrieval mechanism

    NASA Technical Reports Server (NTRS)

    Tewell, J. R.; Spencer, R. A.

    1977-01-01

    An engineering prototype docking and retrieval mechanism (DRM) which enables two spacecraft to dock and be structurally joined on-orbit is described. The joining of two spacecraft or payloads on-orbit supports future planned space activities such as payload servicing, deployment and retrieval, and assembly or large space systems. Advantages of the DRM include: it is a nonimpact docking mechanism; does not require impact absorbing mechanisms or attitude stabilization on the target spacecraft; is capable of docking to a spinning spacecraft; and can spin up and deploy a spinning spacecraft or payload.

  14. Docking and retrieval mechanism

    NASA Technical Reports Server (NTRS)

    Tewell, J. R.; Spencer, R. A.

    1977-01-01

    An engineering prototype docking and retrieval mechanism (DRM) is described which enables two spacecraft to dock and be structurally joined on-orbit. The joining of two spacecraft or payloads on-orbit supports future planned space activities such as payload servicing, deployment and retrieval, and assembly of large space systems. The DRM, as developed, provides advantages over prior approaches because it is a nonimpact docking mechanism, does not require impact absorbing mechanisms or attitude stabilization on the target spacecraft, is capable of docking to a spinning spacecraft, and can spin up and deploy a spinning spacecraft or payload.

  15. Is quantum mechanics exact?

    SciTech Connect

    Kapustin, Anton

    2013-06-15

    We formulate physically motivated axioms for a physical theory which for systems with a finite number of degrees of freedom uniquely lead to quantum mechanics as the only nontrivial consistent theory. Complex numbers and the existence of the Planck constant common to all systems arise naturally in this approach. The axioms are divided into two groups covering kinematics and basic measurement theory, respectively. We show that even if the second group of axioms is dropped, there are no deformations of quantum mechanics which preserve the kinematic axioms. Thus, any theory going beyond quantum mechanics must represent a radical departure from the usual a priori assumptions about the laws of nature.

  16. The neuroprotective effects and possible mechanism of action of a methanol extract from Asparagus cochinchinensis: In vitro and in vivo studies.

    PubMed

    Jalsrai, A; Numakawa, T; Kunugi, H; Dieterich, D C; Becker, A

    2016-05-13

    Extracts of Asparagus cochinchinensis (AC) have antitumor, anti-inflammatory, and immunostimulant effects. The neurobiological mechanisms underlying the effects of AC have not been sufficiently explored. Thus we performed in vivo and in vitro experiments to further characterize potential therapeutic effects and to clarify the underlying mechanisms. In the tail suspension test immobility time was significantly reduced after administration of AC which suggests antidepressant-like activity without effect on body core temperature. Moreover, in animals pretreated with AC infarct size after occlusion of the middle cerebral artery was reduced. In vitro experiments confirmed neuroprotective effects. Total saponin obtained from AC significantly inhibited H2O2-induced cell death in cultured cortical neurons. The survival-promoting effect by AC saponins was partially blocked by inhibitors for extracellular signal-regulated kinase (ErK) and phosphoinositide 3-kinase Akt (PI3K/Akt) cascades, both of which are known as survival-promoting signaling molecules. Furthermore, phosphorylation of Scr homology-2 (SH2) domain-containing phosphatase 2 (Shp-2) was induced by AC, and the protective effect of AC was abolished by NSC87877, an inhibitor for Shp-2, suggesting an involvement of Shp-2 mediated intracellular signaling in AC saponins. Moreover, AC-induced activation of pShp-2 and ErK1/2 were blocked by NSC87877 indicating that activation of these signaling pathways was mediated by the Shp-2 signaling pathway. These effects appear to be associated with activation of the Shp-2, ErK1/2 and Akt signaling pathways. Our results suggest that AC has antidepressant-like and neuroprotective (reducing infarct size) effects and that activation of pShp-2 and pErK1/2 pathways may be involved in the effects. PMID:26947129

  17. Hippocampal protein expression is differentially affected by chronic paroxetine treatment in adolescent and adult rats: a possible mechanism of “paradoxical” antidepressant responses in young persons

    PubMed Central

    Karanges, Emily A.; Kashem, Mohammed A.; Sarker, Ranjana; Ahmed, Eakhlas U.; Ahmed, Selina; Van Nieuwenhuijzen, Petra S.; Kemp, Andrew H.; McGregor, Iain S.

    2013-01-01

    Selective serotonin reuptake inhibitors (SSRIs) are commonly recognized as the pharmacological treatment of choice for patients with depressive disorders, yet their use in adolescent populations has come under scrutiny following reports of minimal efficacy and an increased risk of suicidal ideation and behavior in this age group. The biological mechanisms underlying these effects are largely unknown. Accordingly, the current study examined changes in hippocampal protein expression following chronic administration of paroxetine in drinking water (target dose = 10 mg/kg for 22 days) to adult and adolescent rats. Results indicated age-specific changes in protein expression, with paroxetine significantly altering expression of 8 proteins in adolescents only and 10 proteins solely in adults. A further 12 proteins were significantly altered in both adolescents and adults. In adults, protein changes were generally suggestive of a neurotrophic and neuroprotective effect of paroxetine, with significant downregulation of apoptotic proteins Galectin 7 and Cathepsin B, and upregulation of the neurotrophic factor Neurogenin 1 and the antioxidant proteins Aldose reductase and Carbonyl reductase 3. Phosphodiesterase 10A, a signaling protein associated with major depressive disorder, was also downregulated (-6.5-fold) in adult rats. Adolescent rats failed to show the neurotrophic and neuroprotective effects observed in adults, instead displaying upregulation of the proapoptotic protein BH3-interacting domain death agonist (4.3-fold). Adolescent protein expression profiles also suggested impaired phosphoinositide signaling (Protein kinase C: -3.1-fold) and altered neurotransmitter transport and release (Syntaxin 7: 5.7-fold; Dynamin 1: -6.9-fold). The results of the present study provide clues as to possible mechanisms underlying the atypical response of human adolescents to paroxetine treatment. PMID:23847536

  18. Antihyperglycemic effect of Annona squamosa hexane extract in type 2 diabetes animal model: PTP1B inhibition, a possible mechanism of action?

    PubMed Central

    Davis, Joseph Alex; Sharma, Suchitra; Mittra, Shivani; Sujatha, S.; Kanaujia, Anil; Shukla, Gyanesh; Katiyar, Chandrakant; Lakshmi, B.S.; Bansal, Vinay Sheel; Bhatnagar, Pradip Kumar

    2012-01-01

    Aim: The mechanism of action of Annona squamosa hexane extract in mediating antihyperglycemic and antitriglyceridimic effect were investigated in this study. Materials and Methods: The effects of extract on glucose uptake, insulin receptor-β (IR-β), insulin receptor substrate-1 (IRS-1) phosphorylation and glucose transporter type 4 (GLUT4) and phosphoinositide 3-kinase (PI3 kinase) mRNA expression were studied in L6 myotubes. The in vitro mechanism of action was tested in protein-tyrosine phosphatase 1B (PTP1B), G-protein-coupled receptor 40 (GPR40), silent mating type information regulation 2 homolog 1 (SIRT1) and dipeptidyl peptidase-IV (DPP-IV) assays. The in vivo efficacy was characterized in ob/ob mice after an oral administration of the extract for 21 days. Results: The effect of extract promoted glucose uptake, IR-β and IRS-1 phosphorylation and GLUT4 and PI3 kinase mRNA upregulation in L6 myotubes. The extract inhibited PTP1B with an IC50 17.4 μg/ml and did not modulate GPR40, SIRT1 or DPP-IV activities. An oral administration of extract in ob/ob mice for 21 days improved random blood glucose, triglyceride and oral glucose tolerance. Further, the extract did not result in body weight gain before and after treatment (29.3 vs. 33.6 g) compared to rosiglitazone where significant body weight gain was observed (28.4 vs. 44.5 g; *P<0.05 after treatment compared to before treatment). Conclusion: The results suggest that Annona squamosa hexane extract exerts its action by modulating insulin signaling through inhibition of PTP1B. PMID:22701240

  19. ELECTROMAGNETIC RELEASE MECHANISM

    DOEpatents

    Michelson, C.

    1960-09-13

    An electromagnetic release mechanism is offered that may be used, for example, for supporting a safety rod for a nuclear reactor. The release mechanism is designed to have a large excess holding force and a rapid, uniform, and dependable release. The fast release is accomplished by providing the electromagnet with slotttd polts separated by an insulating potting resin, and by constructing the poles with a ferro-nickel alloy. The combination of these two features materially reduces the eddy current power density whenever the magnetic field changes during a release operation. In addition to these features, the design of the armature is such as to provide ready entrance of fluid into any void that might tend to form during release of the armature. This also improves the release time for the mechanism. The large holding force for the mechanism is accomplished by providing a small, selected, uniform air gap between the inner pole piece and the armature.

  20. Mechanisms for Robust Cognition.

    PubMed

    Walsh, Matthew M; Gluck, Kevin A

    2015-08-01

    To function well in an unpredictable environment using unreliable components, a system must have a high degree of robustness. Robustness is fundamental to biological systems and is an objective in the design of engineered systems such as airplane engines and buildings. Cognitive systems, like biological and engineered systems, exist within variable environments. This raises the question, how do cognitive systems achieve similarly high degrees of robustness? The aim of this study was to identify a set of mechanisms that enhance robustness in cognitive systems. We identify three mechanisms that enhance robustness in biological and engineered systems: system control, redundancy, and adaptability. After surveying the psychological literature for evidence of these mechanisms, we provide simulations illustrating how each contributes to robust cognition in a different psychological domain: psychomotor vigilance, semantic memory, and strategy selection. These simulations highlight features of a mathematical approach for quantifying robustness, and they provide concrete examples of mechanisms for robust cognition. PMID:25352094

  1. MECHANISMS OF PESTICIDE DEGRADATION

    EPA Science Inventory

    This research project was initiated with the overall objective of determining (1) the chemical structures of toxic components of toxaphene, (2) to study anaerobic metabolism to degrade toxaphene and other pesticides, and (3) to understand toxic action mechanism of chlordimeform. ...

  2. Mechanisms flown on LDEF

    NASA Technical Reports Server (NTRS)

    Dursch, Harry; Spear, Steve

    1992-01-01

    A wide variety of mechanisms were flown on the Long Duration Exposure Facility (LDEF). These include canisters, valves, gears, drive train assemblies, and motors. This report will provide the status of the Systems SIG effort into documenting, integrating, and developing 'lessons learned' for the variety of mechanisms flown on the LDEF. Results will include both testing data developed by the various experimenters and data acquired by testing of hardware at Boeing.

  3. Mechanisms for chromosome segregation.

    PubMed

    Bouet, Jean-Yves; Stouf, Mathieu; Lebailly, Elise; Cornet, François

    2014-12-01

    Bacteria face the problem of segregating their gigantic chromosomes without a segregation period restricted in time and space, as Eukaryotes do. Segregation thus involves multiple activities, general or specific of a chromosome region and differentially controlled. Recent advances show that these various mechanisms conform to a “pair and release” rule, which appears as a general rule in DNA segregation. We describe the latest advances in segregation of bacterial chromosomes with emphasis on the different pair and release mechanisms. PMID:25460797

  4. Space Mechanisms Technology Workshop

    NASA Technical Reports Server (NTRS)

    Oswald, Fred B. (Editor)

    2001-01-01

    The Mechanical Components Branch at NASA Glenn Research Center hosted a workshop to discuss the state of drive systems technology needed for space exploration. The Workshop was held Thursday, November 2, 2000. About 70 space mechanisms experts shared their experiences from working in this field and considered technology development that will be needed to support future space exploration in the next 10 to 30 years.

  5. Phase Field Fracture Mechanics.

    SciTech Connect

    Robertson, Brett Anthony

    2015-11-01

    For this assignment, a newer technique of fracture mechanics using a phase field approach, will be examined and compared with experimental data for a bend test and a tension test. The software being used is Sierra Solid Mechanics, an implicit/explicit finite element code developed at Sandia National Labs in Albuquerque, New Mexico. The bend test experimental data was also obtained at Sandia Labs while the tension test data was found in a report online from Purdue University.

  6. Lung Parenchymal Mechanics

    PubMed Central

    Suki, Béla; Stamenovic, Dimitrije; Hubmayr, Rolf

    2014-01-01

    The lung parenchyma comprises a large number of thin-walled alveoli, forming an enormous surface area, which serves to maintain proper gas exchange. The alveoli are held open by the transpulmonary pressure, or prestress, which is balanced by tissues forces and alveolar surface film forces. Gas exchange efficiency is thus inextricably linked to three fundamental features of the lung: parenchymal architecture, prestress, and the mechanical properties of the parenchyma. The prestress is a key determinant of lung deformability that influences many phenomena including local ventilation, regional blood flow, tissue stiffness, smooth muscle contractility, and alveolar stability. The main pathway for stress transmission is through the extracellular matrix. Thus, the mechanical properties of the matrix play a key role both in lung function and biology. These mechanical properties in turn are determined by the constituents of the tissue, including elastin, collagen, and proteoglycans. In addition, the macroscopic mechanical properties are also influenced by the surface tension and, to some extent, the contractile state of the adherent cells. This article focuses on the biomechanical properties of the main constituents of the parenchyma in the presence of prestress and how these properties define normal function or change in disease. An integrated view of lung mechanics is presented and the utility of parenchymal mechanics at the bedside as well as its possible future role in lung physiology and medicine are discussed. PMID:23733644

  7. Docking mechanism for spacecraft

    NASA Technical Reports Server (NTRS)

    Lange, Gregory A. (Inventor); Mcmanamen, John P. (Inventor); Schliesing, John A. (Inventor)

    1989-01-01

    A system is presented for docking a space vehicle to a space station where a connecting tunnel for in-flight transfer of personnel is required. Cooperable coupling mechanisms include docking rings on the space vehicle and space station. The space station is provided with a tunnel structure, a retraction mechanism, and a docking ring. The vehicle coupling mechanism is designed to capture the station coupling mechanism, arrest relative spacecraft motions while limiting loads to acceptable levels, and then realign the spacecraft for final docking and tunnel interconnection. The docking ring of the space vehicle coupling mechanism is supported by linear attentuator actuator devices, each of which is controlled by a control system which receives loading information signals and attenuator stroke information signals from each device and supplies output signals for controlling its linear actuation to attenuate impact loading or to realign the spacecraft for final docking and tunnel interconnection. The retraction mechanism is used to draw the spacecraft together after initial contact and coupling. Tunnel trunnions, cooperative with the latches on the space vehicle constitute the primary structural tie between the spacecraft in final docked configuration.

  8. Nuclear Mechanics in Cancer

    PubMed Central

    Denais, Celine; Lammerding, Jan

    2015-01-01

    Despite decades of research, cancer metastasis remains an incompletely understood process that is as complex as it is devastating. In recent years, there has been an increasing push to investigate the biomechanical aspects of tumorigenesis, complementing the research on genetic and biochemical changes. In contrast to the high genetic variability encountered in cancer cells, almost all metastatic cells are subject to the same physical constraints as they leave the primary tumor, invade surrounding tissues, transit through the circulatory system, and finally infiltrate new tissues. Advances in live cell imaging and other biophysical techniques, including measurements of subcellular mechanics, have yielded stunning new insights into the physics of cancer cells. While much of this research has been focused on the mechanics of the cytoskeleton and the cellular microenvironment, it is now emerging that the mechanical properties of the cell nucleus and its connection to the cytoskeleton may play a major role in cancer metastasis, as deformation of the large and stiff nucleus presents a substantial obstacle during the passage through the dense interstitial space and narrow capillaries. Here, we present an overview of the molecular components that govern the mechanical properties of the nucleus and we discuss how changes in nuclear structure and composition observed in many cancers can modulate nuclear mechanics and promote metastatic processes. Improved insights into this interplay between nuclear mechanics and metastatic progression may have powerful implications in cancer diagnostics and therapy and may reveal novel therapeutic targets for pharmacological inhibition of cancer cell invasion. PMID:24563360

  9. REACTOR CONTROL MECHANISM

    DOEpatents

    Lane, J.A.; Engberg, R.E.; Welch, J.M.

    1959-05-12

    A quick-releasing mechanism is described which may be used to rapidiy drop a device supported from beneath during normal use, such as a safety rod in a nuclear reactor. In accordance with this invention an electrical control signal, such as may be provided by radiation detection or other alarm condition sensing devices, is delivered to an electromagnetic solenoid, the armature of which is coupled to an actuating mechanism. The solenoid is energized when the mechanism is in its upper or cocked position. In such position, the mechanism engages a plurality of retaining balls, forcing them outward into engagement with a shoulder or recess in a corresponding section of a tubular extension on the upheld device. When the control signal to the solenoid suddenly ceases, the armature drops out, allowing the actuating mechanism to move slightly but rapidly under the force of a compressed spring. The weight of the device will urge the balls inward against a beveled portion of the actuating mechanism and away from the engaging section on the tubular extension, thus allowing the upheld device to fall freely under the influence of gravity.

  10. Wear and Tear - Mechanical

    NASA Technical Reports Server (NTRS)

    Swanson, Theodore

    2008-01-01

    The focus of this chapter is on the long term wear and tear, or aging, of the mechanical subsystem of a spacecraft. The mechanical subsystem is herein considered to be the primary support structure (as in a skeleton or exoskeleton) upon which all other spacecraft systems rest, and the associated mechanisms. Mechanisms are devices which have some component that moves at least once, in response to some type of passive or active control system. For the structure, aging may proceed as a gradual degradation of mechanical properties and/or function, possibly leading to complete structural failure over an extended period of time. However, over the 50 years of the Space Age such failures appear to be unusual. In contrast, failures for mechanisms are much more frequent and may have a very serious effect on mission performance. Just as on Earth, all moving devices are subject to normal (and possibly accelerated) degradation from mechanical wear due to loss or breakdown of lubricant, misalignment, temperature cycling effects, improper design/selection of materials, fatigue, and a variety of other effects. In space, such environmental factors as severe temperature swings (possibly 100's of degrees C while going in and out of direct solar exposure), hard vacuum, micrometeoroids, wear from operation in a dusty or contaminated environment, and materials degradation from radiation can be much worse. In addition, there are some ground handling issues such as humidity, long term storage, and ground transport which may be of concern. This chapter addresses the elements of the mechanical subsystem subject to wear, and identifies possible causes. The potential impact of such degradation is addressed, albeit with the recognition that the impact of such wear often depends on when it occurs and on what specific components. Most structural elements of the mechanical system typically are conservatively designed (often to a safety factor of greater than approximately 1.25 on yield for unmanned spacecraft) but do not have backup structure due to the added mass this would impose, and also due to the fact that structural elements can be accurately modeled mathematically and in test. Critical mechanisms or devices may have backups, or alternate work-arounds, since characterization of these systems in a 1g environment is less accurate than structure, and repair in-space is often impossible.

  11. Indications for mechanical ventilation.

    PubMed

    Tung, A

    1997-01-01

    Indications for mechanical ventilation have evolved substantially since widespread use of ventilatory support began in the early 1960s. While the metabolic and blood-gas alterations that mandate institution of ventilatory support have remained unaltered, new noninvasive modes of ventilation have widened the therapeutic options available to patients in acute respiratory failure. An understanding of the effect of mechanical ventilation on other organ systems has clarified the role of mechanical ventilation in the treatment of conditions other than respiratory failure such as stroke or head injury. Studies in patients recovering from major surgery have better defined the benefits and risks of postoperative mechanical ventilation. Finally, a better understanding of disease processes has led to more prognostic information that can help physicians, patients, and families decide on limits to compassionate care. The proper use of mechanical ventilation in disease states that do not involve respiratory failure as their primary manifestation is also important in light of the risks of respiratory support. In patients with CNS injury, the role of hyperventilation is limited to acute control of dangerous elevations of intracranial pressure. Although hypocarbia has been proposed to improve regional cerebral blood flow, studies have not demonstrated an improvement in outcome, suggesting that the risks of intubation, tracheal stimulation, sedation, and inability to examine the mental status outweigh any benefit. Some evidence suggests a detrimental effect from prolonged hyperventilation. The use of mechanical ventilation in postoperative care is another area that requires scrutiny. Numerous studies have shown that with coordination of care between surgeons, anesthesiologists, and nurses, many patients can be extubated significantly sooner than in the past. As techniques for administering anesthesia, performing surgery, and managing pain and mild respiratory insufficiency improve, knowledge in this area will continue to develop. Finally, the relation between mechanical ventilation, quality of life, and patient autonomy has come to play a greater role as the population ages. In many situations, respiratory failure represents the end stage of an irreversible disease. Whereas respiratory failure secondary to pulmonary contusion in young patients does not indicate a poor outcome, progressive respiratory failure in cystic fibrosis or following bone marrow transplantation usually represents a preterminal event. Understanding the epidemiology of respiratory failure in different disease categories is important to physicians, patients, and families in making informed decisions about their care. Mechanical ventilation represents a vital, fundamental form of life support. As the diseases, tools, and treatments change in anesthesia and critical care, careful definition of the role of mechanical ventilation in specific diseases, the route by which it is delivered, and the ability of such a form of life support to affect outcome will continue to be necessary. PMID:9113518

  12. Mechanical Components Branch Overview

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Mr. James Zakrajsek, chief of the Mechanical Components Branch, presented an overview of research conducted by the branch. Branch members perform basic research on mechanical components and systems, including gears and bearings, turbine seals, structural and thermal barrier seals, and space mechanisms. The research is focused on propulsion systems for present and advanced aerospace vehicles. For rotorcraft and conventional aircraft, we conduct research to develop technology needed to enable the design of low noise, ultra safe geared drive systems. We develop and validate analytical models for gear crack propagation, gear dynamics and noise, gear diagnostics, bearing dynamics, and thermal analyses of gear systems using experimental data from various component test rigs. In seal research we develop and test advanced turbine seal concepts to increase efficiency and durability of turbine engines. We perform experimental and analytical research to develop advanced thermal barrier seals and structural seals for current and next generation space vehicles. In space mechanisms, we conduct fundamental research on lubricants, materials, components and mechanisms subjected to deep space and planetary environments.

  13. Fundamentals of Quantum Mechanics

    NASA Astrophysics Data System (ADS)

    Tang, C. L.

    2005-06-01

    Quantum mechanics has evolved from a subject of study in pure physics to one with a wide range of applications in many diverse fields. The basic concepts of quantum mechanics are explained in this book in a concise and easy-to-read manner emphasising applications in solid state electronics and modern optics. Following a logical sequence, the book is focused on the key ideas and is conceptually and mathematically self-contained. The fundamental principles of quantum mechanics are illustrated by showing their application to systems such as the hydrogen atom, multi-electron ions and atoms, the formation of simple organic molecules and crystalline solids of practical importance. It leads on from these basic concepts to discuss some of the most important applications in modern semiconductor electronics and optics. Containing many homework problems and worked examples, the book is suitable for senior-level undergraduate and graduate level students in electrical engineering, materials science and applied physics. Clear exposition of quantum mechanics written in a concise and accessible style Precise physical interpretation of the mathematical foundations of quantum mechanics Illustrates the important concepts and results by reference to real-world examples in electronics and optoelectronics Contains homeworks and worked examples, with solutions available for instructors

  14. Resonant scanning mechanism

    NASA Astrophysics Data System (ADS)

    Wallace, John; Newman, Mike; Gutierrez, Homero; Hoffman, Charlie; Quakenbush, Tim; Waldeck, Dan; Leone, Christopher; Ostaszewski, Miro

    2014-10-01

    Ball Aerospace & Technologies Corp. developed a Resonant Scanning Mechanism (RSM) capable of combining a 250- Hz resonant scan about one axis with a two-hertz triangular scan about the orthogonal axis. The RSM enables a rapid, high-density scan over a significant field of regard (FOR) while minimizing size, weight, and power requirements. The azimuth scan axis is bearing mounted allowing for 30° of mechanical travel, while the resonant elevation axis is flexure and spring mounted with five degrees of mechanical travel. Pointing-knowledge error during quiescent static pointing at room temperature across the full range is better than 100 μrad RMS per axis. The compact design of the RSM, roughly the size of a soda can, makes it an ideal mechanism for use on low-altitude aircraft and unmanned aerial vehicles. Unique aspects of the opto-mechanical design include i) resonant springs which allow for a high-frequency scan axis with low power consumption; and ii) an independent lower-frequency scan axis allowing for a wide FOR. The pointing control system operates each axis independently and employs i) a position loop for the azimuth axis; and ii) a unique combination of parallel frequency and amplitude control loops for the elevation axis. All control and pointing algorithms are hosted on a 200-MHz microcontroller with 516 KB of RAM on a compact 3"×4" digital controller, also of Ball design.

  15. Mechanical regulation of chondrogenesis

    PubMed Central

    2013-01-01

    Mechanical factors play a crucial role in the development of articular cartilage in vivo. In this regard, tissue engineers have sough