title: BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation.
abstract: Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.
authors.affiliation: Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary; MTA-SE Laboratory of Molecular Physiology, Budapest, Hungary.
authors.forename: József T
authors.initials: JT
authors.lastname: Tóth
authors.affiliation: Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Gergő
authors.initials: G
authors.lastname: Gulyás
authors.affiliation: Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Dániel J
authors.initials: DJ
authors.lastname: Tóth
authors.affiliation: Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary; MTA-SE Laboratory of Molecular Physiology, Budapest, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Balla
authors.affiliation: Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA; Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Gerald R V
authors.initials: GR
authors.lastname: Hammond
authors.affiliation: Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary; MTA-SE Laboratory of Molecular Physiology, Budapest, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Hunyady
authors.affiliation: Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Tamás
authors.initials: T
authors.lastname: Balla
authors.affiliation: Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary. Electronic address: varnai.peter@med.semmelweis-univ.hu.
authors.forename: Péter
authors.initials: P
authors.lastname: Várnai
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D019556:COS Cells; D002217:Carbachol / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D025461:Feedback, Physiological; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D008066:Lipolysis; D018721:Muscarinic Agonists / Q000494:pharmacology*; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D015854:Up-Regulation
chemical_list: C000595896:CHRM3 protein, human; D018721:Muscarinic Agonists; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C037178:phosphatidylinositol 4-phosphate; D004815:Epidermal Growth Factor; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D058539:Phosphatidylinositol 3-Kinase; C512478:EGFR protein, human; D066246:ErbB Receptors; D011493:Protein Kinase C; D010738:Type C Phospholipases
keywords: BRET; EGF receptor; GPCR; PI4-kinase; PKC; Phosphoinositides