title: Detecting protein-phospholipid interactions. Epidermal growth factor-induced activation of phospholipase D1b in situ.
abstract: Phospholipase D (PLD) proteins have been identified in secretory and endocytic vesicles, consistent with their proposed role in regulating membrane traffic. However, their sites of catalytic action remain obscure. We have developed here a novel, analytical approach to monitor PLD activation in intact cells employing lifetime imaging microscopy to measure fluorescence resonance energy transfer between protein and membrane phospholipid. Verification and application of this technique demonstrates a dispersed endosomal, epidermal growth factor-induced activation of the PLD1b isoform. Application of this approach will facilitate the spatial resolution of many protein-phospholipid interactions that are key events in the regulation of cellular processes.
authors.affiliation: Protein Phosphorylation Laboratory, Cancer Research United Kingdom London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
authors.forename: William E
authors.initials: WE
authors.lastname: Hughes
authors.forename: Banafshé
authors.initials: B
authors.lastname: Larijani
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Parker
mesh_terms: D015153:Blotting, Western; D002384:Catalysis; D015536:Down-Regulation; D011992:Endosomes / Q000378:metabolism; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010739:Phospholipase D / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms
chemical_list: D008164:Luminescent Proteins; D010743:Phospholipids; D020033:Protein Isoforms; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D010739:Phospholipase D; C106801:phospholipase D1