Detection of novel intracellular agonist responsive pools of phosphatidylinositol 3,4-bisphosphate using the TAPP1 pleckstrin homology domain in immunoelectron microscopy

Stephen A Watt, Wendy A Kimber, Ian N Fleming, Nick R Leslie, C Peter Downes, John M Lucocq

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

PtdIns(3,4) P (2), a breakdown product of the lipid second messenger PtdIns(3,4,5) P (3), is a key signalling molecule in pathways controlling various cellular events. Cellular levels of PtdIns(3,4) P (2) are elevated upon agonist stimulation, mediating downstream signalling pathways by recruiting proteins containing specialized lipid-binding modules, such as the pleckstrin homology (PH) domain. A recently identified protein, TAPP1 (tandem-PH-domain-containing protein 1), has been shown to interact in vitro with high affinity and specificity with PtdIns(3,4) P (2) through its C-terminal PH domain. In the present study, we have utilized this PH domain tagged with glutathione S-transferase (GST-TAPP1-PH) as a probe in an on-section immunoelectron microscopy labelling procedure, mapping the subcellular distribution of PtdIns(3,4) P (2). As expected, we found accumulation of PtdIns(3,4) P (2) at the plasma membrane in response to the agonists platelet-derived growth factor and hydrogen peroxide. Importantly, however, we also found agonist stimulated PtdIns(3,4) P (2) labelling of intracellular organelles, including the endoplasmic reticulum and multivesicular endosomes. Expression of the 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in PTEN-null U87MG cells revealed differential sensitivity of these lipid pools to the enzyme. These data suggest a role for PtdIns(3,4) P (2) in endomembrane function.
Original languageEnglish
Pages (from-to)653-663
Number of pages11
JournalBiochemical Journal
Volume377
Issue number3
DOIs
Publication statusPublished - 1 Feb 2004

Keywords

  • Animals
  • Blood Proteins
  • Carrier Proteins
  • Cell Line
  • Cell Line, Tumor
  • DNA Probes
  • Down-Regulation
  • Endoplasmic Reticulum
  • Humans
  • Hydrogen Peroxide
  • Intracellular Membranes
  • Intracellular Signaling Peptides and Proteins
  • Intracellular Space
  • Membrane Proteins
  • Mice
  • Microscopy, Immunoelectron
  • PTEN Phosphohydrolase
  • Peptides
  • Phosphatidylinositol Phosphates
  • Phosphoproteins
  • Phosphoric Monoester Hydrolases
  • Platelet-Derived Growth Factor
  • Protein Structure, Tertiary
  • Sequence Homology, Nucleic Acid
  • Staining and Labeling
  • Swiss 3T3 Cells
  • Tumor Suppressor Proteins

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