A screen for novel phosphoinositide 3-kinase effector proteins

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

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

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)P(2)). As few molecular targets for PtdIns(3,4)P(2) 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)P(2). A secondary purification of these proteins, optimized using tandem pleckstrin homology domain containing protein-1 (TAPP-1), an established PtdIns(3,4)P(2) 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.
Original languageEnglish
Pages (from-to)M110.003178
JournalMolecular and Cellular Proteomics
Volume10
Issue number4
DOIs
Publication statusPublished - Apr 2011

Keywords

  • Cell Line, Tumor
  • Feasibility Studies
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Peptide Fragments
  • Phosphatidylinositol 3-Kinases
  • Phosphatidylinositol Phosphates
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping
  • Recombinant Fusion Proteins
  • Signal Transduction
  • Surface Plasmon Resonance
  • ras GTPase-Activating Proteins

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