A screen for novel phosphoinositide 3-kinase effector proteins.
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.
The Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dow St., Dundee, DD1 5EH, Scotland, UK.
Miles J
MJ
Dixon
Alexander
A
Gray
François-Michel
FM
Boisvert
Mark
M
Agacan
Nicholas A
NA
Morrice
Robert
R
Gourlay
Nicholas R
NR
Leslie
C Peter
CP
Downes
Ian H
IH
Batty
D045744:Cell Line, Tumor; D005240:Feasibility Studies; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D020703:ras GTPase-Activating Proteins / Q000096:biosynthesis
C101203:IQ motif containing GTPase activating protein 1; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C435927:PLEKHA1 protein, human; D010446:Peptide Fragments; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D019869:Phosphatidylinositol 3-Kinases
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