title: Intracellular segregation of phosphatidylinositol-3,4,5-trisphosphate by insulin-dependent actin remodeling in L6 skeletal muscle cells.
abstract: Insulin stimulates glucose uptake by recruiting glucose transporter 4 (GLUT4) from an intracellular pool to the cell surface through a mechanism that is dependent on phosphatidylinositol (PI) 3-kinase (PI3-K) and cortical actin remodeling. Here we test the hypothesis that insulin-dependent actin filament remodeling determines the location of insulin signaling molecules. It has been shown previously that insulin treatment of L6 myotubes leads to a rapid rearrangement of actin filaments into submembrane structures where the p85 regulatory subunit of PI3-K and organelles containing GLUT4, VAMP2, and the insulin-regulated aminopeptidase (IRAP) colocalize. We now report that insulin receptor substrate-1 and the p110alpha catalytic subunit of PI3-K (but not p110beta) also colocalize with the actin structures. Akt-1 was also found in the remodeled actin structures, unlike another PI3-K effector, atypical protein kinase C lambda. Transiently transfected green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of general receptor for phosphoinositides-1 (GRP1) or Akt (ligands of phosphatidylinositol-3,4,5-trisphosphate [PI-3,4,5-P(3)]) migrated to the periphery of the live cells; in fixed cells, they were detected in the insulin-induced actin structures. These results suggest that PI-3,4,5-P(3) is generated on membranes located within the actin mesh. Actin remodeling and GLUT4 externalization were blocked in cells highly expressing GFP-PH-GRP1, suggesting that PI-3,4,5-P(3) is required for both phenomena. We propose that PI-3,4,5-P(3) leads to actin remodeling, which in turn segregates p85alpha and p110alpha, thus localizing PI-3,4,5-P(3) production on membranes trapped by the actin mesh. Insulin-stimulated actin remodeling may spatially coordinate the localized generation of PI-3,4,5-P(3) and recruitment of Akt, ultimately leading to GLUT4 insertion at the plasma membrane.
authors.affiliation: Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Nish
authors.initials: N
authors.lastname: Patel
authors.forename: Assaf
authors.initials: A
authors.lastname: Rudich
authors.forename: Zayna A
authors.initials: ZA
authors.lastname: Khayat
authors.forename: Rami
authors.initials: R
authors.lastname: Garg
authors.forename: Amira
authors.initials: A
authors.lastname: Klip
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000939:Epitopes; D051275:Glucose Transporter Type 4; D049452:Green Fluorescent Proteins; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D018482:Muscle, Skeletal / Q000166:cytology*; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D050683:R-SNARE Proteins; D013696:Temperature; D013997:Time Factors; D014162:Transfection
chemical_list: D000199:Actins; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D000939:Epitopes; D051275:Glucose Transporter Type 4; D007328:Insulin; D008024:Ligands; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D018129:Phosphatidylinositol Phosphates; D020033:Protein Isoforms; D050683:R-SNARE Proteins; C495692:SLC2A4 protein, human; C495693:Slc2a4 protein, mouse; C060974:phosphatidylinositol 3,4,5-triphosphate; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C