Reconstitution of phosphoinositide 3-kinase-dependent insulin signaling in a cell-free system.
Early insulin signaling events were examined in a novel cell-free assay utilizing subcellular fractions derived from 3T3-L1 adipocytes. The following cellular processes were observed in vitro in a manner dependent on insulin, time of incubation, and exogenous ATP: 1) autophosphorylation and activation of the insulin receptor; 2) tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1); 3) association of tyrosine-phosphorylated IRS-1 with phosphoinositide 3-kinase; 4) activation of the kinase Akt via its phosphorylation on Thr-308 and Ser-473; and 5) phosphorylation of glycogen synthase kinase-3 by activated Akt. The activation of Akt in vitro was abolished in the presence of the phosphoinositide 3-kinase inhibitor, wortmannin, thus recapitulating the most notable regulatory feature of Akt observed in vivo. Evidence is presented indicating that the critical spatial compartmentalization of signaling molecules necessary for efficient signal transduction is likely to be preserved in the cell-free system. Additionally, data are provided demonstrating that full Akt activation in this system is dependent on plasma membrane-associated IRS-1, cannot be mediated by robust cytosol-specific tyrosine phosphorylation of IRS-1, and occurs in the complete absence of detectable IRS-2 phosphorylation in the cytosol and plasma membrane.
Murata
Haruhiko
H
Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.
Hresko
Richard C
RC
Mueckler
Mike
M
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003600:Cytosol / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013347:Subcellular Fractions; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D000077191:Wortmannin
D000730:Androstadienes; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526298:Irs2 protein, mouse; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D013912:Threonine; D012694:Serine; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D000077191:Wortmannin