Differential regulation of insulin receptor substrate-2 and mitogen-activated protein kinase tyrosine phosphorylation by phosphatidylinositol 3-kinase inhibitors in SH-SY5Y human neuroblastoma cells.
Insulin-like growth factor I (IGF-I) is a potent neurotropic factor promoting the differentiation and survival of neuronal cells. SH-SY5Y human neuroblastoma cells are a well characterized in vitro model of nervous system growth. We report here that IGF-I stimulated the tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and insulin receptor substrate-2 (IRS-2) in a time- and concentration-dependent manner. These cells lacked IRS-1. After being tyrosine phosphorylated, IRS-2 associated transiently with downstream signaling molecules, including phosphatidylinositol 3-kinase (PI 3-K) and Grb2. Treatment of the cells with PI 3-K inhibitors (wortmannin and LY294002) increased IGF-I-induced tyrosine phosphorylation of IRS-2. We also observed a concomitant increase in the mobility of IRS-2, suggesting that PI 3-K mediates or is required for IRS-2 serine/threonine phosphorylation, and that this phosphorylation inhibits IRS-2 tyrosine phosphorylation. Treatment with PI 3-K inhibitors induced an increased association of IRS-2 with Grb2, probably as a result of the increased IRS-2 tyrosine phosphorylation. However, even though the PI 3-K inhibitors enhanced the association of Grb2 with IRS-2, these compounds suppressed IGF-I-induced mitogen-activated protein kinase activation and neurite outgrowth. Together, these results indicate that although PI 3-K participates in a negative regulation of IRS-2 tyrosine phosphorylation, its activity is required for IGF-IR-mediated mitogen-activated protein kinase activation and neurite outgrowth.
Kim
B
B
Department of Neurology, University of Michigan, Ann Arbor 48109-0588, USA.
Leventhal
P S
PS
White
M F
MF
Feldman
E L
EL
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D051380:GRB2 Adaptor Protein; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D051380:GRB2 Adaptor Protein; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D019950:Mitogen-Activated Protein Kinase 1; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine; C495948:GRB2 protein, human; C526292:IRS1 protein, human; C526297:IRS2 protein, human; C528024:SHC1 protein, human