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 pr omoting the differentiation and survival of neuronal cells. SH-SY5Y hu man neuroblastoma cells are a well characterized in vitro model of ner vous system growth. We report here that IGF-I stimulated the tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and insulin recept or substrate-2 (IRS-2) in a time- and concentration-dependent manner. These cells lacked IRS-1. After being tyrosine phosphorylated, IRS-2 a ssociated transiently with downstream signaling molecules, including p hosphatidylinositol 3-kinase (PI 3-K) and Grb2. Treatment of the cells with PI 3-K inhibitors (wortmannin and LY294002) increased IGF-I-indu ced 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 wit h PI 3-K inhibitors induced an increased association of IRS-2 with Grb 2, probably as a result of the increased IRS-2 tyrosine phosphorylatio n. 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, t hese 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 n eurite outgrowth.