Insulin-like growth factor I activates insulin receptor substrate 1 and Ras in human osteosarcoma cells

Insulin-like growth factor I (IGF-I) stimulates multiplication of the human osteosarcoma cell line, MG-63, by acting through IGF-I receptor. We have c haracterized IGF-I stimulated phosphorylation of IRS-1, activation of Ras c ycle and phosphorylation of c-Jun in this cell line. Serum starved MG-63 ce lls were (1) IGF-I stimulated and lysates were immunoprecipitated with poly clonal IRS-1 antibody or (2) metabolically labeled with [P-32]orthophosphor ic acid and then cells were treated with IGF-I. Cell lysates were immunopre cipitated with p21Ras antibody (Y13-259) and bound nucleotides were analyse d by thin-layer chromatography. We demonstrated tyrosine phosphorylation of IRS-1/2 immunoprecipitated from MG-63 cells stimulated with IGF-I. We also showed an increased level of GTP in p21Ras immunoprecipitates from IGF-I t reated cells. Nuclear extracts prepared from P-32-labeled cells before and after addition of IGF-I were immunoprecipitated with c-Jun antibody. After electrophoresis and autoradiography, phosphorylation of the c-Jun band was seen to be IGF-I independent. Phosphoamino acid analysis of the c-Jun band showed that phosphoserine was the major species.