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 characterized IGF-I stimulated phosphorylation of IRS-1, activation of Ras cycle and phosphorylation of c-Jun in this cell line. Serum starved MG-63 cells were (1) IGF-I stimulated and lysates were immunoprecipitated with polyclonal IRS-1 antibody or (2) metabolically labeled with [32P]orthophosphoric acid and then cells were treated with IGF-I. Cell lysates were immunoprecipitated with p21Ras antibody (Y13-259) and bound nucleotides were analysed 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 treated cells. Nuclear extracts prepared from 32P-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.
Lopaczynski
W
W
Endocrinology Section, Metabolism Branch, National Cancer Institute, Bethesda, Maryland 20892-1374, USA. WLX@CU.NIH.GOV
Terry
C
C
D001345:Autoradiography; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D014407:Tumor Cells, Cultured
C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D007334:Insulin-Like Growth Factor I; D015689:Oncogene Protein p21(ras)