Identification of Ser(1275) and Ser(1309) as autophosphorylation sites of the human insulin receptor in intact cells

In a previous report we described Ser(1275) and Ser(1309) as autophosphoryl ation sites of the human insulin receptor (IR) tyrosine kinase (TK) in vitr o. The question remained whether the observed phosphorylation was exclusive for the in vitro activated receptor or a more general, mechanism of the ac tivated receptor in situ. In this study, we determined the intrinsic activi ty of the IR to phosphorylate both serine residues in intact cells, For thi s purpose CHO-09 and NIH-3T3 derived cell-lines expressing the human IR wer e metabolically labelled with [P-32]orthophosphate, followed by hormone sti mulation of the receptor. The IR was isolated by immunoprecipitation and SD S-PAGE and subsequently analysed for serine phosphorylation by phosphopepti de mapping of HPLC-purified tryptic phosphopeptides. Activation of the IR i n the intact cell appeared to result in phosphate incorporation into Ser(12 75) and Ser(1309), providing strong evidence that both serine residues are phosphorylation sites of the activated receptor in intact cells, (C) 2001 A cademic Press.