CHARACTERIZATION OF PHORBOL ESTER-STIMULATED SERINE PHOSPHORYLATION OF THE HUMAN INSULIN-RECEPTOR

Phorbol 12-myristate 13-acetate (PMA)-stimulated phosphorylation of th e human insulin receptor (IR) was characterized and compared in two ce ll types of different lineage: normal rat kidney epithelial (NRK) cell s and Chinese hamster ovary (CHO) fibroblasts. PMA stimulation increas ed IR beta-subunit phosphorylation to 252 +/- 43 and 259 +/- 47 % (+/- S.D.) of the unstimulated control in NRK and CHO cells respectively. Tryptic phosphopeptide analysis by Tricine/SDS/PAGE revealed significa nt differences in the PMA-stimulated phosphorylation of the IR in thes e two cell types. This phosphorylation of the IR was predominantly loc ated in two tryptic phosphopeptides, and these phosphopeptides were ab sent in an IR mutant truncated by 43 C-terminal amino acids. The major PMA-stimulated tryptic phosphopeptide from in vivo-labelled CHO/IR wa s immunoprecipitated with an antibody against residues Ser(1315) to Ly s(1329), and this precipitation was blocked with excess unlabelled pep tide containing this sequence. Radiosequencing by manual Edman degrada tion revealed that this tryptic phosphopeptide was phosphorylated at S er(1315). This PMA-stimulated phosphorylation did not inhibit autophos phorylation of the IR in vivo. These results demonstrate that PMA-stim ulated phosphorylation of the IR can exhibit significant differences w hen expressed in different cell types, and that Ser(1315) is a major P MA-stimulated phosphorylation site on the human IR.