STUDIES INTO THE IDENTITY OF THE SITES OF INSULIN-STIMULATED INSULIN-RECEPTOR SERINE PHOSPHORYLATION - CHARACTERIZATION OF SYNTHETIC PEPTIDE-SUBSTRATES FOR THE INSULIN-STIMULATED INSULIN-RECEPTOR SERINE KINASE

The identity of the sites of insulin-stimulated serine phosphorylation in the human insulin receptor was examined by synthesizing peptides t hat together encompassed all the serine residues of the cytosolic port ion of the beta-subunit and testing them as substrates for phosphoryla tion by a preparation of human insulin receptor copurified with insuli n-stimulated insulin receptor serine kinase activity. Of the 14 peptid es studied, only 4 (1071-1080, 1290-1298, 1253-1271, and 1313-1329) we re phosphorylated on serine, with the serine phosphorylation stimulate d 2-4-fold by insulin. Peptides 1071-1080 and 1290-1298 were 3-7-fold better substrates for the serine phosphorylation than the other serine -phosphorylated peptides. Peptides 1071-1080 and 1313-1329 also exhibi ted insulin-stimulated phosphorylation on tyrosine. Two-dimensional th in-layer tryptic mapping of the phosphorylated insulin receptor/insuli n-stimulated insulin receptor serine kinase preparation or of insulin receptor phosphorylated in human Hep G2 cells yielded two major peptid es, called S1 and S2, that ran as a pair of closely migrating spots, a nd other lesser peptides that contained phosphoserine. S1 and S2 also contained some phosphotyrosine and gave phosphoserine/phosphotyrosine ratios of similar to 6 and 0.96-1.50 for the in vivo and in vitro labe led receptor, respectively. S1 and S2 were not cleaved by V8, Of the s erine-phosphorylated peptides, only 1290-1298 and 1071-1080 should be Vs resistant; 1290-1298 contains serine sites 1293/4 and migrated dist inctly from S1 and S2 in tryptic maps. Peptide 1071-1080 mimicked the production of S1 and S2 in tryptic maps yielding a doublet of phosphop eptides, each containing phosphoserine and phosphotyrosine, which comi grated exactly with S1 and S2. Comigration was confirmed at a differen t pH and by mixing experiments. Radiosequenation showed that serine 10 78 was phosphorylated. Tyrosine 1075 was also phosphorylated, but it w as no more than a minor site in vivo, It is concluded that serine 1078 of the insulin receptor is a major site of insulin-stimulated phospho rylation in vivo and in vitro. The peptide sequences provide a range o f substrates to facilitate the study, purification, and characterizati on of the insulin-stimulated insulin receptor serine kinase or kinases , and the identification of a major site of insulin-stimulated serine phosphorylation will help elucidate the function of the insulin recept or serine phosphorylation.