Abstract:
:The identity of the sites of insulin-stimulated serine phosphorylation in the human insulin receptor was examined by synthesizing peptides that together encompassed all the serine residues of the cytosolic portion of the beta-subunit and testing them as substrates for phosphorylation by a preparation of human insulin receptor copurified with insulin-stimulated insulin receptor serine kinase activity. Of the 14 peptides studied, only 4 (1071--1080, 1290--1298, 1253--1271, and 1313--1329) were phosphorylated on serine, with the serine phosphorylation stimulated 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 exhibited insulin-stimulated phosphorylation on tyrosine. Two-dimensional thin-layer tryptic mapping of the phosphorylated insulin receptor/insulin-stimulated insulin receptor serine kinase preparation or of insulin receptor phosphorylated in human Hep G2 cells yielded two major peptides, called S1 and S2, that ran as a pair of closely migrating spots, and other lesser peptides that contained phosphoserine. S1 and S2 also contained some phosphotyrosine and gave phosposerine/phosphotyrosine ratios of approximately 6 and 0.96-1.50 for the in vivo and in vitro labeled receptor, respectively. S1 and S2 were not cleaved by V8. Of the serine-phosphorylated peptides, only 1290--1298 and 1071--1080 should be V8 resistant; 1290--1298 contains serine sites 1293/4 and migrated distinctly from S1 and S2 in tryptic maps. Peptide 1071--1080 mimicked the production of S1 and S2 in tryptic maps yielding a doublet of phosphopeptides, each containing phosphoserine and phosphotyrosine, which comigrated exactly with S1 and S2. Comigration was confirmed at a different pH and by mixing experiments. Radiosequenation showed that serine 1078 was phosphorylated. Tyrosine 1075 was also phosphorylated, but it was 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 phosphorylation in vivo and in vitro. The peptide sequences provide a range of substrates to facilitate the study, purification, and characterization 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 receptor serine phosphorylation.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Carter WG,Asamoah KA,Sale GJdoi
10.1021/bi00029a025subject
Has Abstractpub_date
1995-07-25 00:00:00pages
9488-99issue
29eissn
0006-2960issn
1520-4995journal_volume
34pub_type
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