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 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.
Carter
W G
WG
Department of Biochemistry, School of Biological Sciences, University of Southampton, U.K.
Asamoah
K A
KA
Sale
G J
GJ
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010768:Phosphoserine / Q000032:analysis; D019000:Phosphotyrosine; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011972:Receptor, Insulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012694:Serine; D013379:Substrate Specificity; D014357:Trypsin; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D007328:Insulin; D010748:Phosphopeptides; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine; D012694:Serine; D011972:Receptor, Insulin; D014357:Trypsin