Carter W G, Asamoah K A, Sale G J
Department of Biochemistry, School of Biological Sciences, University of Southampton, U.K.
Biochemistry. 1995 Jul 25;34(29):9488-99. doi: 10.1021/bi00029a025.
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.
通过合成覆盖β亚基胞质部分所有丝氨酸残基的肽段，并将其作为与胰岛素刺激的胰岛素受体丝氨酸激酶活性共纯化的人胰岛素受体制剂的磷酸化底物进行测试，研究了人胰岛素受体中胰岛素刺激的丝氨酸磷酸化位点。在所研究的14个肽段中，只有4个（1071 - 1080、1290 - 1298、1253 - 1271和1313 - 1329）在丝氨酸上发生了磷酸化，胰岛素可使丝氨酸磷酸化增加2 - 4倍。肽段1071 - 1080和1290 - 1298作为丝氨酸磷酸化的底物，比其他丝氨酸磷酸化肽段要好3 - 7倍。肽段1071 - 1080和1313 - 1329在酪氨酸上也表现出胰岛素刺激的磷酸化。对磷酸化的胰岛素受体/胰岛素刺激的胰岛素受体丝氨酸激酶制剂或在人肝癌细胞系Hep G2中磷酸化的胰岛素受体进行二维薄层层析胰蛋白酶图谱分析，得到两个主要的肽段，称为S1和S2，它们以一对紧密迁移的斑点形式出现，还有其他含有磷酸丝氨酸的较小肽段。S1和S2也含有一些磷酸酪氨酸，体内和体外标记受体的磷酸丝氨酸/磷酸酪氨酸比值分别约为6和0.96 - 1.50。S1和S2不能被V8蛋白酶切割。在丝氨酸磷酸化的肽段中，只有1290 - 1298和1071 - 1080应该对V8蛋白酶有抗性；1290 - 1298包含丝氨酸位点1293/4，在胰蛋白酶图谱中与S1和S2明显迁移不同。肽段1071 - 1080在胰蛋白酶图谱中模拟了S1和S2的产生，产生了一对磷酸肽，每个都含有磷酸丝氨酸和磷酸酪氨酸，它们与S1和S2完全共迁移。在不同pH值下以及通过混合实验证实了共迁移。放射性测序表明丝氨酸1078发生了磷酸化。酪氨酸1075也发生了磷酸化，但在体内它只是一个次要位点。结论是胰岛素受体的丝氨酸1078是体内和体外胰岛素刺激的磷酸化的主要位点。这些肽段序列提供了一系列底物，便于研究、纯化和鉴定胰岛素刺激的胰岛素受体丝氨酸激酶，并且确定胰岛素刺激的丝氨酸磷酸化的主要位点将有助于阐明胰岛素受体丝氨酸磷酸化的功能。
