Stadtmauer L, Rosen O M
J Biol Chem. 1986 Jul 25;261(21):10000-5.
Three peptides were synthesized corresponding to potential autophosphorylation sites of the beta subunit of the human insulin receptor. These were peptide 1150 corresponding to amino acids 1142-1153 of the pro-receptor, peptide 960 corresponding to amino acids 952-961 of the proreceptor, and peptide 1316 corresponding to amino acids 1313-1329 of the proreceptor. Peptide 1150 served as a better substrate for the insulin receptor tyrosine protein kinase than either of the other peptides or than the Src peptide (corresponding to the sequence surrounding the autophosphorylation site at Tyr-416). Microsequencing of the phosphorylated peptide 1150 indicated that Tyr-1150 rather than Tyr-1146 or Tyr-1151 was phosphorylated in the in vitro reaction. The insulin receptor was then isolated from 32P-labeled IM-9 cells that had been exposed to insulin. Tryptic digestion of the beta subunit revealed one peptide whose phosphorylation was dependent upon insulin and occurred exclusively on Tyr. This peptide was selectively immunoprecipitated by an antipeptide antibody directed to the Tyr-1150-containing sequence. We conclude that Tyr-1150 is preferentially phosphorylated by the purified receptor kinase and that one of the autophosphorylation reactions elicited by insulin in intact cells occurs in a sequence that contains this residue.
合成了三种与人类胰岛素受体β亚基潜在自磷酸化位点相对应的肽段。它们分别是肽段1150，对应于前体受体的第1142 - 1153位氨基酸；肽段960，对应于前体受体的第952 - 961位氨基酸；以及肽段1316，对应于前体受体的第1313 - 1329位氨基酸。与其他肽段或Src肽段（对应于Tyr - 416处自磷酸化位点周围的序列）相比，肽段1150是胰岛素受体酪氨酸蛋白激酶更好的底物。对磷酸化的肽段1150进行微量测序表明，在体外反应中被磷酸化的是Tyr - 1150，而非Tyr - 1146或Tyr - 1151。随后从暴露于胰岛素的32P标记的IM - 9细胞中分离出胰岛素受体。对β亚基进行胰蛋白酶消化后发现一个肽段，其磷酸化依赖于胰岛素且仅发生在酪氨酸上。该肽段被针对含Tyr - 1150序列的抗肽抗体选择性免疫沉淀。我们得出结论，Tyr - 1150被纯化的受体激酶优先磷酸化，并且胰岛素在完整细胞中引发的自磷酸化反应之一发生在包含该残基的序列中。
