Yu K T, Czech M P
J Biol Chem. 1986 Apr 5;261(10):4715-22.
The phosphorylation characteristics of insulin receptor from control and insulin-treated rat H-35 hepatoma cells 32P-labeled to equilibrium have been documented. The 32P-labeled insulin receptor is isolated by immunoprecipitation with patient-derived insulin receptor antibodies in the presence of phosphatase and protease inhibitors to preserve the native phosphorylation and structural characteristics of the receptor. The unstimulated insulin receptor contains predominantly [32P] phosphoserine and trace amounts of [32P]phosphothreonine in its beta subunit. In response to insulin, the insulin receptor beta subunit exhibits marked tyrosine phosphorylation and a 2-fold increase in total [32P]phosphoserine contents. High pressure liquid chromatography of the tryptic hydrolysates of the 32P-labeled receptor beta subunit from quiescent cells results in the resolution of up to 9 fractions containing [32P]phosphoserine. The insulin-stimulated tyrosine phosphorylation is concentrated in two of these receptor phosphopeptide fractions, whereas the increase in [32P]phosphoserine content is scattered in low abundance over all receptor tryptic fractions. Insulin receptors affinity-purified by lectin- and insulin-agarose chromatographies from insulin-treated, 32P-labeled cells exhibit a 22-fold increase in the Vmax of receptor tyrosine kinase activity toward histone when compared to controls. The elevated kinase activity of the insulin receptor derived from insulin-treated cells is not due to the presence of hormone bound to the receptor because the receptor kinase activity is assayed while immobilized on insulin-agarose. Furthermore, the insulin-activated receptor kinase activity is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase in vitro. The correlation between the insulin-stimulated site specific tyrosine phosphorylation on receptor beta subunit and the elevation of receptor tyrosine kinase activity strongly suggests that the insulin receptor kinase is activated by hormone-stimulated autophosphorylation on tyrosine residues in intact cells, as previously demonstrated for the purified receptor.
已记录了来自对照和胰岛素处理的大鼠H-35肝癌细胞且用32P标记至平衡状态的胰岛素受体的磷酸化特征。在存在磷酸酶和蛋白酶抑制剂的情况下，用患者来源的胰岛素受体抗体通过免疫沉淀分离32P标记的胰岛素受体，以保留受体的天然磷酸化和结构特征。未受刺激的胰岛素受体在其β亚基中主要含有[32P]磷酸丝氨酸和痕量的[32P]磷酸苏氨酸。响应胰岛素时，胰岛素受体β亚基表现出明显的酪氨酸磷酸化，并且总[32P]磷酸丝氨酸含量增加2倍。对静止细胞中32P标记的受体β亚基的胰蛋白酶水解产物进行高压液相色谱分析，可分离出多达9个含有[32P]磷酸丝氨酸的组分。胰岛素刺激的酪氨酸磷酸化集中在其中两个受体磷酸肽组分中，而[32P]磷酸丝氨酸含量的增加则以低丰度分散在所有受体胰蛋白酶组分中。通过凝集素和胰岛素-琼脂糖色谱法从胰岛素处理的32P标记细胞中亲和纯化的胰岛素受体，与对照相比，其对组蛋白的受体酪氨酸激酶活性的Vmax增加了22倍。来自胰岛素处理细胞的胰岛素受体激酶活性升高并非由于受体结合有激素，因为受体激酶活性是在固定于胰岛素-琼脂糖上时进行测定的。此外，体外使用碱性磷酸酶使受体β亚基去磷酸化后，胰岛素激活的受体激酶活性会逆转。受体β亚基上胰岛素刺激的位点特异性酪氨酸磷酸化与受体酪氨酸激酶活性升高之间的相关性强烈表明，胰岛素受体激酶在完整细胞中通过激素刺激的酪氨酸残基自磷酸化而被激活，如先前对纯化受体所证明的那样。
