White M F, Maron R, Kahn C R
Nature. 1985;318(6042):183-6. doi: 10.1038/318183a0.
Phosphotyrosine-containing proteins are minor components of normal cells which appear to be associated primarily with the regulation of cellular metabolism and growth. The insulin receptor is a tyrosine-specific protein kinase, and one of the earliest detectable responses to insulin binding is activation of this kinase and autophosphorylation of its beta-subunit. Tyrosine autophosphorylation activates the phosphotransferase in the beta-subunit and increases its reactivity toward tyrosine phosphorylation of other substrates. When incubated in vitro with [gamma-32P]ATP and insulin, the purified insulin receptor phosphorylates various proteins on their tyrosine residues. However, so far no proteins other than the insulin receptor have been identified as undergoing tyrosine phosphorylation in response to insulin in an intact cell. Here, using anti-phosphotyrosine antibodies, we have identified a novel phosphotyrosine-containing protein of relative molecular mass (Mr) 185,000 (pp185) which appears during the initial response of hepatoma cells to insulin binding. In contrast to the insulin receptor, pp185 does not adhere to wheat-germ agglutininagarose or bind to anti-insulin receptor antibodies. Phosphorylation of pp185 is maximal within seconds after exposure of the cells to insulin and exhibits a dose-response curve similar to that of receptor autophosphorylation, suggesting that this protein represents the endogenous substrate for the insulin receptor kinase.
含磷酸酪氨酸的蛋白质是正常细胞的次要成分，似乎主要与细胞代谢和生长的调节相关。胰岛素受体是一种酪氨酸特异性蛋白激酶，对胰岛素结合最早可检测到的反应之一是该激酶的激活及其β亚基的自磷酸化。酪氨酸自磷酸化激活β亚基中的磷酸转移酶，并增加其对其他底物酪氨酸磷酸化的反应性。当与[γ-32P]ATP和胰岛素在体外孵育时，纯化的胰岛素受体使其酪氨酸残基上的各种蛋白质磷酸化。然而，到目前为止，除了胰岛素受体之外，尚未鉴定出在完整细胞中响应胰岛素而发生酪氨酸磷酸化的其他蛋白质。在此，我们使用抗磷酸酪氨酸抗体，鉴定出一种相对分子质量（Mr）为185,000的新型含磷酸酪氨酸的蛋白质（pp185），它在肝癌细胞对胰岛素结合的初始反应过程中出现。与胰岛素受体不同，pp185不粘附于麦胚凝集素琼脂糖，也不与抗胰岛素受体抗体结合。pp185的磷酸化在细胞暴露于胰岛素后数秒内达到最大值，并呈现出与受体自磷酸化相似的剂量反应曲线，表明该蛋白质代表胰岛素受体激酶的内源性底物。
