Rees-Jones R W, Taylor S I
J Biol Chem. 1985 Apr 10;260(7):4461-7.
Insulin binding to its receptor stimulates a tyrosine-specific protein kinase. This enzyme phosphorylates the insulin receptor, as well as a variety of exogenous substrates in vitro. In the present studies, we have identified an endogenous substrate for the insulin receptor-associated kinase. We studied insulin-stimulated protein phosphorylation in partially purified insulin receptor preparations from the livers of dexamethasone-treated rats. In this cell-free system, insulin stimulated the phosphorylation of its own receptor as well as of a phosphoprotein of apparent Mr = 120,000 (termed pp120). pp120 was not immunoprecipitated by three anti-receptor antisera, nor was the receptor immunoprecipitated by antisera raised against pp120, suggesting that pp120 is not antigenically related or tightly bound to the insulin receptor. Dose-response curves for receptor and pp120 phosphorylation stimulated by pork insulin were essentially identical, and showed the appropriate specificity (insulin much greater than proinsulin) for a receptor-mediated event. Phosphoamino acid analysis revealed that insulin stimulated the incorporation of 32P predominantly into tyrosine residues of pp120. Casein, an artificial substrate for the insulin receptor kinase, competed with pp120 for insulin-stimulated phosphorylation. Phosphorylation of pp120 was rapid (half-maximal effect within 2 min at 24 degrees C) and, like receptor phosphorylation, was supported with Mn2+ or Mg2+ as divalent cation and ATP as the phosphate donor. While receptor autophosphorylation and artificial substrate phosphorylation were not altered by prior treatment of the rats with dexamethasone, insulin-stimulated pp120 phosphorylation was enhanced in preparations derived from dexamethasone-treated rats, suggesting an alteration of pp120, not the receptor, as a result of dexamethasone-treatment. Further studies of this newly identified endogenous substrate may help clarify the physiologic role of the insulin receptor-associated kinase.
胰岛素与其受体结合会刺激一种酪氨酸特异性蛋白激酶。这种酶在体外可使胰岛素受体以及多种外源底物磷酸化。在本研究中，我们鉴定出了胰岛素受体相关激酶的一种内源性底物。我们研究了地塞米松处理大鼠肝脏中部分纯化的胰岛素受体制剂中胰岛素刺激的蛋白磷酸化情况。在这个无细胞系统中，胰岛素刺激了其自身受体以及一种表观分子量为120,000的磷蛋白（称为pp120）的磷酸化。三种抗受体抗血清均未免疫沉淀pp120，针对pp120产生的抗血清也未免疫沉淀受体，这表明pp120与胰岛素受体在抗原性上无关或未紧密结合。猪胰岛素刺激受体和pp120磷酸化的剂量反应曲线基本相同，并且显示出受体介导事件所具有的适当特异性（胰岛素远大于胰岛素原）。磷酸氨基酸分析表明，胰岛素刺激32P主要掺入pp120的酪氨酸残基中。酪蛋白是胰岛素受体激酶的一种人工底物，它与pp120竞争胰岛素刺激的磷酸化。pp120的磷酸化很快（在24℃下2分钟内达到最大效应的一半），并且与受体磷酸化一样，以Mn2+或Mg2+作为二价阳离子以及ATP作为磷酸供体时能够发生。虽然大鼠预先用地塞米松处理并未改变受体自身磷酸化和人工底物磷酸化，但地塞米松处理大鼠来源的制剂中胰岛素刺激的pp120磷酸化增强，这表明地塞米松处理导致的是pp120而非受体发生了改变。对这种新鉴定出的内源性底物的进一步研究可能有助于阐明胰岛素受体相关激酶的生理作用。
