Insulin receptor kinase domain autophosphorylation regulates receptor enzymatic function.
Insulin receptor kinase domain autophosphorylation regulates receptor enzymatic function.
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DOI：
10.1016/s0021-9258(18)42053-4
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发表时间：
1992-08
期刊：
影响因子：
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通讯作者：
P. Wilden;C. Kahn;K. Siddle;M. White
中科院分区：
文献类型：
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作者：
P. Wilden;C. Kahn;K. Siddle;M. White
我们研究了一系列胰岛素受体分子，其中β亚基激酶结构域中发生自磷酸化的3个酪氨酸残基（Tyr 1158，Tyr 1162和Tyr 1163）通过体外诱变单独、成对或全部被苯丙氨酸或丝氨酸取代。在这三个位置中的每一个处的单个Phe替换减少了用野生型受体观察到的45-60%的胰岛素刺激的溶解受体的自磷酸化。双Phe取代显示出60-70%的减少，并且用Phe或Ser取代所有3个酪氨酸残基使胰岛素刺激的酪氨酸自磷酸化减少超过80%。每个突变体的磷酸肽图谱显示，所有剩余的酪氨酸自磷酸化位点在胰岛素刺激后正常磷酸化，并且没有新的位点出现。与野生型受体激酶活性相比，单苯丙氨酸突变体对合成肽底物的胰岛素刺激激酶活性为50-75%。胰岛素刺激的激酶活性在双苯丙氨酸突变体中进一步降低，在三苯丙氨酸突变体中几乎检测不到。与野生型受体相反，所有的突变体受体激酶在体外胰岛素刺激的自磷酸化后的活化显着减少。当在完整的中国仓鼠卵巢细胞中研究时，胰岛素刺激的受体自磷酸化和酪氨酸磷酸化的细胞底物pp 185在单苯丙氨酸和双苯丙氨酸突变体是逐步降低，增加酪氨酸替代，并没有超过基础水平的三苯丙氨酸突变体。然而，所有突变受体，包括三重Phe突变体，都保留了接受胰岛素刺激的Ser和Thr磷酸化的能力。因此，胰岛素受体酪氨酸激酶的完全活化依赖于激酶结构域的胰岛素刺激的Tris磷酸化，并且激酶结构域中的自磷酸化水平提供了在胰岛素刺激后调节胰岛素受体激酶活性的机制。相比之下，胰岛素刺激受体磷酸化的丝氨酸和苏氨酸残基的细胞丝氨酸/苏氨酸激酶可以发生，尽管显着减少酪氨酸自磷酸化。
We have studied a series of insulin receptor molecules in which the 3 tyrosine residues which undergo autophosphorylation in the kinase domain of the beta-subunit (Tyr1158, Tyr1162, and Tyr1163) were replaced individually, in pairs, or all together with phenylalanine or serine by in vitro mutagenesis. A single-Phe replacement at each of these three positions reduced insulin-stimulated autophosphorylation of solubilized receptor by 45-60% of that observed with wild-type receptor. The double-Phe replacements showed a 60-70% reduction, and substitution of all 3 tyrosine residues with Phe or Ser reduced insulin-stimulated tyrosine autophosphorylation by greater than 80%. Phosphopeptide mapping each mutant revealed that all remaining tyrosine autophosphorylation sites were phosphorylated normally following insulin stimulation, and no new sites appeared. The single-Phe mutants showed insulin-stimulated kinase activity toward a synthetic peptide substrate of 50-75% when compared with wild-type receptor kinase activity. Insulin-stimulated kinase activity was further reduced in the double-Phe mutants and barely detectable in the triple-Phe mutants. In contrast to the wild-type receptor, all of the mutant receptor kinases showed a significant reduction in activation following in vitro insulin-stimulated autophosphorylation. When studied in intact Chinese hamster ovary cells, insulin-stimulated receptor autophosphorylation and tyrosine phosphorylation of the cellular substrate pp185 in the single-Phe and double-Phe mutants was progressively lower with increased tyrosine replacement and did not exceed the basal levels in the triple-Phe mutants. However, all the mutant receptors, including the triple-Phe mutant, retained the ability to undergo insulin-stimulated Ser and Thr phosphorylation. Thus, full activation of the insulin receptor tyrosine kinase is dependent on insulin-stimulated Tris phosphorylation of the kinase domain, and the level of autophosphorylation in the kinase domain provides a mechanism for modulating insulin receptor kinase activity following insulin stimulation. By contrast, insulin stimulation of receptor phosphorylation on Ser and Thr residues by cellular serine/threonine kinases can occur despite markedly reduced tyrosine autophosphorylation.