Van der Zon G C, Ouwens D M, Dorrestijn J, Maassen J A
Department of Medical Biochemistry, Sylvius Laboratory, Leiden University, The Netherlands.
Biochemistry. 1996 Aug 13;35(32):10377-82. doi: 10.1021/bi960350r.
The families of tyrosine and serine/threonine kinases exhibit shared clusters of conserved amino acid residues. Some conserved residues are confined to the family of tyrosine kinases (TKs), like Tyr at position 1210 in the insulin receptor. Nearly all TKs have at this position Tyr, whereas Ser/Thr kinases generally have Phe at this site. The three-dimensional structure of the insulin receptor TK domain shows Tyr1210 to be located in the cleft, below bound ATP, in a region which potentially contributes to substrate binding. We have examined whether this specific Tyr residue contributes to the generation of TK-specific responses, such as Tyr phosphorylation of Shc, activation of Ras and Erk1,2, and stimulation of DNA synthesis. In addition, we have examined the contribution of Tyr1210 to insulin receptor-specific responses as Tyr phosphorylation of IRS1, stimulation of glycogen synthesis, and dephosphorylation of focal adhesion kinase (FAK). Wild-type and a mutant insulin receptor, in which Tyr1210 was replaced by Phe, were stably expressed in CHO cells, and clones expressing similar numbers of insulin receptors were selected. It was found that replacement of Tyr1210 by Phe resulted in a receptor which was nearly inactive in inducing dephosphorylation of FAK. The mutant receptor was able to induce RasGTP formation, glycogen synthesis, and activation of phosphatidylinositol 3-kinase, though the magnitude of stimulation of some responses was decreased. These findings indicate that Tyr1210 is not essential for the induction of tyrosine kinase-specific responses, such as activation of the Shc/Ras/Erk1,2 pathway and mitogenicity. On the other hand, the abrogation of insulin-induced FAK dephosphorylation indicates that Tyr1210 is involved in coupling of the activated receptor to some downstream targets. Thus, Tyr1210 may fine tune the signal generated by the activated insulin receptor.
酪氨酸激酶家族和丝氨酸/苏氨酸激酶家族展现出保守氨基酸残基的共享簇。一些保守残基局限于酪氨酸激酶（TKs）家族，如胰岛素受体中第1210位的酪氨酸。几乎所有的酪氨酸激酶在该位置都是酪氨酸，而丝氨酸/苏氨酸激酶在这个位点通常是苯丙氨酸。胰岛素受体TK结构域的三维结构显示，酪氨酸1210位于裂隙中，在结合的ATP下方，处于一个可能有助于底物结合的区域。我们研究了这个特定的酪氨酸残基是否有助于产生酪氨酸激酶特异性反应，如Shc的酪氨酸磷酸化、Ras和Erk1、2的激活以及DNA合成的刺激。此外，我们还研究了酪氨酸1210对胰岛素受体特异性反应的作用，如IRS1的酪氨酸磷酸化、糖原合成的刺激以及粘着斑激酶（FAK）的去磷酸化。野生型和酪氨酸1210被苯丙氨酸取代的突变型胰岛素受体在CHO细胞中稳定表达，并选择表达相似数量胰岛素受体的克隆。结果发现，酪氨酸1210被苯丙氨酸取代导致受体在诱导FAK去磷酸化方面几乎没有活性。突变型受体能够诱导RasGTP形成、糖原合成以及磷脂酰肌醇3激酶的激活，尽管某些反应的刺激程度有所降低。这些发现表明，酪氨酸1210对于诱导酪氨酸激酶特异性反应，如Shc/Ras/Erk1、2途径的激活和有丝分裂原性并非必不可少。另一方面，胰岛素诱导的FAK去磷酸化的消除表明，酪氨酸1210参与了活化受体与一些下游靶点的偶联。因此，酪氨酸1210可能对活化的胰岛素受体产生的信号进行微调。
