Ouwens D M, van der Zon G C, Pronk G J, Bos J L, Möller W, Cheatham B, Kahn C R, Maassen J A
Department of Medical Biochemistry, University of Leiden, The Netherlands.
J Biol Chem. 1994 Dec 30;269(52):33116-22.
The activation of p21ras by receptor tyrosine kinases involves the translocation of the growth factor receptor bound protein 2-mammalian son of sevenless protein (Grb2-SOS) complex to the plasma membrane where p21ras is localized. Insulin receptors induce p21ras-GTP formation by two possible mechanisms: tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent association with Grb2, or Shc phosphorylation and its subsequent association with Grb2. We investigated the contribution of the major tyrosine autophosphorylation sites Tyr1158, Tyr1162, and Tyr1163 of the insulin receptor to IRS1.Grb2 and Shc.Grb2 association and the formation of p21ras-GTP. Chinese hamster ovary-derived cell lines were used overexpressing mutant insulin receptors in which the major tyrosine autophosphorylation sites were stepwise replaced by phenylalanines. In cell lines expressing wild type or mutant Y1158F,Y1162,Y1163 (FYY) receptors, insulin stimulated tyrosine phosphorylation of IRS1 and Shc and the formation of IRS1.Grb2 and Shc.Grb2 protein complexes, together with an increase in p21ras-GTP. Cell lines expressing mutant Y1158,Y1162F,Y1163F (YFF) receptors showed insulin-induced tyrosine phosphorylation of Shc, Shc.Grb2 complex formation, and p21ras-GTP formation, whereas tyrosine phosphorylation of IRS1 was strongly decreased and formation of IRS1.Grb2 complexes was undetectable. The activity of FYY and YFF receptors to mediate p21ras-GTP formation correlated with their activity to induce Shc phosphorylation and Shc.Grb2 association. The mutant insulin receptors Y1158F,Y1162F,Y1163 and Y1158F,Y1162F,Y1163F were inactive in inducing any of these responses. We conclude that phosphorylation of Tyr1158 and Tyr1162 of the insulin receptor is linked to distinct post-receptor processes and that YFF receptors generate p21ras-GTP via the Shc.Grb2 pathway rather than one involving IRS1.Grb2 interaction.
受体酪氨酸激酶对p21ras的激活涉及生长因子受体结合蛋白2-哺乳动物七号缺失蛋白之子（Grb2-SOS）复合物向p21ras所在的质膜转位。胰岛素受体通过两种可能的机制诱导p21ras-GTP形成：胰岛素受体底物1（IRS1）的酪氨酸磷酸化及其随后与Grb2的结合，或Shc磷酸化及其随后与Grb2的结合。我们研究了胰岛素受体主要酪氨酸自磷酸化位点Tyr1158、Tyr1162和Tyr1163对IRS1.Grb2和Shc.Grb2结合以及p21ras-GTP形成的作用。使用了过表达突变胰岛素受体的中国仓鼠卵巢来源细胞系，其中主要酪氨酸自磷酸化位点被苯丙氨酸逐步取代。在表达野生型或突变型Y1158F、Y1162、Y1163（FYY）受体的细胞系中，胰岛素刺激了IRS1和Shc的酪氨酸磷酸化以及IRS1.Grb2和Shc.Grb2蛋白复合物的形成，同时p21ras-GTP增加。表达突变型Y1158、Y1162F、Y1163F（YFF）受体的细胞系显示胰岛素诱导的Shc酪氨酸磷酸化、Shc.Grb2复合物形成和p21ras-GTP形成，而IRS1的酪氨酸磷酸化强烈降低且未检测到IRS1.Grb2复合物的形成。FYY和YFF受体介导p21ras-GTP形成的活性与其诱导Shc磷酸化和Shc.Grb2结合的活性相关。突变胰岛素受体Y1158F、Y1162F、Y1163和Y1158F、Y1162F、Y1163F在诱导这些反应中的任何一种时均无活性。我们得出结论，胰岛素受体Tyr1158和Tyr1162的磷酸化与不同的受体后过程相关，并且YFF受体通过Shc.Grb2途径而非涉及IRS1.Grb2相互作用的途径产生p21ras-GTP。
