Kim B, Cheng H L, Margolis B, Feldman E L
Neuroscience Program and Department of Neurology, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1998 Dec 18;273(51):34543-50. doi: 10.1074/jbc.273.51.34543.
The major substrates for the type I insulin-like growth factor (IGF-I) receptor are Shc and insulin receptor substrate (IRS) proteins. In the current study, we report that IGF-I induces a sustained tyrosine phosphorylation of Shc and its association with Grb2 in SH-SY5Y human neuroblastoma cells. The time course of Shc tyrosine phosphorylation parallels the time course of IGF-I-stimulated activation of extracellular signal-regulated kinase (ERK). Transfection of SH-SY5Y cells with a p52 Shc mutant decreases Shc tyrosine phosphorylation and Shc-Grb2 association. This results in the inhibition of IGF-I-mediated ERK tyrosine phosphorylation and neurite outgrowth. In contrast, IGF-I induces a transient tyrosine phosphorylation of IRS-2 and an association of IRS-2 with Grb2. The time course of IRS-2 tyrosine phosphorylation and IRS-2-Grb2 and IRS-2-p85 association closely resembles the time course of IGF-I-mediated membrane ruffling. Treating cells with the phosphatidylinositol 3'-kinase inhibitors wortmannin and LY294002 blocks IGF-I-induced membrane ruffling. The ERK kinase inhibitor PD98059, as well as transfection with the p52 Shc mutant, has no effect on IGF-I-mediated membrane ruffling. Immunolocalization studies show IRS-2 and Grb2, but not Shc, concentrated at the tip of the extending growth cone where membrane ruffling is most active. Collectively, these results suggest that the association of Shc with Grb2 is essential for IGF-I-mediated neurite outgrowth, whereas the IRS-2-Grb2-phosphatidylinositol 3'-kinase complex may regulate growth cone extension and membrane ruffling.
I型胰岛素样生长因子（IGF-I）受体的主要底物是Shc和胰岛素受体底物（IRS）蛋白。在本研究中，我们报告IGF-I在SH-SY5Y人神经母细胞瘤细胞中诱导Shc持续的酪氨酸磷酸化及其与Grb2的结合。Shc酪氨酸磷酸化的时间进程与IGF-I刺激的细胞外信号调节激酶（ERK）激活的时间进程平行。用p52 Shc突变体转染SH-SY5Y细胞可降低Shc酪氨酸磷酸化和Shc-Grb2结合。这导致IGF-I介导的ERK酪氨酸磷酸化和神经突生长受到抑制。相反，IGF-I诱导IRS-2短暂的酪氨酸磷酸化以及IRS-2与Grb2的结合。IRS-2酪氨酸磷酸化以及IRS-2-Grb2和IRS-2-p85结合的时间进程与IGF-I介导的膜皱襞的时间进程非常相似。用磷脂酰肌醇3'-激酶抑制剂渥曼青霉素和LY294002处理细胞可阻断IGF-I诱导的膜皱襞。ERK激酶抑制剂PD98059以及用p52 Shc突变体转染对IGF-I介导的膜皱襞没有影响。免疫定位研究显示IRS-2和Grb2，而不是Shc，集中在延伸生长锥的尖端，那里膜皱襞最活跃。总的来说，这些结果表明Shc与Grb2的结合对于IGF-I介导的神经突生长至关重要，而IRS-2-Grb2-磷脂酰肌醇3'-激酶复合物可能调节生长锥延伸和膜皱襞。
