Ishihara H, Sasaoka T, Wada T, Ishiki M, Haruta T, Usui I, Iwata M, Takano A, Uno T, Ueno E, Kobayashi M
Toyama Medical & Pharmaceutical University, Toyama, 930-0194, Japan.
Biochem Biophys Res Commun. 1998 Nov 9;252(1):139-44. doi: 10.1006/bbrc.1998.9621.
Shc is phosphorylated on Tyr-239/240 and/or Tyr-317, which serves as a docking site for Grb2. To clarify the relative involvement of Shc Tyr-239/240 and Tyr-317 in insulin-induced mitogenesis, we generated expression vectors for Y317F (1F)-Shc, Y239/240F (2F)-Shc, and Y239/240/317F (3F)-Shc, and stably transfected them into Rat1 fibroblasts expressing insulin receptors (HIRc). Insulin-induced Shc phosphorylation and subsequent association with Grb2 was enhanced in wild-type (WT)-Shc cell. In contrast, insulin-stimulated Shc phosphorylation and Shc.Grb2 association were significantly decreased in 1F-Shc and 3F-Shc cells, while these were only slightly affected and almost comparable in 2F cells compared with those in parental HIRc cells. The kinetics of MAP kinase activation closely paralleled the kinetics of Shc phosphorylation and Shc.Grb2 association. Thus, insulin stimulation of MAP kinase activation occurred more rapidly in WT-Shc cells, and the activation was delayed in 1F-Shc and 3F-Shc cells, while it was comparable in 2F-Shc cells compared with that in HIRc cells. Furthermore, WT-Shc cells displayed enhanced sensitivity to insulin stimulation of thymidine incorporation. Importantly, the sensitivity was significantly decreased in 1F-Shc and 3F-Shc cells, while it was almost comparable in 2F-Shc cells compared with that in HIRc cells. These results indicate that Shc Tyr-317 is more predominant insulin-induced phosphorylation site than Tyr-239/240 for coupling with Grb2 leading to MAP kinase activation and mitogenesis in Rat1 fibroblasts.
Shc在酪氨酸239/240和/或酪氨酸317位点发生磷酸化，该位点作为Grb2的停靠位点。为了阐明Shc酪氨酸239/240和酪氨酸317在胰岛素诱导的有丝分裂中的相对作用，我们构建了Y317F（1F）-Shc、Y239/240F（2F）-Shc和Y239/240/317F（3F）-Shc的表达载体，并将它们稳定转染到表达胰岛素受体的大鼠1型成纤维细胞（HIRc）中。在野生型（WT）-Shc细胞中，胰岛素诱导的Shc磷酸化及随后与Grb2的结合增强。相比之下，在1F-Shc和3F-Shc细胞中，胰岛素刺激的Shc磷酸化及Shc与Grb2的结合显著降低，而在2F细胞中，与亲代HIRc细胞相比，这些仅受到轻微影响且几乎相当。MAP激酶激活的动力学与Shc磷酸化及Shc与Grb2结合的动力学密切平行。因此，在WT-Shc细胞中，胰岛素刺激MAP激酶激活发生得更快，而在1F-Shc和3F-Shc细胞中激活延迟，而在2F-Shc细胞中与HIRc细胞相比相当。此外，WT-Shc细胞对胰岛素刺激胸苷掺入表现出增强的敏感性。重要的是，在1F-Shc和3F-Shc细胞中敏感性显著降低，而在2F-Shc细胞中与HIRc细胞相比几乎相当。这些结果表明，在大鼠1型成纤维细胞中，对于与Grb2偶联导致MAP激酶激活和有丝分裂而言，Shc酪氨酸317是比酪氨酸239/240更主要的胰岛素诱导磷酸化位点。
