Yonezawa K, Ando A, Kaburagi Y, Yamamoto-Honda R, Kitamura T, Hara K, Nakafuku M, Okabayashi Y, Kadowaki T, Kaziro Y
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
J Biol Chem. 1994 Feb 11;269(6):4634-40.
We have examined the involvement of insulin receptor (IR) substrate-1 (IRS-1) and/or Shc in the upstream of Ras activation in insulin signaling using Chinese hamster ovary (CHO) cell lines overexpressing wild-type (CHO-IR) cells) or mutant insulin receptors. In CHO-IR cells, insulin rapidly phosphorylated IRS-1 and Shc at tyrosine residues and stimulated the formation of the active GTP-bound Ras (Ras.GTP). In contrast, a CHO cell line overexpressing the kinase-negative mutant insulin receptor substituting Arg1018 for Lys1018 was unable to tyrosine-phosphorylate IRS-1 and Shc and failed to activate Ras in response to insulin. A CHO cell line overexpressing the mutant insulin receptor, substituting Ala960 for Tyr960 and which was known to exhibit impaired tyrosine phosphorylation of IRS-1 and biological effects evoked by insulin, showed severely impaired insulin-dependent tyrosine phosphorylation of Shc and moderately impaired activation of Ras. Another cell line overexpressing the mutant insulin receptor, lacking 82 amino acids of the C terminus of beta-subunit and which was recently reported to retain normal insulin-dependent tyrosine phosphorylation of IRS-1, showed slightly impaired Ras activation at 10(-7) M insulin with severely reduced tyrosine phosphorylation of Shc protein. Furthermore, insulin did not induce the association of tyrosine-phosphorylated IRS-1 and Shc in CHO-IR cells. These results suggest that Shc and IRS-1 lie in the separate signaling pathways and that the tyrosine phosphorylation of IRS-1 with or without some low level of Shc phosphorylation may be enough to stimulate the submaximal accumulation of Ras.GTP complex and may need synergistically the higher level of tyrosine phosphorylation of Shc to induce the full activation of Ras in insulin signaling.
我们利用过表达野生型（CHO-IR细胞）或突变型胰岛素受体的中国仓鼠卵巢（CHO）细胞系，研究了胰岛素受体（IR）底物-1（IRS-1）和/或Shc在胰岛素信号转导中Ras激活上游的作用。在CHO-IR细胞中，胰岛素能迅速使IRS-1和Shc的酪氨酸残基磷酸化，并刺激活性GTP结合型Ras（Ras.GTP）的形成。相比之下，过表达将赖氨酸1018替换为精氨酸1018的激酶负性突变型胰岛素受体的CHO细胞系，无法使IRS-1和Shc酪氨酸磷酸化，且对胰岛素无反应，无法激活Ras。过表达将酪氨酸960替换为丙氨酸且已知IRS-1酪氨酸磷酸化受损及胰岛素诱发的生物学效应受损的突变型胰岛素受体的CHO细胞系，Shc的胰岛素依赖性酪氨酸磷酸化严重受损，Ras激活中度受损。另一个过表达突变型胰岛素受体的细胞系，其β亚基C末端缺失82个氨基酸，最近报道该细胞系IRS-1的胰岛素依赖性酪氨酸磷酸化正常，在10^(-7) M胰岛素作用下，Ras激活略有受损，Shc蛋白酪氨酸磷酸化严重降低。此外，胰岛素未诱导CHO-IR细胞中酪氨酸磷酸化的IRS-1与Shc结合。这些结果表明，Shc和IRS-1位于不同的信号通路中，IRS-1的酪氨酸磷酸化无论有无一定程度的Shc磷酸化，可能足以刺激Ras.GTP复合物的次最大积累，且可能需要Shc更高水平的酪氨酸磷酸化协同作用，以诱导胰岛素信号转导中Ras的完全激活。
