Sasaoka T, Ishiki M, Sawa T, Ishihara H, Takata Y, Imamura T, Usui I, Olefsky J M, Kobayashi M
First Department of Medicine, Toyama Medical & Pharmaceutical University, Japan.
Endocrinology. 1996 Oct;137(10):4427-34. doi: 10.1210/endo.137.10.8828504.
We compared the intracellular insulin-like growth factor-1 (IGF-1) and insulin signaling pathways in Rat1 fibroblasts expressing the equivalent number of insulin receptors and endogenous IGF-1 receptors. Insulin and IGF-1 stimulated tyrosine phosphorylation of IRS-1 and Shc in a similar dose- and time-dependent manner. The time course of Shc phosphorylation by both IGF-1 and insulin was slower than that of IRS-1. Both phosphorylated IRS-1 and Shc associated with Grb2.Sos complexes, leading to p21ras activation. To compare the functional importance of p21ras for IGF-1-and insulin-induced DNA synthesis, single cell microinjection studies were performed. BrdU incorporation into newly synthesized DNA was measured by immunofluorescence microscopy to assess the functional importance of p21ras. Both IGF-1 and insulin stimulated BrdU incorporation, but the effect of IGF-1 was greater. Microinjection of anti-p21ras antibody completely inhibited both IGF-1-and insulin-induced DNA synthesis, indicating the central role of p21ras in signaling by both hormones. Signal transduction from these receptors to Grb2.Sos complexes can occur through IRS-1 and/or Shc. To assess these two possible pathways, we performed Western blots for Grb2 in anti-Shc and anti-IRS-1 immunoprecipitates and found that 5-fold more Grb2 was associated with Shc than with IRS-1 after either IGF-1 or insulin stimulation. Microinjection of anti-Shc antibody inhibited IGF-1 and insulin stimulation of DNA synthesis by 78% and 74%, respectively. By microinjecting Shc subdomains of GST fusion proteins, we found that Shc N-terminus, but not the Shc SH2, was the functionally important domain through which Shc interacts with IGF-1 and insulin receptors. Insulin stimulation caused hyperphosphorylation and decreased electrophoretic mobility of Sos, and a similar effect was seen with IGF-1, although the time course was delayed compared with insulin. Finally, IGF-1 activated mitogen-activated proten kinase activity more effectively than insulin. These data indicate that Shc, rather than IRS-1, appears to be the predominant functional link to Grb2.Sos complexes from the IGF-1 receptor, as it is from the insulin receptor. Although IGF-1 and insulin stimulate cell cycle progression with similar coupling mechanisms from the receptor to Shc, to Grb2.Sos, to p21ras, the delayed IGF-1 induced mobility shift of Sos could lead to, at least in part, more efficient coupling to mitogen-activated protein kinase. These findings might explain the greater mitogenic activity of IGF-1 compared with insulin.
我们比较了在表达等量胰岛素受体和内源性胰岛素样生长因子-1（IGF-1）受体的大鼠1成纤维细胞中，细胞内胰岛素样生长因子-1（IGF-1）和胰岛素信号通路。胰岛素和IGF-1以相似的剂量和时间依赖性方式刺激胰岛素受体底物-1（IRS-1）和Shc的酪氨酸磷酸化。IGF-1和胰岛素对Shc的磷酸化时间进程均慢于IRS-1。磷酸化的IRS-1和Shc均与生长因子受体结合蛋白2（Grb2）-七号染色体失活蛋白（Sos）复合物结合，导致p21ras激活。为比较p21ras对IGF-1和胰岛素诱导的DNA合成的功能重要性，进行了单细胞显微注射研究。通过免疫荧光显微镜测量5-溴脱氧尿嘧啶核苷（BrdU）掺入新合成的DNA中，以评估p21ras的功能重要性。IGF-1和胰岛素均刺激BrdU掺入，但IGF-1的作用更强。显微注射抗p21ras抗体完全抑制了IGF-1和胰岛素诱导的DNA合成，表明p21ras在两种激素的信号传导中起核心作用。从这些受体到Grb2-Sos复合物的信号转导可通过IRS-1和/或Shc发生。为评估这两种可能的途径，我们对抗Shc和抗IRS-1免疫沉淀物中的Grb2进行了蛋白质免疫印迹分析，发现在IGF-1或胰岛素刺激后，与Shc结合的Grb2比与IRS-1结合的多5倍。显微注射抗Shc抗体分别抑制了IGF-1和胰岛素对DNA合成的刺激作用的78%和74%。通过显微注射谷胱甘肽S-转移酶（GST）融合蛋白的Shc亚结构域，我们发现Shc的N末端而非Shc的Src同源2（SH2）结构域是Shc与IGF-1和胰岛素受体相互作用的功能重要结构域。胰岛素刺激导致Sos过度磷酸化并降低其电泳迁移率，IGF-1也有类似作用，尽管与胰岛素相比时间进程延迟。最后，IGF-1比胰岛素更有效地激活丝裂原活化蛋白激酶活性。这些数据表明，与胰岛素受体一样，Shc而非IRS-1似乎是IGF-1受体与Grb2-Sos复合物的主要功能连接。尽管IGF-1和胰岛素通过从受体到Shc、到Grb2-Sos、到p21ras的相似偶联机制刺激细胞周期进程，但IGF-1诱导的Sos迁移率变化延迟可能至少部分导致与丝裂原活化蛋白激酶的偶联更有效。这些发现可能解释了IGF-1比胰岛素更强的促有丝分裂活性。
