Harada S, Smith R M, Smith J A, White M F, Jarett L
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
J Biol Chem. 1996 Nov 22;271(47):30222-6. doi: 10.1074/jbc.271.47.30222.
Many studies suggest that insulin utilizes multiple signal transduction pathways. Insulin's effects are initiated by insulin binding to the insulin receptor, resulting in tyrosine phosphorylation of insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1), IRS-2, or Shc. We recently demonstrated that immediate-early gene egr-1 transcription was fully induced without phosphorylation of IRS-1 in Chinese hamster ovary cells (Harada, S., Smith, R. M., Smith, J. A., Shah, N. , Hu, D.-Q. & Jarett, L. (1995) J. Biol. Chem. 270, 26632-26638). In the present study, we examined the effects of insulin on immediate-early gene egr-1 and c-fos expression in 32D cells overexpressing the insulin receptor (32D/IR), IRS-1 (32D/IRS), or both (32D/IR+IRS) and compared these effects with insulin-induced tyrosine phosphorylation. Insulin (17 nM) increased egr-1 and c-fos expression in 32D/IR and 32D/IR+IRS cells, but not in parental cells or 32D/IRS cells, as determined by Northern blot analysis. Insulin treatment (5 min at 37 degrees C) markedly increased tyrosine phosphorylation of several proteins, including the insulin receptor, IRS-1, and Shc, in 32D/IR+IRS cells as determined by immunoprecipitation and Western blot analysis with anti-phosphotyrosine antibody. In contrast, only two tyrosine-phosphorylated proteins, i.e. insulin receptor and Shc, were detected in 32D/IR cells. These data suggest that insulin receptor and Shc phosphorylation is necessary for insulin-induced egr-1 and c-fos expression, but IRS-1 phosphorylation is not necessary or sufficient for the expression of these genes. Furthermore, the effect of specific inhibitors on insulin-induced egr-1 expression was examined. Wortmannin (25 nM), a phosphatidylinositol 3-kinase inhibitor, had no effect on insulin-induced egr-1 expression. In contrast, PD 98059 (30 microM), a mitogen-activated protein kinase kinase inhibitor, totally blocked egr-1 expression induced by insulin. These data indicate that mitogen-activated protein kinase activation, but not phosphatidylinositol 3-kinase activation, is involved in insulin-induced egr-1 expression. Taken together, insulin receptor tyrosine phosphorylation, Shc tyrosine phosphorylation, and mitogen-activated protein kinase activation appear to be the signal transduction pathway responsible for insulin-induced egr-1 expression in 32D cells. These data demonstrate that insulin has multiple signal transduction pathways that vary from cell to cell.
许多研究表明，胰岛素利用多种信号转导途径。胰岛素的作用通过胰岛素与胰岛素受体结合而启动，导致胰岛素受体及细胞内底物如胰岛素受体底物-1（IRS-1）、IRS-2或Shc发生酪氨酸磷酸化。我们最近证明，在中国仓鼠卵巢细胞中，即时早期基因egr-1转录在IRS-1未磷酸化的情况下也能被完全诱导（原田，S.，史密斯，R.M.，史密斯，J.A.，沙阿，N.，胡，D.-Q.和贾勒特，L.（1995年）《生物化学杂志》270，26632 - 26638）。在本研究中，我们检测了胰岛素对过表达胰岛素受体（32D/IR）、IRS-1（32D/IRS）或两者（32D/IR + IRS）的32D细胞中即时早期基因egr-1和c-fos表达的影响，并将这些影响与胰岛素诱导的酪氨酸磷酸化进行比较。通过Northern印迹分析确定，胰岛素（17 nM）增加了32D/IR和32D/IR + IRS细胞中egr-1和c-fos的表达，但在亲本细胞或32D/IRS细胞中未增加。通过用抗磷酸酪氨酸抗体进行免疫沉淀和Western印迹分析确定，胰岛素处理（37℃下5分钟）显著增加了32D/IR + IRS细胞中几种蛋白质的酪氨酸磷酸化，包括胰岛素受体、IRS-1和Shc。相比之下，在32D/IR细胞中仅检测到两种酪氨酸磷酸化蛋白，即胰岛素受体和Shc。这些数据表明，胰岛素受体和Shc磷酸化对于胰岛素诱导的egr-1和c-fos表达是必要的，但IRS-1磷酸化对于这些基因的表达既不是必要的也不是充分的。此外，还检测了特异性抑制剂对胰岛素诱导的egr-1表达的影响。渥曼青霉素（25 nM），一种磷脂酰肌醇3-激酶抑制剂，对胰岛素诱导的egr-1表达没有影响。相比之下，PD 98059（30 microM），一种丝裂原活化蛋白激酶激酶抑制剂，完全阻断了胰岛素诱导的egr-1表达。这些数据表明，丝裂原活化蛋白激酶的激活而非磷脂酰肌醇3-激酶的激活参与了胰岛素诱导的egr-1表达。综上所述，胰岛素受体酪氨酸磷酸化、Shc酪氨酸磷酸化和丝裂原活化蛋白激酶激活似乎是32D细胞中胰岛素诱导的egr-1表达所涉及的信号转导途径。这些数据表明胰岛素具有多种因细胞而异的信号转导途径。
