Häring H U, White M F, Machicao F, Ermel B, Schleicher E, Obermaier B
Proc Natl Acad Sci U S A. 1987 Jan;84(1):113-7. doi: 10.1073/pnas.84.1.113.
It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, we labeled intact rat fat cells with [32P]orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, we found, in addition to the 95-kDa beta-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor beta-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. The insulin effect starts after 30 sec, is maximal at 150 sec, and declines to almost basal values by 5 min. Furthermore, the antiphosphotyrosine antibody precipitated at least five proteins in the soluble fraction of the fat cell. Insulin (0.5 nM, 100 nM) stimulated within 2 min the 32P incorporation into a 116-kDa band, a 62-kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. We suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission.
据推测，胰岛素信号穿过细胞质膜的传递是通过酪氨酸特异性受体激酶的激活、受体的自身磷酸化以及随后细胞中未鉴定底物的磷酸化来实现的。为了鉴定可能的底物，我们用[32P]正磷酸盐标记完整的大鼠脂肪细胞，并使用抗磷酸酪氨酸抗体来鉴定以胰岛素刺激方式在酪氨酸残基上发生磷酸化的蛋白质。在脂肪细胞的膜部分，我们发现，除了受体的95 kDaβ亚基外，还有一种46 kDa的磷蛋白，它仅在酪氨酸残基上发生磷酸化。该蛋白不能被针对胰岛素受体不同区域的抗体免疫沉淀，其HPLC胰蛋白酶肽图与胰岛素受体的胰蛋白酶肽图不同，这表明它不是来自受体β亚基。在完整细胞中，胰岛素在150秒内以剂量依赖方式刺激46 kDa蛋白的酪氨酸磷酸化，在0.5 nM至100 nM的胰岛素浓度下增加3至4倍。胰岛素作用在30秒后开始，在150秒时达到最大值，并在5分钟时降至几乎基础值。此外，抗磷酸酪氨酸抗体在脂肪细胞的可溶性部分沉淀了至少五种蛋白质。胰岛素（0.5 nM，100 nM）在2分钟内刺激32P掺入一条116 kDa带、一条62 kDa带以及45 kDa至50 kDa之间的三条带，增加2至10倍。我们认为，46 kDa膜蛋白以及可能还有可溶性蛋白是脂肪细胞中受体酪氨酸激酶的内源性底物，它们的磷酸化是胰岛素信号传递的早期步骤。
