Ray L B, Sturgill T W
Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville 22908.
Proc Natl Acad Sci U S A. 1988 Jun;85(11):3753-7. doi: 10.1073/pnas.85.11.3753.
Exposure of 3T3-L1 cells to insulin stimulates a soluble, serine(threonine)-specific protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) in vitro. The enzyme, termed MAP kinase, was isolated from insulin-treated or control cells radiolabeled with 32Pi. A 40-kDa phosphoprotein was found to elute in exact correspondence with enzymatic activity during hydrophobic interaction and gel filtration chromatography of extracts from cells stimulated with insulin. Both MAP kinase activity and the phosphoprotein were absent in fractions prepared from untreated cells. The 32P incorporated into the 40-kDa protein was stable during treatment with alkali. Phospho amino acid analysis confirmed that the radiolabel was primarily incorporated into phosphotyrosine and to a lesser extent phosphothreonine. In addition, MAP kinase was incompletely but specifically adsorbed by antibodies to phosphotyrosine. We conclude, based on these data and additional studies from this laboratory, that MAP kinase is phosphorylated on tyrosine in vivo. The data are consistent with the possibility that MAP kinase may be a substrate for the insulin receptor or another insulin-regulated tyrosine kinase.
将3T3-L1细胞暴露于胰岛素会刺激一种可溶性的丝氨酸（苏氨酸）特异性蛋白激酶，该激酶在体外可使微管相关蛋白2（MAP-2）磷酸化。这种酶被称为MAP激酶，是从用32Pi进行放射性标记的胰岛素处理细胞或对照细胞中分离出来的。在用胰岛素刺激的细胞提取物进行疏水相互作用和凝胶过滤层析时，发现一种40 kDa的磷蛋白与酶活性完全对应地洗脱出来。从未经处理的细胞制备的组分中既没有MAP激酶活性，也没有这种磷蛋白。掺入40 kDa蛋白中的32P在用碱处理时是稳定的。磷酸氨基酸分析证实，放射性标记主要掺入磷酸酪氨酸，其次是磷酸苏氨酸。此外，MAP激酶被抗磷酸酪氨酸抗体不完全但特异性地吸附。基于这些数据以及本实验室的其他研究，我们得出结论，MAP激酶在体内酪氨酸位点被磷酸化。这些数据与MAP激酶可能是胰岛素受体或另一种胰岛素调节的酪氨酸激酶的底物这一可能性是一致的。
