Tobe K, Matuoka K, Tamemoto H, Ueki K, Kaburagi Y, Asai S, Noguchi T, Matsuda M, Tanaka S, Hattori S
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
J Biol Chem. 1993 May 25;268(15):11167-71.
Insulin activates the ras proto-oncogene product p21ras (Ras) by stimulating conversion of the inactive GDP-bound form of Ras to the active GTP-bound form. The protein ASH (for abundant Src homology) (Matuoka, K., Shibata, M., Yamakawa, A., and Takenawa, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9015-9019) is composed of one Src homology (SH)2 and two SH3 domains and highly homologous to the Caenorhabditis elegans protein sem-5 that couples a tyrosine kinase to a Ras protein. We have studied an interaction of ASH with insulin-stimulated tyrosine-phosphorylated proteins in Chinese hamster ovary cells overexpressing human insulin receptors (CHO-HIR cells). In an anti-ASH (alpha ASH) immunoprecipitates, we detected a 170-kDa phosphoprotein that was recognized by an anti-phosphotyrosine antibody and an anti-insulin receptor substrate 1 antibody (alpha IRS-1) from the insulin-stimulated [32P]orthophosphate-labeled CHO-HIR cells. We failed to detect the tyrosine phosphorylation of the protein ASH. These data suggested that insulin stimulates IRS-1.ASH complex formation in intact cells. Incubation of an ASH fusion protein with the lysates of insulin-stimulated CHO-HIR cells revealed that the fusion protein of ASH was able to bind the tyrosine-phosphorylated 170-kDa protein that was recognized by alpha IRS-1. We also demonstrated that fusion protein of ASH was able to bind the fusion protein of tyrosine-phosphorylated IRS-1 fragments, suggesting that ASH is able to bind tyrosine-phosphorylated IRS-1 directly. These data suggest that IRS-1.ASH complex formation may play a role in coupling the insulin receptor kinase to a Ras signaling pathway. Furthermore, we observed an insulin-stimulated phosphatidylinositol (PI) 3-kinase activity in alpha ASH immunoprecipitates, suggesting the formation of an ASH.IRS-1.PI 3-kinase complex. This complex formation was detected as early as 10 s after insulin stimulation in intact CHO-HIR cells. This is the first report that supports the notion that IRS-1 binds several signal transducing molecules containing SH2 domains, thus serves as an SH2 docking protein that transduces insulin's signal multidirectionally.
胰岛素通过刺激将无活性的结合GDP形式的Ras转化为有活性的结合GTP形式，从而激活原癌基因产物p21ras（Ras）。ASH蛋白（富含Src同源结构域）（Matuoka, K., Shibata, M., Yamakawa, A., and Takenawa, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9015 - 9019）由一个Src同源（SH）2结构域和两个SH3结构域组成，与秀丽隐杆线虫中连接酪氨酸激酶和Ras蛋白的蛋白sem - 5高度同源。我们研究了在过表达人胰岛素受体的中国仓鼠卵巢细胞（CHO - HIR细胞）中ASH与胰岛素刺激的酪氨酸磷酸化蛋白之间的相互作用。在抗ASH（αASH）免疫沉淀中，我们从胰岛素刺激的[32P]正磷酸盐标记的CHO - HIR细胞中检测到一种170 kDa的磷蛋白，它能被抗磷酸酪氨酸抗体和抗胰岛素受体底物1抗体（αIRS - 1）识别。我们未能检测到ASH蛋白的酪氨酸磷酸化。这些数据表明胰岛素在完整细胞中刺激IRS - 1 - ASH复合物的形成。将ASH融合蛋白与胰岛素刺激的CHO - HIR细胞裂解物一起孵育发现，ASH融合蛋白能够结合被αIRS - 1识别的酪氨酸磷酸化的170 kDa蛋白。我们还证明ASH融合蛋白能够结合酪氨酸磷酸化的IRS - 1片段的融合蛋白，这表明ASH能够直接结合酪氨酸磷酸化的IRS - 1。这些数据表明IRS - 1 - ASH复合物的形成可能在将胰岛素受体激酶与Ras信号通路偶联中起作用。此外，我们在αASH免疫沉淀中观察到胰岛素刺激的磷脂酰肌醇（PI）3激酶活性，提示形成了ASH - IRS - 1 - PI 3激酶复合物。在完整的CHO - HIR细胞中，胰岛素刺激后最早10秒就能检测到这种复合物的形成。这是第一份支持以下观点的报告：IRS - 1结合多个含有SH2结构域的信号转导分子，因此作为一种SH2对接蛋白，多向传递胰岛素信号。
