An Mr 180,000 protein is an endogenous substrate for the insulin-receptor-associated tyrosine kinase in human placenta.
An Mr 180,000 protein is an endogenous substrate for the insulin-receptor-associated tyrosine kinase in human placenta.
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Mr 180,000 蛋白是人胎盘中胰岛素受体相关酪氨酸激酶的内源性底物。
DOI：
10.1042/bj2430797
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
O. Wieland
中科院分区：
文献类型：
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作者：
F. Machicao;H. Häring;M. White;J. Carrascosa;B. Obermaier;O. Wieland
胰岛素受体的β亚基含有一种酪氨酸特异性的蛋白激酶。胰岛素结合激活该激酶，导致胰岛素受体的β-亚基的磷酸化。人们认为，其他蛋白质的磷酸化可能会将胰岛素信号从受体传递到细胞。在本研究中，我们使用了一种多克隆抗酪氨酸抗体来检测在胰岛素刺激下酪氨酸磷酸化的其他蛋白质。人胎盘膜糖蛋白经麦芽凝集素层析浓缩后，用[γ-~(32)P]-ATP和胰岛素进行体外磷酸化研究。用抗胰岛素受体抗体和含抗磷酸酪氨酸抗体的血清免疫沉淀磷酸化蛋白。除了胰岛素刺激的胰岛素受体95 kDaβ亚基的磷酸化外，还发现了胰岛素刺激的180 kDa蛋白的磷酸化。这两种蛋白质的磷酸化只发生在酪氨酸残基上。胰岛素使~(32)P掺入180 kDa条带的量增加2.7倍(S.E.M.+/-0.3，n=5)。180 kDa蛋白不被胰岛素受体抗体沉淀。H.P.L.C.磷酸化的180 kDa蛋白质的胰酶片段和胰岛素受体的β亚基的色谱图显示，这两种蛋白质的模式不同。胰岛素刺激的180 kDa蛋白的磷酸化也可以在未分离的洗涤剂增溶的膜中检测到。180 kDa蛋白的磷酸化被胰岛素以与β亚基磷酸化相同的剂量-反应曲线刺激，提示该蛋白可能是胰岛素受体激酶的另一种内源性底物。
The beta-subunit of the insulin receptor contains a tyrosine-specific protein kinase. Insulin binding activates this kinase and causes phosphorylation of the beta-subunit of the insulin receptor. It is believed that phosphorylation of other proteins might transmit the insulin signal from the receptor to the cell. In the present study we used a polyclonal anti-phosphotyrosine antibody to detect other proteins that become tyrosine phosphorylated upon insulin stimulation. Glycoproteins from human placenta membranes were enriched by wheat germ agglutinin chromatography and phosphorylation was studied with [gamma-32P]ATP and insulin in vitro. Phosphorylated proteins were immunoprecipitated by antibodies against the insulin receptor and by serum containing the anti-phosphotyrosine antibody. Beside the insulin-stimulated phosphorylation of the 95 kDa beta-subunit of the insulin receptor, an insulin-stimulated phosphorylation of a 180 kDa protein was found. The phosphorylation of both proteins occurred only on tyrosine residues. Insulin increased 32P incorporation into the 180 kDa band 2.7-fold (S.E.M. +/- 0.3, n = 5). The 180 kDa protein was not precipitated by antibodies against the insulin receptor. H.p.l.c. chromatograms of tryptic fragments of the phosphorylated 180 kDa protein and of the beta-subunit of the insulin receptor revealed different patterns for both proteins. Insulin-stimulated phosphorylation of the 180 kDa protein was also detectable in unfractionated detergent-solubilized membranes. The phosphorylation of the 180 kDa protein was stimulated by insulin with the same dose-response curve as the phosphorylation of the beta-subunit, suggesting that this protein might be another endogenous substrate of the insulin receptor kinase.