转化淋巴细胞胰岛素受体酪氨酸蛋白激酶活性及细胞内源性底物的研究

Changes in Activity of Insnlin Receptor Tyrosine Kinase and Characterization of Its Endogenous Substrate in Transformed Human Lymphocytes

本文对增殖期的淋巴细胞胰岛素依赖性酪氨酸蛋白激酶活性及内源性废物进行了分析研究。在纯化的健康人淋巴细胞中加入适量的植物血凝素(PHA),经过72h培养即成为转化淋巴细胞(增殖期淋巴细胞)。应用^(32)P参入实验,证实转化淋巴细胞胰岛素受体具有胰岛素依赖性的酪氨酸蛋白激酶活性,与未转化的对照组相比其活性增加约9倍。Scatchard分析表明转化后淋巴细胞膜表面胰岛素受体数增加3.5倍。应用抗酪氨酸磷酸酯抗体,对胰岛素作用前后的转化与未转化淋巴细胞内,酪氨酸残基磷酸化的蛋白进行了鉴定,结果表明:除了95kD受体β亚基自身磷酸化外,45kD蛋白质也明显磷酸化,我们命名它为PP45。我们认为PP45可能是淋巴细胞中胰岛素受体酪氨酸蛋白激酶的主要内源性废物,它的磷酸化是胰岛素信息传递过程级联反应的初始步骤。

The cascade reaction of tyrosine phosphorylation provides an attractive mechanism to regulate the metabolism and growth of normal and transformed cells.Thus,considerable effort has been made to identify the cellular substrates of the tyrosine kinases.In this work,the activity of the insulin receptor tyrosine kinase and endogenous substrate in transformed lymphocytes were studied.Purified human lymphocytes incubated with phytohemagglutinin (PHA) for 72h served as transformed cells,when labeled with (32P)-orthophosphate it appeared that the insulin-dependent protein kinase activity in the transformed cells increased 9-fold.In search for the physiologically significant substrates by using polyclonal antiphosphotyrosine antibody to imm-unoprccipitate phosphotyrosine-containing proteins that produced in the intact cell during insulin stimulation,a protein with molecular weight of 45kD was identified and designated as PP45,whicn occured during the 5 min response of lymphocytes to insulin.