Giordano S, Di Renzo M F, Ferracini R, Chiadò-Piat L, Comoglio P M
Department of Biomedical Sciences and Oncology, University of Torino Medical School, Turin, Italy.
Mol Cell Biol. 1988 Aug;8(8):3510-7. doi: 10.1128/mcb.8.8.3510-3517.1988.
A protein with an Mr of 145,000 (p145) was detected by antibodies to phosphotyrosine by Western blot (immunoblot) analysis. This protein was phosphorylated on tyrosine in a gastric carcinoma cell line. In cells that were metabolically labeled with 32Pi, this protein was phosphorylated on tyrosine and serine. p145 is a cysteine-rich transmembrane glycoprotein. The extracellular domain could be labeled by 125I under nonpermeating conditions and was cleaved by mild trypsin treatment of intact cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed a shift of p145 mobility to an apparent Mr of 190,000. After immunoprecipitation with phosphotyrosine antibodies, p145 displayed a strong associated protein kinase activity in vitro, becoming phosphorylated on tyrosine. There was no immunological cross-reaction between p145 and known tyrosine kinases. Both in vivo and in vitro tyrosine phosphorylations were unaffected by the addition of known growth factors. However, p145 was rapidly dephosphorylated in vivo when cells were exposed to low pH, a condition that is known to dissociate ligands from their receptors. These data suggest that p145 is associated with a protein tyrosine kinase activity which, in the tumor cell line studied, is activated by an as yet unidentified factor.
通过蛋白质免疫印迹（免疫印迹）分析，用抗磷酸酪氨酸抗体检测到一种分子量为145,000的蛋白质（p145）。该蛋白质在胃癌细胞系中酪氨酸发生了磷酸化。在用32Pi进行代谢标记的细胞中，该蛋白质的酪氨酸和丝氨酸均发生了磷酸化。p145是一种富含半胱氨酸的跨膜糖蛋白。在非通透条件下，其细胞外结构域可被125I标记，完整细胞经温和胰蛋白酶处理后可被切割。在非还原条件下进行的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示，p145的迁移率发生改变，表观分子量变为190,000。用磷酸酪氨酸抗体进行免疫沉淀后，p145在体外显示出强烈的相关蛋白激酶活性，酪氨酸发生了磷酸化。p145与已知的酪氨酸激酶之间没有免疫交叉反应。体内和体外的酪氨酸磷酸化均不受已知生长因子添加的影响。然而，当细胞暴露于低pH环境（已知该条件会使配体与其受体解离）时，p145在体内会迅速去磷酸化。这些数据表明，p145与一种蛋白质酪氨酸激酶活性相关，在所研究的肿瘤细胞系中，该活性由一种尚未确定的因子激活。
