Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton.
Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton.
复制标题
DOI：
10.1083/jcb.108.6.2401
复制
发表时间：
1989-06
影响因子：
7.8
通讯作者：
Zokas, L
中科院分区：
文献类型：
--
作者：
Glenney, J R Jr;Zokas, L
我们之前曾报道过针对磷酸酪氨酸的单克隆抗体的生产，磷酸酪氨酸是许多癌基因产物和生长因子受体诱导的修饰。其中一种抗磷酸酪氨酸抗体 (py20) 用于亲和层析，从劳斯肉瘤病毒转化的鸡胚成纤维细胞 (RSV-CEF) 中分离出含有磷酸酪氨酸 (PY) 的蛋白质。用这些 PY 蛋白对小鼠进行免疫，以产生针对各个底物的单克隆抗体。以这种方式产生了十五种针对分子量为215、76、60和22 kD的抗原的抗体。使用针对各个底物的抗体来分析正常和 RSV-CEF 中的亚细胞位置。当用于免疫荧光显微镜时，215-和76-kD抗原的抗体对焦点接触染色，而抗22-kD蛋白抗体导致点状染色集中在细胞边缘和平行阵列中。两种分布在转化细胞中都发生了改变。当在稳定细胞骨架的条件下用非离子去污剂提取细胞时，50% 的 76-kD 蛋白质和大于 90% 的 22-kD 蛋白质与细胞骨架分离。这项研究为鉴定成纤维细胞中的膜骨架蛋白以及激活酪氨酸激酶转化后发生的变化提供了一种新方法。
We have previously reported the production of monoclonal antibodies directed against phosphotyrosine, which is the modification induced by many oncogene products and growth factor receptors. One of these antiphosphotyrosine antibodies (py20) was used in affinity chromatography to isolate phosphotyrosine (PY)-containing proteins from Rous sarcoma virus-transformed chick embryo fibroblasts (RSV-CEFs). Mice were immunized with these PY-proteins for the production of monoclonal antibodies to individual substrates. Fifteen antibodies were generated in this way to antigens with molecular masses of 215, 76, 60, and 22 kD. Antibodies to individual substrates were used to analyze the subcellular location in normal and RSV-CEFs. Antibodies to the 215- and 76-kD antigen stained focal contacts when used in immunofluorescence microscopy while anti-22-kD protein antibodies resulted in punctate staining concentrated in the margins of cells and in parallel arrays. Both distributions were altered in transformed cells. When cells were extracted with nonionic detergent under conditions that stabilize the cytoskeleton, 50% of the 76-kD protein and greater than 90% of the 22- kD protein fractionated with the cytoskeleton. This study offers a new approach to both the identification of membrane skeletal proteins in fibroblasts and changes that occur upon transformation by an activated tyrosine kinase.