Naldini L, Stacchini A, Cirillo D M, Aglietta M, Gavosto F, Comoglio P M
Mol Cell Biol. 1986 May;6(5):1803-11. doi: 10.1128/mcb.6.5.1803-1811.1986.
Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene.
抗磷酸酪氨酸抗体是一种强大的工具，可用于鉴定在酪氨酸残基上磷酸化的蛋白质，例如病毒癌基因编码的转化蛋白及其细胞蛋白底物。在用人类白血病细胞系进行检测时，磷酸酪氨酸抗体在K562细胞（一种源自费城染色体（Ph）阳性慢性粒细胞白血病（CML）的细胞系）中识别出一种分子量为210,000的蛋白质（p210），但在对照的Ph阴性非CML白血病细胞中未识别出任何蛋白质。在免疫复合物激酶分析中，p210蛋白也被抗v-abl编码多肽的抗血清识别，并显示出激酶活性，可使自身酪氨酸磷酸化。这些数据与报道的在Ph阳性CML细胞中重组bcr-abl基因表达的结果一致，导致合成具有酪氨酸激酶活性的改变的p210c-abl蛋白。磷酸酪氨酸抗体还检测到在急变期CML患者采集的新鲜骨髓细胞中p210c-abl蛋白的表达。除了p210c-abl蛋白激酶外，磷酸酪氨酸抗体在K562细胞中还识别出分子量为110,000、68,000和36,000的其他蛋白质（p110、p68和p36）。当将[γ-32P]ATP添加到非离子去污剂提取的细胞中时，经磷酸氨基酸分析证实，这些蛋白质在酪氨酸上发生了磷酸化。与由v-abl、v-src和v-fps癌基因转化的成纤维细胞进行比较表明，p36蛋白与病毒癌基因编码的酪氨酸激酶的常见36千道尔顿蛋白底物相同。因此，细胞蛋白酪氨酸磷酸化增强是v-abl转化细胞和表达重排bcr-abl基因的细胞共有的特征。
