Detection of phosphotyrosine‐containing proteins in the detergent‐insoluble fraction of RSV‐transformed fibroblasts by azobenzene phosphonate antibodies.
Detection of phosphotyrosine‐containing proteins in the detergent‐insoluble fraction of RSV‐transformed fibroblasts by azobenzene phosphonate antibodies.
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通过偶氮苯膦酸抗体检测 RSV 转化的成纤维细胞的去污剂不溶部分中含有磷酸酪氨酸的蛋白质。
作者：
P. Comoglio;M. F. Renzo;G. Tarone;F. Giancotti;Luigi Naldini;Pier Car lo Marchisio
已知劳斯肉瘤病毒 (RSV) 癌基因产物 pp60src 通过在酪氨酸残基处磷酸化宿主细胞靶分子来触发转化表型的获得。为了鉴定含磷酸酪氨酸的蛋白质，针对合成半抗原对偶氮苯膦酸酯 (ABP) 产生了兔抗体，该半抗原与磷酸化酪氨酸发生特异性交叉反应。通过对从 RSV 转化的小鼠成纤维细胞中提取的蛋白质进行免疫修饰并将其转移到硝酸纤维素片上，鉴定出 130、70 和 60 kd 的磷蛋白。发现这些分子与不溶于非离子洗涤剂的细胞部分相关。此外，ABP 抗体沉淀了 130、70 和 60 kd 的去污剂不溶性蛋白质，以及 85 和 65 kd 的两种附加成分，这些成分在体外被 [gamma-32P]ATP 在允许 pp60src 催化激酶反应的条件下磷酸化。密切相关分子的磷蛋白。重量。由 RSV 转化的禽类成纤维细胞进行免疫沉淀。在二维色谱中，放射性与真实的磷酸酪氨酸共同迁移。 60-kd 蛋白与 pp60src 共迁移，而 130-kd 蛋白与纽蛋白之间的同一性因缺乏与适当抗血清的交叉反应而被反驳。在转化的小鼠和鸭成纤维细胞中，间接免疫荧光显微镜中使用的 ABP 抗体对细胞质进行了广泛染色，并强烈装饰了腹侧细胞质膜的限制区域。这些数据表明，与磷酸酪氨酸反应的抗体可有效用于识别和细胞内定位作为pp60src蛋白激酶的潜在靶标的分子。
The Rous sarcoma virus (RSV) oncogene product pp60src is known to trigger the acquisition of the transformed phenotype by phosphorylating host cell target molecule(s) at tyrosine residues. To identify phosphotyrosine‐containing proteins, rabbit antibodies were raised against the synthetic hapten p‐azobenzene‐phosphonate (ABP) that specifically cross‐reacts with phosphorylated tyrosine. By immuno‐decoration of proteins extracted from RSV‐transformed mouse fibroblasts and transferred to nitrocellulose sheets, phosphoproteins of 130, 70 and 60 kd were identified. These molecules were found to be associated with the cellular fraction insoluble in non‐ionic detergent. Moreover, ABP antibodies precipitated detergent‐insoluble proteins of 130, 70 and 60 kd, plus two additional components of 85 and 65 kd, that had been phosphorylated in vitro by [gamma‐32P]ATP under conditions allowing the kinase reaction catalyzed by pp60src. Phosphoproteins of closely related mol. wts. were immunoprecipitated from RSV‐transformed avian fibroblasts. The radioactivity co‐migrated with authentic phosphotyrosine in two‐dimensional chromatography. The 60‐kd protein comigrated with pp60src, while the identity between the 130‐kd protein and vinculin was disproved by the lack of cross‐reaction with appropriate antisera. In transformed mouse and duck fibroblasts ABP antibodies, employed in indirect immunofluorescence microscopy, stained diffusely the cytoplasm and intensely decorated restricted areas of the ventral cell plasma membrane. These data show that antibodies reacting with phosphotyrosine may be usefully employed in the identification and in the intracellular localization of molecules that are potential targets of the pp60src protein kinase.