Yee S P, Branton P E
J Cell Physiol. 1981 Jan;106(1):149-63. doi: 10.1002/jcp.1041060116.
Normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts growing on plastic dishes were incubated with ATP (gamma 32P) in situ to detect external cell surface protein kinase activity. Under the conditions employed, 32P was incorporated exclusively into proteins, specifically those at the external cell surface, as radioactivity was removed by trypsin treatment of labeled whole cells. In addition, exogenous histones were phosphorylated when added to the reaction mixture. Cyclic nucleotides had virtually no effect on 32P incorporation, suggesting that little or no cyclic nucleotide-dependent protein kinase activity was present at the external cell surface. Cell surface protein kinase activity was higher in transformed than in normal cells, and, using a temperature-sensitive RSV src mutant, this difference was shown to be transformation-specific. Several differences were observed in the cell surface proteins phosphorylated in normal and transformed cells and at least two of these were transformation-specific. These data suggest that changes in external cell surface protein phosphorylation are associated with RSV transformation and thus could play a role in the formation of the transformed cell phenotype.
将生长在塑料培养皿上的正常鸡胚成纤维细胞和劳斯肉瘤病毒（RSV）转化的鸡胚成纤维细胞原位与ATP（γ-32P）一起温育，以检测细胞外表面蛋白激酶活性。在所采用的条件下，32P仅掺入蛋白质中，特别是那些细胞外表面的蛋白质，因为用胰蛋白酶处理标记的完整细胞可去除放射性。此外，当向反应混合物中加入外源性组蛋白时，其会被磷酸化。环核苷酸对32P掺入几乎没有影响，这表明在细胞外表面几乎不存在或不存在环核苷酸依赖性蛋白激酶活性。转化细胞的细胞表面蛋白激酶活性高于正常细胞，并且使用温度敏感的RSV src突变体，这种差异显示为转化特异性。在正常细胞和转化细胞中磷酸化的细胞表面蛋白中观察到了几个差异，其中至少两个是转化特异性的。这些数据表明，细胞外表面蛋白磷酸化的变化与RSV转化有关，因此可能在转化细胞表型的形成中起作用。
