Cooper J A, Hunter T
Salk Institute, San Diego, California 92138.
Mol Cell Biol. 1981 Feb;1(2):165-78. doi: 10.1128/mcb.1.2.165-178.1981.
Rous sarcoma virus encodes a tyrosine-specific protein kinase (p60src) which is necessary for cell transformation. To identify substrates for this kinase, we set out to detect phosphotyrosine-containing proteins in Rous sarcoma virus-transformed chicken embryo cells, making use of the known alkali stability of phosphotyrosine. 32P-labeled phosphoproteins were separated by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were then incubated in alkali. Using this procedure with normal cells, we detected a total of about 190 alkali-resistant phosphoproteins. In Rous sarcoma virus-transformed cells, five phosphoproteins were found which were not detectable in normal cells. Two of these are probably structural proteins of the virus. The other three transformation-dependent phosphoproteins, and four other phosphoproteins which were elevated by transformation, all contained phosphotyrosine. Increased phosphorylation of these proteins did not occur with cells infected with a mutant Rous sarcoma virus, temperature sensitive for transformation, grown at the restrictive temperature. We conclude that these seven proteins are probably substrates of p60src, although they may be substrates for other tyrosine-specific protein kinases activated by p60src.
劳氏肉瘤病毒编码一种酪氨酸特异性蛋白激酶（p60src），它是细胞转化所必需的。为了鉴定这种激酶的底物，我们利用磷酸酪氨酸已知的碱稳定性，着手检测劳氏肉瘤病毒转化的鸡胚细胞中含磷酸酪氨酸的蛋白质。用等电聚焦和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离32P标记的磷蛋白。然后将凝胶在碱中孵育。用这种方法处理正常细胞时，我们总共检测到约190种耐碱磷蛋白。在劳氏肉瘤病毒转化的细胞中，发现了五种在正常细胞中无法检测到的磷蛋白。其中两种可能是病毒的结构蛋白。另外三种依赖转化的磷蛋白，以及另外四种因转化而增加的磷蛋白，都含有磷酸酪氨酸。用对转化温度敏感的突变劳氏肉瘤病毒感染细胞，并在限制温度下培养，这些蛋白质的磷酸化没有增加。我们得出结论，这七种蛋白质可能是p60src的底物，尽管它们可能是被p60src激活的其他酪氨酸特异性蛋白激酶的底物。
