Stürzbecher H W, Montenarh M, Henning R
Department of Biochemistry, University of Ulm, Federal Republic of Germany.
Virology. 1987 Oct;160(2):445-55. doi: 10.1016/0042-6822(87)90016-x.
We have studied the phosphorylation of cellular phosphoproteins and, in more detail, of SV40 T antigen and the cellular protein p53 in SV40 tsA-transformed cells. As detected by radiolabeling cold-sensitive tsA1499- or heat-sensitive tsA58-transformed rat fibroblasts with [32P]orthophosphate or by in vitro labeling extracts with [gamma-32P]ATP the hyperphosphorylation of certain cellular phosphoproteins including p53 and also of free SV40 large T antigen and T antigen complexed with p53 is strictly correlated with the expression of the transformed phenotype. This hyperphosphorylation can be observed as early as 30 min after shifting to the temperature where the cells expressed the transformed phenotype and, furthermore, it is dependent on protein synthesis. To evaluate the influence of a functional T antigen and to exclude properties of individual transformants we 32P labeled in vitro cellular proteins from rat F111, mouse NIH 3T3, and monkey TC-7 cells infected with tsA58 or tsA1499. In tsA58-infected cells we found a heat-sensitive enhancement of protein phosphorylation just as in tsA58 transformants. In tsA1499-infected monkey cells we observed a heat-sensitive and in abortively infected rat or mouse cells a cold-sensitive hyperphosphorylation of proteins. Thus in tsA-transformants and in various tsA-infected cells we found a strong correlation among the transformed phenotype, functions of T antigen, and the phosphorylation of various cellular proteins and in particular T antigen and p53.
我们研究了细胞磷蛋白的磷酸化情况，更详细地研究了SV40 tsA转化细胞中SV40 T抗原和细胞蛋白p53的磷酸化。通过用[32P]正磷酸盐对冷敏感的tsA1499或热敏感的tsA58转化的大鼠成纤维细胞进行放射性标记，或用[γ-32P]ATP对体外标记提取物进行检测，发现某些细胞磷蛋白（包括p53）以及游离的SV40大T抗原和与p53复合的T抗原的过度磷酸化与转化表型的表达密切相关。这种过度磷酸化最早可在转移到细胞表达转化表型的温度后30分钟观察到，此外，它还依赖于蛋白质合成。为了评估功能性T抗原的影响并排除单个转化体的特性，我们对感染了tsA58或tsA1499的大鼠F111、小鼠NIH 3T3和猴TC-7细胞的细胞蛋白进行了体外32P标记。在感染tsA58的细胞中，我们发现蛋白质磷酸化有热敏感增强，就像在tsA58转化体中一样。在感染tsA1499的猴细胞中，我们观察到蛋白质有热敏感过度磷酸化，而在流产感染的大鼠或小鼠细胞中，蛋白质有冷敏感过度磷酸化。因此，在tsA转化体和各种tsA感染的细胞中，我们发现转化表型、T抗原的功能以及各种细胞蛋白（特别是T抗原和p53）的磷酸化之间存在很强的相关性。
