Enhanced phosphorylation of many endogenous protein substrates in human fibroblasts transformed by simian virus 40

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Abstract
Protein phosphorylation in normal and in SV-40-transformed human skin fibroblasts was assessed by 2 different methods: incubation of whole-cell homogenates with [.gamma.-32P]ATP or labeling of intact cells with Na2H32PO4. Phosphorylated proteins were detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. With both methods, the Coomassie-blue-stained protein patterns of the 3 transformed cell lines studied were similar to the patterns of the nontransformed normal human cells. Although the phosphoprotein autoradiograms of the 3 transformed cell lines were nearly identical, their patterns were strikingly different from those of the nontransformed cells. Each of the 3 transformed lines tested showed approximately 25-30 phosphoprotein bands that were significantly enhanced when compared to the patterns of the nontransformed cells. Quantitation of 12 of the enhanced phosphoprotein bands in one of the transformed cell lines showed an average of 4.4 times as much phopshorylation as in the normal cells. The enhanced phosphorylation observed in the transformed cell lines was not dependent on the growth rate of the cells or on cyclic AMP. When homogenates of transformed and nontransformed cells were mixed prior to incubation with [.gamma.-32P]ATP, the resultant phosphoprotein patterns resembled those obtained with transformed cells alone. An evaluation of the time course of protein phosphorylation revealed that the initial reaction rate was greater in the transformed than in the normal cells, although in both cell types the reaction was complete after 1 min. The SV-40-transformed human fibroblasts possess an increased ability to phosphorylate proteins rather than that the normal cells possess a diffusible inhibitor. There appear to be many endogenous cellular substrates for this increased activity.