Selective phosphorylation of cytosol proteins associated with transformation and restoration of normal phenotype in AKR mouse embryo fibroblasts.

Phosphoproteins from cytosol preparations of methylcholanthrene-transformed AKR mouse (AKR-MCA) cells were compared to those of their untransformed counterparts, AKR-2B cells, by two-dimensional electrophoresis following an in vitro 32P phosphorylation procedure using endogenous kinases and substrates. Five proteins were phosphorylated in the AKR-MCA cells which were not observed in the AKR-2B cells, while six proteins were phosphorylated in the untransformed cells which were not observed in the malignant cells. Treatment of AKR-MCA cells with 1% N,N-dimethylformamide induced the reversion of the malignant cells to a phenotype similar to that of untransformed AKR-2B cells (S. Chakrabarty et al., Cancer Res., 44: 2181, 1984). Treatment of AKR-MCA cells with dimethyl formamide resulted in the restoration of five of the AKR-2B-associated phosphorylations and abolished 2 of the AKR-MCA-associated phosphorylations. AKR-2B cells have been shown to respond to transforming growth factors with reversible phenotypic transformation (R. F. Tucker et al., Cancer Res., 43: 1581, 1983). Transforming growth factor treatment of AKR-2B cells induced all five of the AKR-MCA-associated phosphoproteins and the loss of all six of the AKR-2B phosphoproteins. Epidermal growth factor treatment of AKR-2B cells resulted in the phosphorylation of several proteins which were not observed in either AKR-MCA or untreated AKR-2B cells. Some, but not all, of the AKR-2B-associated phosphorylations were also observed in epidermal growth factor-treated cells. The results of these studies demonstrated qualitative and/or quantitative changes in cytosolic protein kinase-phosphatase activities between transformed and normal AKR-2B cells. Treatment of AKR-MCA cells with dimethylformamide resulted in the restoration of some of the normal AKR-2B cell-associated protein kinase-phosphatase activities.