Effect of transformation of chicken cells by Rous sarcoma virus on in vitro phosphorylation of nuclear non-histone proteins

Abstract

The use of virus temperature-sensitive (ts) mutants for transformation allows us to determine the effect of transformation on different alterations in cell morphology and cell functions. We studied the kinase activity of nuclear non-histone proteins (NHP) extracted from chicken embryo fibroblasts (CEF), both infected and non-infected with a ts mutant of Rous sarcoma virus (Schmidt Ruppin strain, subgroup A, T class) and cultivated at the restrictive (41°C) and permissive (37 °C) temperature for transformation.The endogenous in vitro phosphorylation of NHP extracted from stationary ts-infected cultures increased by 300% when the cells were cultivated at 37 instead of 41°C. This increase is probably a consequence of the expression of transformation, since it was not observed when NHP were extracted from non-infected CEF maintained either at 41 or 37 °C. The increase in endogenous phosphorylation of NHP does not seem to be entirely due to a change in the density-dependent inhibition of cell metabolism in transformed cells, as an increase in protein phosphorylation was observed when the ts cultures were in both exponential growth phase and plateau phase.The urea-polyacrylamide gel electrophoresis of 32P-phosphorylated proteins showed that the increase in phosphorylation observed when growing ts cells were maintained at 37 instead of 41 °C was mostly due to the stimulation of one fraction (fraction d), as if the increase in phosphorylation of this fraction was linked to the expression of transformation. When the ts cultures were in stationary phase, the enhancement of the phosphorylation of proteins observed when cells were maintained at 37 instead of 41 °C was due to stimulation of the phosphorylation of all protein fractions, and particularly of fractions d and b.When phosvitin was used as a substrate, the kinase activity of NHP extracted from ts-infected cells was increased 300% when the cells were cultivated at 37 instead of 41 °C. On the other hand, the phosvitin kinase activity of NHP extracted from non-infected cells decreased when the cells were maintained at 37 instead of 41 °C. Thus, the transformation of cells induced an increase in the phosvitin kinase activity of nuclear NHP.When ts cells were cultivated at 41 °C until saturation density and then transferred to 37 °C for 3 h before extraction of proteins, a 37% increase in phosvitin kinase activity of NHP was observed, as compared with the activity of proteins extracted from cells left at 41 °C. Thus, the change in kinase activity is an early event in the process of transformation.