Transformation by Rous sarcoma virus: Effects of src-gene expression on the synthesis and phosphorylation of cellular polypeptides

To investigate the effects of transformation on the synthesis and phosphorylation of cellular polypeptides, we have used two-dimensional polyacrylamide gel electrophoresis to examine the radioactively labeled polypeptides of chick embryo fibroblasts infected either with nondefective (nd) RSV or with transformation-defective (td) or temperature-sensitive mutants. Infection by RSV does not appear to induce the de novo synthesis or the complete suppression of any of the [³⁵S]methionine-labeled cellular polypeptides that can be resolved with this technique; however, there are quantitative changes in a minor fraction (approximately 4%) of the [³⁵S]methionine-labeled polypeptides. When cells labeled with [³²P]orthophosphate were examined, a phosphorylated polypeptide (M(r)=36,000) was detected in transformed cells, in addition to a poorly resolved streak of spots (M(r)=60,000), which may represent the product of the src gene (pp60(src)). the 36,000-M(r) phosphorylated polypeptide appears within 20 minutes when cells infected by a temperature-sensitive mutant of RSV are shifted from the nonpermissive to the permissive temperature; this change occurs even when protein synthesis is inhibited by cycloheximide. Phosphorylation of the 36,000-M(r) polypeptide thus represents an early event in the process of transformation, and it is possible that this polypeptide is a target for the kinase activity associated with pp60(src).