Rous sarcoma virus-induced phosphorylation of a 50,000-molecular weight cellular protein

Abstract

Transformation by Rous sarcoma virus (RSV) is mediated by a single virus-encoded polypeptide, pp60^src (refs 1–3), which has protein kinase activity specific for tyrosine residues^4–7. The identification of the proteins phosphorylated by pp60^src is important in understanding the events involved in RSV-mediated transformation. One potential substrate is a 50,000-molecular weight (MW) phosphotyrosine-containing protein (pp50) which is associated in a complex with pp60^src and a cellular 90,000-MW protein (pp90; ref. 8) and thus is immunoprecipitated by antibody to pp60^src (refs 6, 9, 10). We have performed further studies to determine whether this 50,000-MW protein (pp50) is present in uninfected cells and whether its phosphorylation at tyrosine depends on RSV-induced transformation. Here we demonstrate that a phosphoserine-containing form of pp50 is present in uninfected chicken cells and that on transformation by RSV, a second form of pp50, detectable by two-dimensional analysis of ³²P-labelled proteins, contains phosphotyrosine as well as phosphoserine. This phosphorylation of pp50 at tyrosine is not temperature sensitive (ts) in cells infected with mutant viruses containing a ts defect in the src gene. Previous results⁸ have suggested that the temperature stability of this phosphorylation might be a result of the strong interaction between the mutant pp60^src protein and pp50.