Pp60 src-Dependent protein phosphorylation in membranes from Rous sarcoma virus-transformed chicken embryo fibroblasts

Abstract

The Rous sarcoma virus (RSV)-transforming protein, pp60 src , is a plasma membrane-associated tyrosine-specific protein kinase. A 36,000-Da cellular polypeptide (p36) which is phosphorylated at tyrosine in RSV-transformed chicken embryo fibroblasts (RSV-CEF) is also plasma membrane associated. To determine if p36 is directly phosphorylated by pp60 src , and to examine the effects of mutations within src on pp60 src phosphorylation and kinase activity in situ in the plasma membrane, src-dependent protein phosphorylation in membranes isolated from RSV-CEF has been characterized. These membrane preparations contained high ATPase and phosphoprotein phosphatase activities; but when sufficient concentrations of [γ- 32P]ATP were used, the phosphorylation of pp60 src and the phosphorylation of p36 were linear for 1 min or more, and the initial rates of phosphorylation could therefore be determined. In membranes from RSV-CEF pp60 src and p36 became phosphorylated predominantly at tyrosine, while in membranes from uninfected cells p36 was phosphorylated at low levels at serine. When membranes from RSV-CEF were preincubated with tumor-bearing rabbit (TBR) serum, the IgG became phosphorylated while the phosphorylation of p36 was inhibited, suggesting that p36 is directly phosphorylated by pp60 src . Phosphorylation of pp60 src , p36, and TBR-IgG was dependent on growth temperature in membranes from cells infected by a temperature-sensitive mutant, tsNY68, although some dependence on growth temperature was observed even with membranes from wild-type RSV-infected cells. However, at the nonpermissive temperature, tsNY68 pp60 src retained 20–40% of its kinase activity, providing supporting for the proposal (B. M. Sefton, T. Hunter, and K. Beemon (1980, J. Virol, 33, 220–229) that transformation may result from a small quantitative change in pp60 src activity. The phosphorylation of pp60 src and its kinase activity were not coordinately affected by growth temperature or mutations within src, indicating that different factors affect the phosphoacceptor capacity and kinase activity of the protein.