Rous sarcoma virus transforming protein, p60^src, expressed in E. coli, functions as a protein kinase

Abstract

The product of the Rous sarcoma virus (RSV) transforming gene, src, is a phosphoprotein of molecular weight (M_r) 60,000 (pp60^src)^1–3 that is responsible for cell transformation as well as fibrosarcoma formation in a variety of animals^4,5. Several experiments suggest that pp60^src is a protein kinase^6–10 with the unusual capacity of phosphorylating tyrosine residues^11,12, but additional evidence would be of value. Earlier studies had suggested that bacteria were unable to carry out protein phosphorylation¹³, although more recent findings indicate that distinct protein kinases and phosphatases are present in Salmonella typhimurium¹⁴. Despite this observation, because of the evolutionary distance, the enzymatic activities observed in bacteria are likely to be carried out by proteins unrelated to those expressed in eukaryotic cells. Thus, to study the RSV transforming protein synthesized in the absence of the variety of protein kinases expressed in normal host cells (for reviews see refs 15, 16), we have now constructed plasmids that express p60^src in Escherichia coli. Analysis of p60^src produced in E. coli and selected by immunoaffinity chromatography, shows that it has the capacity to phosphorylate proteins at tyrosine residues; this activity is specifically inhibited by anti-p60^src IgG. Extracts from E. coli carrying identical plasmids but lacking the src gene yield no detectable enzyme activity. These data, taken with those previously published^9,10,17, lead to the conclusion that the RSV src gene encodes a protein kinase.