Two cellular proteins that immunoprecipitate with the transforming protein of Rous sarcoma virus

The transforming gene (src) of Rous sarcoma virus encodes a 60,000-dalton phosphoprotein (pp60^src) with the ability to phosphorylate tyrosine in certain protein substrates. The enzymatic activity of pp60^src is thought to mediate neoplastic transformation by src. It would therefore be useful to identify cellular proteins that interact with pp60^src on the chance that these proteins might be substrates for the kinase activity of the viral protein or be otherwise involved in neoplastic transformation of the host cell. In pursuit of this objective, we characterized the proteins that coprecipitate with pp60^src in immune complexes. These proteins proved to be of two types. (i) Most immune complexes contained a series of proteins (50,000 to 58,000 daltons) that were apparently derived from pp60^src by sequential degradation from the amino terminus. We do not know if this degradation has a physiological purpose in the infected cell, but it has at least two practical implications: it has proved useful in the analysis of the functional topography of pp60^src; and it can give rise to experimental artifacts in the analysis of proteins obtained from cells infected with Rous sarcoma virus. (ii) Two proteins (50,000 and 89,000 daltons) coprecipitated with pp60^src, probably by virtue of their ability to bind to the viral protein. Both proteins are phosphorylated, both are encoded by the cellular genome, and both can be recovered from either avian or mammalian cells transformed by Rous sarcoma virus. The 89,000-dalton protein contains phosphoserine, irrespective of its source, and its structure is otherwise highly conserved among widely diverged vertebrate species. By contrast, the forms of the 50,000-dalton protein recovered from chicken and rat cells can be readily distinguished by their peptide maps and by their phosphoamino acids (the avian form of the protein contains both phosphoserine and phosphotyrosine, whereas the mammalian form contains only phosphoserine). We used temperature-sensitive mutants in src to explore the possibility that the two cellular proteins might be substrates for the protein kinase activity of pp60^src: propagation of infected cells at the nonpermissive temperature failed to affect the phosphorylation of either of the proteins. We conclude that at least two cellular proteins are associated with pp60^src prior to immunoprecipitation with antisera directed against the viral protein. It is possible that neither of these proteins is a substrate for the protein kinase activity of pp60^src, however, and their role in neoplastic transformation by src (if any) remains moot.