Guanine nucleotide-binding activity as an assay for src protein of rat-derived murine sarcoma viruses

Abstract

We have recently identified a 21,000-dalton protein, p21, coded for by Kirsten or Harvey murine sarcoma virus. On the basis of the results obtained with the p21 of a mutant of Kirsten sarcoma virus, temperature sensitive for the maintenance of transformation, we concluded that the p21 was required for the maintenance of transformation induced by either virus. We report herein that when extracts from cells transformed by Kirsten or Harvey sarcoma virus are incubated with [(3)H]GDP or [alpha-(32)P]GTP, picomole quantities of guanine nucleotide can be immunoprecipitated with antisera that contain antibodies to the p21. Previously we have shown that the immunoprecipitability of [(35)S]methionine-labeled p21 of the temperature-sensitive mutant of Kirsten sarcoma virus is thermolabile. The binding of guanine nucleotide is shown herein also to be thermolabile in extracts of cells transformed by the same mutant. However, the immunoprecipitability of the [(35)S]methionine-labeled p21 in such extracts of the temperature-sensitive mutant can be preserved if the extracts containing labeled p21 are incubated with added GDP or GTP prior to heating. The results suggest an interaction between p21 and certain guanine nucleotides, and the possible roles of guanine nucleotides and p21 in the maintenance of transformation are discussed.