Sera from certain rabbits bearing SchmidtRuppin strain Rous sarcoma virus (RSV)induced tumors precipitated p6Osrc from chicken cells transformed by the homologous virus as well as by other strains [Prague strain RSV, Bryan high-titer strain RSV, and Bratislava 77 strain of avian sarcoma virus (ASV)J, the molecular weights (Mrs) In order to test whether the protein kinase reaction is directly catalyzed by p6Osrc, we compared the in vitro temperature sensitivities of the kinase activities from cells infected by transformation-temperature-sensitive mutant and parental wild-type virus. The first-order rate constant for the inactivation of the kinase from extracts of cells infected by the mutant virus was 2-fold greater than that from cells infected by wild-type virus. This result implicates the protein kinase as an enzymatic activity of the src gene product, the p60src. Concomitant with the loss of the kinase activity by heat inactivation, p60src loses 60-70% of its phosphate content. The kinetics of dephosphorylation exactly parallel those for the inactivation of the kinase activity, suggesting that the p6osrc kinase is itself dependent on phosphorylation for its activity. In recent papers, Erikson and coworkers have shown that sera from rabbits bearing tumors induced by the Schmidt-Ruppin (SR) strain of Rous sarcoma virus (RSV) precipitated a sarcoma virus-specific protein of molecular weight (Mr) 60,000 from extracts of SR-transformed chicken embryo cells (1), as well as from various mammalian cells transformed by the same virus (2). The in vitro cell-free translation of the 3' one-third of the RSV genome, the region known to. contain the viral transforming information [the src gene (3, 4)1, yielded a protein of the same molecular weight and similar peptide composition (5, 6). Hence, this protein is thought to be responsible for RSVinduced cell transformation and has been named p6Osrc. A protein kinase activity is associated with the p6Osrc, because immunoprecipitates of p60src incorporate 32P from [y-32P]ATP into the Mr 53,000 subunit of IgG (7). This activity was greatly reduced (though not completely lost) in immunoprecipitates prepared from cells that were infected by a transformationdefective temperature-sensitive (ts) mutant of avian sarcoma virus (ASV) when the cells were grown at the nonpermissive temperature for transformation. These data were interpreted to indicate that the kinase activity found in immunoprecipitates of p60src is an enzymatic activity of the protein and not of a contaminant (7).In the present study we show that p60srC can be precipitated by certain rabbit sera from extracts of cells infected by several different strains of ASV and that all these immunoprecipitates exhibit a kinase activity. p60src proteins from cells infected with wild-type and transformation-defective (T class) ts mutants of RSV were compared with respect to protein kinase inactivation kinetics at 42°C. The results obtained show that, after in vitro incubations, the protein kinase activity from cells infecte...

Using chicken embryo fibroblasts infected with the NY68 transformation‐defective temperature‐sensitive mutant of Rous sarcoma virus, the phosphorylation and enzyme kinetic properties of enolase have been studied before, and at different stages after, the onset of transformation. A method for purification of enolase was developed, which minimized dephosphorylation. Two enolase (EC 4.2.1.11) isoenzymes were separated by isoelectric focussing revealing that it was the gammagamma form (pI 5.2‐6.7) which had become phosphorylated at tyrosine residues after transformation. The phosphorylation of enolase in tyrosine occurred slowly after shift to the permissive temperature, rising from undetectable levels in phenotypically normal cells, to < 10% of the total phosphoamino acid after 3 h, and reaching 30‐50% of the total phosphoamino acid by 16 h. Interestingly, the fraction of phosphorylated enolase molecules declined during transformation from 8% in normal cells to 5% by 16 h after temperature shift, due to a 3‐ to 5‐fold increase in the total amount of enolase present in the transformed cultures. Although transformation had no apparent effect on the K0.5 of enolase (26 +/‐ 4 microM for 2‐phosphoglycerate), its specific activity was reduced by about one third.

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A cellular protein of apparent Mr 34,000-36,000 was suggested as a possible physiological substrate for the protein kinase (EC 2.7.1.37) activity associated with the transforming gene product of Rous sarcoma virus. We find this protein to migrate with an apparent Mr of38,000 in NaDodSO4polyacrylamide gels.