src gene product from different strains of avain sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective temperature-sensitive mutant and wild-type virus

摘要

Sera from certain rabbits bearing Schmidt-Ruppin strain Rous sarcoma virus (RSV)-induced tumors precipitated p60^src 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 avain sarcoma virus (ASV)], the molecular weights (Mrs) ranging from 60,000 to 64,000. The p60^src immunoprecipitated from cells transformed by each of these strains incorporated [γ-³²P]ATP into the Mr 53,000 subunit of IgG, though with differing activities. No such protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) was observed when the following immunoprecipitates were used: from uninfected cells, from untransformed cells infected by Rous-associated virus, or from cells transformed by acute leukosis viruses, avian erythroblastosis virus, or myelocytoma virus 29. The kinase reaction had a pH optimum at pH 5.9 and an apparent Km for ATP of 4.9 ± 2 μM, and was dependent on Mg²⁺ (Kb = 46 ± 12 mM), for which Ca²⁺ was no substitute. The kinase was cyclic AMP independent.In order to test whether the protein kinase reaction is directly catalyzed by p60^src, 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 p60^src. Concomitant with the loss of the kinase activity by heat inactivation, p60^src loses 60-70% of its phosphate content. The kinetics of dephosphorylation exactly parallel those for the inactivation of the kinase activity, suggesting that the p60^src kinase is itself dependent on phosphorylation for its activity.