CHARACTERIZATION OF THE PROTEIN-KINASE ACTIVITY OF AVIAN-SARCOMA VIRUS SRC GENE-PRODUCT

The avian sarcoma virus src gene product, p60(src), has been purified 650-fold from cytoplasmic extracts of the rat tumor cell line RR1022 by using ammonium sulfate fractionation, hydrophobic chromatography on ω-aminohexyl agarose, and ion exchange chromatography on phosphocellulose. Partially purified p60(src) is a monomer, with a native molecular weight of about 60,000 and an apparent pI of 6.0. In immunoprecipitates, p60(src) catalyzed phosphorylation of anti-p60(src) IgG heavy chains within the variable (V(H)) domain, which contains the heavy chain portion of the antigen combining site. Crude preparations of p60(src) contained phosphatase activity able to cleave phosphate from Igg heavy chains; this activity was removed by the purification procedure, and partially purified p60(src) could phosphorylate the heavy chain of specific antibody in solution. Furthermore, purified p60(src) catalyzed phosphorylation in solution of the general protein kinase substrate, α-casein, strengthening the hypothesis that it may in fact function as a protein kinase in vitro.