Characterization of the protein kinase activity of avian sarcoma virus src gene product.

Abstract

The avian sarcoma virus [ASV] src gene product, p60src, was purified 650 fold from cytoplasmic extracts of the rat tumor cell line RR1022 [ASV induced cell line] by using ammonium sulfate fractionation, hydrophobic chromatography on .omega.-aminohexyl agarose and ion exchange chromatography on phosphocellulose. Partially purified p60src is a monomer with a native MW of about 60,000 and an apparent pI [isoelectric point] of 6.0. In immunoprecipitates, p60src catalyzed phosphorylation of anti-p60src Ig[immunoglobulin]G H chains within the variable (VH) domain, which contains the H chain portion of the antigen combining site. Crude preparations of p60src contained phosphatase activity able to cleave phosphate from IgG H chains; this activity was removed by the purification procedure and partially purified p60src could phosphorylate the H chain of specific antibody in solution. Purified p60src catalyzed phosphorylation in solution of the general protein kinase substrate, .alpha.-casein, strengthening the hypothesis that it may in fact function as a protein kinase in vivo.