Evidence that the src gene product of Rous sarcoma virus is membrane associated

We have investigated the intracellular localization of the src phosphotransferase and of pp60^src by cellular fractionation. Fractionation of cell lysates by differential centrifugation showed that both the phosphotransferase activity, measured using an immune complex assay, and pp60^src cosedimented with particulate fractions enriched in cellular membranes. Upon further fractionation of particulate fractions by equilibrium centrifugation in discontinuous sucrose gradients the src phosphotransferase and pp60^src fractionated in parallel with plasma membranes. The phosphotransferase activity remained associated with cellular membranes under conditions of high ionic strength or of high concentrations of divalent cation chelators further supporting a direct membrane association. The src phosphotransferase activity is higher in buffer containing only Triton X-100 detergent as compared to a more complicated detergent mixture, but under these assay conditions the pp60^scr appears to undergo proteolysis to a 52K-dalton polypeptide (pp52^src) which is active as a phosphotransferase. Pyrimidine triphosphates were shown to act as donors in the immune complex assay of src phosphotransferase activity. [γ-³²P]CTP was about one-third as efficient as [γ-³²P]ATP in serving as a donor. Both CTP and UTP were effective inhibitors of src phosphotransferase activity when either radiolabeled ATP or GTP were used as substrates. The ability of src to interact efficiently with both purine and pyrimidine triphosphates contrasts markedly with most well-studied protein kinases which have been shown to use almost exclusively purine triphosphates.