Protein kinase activity associated with the avian sarcoma virus src gene product.

Abstract

Incorporation of phosphorus from [y-30PJATP into protein was catalyzed by specific immunoprecipitates from avian sarcoma virus (ASV-transformed avian and mammalian cells. This incorporation was observed only when antiserum from tumor-bearing rabbits able to specifically precipitate the ASV sarcoma gene product, p~o srC was used to immunoprecipitate antigens from transformed cell lysates. Immunoprecipitates of extracts from normal cells or cells infected with a transformation-defective ASV mutant showed no activity in this assay, nor did any immune complexes formed with normal rabbit serum and any of the cell extracts tested. The expression of the protein-kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) was growth temperature-dependent in cells infected with an ASV mutant temperature-sensitive for transformation. These results on an enzymatic activity associated with the ASV transforming protein are discussed in terms of protein phosphorylation as a mechanism for viral transformation.Four genes have been identified and mapped on the avian sarcoma virus (ASV) genome (1, 2). Three of these genes-gag, pol, and env-code for the virion structural proteins, including the group-specific (gs) viral core proteins, the RNA-directed DNA polymerase, and the envelope glycoproteins, respectively. The fourth gene, designated src, has been identified through the isolation of temperature-sensitive (ts) and deletion mutants defective in transformation in vitro and sarcoma induction in vivo (3-7).Recent work from this laboratory has resulted in the identification of a protein of molecular weight (Mr) 60,000 that appears to be the product of the ASV src gene (8-10). Determination that this protein is actually the product of the src gene is based on the following data: (i) It was detected as a nonstructural, transformation-specific antigen in ASV-transforned chicken cells, ASV-transformed mammalian cells, and ASVinduced mammalian tumor cells, by immunoprecipitation of radiolabeled cell extracts with serum from ASV-tumor-bearing rabbits. (ii) In vitro cell-free translation of the 3' third of nondefective ASV viral RNA, the region of the genome that contains the src gene, resulted in the synthesis of a polypeptide of Mr 60,000. (iii) The polypeptide of Mr 60,000 made in vitro by cell-free translation and the transformation-specific antigen isolated by immunoprecipitation of all types of ASV-infected cell extracts tested are identical as determined by peptide analyses (ref. 10; J. S. Brugge, E. Erikson, M. S. Collett, and R. L. Erikson, unpublished results). We feel that it is consistent with these data to conclude that the protein of Mr 60,000 is the product of the ASV src gene and consequently give it the following designation: p60src.In order to further elucidate the mechanism of ASV-induced Preparation of Antiserum and Immunoprecipitation. Antiserum was obtained from New Zealand rabbits in which tumors had been induced by injection of purified SR-ASV as previously described (8, 13). Several preparations of antis...

Kinase Inhibitors: Not Just for Kinases Anymore

“…[1][2][3] Small-molecule kinase inhibitors are the subject of much interest, both as potential therapeutics and as experimental tools for understanding the physiological roles of these enzymes. Accordingly, much research over the past 2 decades has been devoted to the identification and development of such compounds; currently, 20-30% of pharmaceutical discovery programs are focused on kinases.…”

Src Points the Way to Biomarkers and Chemotherapeutic Targets

“…15 Both v-Src and c-Src encode a 60-kDa phosphoprotein with tyrosine kinase activity. [16][17][18][19] However, unlike v-Src, c-Src has low transforming ability unless it is overexpressed and/or mutated. 10 As with most tumor cells, Srctransformed cells display characteristic phenotypes that include rounded morphology, increased motility, uncontrolled proliferation, anchorage independence, and growth factor independence.…”

Comparative studies on the src proto-oncogene and its gene product pp60c-src in normal and neoplastic tissues of lower vertebrates

“…Soluble protein (0.2 mg) was incubated with 5 ,ul TBR-serum for at least 60 min at 4°C and precipitated with the protein Acontaining bacteria Staphylococcus aureus. The bacterial bound immunocomplex was washed and the protein kinase assay was carried out by a modification (Barnekow and Bauer, 1984) of the method of Collett and Erikson (1978). The assay is based on the phosphorylation of the heavy chain (53 K) of IgG in the immune complex by the precipitated pp60srr after addition of y( 32 P)ATP in the presence of bivalent cations.…”