Antibodies to a defined region of pp60src neutralize the tyrosine-specific kinase activity.
Site-specific antibodies to pp60src, the transforming protein of Rous sarcoma virus (RSV), have been prepared by immunizing rabbits with a chemically synthesized pentadecapeptide corresponding to residues 498-512 (Cys-Trp-Arg-Lys-Asp-Pro-Glu-Glu-Arg-Pro-Thr-Phe-Lys-Tyr-Leu) as deduced from the nucleotide sequence of the Prague C src gene. Antibodies specific for the synthetic peptide were purified from immune sera by affinity chromatography on peptide-bound Sepharose and characterized by a number of immunocytochemical techniques. Immunoprecipitation and Western blot analyses of normal and RSV-transformed cell lines revealed that this peptide antibody identified the authentic viral src gene product. This finding was further supported by indirect immunofluorescence on RSV-transformed rat kidney cells. The anti-peptide antibodies produced dramatic intracellular staining patterns characteristic of the src protein. Although able to immunoprecipitate pp60src, in vitro kinase reactions indicated that, unlike sera from RSV-induced tumor-bearing rabbits, the peptide antibody did not serve as a phosphate acceptor in the immunocomplex. Moreover, immunoprecipitates of pp60src prepared from this site-specific immune reagent were unable to phosphorylate exogenously added casein or the synthetic peptide substrate, Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly. In contrast, pp60src-containing immunoprecipitates made from an anti-peptide serum specific for the COOH-terminal six amino acids (residues 521-526), a region only eight amino acids removed, readily phosphorylated both substrates. This evidence indicates that an antibody directed against residues 498-512 neutralizes the kinase activity of pp60src and suggests that this region may be functionally necessary for the tyrosine-specific kinase activity of this transforming protein.
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002364:Caseins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007668:Kidney / Q000201:enzymology; D009842:Oligopeptides / Q000378:metabolism; D015688:Oncogene Protein pp60(v-src); D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D011817:Rabbits; D051381:Rats; D014764:Viral Proteins / Q000276:immunology*
D000906:Antibodies; D002364:Caseins; D009842:Oligopeptides; D014764:Viral Proteins; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)