Visualization of phosphotyrosine containing molecules within the detergent insoluble cell matrix of v-src transformed fibroblasts

The RSV oncogene v-src is known to transform host cells through the action of a single gene protein product (pp60src) endowed with tyrosine specific kinase activity. The nature and cellular localization of substrates of pp60src kinase are largely unknown. Detergent insoluble cell matrix was prepared by treating RSV-transformed mouse fibroblasts with the non-ionic detergent NP-40. These preparations, highly enriched in cytoskeletal proteins, when observed in the E M consisted mainly of a meshwork of filaments; fragments of plasma membrane and nuclear "ghosts" were also present. Antibodies against phosphotyrosine -previously shown to be reactive with protein phosphorylated at tyrosine residues- were prepared and affinity purified using a synthetic hapten (azobenzyl phosphonic acid, ABP). By means of the immunogold techniques applied to electron microscopy, phosphotyrosine containing molecules were found to be present in RSV transformed, but not in control fibroblasts. Gold particles were mostly bound to electron dense granular material associated with the filaments. These results are consistent with the idea that pp60src itself and some tyrosine phosphorylated proteins may be found among detergent-insoluble cell structures.