Immunological detection of proteins phosphorylated at tyrosine in cells stimulated by growth factors or transformed by retroviral-oncogene-coded tyrosine kinases.
The receptors for polypeptide growth factors and proteins coded by oncogenes of the src family are endowed with protein kinase activity and share the uncommon property of autophosphorylating at tyrosine residues. It is unclear whether the tyrosine kinase activity is also directed towards other targets of physiological significance. In this work, phosphotyrosine antibodies were used to detect, by Western blots and immunoprecipitation, proteins phosphorylated at tyrosine in fibroblasts either stimulated by growth factors (PDGF and EGF) or transformed by oncogene-coded tyrosine kinases. In stimulated cells the antibodies detected the autophosphorylated receptors, but only trace amounts of other proteins phosphorylated at tyrosine. In fibroblasts transformed by retroviral oncogenes (v-src, v-abl, v-fps or v-fes) proteins other than the corresponding oncogene-coded kinase, were found. A p70 was found to be heavily phosphorylated in fibroblasts transformed by v-src, v-fes and v-fps. A p130 and a p36 were found in cells transformed by v-src and v-abl. A unique p70 was phosphorylated in v-abl-transformed fibroblasts. These proteins were also phosphorylated in vitro in an immunocomplex kinase reaction. This reaction was blocked by the specific kinase inhibitors. These data strongly suggest that tyrosine kinases phosphorylate protein targets other than themselves. These targets are barely detectable in normal cells stimulated by growth factors, where the kinase activity is triggered rapidly and transiently. By contrast, a number of intracellular proteins phosphorylated at tyrosine accumulate in cells transformed by v-onc-coded kinases, endowed with constitutive and non-regulated enzymatic activity.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D066246:ErbB Receptors; D006133:Growth Substances / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D015688:Oncogene Protein pp60(v-src); D009856:Oncogene Proteins, Viral / Q000032:analysis*; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000032:analysis*; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D011956:Receptors, Cell Surface / Q000032:analysis; D017479:Receptors, Platelet-Derived Growth Factor; D012191:Retroviridae Proteins / Q000032:analysis; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology
D000906:Antibodies; D006133:Growth Substances; D009856:Oncogene Proteins, Viral; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; D012191:Retroviridae Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D015688:Oncogene Protein pp60(v-src)