Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.
Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene.
D000596:Amino Acids / Q000032:analysis; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001853:Bone Marrow / Q000737:chemistry / Q000201:enzymology; D002460:Cell Line; D016044:Fusion Proteins, bcr-abl; D006801:Humans; D007951:Leukemia, Myeloid / Q000201:enzymology*; D009363:Neoplasm Proteins / Q000032:analysis*; D010761:Phosphorus Radioisotopes; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis*; D012016:Reference Values; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D000596:Amino Acids; D000906:Antibodies; D000936:Antigen-Antibody Complex; D009363:Neoplasm Proteins; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl